Telomeres, Telomerase and Disease

An EMBO Conference Series
Telomeres, Telomerase and Disease
30 April - 4 May 2014
Brussels, Belgium
Organizers
Peter Baumann
HHMI, Stowers Institute for Medical Research
Anabelle Decottignies
de Duve Institute
Jan Karlseder
The Salk Institue for Biological Studies
Telomeres, Telomerase and Disease
Schedule Outline
Wednesday 30th April
15:00
17:30
19:30
19:45
21:00
-
18:00
19:30
19:45
20:45
22:00
Arrival and registration
Cocktails and dinner
Opening remarks
Keynote Session: Titia de Lange
Session 1: Telomere dynamics
Thursday 1st May
09:00
12:30
14:00
16:00
18:30
20:00
-
12:30
13:30
16:00
18:00
19:45
21:30
Session 2: Telomere dysfunction and disease
Lunch
Session 3: Telomerase assembly and activation
Poster Session 1
Dinner
Session 4: Telomere replication
Friday 2nd May
09:00
12:45
14:00
16:00
18:00
-
12:45
14:00
16:00
18:00
Session 5: Alternative mechanisms of telomere maintenance
Lunch
Poster Session 2
Free time
Belgium beer tasting and off-site dinner
Saturday 3rd May
09:00
12:30
13:45
16:00
18:00
19:00
19:30
-
12:30
13:30
15:30
17:45
19:00
19:30
22:00
Session 6: DNA damage response at telomeres
Lunch
Session 7: Telomeres and non-coding RNAs
Session 8: Structure and function
Keynote Session: Elizabeth Blackburn
Meeting discussion and closing remarks
Banquet dinner
Sunday 4th May
Breakfast and departure
1
Table of Contents
Schedule Summary
1
Sponsors
3
Meeting Details
Arrival Information
Departure Information
Breakfast
Social Event
4
5
5
6
Meeting Policy
7
Complete Schedule
8
Abstracts
Speaker Abstracts
Poster Abstracts
23
73
Participant List
205
Reference Maps
217
2
Sponsors
We would like to thank the following organizations for their generous support of this conference:
We would also like to thank the following institutions for lending administrative time and support:
de Duve Institute
Salk Institute for Biological Studies
Stowers Institute for Medical Research
3
Arrival Information
Husa President Park Hotel
Blvd du Roi Albert II
44-1000 Brussels, Belgium
Tel: +32 (2) – 203 20 20
Fax: +32 (2) – 203 24 40
[email protected]
For those arriving at Brussels International Airport:
th
We are offering bus transportation between Brussels International Airport and Husa President Park on April 30
during peak arrival times. To take the bus from the airport to the hotel, you will meet near the Java Café on Level
2. A map of the airport is shown below for your convenience. Someone holding a sign reading “Telomeres,
Telomerase & Disease” near the café will be able to direct you from there.
The first bus will depart for Husa around 10:30. Buses will also leave the airport around 12:30, 14:30, and 16:30,
but exact departure times will depend on when a bus is full. Note that the last bus is scheduled to depart around
16:30. If you arrive later than this, you will need to use public transportation (see below) or a taxi.
If you are lost in the airport, you can call Florian Poulain (member of the Decottignies lab): 32-497-795930.
4
Additional Transportation Information:
If you need to arrange your own transportation, you can find additional information here:
http://www.brusselsairport.be/en/passngr/to_from_brussels_airport/train/
The airport train station is located below the terminal (basement level-1). Up to four trains an hour connect the
airport to Brussels North, Brussels Central and Brussels Midi stations.
You should take the train to Brussels North. Then, the hotel is only a 8-9 minute walk away. Please refer to the
‘Reference Maps’ section at the end of the abstract book for a map.
Departure Information
Buses will also be provided to shuttle participants between Husa President Park Hotel and Brussels International
th
Airport on Sunday, May 4 during peak departure times. The first bus will depart for the airport around 5:30.
Buses will also leave for the airport around 7:30, 9:30, and 11:30.
Breakfast
Breakfast will be offered each morning of the conference at Husa President Park. There are two restaurants
serving breakfast at Husa President Park, Maison Blanche and St-Roch. Both restaurants will offer the same
breakfast.
5
Social Event
nd
Friday, 2 May | Visit of the Grand-Place, Beer-tasting session, and Dinner
Transportation
There will be 2 bus transfers between Husa President Park and the Grand-Place. These will occur at 16:30 and
17:00. Room is limited to 50 people per bus. We expect that many participants will want to walk the short distance
(about 2 km) or take the metro (from “Gare du Nord” to “Rogier” – Line 3).
Beer-tasting sessions
There will be 5 consecutive beer-tasting sessions, at the times listed below. There is a maximum of 50 people at
each session.
17:30 18:00 18:30 19:00 19:30
The beer-tasting session will take place on the Grand-Place itself, in a place called the “Museum of the Beer”. This
is indicated as Arbre d’Or on the picture below.
Dinner
The off-site dinner will be at La Manufacture, located near the Grand-Place.
Restaurant La Manufacture
Rue Notre Dame du Sommeil 12
1000 Bruxelles
Tel: +32 (0)2 502 25 25
There will be bus transfers between La Manufacture and Husa President Park at the following times:
22:30
23:00
23:30
Participants may also like to have an after-dinner drink in one of the many nearby bars and return to the hotel on
foot or by taxi in time for the morning session. Please refer to the ‘Reference Maps’ section at the end of the
abstract book for a map.
6
Meeting Policy
Information presented during the meeting is considered private information.
Therefore, the use of audio, photographic, or video recording equipment in any
of the sessions is strictly prohibited.
Please make sure that cell phones, computers, and PDAs taken into the meeting
room are kept in ‘silent’ mode during the sessions.
Meeting participants will be given name badges at the start of the conference.
These must be worn to all sessions, meals, and social activities. Participants not
wearing name badges may not be allowed into the meeting room. Accompanying
guests will not be allowed into the meeting room. However, guests registered with
the conference are welcome to attend the meals and social events.
7
Wednesday, April 30th
15:00 - 18:00
Lobby
Registration
17:30 - 19:30
Mathilde
Cocktails and Dinner
19:30 - 19:45
Fabiola
Opening Remarks
19:45 - 20:45
Fabiola
Keynote Session, Titia de Lange
The repression of PARP1 at telomeres
Lecture sponsored by Abcam
21:00 - 22:00
Fabiola
Session 1: Telomere dynamics
Chair: Anabelle Decottignies
Julie Cooper
Riff’ing on the final step of telomere segregation at anaphase
Susan Smith
The role of telomere cohesion in aging and ALT cells
Woody Wright
Telomere looping: A new paradigm for the regulation of gene expression with
progressive telomere shortening
Thursday, May 1st
09:00 - 10:30
Fabiola
Session 2a: Telomere dysfunction and disease
Chairs: Sara Selig and Martin Kupiec
Steven Artandi
A TERT promoter gradient enables the isolation and purification of
spermatogonial stem cells
Duncan Baird
Telomere dysfunction accurately predicts clinical outcome in two common
cancers
Nya Nelson
The complex and differing roles of TIN2 isoforms in telomere regulation
Simon Boulton
The function and execution of RTEL1 activities at vertebrate telomeres
10:30 - 11:00
Mat, Fab,
Fab II
Coffee break
8
11:00 - 12:30
Fabiola
Session 2b: Telomere dysfunction and disease
Sharon Savage
Dyskeratosis congenita caused by an OB fold mutation in TPP1
Maria Blasco
Role of telomeres in disease and longevity
Mary Armanios
Disease-specific manifestations of the telomere syndromes
12:30 - 13:30
Mathilde
Lunch
14:00 - 16:00
Fabiola
Session 3: Telomerase assembly and activation
Chairs: Tracy Bryan and Daniela Rhodes
Vicky Lundblad
Regulated assembly and disassembly of the yeast telomerase quaternary complex
Kazunori Tomita
Two-step mechanism of telomerase recruitment and activation in fission yeast
Kathleen Collins
Assembly, Architecture and Function of Human Telomerase
Dirk Hockemeyer
The essential role of TPP1 in telomerase dependent telomere maintenance
Jens Schmidt
Identification of the site on telomerase that binds TPP1 to promote recruitment to
telomeres
Julian Chen
A self-regulating template in human telomerase
16:00 - 18:00
VIP, Fab II
Poster Session 1, odd numbered posters, refreshments will be served
18:30 - 19:45
Mathilde
Dinner
20:00 - 21:30
Fabiola
Session 4: Telomere replication
Chairs: Tracy Bryan and Daniela Rhodes
Toru Nakamura
Negative regulation of telomerase by SUMOylation-dependent Tpz1(TPP1) – Stn1
interaction in fission yeast
Carolyn Price
Roles of human CST in telomere replication and genome-wide replication rescue
David Shore
Rif1 controls DNA replication timing and anti-checkpoint functions through
recruitment of the PP1 phosphatase, Glc7
9
Friday, May 2nd
09:00 - 10:30
Fabiola
Session 5a: Alternative mechanisms of telomere maintenance
Chairs: Alessandro Bianchi and Lee Wong
Roger Reddel
Long-term proliferation of human cancer cells in the absence of telomere length
maintenance
Miguel Godinho Ferreira
Absence of telomerase causes the early onset of cancer in zebrafish
Vincent Géli
The role of SUMO pathway in telomere recombination
Roger Greenberg
Interchromosomal homology searches drive directional ALT telomere movement
and synapsis
10:30 - 11:00
11:00 - 12:45
Mathilde,
Fab, Fab II
Fabiola
Coffee break
Session 5b: Alternative mechanisms of telomere maintenance
Paulina Marzec
Genome instability driven by targeted telomere insertion in ALT cells
Harikleia Episkopou
Alternative Lengthening of Telomeres is characterized by reduced compaction of
telomeric chromatin
Rajika Arora
A complex interplay between telomere transcription, stability and recombination
in human ALT cells
Hilda Pickett
NuRD-ZNF827 recruitment to telomeres creates a molecular scaffold for
homologous recombination
Aaron Mendez Bermudez
TRF2 is required to protect heterochromatic pericentromeric regions from
topological and replicative DNA damage
Geneviève Almouzni
The multifaces of chromatin assembly, a recipe that mixes new with old partners
12:45 - 14:00
Mathilde
Lunch
14:00 - 16:00
VIP, Fab II
Poster Session 2, even numbered posters, refreshments will be served
16:00
18:00
Free time
Belgian beer tasting and off-site dinner
10
Saturday, May 3rd
09:00 - 10:30
Fabiola
Session 6a: DNA damage response at telomeres
Chairs: Maria Teresa Teixeira and Ted Weinert
Eros Lazzerini Denchi
Characterization of the changes in telomeric chromatin upon DNA damage
induction
Makoto Hayashi
Mitotic telomere deprotection dictates cellular fate upon mitotic arrest
Lea Harrington
An optimal telomere length protects human tumor cells from ionizing radiation
Kate Clark
Interactions between CST, DDR, and NMD genes show that Stn1 and Ten1 are the
key components of the CST complex
10:30 - 11:00
11:00 - 12:30
Mathilde,
Fab, Fab II
Fabiola
Coffee break
Session 6b: DNA damage response at telomeres
Karl Lenhard Rudolph
Delineating the role of upstream DNA damage responses in cell and tissue aging
induced by telomere shortening
Jacqueline Jacobs
Identification of a critical regulator of DNA repair activity at mammalian
telomeres
Agnel Sfeir
DNA polymerase theta promotes alternative-NHEJ at dysfunctional telomeres
Brian Luke
Rapamycin prevents checkpoint adaptation via autophagy and Cdc5 (PLK1),
which increases proliferative potential following telomere dysfunction
Francesca Rossiello
A role for DNA damage response RNAs (DDRNA) at dysfunctional telomeres
12:30 - 13:30
Mathilde
Lunch
13:45 - 15:30
Fabiola
Session 7: Telomeres and non-coding RNAs
Chairs: Jiri Fajkus and Eric Gilson
Paul Lieberman
Telomere Response to Viral Infection
Joachim Lingner
Regulation and Functions of TERRA ncRNA
11
Antonis Kirmizis
Identification of novel telomere regulators through systematic genetic screens in
yeast
Arturo Londono-Vallejo
Human RTEL1 regulates the export and cytoplasmic trafficking of ncRNAs,
including telomerase RNA
Mattia la Torre
AKTIP, an E2 variant enzyme that interacts with lamin, is required for correct
telomere maintenance
15:30 - 16:00
16:00 - 17:45
Mathilde,
Fab, Fab II
Fabiola
Coffee break
Session 8: Structure and function
Chairs: Jiri Fajkus and Eric Gilson
Lili Pan
A billion years of shelterin – conservation of interaction modules and the
potential to form higher order structures
Nicolas Thomä
Examining telomere architecture in yeast
Peter Lansdorp
Guanine quadruplex structures revisited
Omesha Perera
A telomere protective function of human telomerase reverse transcriptase
(hTERT), which is independent of catalytic activity
Ming Lei
Structural basis for protein-RNA recognition in telomerase
18:00 - 19:00
Fabiola
Keynote Session, Elizabeth Blackburn
Regulation of human immune system CD4+ T cell apoptosis specifically by the
RNA component of telomerase
Lecture sponsored by Active Motif
19:00 - 19:30
Fabiola
Meeting discussion and closing remarks
19:30 - 22:00
Mathilde
Banquet dinner
Sunday, May 4th
Breakfast and departure
12
Poster Session 1 | Thursday, May 1st, 16:00 – 18:00
[1]
Detection of DNA damage response RNAs (DDRNAs) at dysfunctional telomeres
Julio Aguado, Francesca Rossiello, Fabrizio d'Adda di Fagagna
[3]
A simple model for tandem telomeric G-quadruplexes
Patrizia Alberti
[5]
Identification of telomere dysfunction in Friedreich ataxia
Sara Anjomani Virmouni, Sahar Al-Mahdawi, Chiranjeevi Sandi, Hemad Yasaei, Predrag Slijepcevic, Mark
Pook
[7]
Retroviral activation of TERT expression in chicken B-cell lymphomas
Karen Beemon, James Justice
[9]
Impact of cancer treatments in telomere length: A prospective study
Carlos Benitez-Buelga, Lara Sanchez-Barroso, Maria Mercedes Gallardo, Maria Apellaniz, Lucia Inglada
Perez, K. Yanowski, Miguel Urioste, Ana Osorio, Maria Antonia Blasco, Cristina Rodriguez-Antona, Javier
Benitez
[11]
Control of telomere replication by phosphorylation
Carol Cooley, Anoushka Dave, Mansi Garg, Resham Lal Gurung, Aziz Muhamad, Alessandro Bianchi
[13]
Mutations in Telomerase Reverse Transcriptase Promoter Contribute to Urothelial Cancer: Mechanism
and Therapeutic Implications
Sumit Borah, Arthur Zaug, Linghe Xi, Natasha Powell, James Costello, Dan Theodorescu, Thomas Cech
[15]
Beneficial testosterone treatment in a mouse model of aplastic anaemia
Christian Bӓr, Fabian Beier, Miguel Foronda, Maria Blasco
[17]
The Effect of Polymerase-alpha on Telomerase Processivity
Chris Caridi, Connie Nugent
[19]
Identification and validation of genome instability drivers in a panel of human cancers
Maria Antonietta Cerone, Nnenna Kanu, Mcgranahan Nicholas, Pierre Martinez, Charles Swanton
[21]
Telomere and mitochondrial dysfunction in Duchenne Muscular Dystrophy
Alex AC Chang, Foteini Mourkioti, Sang-Ging Ong, Joseph Wu, Helen M Blau
[23]
Anti-metastatic factor inhibits telomerase activity in aggressive cancer cells - emerging connection
between telomerase activity and metastasis suppression
Vivek Srivastava, Anirban Kar, Maneesh Kumar, Dhurjhoti Saha, Ankita Singh, Vinod Kumar Yadav,
Shantanu Chowdhury
[25]
Separase is required for telomere protection
Francesca Cipressa, Patrizia Morciano, Giuseppe Bosso, Linda Mannini, Grazia Daniela Raffa, Antonio
Musio, Giovanni Cenci
13
[27]
ALT-specific recruitment of NuRD-ZNF827 to telomeres promotes homologous recombination and
protects against senescence and apoptosis
Dimitri Conomos, Roger Reddel, Hilda Pickett
[29]
RPA and Pfh1(Pif1) helicase prevent secondary structures formation during telomere replication
Julien Audry, Pierre Luciano, Toru Nakamura, Vincent Geli, Stephane Coulon
[31]
Dissecting the link between the long non-coding RNA TERRA and the alternative lengthening of
telomeres mechanism
Katharina Deeg, Karsten Rippe
[33]
Pervasive hallmarks of Alternative Lengthening of Telomeres (ALT) in diploid Sorex granarius (Soricidae,
Eulipotyphla) fibroblast cells
Irena Draskovic, Natalia Zhdanova, Julia Minina, Tatiana Karamysheva, Clara Novo-Lopes, Win-Yan Liu,
Maria Zvereva, Andrey Skvo
-Vallejo
[35]
Molecular Dynamics of Telomeric Heterochromatin Formation
Yi-Min Duan, Yang Zhang, Ting Gong, Jin-Qiu Zhou
[37]
Ssu72 phosphatase regulates telomere length in S. pombe
Jose Miguel Escandell, Clara Reis, Maria Gallo, Miguel Godinho-Ferreira
[39]
HSF1 regulates TERRA transcription upon heat shock
Sivan Koskas, Claire Vourc'h, Virginie Faure
[41]
CBX1 buffers DNA damage responses and senescence induction in response to oncogene activation, γirradiation and telomere dysfunction
T. K. Leucht, S. Foersch, S. Tao, A. Lechel, K. L. Rudolph
[43]
A high content screen for telomerase trafficking regulators uncovers the cytosolic chaperonin TRiC as
essential for TCAB1 folding and telomerase function
Adam Freund, Steven Artandi
[45]
Chromosomal instability and cancer cell stemness in the alternative lengthening of telomeres
Fani-Marlen Roumelioti, Maria Chiourea, Christina Raftopoulou, Sarantis Gagos
[47]
Telomere uncapping impairs K-RasG12V-induced lung carcinogenesis
Maria Garcia-Beccaria, Paula Martinez, Marta Cañamero, Francisca Mulero, Maria Blasco
[49]
Telomeric Protein TRF2 Is an Angiogenic Target of WT1 That Regulates the Expression of PDFGRβ
Mounir El Mai, Kay Wagner, Jean-François Michiel, Valerie Renault, Nicole Wagner, Eric Gilson
[51]
RTEL1 mutations cause Hoyeraal-Hreidarsson syndrome by disrupting an essential function of RTEL1 in
telomerase-dependent telomere maintenance
Galina Glousker, Zhong Deng, Arturo Londoño-Vallejo, Paul M. Lieberman, Yehuda Tzfati
[53]
PARP1 and ATRX are Required for Proper Telomere Length Maintenance
Adam Harvey, Duncan Baird, Eric Hendrickson
14
[55]
Which domains are critical for TRF2-REST interaction?
Milan Hluchý J P eče C
H f
[57]
Telomere transcripts without subtelomere sequences improve targeting of telomerase in cancer cells
Sandra Sampl, Christian Stern, Doris Mejri, Klaus Holzmann
[59]
Human Rap1 Induces TRF2 Release from Telomeric DNA
E š J
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á M ch Z mme m
M
H chý C
H f
[61]
Sequence variants of the telomerase reverse transcriptase (TERT) gene in Nicotiana tabacum
Jana Jureckova, Eva Sykorova, Miloslava Fojtova
[63]
Multiple interactions within a primer influences Saccharomyces castellii telomerase activity in vitro
Cecilia Gustafsson, Ahu Karademir, Roopesh Krishnankutty, Marita Cohn
[65]
Interfacial inhibition of human telomerase reconstitution
Guillaume Kellermann, Markus Kaiser, Florent Dingli, Olivier Lahuna, Florence Mahuteau-Betzer, Damarys
Loewd, Evelyne Ségal-Bendirdjian, Marie-Paule Teulade-Fichou, Sophie Bombard
[67]
Telomerase RNP complex assembly in disease background
Elzbieta Kowalska, Georgeta Zemora, Michael Wildauer, Christina Waldsich
[69]
Telomere Length Kinetics Assay (TELKA) sorts the Telomere Length Maintenance (tlm) mutants into
functional groups
Martin Kupiec, Linda Rubinstein, Yaniv Harari, Vera Babin
[71]
Anticancer mechanism of TMPyP4 in breast cancer cell lines MCF7 and MDA-MB-231
Natalia Lipinska, Hanna Holysz, Aleksandra Romaniuk, Mariusz Kaczmarek, Blazej Rubis
[73]
Identification of the subtelomeric sequence of the long non-coding RNA TERRA unveils its role at
telomeres
Isabel López de Silanes, Osvaldo Graña, Maria Luigia De Bonis, Orlando Domínguez, David G. Pisano, Maria
Antonia Blasco
[75]
Unraveling the changes in telomere composition during senescence, immortalization, and tumorigenic
conversion of normal human fibroblasts
Jana Majerská, Joachim Lingner
[77]
Bridging ageing and metabolism through RAP1?
Paula Martinez, David Pisano, Maria A. Blasco
[79]
HOMOLOGY-DEPENDENT REPAIR IS INVOLVED IN 45S rDNA LOSS IN FAS MUTANTS
Veronika Muchova S m
m
I M zg á M
D řáč á M
E. G eg
Fajkus
Ch
e Wh e J ří
15
[81]
Expression of ATRX represses the Alternative Lengthening of Telomeres phenotype
Christine Napier, Jane Noble, Roger Reddel
[83]
TRF2 Overexpression Induces Telomeric Ultrafine Anaphase Bridges
Bernadette Nera, Hui-Shun Huang, Thao Lai, Lifeng Xu
[85]
Selective Bioluminogenic HDAC Activity Assays for Profiling HDAC Inhibitors
Andrew L. Niles, Nathan J. Evans, Kevin R. Kupcho, Thomas A. Kirkland, and Dan F. Lazar
[87]
Androgenetic fish as models in studies concerning healing of chromosome breaks by de novo telomere
addition
Konrad Ocalewicz
[89]
Unusual telomeres in plants
Vratislav Peska, Jiri Fajkus
[91]
Long-term particulate air pollution in association with the mitochondrial-telomere axis of ageing
Nicky Pieters, Bram G. Janssen, Karen Smeets, Harrie Dewitte, Bianca Cox, Michelle Plusquin, Ann
Cuypers, Tim S. Nawrot
[93]
Preclinical assessment of the telomerase inhibitor Imetelstat combined with radiotherapy in an
orthotopic model of human glioblastoma
Sylvain Ferrandon, Céline Malleval, Priscillia Battiston-Montagne, Badia El Hamdani, Radu Bolbos, Patrick
Manas, Sergei M Gryaznov, Claire Rodriguez-Lafrasse, Jérôme Honnorat, Delphine Poncet, et al.
[95]
Telomere Repeat Binding (TRB) proteins serve as functional components of telomeres and interact with
telomerase (TERT)
Petra Prochazkova Schrumpfova, Ivona Vychodilova, Martina Dvorackova, Jana Majerska, Ladislav
Dokladal, Sarka Schorova, Jiri Fajkus
[97]
Effects of dyskeratosis congenita patient mutations in hTERT on telomerase dimerization and activity
Alix Christen, Sophie Redon, Sascha Feuerhahn, Joachim Lingner
[99]
MTV, a telomeric ssDNA-binding complex, protects Drosophila telomeres and recruits retro-transposon
to chromosome ends
yi zhang, liang zhang, guanjun gao, yikang rong
[101]
Induced pluripotent stem cells as a model for telomeric abnormalities in ICF type I syndrome
Shira Sagie , Erika Ellran, Hagar Katzir, Rony Shaked, Alaa Ghanayim, Maty Tzukerman, Sara Selig
[103]
The inhibition of protein-protein interactions within telomeres: a route to cancer chemotherapy?
Twana Salih, Weng Chan, Lodewijk Dekker, Charles Laughton
[105]
Telomere maintenance mechanisms without telomerase are of clinical relevance in colorectal
carcinoma
Sandra Sampl, Stefan Stättner, Fabienne Bastian, Friedrich Wrba, Brigitte Wolf, Jeremy Henson, Loretta
Lau, Roger Reddel, Klaus Holzmann
[107]
A Shelterin-RTEL1 interaction required for maintaining telomere integrity
Grzegorz Sarek, Stephanie Panier, Jean-Baptiste Vannier, Simon Boulton
16
[109]
A mouse model for ICF syndrome does not recapitulate the telomeric phenotype of human ICF
syndrome patients
Gal Larom, Shiran Yehezkel, Robert J. Duszynski, Lucy A. Godley, Claire Francastel, Guillaume Velasco, Sara
Selig
[111]
Subtelomeric elements of the shortest telomere are required for TERRA expression and buffer
senescence onset
Kamar al zaman Serhal, Marco Graf, Pascale Jolivet, Maria Teresa Teixeira, Brian Luke
[113]
Development of a high-throughput, fluorescence-based, PCR-free method of C-circle detection to
screen drugs and genes targeting the alternative lengthening of telomeres (ALT) pathway in human
cancers
David Halvorsen, Haroldo Silva
[115]
The role of DAXX and ATRX in telomere maintenance
Zhou Songyang, Quanyuan He, Mengfan Tang
[117]
Evidence for telomerase- and Rad52-independent sequence alterations at yeast telomeres
Clémence Claussin, Michael Chang, Sonia Stinus
[119]
in vivo isolation of telomeric proteins in the nematode Caenorhabditis elegans
Sanghyun Sung, Beomseok Seo, Junho Lee
[121]
DNA DAMAGE SIGNALLING IN THE RECRUITMENT OF TELOMERASE TO TELOMERES
Adrian Tong, Josh Stern, Scott Cohen, Xu-Dong Zhu, Tracy Bryan
[123]
Single-molecule imaging of RTEL1 in embryonic stem cells
Evert-Jan Uringa, Andrew Robinson, Karl Duderstadt, Antoine Van Oijen, Peter Lansdorp
[125]
Depression and shortened telomeres: implication on shelterin and telomerase
Yabin Wei, Lena Backlund, Lina Martinsson, Aleksander A. Mathé, Gregers Wegener, Martin Schalling,
Catharina Lavebratt
[127]
Structure-Function studies on Cdc13
Sofiane Y. Mersaoui, Raymund J. Wellinger
[129]
Roles of ATRX and H3.3 to assemble a repressive chromatin state at telomeres
Maheshi Udugama, Fiona Chang, Lyn Chan, James McGhie, Philippe Collas, Jeffrey Mann, Lee Wong
[131]
TPP1 is a substrate for the deubiquitinating enzyme USP7
Ivo Zemp, Patrick Reichenbach, Joachim Lingner
17
Poster Session 2 | Friday, May 2nd, 14:00 – 16:00
[2]
RTEL-1 promotes telomere-ITS recombination events
Megan Brady, Shawn Ahmed
[4]
Severe liver fibrosis caused by S. mansoni infection in Dkc1m hypomorphic mice
Raquel Alves-Paiva, Leandra Ramalho, Cleide Araujo-Silva, Olinda Mara, Vanderlei Rodrigues, Eduardo
Rego, Rodrigo Calado
[6]
Longer telomeres lead to Type II survivors in the absence of telomerase in yeast
Usha Aryal, Phillipa Muston, Edward J Louis
[8]
A novel method of analysis of TANK1 enzymatic activity influence on TRF1 DNA-binding
Petra Bencúrová P
cj
Kł I
eč
á C
H f
[10]
Implication of INT6 in telomere stability
Maname Benyelles, Vincent Mocquet, Serge Bauwens, Pierre Jalinot
[12]
PLATINATION OF TELOMERES BY CIS-PLATINE IS NOT AT THE ORIGINE OF TRF2 DISPLACEMENT
Lina Saker, Samar Ali, Guillaume Kellermann, Evelyne Ségal-Bendirdjian, Joël Poupon, Sophie Bombard
[14]
Regulation of human telomere transcription
Joanna Boros, Anabelle Decottignies
[16]
The putative Leishmania spp. telomerase RNA (TER) undergoes trans-splicing and contains a conserved
template sequence
Elton José Vasconcelos, Vinicius Santana Nunes, Marcelo Santos da Silva, Marcela Segatto, Peter Myler,
Maria Isabel Nogueira Cano
[18]
EXPRESSION OF A SMALL INTERNAL FRAGMENT OF DYSKERIN (GSE24.2), DECREASES DNA DAMAGE,
OXIDATIVE STRESS AND SENESCENCE IN ATAXIA TELANGIECTASIA CELLS
Jaime Carrillo, Laura Pintado Berninches, Cristina Manguán García, Laura Iarriccio, Guillermo Güenechea
Amurrio, Juan A. Bueren, Leandro Sastre, Rosario Perona
[20]
The Ctc1/Stn1/Ten1 complex localizes in APB and regulates C-circle formation in ALT cells
Chenhui Huang, Xueyu Dai, Weihang Chai
[22]
CTC1 Mutations in Telomere Syndromes Impair Telomere Replication
Liuh-yow Chen, Jana Majerská, Joachim Lingner
[24]
Characterization of the binding of the Drosophila telomeric protein Verrocchio to single-stranded DNA
Alessandro Cicconi, Emanuela Micheli, Domenico Raimondo, Giovanni Cenci, Maurizio Gatti, Stefano
Cacchione, Grazia D. Raffa
[26]
TELOMERE DAMAGE AND CHROMOSOME INSTABILITY INDUCED BY ACUTE OXIDATIVE STRESS
Elisa Coluzzi M c C m
e
C zz S ef
e e
h O’C gh
e
Sgura
18
[28]
Shading the shelterin TRFH TRF2 domain through cell-permeable chemical tools: rational design,
synthesis and biological effects at telomeres
Salvatore Di Maro, Pasquale Zizza, Erica Salvati, Bruno Pagano, Clemente Capasso, Luciana Marinelli,
Ettore Novellino, Annamaria Biroccio, Sandro Cosconati
[30]
Cross talk between EBV and telomerase: the role of TERT in the switch of latent/lytic cycle of the virus
Silvia Giunco, Andrea Celeghin, Stefano Indraccolo, Riccardo Dolcetti, Anita De Rossi
[32]
Influence of telomere dynamics on disease progression and response to therapeutics in bone marrow
failure syndromes
Erin Degelman, Nicholas Ting, Tara Beattie
[34]
Telomere Maintenance in Pediatric Medulloblastoma
Matthew Sobo, Kathleen Dorris, Ashley Margol, Charles Stevenson, Shahab Asgharzadeh, Stewart
Goldman, Lili Miles, Arzu Onar-Thomas, Maryam Fouladi, Rachid Drissi
[36]
Role of DNA end-binding in Ku heterodimer function
Charlene Emerson, Christopher Lopez, Albert Ribes-Zamora, Alison Bertuch
[38]
Roles of DNA repair factors in plant telomere maintenance
Lucie Najdekrova, Miloslava Fojtova, Dagmar Zachova, Karel J. Angelis, Jiri Fajkus
[40]
Telomerase is essential for zebrafish heart regeneration
Dorota Bednarek, Juan Manuel González-Rosa, Oscar Gutiérrez-Gutiérrez, Tania Aguado, Gabriela
Guzmán, Alfonso Cortés, Agustín Zapata, Jesús Jiménez-Borreguero, Nadia Mercader, Ignacio Flores
[42]
Epigenetic regulation of telomeres and telomerases in different model plants
Miloslava Fojtova, Pavla Polanska, Eva Majerova, Jana Jureckova, Anna Ogrocka, Jiri Fajkus
[44]
The Diversity and Evolution of Telomeres in Algae
Jana Fulneckova, Tereza Sevcikova, Jiri Fajkus, Alena Lukesova, Marek Elias, Eva Sykorova
[46]
Yeast Rap1 binds dsDNA in multiple binding modes
Erik Feldmann, Roberto Galletto
[48]
Interactions between the Elg1 clamp-unloader and the DNA damage checkpoint kinase Dun1
Inbal Gazy, Martin Kupiec
[50]
Keeping telomere under wraps to avoid DNA damage: a job for TRF2
Delphine Bennaroch-Popivker, Sabrina Pisano, Alexandre Gay, Aaron Mendez-Bermudez, Chrysa Latrick,
Bei Pei, Simona Miron, Marie-Hélène Le Du, Eric Gilson, Marie-Josèphe Giraud-Panis
[52]
Development of a high-content, automated platform for rapid analysis of alternative lengthening of
telomeres (ALT)-associated promyelocytic leukemia nuclear bodies (APBs) in human cancer cells
David Halvorsen, Thomas Hunt, Manali Aggrawal, Haroldo Silva
[54]
Local telomerase over-expression promotes cancer in a tissue-dependent manner
Catarina M. Henriques, Miguel Godinho Ferreira
19
[56]
A rabbit ear mechanism by which human Rap1 modulates TRF2 attraction to telomeric DNA
E š J
š á I
eč
á M ch Z mme m
M
H chý Ctirad Hofr
[58]
Telomerase modulates cyclinD1 gene in concert with NOL1
Juyeong Hong, Jihoon Lee, In Kwon Chung
[60]
Influence of environmental factors on telomerase-independent survivor formation in fission yeast
Kristi Jensen, Baumann Peter
[62]
Fission yeast without subtelomeres
Sanki Tashiro, Yuki Nishihara, Junko Kanoh
[64]
Dissecting the telomeric and non-telomeric roles of human CTC1 through gene disruption
Christopher Kasbek, Jason Stewart, Mary Chaiken, Caitlyn Goodwin, Carolyn Price
[66]
Therapeutic agents influence the TRF2-TIN2 interactions and their cellular distribution: study in human
cancer cell lines
Patrycja Klos, Ctirad Hofr, Ivona Necasova, Jiri Fajkus
[68]
Alternative lengthening of telomeres detected in telomerase-positive canine histiocytic sarcoma
Theresa Kreilmeier, Sandra Sampl, Marlene Hauck, Ingrid Walter, Klaus Holzmann, Miriam Kleiter
[70]
Involvement of SRSF11 in cell cycle-specific recruitment of active telomerase to telomeres at nuclear
speckles
Jihoon Lee, Sun Ah Jeong, Prabhat Khadka, Juyeong Hong, In Kwon Chung
[72]
The Mechanism of Epigenetic Behavior at the Mre11/Rad50 Interface
Danielle Tatum, In-Joon Baek, Heewon Park, Arthur Lustig, et al.
[74]
Epigenetic landscape of terminal and internal telomeric DNA using different plant models
Eva Majerova, Terezie Mandakova, Miloslava Fojtova, Jiri Fajkus
[76]
Differential processing of double strand breaks and telomeres at the nuclear envelope in S. cerevisiae
Isabella Marcomini, Chihiro Horigome, Susan M. Gasser
[78]
Telomere shortening induces skeletal and cardiac muscle failure in Duchenne Muscular Dystrophy
Foteini Mourkioti, Alex Chang, Alan Meeker, Helen Blau
[80]
The effects of lifestyle factors on leukocyte telomere length dynamics: Results from the ESTHER Cohort
Aysel Muezzinler, Aida Karina Dieffenbach, Katja Butterbach, Hermann Brenner
[82]
In vitro assessment of the protective role of Cdc13 against degradation of telomeric single-stranded 3’
overhangs
Saishyam Narayanan, Cecilia Gustafsson, Marita Cohn
[84]
How specific is TRF2 binding to telomeric DNA?
I
eč
á, Michal Zimmermann, Ctirad Hofr
[86]
IDENTIFICATION OF NEW TERT GENE MUTATIONS IN FAMILIAL STUDIES OF PATIENTS PRESENTING
WITH APLASTIC ANEMIA/HYPOCELLULAR MYELODYSPLASTIC SYNDROME
Valeria Nofrini, Valentina Pierini, Francesco Berardinelli, Caterina Matteucci, Valeria Di Battista, Antonella
Sgura, Tamara Iannotti, Alessandro Brozzi, Roberta La Starza, Cristina Mecucci
20
[88]
A new role for histone deacetylase 5 in the maintenance of long telomeres
Catherine Polese, Clara Lopes Novo, Nicolas Matheus, Anabelle Decottignies,
Arturo Londono-Vallejo, Vincent Castronovo, and Denis Mottet
[90]
A study of telomere-related biomarkers in an arsenic-exposed Bangladeshi population
Brandon Pierce, Shantanu Roy, Farzana Jasmine, Muhammad Kibriya, Habibul Ahsan
[92]
Mitochondria dysfunction in telomerase zebrafish mutants
Inês Pimenta de Castro, Madalena Carneiro, Miguel Godinho Ferreira
[94]
Human RTEL1 is required for the maintenance of long telomeres
Rosa Maria Porreca, Christian Naucke, Mike Schertzer, Mylène Perderiset, Irena Draskovic, Arturo
Londoño-Vallejo
[96]
In vivo selection for new telomerase RNA template regions in S. pombe
Margaret Pruitt, Richard Dannebaum, Peter Baumann
[98]
Quantile regression for flow-FISH telomere length measurement as a diagnostic tool for telomere
diseases
Fernanda Gutierrez Rodrigues, Priscila Santos Scheucher, Edson Zangiacomi Martinez, Bárbara Amélia
Aparecida Santana, Rodrigo Tocantins Calado
Telomere length dynamics and hematopoietic differentiation in human DKC1-mutant iPSCs
Flavia Sacilotto Donaires, Thomas Winkler, Cynthia Dunbar, Rodrigo Tocantins Calado
[100]
[102]
Studies of G quadruplex-hRPA interactions in the telomere maintenance context
Layal Safa, Jean-François Riou, Carole Saintomé
[104]
Uncapped telomeres as determinant for cell sensitivity to G-quadruplex ligands
Erica Salvati, Angela Rizzo, Sara Iachettini, Pasquale Zizza, Chiara Cingolani, Carmen D'Angelo, Manuela
Porru, Annamaria Biroccio
[106]
Telomere Structure and Maintenance in Trypanosoma brucei
Ranjodh Sandhu, Bibo Li
[108]
Associations between five-factor model of personality and leukocyte telomere length in elderly men
and women – the Helsinki Birth Cohort Study (HBCS)
Katri Savolainen, Johan Eriksson, Eero Kajantie, Anu-Katriina Pesonen, Katri Räikkönen
[110]
Dissecting Alternative Lengthening of Telomeres in the Nematode Caenorhabditis elegans
Beomseok Seo, Chuna Kim, Mark Hills, Sung Sanghyun, Stephane Flibotte, Donald Moerman, Lee Junho
[112]
Telomere alterations and chromosome segregation defects induced by prolonged oxidative stress
treatment
Elisa Coluzzi, Rossella Buonsante, Anthony J. Asmar, Kelley L. Miller, Daniela Cimini, Antonella Sgura
21
[114]
TERT Promoter Mutations Are a Major Indicator of Poor Outcome in Differentiated Thyroid Carcinomas
Paula Soares, Miguel Melo, Adriana Gaspar da Rocha, João Vinagre, et al.
[116]
Choice of processing dysfunctional telomeres influences stem cell aging and survival
Tobias Sperka, Satjavani Ravipati, Omid Omrani, Lenhard Rudolph
[118]
In vivo Interaction of Telomerase, Telomere homeostasis and the Human Papillomavirus Oncogenes
Charis Achilleos, Stella Michael, Katerina Strati
[120]
The DNA-end-replication problem and the establishment of replicative senescence
Julien Soudet, Emilie Fallet, Pascale Jolivet, Michael Lisby, Eric Gilson, Maria Teresa Teixeira
[122]
Effects of bilirubin on telomere integrity and dynamics
Anela Tosevska, Christine Moelzer, Karl-Heinz Wagner
Insights into telomere protection by Ku heterodimer
Sona Valuchova, Eliska Janouskova, Ctirad Hofr, Karel Riha
[124]
[126]
Chromosome Instability: Major Events Likely Start in the Telomeres, Stupid
Ted Weinert, Tracey Beyer, Pete Vinton, Rachel Langston, Margherita Paschini, Vicki Lundblad
[128]
The structural organization of the human telomerase
Michael Wildauer, Christina Waldsich
[130]
A cryptic route to senescence reveals a constitutive protective function of telomerase
Zhou Xu, Emilie Fallet, Camille Paoletti, Steffen Fehrmann, Gilles Charvin, Maria Teresa Teixeira
[132]
SUMOylation of Tpz1 controls telomerase action in fission yeast
Mansi Garg, Sahar Mansoubi, Resham Lal Gurung, Steve Plumb, Felicity Watts, Alessandro
Bianchi
22
Keynote Session 1
The repression of PARP1 at telomeres
Titia de Lange, Isabelle Schmutz
The Rockefeller University; New York, USA
Mammalian telomeres have structural features that could potentially result in the binding and
activation of PARP1 and PARP2. Presumably, activation of these PARPs would be deleterious and would
require repression. Our prior work with shelterin-free telomeres had revealed the activation of
alternative non-homologous end-joining (alt-NHEJ), a pathway that was shown to be promoted by
PARP1 and DNA ligase 3 and inhibited by Ku70/80 (Sfeir and de Lange, Science 2012). Initial data
showed that while TRF2 is one of the shelterin subunits required for the repression of alt-NHEJ, it is not
the only shelterin factor with this function.
We were able to directly examine the localization and activation of PARP1 at telomeres. With these
assays for PARP1 activation, we queried conditional mouse embryo fibroblasts to determine which
components of shelterin contribute to the repression of PARP1 signaling at chromosome ends. The
data indicate that TRF2 and TIN2 act independently to minimize the localization of PARP1 to telomeres
and reduce its activation by dysfunctional telomeres. In contrast, TRF1, Rap1, TPP1, or POT1a/b were
not needed for the control of PARP1. We will present data on the mechanism by which TRF2 represses
PARP1 signaling.
Presented by: de Lange, Titia
23
Session 1-1
Riff’ing on the final step of telomere segregation at anaphase
Sophie Zaaijer, Nadeem Shaikh, Julia Promisel Cooper
National Cancer Institute, NIH, Bethesda, MD and Cancer Research UK, London
Rif1 has been implicated as a regulator of several processes fundamental to telomere maintenance –
chromosome replication, DSB resection and telomerase-mediated telomere extension. We have
uncovered an additional telomeric role for Rif1, later in the cell cycle, that prompts an assessment of
Rif1’s fundamental mode of action and suggests a final, regulated step in chromosome segregation.
Cells lacking Taz1 suffer stalled telomeric replication forks that result in telomere entanglements that
fail to be resolved at mitosis at cold temperatures. Hence, taz1 cells are cold sensitive. Entangled
telomeres become apparent as DNA masses persisting between the separating sister chromatids
during M-phase. These masses are associated with persistent DNA polymerase alpha foci as well as
Rad11RPA and Rad22Rad52, indicating ongoing attempts to deal with entanglements as mitosis
progresses. Rif1 sparked our interest when we observed that rif1+ deletion suppresses taz1 cold
sensitivity. We have found that Rif1 plays a role in removal of telomeric entanglements rather than
the fork-stalling events that generate them, and while Rif1’s telomere binding is reported to depend on
Taz1, we find that Rif1 co-localizes with telomeres in the anaphase mid-zone in both wild type and
taz1∆ cells. This localization is independent of the presence of spindle microtubules. We present a
model in which Rif1 regulates the fate of ssDNA overhangs generated by prolonged replication fork
stalling and in turn, the final steps of sister chromatid resolution.
Presented by: Cooper, Julia Promisel
24
Session 1-2
The role of telomere cohesion in aging and ALT cells
Susan Smith, Mahesh Ramamoorthy
The Skirball Institute, NYU School of Medicine, New York, USA
Resolution of telomere cohesion in human cells relies on the poly(ADP-ribose) polymerase tankyrase1
that localizes to telomeres in late G2/early mitosis to resolve cohesion and allow progression through
mitosis. We found that this “normal” system of telomere resolution is corrupted in aging and ALT cells;
they display excess persistent cohesion in mitosis and subsequent anaphase delay. Since a hallmark of
ALT is loss of the chromatin remodeler ATRX, we assessed its role in telomere cohesion. Reintroduction
of ATRX expression in ALT cells restored normal resolution of telomere cohesion. Conversely, ATRX
depletion (like that of tankyrase1) in telomerase positive cancer cells induced persistent telomere
cohesion. ATRX is in complex with the free pool of macroH2A histone variants. We show that,
dependent on its catalytic PARP activity, tankyrase1 binds specifically to macroH2A1.1, the poly(ADPribose) polymer-binding isoform. In ALT cells (in the absence of ATRX) the free pool of macroH2A1.1
sequesters tankyrase1, rendering it unable to resolve cohesion. Overexpression of tankyrase1
overrides the macroH2A1.1 inhibition and rescues persistent telomere cohesion in ALT cells, but with
dire consequences: a dramatic and immediate increase in telomere copying to other chromosomes and
impaired cell growth. A similar effect on cell growth was observed in aging fibroblasts. Our studies
suggest that keeping sister telomeres in close proximity into mitosis, not only promotes recombination
between sister telomeres, but also prevents excessive illegitimate recombination between nonhomologous chromosomes. This may be particularly important in the recombination permissive ALT
cell setting and in aging cells as telomeres become critically short.
Presented by: Smith, Susan
25
Session 1-3
Telomere Looping: A new paradigm for the regulation of gene expression with progressive
telomere shortening
Jérôme D. Robin1, Andrew T. Ludlow1, Min Chen2, Frédérique Magdinier3, Kimberly Batten1, Brody
Holohan1, Guido Stadler1, Kathyrin R. Wagner4, Jean-Marie Rouillard5, Jerry W. Shay1, Woodring E
Wright1
1
Dept. Cell Biology, UT Southwestern Medical Center, Dallas TX 75390 USA
Dept. Molecular Biophysics, UT Southwestern Medical Center, Dallas TX 75390 USA
3
Aix Marseille University, INSERM, UMRS 910. Epigenetics, Chromatin and Diseases team, 27 Bd Jean
Moulin, Marseille 13385 cedex 05 France
4
Center for Genetic Muscle Disorders, Kennedy Krieger Institute, Departments of Neurology and
Neuroscience, Johns Hopkins School of Medicine, Baltimore MD 21205 USA
5
Biodiscovery, LLC, Ann Arbor, MI 48105
2
Studies of human telomeres have focused on their role as a tumor-suppressor dependent on DNA
damage signals from too-short telomeres. We here describe a new paradigm for how telomere
shortening can progressively affect gene expression during the human lifespan, long before telomeres
become short enough to generate damage signals. ISG15 (Interferon Stimulated Gene 15 kda) is 1 Mb
from the telomere and its expression is regulated by telomere length. Many genes between it and
the telomere are not affected by telomere shortening. We here demonstrate that the distance
between the telomere and ISG15 is small when telomeres are long but the loci separate when
telomeres are short. We call this phenomenon TPE-OLD (Telomere Position Effects-Over Long
Distances). We next developed high-resolution Hi-C to directly map TPE-OLD interactions in a disease
whose locus is adjacent to the telomere of 4q and which exhibits an age-associated phenotype
(FacioScapuloHumeral muscular Dystrophy: FSHD) and show multiple interactions, which can extend
at least 10 Mb into the subtelomeric region. Global analysis of expression then established that 100s
of changes can be regulated by telomere length. Our results suggest new mechanisms for how
telomere shortening could influence human aging and disease initiation/progression long before
shortening produced DNA damage signals. These changes could be local, affecting stem cell
behavior, inflammation and repair. They could also be global, for example where secretory signals
from glial cells dividing on a regular basis every seven years could influence hormonal signals
governing energy distributions throughout the body. We have found TPE-OLD effects in both
myoblasts and fibroblasts, and are investigating its manifestations in epithelial cells. Length- regulated
long-range (Mb) telomere chromatin conformation changes may alter gene expression to optimize
fitness in species such as humans that live for many decades, and where the optimization of energy
utilization may differ significantly between newly mature and aged individuals. These changes may
profoundly affect human physiology, aging and disease.
Presented by: Wright, Woodring E.
26
Session 2-1
A TERT promoter gradient enables the isolation and purification of spermatogonial stem cells
Steven Artandia,*, Matthew Pecha, Alina Garbuzova, Bérénice Benayouna, Meena Sukhwania,
Shengda Lina, Stephanie Brockmana, Anne Bruneta, Kyle Orwigb, Jenny Zhanga
a
b
Stanford University
University of Pittsburgh
Germline mutations in the telomerase pathway cause a diverse set of diseases, including
dyskeratosis congenita, pulmonary fibrosis, aplastic anemia and liver cirrhosis. Telomerase
expression is controlled in part at the level of transcription of TERT. The ability to identify and
isolate TERT-positive cells in vivo would significantly advance our understanding of telomerase
function. To address these issues, we have created knock-in transcriptional reporters of TERT
expression by replacing the TERT open reading frame with genes encoding fluorescent proteins.
Among mouse tissues, telomerase activity is most strongly expressed in testis, a tissue in which
resident stem cells fuel the continuous generation of transit amplifying cells, which in turn enter
meiosis to produce the male gametes. In the human testis, telomere lengths are preserved with
age, although how this is achieved, in contrast to the aging-dependent telomere shortening seen
in somatic tissues, remains unresolved. We determined the TERT-reporter expression in the
stem cell, transit-amplifying cell and meiotic populations of the testis by wholemount and FACS
analysis. The combination of TERT-Reporter activity together with other surface markers allows
for purification of each of these cell populations in spermatogenesis by FACS, enabling each of
these populations to be isolated for molecular analysis. Our data provide important new insights
into how telomeres are maintained in the mammalian germline, preventing telomere attrition
that might otherwise precipitate telomere shortening disease phenotypes. This approach may
enable a greater understanding of telomerase regulation during carcinogenesis and in normal
tissues during aging.
Presented by: Artandi, Steven
27
Session 2-2
Telomere dysfunction accurately predicts clinical outcome in two common cancers
Duncan Baird, Thet Lin, Kevin Norris, Nicole Heppel, Kate Simpson, Guy Pratt, Robert Hills, Rhiannon
Jones, Julia Grimstead, Bethan Britt-Compton, et al.
Cardiff University
Defining the prognosis of individual cancer sufferers remains a significant clinical challenge. Telomere
length has been identified as a prognostic marker in a number of human cancers but it is not routinely
used in clinical practice. Here we provide a definition of the threshold length at which telomeres
become dysfunctional and show that this transforms telomere analysis into the most prognostic
marker of survival in patients with two common malignancies.
Using a cohort of chronic lymphocytic leukaemia (CLL) patients we undertook telomere fusion analysis
to define the upper telomere length limit at which fusion could be detected. We found that CLL
patients with telomeres within the fusogenic range had a significantly shorter overall survival
(P<0·0001; HR=13.2 (11.6-106.4)) and importantly this was particularly prognostic in early-stage
disease patients (HR=19.3 (17.8-802.5), P<0·0001). Remarkably, the same threshold was also highly
prognostic in a cohort of breast cancer patients (HR=18.2 (13.9-242.0), P<0·0001). In both cohorts the
fusogenic mean was the dominant variable in multivariate analysis. Finally we investigated whether
telomere dysfunction could predict response to therapy. Using the fludarabine/cyclophosphamide arm
of the LRF CLL4 clinical trial cohort we showed that the upper limit of telomere dysfunction strongly
predicted for shorter progression-free survival and overall survival.
Our findings identify the loss of telomeric functional integrity as a critical factor in the pathology of two
unrelated malignancies. Routine assessment of this parameter should provide more reliable prognostic
information for individual patients suffering from these diseases.
Presented by: Baird, Duncan
28
Session 2-3
The complex and differing roles of TIN2 isoforms in telomere regulation
Nya Nelson, Alison Bertuch
Baylor College of Medicine, Department of Molecular and Human Genetics
TIN2 is a central component of the shelterin complex, linking the telomeric DNA binding proteins TRF1
and TRF2 with TPP1/POT1. Heterozygous mutations in TINF2, which encodes TIN2, are found in the
telomere disorder dyskeratosis congenita (DC). The mutations, including truncations, cluster in a region
shared by both the short (TIN2S) and long (TIN2L) TIN2 isoforms. We hypothesized that TIN2L has
functions not shared with TIN2S that could be impacted by DC-cluster mutations. We found that, in
contrast to TIN2S, TIN2L was predominantly phosphorylated in vivo and was phosphorylated in vitro by
CK2, at a conserved residue present only in TIN2L. While telomeres progressively elongated in HT1080
cells overexpressing TIN2L or TIN2L with a phosphomimetic mutation, telomere length remained static
with overexpression of TIN2S or TIN2L with a phosphodead mutation. In co-immunoprecipitation
assays, we found that TIN2L had increased interaction with TRF2 compared to TIN2S, and the DCcluster and TIN2L phosphorylation cooperated to promote TRF2 interaction. In contrast to TIN2S, TIN2L
bound TRF2 via residue F120, which is also required for TRF2-Apollo interaction. This suggests TIN2L
may regulate Apollo recruitment. Conversely, TRF1 interacted more robustly with TIN2S than TIN2L.
DC-cluster mutations had no effect on TIN2S-TRF1 interaction, but further reduced TIN2L-TRF1
interaction. Both isoforms interacted similarly with TPP1. These results indicate that the composition
of shelterin is likely complex, not only because of stoichiometric differences as previously described,
but also due to differences in TIN2S and TIN2L activity, and that this composition may be
fundamentally different in patients with TINF2 mutations.
Presented by: Nelson, Nya
29
Session 2-4
The function and execution of RTEL1 activities at vertebrate telomeres
Grzegorz Sarek, Jean-Baptiste Vannier, Steffi Panier, Simon Boulton
London Research Institute, Clare Hall
Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles T-loops and
suppresses telomere fragility to maintain the integrity of chromosome ends. The emergence of RTEL1
variants that confer increased susceptibility to high-grade glioma, astrocytomas and glioblastomas has
highlighted the importance of RTEL1 for genome stability. Mutations in RTEL1 have also been
implicated in Hoyerall-Hreidarsson syndrome (HHS), a severe form of the bone marrow failure and
cancer predisposition disorder, Dyskeratosis Congenita. We previously reported that RTEL1 binds to the
replisome via a PIP-box dependent interaction with PCNA. Disruption of the RTEL1-PCNA interaction in
mice compromised genome-wide and telomere replication, and accelerated tumourigenesis.
Unexpectedly, the RTEL1-PCNA interaction was found to be dispensable for T-loop disassembly, which
suggested the existence of a distinct mechanism for recruiting RTEL1 to telomeres. Indeed, we have
discovered that RTEL1 is recruited to telomeres to promote T-loop disassembly via an S-phase specific
interaction with Shelterin. We identify a single point mutation within Shelterin that abrogates the
interaction with RTEL1 and phenocopies the Rtel1 null phenotype. Finally, we show that the RTEL1Shelterin interaction is compromised by a mutation in RTEL1 present in a subset of HHS patients. These
results establish a dynamic interplay between RTEL1 and Shelterin, which is critical for the timely
disassembly of T-loops during S-phase of the cell cycle.
Presented by: Boulton, Simon
30
Session 2-5
Dyskeratosis congenita caused by an OB fold mutation in TPP1
Hande Kocaka, Bari Ballewb, Kamlesh Bishta, Joseph Bolandc, Belynda Hicksc, Neelam Girib, Blanche
b,
Alterb, Catherine Keegana, Jayakrishnan Nandakumara, Sharon Savage
, et al.
*
a
University of Michigan
National Cancer Institute
c
Cancer Genomics Research Laboratory, NCI-Frederick
b
Dyskeratosis congenita (DC), the prototypic telomere biology disorder, is diagnosed by the triad of
dysplastic nails, skin pigmentation, and oral leukoplakia, or by leukocyte telomere lengths <1st
percentile for age. Patients are at high risk of bone marrow failure, cancer, pulmonary fibrosis, liver
disease, and other complications. Causative germline mutations in one of nine telomere biology
genes (DKC1, TERC, TERT, TINF2, NOP10, NHP2, WRAP53, CTC1, or RTEL1) are present in ~70% of
DC.
We performed whole exome sequencing on mutation-negative DC families participating in our IRBapproved longitudinal cohort study using the Illumina HiSeqTM platform. We focused on low
frequency, high quality coding variants, and explored all inheritance patterns. The likelihood of a
nonsynonymous variant being deleterious was predicted using information from multiple algorithms.
The proband of family NCI-275 is a 5 year-old boy with Hoyeraal Hreidarsson syndrome, a severe
variant of DC that includes cerebellar hypoplasia, immunodeficiency and intra-uterine growth
retardation in addition to DC features. The proband, his older sister, and father have telomeres
significantly <1st percentile; all three also harbor a single-amino acid deletion within the TEL patch of
the shelterin component TPP1. His healthy twin sisters and mother have telomeres at the 1st
percentile and 10th to
50th percentiles, respectively, and do not have the TEL patch mutation. This deletion severely
compromises the processivity of telomerase and its recruitment to telomeres. Structural modeling
of the mutation using the crystal structure of TPP1-OB as a template reveals disruptive
conformational changes, supporting a causative role of this mutation in DC.
Presented by: Savage, Sharon
31
Session 2-6
Role of telomeres in disease and longevity
Maria A. Blasco
Molecular Oncology Program, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain
Over the past years our laboratory has contributed to dissect the role of telomerase and telomere
length as key molecular pathways underlying cancer and aging, as well as has addressed the potential
use of telomerase activation as a therapeutic strategy for telomere syndromes and age-related
diseases (Blasco et al., Cell, 1997; Tomás-Loba, Cell, 2008). More recently, we have developed a
telomerase-based gene therapy strategy that allows telomerase activation in adult organisms and that
has shown beneficial effects in a variety of age-related pathologies in mice (Bernardes de Jesus et al.,
EMBO Molecular Medicine, 2012). I will present recent findings showing the efficacy of this telomerase
gene therapy in mouse models of disease.
Finally, I will discuss recent findings from our group regarding generation of mice with long telomeres
in the absence of genetic modifications.
Presented by: Blasco, Maria A.
32
Session 2-7
Disease-specific manifestations of the telomere syndromes
Mary Armanios
Department of Oncology and McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins
University School of Medicine, Baltimore MD USA
The telomere syndromes encompass a group of inherited degenerative disease states that share short
telomere length as a defect. These disorders affect both children and adults and have overlapping
phenotypes. In children, the predominant manifestation of telomere syndromes is bone marrow
failure which is at times seen in the context of features of dyskeratosis congenita. In adults, lung
disease usually manifests alone as idiopathic pulmonary fibrosis and, because of its prevalence in the
population, it is estimated to account for at least 90% of telomere syndrome cases. The biology that
underlies these differential presentations and the mechanisms that underlie the telomere-mediated
pulmonary phenotype will be the topic of this presentation.
Presented by: Armanios, Mary
33
Session 3-1
Regulated assembly and disassembly of the yeast telomerase quaternary complex
Timothy Tucey, Vicki Lundblad
Salk Institute for Biological Studies
Telomerase from budding yeast consists of the catalytic Est2 protein and two regulatory subunits (Est1
and Est3) in association with the TLC1 RNA, with each of the four subunits essential for in vivo
function. Using a biochemical approach that permits quantitative assessment of each of the protein
subunits of telomerase, we have monitored the holoenzyme, as well as sub-complexes, through the
cell cycle. This has revealed a hierarchy of assembly and disassembly, which results in limiting amounts
of the telomerase quaternary complex late in the cell cycle. Formation of the quaternary complex is
dependent on interaction of Est3 in late S phase/early G2 with a previously formed Est1-TLC1-Est2
sub-complex.
Est3 association is dependent on separate binding sites on both Est1 and Est2, suggesting that Est1
and Est2 form a single interface that is bound by Est3, as well as a regulatory surface on Est3 itself
that functions as an apparent toggle switch. The involvement of multiple surfaces that regulate Est3
binding suggests that it sits at a regulatory node for assembly. Surprisingly, once formed, the
quaternary complex disassociates through loss of the catalytic Est2 subunit, indicating that assembly
and disassembly of the telomerase complex proceed by different pathways. These studies reveal a
previously unappreciated means by which telomere homeostasis can be modulated, through a
complex and apparently highly regulated process of telomerase assembly and disassembly.
Presented by: Lundblad, Vicki
34
Session 3-2
Two-step mechanism of telomerase recruitment and activation in fission yeast
Christine Armstrong, Siân Pearson, Kazunori Tomita
UCL Cancer Institute, University College London, London, WC1E 6DD, United Kingdom.
Current models depict telomerase activation to be achieved by the presence of telomerase at
telomeres. In fission yeast, telomerase recruitment is achieved through interaction of the telomerase
subunit Est1 with Ccq1, a component of the Pot1 telomere binding complex. Our data show that the
association of telomerase with Ccq1 alone is not sufficient to fully activate telomerase. The OB fold
domain of Tpz1, a human TPP1 ortholog, positively regulates telomerase activity/processivity after
association of telomerase with the Ccq1 complex. In tpz1-K75A mutants, telomeres are critically short
even though telomerase is recruited. However, telomerase activity can be restored by fusing Tpz1K75A with the catalytic subunit of telomerase, Trt1. We therefore propose the Ccq1-Est1 interaction to
be the recruitment process and the OB-fold of Tpz1 to regulate telomerase activity, presumably
through the interaction of Trt1 with Tpz1. These two processes are separable. These findings reveal a
layer of telomerase regulation that controls processivity after recruitment.
Presented by: Tomita, Kazunori
35
Session 3-3
Assembly, Architecture and Function of Human Telomerase
Kathleen Collins
University of California, Berkeley
Reconstitution of minimal recombinant active telomerase in vitro can require only the templatecontaining telomerase RNA subunit (TER) and the telomerase reverse transcriptase protein (TERT). In
cells, additional chaperone and telomerase holoenzyme proteins direct the process of TER biogenesis
and folding, catalytic activity and telomere elongation. Dissecting the cellular requirements for TER
and TERT structure and associated factors has been complicated by overlapping and interlaced subunit
roles. We are using a wide array of approaches to investigate the biochemical specificity and
mechanisms that underlie unique features of the telomerase catalytic cycle, intimate protein and RNA
domain co-folding, enzyme activity on telomere substrates, and RNP regulation over the cell cycle.
Presented by: Collins, Kathleen
36
Session 3-4
The essential role of TPP1 in telomerase dependent telomere maintenance
Alec Sexton, Kathy Collins, Dirk Hockemeyer
University of California, Berkeley
Department of Molecular & Cell Biology
Telomeric binding proteins regulate the recruitment of telomerase to telomeric DNA in human stem
and tumor cells, thereby establishing telomere length homeostasis as necessary for their immortal
phenotype. TPP1 has been previously implicated in facilitating this interaction. Specifically, a region in
the OB-fold of TPP1, called the TEL patch, mediates TPP1’s ability to interact with the telomerase
holoenzyme and to stimulate its catalytic processivity in vitro. However, the physiological role of this
interaction in telomere maintenance is still unclear. Here, we use genome editing of the endogenous
TPP1 locus to demonstrate that the TEL patch of TPP1 is essential for telomere maintenance in
pluripotent stem cells (hPSCs). Genetically engineered hPSCs carrying homozygous substitutions in the
TEL patch of TPP1 (TPP1ΔL) phenocopy telomerase deficient TERT-/- hESCs, undergoing telomere
shortening and eventually cell death. TERT-/- cells can be complemented by ectopic TERT expression
but not TERTR132D, an allele which fails to interact with TPP1. Conversely, TPP1ΔL/ΔL hPSCs can be
rescued by wild type TPP1. We used this complementation assay to test a panel of TPP1 sequence
substitutions and fusion proteins to understand the overall pathway of telomerase function at
telomeres. By demonstrating the essential role of TPP1 in the telomere maintenance pathway this data
provides a unique target to interfere with telomerase activity in tumor cells.
Presented by: Hockemeyer, Dirk
37
Session 3-5
Identification of the site on telomerase that binds TPP1 to promote recruitment to telomeres
Jens C Schmidta,*, Andrew B Dalbyb, Thomas R Cechc
a
BioFrontiers Institute University of Colorado Boulder, Damon Runyon Cancer Research Foundation
BioFrontiers Institute University of Colorado Boulder
c
BioFrontiers Institute University of Colorado Boulder, Howard Hughes Medical Institute
b
Human chromosomes end in protein-DNA complexes termed telomeres, containing G-rich repetitive
DNA sequences, which bind the six protein shelterin complex. The shelterin complex carries out
multiple functions: it protects chromosome ends, prevents them from being recognized as sites of DNA
damage and recruits telomerase, an RNA-containing reverse transcriptase that synthesizes telomeric
repeat DNA. Recently, a group of amino acids on the OB-fold domain of the shelterin component TPP1,
termed the TEL patch, was demonstrated to be essential to recruit telomerase to telomeres
(Nandakumar et al 2012, Zhong et al 2012). In contrast, the site on telomerase that interacts with the
TPP1 OB-fold has remained poorly defined. Here we identify separation-of-function mutations in the
telomerase protein component hTERT that disrupt the interaction of telomerase with the OB-fold
domain of TPP1 in vivo and in vitro but have no effect on the catalytic activity of telomerase. The
mutant telomerases fail to localize to telomeres in vivo, their processivity is not stimulated by
POT1/TPP1 in vitro and they are incapable of maintaining telomere length in cultured cells. We will
provide evidence that these residues define the site on telomerase that directly binds to the TPP1-OB
fold.
Presented by: Schmidt, Jens C
38
Session 3-6
A self-regulating template in human telomerase
Julian J.-L. Chen, Andrew F. Brown, Xiaodong Qi, Yinnan Chen, Joshua D. Podlevsky, Mingyi Xie
Arizona State University, Department of Chemistry and Biochemistry, Tempe, Arizona 85287, USA
Telomerase is a specialized reverse transcriptase (RT) containing an intrinsic telomerase RNA (TR). It
synthesizes telomeric DNA repeats, (GGTTAG)n in humans, by reiteratively copying a precisely defined,
short template sequence from the integral TR component. The specific mechanism of how the
telomerase active site utilizes this short template region accurately and efficiently during processive
DNA repeat synthesis has remained elusive. Here we report that the human TR template, in addition to
specifying the repeat sequence, is embedded with a single-nucleotide signal to pause DNA synthesis.
After the addition of a dT residue to the DNA primer, specified by the signaling residue 49 rA in the
template, telomerase extends the DNA strand with three additional nucleotides and then pauses DNA
synthesis. This sequence-defined pause site coincides precisely with the structurally defined template
boundary, and cooperatively precludes incorporation of non-telomeric nucleotides from residues
outside the template region. Additionally, this sequence-defined pausing mechanism prevents
premature arrest of nucleotide synthesis and is the predominate mechanism for generating the
characteristic 6-nt ladder banding-pattern of telomeric DNA products in vitro. In the absence of the
pausing signal, telomerase stalls nucleotide addition at multiple sites along the template, generating
DNA products with diverse repeat registers at the termini. Our findings demonstrate a unique selfregulating mechanism of the human TR template for high fidelity synthesis of DNA repeats.
Presented by: Chen, Julian J.-L.
39
Session 4-1
Negative regulation of telomerase by SUMOylation-dependent Tpz1(TPP1)-Stn1 interaction in
fission yeast
Ross Lowa, Bettina Mosera, Alice Hoylea, Ya-Ting Changa, Keisuke Miyagawab, Katsunori Tanakab, Toru
Nakamura*a,
a
Department of Biochemistry & Molecular Genetics, University of Illinois at Chicago, Chicago, IL
60607, USA
b
Department of Bioscience, Kwansei Gakuin University, Sanda 669-1337, Japan
Studies in fission yeast have previously identified conserved shelterin and Stn1-Ten1 complexes, and
established Rad3(ATR)/Tel1(ATM)-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93
as the critical modification for telomerase recruitment. The shelterin subunits Poz1 and Rap1
negatively regulate Thr93 phosphorylation and telomerase recruitment by promoting the timely
recruitment of the lagging strand DNA polymerase to limit Rad3 checkpoint kinase accumulation.
Intriguingly, ChIP analyses have also revealed that Poz1 and Stn1 show nearly identical temporal
recruitment patterns with slightly delayed peak binding compared to Tpz1(TPP1)/Ccq1/Trt1(TERT). We
have also found that the shelterin subunit Tpz1(TPP1) interacts with Stn1-Ten1, based on yeast threehybrid (Y3H) assay. However, it remained unclear if Tpz1(TPP1)-Stn1 interaction plays a role in
telomere length regulation, and if Poz1’s ability to enforce the timely lagging-strand synthesis might
be dependent on Tpz1(TPP1)-Stn1 interaction. To address these questions, we have identified a tpz1
mutation (tpz1-K242R) that significantly weakens the Tpz1(TPP1)-Stn1 interaction, and determined
that this mutation causes significant telomere elongation, major reduction in late S/G2-phase binding
of Stn1, and massive increase in late S/G2- phase binding of Trt1(TERT). We also established that
Lysine 242, which occurs within a SUMOylation consensus sequence motif, is indeed SUMOylated in
S/G2-phase. Southern blot analysis also found that pmt3D (SUMO deletion mutant), tpz1-K242R, and
pmt3D tpz1-K242R cells show similar extent of telomere elongation, indicating that SUMOylation of
Tpz1 at K242 negatively regulates telomere extension. Our results thus suggest that SUMOylationdependent Tpz1(TPP1)-Stn1 interaction facilitates Stn1 recruitment to telomeres and timely
dissociation of telomerase to limit telomere extension in late S/G2-phase.
Presented by: Nakamura, Toru
40
Session 4-2
Roles of human CST in telomere replication and genome-wide replication rescue
Jason Stewarta, Feng Wanga, Anukana Bhattacharjeea, Carolyn Priceb,*
a
b
University of Cincinnati
Dept. Cancer Biology, University of Cincinnati
CST (CTC1-STN1-TEN1) is the mammalian homolog of the Cdc13-Stn1-Ten1 complex from S.
cerevisiae. Human CST has evolved various roles in telomere and non-telomeric DNA replication.
During telomere replication, CST first facilitates replication of the duplex DNA and then enables fill-in
synthesis of the C- strand following telomerase action. CST also aids is replication recovery following
genome-wide replication stress. Cells depleted of CST display decreased nucleotide uptake and
reduced firing of late or dormant origins after HU-induced replication fork stalling. In the absence of
exogenous replication stress, cells show increased anaphase bridges without telomere fusions. We
have developed a separation of function mutant that rescues CST-depleted cells from the defects in
C-strand fill-in and new origin firing in response to HU but not defects in telomere duplex replication
or anaphase bridge
formation. Our results demonstrate that CST is needed to rescue cells from multiple forms of
replication stress and that the transactions involved in this rescue differ with the type of replication
problem. We have also shown that CST helps cells recover after treatment with diverse DNA damaging
agents that impair replication. Cells with reduce CST show decreased recovery while cells that overexpress CST have enhanced recovery. Since the agents evoke different repair pathways, our results are
consistent with
CST acting at a common step during subsequent re-start of DNA replication. Overall, our findings
indicate that while CST may use the same final mechanism to rescue replication following
diverse challenges to the replication machinery, the actual pathway to this replication rescue
varies.
Presented by: Price, Carolyn
41
Session 4-3
Rif1 controls DNA replication timing and anti-checkpoint functions through recruitment of the
PP1 phosphatase, Glc7
Maksym Shyiana, Stefano Mattaroccia, Laure Lemmensa, Tianlai Shib, Nicolas Thomäb, David Shorea,*
a
b
Department of Molecular Biology, University of Geneva
Friedrich Miescher Institute for Biomedical Research, Basel
The Rif1 protein, originally identified as a budding yeast telomere-binding factor controlling the
telomerase pathway, has more recently been implicated in conventional DNA replication control, and
in the DNA damage response, from yeast to mammals. Here we will describe experiments showing
that budding yeast Rif1 interacts through two short N-terminal motifs (RVxF and SILK) with the PP1
phosphatase (Glc7 in yeast) to carry out both replication and anti-checkpoint functions. More
specifically, we will show how Rif1-Glc7 inhibits the activation of pre-replication complexes (pre-RCs)
through regulation of two different Dbf4-dependent kinase (DDK) substrates. In addition, we will
describe experiments that implicate Rif1-Glc7 in an anti-checkpoint function at both telomeric and
non- telomeric double-strand breaks (DSBs). Given the apparent conservation of the Rif1-PP1
interaction, we propose that these studies in yeast will be highly relevant to Rif1 function in higher
eukaryotes.
Presented by: Shore, David
42
Session 5-1
Long-term proliferation of human cancer cells in the absence of telomere length maintenance
Loretta Laua, Rebecca Dagga, Jeremy Hensonb, Hilda Pickettb, Axel Neumannb, Roger Reddelb,*
a
b
The Children's Hospital at Westmead, Westmead, NSW 2145, Australia
Children's Medical Research Institute, Westmead, NSW 2145, Australia
Cellular immortalization is a hallmark of cancer, and requires an active telomere length maintenance
mechanism (TMM). Every immortalized cancer cell line studied to date has telomerase or Alternative
Lengthening of Telomeres (ALT) activity. We have now identified two neuroblastoma (NB) cell lines
that proliferate extensively (>300 population doublings [PDs]) despite continuous telomere
shortening. Their telomeres start out very long (mean terminal restriction fragment [TRF] length of 31
to 37 kb) and steadily decrease in length at a rate of 50 – 80 base pairs/PD without any evidence of
compensatory lengthening events. The telomeres appear very similar to the long and heterogeneous
telomeres of ALT cell lines, with the exception that individual telomere bands are visible on Southern
blot analysis, in contrast to the typical smeared TRF pattern of ALT cell lines. This unique pattern has
only been observed previously in ALT cell lines where ALT activity has been switched off by genetic
manipulation. These cells are telomerase-negative, but, unlike ALT cells, they lack mutations in p53,
ATRX, DAXX, H3.3 and IDH1/2, lack T-circles and ALT-associated PML nuclear bodies, and have very
low C-circle levels. Moreover, we have identified a subset of NB tumours (~10% of high-risk NBs) which
have characteristics similar to
these cell lines, and found that patients with these tumours have better prognosis than those
with telomerase or ALT. These cell lines (and, most likely, cancers) therefore undergo very longterm proliferation despite failing to maintain telomere length.
Presented by: Reddel, Roger
43
Session 5-2
Absence of telomerase causes the early onset of cancer in zebrafish
Madalena Carneiro, Joana Nabais, Tania Carvalho, Miguel Godinho Ferreira
Instituto Gulbenkian de Ciência, Oeiras, Portugal
Does telomere-shortening act as a tumour suppressor or an oncogene-type mechanism? In human
somatic cells, telomerase expression is restricted and age-progressive telomere attrition occurs with
each cell division. As tumour suppressors, critically short telomeres trigger p53 activation and restrain
cell proliferation. As “oncogenes”, short telomeres are themselves sources of genome instability and
tumorogenesis. Humans with telomerase deficiencies and shorter telomeres are tumour-prone
attesting to the possibility that short telomeres may be conducive to cancer.
Zebrafish has recently emerged as a complementary cancer model exhibiting increased
tumourogenesis in later life. Unlike lab mice, zebrafish have human-size telomeres that decline with
age and we have shown that ztert-/- fish die prematurely displaying several ageing phenotypes.
We now report that ztert-/- fish exhibit an anticipation of tumourogenesis that mimics both the rates
and types of older fish. Spontaneous tumours arise in young ztert-/- fish in a context of an overall
decline in cell proliferation, increased cell senescence and inflammation. These tumours exhibit
characteristics of ALT.
In parallel, we are characterizing the role of telomerase in an established model for melanoma in
zebrafish. Transgenic fish expressing human BRAF-V600E in melanocytes develop invasive melanoma in
a p53-/- mutant background within the first year of age. Absence of telomerase in this model
significantly reduces the incidence of melanoma. However, in contrast to spontaneous tumours, the
timing of melanoma formation in this model is unaffected. Our results thus suggest that exceedingly
short telomeres provoke the early onset of tumours in zebrafish depending on the status of p53.
Presented by: Ferreira, Miguel Godinho
44
Session 5-3
The role of SUMO pathway in telomere recombination
Ferose Charifia, Dmitri Churikova, Nadine Eckert-Bouletb, Sonia Silvab, Marie-Noelle Simona, Michael
Lisbyb, Vincent Géli*a,
a
b
Cancer Research Center of Marseille
University of Coppenhaguen
In budding yeast, sumoylation of the telomere-associated and DNA repair proteins is thought to
promote sequestration of the telomeres and persistent DNA double-strand breaks to the nuclear
periphery, where canonical homologous recombination (HR) is repressed. We have previously shown
that eroded telomeres that cannot be extended are shifted from their perinuclear location to the
nuclear pore complexes (NPCs). The SUMO protease Ulp1 also localizes at the NPC and may
contribute to channelling damage to alternative repair. We have analyzed the dependence of the two
HR- dependent survivor pathways in telomerase-negative cells on Rad52 sumoylation and telomere
relocalization to NPC. We found that sumoylated Rad52 promotes Rad51-dependent type I, whereas
Rad52 desumoylation (presumably by Ulp1 at the NPC) favours type II survivor formation.
Interestingly, we found that telomere shift to NPC was dependent on Rad59, whereas Rad51 played
no role in this
process. To test the importance of telomere relocalization to NPC for the type II pathway, we
introduced
LexA binding sites in the vicinity of telomere VI-R, and expressed LexA-fused proteins to either target
VI- R to the NPC or to locally modulate sumoyalation at VI-R. Our results suggest that NPC targeting
affects the dynamics of telomere recombination. Also, targeting Ulp1 catalytic domain to a specific
telomere was found to be sufficient to induce its recombination even in the presence of telomerase.
We will present our current view of the role of the SUMO pathway in telomere recombination.
Presented by: Géli, Vincent
45
Session 5-4
Interchromosomal homology searches drive directional ALT telomere movement and synapsis
Roger Greenberg
Department of Cancer Biology
Perelman School of Medicine, University of Pennsylvania
Telomere length maintenance is a requisite feature of cellular immortalization and a hallmark of
human cancer. While most human cancers express telomerase activity, approximately 10-15% employ
a recombination-dependent telomere maintenance pathway known as Alternative Lengthening of
Telomeres (ALT), an incompletely understood process that is characterized by multi-telomere clusters
and associated promyelocytic leukemia protein bodies. Here, we show that a DNA double-strand break
(DSB) response at ALT telomeres triggers long-range movement and clustering between chromosome
termini, resulting in homology-directed telomere synthesis. Damaged telomeres initiate increased
random surveillance of nuclear volumes before displaying rapid directional movement and association
with recipient telomeres over micron-range distances. This phenomenon required homologous
recombination proteins and the Hop2-Mnd1 heterodimer, which is essential for homologous
chromosome synapsis during meiosis. These findings implicate a meiotic-like homology searching
mechanism in ALT dependent telomere maintenance and provide a molecular basis underlying the
preference for recombination between non-sister telomeres during ALT.
Presented by: Greenberg, Roger
46
Session 5-5
Genome instability driven by targeted telomere insertion in ALT cells
Paulina Marzeca,*, Claudia Armenisea, Gaelle Perotb, Christina Raftopoulouc, Sarantis Gagosc, Frederic
Chibonb, Jerome Dejardina
a
Institute of Human Genetics, CNRS UPR1142,141 rue de la Cardonille,34090 Montpellier, FRANCE
Translational Research, Department of Biopathology, Institut Bergonié, 229 cours de l’Argonne, 33076
Bordeaux, FRANCE
c
Laboratory of Genetics and Gene Therapy, Center of Basic Research II, Biomedical Research
Foundation, of the Academy of Athens, GREECE
b
The Breakage-Fusion Bridge cycle is a classical mechanism of telomere driven genome instability, in
which critically short or unprotected telomeres are fused to other chromosomal extremities, creating
dicentric chromosomes which eventually break at mitosis. We uncover a new mechanism of telomere
driven genome instability, specifically occurring in cells which have activated the Alternative
Lengthening of Telomeres pathway. In these cells, we show that telomeric DNA is added to multiple
discrete sites throughout the genome. These sites correspond to regions usually regulated by NR2C/F
transcription factors, and we show that these factors drive local telomere DNA addition by recruiting
telomeric chromatin. This new mechanism, which we name targeted telomere insertion (TTI),
generates potential common fragile sites that destabilize the genome. We propose that targeted
telomere insertion driven by NRC2/F proteins creates complex karyotypes in ALT tumors.
Presented by: Marzec, Paulina
47
Session 5-6
Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric
chromatin
Harikleia Episkopoua,*, Irena Draskovicb, Amandine Van Benedena, Gaëlle Tilmana, Marina Mattiussia,
Matthieu Gobina, Nausica Arnoulta, Arturo Londoño-Vallejob, Anabelle Decottigniesa
a
Genetic and Epigenetic Alterations of Genomes, de Duve Institute, Catholic University of Louvain,
Brussels 1200, Belgium
b
Telomeres and Cancer Laboratory, Equipe Labellisée Ligue, UMR3244-UPMC-Institut Curie, Paris
75248, France
Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and
stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might
favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous
recombination. However, analysis of chromatin status at human ALT telomeres has never been
reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or
ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is
associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of
telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through
telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further
show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not
sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric
RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support
the hypothesis that telomeric chromatin decondensation is important for ALT pathway.
Presented by: Episkopou, Harikleia
48
Session 5-7
A complex interplay between telomere transcription, stability and recombination in human ALT cells
Rajika Aroraa,*, Yongwoo Leea, Harry Wischnewskia, Catherine Brunb, Claus Azzalina
a
b
Eidgenössische Technische Hochschule Zürich (ETHZ), Institute of Biochemistry
The Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario
We previously generated telomerase-positive HeLa cell lines where transcription of one specific
telomere (‘transcriptionally inducible telomere’: tiTEL) can be induced experimentally. Transcription
induction did not alter tiTEL length or stability, nor did it affect telomerase-mediated elongation of
tiTELs. We have now generated tiTEL-containing U2OS cells, which maintain their telomeres through
the ‘alternative lengthening of telomeres’ (ALT) pathway. Transcription induction in U2OS cells leads to
increased rates of tiTEL fragility, sister tiTEL exchanges and accumulation of RNA:DNA hybrids at tiTELs.
These data suggest that in an ALT background, TERRA transcription compromises telomere replication
and promotes homologous recombination between telomeric sequences, possibly by engaging in
RNA:DNA hybrids at telomeres (‘telomeric R-loops’; telR-loops). Consistent with this hypothesis,
different ALT cell lines exhibit increased rates of TERRA transcription from CpG island subtelomeric
promoters and of telomere fragility as compared to telomerase-positive cells. Over-expression of TRF1
in U2OS cells diminishes telR-loops and telomere fragility, while siRNA-mediated depletion of RNaseH1
increases telR-loops levels, telomere fragility, telomere loss, sister telomere exchanges and
extrachromosomal telomeric circles. Strikingly, depletion of RNaseH1 in HeLa cells has only a minor
impact on telomere homeostasis. We propose that telR-loops are dynamic structures appearing at
human telomeres and their improper resolution, possibly during cell cycle progression, might
compromise replication of telomeric sequences and trigger homologous recombination or telomere
loss. TERRA transcription-mediated telomere recombination may be a mechanism utilized by ALT cells
to maintain their telomeres in the face of telomere integrity.
Presented by: Arora, Rajika
49
Session 5-8
NuRD-ZNF827 recruitment to telomeres creates a molecular scaffold for homologous recombination
Dimitri Conomos1,2, Roger R. Reddel2,3, and Hilda A. Pickett1,2
1
Telomere Length Regulation Group, Children’s Medical Research Institute, Westmead, New South
Wales, Australia
2
Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
3
Cancer Research Unit, Children’s Medical Research Institute, Westmead, New South Wales, Australia
Alternative lengthening of telomeres (ALT) is an homologous recombination (HR)-dependent
mechanism for de novo synthesis of telomeric DNA. Nuclear receptors are bound to the telomeres of
cells that utilize ALT. Here we demonstrate that nuclear receptors recruit ZNF827, a zinc finger protein
of unknown function, which recruits the nucleosome remodeling and histone deacetylation (NuRD)
complex via binding to an N-terminal RRK motif within ZNF827. This results in hypoacetylation of
telomeric chromatin, decreased shelterin binding, a diminished telomeric DNA damage response (DDR)
as well as enhanced telomere-telomere interactions and recruitment of HR proteins, and is critically
important for cell viability and proliferation. We propose that NuRD-ZNF827 recruitment to telomeres
establishes a molecular platform which remodels telomeric chromatin and creates an environment
which promotes telomere-telomere recombination, and integrates and controls multiple mechanistic
elements of ALT activity.
Presented by: Pickett, Hilda
50
Session 5-9
TRF2 is required to protect heterochromatic pericentromeric regions from topological and replicative
DNA damage
Aaron Mendez Bermudeza,*, Karine Jameta, Jing Yeb, Eric Gilsona
a
Institute for Research on Cancer and Aging, Nice (IRCAN), CNRS UMR 7284 - INSERM U1081 Medical
School, University of Nice, France
b
Shanghai Ruijin Hospital affiliated to Shanghai Jiaotong University, School of Medicine, China
Previous results from our lab showed that TRF2 binds to a subset of interstitial telomeric sequence
(ITS) and pericentromeric satellite III (SatIII) DNA (Simonet et al, 2011) and that it is required to protect
a stretch of telomeric sequences artificially inserted in the interior of chromosome 4 against replicative
DNA damage (Ye et al, 2010).
In this work, we investigate whether TRF2 could also be involved in the protection of naturally
occurring extratelomeric sequences. First, chromatin immunoprecipitation coupled with deep
sequencing (ChIP-seq) analysis revealed that, in addition to telomeric sequences, a reduced expression
of TRF2 triggers γ-H2AX accumulation at pericentromeric SatIII regions. In addition, we showed by
immunofluorescence that cells suffering from topological stress caused by topoisomerase
downregulation (either Top1 or TopIIa or TopIIB) or by treatment with the topoisomerase II inhibitor
ICRF193 exhibit an increased binding of TRF2 to the pericentromeric SatIII region of chromosome 9 and
also an increased in DNA damage. Conversely, in cells treated with TSA or compromised for the histone
methyltransferase responsible for H3K9me3, Suv39H1, the binding of TRF2 to pericentromeric SatIII
repeats is reduced. Finally, in support of a role for TRF2 in the protection of pericentromeric chromatin
against replicative DNA damage, treatment of cells with hydroxyurea and aphidicolin as well as
inhibition of the BLM and WRN helicases increases the binding of TRF2 to SatIII regions.
Together, these findings suggest that TRF2 is recruited to heterochromatic pericentromeric regions to
alleviate the topological problems occurring during their replication.
Presented by: Mendez Bermudez, Aaron
51
Session 5-10
The multifaces of chromatin assembly, a recipe that mixes new with old partners
Genevieve
ALMOUZNI Institut
Curie / CNRS
Our dissection of the mechanisms of chromatin assembly, from the basic structural unit, the
nucleosome, up to higher-order structures in the nucleus has enabled to characterize key chaperones
involved in nucleosome assembly as well as to define the dynamics of new histone incorporation in
chromatin. We have examined specific nuclear domains: non-coding centromeric heterochromatic
regions, which are of major importance for chromosome segregation.Our findings have shed light on
the fundamental issues of the dynamics, fate, and inheritance of histones, with their specific marks
typical
of particular chromatin domains.
Our current hypothesis is that histone chaperones function in an ‘assembly line’ with specificity for
individual histone variants to mark defined regions of the genome. Remarkably, we have found that
misregulation of specific histone chaperones is a common feature of aggressive breast cancers. Our
recent studies show that the depletion of the histone chaperone ASF1 leads to the induction of the
characteristics of Alternative Lengthening of Telomeres. We will discuss our most recent advances in
the regulatory pathways that target histone chaperones and variants to control the assembly line and
its connecting network.
Casanova M. et al. (2013) Cell Rep., 4, 1156-1167.
Abascal F. et al. (2013) Mol. Biol. Evol., 30, 18531866. Adam S. et al. (2013) Cell, 155, 94-106.
Szenker E. et al. (2013) In Fundamentals of Chromatin, J. Workman & Abmayr S. Eds., Springer
Verlag, pp. 375-427.
O'Sullivan R.J. et al. (2014) Nature Struct. Mol. Biol., 21, 167-174.
Lacoste N. et al. (2014) Mol. Cell, 53, 631-644.
Presented by: ALMOUZNI, Genevieve
52
Session 6-1
Characterization of the changes in telomeric chromatin upon DNA damage induction
Cristina Bartoccia, Jolene Diedrichb, Yates Johnb, Eros Lazzerini Denchia,*
a
b
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA
When telomeres become critically short DNA damage response factors are recruited at
chromosome ends initiating a cellular response to DNA damage. Here, we performed Proteomic
Isolation of Chromatin fragments (PICh) to define the changes in chromatin composition that occur
upon onset of acute telomere dysfunction triggered by depletion of the telomere-associated factor
TRF2. This unbiased isolation and purification of telomere-associated proteins in functional or
dysfunctional conditions revealed the dynamic changes in chromatin composition that take place at
telomeres upon DNA damage induction. Our analysis identified several proteins that are lost from
telomeres following DNA damage induction, as well as several proteins that localize to dysfunctional
telomeres. Based on
our chromatin purification and mass spectrometry analyses, we describe a critical role for the polycomb
group protein Ring1b in NHEJ-mediated end-to-end chromosome fusions in response to telomere
dysfunction. Our data suggest that cells with reduced levels of Ring1b have a reduced ability to repair
uncapped telomeric chromatin. Our data represent the first unbiased isolation of chromatin
undergoing DNA damage and represent a valuable resource to map the changes at the level of
chromatin composition in response to DNA damage activation.
Presented by: Lazzerini Denchi, Eros
53
Session 6-2
Mitotic telomere deprotection dictates cellular fate upon mitotic arrest
Makoto T Hayashia,*, Anthony J Cesareb, Jan Karlsedera
a
b
Salk Institute for Biological Studies. La Jolla, California 92037
Children’s Medical Research Institute. Sydney, Australia. 2145
The DNA damage response activated at shortened telomeres avoids tumorigenesis by inducing
senescence. Upon the bypass of senescence, cells enter a crisis, during which further telomere
shortening acts both tumor suppressive and tumor promoting, by inducing cell death and chromosome
instability, respectively. Although telomere fusions and the resulting genome instability are wellcharacterized features of the crisis, the mechanism underlying cell death has not been fully
understood. Our finding that prolonged mitotic arrest induces telomere deprotection prompted us to
ask whether mitotic telomere deprotection has a physiological function.
Here, we found that IMR-90 E6E7 cells that bypassed senescence display a spontaneous mitotic arrest
phenotype in the pre-crisis cells. The arrest phenotype was induced by loss of p53, and suppressed by
telomerase overexpression, indicating extreme telomere shortening as the underlying cause. However,
telomere fusions induced by overexpressing dominant-negative TRF2 did not cause a mitotic arrest
phenotype in middle-aged IMR-90 E6E7, suggesting fusions do not directly trigger mitotic arrest. The
actual cause of the spontaneous mitotic arrest is under investigation.
Pre-crisis cells with premature sister chromatid separation, which is a consequence of mitotic arrest,
suffer from increased telomere deprotection, suggesting pre-crisis cells also undergo mitotic telomere
deprotection. Exacerbation of mitotic telomere deprotection by partial knockdown of TRF2 increased
the cell death ratio during mitosis and sensitized cells to drugs that induce mitotic arrest. We propose
that mitotic telomere deprotection functions to promote cell death during mitotic arrest in both cells
that bypassed senescence, and cells that are exposed to mitotic drugs.
Presented by: Hayashi, Makoto T
54
Session 6-3
An optimal telomere length protects human tumor cells from ionizing radiation
Jennifer Fairliea, Lea Harrington*b,
a
University of Edinburgh, School of Biological Sciences, Wellcome Trust Centre for Cell Biology
University of Montreal, Institute for Research in Immunology and Cancer
Visiting Professor, University of Edinburgh, School of Biological Sciences
b
The acquisition of telomerase reverse transcriptase (TERT) expression and its ability, together with
the telomerase RNA, to maintain telomeres at chromosome ends is one of the hallmarks of cancer.
More than 85% of all human cancers possess telomerase activity, and telomerase inhibition has
been established in several model systems to be a tractable target for cancer therapy. As human
tumor cells typically maintain short equilibrium telomere lengths, we wondered if excessive
telomere elongation would impact the fitness of human tumor cells. Indeed, we found that telomere
elongation beyond a certain length significantly decreased cell clonogenic survival after gamma
irradiation. Susceptibility to irradiation was dosage-dependent and increased at telomere lengths
>17 kbp despite the fact that all chromosome ends retained telomeric DNA. We postulate that
human cancer cell survival may be selected around an optimal telomere length that balances the
maintenance of telomere integrity and efficient DNA replication.
Presented by: Harrington, Lea
55
Session 6-4
Interactions between CST, DDR and NMD genes show that Stn1 and Ten1 are the key components of
the CST complex
Eva Holstein, Kate Clark, David Lydall
Institute for Cell and Molecular Biosciences, Newcastle University Medical School, Newcastle upon
Tyne, UK
A large and diverse set of proteins, including CST complex, Nonsense Mediated Decay (NMD) and DNA
Damage Response (DDR) proteins play important roles at the telomere in mammals and yeast. Here we
show that NMD, like the DDR, affects single stranded DNA (ssDNA) production at uncapped telomeres.
We also show that the requirement for Cdc13, one of the components of CST, can be efficiently
bypassed when aspects of DDR and NMD pathways are inactivated. However, identical genetic
interventions do not bypass the need for the other CST components, Stn1 and Ten1. We show that
disabling NMD alters the stoichiometry of CST components at telomeres and permits Stn1 to bind
telomeres in the absence of Cdc13. Our data supports a model that Stn1 and Ten1 cap the telomere in
a Cdc13-independent manner and have implications for the function of CST components across
eukaryotes.
Presented by: Clark, Kate
56
Session 6-5
Delineating the role of upstream DNA damage responses in cell and tissue aging induced by telomere
shortening
Karl Lenhard Rudolph
Leibniz Institute for Age Research, Fritz-Lipmann-Institute (FLI), Jena, Germany
Telomere shortening limits the proliferative capacity of cells and the regenerative capacity of tissues by
induction of DNA damage checkpoints. Telomere dysfunction can also contribute to cancer initiation by
inducing chromosomal fusions, fusion-bridge-breakage cycles and chromosomal instability (CIN). Late
generation telomerase knockout mice (G3 Terc-/-) with shortened telomeres exhibit premature aging
and chromosomal instability. In yeast, Exo1-dependent DNA end resection at dysfunctional telomeres
mediates induction of DNA damage responses. The deletion of Exo1 improved tissue maintenance and
prolonged lifespan of telomere dysfunctional mice. The study indicated that Exo1 dependent end
resection contributes to both the induction of DNA damage checkpoints and chromosomal fusions in
response to telomere shortening in mammalian cells. It is currently not known which process – endresection induced DNA damage checkpoints or chromosomal fusions - is mediating the adverse effect
of telomere shortening on tissue aging. In my talk I will present how specific genes that regulate the
upstream induction of DDRs influence the proliferative capacity of cells, tissue maintenance, and
oncogenic transformation in response to telomere shortening.
Presented by: Rudolph, Karl Lenhard
57
Session 6-6
Identification of a critical regulator of DNA repair activity at mammalian telomeres
Vera Boersmaa, Sandra Segura-Bayonaa, Marieke Peuschera, Jaco van der Torrea, Nathalie Moattia,
Alexandre Orthweinb, Daniel Durocherb, Jacqueline Jacobsa,*
a
The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON
M5G 1X5, Canada
b
Appropriate repair of DNA lesions and inhibition of repair activities at telomeres are critical for the
maintenance of genome integrity and prevention of genomic instability. Upon loss of telomere
protection natural chromosome ends are processed by DNA repair factors that cause chromosome
end-to-end fusions. Upon cell division these fusions result in missegregation of chromosomes and
unbalanced chromosomal rearrangements that can compromise cell viability and promote cancer
development. Similarly, inaccurate or inappropriate repair of DNA lesions can contribute to aging or
tumorigenesis by fueling genetic alteration. The mechanisms underlying control of DNA repair activities
are not completely understood. Therefore, our laboratory undertakes candidate-driven and unbiased
approaches to identify factors with important roles in the responses to telomere dysfunction. In
particular, we performed loss-of-function genetic screens in p53-deficient mouse embryo fibroblasts in
which we inactivate TRF2 to induce telomere uncapping. While this results in a chromosome fusion
phenotype that becomes so severe that cells arrest or die, we identified multiple shRNAs that allow
cells to survive and proliferate despite prolonged telomere uncapping. We have focused our validation
and follow-up efforts on several screen hits not previously known to act at telomeres and linked to
diverse activities, including ubiquitylation, methylation, control of mitosis and DNA damage tolerance.
In this process we identified a new factor that promotes NHEJ-mediated chromosome end-to-end
ligation and telomere-induced genomic instability by inhibiting 5’ DNA-end-resection. Moreover, this
factor is also essential for efficient end-joining of DNA DSBs during immunoglobulin class switch
recombination.
Presented by: Jacobs, Jacqueline
58
Session 6-7
DNA polymerase theta promotes alternative-NHEJ at dysfunctional telomeres
Pedro Mateos-Gomeza, Fade Gongb, Kyle Millerb, Eros Lazzerini-Denchic, Agnel Sfeira,*
a
Skirball Institute, NYU Langone Medical Center, New York, NY, USA
Institute for Cell and Molecular Biology, UT Austin, Austin, Tx, USA
c
The Scripps Research Institute, San Diego, CA, USA
b
Mammalian cells have evolved complex mechanisms to repair double strand breaks (DSBs). In addition
to the error-free homology-directed repair (HDR), DSBs can be rejoined by the two error-prone
pathways of classical Non-Homologous End-Joining (NHEJ), and alternative-NHEJ. The engagement of
end-joining pathways at the end of mammalian chromosomes is detrimental, and is inhibited by the
six-subunit shelterin complex. While classical-NHEJ is primarily blocked by TRF2, alt-NHEJ is only
unleashed when telomeres loose the binding of the entire shelterin complex (shelterin-free), and in
cells lacking Ku70/80.
To define the molecular differences between the two distinct end-joining pathways, we applied deep
sequencing technology on telomeric DNA isolated either from TRF2 null mouse embryonic fibroblasts
(MEFs), or shelterin-free/Ku80 deficient MEFs. Telomeres fused by alt-NHEJ were marked by nontelomere insertions that scar the junction between fused ends. Based on this analysis, we tested the
function of a number of translesion synthesis DNA polymerases and identified polymerase theta (PolQ)
as a key alternative-NHEJ factor. PolQ is a poor fidelity A-family DNA polymerase with an activity that
appears to be template independent, and known to be preferentially expressed in cancers cells.
Inhibiting PolQ blocked telomere fusions in shelterin-free/Ku80 deficient MEFs, but did no impact
classical-NHEJ fusions in cells lacking TRF2. In addition to its role at telomeres, PolQ depletion impaired
alternative-NHEJ repair when tested in the context of non-reciprocal chromosomal translocation. In
summary, our study underscores a novel function for the highly promiscuous PolQ, potentially
accounting for the increased mutagenicity of the alternative-NHEJ pathway of DNA repair.
Presented by: Sfeir, Agnel
59
Session 6-8
Rapamycin prevents checkpoint adaptation via autophagy and Cdc5 (PLK1), which increases
proliferative potential following telomere dysfunction
Julia Klermund, Katharina Bender, Brian Luke
University of Heidelberg, ZMBH
The DNA damage checkpoint provides a quality control function for the genome by preventing cells
with damaged DNA from continuing in the cell cycle. In response to dysfunctional telomeres, yeast cells
arrest at the G2/M border due to the activation of a Mec1 (ATR) / Rad53 (Chk2) – mediated arrest,
which prevents the onset of mitosis. When damaged telomeres persist for longer periods without
repair, the checkpoint is eventually extinguished and cells continue to cycle even in the presence of
damage – a phenomenon referred to as checkpoint adaptation. Checkpoint adaptation is typically
accompanied by the de-phosphorylation of Rad53 and the progression of cells into the following G1
phase of the cell cycle. Surprisingly, the inhibition of the conserved TOR signaling pathway, through
rapamycin addition, is able to prevent cells with dysfunctional telomeres from adapting, thereby
establishing crosstalk between the cellular nutritional status and the DNA damage response. Indeed, in
the presence of rapamycin Rad53 phosphorylation remains high and cells do not enter the following
G1 phase. Importantly, the rapamycin-mediated inhibition of checkpoint adaptation increases cell
viability following long term telomere dysfunction from 30% to over 80%. We have found that
rapamycin prevents checkpoint adaptation via two parallel pathways 1. Through the induction of
autophagy and 2. Via inhibition of Cdc5 (PLK1) – mediated inactivation of Rad53. Finally, we were able
to demonstrate that rapamycin extends chronological lifespan, in yeast, in a DNA damage checkpointdependent manner. Therefore nutrient status has a critical influence on cell cycle regulation following
telomere dysfunction.
Presented by: Luke, Brian
60
Session 6-9
A role for DNA damage response RNAs (DDRNA) at dysfunctional telomeres
Francesca Rossielloa,*, Julio Aguadoa, Fabrizio d'Adda di Fagagnab
a
IFOM – the FIRC Institute for Molecular Oncology Foundation, Milan, Italy
IFOM – the FIRC Institute for Molecular Oncology Foundation, Milan, Italy
CNR – Consiglio Nazionale delle Ricerche, Pavia, Italy
b
We have recently shown that small non-coding RNAs are novel components of the DNA damage
response (DDR) machinery (Francia et al. Nature 2012). These small RNAs, named DDR RNAs or
DDRNAs, have the sequence of the damaged locus and their biogenesis depends on Dicer and Drosha
endoribonucleases. DDRNAs are necessary for DDR activation at DNA double-strand breaks (DSBs)
following ionizing radiations, endonuclease cleavage or oncogene-induced DNA replication stress.
Moreover, a role for Dicer and Drosha has been proposed for DSB repair (Wei et al. Cell 2012).
Deprotected telomeres resemble DSBs as they lead to DDR signaling activation and DNA repair events.
We investigated the potential role of DDRNAs in DDR activation at telomeres. Upon TRF2 inactivation,
we show that transient RNase A treatment of permeabilized mouse cells impairs DDR foci at telomeres
and DDR signaling can be restored by reintroducing cellular RNA. Consistently with the DDRNA
biogenesis, Dicer or Drosha knock down prevents DDR activation at dysfunctional telomeres. In
addition, knock down of either of these two endoribonucleases impairs telomeric fusions upon
telomere uncapping. Furthermore the inhibition of telomeric DDRNAs by the use of oligonucleotide
molecules with a complementary sequence can prevent accumulation of DDR markers at telomeres
and senescent-associated cell cycle arrest in human cells. These data suggest that the full activation of
the DDR cascade at uncapped telomeres is RNA-dependent and is controlled by Dicer and Drosha.
Presented by: Rossiello, Francesca
61
Session 7-1
Telomere Response to Viral Infection
Zhong Denga, Eui Tae Kimb, Nick Stonga, Jayaraju Dheekollua, Zhuo Wanga, Harold Riethmana, Nigel
Fraserb, Matthew Weitzmanb, Paul Lieberman*a,
a
b
The Wistar Institute
University of Pennsylvania, School of Medicine
Telomeres have well-established functions in chromosome-end protection in response to DNA
replication stress, but it is not yet known whether telomeres provide protection against other forms of
nuclear stress, like viral infection. Viruses typically subvert host-cell regulatory mechanisms that
function in anti-viral capacities. Here, we show that acute infection by herpes simplex virus type 1
(HSV-1) causes massive changes in telomere activity and structure. We found that HSV-1 induces a
rapid increase in telomere repeat-containing RNA (TERRA) transcripts. This TERRA induction is
accompanied by dissociation of telomere repeat binding factors (shelterin), and ultimately the
selective loss of telomere repeat DNA. The HSV-1 encoded E3 ubiquitin ligase ICP0 contributes to
TERRA induction, telomere
repeat factor dissociation, and proteasome-dependent degradation of telomere protection protein
TPP1. We also find that the HSV-1 replication factor ICP8, a single-stranded DNA binding protein,
colocalizes with telomere DNA foci and binds to subtelomere DNA proximal to telomere repeats. In
addition, ICP8-mediates viral replication compartments assembly at sites of telomere DNA foci.
Furthermore, shRNA depletion of TPP1 accelerates viral replication. These findings reveal that viral
infection can extensively remodel cellular telomeres, and further suggest that telomeric factors can
play a broader role in restricting and regulating viral infection in the nucleus.
Presented by: Lieberman, Paul
62
Session 7-2
Regulation and Functions of TERRA ncRNA
Antonio Porro, Sascha Feuerhahn, Joachim Lingner, et
al. EPFL
The long noncoding RNA TERRA sustains several telomere functions. In Saccharomyces cerevisiae,
TERRA
transcription promotes telomere end shortening by exonuclease 1. In this organism, TERRA has also
been implicated in seeding the formation of nucleoplasmic telomerase clusters, which precedes
association of these clusters with short telomeres for their extension. On the other hand, TERRA is a
potent inhibitor of at least human telomerase in vitro but this effect may be prevented in vivo by
TERRA binding proteins such as hnRNPA1 or by downregulation of TERRA during S phase. TERRA has
also been suspected to mediate the exchange of single strand telomere binding proteins upon
telomere replication. Finally, TERRA may help recruiting proteins that modulate telomeric chromatin
and DNA processing enzymes. By RNA-IP, we recently identified the LSD1 lysine demethylase as TERRA
binder and in ChIP and Q-TIP experiments we see LSD1 at telomeres. We provided evidence that
TERRA and LSD1
cooperate with the MRE11 nuclease at telomeres that are uncapped by depletion of TRF2. We propose
a neat 3-step mechanism that guides removal of single-stranded terminal telomeric DNA at uncapped
telomeres. The mechanism involves telomere transcription into TERRA, TERRA-guided recruitment of
the LSD1 demethylase to MRE11 at uncapped telomeres and LSD1-mediated activation of the
DNA nuclease activity of MRE11. I will report on the mechanisms that lead to upregulation of the
TERRA transcriptome when TRF2 is depleted and TERRA interactions with chromatin modifying
enzymes.
Presented by: Lingner, Joachim
63
Session 7-3
Identification of novel telomere regulators through systematic genetic screens in yeast
Dimitris Kyriakoua, Emmanouil Stavroua, Panayiota Demosthenousa, Bryan-Joseph San Luisb, Charlie
Booneb, Antonis Kirmizisa,*
a
Department of Biological Sciences, University of Cyprus, Nicosia, Cyprus
Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto,
Canada
b
Pervasive transcription is a feature of eukaryotic genomes resulting in numerous non-coding
transcripts. Recent high-throughput studies in S. cerevisiae using hybridization to DNA-tiling arrays
have uncovered the expression of several long intergenic non-coding RNAs (lincRNAs). In an attempt to
illuminate the biological function of selected lincRNAs we have employed a synthetic genetic array
(SGA) technique. Using this assay we expect to identify genetic interactions by generating
systematically double mutants of a lincRNA with a genome-wide library of protein-coding genes. Since
the SGA procedure has been previously applied only on protein-coding genes we first validated our
approach by performing a screen for the yeast telomerase RNA (TLC1) that represents an example of a
lincRNA with a known function. Indeed, we identified and validated several synthetic genetic
interactions between TLC1 and genes whose biological functions associate with telomere organization.
In particular, we identified genetic interactions between TLC1 and the telomeric Ku complex, DNA
recombination factors that maintain telomere length and the INO80 nucleosome-remodeling complex,
which has been recently implicated in the control of chromatin structure at telomeres. Interestingly,
TLC1 also genetically interacts with other chromatin regulators, such as various histone modifiers
whose role in telomere organization remains unknown. Among the other lincRNAs screened, one of
them (SUT457), genetically interacts with genes involved in telomere maintenance, implicating this
lincRNA within this process. Overall, our findings demonstrate that SGA can be utilized to point
towards the biological function of lincRNAs in yeast and in the above examples identify new candidates
that control telomere structure.
Presented by: Kirmizis, Antonis
64
Session 7-4
Human RTEL1 regulates the export and cytoplasmic trafficking of ncRNAs, including telomerase RNA
Michael Schertzera, Rosa Maria Porrecaa, Karina Jouravlevaa, Mylène Perderiseta, Florent Dinglib,
Damarys Loewb, Tangui Le Guenc, Jean Pierre De Villartayc, Patrick Revyc, Arturo Londono-Vallejoa,*
a
Telomeres and Cancer lab, Institut Curie
Laboratory of Proteomic Mass Spectrometry, Institut Curie
c
INSERM, U768
b
Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by
developmental defects, bone marrow failure, and immunodeficiency and has been associated with
telomere dysfunction. Recently, mutations in RTEL1, a helicase first identified in Mus musculus as being
responsible for the maintenance of long telomeres, have been identified in several HHS patients. We
show that RTEL1 interacts with exportins XPO1, XPO5, and XPOT and is required for the export and
correct cytoplasmic trafficking of non-coding (nc) RNAs, including pre-U2, a component of the major
spliceosome complex, and TERC, the telomerase RNA. While reconstitution of telomerase activity is
deficient in RTEL1-HHS cells, these cells show abnormal subcellular partitioning of TERC and pre-U2,
defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm, phenotypes that can be
experimentally induced by the expression of RTEL1 variants carrying mutations in the helicase and Cterminal domains. Our work unravels broad and totally unanticipated roles for RTEL1 in RNP export
and trafficking, thus leaving open the possibility that general defects in RNP biogenesis contribute to
the pathology of HHS.
Presented by: Londono-Vallejo, Arturo
65
Session 7-5
AKTIP, an E2 variant enzyme that interacts with lamin, is required for correct telomere maintenance
Mattia La Torrea,*, Romina Burlaa, Mariateresa Carcuroa, Grazia Daniela Raffaa, Alessandra Galatia,
Domenico Raimondob, Angela Rizzoc, Laura Ciapponia, Giovanni Cencid, Enrico Cundarie, et al.
a
Dipartimento di Biologia e Biotecnologie, Sapienza, Università di Roma, 00185, Italy
Istituto Pasteur Fondazione Cenci Bolognetti, Sapienza, Università di Roma, 00185, Italy
b
Dipartimento di Fisica, Sapienza, Università di Roma, 00185, Italy
c
Istituto Nazionale Tumori Regina Elena, Rome 00144, Italy
d
Dipartimento di Biologia e Biotecnologie, Sapienza, Università di Roma, 00185, Italy
e
Istituto di Biologia e Patologia Molecolari del CNR, Sapienza, Università di Roma, 00185, Italy
Telomeric DNA challenges cell metabolism at multiple levels requiring the shelterin complex and the
telomere accessory factors for chromosome end protection and for correct telomere replication. We
report here on AKTIP, an ubiquitin E2 variant enzyme, that we have identified as required for
mammalian telomere metabolism. AKTIP was investigated on the basis of its homology with the
Drosophila telomere-capping protein Pendolino. AKTIP interacts with purified TRF1 and TRF2, and
immunoprecipitates telomeric DNA. Loss of AKTIP/Ft1 results in fragile telomeres and sister telomere
associations, which, in turn, have been associated to telomere replication problems, as clearly assessed
in TRF1-depleted cells. In doubly depleted TRF1/AKTIP cells, the TRF1-induced fragile telomere
phenotype was epistatic to that of AKTIP, suggesting that TRF1 and AKTIP are involved in a common
molecular pathway. In fact, in AKTIP depleted cells, telomeric replication is impaired, and AKTIP
interacts with the DNA replication factors PCNA and RPA70. Intriguingly, AKTIP locates at the nuclear
periphery, the site of late replicating heterochromatic DNA, and interacts with lamins. In vivo, the
depletion of the mouse homologue of AKTIP, Ft1, causes severe developmental abnormalities and
premature death. Our results suggest that AKTIP/Ft1 plays a crucial role in telomere maintenance
presumably acting in lamin-associated replication factories.
Presented by: La Torre, Mattia
66
Session 8-1
A billion years of shelterin – conservation of interaction modules and the potential to form higher
order structures.
Lili Pan2,3, Cian Stutz4, Nicolas Thomä4, Peter Baumann1,2,3
1
Howard Hughes Medical Institute
Stowers Institute for Medical Research, Kansas City, MO 64110
3
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City,
KS 66103
4
Friedrich Miescher Institute for Biomedical Research, Basel, 4058, Switzerland
2
In mammalian cells, the shelterin proteins (TRF1, TRF2, RAP1, TIN2, POT1 and TPP1) are central to
telomere protection and length maintenance. The fission yeast Schizosaccharomyces pombe telomere
maintenance machinery shares fundamental similarities with the mammalian factors. Taz1 (hTRF1/2
ortholog) interacts with double-stranded telomeric repeats; Rap1 binds to Taz1 as hRAP1 binds to
hTRF2 and the Pot1-Tpz1 complex (hPOT1-hTPP1 homologs) bind single stranded telomeric DNA.
Finally, Poz1, functionally similar to hTIN2, forms a bridge between Taz1/Rap1 and Pot1/Tpz1. All five
proteins play critical yet different roles in telomere length regulation. Deletion of Taz1, Rap1 and Poz1
leads to telomere elongation, while deletion of Tpz1 and Pot1 results in rapid telomere loss. How these
proteins work together to maintain telomere length equilibrium is not well understood. The Poz1-Tpz1
interface is of particular interest as it forms the boundary between negative and positive regulators of
telomere length.
To understand the role of this interaction, we solved the crystal structure of Poz1 bound to Tpz1 at
2.4Å resolution. Surprisingly, the Poz1-Tpz1 complex shares remarkable similarities with the docking
motifs in human TRF1 and TRF2 that bind TIN2 and Apollo, respectively. Indeed, the corresponding
regions of TRF2 and Apollo can functionally substitute for the fission yeast proteins. Structural and
biochemical analysis further revealed a homodimerization interface in Poz1. Mutational analysis
indicates that Poz1 homodimerization is critical for telomere length regulation. We speculate that the
complexity of interactions between telomeric proteins forms the basis for a higher order architecture
with implications for telomere function.
Presented by: Pan, Lili
67
Session 8-2
Examining telomere architecture in yeast
Nicolas Thomä
Friedrich Miescher Institute for Biomedical Research
Maulbeerstrasse 66
4051 Basel - Switzerland
Budding yeast telomeres comprise irregular TG₁₋₃ DNA repeats bound by the general transcription
factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits
telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition
of telomeres as DNA double-strand breaks. We provide a molecular, biochemical, and functional
dissection of the
protein backbone at the core of the yeast telosome. The X-ray structures of Rif1 and Rif2 bound to
the Rap1 C-terminal domain and that of the Rif1 C terminus are presented. Both Rif1 and Rif2 have
separable and independent Rap1-binding epitopes, allowing Rap1 binding over large distances (42110
Å). We identify tetramerization (Rif1) and polymerization (Rif2) modules that, in conjunction with the
long-range binding, give rise to a higher-order architecture that interlinks Rap1 units. The Rif1 Nterminus exhibits high apparent affinity for DNA, and is expected to engage telomeric single-stranded
and double stranded regions once gaps, or overhangs, arise that that are longer than 50nt. This
molecular Velcro relies on Rif1 and Rif2 to recruit and stabilize Rap1 on telomeric arrays and is
required for telomere homeostasis in vivo. Parallels and differences between the protein architecture
of S.cerevisiae and S.pombe telomeres will be discussed.
Presented by: Thomä, Nicolas
68
Session 8-3
Guanine quadruplex structures revisited
Peter Lansdorp
European Research Institute for the Biology of Ageing, University Medical Center Groningen,
University of Groningen, Groningen, The Netherlands and Terry Fox Laboratory, BC Cancer Research
Centre and University of British Columbia, Vancouver, Canada
Reduced telomerase levels in humans result in very short telomeres. Other factors that regulate
telomere length were studied by crossing mice with long telomeres (Mus musculus) to mice with short
telomeres (Mus spretus). A “telomere elongation” trait was mapped to a region at the tip of
chromosome 2. One candidate gene in this region, “novel helicase-like” was studied in C.elegans.
Animals without this helicase showed a remarkable “signature” phenotype: genome-wide deletions
that start at the 3’end of guanine tracks > 18 nucleotides. We dubbed the gene Deletion of G-rich DNA1
(dog-1) and proposed that the DOG-1 helicase could be required to unwind intramolecular guanine
quadruplex (G4) DNA structures arising during lagging strand DNA synthesis. More recent studies in
yeast have shown that G-rich DNA is unstable if replicated by leading strand DNA synthesis in the
absence of PIF1. The human homolog of dog-1 is FANCJ and “novel helicase-like“ was renamed
"Regulator of telomere length-1” or RTEL-1. All these studies implicate G4 structures in genome
stability and telomere regulation. Both FANCJ and RTEL-1 are FeS helicases that unwind G4 structures.
In order
to further study the role of guanine quadruplex structures in biology we recently described
monoclonal antibodies to synthetic G4 DNA structures. Surprisingly, one of these antibodies, 1H6,
shows bright nuclear staining of most cells suggesting that G4 structures are not formed during
replication but are already present in some cells prior to replication. The emerging role of G4
structures in nuclear organization and cell biology will be discussed.
Presented by: Lansdorp, Peter
69
Session 8-4
A telomere protective function of human telomerase reverse transcriptase (hTERT), which is
independent of catalytic activity
Omesha N. Pereraa,*, Michelle F. Maritzb, Anthony J. Cesarea, Karen L. MacKenzieb, Tracy M. Bryana
a
Children’s Medical Research Institute, Westmead, NSW 2145, Australia
Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, Sydney
NSW 2031, Australia
b
The majority (>85%) of cancers rely on the ribonucleoprotein enzyme telomerase to catalyse the
addition of telomeric DNA repeats to counteract telomere shortening, thus conferring cells with an
unlimited replicative lifespan. There is growing evidence for a number of non-canonical functions of
telomerase, and more specifically the protein hTERT, that are independent of telomere maintenance
and catalytic activity. One of these includes a protective function for telomerase in protecting
telomeres from DNA damage.
We have observed that over-expression of a catalytically inactive (D712A) hTERT in immortal GM639
fibroblasts can reduce the number of telomere dysfunction-induced foci (TIF) in comparison to cells
transfected with an empty vector control, to a level similar to that observed in cells over-expressing
wild-type hTERT. Interestingly, over-expression of a mutant hTERT which does not localise to telomeres
also demonstrated a similar rescue of TIF. This suggests that hTERT potentially activates a telomereindependent signalling pathway that provides protection indirectly.
A potential mechanism involves the oxidative stress pathway. Upon increasing intracellular reactive
oxygen species (ROS) in HT1080 cells, we observed a doubling in the number of TIF, which was rescued
upon over-expression of D712A. Furthermore, depletion of endogenous hTERT decreased levels of the
antioxidant, superoxide dismutase 2 (SOD2), which corresponded to increased mitochondrial
superoxide levels, as well as a further increase in ROS-induced TIF. These data suggest that hTERT may
possess a protective function independent of its catalytic activity, to protect immortal cells from
telomeric DNA damage through activation of a telomere-independent signalling pathway.
Presented by: Perera, Omesha N.
70
Session 8-5
Structural basis for protein-RNA recognition in telomerase
Jing Huang1,2,3, Andrew F. Brown4, Jian Wu1, Jing Xue1, Christopher J. Bley4, Dustin P. Rand4, Lijie Wu1,
Rongguang Zhang1, Julian J.-L. Chen4, and Ming Lei1,2,3
1
National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of
Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
2
Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI 48109, USA
3
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA
4
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA
Telomerase is a large ribonucleoprotein complex minimally composed of a catalytic telomerase reverse
transcriptase (TERT) and an RNA component (TR) that provides the template for telomeric DNA
synthesis. However, it remains unclear how TERT and TR assemble into a functional telomerase. Here
we report the first crystal structure of the conserved regions 4 and 5 (CR4/5) of TR in complex with the
TR-binding domain (TRBD) of TERT from the teleost fish Oryzias latipes. The structure shows that CR4/5
adopts an L-shaped three-way-junction conformation with its two arms clamping onto TRBD. Both the
sequence and conformation of CR4/5 are required for the interaction. Our structural and mutational
analyses suggest that the observed CR4/5-TRBD recognition is common to species from yeast to
humans, and CR4/5 in vertebrate TR might play a similar role in telomerase regulation as stem-loop IV
in Tetrahymena TR.
Presented by: Lei, Ming
71
Keynote Session 2
Regulation of human Immune system CD4+ T cell apoptosis specifically by the RNA component
of telomerase
Elizabeth Blackburn, Francesca Gazzaniga
Department of Biochemistry and Biophysics, University of California San Francisco, CA, USA
Genetic deficiencies in telomerase RNA (hTR) levels in humans exacerbate telomere shortening and
cause a spectrum of diseases. While loss of telomere protective function can account for the
pathology of these inherited diseases, no previous studies have identified any other consequences of
the diminished levels of hTR in these patients.
T cells are differentiated adult human cells that rapidly upregulate telomerase activity from low basal
levels upon mitotic stimulation. We tested whether this upregulation is a consequence of the
stimulation response or, conversely, is necessary for T cell proliferation and survival. Surprisingly,
knocking down telomerase enzymatic activity level in stimulated CD4+ T cells to an equal extent by
depleting either hTR or hTERT produced very different short-term responses. First, the hTERT
knockdown caused no effect in the short time frame of the experiments. In contrast, the hTR
knockdown reproducibly induced Bim-mediated apoptosis. This apoptosis occurred despite the
absence throughout the experimental time-course of detectable p53 activation or telomere
shortening or damage. Experiments including utilizing hTR mutated in various known functional
regions provided evidence that hTR specifically in a telomerase enzymatically inactive form protects
CD4+ T cells from apoptosis, including dexamethasone-induced apoptosis.
This is the first report suggesting a new protective role for the non-coding telomerase RNA in normal
human cells through a telomere- and telomerase enzymatic-independent pathway. We propose that
in addition to having long-term effects on telomere shortening, reduced hTR potentially impacts on
immune system functionality through a new route: by perturbing short-term survival of differentiated
T cells.
Presented by: Blackburn, Elizabeth
72
Poster 1
Detection of DNA damage response RNAs (DDRNAs) at dysfunctional telomeres
Julio Aguadoa,*, Francesca Rossielloa, Fabrizio d'Adda di Fagagnab
a
The FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy
-The FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy
-Istituto di Genetica Molecolare – Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100
Pavia, Italy
b
Our lab has recently shown that small non-coding RNAs are novel components of the DNA damage
response (DDR) machinery (Francia et al. Nature 2012). These small RNAs, named DDRNAs, are
involved in the control of the DNA damage response (DDR) activation, have the sequence of the
damaged locus and their biogenesis depends on Dicer and Drosha endoribonucleases. Upon TRF2
inactivation telomeres are uncapped and a DDR is triggered. Different approaches are being
undertaken to detect the potential presence of putative DDRNAs upon telomere uncapping. A method
based on qRT-PCR is being developed to detect and characterize DDRNAs at dysfunctional telomeres.
We will report on our progresses in our attempts to monitor telomeric DDRNAs accumulation.
Presented by: Aguado, Julio
73
Poster 2
RTEL-1 promotes telomere-ITS recombination events
Megan Brady, Shawn Ahmed
Department of Genetics, Curriculum in Genetics and Molecular Biology, University of North Carolina,
Chapel Hill NC 27599, USA
Telomerase deficiency ultimately results in critically shortened telomeres that are joined to yield endto-end chromosome fusions. Though critically shortened telomeres can be directly ligated via NHEJ or
single-strand annealing, they can also recombine with interstitial telomere sequence (ITS) tracts
thereby duplicating subtelomeric segments of the genome prior to fusion. We developed a PCR-based
assay to study telomere-ITS recombination and found that recombination events occurred within ITS
tracts, which are composed of degenerate telomere repeats, irrespective of orientation to the
telomere, and at sites of longest perfect homology to the telomere, typically 12 to 30 nt in length.
RTEL1 promotes telomere stability, regulates homologous recombination, and is essential for
development and cell proliferation in mammals. Deficiency for the C. elegans homolog of RTEL1
helicase, RTEL-1, does not abrogate viability or fertility, allowing us to assess the consequences of
RTEL-1 dysfunction in the context of telomerase deficiency. We found that RTEL-1 plays a central role
telomere-ITS recombination events, indicating that this helicase not only plays a role in normal
telomere homeostasis but also promotes illegitimate recombination events at critically shortened
telomeres. Our results are consistent with the proposal that RTEL-1 may promote synthesis-dependent
strand annealing events at sites of DNA damage, and imply that telomere-ITS recombination events
reflect inappropriate engagement of a major homologous recombination pathway.
Presented by: Ahmed, Shawn
74
Poster 3
A simple model for tandem telomeric G-quadruplexes
Patrizia Alberti
Muséum national d'Histoire naturelle, Paris (France)
In human, telomeric DNA is composed of tandem repeats of the GGGTTA motif. The average length of
the telomeric 3’-overhang in human chromosomes (100-200 nt) makes this overhang prone to fold into
tandem G-quadruplexes (G4), like beads on a string. While the in vivo formation and the role of
tandem G4 at telomeres still remain elusive, evidences of the in vitro formation of this kind of
structures have been reported in several studies.
How stable are these structures? When considering (GGGTTA)7,11,15GGG sequences, able to fold into
two, three and four consecutive G4, respectively, I found that the global stability of the formed
structures decreased with the number of repeats. This behaviour has already been reported for similar
sequences (1), nevertheless it appears to depend on boundary conditions. For exemple, the two- and
three-tandem G4 structures formed by the sequences TTA-(GGGTTA)7,11GGG display the same
thermal stability (2). Results obtained from an analysis of melting transitions suggested that the G4
units in a tandem structure were not identical nor independend and revealed an unfavourable coupling
free energy in the formation of three-tandem G4 structures (2).
What makes G4 units in a tandem structure not identical? do the G4 units interact each other? Despite
the accumulation of data, a systematic approach to the comprehension of the stability of these kind of
structures is still lacking. In this poster, I present a simple model to explain the observed stability of
tandem telomeric G4 and its dependence on boundary sequences.
(1) Vorlickova et al. (2005) NAR 33, 5851
(2) Petraccone et al. (2011) JACS 133, 20951
Presented by: Alberti, Patrizia
75
Poster 4
Severe liver fibrosis caused by S. mansoni infection in Dkc1m hypomorphic mice
Raquel Alves-Paiva, Leandra Ramalho, Cleide Araujo-Silva, Olinda Mara, Vanderlei Rodrigues, Eduardo
Rego, Rodrigo Calado
University of Sao Paulo
Mutations in telomerase complex genes cause telomere shortening and are associated with bone
marrow failure, dyskeratosis congenita, aplastic anemia, and liver diseases. Clinically, approximately
7% of DC patients present hepatic disease, such as hepatic fibrosis, cirrhosis, and HCC. We evaluated
Dkc1 deficiency, based on Dkc1m hypomorphic mouse model (kindly provided by Davide Ruggero,
Sloan-Kettering Institute, USA), to chronic liver damage in a schistosomiasis experimental model. The
immune response to eggs of S. mansoni trapped in human tissues causes hepatosplenic disease and
liver fibrosis. Telomere length was performed by qPCR and Dkc1m mice had slitghly shorter telomeres
in comparison to WT mice. Dkc1m mice WT B6 controls were infected with 30 cercaries by SC injection
in the dorsal region. Over the course of infection, Dkc1m mice presented anemia and diarrhea,
although gain of body weight was noted. Mice were euthanized 45 days after S. mansoni infection and
liver fibrosis intensity was assessed. S. mansoni infection caused liver damage as fibrosis and
hepatomegaly and splenomegaly in both infected groups. S. mansoni exposure caused significantly
more hepatic fibrosis in Dkc1m mice in comparison to infected WT B6 mice (P<0.0001), as evidenced
by Sirius red staining (quantification by ImageJ software). Our findings suggest that Dkc1m mice are
more susceptible to liver damage mediated by S. mansoni infection in comparison to WT B6 mice.
Mouse model for chronic liver injury using animals with telomerase complex mutations may represent
a tool to investigate liver damage in response to chronic liver disease.
Presented by: Alves-Paiva, Raquel
76
Poster 5
Identification of telomere dysfunction in Friedreich ataxia
Sara Anjomani Virmouni, Sahar Al-Mahdawi, Chiranjeevi Sandi, Hemad Yasaei, Predrag Slijepcevic,
Mark Pook
Biosciences, SHSSC, Brunel University, Uxbridge, UK
In Friedreich ataxia (FRDA), GAA repeat expansion within the first intron of the frataxin (FXN) gene
leads to downregulation of FXN, resulting in oxidative stress, mitochondrial iron accumulation and
neuronal atrophy. We hypothesised that telomere length might be shortened as a result of oxidative
damage in FRDA. To investigate telomere function in FRDA, we initially assessed the telomere length in
human and mouse FRDA fibroblasts and we found that both cell types had chromosomes with
relatively longer telomeric repeats compared to controls. In contrast, we noted a significant telomere
shortening in FRDA leukocyte cells compared to control cells. Consequently, we screened the FRDA
fibroblasts for expression of telomerase activity and we identified that the telomerase activity was not
present in these cells. We then assessed the co-localisation of PML bodies with telomeres and
frequencies of telomere sister chromatid exchange (T-SCE) in these cells. Our results showed
significantly higher co-localised PML foci with telomeric DNA and substantially higher T-SCE levels in
the FRDA cell lines relative to the controls suggesting activation of an alternative lengthening of
telomeres (ALT)-like mechanism. In addition, further analysis revealed an accelerated rate of telomere
shortening and a higher frequency of telomere dysfunction-induced γ-H2AX foci in FRDA fibroblasts
compared to control cells. Thus, we report the novel finding of telomere dysfunction in FRDA that is
characterised by both ALT-like activation and accelerated telomere shortening. Our findings provide
novel insights into mechanisms of FRDA molecular disease progression with implications for future
FRDA therapy.
Presented by: Anjomani Virmouni, Sara
77
Poster 6
Longer telomeres lead to Type II survivors in the absence of telomerase in yeast
Usha Aryala,*, Phillipa Mustonb, Edward J Louisa
a
b
University of Leicester
University of Nottingham
Telomeres in budding yeast Saccharomyces cerevisiae are maintained by telomerase. Loss of
telomerase leads to progressive shortening of the telomeres and eventually cellular senescence.
However, a few cells adapt and become survivors by lengthening their telomeres via recombination, a
process known in humans as the alternative lengthening of telomeres (ALT). Survivors can be of Type I
and Type II, characterized by the amplification of the sub-telomeric Y’ repeats or the telomeric TG1-3
repeats respectively. We have shown that starting with longer telomeres at the initiation of telomerase
loss leads to a higher proportion of Type II survivors and vice-versa. Here we show that telomerase
negative cells with artificially induced long telomeres can produce Type II survivors in the absence of
RAD59, a recombination protein thought to be required for Type II survivor pathway and in the
absence of the endonuclease SAE2, loss of which predominantly produces Type I survivors from wild
type length telomeres. Other gene deletions such as sgs1Δ do not respond in this way. Telomere
elongation was achieved by growing RAD59/rad59Δ and SAE2/sae2Δ diploids, heterozygous for
telomerase catalytic subunit Est2/est2Δ, in ethanol cultures for a week prior to sporulation for
generating telomerase negative survivors with the respective gene knockouts. Cells treated with
ethanol display late senescence and a more efficient recovery from crisis. This suggests that longer
telomeres favour the Type II survivor pathway, resulting in faster growth recovery after crisis in the
absence of telomerase, overcoming some of the previously thought genetic requirements for Type II
survival.
Presented by: Aryal, Usha
78
Poster 7
Retroviral activation of TERT expression in chicken B-cell lymphomas
Karen Beemon, James Justice
Johns Hopkins University
We are studying the role of TERT at early stages of tumorigenesis in chicken B- cells. Analysis of avian
leukosis virus (ALV) proviral integration sites in chicken lymphomas can lead to the discovery of
activated oncogenes and inactivated tumor suppressors. Deep sequencing studies of a series of
metastatic lymphomas showed that the most common integration sites were in the TERT promoter.
Several-fold increases in TERT mRNA levels and telomerase activity were observed in the tumors. In
some tumors, these integrations were clonal, suggesting that TERT activation is an early step in tumor
induction. Extensive sequencing to identify other ALV integrations in these tumors has been carried
out. We are currently asking whether the ALV integration is changing the alternative splicing pattern of
TERT in the tumors. We are also asking whether TERT over-expression from a retroviral vector will lead
to rapid tumorigenesis in chickens. We will also analyze cooperating genes in these TERT-induced
tumors.
In the past year, a number of investigators reported point mutations in the human TERT promoter in
melanomas, gliomas, and many other types of tumors, especially those derived from cells with low
self-renewal (Horn et al, Huang et al, Killela et al., all 2013). These mutations also led to slightly
increased expression of TERT mRNA and were thought to be an early event in oncogenesis. Thus, we
think our chicken tumor system is a good model for study of the role of TERT in initiation of many types
of tumors.
Presented by: Beemon, Karen
79
Poster 8
A novel method of analysis of TANK1 enzymatic activity influence on TRF1 DNA-binding
Petra Bencúrová, Patrycja Anna Kłos, Ivona Nečasová, Ctirad Hofr
Chromatin Molecular Complexes, CEITEC and Faculty of Science, Masaryk University, Brno, CZ-62500,
Czech Republic
Mammalian telomeres are composed of long TTAGGG tandem repetitive sequences. TRF1 is one of key
shelterin proteins involved in telomere maintenance. TRF1 along with TRF2 recruit various proteins to
telomeric sites via specific binding to telomeric repeats. Previous works have shown that TRF1 is a
strong negative regulator of the length of telomeric DNA. Tankyrase 1 (TANK1) is a protein containing
domains with homology to ankyrins and to the catalytic domain of poly (ADP-ribose) polymerase
(PARP). Among various functions of TANK1, the best studied is its function at telomeres. TANK1
unprotects telomeres via poly(ADP)-ribosylation of TRF1 which results in an eventual telomeric DNA
elongation. Our study is focused on the development of novel fluorescence based method that
explores TANK1 influence on protein TRF1 equilibrium DNA-binding and TANK1 reaction kinetics. This
work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), the European Social Fund
(CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550), and KONTAKT II (LH13054).
Presented by: Bencúrová, Petra
80
Poster 9
Impact of cancer treatments in telomere length: A prospective study
Carlos Benitez-Buelgaa,*, Lara Sanchez-Barrosob, Maria Mercedes Gallardoc, Maria Apellanizb, Lucia
Inglada Perezb,e, K. Yanowskia, Miguel Uriosted,e, Ana Osorioa,e, Maria Antonia Blascoc, Cristina
Rodriguez-Antonab,e, Javier Beniteza,e
a
Human Genetic Group, Spanish National Cancer Research Center (CNIO)
Endocrine Cancer Group, Spanish National Cancer Research Center (CNIO)
c
Telomeres and Telomerase Group, Spanish National Cancer Research Center (CNIO)
d
Familial Cancer Group, Spanish National Cancer Research Center (CNIO), Network of Research in Rare
Diseases (CIBERER)
e
Network of Research in Rare Diseases (CIBERER), Spanish National Cancer Research Center (CNIO),
Madrid 28029, Spain
b
BRCA1 and BRCA2 play a role in telomere stability. Recently we demonstrated by q-RT-PCR that
BRCA1/2 mutation carriers present shorter telomeres in peripheral blood than controls or sporadic
breast cancer patients, suggesting that the mutations in these genes are responsible for this event.
To test this hypothesis, we are currently performing a prospective study in a series of 241 familial
breast and ovarian cancer patients that includes affected and non affected BRCA1/2 mutation carriers,
analyzing different parameters (percentage of short telomeres by high throughput quantitative
telomere FISH, telomerase activity, oxidative stress, treatment, exogenous factors) that could alter the
telomere length. We have observed that, in addition to the BRCA1/2 mutation, telomere shortening is
mainly associated with the treatment.
To confirm these findings, we have measured telomere length in DNA from peripheral blood of 265
sporadic breasts cancer patients treated since diagnosis with chemotherapy. We have demonstrated
that since the beginning there is a telomere shortening that becomes statistically significant around 1216 weeks of treatment. Then we observe that telomeres remain significantly shortened over a period
of 1.5 years, after which they start to elongate, back to the telomere length values of the control group
with a speed of 0.06 T/S per year.
Our results stress the need to perform prospective studies taking in account the treatment status of
the patients to get conclusive results about the relation between telomeres and cancer.
Presented by: Benitez-Buelga, Carlos
81
Poster 10
Implication of INT6 in telomere stability
Maname Benyelles, Vincent Mocquet, Serge Bauwens, Pierre Jalinot
Laboratoire de Biologie Moléculaire de la Cellule, Unité Mixte de Recherche 5239, Centre National de
la Recherche Scientifique, Ecole Normale Supérieure de Lyon, Lyon, France
The INT6 protein has been implicated in the breast cancer developpement. As part of the translation
initiation factor eiF3, INT6 is necessary for the translation of specific mRNA such as histone mRNA. It
has also been involved in the Nonsense Mediated mRNA Decay process. In this latter one, INT6 could
be a link between eiF3 and the NMD complex including UPF2 and UPF1. In parallel, INT6 is necessary
for a correct replication by favoring the ubiquitination of the MCM7 helicase. Recent data also showed
that at the telomeres, UPF1 plays a role in the adequate replication fork progression. Then, UPF1
defects lead to the accumulation of the telomere repeat containing RNA (TERRA) at the telomeres.
Based on those data, we hypothesized that INT6 could influence the telomere integrity. Here, by
treating U2OS cells with siRNA, we found that INT6 silencing increased total TERRA molecules and that
was confirmed by RT-qPCR. These variations were not due to TERRA stability modification. So, we
hypothesis that this accumulation could be explained by a telomere instability because INT6 silencing
generates telomere dysfunction induced focis (Tifs). Specifically, using a PNA-FISH approach, we
observed that the inhibition of INT6 induced telomeres free ends (TFEs) which correlate with telomere
length decreasing (Teloblot). In addition, we found fragile telomeres (FTs) which could correspond to
replication defects. From all these observations, we propose that the protein INT6 is important for
telomere integrity through a replication defect that we have not yet elucidate.
Presented by: Benyelles, Maname
82
Poster 11
Control of telomere replication by phosphorylation
Carol Cooley, Anoushka Dave, Mansi Garg, Resham Lal Gurung, Aziz Muhamad, Alessandro Bianchi
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK
The action of telomerase is strictly linked to conventional DNA replication. We and others have
previously shown that telomere length affects replication timing in yeast. In addition, Rif1 is required
to specify the replication timing of telomeres. We demonstrate, in both buddying and fission yeast,
that the function of Rif1 in DNA replication, at telomeres and elsewhere in the genome, is due to its
ability to interact with PP1 phosphatases. In both organisms Rif1 is at least partly responsible for
recruiting PP1 to telomeres. We show that, in budding yeast, Rif1-bound PP1 acts to reverse the action
of DDK on the MCM helicase. Intriguingly, we find that the early-replication phenotype of short
budding yeast telomeres is due to the action of the Tel1 kinase. Several lines of evidence indicate that
the role of Tel1 in DNA replication at short telomeres is not to affect Rif1 itself. Because we have yet to
identify a role of Mcms in the Tel1 pathway, it remains to be established whether Tel1 and Rif1
opposing activities converge on the same target(s) in affecting replication timing.
The PP1-recruiting function of S. pombe Rif1 extends beyond the control of replication timing: we find
that Rif1 alleles defective in the ability to interact with PP1 have elongated telomeres, implicating PP1
phosphatases in the control of telomerase action.
Presented by: Bianchi, Alessandro
83
Poster 12
PLATINATION OF TELOMERES BY CIS-PLATINE IS NOT AT THE ORIGINE OF TRF2 DISPLACEMENT
Lina Sakera, Samar Alia, Guillaume Kellermanna, Evelyne Ségal-Bendirdjiana, Joël Pouponb, Sophie
Bombarda,*
a
b
INSERM UMR-S 1007, Université Paris Descartes; Paris, France
Laboratoire de Toxicologie-Biologie, Hôpital Lariboisière, Paris, France
Telomeres are DNA-protein structures that cap chromosome ends protecting them from recognition by
the DNA repair machinery. In mammalians, among the six telomeric proteins belonging to the
shelterin, TRF1 and TRF2 are both proteins able to bind directly to the double stranded telomeric DNA
and are involved in telomere length regulation and chromosome end protection. Since telomeric DNA
consists of highly repetitive short sequences of adjacent guanine residues (TTAGGG)n, it is a potential
target for platinum complexes and especially for the anti-tumor drug cis-platin. In vitro, we have
shown that adducts of cis-platin on telomeric DNA prevent the fixation of TRF2 and in a lesser extent
the one of TRF1 to telomeres (1). Our aim was to determine if it could be the case in cellulo. Telomeres
from two cancerous cells lines treated by cis-platin were purified and their platinum content has been
quantified using ICP-mass. The amount of remaining telomeric proteins bound to telomeres has been
also evaluated. As expected, we found that treatment of cells by cis-platin led to the platination of
their telomeres. However, the amount of platinum bound to telomeres cannot explain, alone, the
specific displacement of TRF2 from telomeres, suggesting that another mechanism is at the origin of
TRF2 displacement.
1. Ourliac-Garnier, I. et al (2010) J. Biol. Inorg. Chem. 15,. 641-654
Presented by: Bombard, Sophie
84
Poster 13
Mutations in Telomerase Reverse Transcriptase Promoter Contribute to Urothelial Cancer:
Mechanism and Therapeutic Implications
Sumit Boraha,*, Arthur Zauga, Linghe Xia, Natasha Powella, James Costellob, Dan Theodorescuc, Thomas
Cecha
a
University of Colorado Boulder BioFrontiers Institute, Dept of Chemistry and Biochemistry and HHMI
University of Colorado Denver Anschutz Medical Campus, Dept of Pharmacology
c
University of Colorado Denver Anschutz Medical Campus, Cancer Center
b
Although telomerase activity is upregulated in 85% - 90% of cancer cells, the molecular mechanisms by
which this happens are incompletely understood. The recent finding of highly recurrent point
mutations in the TERT promoter in several types of cancer suggests their importance for oncogenesis.
For some cancers, such as urothelial cancer (UC), these mutations are more frequently observed than
any other. Although the promoter mutations were hypothesized to increase transcription by further
recruitment of E-twenty-six transcription factors, very mild increases were observed in luciferase
reporter assays. We seek to more thoroughly understand the significance of these mutations for
oncogenesis by specifically focusing on UC – the fifth most common cancer in the Western world. We
have collected 23 UC cell lines, sequenced the TERT promoters of each, and measured the levels of
TERT protein, TR RNA, telomere lengths and, using direct assays, endogenous telomerase activity.
Although heterogeneous with respect to these parameters, the five cell lines with the greatest
telomerase activity harbor the -124 promoter mutation. Furthermore, we have collected genome-wide
data to identify exon mutations and to measure mRNA expression levels in each cell line, to identify
genes whose altered expression and/or mutation exacerbate or substitute for TERT promoter
mutations. Finally, the susceptibility of several of these cell lines to the telomerase inhibitor BIBR1532
may reveal whether the promoter mutations might provide a biomarker for anti-telomerase therapy.
Our goal is to elucidate the molecular mechanisms by which these mutations contribute to
oncogenesis, and potentially reveal unexplored aspects of telomerase biology.
Presented by: Borah, Sumit
85
Poster 14
Regulation of human telomere transcription
Joanna Boros, Anabelle Decottignies
Genetic and Epigenetic Alterations of Genomes, de Duve Institute, Catholic University of Louvain,
Brussels, Belgium
Telomeres, despite their highly heterochromatic nature, are transcribed into TElomeric Repeatcontaining RNAs (TERRA). TERRA molecules are transcribed by RNA polymerase II, initiate from
subtelomeric promoters and are controlled by cell cycle and telomere length. Ensuring proper levels of
TERRA in cells is vital for telomere maintenance, as recent studies suggested an involvement of these
non-coding RNAs in various aspects of telomere biology, including heterochromatin formation or Tloop regulation. In addition, a recent quantitative SILAC-based screen identified additional putative
TERRA interacting proteins involved in a variety of cellular processes. All these data suggest that
generation of TERRA RNA has to be subjected to tight transcriptional regulation.
To obtain more insight into TERRA regulation, we have performed an in silico analysis of human
subtelomeric promoter regions. Distribution patterns of general core promoter elements within the
CpG islands containing promoters imply, that transcription initiates from multiple sites. Interestingly, in
promoter regions lacking the CpG islands, we identified TATA-box like elements indicative of single-site
transcription initiation. In addition, we also identified putative sites for specific transcription factor and
verified its presence on human TERRA promoters by ChIP. Moreover, we demonstrate that TERRA can
be a downstream effector of a signaling pathway, which can provide potential link between
mitochondria maintenance and telomere integrity.
Presented by: Boros, Joanna
86
Poster 15
Beneficial testosterone treatment in a mouse model of aplastic anaemia
Christian Bӓr, Fabian Beier, Miguel Foronda, Maria Blasco
Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre
(CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain
Aplastic anaemia (AA) is a rare but serious disorder which can develop at any stage of life and which is
characterised by cytopenia in one or more of the three blood lineages. Most cases of AA are due to
autoimmune processes against the hematopoietic stem cell compartment. One general features of AA
is the presence of shortened telomeres. In this regard, recent studies demonstrated that a small but
significant subset of patients carry mutations in the core telomerase components Tert or Terc. Haploinsufficiencies in components of the telomerase complex lead to accelerated telomere attrition and
subsequently to stem cell exhaustion and bone marrow failure. Interestingly, testosterone has been
used for over half a century in the treatment of AA. Despite of frequent induction of remission
following hormone therapy, the underlying mechanism and the cause for treatment response
remained obscure. However, recent studies showed that sex hormones increase telomerase activity in
primary human haematopoietic cells in vitro. It is therefore conceivable that testosterone-mediated
telomerase activation could attenuate or reverse AA disease progression associated with impaired
telomere maintenance.
To address this question, here we use a mouse model of aplastic anaemia recently developed in our
laboratory in which we can shorten telomeres at will specifically in the bone marrow compartment.
We find that testosterone therapy increases blood counts and extends life-span under conditions of
continuous telomere erosion. Importantly, longitudinal follow-up studies revealed a positive
correlation between telomere length and hormone treatment in peripheral blood.
Presented by: Bӓr, Christian
87
Poster 16
The putative Leishmania spp. telomerase RNA (TER) undergoes trans-splicing and contains a
conserved template sequence
Elton José Vasconcelosa, Vinicius Santana Nunesb, Marcelo Santos da Silvac, Marcela Segattod, Peter
Mylera, Maria Isabel Nogueira Canob,*
a
Seattle Biomedical Research Institute, Seattle, Washington, United States of America
Genetics Dept., Bioscience Institute, Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP,
Botucatu, Sao Paulo, Brazil
c
Genetics Dept., Bioscience Institute, Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP,
Botucatu,Sao Paulo, Brazil
d
Genetica Dept., Bioscience Institute, Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP,
Botucatu, Sao Paulo, Brazil
b
Telomerase RNAs (TERs) are highly divergent among different species, varying both in size and
sequence composition. Recent studies have unraveled a 993 nt transcript that is part of the
trypanosomatid protozoa Trypanosoma brucei TERT holoenzyme (TbTER). Due to the existence of
alternative polyA sites, TbTER might also achieve a length of about 1,500 nt. Here we present LeishTER,
a candidate to the telomerase RNA component of Leishmania spp., another pathogenic
trypanosomatid that causes leishmaniasis, a neglected tropical multi-symptoms disease. Merging a
thorough computational screening together with RNA-Seq evidences and target-driven molecular
biology assays, we have detected a ~2,150 nt-long transcript assigned as LeishTER, one of the longest
TERs described so far. Similarly to TbTER, LeishTER is also trans-spliced, probably polyadenylated and
bears a C/D box snoRNA domain. LeishTER was co-purified with the telomerase protein component
(TERT) from TERT-IP nuclear extracts. Using RNA FISH coupled with immunofluorescence, it was
possible to see that LeishTER co-localizes with TERT in a cell cycle-dependent manner. Our in silico
prediction of the LeishTER secondary structure strongly suggests the existence of a bona fide singlestranded template sequence similar to the TbTER template and a C[U/C]GUCA motif-containing Helix II,
which is also conserved in Tetrahymena thermophila TER and represents the template boundary
element (TBE). This study is the first report on a candidate to Leishmania spp TER component, paving
the way to further investigations on the biogenesis of parasite TERT RNP and, consequently, its role in
parasite telomere biology and as a future target for chemotherapeutic intervention.
Presented by: Cano, Maria Isabel Nogueira
88
Poster 17
The Effect of Polymerase-alpha on Telomerase Processivity
Chris Caridi, Connie Nugent
University of California, Riverside
Telomeres, the ends of linear chromosomes, serve three functions; protecting DNA from nucleolytic
degradation, blocking the DNA damage checkpoint and ensuring replication of chromosome ends.
Telomerase, a reverse transcriptase that extends the 3’ ends of chromosomes, is important for
maintaining sufficient telomere length ensuring genome stability and the replicative lifespan of cells.
One question regarding telomere biology is how telomerase mediated extension is terminated.
Telomere binding proteins promote the selection of ends for telomerase extension, but once
telomerase engages an end, little is known about what limits the number of repeats that it synthesizes.
We investigated the role of the polymerase alpha-primase complex (Pol-α) in regulation of telomerase
access and repeat addition processivity in S. cerevisiae. To test the effect of Pol-α on telomerase
activity an assay developed by Linger et al was used. Using a Two Template-Single Telomere Extension
Assay (2T-STEX, Chang et al. 2007) we have determined that a temperature sensitive allele of POL1
(cdc17-2) results in increased telomerase processivity at both the permissive and non-permissive
temperatures. In addition, telomerase is more promiscuous in extending ends, lacking a preference for
extending shorter ends. Thus, Pol-α may play a role in stopping telomerase extension and allowing for
“fill-in” synthesis of the complementary strand. This mis-regulation of telomerase may account for the
elongated telomeres observed in cdc17-2 strains. Whether the impact of Pol-α on telomerase is
mediated through its contacts with the Cdc13-Stn1-Ten1 proteins is being determined by testing alleles
more specifically deficient for interaction among the complexes.
Presented by: Caridi, Chris
89
Poster 18
EXPRESSION OF A SMALL INTERNAL FRAGMENT OF DYSKERIN (GSE24.2), DECREASES DNA DAMAGE,
OXIDATIVE STRESS AND SENESCENCE IN ATAXIA TELANGIECTASIA CELLS
Jaime Carrilloa,*, Laura Pintado Berninchesa, Cristina Manguán Garcíaa, Laura Iarriccioa, Guillermo
Güenechea Amurriob, Juan A. Buerenb, Leandro Sastrea, Rosario Peronaa
a
Instituto de Investigaciones Biomédicas CSIC/UAM, IDIPaz and CIBER de enfermedades raras. Madrid,
Spain.
b
Gene therapy in haematopoyetic system department. CIEMAT and CIBER de enfermedades raras.
Madrid, Spain.
The ATM gene is mutated in Ataxia telangiectasia (AT), although the mechanisms underlying AT disease
are still not fully understood. ATM protein senses and regulates cellular redox status, using a complex
network of downstream signaling pathways, and is also a key transducer of DNA damage signals to
downstream effectors. ATM deficient cells show increased ROS accumulation, activation of the p38
protein kinase and increased basal levels of DNA damage.
GSE24.2 a peptide corresponding to an internal domain of Dyskerin has proved to induce telomerase
activity by stabilizing TERC and increasing expression of TERT (Machado-Pinilla, R. et al. (2008) Blood,
111:2606-14). GSE24.2 (Gestelmir) has been recently approved by EMA for the treatment of
Dyskeratosis congenita. Due to these findings, GSE24.2 was expressed in AT human primary fibroblasts
and lymphoblastoid cell lines, which harbor different mutations in the ATM gene, to study the effect in
DNA damage, ROS production, expression of proinflammatory cytokines, survival to radiomimetic
drugs, activation of p38 protein kinases and senescence induction. Infection of these cells with a
lentiviral vector expressing GSE24.2 showed a reduction in basal levels of DNA damage, decreased
levels of ROS, lower levels of p38 phosphorylation and pro-inflammatory cytokines, higher resistance
to bleomycin induced DNA damage and lower senescence than cells infected with control lentivirus
expressing GFP. Furthermore, treatment of AT fibroblasts with nanoparticles loaded with synthetic
GSE24.2 or a shorter derived peptide GSE4 was able to rescue from DNA damage and senescence.
Financial Support: (FIS-P11-00949 and INNPACTO IPT-2012-0674-090000 Cristina Manguan is
contracted by CIBERER.
Presented by: Carrillo, Jaime
90
Poster 19
Identification and validation of genome instability drivers in a panel of human cancers
Maria Antonietta Ceronea,*, Nnenna Kanua, Mcgranahan Nicholasb, Pierre Martinezb, Charles Swantonc
a
UCL Cancer Institute, Paul O'Gorman Building, Huntley St., London WC1E 6BT, UK
Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
c
Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
UCL Cancer Institute, Paul O'Gorman Building, Huntley St., London WC1E 6BT, UK
b
Genome instability is a hallmark of all cancers. It is characterized by alterations and/or loss of part or
whole chromosomes and often results in aneuploidy. High genome instability has been associated with
a more malignant phenotype, resistance to treatment and worse prognosis for the patients.
The molecular mechanisms underlying genomic instability are still not completely understood. Several
large-scale genomic studies have been performed to identify gene mutations that cause genome
instability. The aim of this study was to evaluate the functional impact of a subset of gene mutations
on inducing and/or enhancing genome instability.
Using the data obtained by exome sequencing of hundreds of human tumors recently published in the
literature, we have selected 65 unique genes significantly mutated in four cancer types: breast, colon,
lung and kidney. These genes are all potential tumor suppressor genes, whose alterations result in loss
of function of the proteins of interest. They include 30 genes mutated in breast cancer, 25 in colon, 12
in kidney and 24 in lung. To analyze their function, we will use three cell lines per tumor type and
perform a siRNA screen using as read-out a panel of assays to monitor several aspects of genome
stability, including induction of DNA damage, presence of segregation errors, alteration of the cell cycle
profile, cytokinesis defects, telomere maintenance, viability and cell death. Preliminary results will be
presented at the meeting.
Presented by: Cerone, Maria Antonietta
91
Poster 20
The Ctc1/Stn1/Ten1 complex localizes in APB and regulates C-circle formation in ALT cells
Chenhui Huang, Xueyu Dai, Weihang Chai
Washington State University
Telomerase-negative cancer cells counteract telomere loss by activating the telomerase-independent
ALT pathway. However, the molecular details of the ALT pathway are largely elusive. The
Ctc1/Stn1/Ten1 complex (CST) was previously reported to play an important role in telomere
maintenance through promoting efficient telomere replication, mediating C-strand fill-in, and
regulating telomerase activity in telomerase-expressing non-ALT cancer cells. In this study, we report
that human CST forms a functional complex localizing in the ALT-associated PML bodies (APBs) in ALT
cells. Suppression of CST inhibits ALT cell proliferation and significantly diminishes C-circle abundance
without affecting the overall telomere length. The impact of CST on C-circle homeostasis is unlikely due
to replication defect induced by CST defection. Using the separation-of-function mutations of Ctc1, we
also find that CST binding to telomeres is important for maintaining the CC levels in ALT cells, while the
CST/polalpha interaction is dispensable for regulating CC production. Our findings therefore reveal a
role of CST in regulating C-circle formation that is independent of its function in telomere replication,
suggesting that the function of CST at telomeres is multifaceted.
Presented by: Chai, Weihang
92
Poster 21
Telomere and mitochondrial dysfunction in Duchenne Muscular Dystrophy
Alex AC Changa,*, Foteini Mourkiotia, Sang-Ging Ongb, Joseph Wub, Helen M Blaua
a
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for
Stem Cell Biology and Regenerative Medicine, and Stanford Cardiovascular Institute, Stanford, CA, USA
b
Division of Cardiology, Department of Medicine and Stanford Cardiovascular Institute, Stanford, CA,
USA
Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disease that is result of mutations in
the dystrophin gene and is the most common myopathic disease in humans with a prevalence of one in
every 3500 males. Dystrophin is crucial for the formation of a dystrophin-glycoprotein complex (DGC),
which connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix in both
skeletal and cardiac muscles. In the heart, loss of dystrophin leads to increased fibrosis and death in
the third decade of life due to dilated cardiomyopathy. A conundrum in studying and developing
therapies for DMD has been the lack of a mouse model that fully recapitulates the clinical phenotype,
as mice that lack dystrophin (mdx model), unlike patients, exhibit only mild skeletal muscle defects,
essentially no cardiac defects and have a relatively normal lifespan. Our lab reasoned that the
difference in the manifestation of the disease in mice and humans could be telomere length, as mice
have substantially longer telomeres than humans. We created a novel mouse model with shortened
telomere lengths (similar to humans) that fully recapitulates the skeletal muscle (Cell. 2010;143:10591071; the mdx/mTRKO model) and cardiac muscle phenotype of DMD (Nat Cell Biol. 2013; 15:895-904;
dilated cardiomyopathy). Interestingly, we observed a 45% reduction in cardiomyocyte telomere
length in our mdx/mTRKO animals as well as patient samples. Here we present new evidence of
mitochondrial dysfunction and telomere dysfunction.
Presented by: Chang, Alex AC
93
Poster 22
CTC1 Mutations in Telomere Syndromes Impair Telomere Replication
Liuh-yow Chen, Jana Majerská, Joachim Lingner
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Ecole Polytechnique
Fédérale de Lausanne (EPFL), Switzerland
Mutations in CTC1 lead to the telomere syndromes Coats Plus and Dyskeratosis Congenita, but the
involved molecular mechanisms remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex
termed CST, which promotes telomere DNA synthesis and inhibits telomerase mediated telomere
elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical
interaction with DNA polymerase α-primase, telomeric ssDNA binding in vitro or/and nuclear
localization and telomere association in vivo. While having diverse molecular defects, expression of
CTC1 mutant proteins commonly leads to the accumulation of internal single-stranded gaps of
telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease.
Furthermore, cytological analysis indicates that telomerase inhibition enhances telomere structure loss
upon expression of CTC1 mutant proteins, suggesting that telomerase may be able compensate the
telomere replication defect. Hence the telomere defect initiated by CTC1 mutations is distinct from the
telomerase deficiency seen in classical forms of telomere syndromes, which cause short telomeres due
to reduced maintenance of distal telomeric ends by telomerase. In addition, our analysis provides
direct molecular evidence that CST collaborates with DNA polymerase α-primase to promote faithful
telomere DNA replication.
*Current Address
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan
Presented by: Chen, Liuh-yow
94
Poster 23
Anti-metastatic factor inhibits telomerase activity in aggressive cancer cells - emerging connection
between telomerase activity and metastasis suppression
Vivek Srivastavaa, Anirban Kara, Maneesh Kumara, Dhurjhoti Sahaa, Ankita Singha, Vinod Kumar Yadavb,
Shantanu Chowdhuryc,*
a
Proteomics and Structural Biology Unit, CSIR-Institute of Genomics and Integrative Biology, Mathura
Road, New Delhi 110 020, India
b
G.N.R. Knowledge Centre for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology,
Mathura Road, New Delhi 110 020, India
c
G.N.R. Knowledge Centre for Genome Informatics, Proteomics and Structural Biology Unit, CSIRInstitute of Genomics and Integrative Biology, Mathura Road, New Delhi 110 020, India
Anti-metastatic function of NME2 has been demonstrated in several human cancers, however
mechanisms of metastasis control by NME2 are poorly understood. In genomewide location (ChIP-seq)
experiments we found presence of NME2 on telomeres in multiple human cancer cells. This led to the
finding that NME2 associates with telomerase and inhibits telomerase activity in vitro and in cellulo
(Kar et al., Nucleic Acids Research). Recent results, using a loss of function mutant, confirm interaction
of NME2 with hTERT is through direct protein-protein association in a particular domain of telomerase.
Mechanistic evidence supports inhibition of telomerase reverse transcription function in presence of
NME2. Furthermore, we found that telomerase-inhibition is dependent on NME2-telomerase
association. The impact of NME2-mediated inhibition of telomerase activity on metastatic potential of
aggressive cancer cells has also been studied. These results will be presented. Together these novel
findings present an unique opportunity to explore telomerase function in metastasis.
Presented by: Chowdhury, Shantanu
95
Poster 24
Characterization of the binding of the Drosophila telomeric protein Verrocchio to single-stranded
DNA
Alessandro Cicconia,*, Emanuela Michelia, Domenico Raimondob, Giovanni Cencia, Maurizio Gattia,
Stefano Cacchionea, Grazia D. Raffaa
a
b
Dipartimento di Biologia e Biotecnologie Charles Darwin, Sapienza University, Roma, Italy
Dipartimento di Fisica, Sapienza University, Roma, Italy
Drosophila telomeres are composed of three specialized non-LTR retrotransposons. These elements
are recognized by a specific telomeric complex, called terminin, whose subunits are not conserved in
other eukaryotic organisms. The terminin proteins HOAP and HipHop bind double stranded DNA, and
are enriched over a large telomeric domain, while Modigliani (Moi) and Verrocchio (Ver) localize only
at the very end of the chromosome. Ver has an OB-fold domain, a structural motif present also in other
proteins that are capable of binding single-stranded DNA, such as RPA and POT1. Moi is not able to
bind single-stranded DNA on its own, but it’s responsible for Ver recruitment at telomeres, through
interaction with HOAP. Moi and Ver closely interact with each other and are mutually dependent for
their stability, suggesting that their function could be analogous to TPP1-POT1 function. Using EMSA
assay, we showed that Ver binds several single-stranded probes, in a sequence-independent manner
and with single-stranded DNA specificity. We have further analyzed Ver binding to a telomere model
system using AFM imaging. We found that Ver binds DNA in dimeric and tetrameric form, in agreement
with the fact that OB-fold containing proteins bind DNA with two or more OB-folds. Our results
reinforce the model of telomere protection in Drosophila based on the formation of the terminin
complex, that is functionally analogous to the mammalian shelterin complex, but lacks sequence
specific binding activity.
Presented by: Cicconi, Alessandro
96
Poster 25
Separase is required for telomere protection
Francesca Cipressaa,*, Patrizia Morcianoa, Giuseppe Bossoa, Linda Manninib, Grazia Daniela Raffaa,
Antonio Musiob, Giovanni Cencia
a
Department of Biology and Biotechnology "Charles Darwin"
Section of Genetics, SAPIENZA University of Rome
00185 Rome, Italy
b
Istituto di Ricerca Genetica e Biomedica, Consiglio Nazionale delle Ricerche, Pisa
In most organisms, telomeres are extended by telomerase and contain GC-rich repeats. Drosophila
telomeres are elongated by transposition events rather than telomerase activity and are assembled in
a sequence independent fashion. Drosophila telomeres are capped by terminin, a complex that
includes the non-conserved and fast evolving proteins HOAP, HipHop, Moi and Ver, which localize and
function only at telomeres, protecting them from fusion events. The other proteins that protect
Drosophila telomeres from fusion (Eff, HP1, ATM, Rad50, Mre11,Nbs, and Woc) are evolutionarily
conserved, do not localize only at telomeres and play several functions in addition to telomere
protection. Here, we show that mutations in the Drosophila Separase coding gene (Sse) lead to
endoreduplication and telomeric fusion (TFs). We also demonstrate that Sse physically binds both the
terminin proteins and HP1 and that it is enriched at telomeres. While localization of terminin and Sse
are not interdependent, loss of Sse strongly reduces HP1 levels. Overexpression of HP1 in Sse mutants
suppresses telomere fusion but does not rescue the endoreduplication phenotype, suggesting that the
TFs seen in Sse mutants are due to a reduction of the HP1 level. A catalytically inactive SSE failed to
restore the HP1 level and to reduce TF frequency indicating that the SSE endopeptidase activity is
ultimately required for telomere protection. Finally, siRNA-induced depletion of ESLP1, the SSE human
ortholog, caused dysfunctional telomeres and HP1 reduction in human primary fibroblasts, highlighting
and evolutionarily conserved role of separase in telomere protection.
Presented by: Cipressa, Francesca
97
Poster 26
TELOMERE DAMAGE AND CHROMOSOME INSTABILITY INDUCED BY ACUTE OXIDATIVE STRESS
Elisa Coluzzia,*, Monica Colamartinoa, Renata Cozzia, Stefano Leonea, Nathan O’Callaghanb, Antonella
Sguraa
a
b
Dept. of Science, University of “ Roma Tre”, Viale Guglielmo Marconi, 446, 00146 Rome, Italy.
CSIRO Food and nutritional Sciences, Nutritional Genomics Laboratory, Adelaide, Australia.
One of the main role of telomere is to maintain chromosome and genome stability. In this work, our
interest was focused on the role of telomere in chromosome instability induced by oxidative stress.
With the aim to evaluate if the chromosome instability induced by oxidative stress is related
specifically to telomeric damage, we used human primary fibroblasts (MRC-5) treated with two doses
of H2O2 (100 and 200 μM) for 1 hour. The evaluation of the cell cycle by citofluorimetric analysis
showed no cell cycle effects after treatments. The analysis of chromosome instability (CIN)
demonstrated a significant telomere shortening 48 hours after treatment and an increase of CIN
(micronuclei, nuclear buds and nucleoplasmic bridges) at the same time. At the subsequent times (72
and 96 hrs) we can observe a restoring of telomere length and a reduction of CIN, leaving us to
suppose a correlation between telomere shortening/dysfunction and chromosome instability.
To understand the mechanism leading to chromosome instability, we evaluate the persistence of
oxidative stress-induced damage up to 24 hours after treatment analyzing genomic and specifically
telomeric FPG-sensitive sites by modified comet assay and qPCR, respectively. The results obtained
demonstrated that after 24 hrs genomic damage was completely repaired while telomeric oxidative
damage persist up to 24 hrs leading us to hypothesize that telomere dysfunction could be responsible
of chromosome instability observed 48 hrs from treatment. Furthermore, the analysis of nucleoplasmic
bridges hybridized with telomeric probe will give us additional information on the role of telomere in
the observed aberration.
Presented by: Coluzzi, Elisa
98
Poster 27
ALT-specific recruitment of NuRD-ZNF827 to telomeres promotes homologous recombination and
protects against senescence and apoptosis
Dimitri Conomosa,*, Roger Reddelb, Hilda Picketta
a
Telomere Length Regulation Group, Children’s Medical Research Institute, Westmead, NSW,
Australia.
b
Cancer Research Unit, Children’s Medical Research Institute, Westmead, NSW, Australia.
It has recently been shown that nuclear receptors are recruited to the telomeres of cells that utilize
ALT (1,2). We proposed that this contributes to remodeling of the telomeric architecture of ALT cells to
promote ALT activity (3), but the mechanism remained to be determined. Here we demonstrate that
the nucleosome remodeling and histone deacetylation (NuRD) complex is recruited to nuclear
receptors by an N-terminal RRK motif within ZNF827, a zinc finger protein of unknown function. We
show that the NuRD-ZNF827 complex compacts ALT telomeric chromatin and is required for ALT, and
alteration of its expression level affects numerous ALT phenotypic characteristics and recruitment of
homologous recombination (HR) proteins. NuRD-ZNF827 binding to telomeres promotes interactions
of telomeres with telomeric DNA in ALT-associated PML bodies (APBs). ZNF827 depletion increases the
telomeric DNA damage response and results in senescence and/or apoptosis, in an ALT-specific
manner. We propose that NuRD-ZNF827 provides an environment that fosters HR and chromatin
compaction and thereby inhibits recognition of DNA damage at the telomeres of ALT cells.
(1) Dejardin, J. and Kingston, R. E. Purification of proteins associated with specific genomic loci. Cell
136: 175-186, 2009.
(2) Conomos, D., et al. Variant repeats are interspersed throughout the telomeres and recruit nuclear
receptors in ALT cells. J. Cell Biol. 199: 893-906, 2012.
(3) Conomos, D., et al. Alternative lengthening of telomeres: remodeling the telomere architecture.
Front. Oncol. 3: 1-7, 2013.
Presented by: Conomos, Dimitri
99
Poster 28
Shading the shelterin TRFH TRF2 domain through cell-permeable chemical tools: rational design,
synthesis and biological effects at telomeres
Salvatore Di Maroa, Pasquale Zizzab, Erica Salvatib, Bruno Paganoa, Clemente Capassoc, Luciana
Marinellia, Ettore Novellinoa, Annamaria Birocciob, Sandro Cosconatid,*
a
Department of Pharmacy, University of Naples “Federico II”, Naples, Italy
Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy
c
Istituto di Biochimica delle Proteine—CNR, Naples, Italy
d
DiSTABiF, Second University of Naples, Caserta, Italy
b
In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain–containing
telomeric protein TRF2 associates with different downstream factors necessary for telomere
maintenance and cell-cycle progression. While cogent experimental evidences demonstrate that the
TRF2 recruiting activity is a critical arbiter of cell fate decisions, no chemical tools have ever been
described to modulate this function.
Based upon the crystal structure of TRF2 in complex with the Apollo C-terminal peptide, we have
designed and synthesized a series of peptides through mutations and cyclization. Aided by computer
simulations we were able to identify a potent TRF2 TRFH binder with a Kd value of 125 nM and two
times more affine that the wild-type Apollo peptide (250 nM). By using human transformed BJ
fibroblasts (BJ-EHLT), we demonstrate that, differently to the linear Apollo peptide, our chemotype is
able to induce marked DNA damage response and deconvolution microscopy analysis showed that the
damaged foci colocalized with TRF1, a good marker for interphase telomeres forming the so-called TIF.
Future experimental studies will involve the employment of this pharmacological tool to study the role
of TRF2 TRFH protein hub in shaping and safeguarding telomeres.
Presented by: Cosconati, Sandro
100
Poster 29
RPA and Pfh1(Pif1) helicase prevent secondary structures formation during telomere replication
Julien AUDRYa, Pierre LUCIANOa, Toru NAKAMURAb, Vincent GELIa, Stephane COULONa,*
a
b
CRCM-Marseille
University of Illinois at Chicago
In fission yeast, the telomeric complex, also named shelterin complex, ensures the protection of
chromosomes ends and contributes to the recruitment of telomerase. It is established that the nontelomeric factor ATRRad3 kinase phosphorylates Ccq1 to promote recruitment and activation of
telomerase when replication is completed. Lately, we have shown that the Replication Proteine A
(RPA) participates to telomere maintenance in a telomerase-dependent manner but independently of
ATRRad3 pathway. RPA recruitment at telomeres is cell cycle regulated and peaks in late S-phase. A
point mutation (D223Y) provokes telomeres shortening. This mutation does not impede RPA
recruitment at telomeres, but the presence of RPAD223Y at telomeres is prolonged. ChIP of replicative
polymerases have shown that Polε is recruited at telomeres indifferently in wild-type and D223Y
strains while Polα remains longer at telomeres after completion of S-phase in RPA1D223Y mutant.
These observations suggest that D223Y allele causes defects in replication of the G-rich lagging strand
telomere. In agreement, 2D-gel analysis showed that replication of telomeric regions is severely
altered in D223Y strain. Our results suggest that D223Y mutation impedes the proper replication of the
G-rich telomeric DNA and provokes accumulation of secondary DNA structures like G-quadruplex. DNA
helicases are known to unwind these structures. Strikingly, we have shown that over-expression of
either Pfh1(Pif1) or S. cerevisae Pif1 are able to rescue the short telomere phenotype of RPAD223Y. We
propose that RPA and Pfh1Pif1 act at telomeres to prevent accumulation of DNA secondary structures
for efficient telomere elongation.
Presented by: COULON, Stephane
101
Poster 30
Cross talk between EBV and telomerase: the role of TERT in the switch of latent/lytic cycle of the
virus
Silvia Giuncoa, Andrea Celeghinb, Stefano Indraccoloa, Riccardo Dolcettic, Anita De Rossid,*
a
Istituto Oncologico Veneto-IRCCS, Padova, Italy
Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology,
University of Padova; Padova; Italy
c
Cancer Bio-Immunotherapy Unit, CRO-IRCCS, National Cancer Institute, Aviano, Italy
d
Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology,
University of Padova; Padova; Italy;
Istituto Oncologico Veneto-IRCCS, Padova, Italy
b
Epstein-Barr virus (EBV)-associated malignancies in vivo, as well as lymphoblastoid cell lines (LCLs),
obtained in vitro by EBV infection of B cells, selectively express latent viral proteins and maintain ability
to grow indefinitely through inappropriate activation of TERT, the catalytic component of telomerase.
Our studies demonstrated that high level of TERT expression in LCLs prevented the EBV lytic cycle,
which is instead triggered by TERT silencing (Giunco et al, Clin Cancer Res 2013;19:2036). As the lytic
infection promotes the death of EBV-positive tumor cells, understanding the mechanism(s) by which
TERT affects the latent/lytic status of EBV may be important for setting new therapeutic strategies.
BATF, a transcription factor activated by NOTCH2, the major NOTCH family member in B cells,
negatively affects the expression of BZLF1, the master protein of the viral lytic cycle; therefore we
analyzed the interplay between TERT, BATF, and NOTCH2 in LCLs. We found that LCLs with high TERT
levels showed high NOTCH2 and BATF expression, while LCLs with low TERT levels did not. Infection of
TERT-negative LCLs with a retroviral vector expressing TERT(pBABE-hTERT) increased NOTCH2 and
BATF at both mRNA and protein levels. By contrast, infection of TERT-negative LCLs with retroviral
vectors expressing NOTCH2(NOTCH2-IC) did not induce TERT transcription. By using luciferase reporter
assay we demonstrated that ectopic expression of TERT (pEGFP-hTERT) activated NOTCH2 promoter
(pGL3-TATA-N2PR-2327/-99) in a dose-dependent manner; while NOTCH2 did not activate the TERT
promoter.
Taken together, these results suggest that TERT modulates the lytic/latent status of EBV through the
NOTCH2 pathway.
Presented by: De Rossi, Anita
102
Poster 31
Dissecting the link between the long non-coding RNA TERRA and the alternative lengthening of
telomeres mechanism
Katharina Deeg, Karsten Rippe
Research Group Genome Organization and Function, German Cancer Research Center (DKFZ) &
BioQuant, Heidelberg, Germany
To counteract the continuous shortening of the telomeres, cancer cells activate a telomere
maintenance mechanism and thereby acquire unlimited proliferative potential. A fraction of human
cancers utilizes the alternative lengthening of telomeres (ALT) mechanism that involves recombination
and repair processes. Hallmarks of the ALT pathway are colocalizations of telomeres and promyelocytic
leukemia protein (PML), termed ALT-associated PML-bodies (APBs) and the presence of
extrachromosomal telomere repeats. In addition, ALT-positive cells have elevated levels of the
telomeric repeat-containing RNA (TERRA), a long non-coding RNA that is transcribed from the
telomeres. Notably, TERRA has been shown to stimulate telomere recombination in yeast cells lacking
telomerase. However, it remains elusive whether TERRA is essential for the ALT pathway in cancer
cells.
We performed a comprehensive analysis of telomere maintenance features in glioblastoma cell lines
with different genetic background and reveal a strong correlation between TERRA levels and the
presence of ALT in these tumors. Yet, APB depletion by stable knockdown of PML did not alter TERRA
levels. To investigate whether TERRA is a functional component of the ALT pathway, we depleted the
telomeric part of TERRA by antisense oligonucleotide transfection and analyzed for changes in the
number of APBs and C-circle levels. TERRA knockdown in the ALT-positive U2OS cell line did not
significantly affect any of the characteristic ALT features tested. This suggests that TERRA is not needed
for the maintenance of ALT, but may still be essential for ALT initiation.
Presented by: Deeg, Katharina
103
Poster 32
Influence of telomere dynamics on disease progression and response to therapeutics in bone
marrow failure syndromes
Erin Degelman, Nicholas Ting, Tara Beattie
The University of Calgary
Aplastic Anemia (AA) is a bone marrow failure disease where insufficient levels of hematopoietic cells
are produced. In approximately 10-15% of patients, the AA evolves into myelodysplastic syndrome
(MDS) or acute myeloid leukemia (AML). Studies have demonstrated an association between
shortened chromosome ends, advanced AA and increased risk of developing blood cancers.
Heterozygous mutations in the gene encoding the telomerase protein component hTERT, are seen in
approximately 10% of patients, resulting in shortened telomeres. However, how telomerase mutations
and shortened telomeres impact disease progression and response to treatment is not well
understood.
To understand the biochemical properties and cellular consequences of mutant hTERT expression we
have generated expression constructs corresponding to four hTERT mutations. These mutations have
been identified in patients with familial AA and AML and family history of evolution of disease and are
all located in functionally distinct regions of the protein. We have demonstrated that the hTERT
mutants retain telomerase activity in vitro and we are investigating the consequences of their
expression in leukemic cells lines. Preliminary data suggests that expression of certain mutant hTERT
proteins in THP-1 cells, results in a delay of the G1/S transition, which may have profound implications
during hematopoiesis. In addition, THP-1 cells expressing mutant hTERT proteins are more resistant to
chemotherapeutic agents suggesting that treatment protocols might have differential efficacies
depending whether cells express wild-type or mutant hTERT.
By defining the role of telomeres in hematological disorders, it may be possible to alter treatment
strategies based on predicted outcomes from our investigations.
Presented by: Degelman, Erin
104
Poster 33
Pervasive hallmarks of Alternative Lengthening of Telomeres (ALT) in diploid Sorex granarius
(Soricidae, Eulipotyphla) fibroblast cells
Irena Draskovica,*, Natalia Zhdanovab, Julia Mininab, Tatiana Karamyshevab, Clara Novo-Lopesa, Win-Yan
Liua, Maria Zverevac, Andrey Skvortsovc, Nikolay Rubtsovb, Arturo Londo o-Vallejoa
a
Telomeres Cancer Laboratory, “E uipe Labellis e Ligue Contre le Cancer”, Institut Curie, 26 rue
d'Ulm, 75248 Paris, France; UPMC - University Paris 06, 75005 Paris, France.
b
Institute of Cytology and Genetics of Siberian Branch of Russian Academy of Sciences, Novosibirsk,
Russia.
c
Moscow State University, Faculty of Chemistry, Moscow, Russia.
The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking
features. While the closely related S. araneus bears telomeres very similar in structure to those found
in humans, the short arms of S. granarius acrocentric chromosomes carry extremely long telomeres (up
to 300 kb) with interspersed rDNA repeat blocks. In this work, we investigate the telomere physiology
of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and
that there is no telomere-dependent senescence mechanism, with telomere lengths being maintained
in long-term cultures. Although telomerase activity is detectable all along the cell culture, we also
discovered that hallmarks of Alternative Lengthening of Telomeres (ALT) mechanism are omnipresent,
including telomere length heterogeneity, presence of telomere sister chromatid exchanges (T-SCE),
formation of ALT-associated PML bodies (APBs), production of telomere circles (T-circles) and high
frequency of telomeres carrying marks of DNA damage response. Our results indicate that both
telomerase-dependent and ALT-like mechanisms participate in telomere length homeostasis of normal
S. granarius fibroblasts.
Presented by: Draskovic, Irena
105
Poster 34
Telomere Maintenance in Pediatric Medulloblastoma
Matthew Soboa, Kathleen Dorrisa, Ashley Margolb, Charles Stevensona, Shahab Asgharzadehb, Stewart
Goldmanc, Lili Milesa, Arzu Onar-Thomasd, Maryam Fouladia, Rachid Drissia,*
a
Cincinnati Children's Hospital Medical Center
The Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California
c
Ann Robert H. Lurie Children’s Hospital of Chicago
d
St. Jude Children’s Research Hospital
b
The prognostic significance of telomerase and telomere maintenance mechanisms in pediatric
embryonal tumors has not been well defined. We, therefore, conducted a multi-institutional
retrospective study of telomerase expression and telomere maintenance in 79 newly-diagnosed
pediatric medulloblastomas and 15 primitive neuroectodermal tumors (PNET).
Telomerase activity, hTERT promoter methylation, expression levels of hTERT, NPR3, and c-MYC mRNA
and TERC RNA, and presence of ALT were evaluated. Cox proportional hazard regression analyses
evaluated the association of clinical factors, NPR3 and c-MYC expression levels and telomere
maintenance variables with progression-free survival (PFS) or overall survival (OS). Median age at
diagnosis was 6.5 and 6.0 years for medulloblastoma and PNET cohorts, respectively. Seventy-three
percent of medulloblastoma patients and 67% of PNET patients were M0 at diagnosis.
hTERT expression levels were increased compared to non-neoplastic brain controls in 91.0% of
medulloblastoma and 69.2% PNET samples. Ninety-three percent of medulloblastoma and 50% PNET
samples assessed demonstrated telomerase activity. Median telomerase activity was 3-fold higher in
anaplastic versus non-anaplastic medulloblastoma cohorts. All medulloblastoma samples that used ALT
also exhibited evidence of telomerase use. In multivariable analysis of all medulloblastoma subjects,
increased hTERT expression level was associated with worse PFS and OS in models that also included
risk status or age. Similarly, in patients ≥ 36 months at diagnosis, increased hTERT expression level was
associated with worse PFS and OS when simultaneously modeling for risk status.
The relation between hTERT promoter methylation, hTERT expression and outcome will be discussed.
M.S. and K.D. contributed equally
Presented by: Drissi, Rachid
106
Poster 35
Molecular Dynamics of Telomeric Heterochromatin Formation
Yi-Min Duan, Yang Zhang, Ting Gong, Jin-Qiu Zhou
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, 320 Yueyang Road, Shanghai 200031, China
In budding yeast Saccharomyces cerevisiae, heterochromatin structure is found at three well defined
chromosome regions, i.e. HM Loci, rDNA region and telomere region. The former two regions exhibit
relatively stable DNA sequence, which do not shorten or lengthen through cycles of cell division.
Telomeres are rather more dynamic: elongated through telomerase pathway and/or attacked/trimmed
by nucleases. To understand how telomeric heterochromatin is assembled, we adopted a de novo
telomere addition system, and analyzed whether telomere lengthening and heterochromatin
formation correlate with each other. Our results indicated that telomere lengthening takes more cycles
of cell division to reach a stable length, while Sir protein spreading proceeds much faster. We also
monitored the dynamics of histone modification and nucleosome positioning, and found that both
levels of H4K16 acetylation and H3K79 methylation declined, and telomere proximal nucleosomes
become more stable during the prosess of heterochromatin assembling.
Presented by: Duan, Yi-Min
107
Poster 36
Role of DNA end-binding in Ku heterodimer function
Charlene Emersona,*, Christopher Lopezb, Albert Ribes-Zamorab, Alison Bertuchc
a
Molecular and Human Genetics, Baylor College of Medicine
Pediatrics, Baylor College of Medicine
c
Pediatrics, Molecular and Human Genetics, Baylor College of Medicine
b
The evolutionarily conserved Ku heterodimer is a DNA end-binding (DEB) complex involved in both
telomere maintenance and non-homologous end-joining (NHEJ). In Saccharomyces cerevisiae, the
Yku70/Yku80 subunits bind DNA ends via a channel structure. We previously generated DEB defective
Ku mutants by mutating residues in the DNA-binding channel, revealing that DEB is re uired for Ku’s
telomeric functions. Paradoxically, we also found that these mutants demonstrated increased colony
survival in an assay that probes for imprecise NHEJ (Lopez et al. 2011, PLoS Genet). A subsequent study
also generated DEB defective mutants of Ku by deleting residues to constrict the DNA binding channel
(Pfingsten et al. 2013, Cell). These mutants similarly demonstrated a re uirement for Ku’s DEB activity
at telomeres. However, these mutants were defective for imprecise NHEJ. To reconcile the differences
between the two studies, we generated additional DEB defective Ku heterodimers and found that they
too had increased imprecise NHEJ and defective telomere function. We also identified a novel region in
Yku70 that is required solely for NHEJ and is predicted to associate with residues deleted in the DEB
mutants reported by Pfingsten et al. We propose that these results reconcile the imprecise NHEJ
differences observed and support our hypothesis that Ku heterodimers solely defective for DEB can
modulate NHEJ off DNA ends. Currently, we are assaying whether the DEB mutants bind to double
strand breaks (DSBs) in vivo. We expect that DEB mutants will be unable to bind DSBs, suggesting a
previously unknown role for Ku in imprecise NHEJ.
Presented by: Emerson, Charlene
108
Poster 37
Ssu72 phosphatase regulates telomere length in S. pombe
Jose Miguel Escandell, Clara Reis, Maria Gallo, Miguel Godinho-Ferreira
Instituto Gulbenkian de Ciência. Oeiras, Portugal
Telomeres protect chromosome-ends from being recognized as deleterious double strand breaks.
Paradoxically, the DNA damage recognition machinery is, itself, required for normal telomere
homeostasis. In fission yeast, Rad3 and Tel1 are required for telomerase recruitment through the
phosphorylation of Ccq1 on T93. In contrast, shelterin components are negative regulators of telomere
elongation by counteracting Rad3/Tel1 dependent Ccq1 phosphorylation. The precise mechanism
whereby telomere length regulation is achieved is not fully understood.
We have identified a new member of telomere length regulation in S. pombe. We screened the fission
yeast whole genome deletion library looking for new telomere regulators. We found that cells devoid
of Ssu72 exhibit longer telomeres. Ssu72 is a highly conserved phosphatase previously identified in
humans as an RNA polII regulator and a cohesin-binding factor involved in sister-chromatid cohesion.
Telomere elongation in ssu72Δ cells is both trt1+ and rad3+ -dependent, consistent with a role as a
negative regulator of telomerase. Telomere elongation in ssu72Δ is similar to the one observed in rif1Δ
mutants. However, ssu72Δ rif1Δ double mutants have longer telomeres than any of the single mutants,
suggesting that these genes are involved in independent pathways. Surprisingly, deletion of ssu72+ in
taz1Δ, rap1Δ or poz1Δ leads to telomeres shortening upon progressive cell divisions. Thus, in
combination with these shelterin components, Ssu72 appears to work as a positive regulator of
telomerase. To uncover the role of Ssu72 in telomere homeostasis, we are currently investigating
Ssu72 control of G-overhang formation and Ccq1 phosphorylation.
Presented by: Escandell, Jose Miguel
109
Poster 38
Roles of DNA repair factors in plant telomere maintenance
Lucie Najdekrovaa, Miloslava Fojtovaa, Dagmar Zachovaa, Karel J. Angelisb, Jiri Fajkusa,*
a
Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology (CEITEC)
and Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic
b
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, v.v.i., Na Karlovce 1,
160 00 Praha 6, Czech Republic
Plant telomeres have been investigated during past 20 years mainly in the most common model plant,
Arabidopsis thaliana. Although our previous studies focused on evolutionary structural diversity of
telomeres in a broad phylogenetic radiation of monocotyledonous and dicotyledonous plants showed
interesting changes in telomere players, including telomeric DNA sequences and respective
telomerases for their replenishment, functional studies have almost exclusively been limited to
Arabidopsis. Here we set to perform comparative investigation of telomere maintenance in the moss
model, Physcomitrella patens. Unlike its 450 million years younger relatives – land plants,
Physcomitrella is one of a few known multicellular organisms, with highly efficient system of
homologous recombination. Although its basic telomere maintenance machinery is apparently similar
to that of most land plants, the crosstalk between DNA repair factors and telomeres may result in
telomere phenotypes different from those observed in A. thaliana, thus challenging the generalized
views on the role of repair factors on telomere maintenance. We will present our current results on
telomere dynamics obtained in P. patens strains deficient in DNA repair factors.
Funding: Czech Science Foundation (13-06595S), CEITEC (CZ.1.05/1.1.00/02.0068) and the European
Social Fund (CZ.1.07/2.3.00/30.0019).
Presented by: Fajkus, Jiri
110
Poster 39
HSF1 regulates TERRA transcription upon heat shock
Sivan Koskas, Claire Vourc'h, Virginie Faure
Albert Bonniot Institute- University of Grenoble- INSERM U823
38042 Grenoble cedex 9
FRANCE
In response to metabolic or environmental stress, the key transcription factor named Heat Shock
Factor 1 (HSF1) is activated and coordinates a cellular protective response. In heat-shocked human
cells, HSF1 induces the transcription of heterochromatin pericentric regions resulting in the
accumulation of non coding RNA satIII. A global transcriptional analysis of repetitive sequences
revealed that telomeric UUAGGG repeat-containing RNA (TERRA) expression is up-regulated upon heat
shock. Recently, it has been reported that heat shock induces also the dissociation of TRF2 from
telomeres. TRF2 is a well-known component of the shelterin complex protecting telomeres. These data
prompted us to investigate the role of HSF1 on TERRA regulation and on telomere stability upon heat
shock.
Here, we clearly demonstrate for the first time that HSF1 upregulates TERRA expression upon heat
shock. We show that HSF1 is recruited on subtelomeric regions under stress. Loss of HSF1 leads to a
decrease in RNA polymerase II binding at subtelomeric regions and reduces the level of TERRA without
impacting TERRA stability.
We also bring evidence that TERRA upregulation is not accompanied by major changes in the
epigenetic status of subtelomeric regions. HSF1 also has not impact on telomerase activity. In contrast,
we show that HSF1 is involved in the telomeric localization of telomeric repeat factor 2 (TRF2) upon
heat shock. We are currently examining the impact of HSF1 on telomere stability.
Our work shades light on the role of HSF1 in the upregulation of TERRA transcription, which may
participate in telomere stability in stressed cells.
Presented by: Faure, Virginie
111
Poster 40
Telomerase is essential for zebrafish heart regeneration
Dorota Bednareka, Juan Manuel González-Rosaa, Oscar Gutiérrez-Gutiérreza, Tania Aguadoa, Gabriela
Guzmána, Alfonso Cortésb, Agustín Zapatab, Jesús Jiménez-Borregueroa, Nadia Mercadera, Ignacio
Floresa
a
Spanish National Centre for Cardiovascular Research (CNIC-ISCIII), Melchor Fernández Almagro 3,
28029 Madrid, Spain
b
Electron Microscopy Center, Complutense University, 28040 Madrid, Spain
Adult mammalian hearts regenerate very poorly after infarction. By contrast, zebrafish hearts
efficiently regenerate after an insult. An essential step in the regeneration process that distincts
zebrafish from adult mammalian hearts is the strong proliferation response of zebrafish
cardiomyocytes to injury. One route to proliferation is thought to be rejuvenation, whereby aged cells
regain characteristics of younger cells. Telomerase can be considered a rejuvenation enzyme since its
reactivation in artificially aged tissues restores youthful characteristics and delays the aging process.
Using a model of heart regeneration in zebrafish, we show that, after injury, telomerase is
hyperactivated and telomeres transiently rejuvenate. Subsequently a subset of cardiomyocytes marked
with long telomeres strongly proliferates. In the absence of telomerase activity, cardiomyocytes
shorten their telomeres, accumulate DNA damage and are unable to efficiently expand, which
eventually leads to a blockade of the regeneration process. These findings highlight the importance of
rejuvenation in organ regeneration, establish long telomeres as a proliferation-competent marker for
zebrafish cardiomyocytes and points to telomerase therapy as a potential approach to awaken
hibernating cardiomyocytes found after myocardial infarction.
Presented by: Flores, Ignacio
112
Poster 41
CBX1 buffers DNA damage responses and senescence induction in response to oncogene activation,
γ-irradiation and telomere dysfunction
T. K. Leuchta, S. Foerschb,*, S. Taoc, A. Lechela, K. L. Rudolphc
a
Cooperation group of the Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena and the
University of Ulm, Ulm, Germany
b
1. Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany
2. Department of Medicine 1, University of Erlangen-Nuremberg, Erlangen, Germany
c
Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany
DNA damage responses (DDRs) impact on aging. CBX1 is a member of the heterochromatin-protein-1family, which binds to H3K9me. Upon DNA damage, CBX1 is phosphorylated leading to the mobilization
of CBX1 and the activation of DDRs. It is unknown whether deletion of CBX1 would alter DDRs and how
this would affect cellular aging and transformation. Here, we studied consequences of CBX1 gene
deletion on DDRs, cellular proliferation and senescence in human fibroblasts. The study shows that
CBX1 buffers the induction of DDRs in cells with shortened telomeres and in response to oncogene
activation. CBX1 deletion limits the proliferation of damaged cells by induction of cellular senescence.
Importantly, CBX1 deletion has no effects on the proliferation of undamaged cells with long telomeres.
Foci of p-CBX1 and decreases in CBX1 levels appear in senescent cells and in aging tissues with short
telomeres indicating that CBX1 degradation represents an integral process of telomere dysfunction
induced aging. Additionally, the expression of endogenous CBX1 is essential to prevent premature
formation of γH2AX foci at critically short telomeres. Accordingly, the knockdown of CBX1 significantly
shortens the proliferative lifespan of human fibroblasts. The negative effects of CBX1-deletion on
proliferative lifespan of non-transformed cells, are contrasted by improved protection of human cells
from oncogene-induced transformation. First data on conditional KO-mice confirm that CBX1 is
essential to impair the activation of DDRs in vivo. Together, we provide first evidence for a CBX1dependent molecular buffer alleviating the activation of DDRs in the context of telomere shortening,
irradiation and oncogenic stress.
Presented by: Foersch, S.
113
Poster 42
Epigenetic regulation of telomeres and telomerases in different model plants
Miloslava Fojtovaa,*, Pavla Polanskab, Eva Majerovab, Jana Jureckovab, Anna Ogrockab, Jiri Fajkusc
a
Central European Institute of Technology (CEITEC), Masaryk University Brno, Czech Republic
Faculty of Science and CEITEC, Masaryk University Brno, Czech Republic
c
CEITEC, Masaryk University Brno, Czech Republic and Institute of Biophysics, ASCR, Brno, Czech
Republic
b
Telomeres, as chromatin structures, are targets for epigenetic molecular mechanisms. In this case, the
result of regulation processes in not a change of gene expression but rather a change of telomere
length or structure potentially modulating telomere protective function. We analyzed maintenance of
telomeres in hypomethylated plant models; Nicotiana tabacum (tobacco) culture cells cultivated in the
presence of hypomethylating drugs, and Arabidopsis thaliana with loss of function of enzymes crucial
for the maintenance of stable methylation pattern or wild-type plants germinated in the presence of
hypomethylation drugs. Surprisingly, opposed results were obtained. While in tobacco telomere length
in hypomethylated cells was mainteined despite significantly increased telomerase activity,
Arabidopsis telomeres were markedly shortened while telomerase activity and transcription were
unchanged in hypomethylated individuals. Correspondingly, AtTERT (telomerase reverse transcriptase)
chromatin was of euchromatic nature in both telomerase-positive and telomerase-negative tissues
except of H3K27me3 loading, which was higher in telomerase-negative mature leaves. Analysis of
epigenetic state of tobacco TERT chromatin (DNA methylation, histone modifications) is in progress.
Majerova E, Fojtova M, Mozgova I, Bittova M, Fajkus J: Hypomethylating drugs efficiently decrease
cytosine methylation in telomeric DNA and activate telomerase without affecting telomere lengths in
tobacco cells. Plant Mol Biol 77:371–380 (2011).
Ogrocka A, Polanska P, Majerova E, Janeba Z, Fajkus J, Fojtova M: Compromised telomere maintenance
in hypomethylated Arabidopsis thaliana plants. Nucleic Acids Res, in press (2014).
Funded by Czech Grant Agency (P501/11/0596), CEITEC (CZ.1.05/1.1.00/02.0068) and by the European
Social Fund (CZ.1.07/2.3.00/20.0189).
Presented by: Fojtova, Miloslava
114
Poster 43
A high content screen for telomerase trafficking regulators uncovers the cytosolic chaperonin TRiC as
essential for TCAB1 folding and telomerase function
Adam Freund, Steven Artandi
Stanford University School of Medicine
The identification of telomerase regulators is difficult due to the low levels of telomerase in most cells
and subsequent challenges developing high-throughput screening assays for telomerase activity. The
identification of TCAB1, a telomerase component required for the trafficking of telomerase to
telomeres, offers a unique opportunity to develop a cell-based readout for telomerase activity, due to
the easily detectable localization of TCAB1 to Cajal bodies. In a whole genome, high-content siRNA
screen for inhibitors of TCAB1 localization, we identified the cytosolic chaperonin CCT/TRiC as required
for TCAB1 protein homeostasis. We determined that TCAB1 is an obligate TRiC substrate: without TRiC,
TCAB1 cannot properly fold, cannot bind hTERC, and telomerase localization is perturbed. This
TRiC:TCAB1 interaction is mediated by the WD40 repeat domain of TCAB1. Furthermore, we found that
all known disease-causing TCAB1 mutants are defective in precisely this folding step, providing a
molecular explanation for these cases of human telomere dysfunction.
Presented by: Freund, Adam
115
Poster 44
The Diversity and Evolution of Telomeres in Algae
Jana Fulneckovaa,*, Tereza Sevcikovab, Jiri Fajkusa, Alena Lukesovac, Marek Eliasb, Eva Sykorovaa
a
CEITEC MU Kamenice 5/A2 CZ-62500 Brno
& Inst. of Biophysics, ASCR,v.v.i., Královopolská 135, CZ-61265 Brno, Czech Republic
b
Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho10, CZ-71000
Ostrava, Czech Republic
c
Institute of Soil Biology, Biology Centre ASCR,v.v.i.,Na Sadkach 7, CZ-37005 Ceske Budejovice, Czech
Republic
Algae are of interest for telomere biology, because they represent a substantial portion of the
eukaryotic phylogenetic diversity. We employed an experimental approach to probe telomeric
minisatellites. Our results support the view that there are few variants of the telomeric motifs that are
conserved in particular groups of algae. Telomeric motif TTTAGGG, conserved in most green algal
lineages, is ancestral for Chloroplastida. However, an interesting diversity of telomeres was described
in the order Chlamydomonadales, where at least two independent evolutionary transitions from the
ancestral type to TTTTAGGG occurred; moreover a change to TTAGGG was found in a subclade of
Caudivolvoxa. Algae of the streptophyte class Klebsormidiophyceae possess TTTTAGGG telomeric
repeat or a novel TTTTAGG motif. The TTTAGGG telomeric sequence is also present in telomeres of the
photosynthetic alveolate Chromera velia and of Xanthophyceae, in contrast to the presence of the
TTAGGG motif in other ochrophytes studied. Glaucophytes and haptophytes exhibit TTAGGG telomeric
type. To expand knowledge about the distribution of different telomere types in framework of the
eukaryotic phylogeny, we performed in silico analyses of genomic data from major eukaryotic lineages.
These analyses confirm the TTAGGG telomeric repeat as the most common and possibly ancestral in
eukaryotes, but alternative motifs replaced it along the phylogeny of diverse eukaryotic lineages.
This work was supported by research grant from Czech Science Foundation 13-06595S, and by the
European Social Fund (CZ.1.07/2.3.00/20.0189).
Presented by: Fulneckova, Jana
116
Poster 45
Chromosomal instability and cancer cell stemness in the alternative lengthening of telomeres
Fani-Marlen Roumelioti, Maria Chiourea, Christina Raftopoulou, Sarantis Gagos
Laboratory of Genetics, Biomedical Research Foundation of the Academy of Athens Greece
Extensive chromosomal instability in neoplasia (CIN) leads towards the selection of highly imbalanced
cancer genomes. Many cancers are driven by cancer stem cells (CSCs) that may differentiate into a
variety of cell types while maintaining the ability to self-renew. Human immortalized cell lines utilizing
the Alternative Lengthening of Telomeres (ALT), lack telomerase activity and exert high rates of
ongoing telomere dysfunction and CIN. To identify putative CSCs in the ALT-pathway, we combined
single cell analysis by M-FISH/SKY, with a-CGH and Immunocytochemistry, in two human ALT cell lines,
before and after exposure to ionizing radiation, topoisomerase inhibition, or DNA hyper-replication.
Inducible genotoxic stress was convoyed by increased rates of random structural chromosome
aberrations, polyploidization, and elevated frequencies of cells expressing the mesenchymal CSC
marker CD133. Molecular karyotyping revealed several DNA damage-induced novel clonal structural
chromosomal rearrangements. However by aCGH, the insulted ALT genome displayed remarkable
propensity to maintain the major genomic imbalances of the control cells. Structural CIN was unequally
dispersed between co-dividing cells both in control and challenged cell cultures. Mitotic survivors of
structural CIN were products of polyploidization or polyploidy reduction and their percentages
corresponded to the fraction of CD133+ cells/population. Furthermore, enrichment of CD133+ cells in
culture, showed significant decrease at the frequencies of telomere dysfunction foci (TIF) and in
random structural CIN, whereas the rates of whole genome endoreduplication or polyploidy reduction
were highly elevated. Our results demonstrate a continuous process of ALT cancer genome
homeostasis that is related to genotoxic therapy resistance and cancer cell stemness.
Presented by: Gagos, Sarantis
117
Poster 46
Yeast Rap1 binds dsDNA in multiple binding modes
Erik Feldmann, Roberto Galletto
Department of Biochemistry and Molecular Biophysics
Washington University School of Medicine, Saint Louis, MO 63110
Yeast Repressor activator protein 1 (Rap1) is a central player in establishing the shelterin-like complex
at telomeres. In addition, Rap1 plays fundamental roles both as a transcriptional activator at ribosomal
protein genes and glycolytic genes and as a repressor at the silencing mating-type loci. The data
currently available provide a relatively simple picture where Rap1 binds with high affinity as a one-toone complex to its recognition sequences. In this work we show that on model DNA substrates
containing a Rap1 recognition sequence, the DNA-binding domain of Rap1 (DBD) is able to access
higher stoichiometries than expected. The data strongly suggest that the DBD is able to bind in multiple
DNA-binding modes. We further show that the ability of the DBD to bind with higher stoichiometry
than a 1:1 is modulated by the spacing between the two half-sites of the recognition sequence.
Moreover, the data show that the ability to access different binding modes on DNA is strongly
regulated by the C-terminal wrapping loop, observed in the crystal structure of the DBD. Also, access to
different binding modes is not specific to the DBD but is conserved in larger Rap1 constructs, albeit the
presence of additional domains appears to provide regulative regulatory roles. These novel findings
suggest that the DNA-binding mode of Rap1 is dynamic and this may offer an additional mechanism of
regulation for Rap1 that was previously unanticipated. Possible functional implications of our
observations are discussed.
Presented by: Galletto, Roberto
118
Poster 47
Telomere uncapping impairs K-RasG12V-induced lung carcinogenesis
Maria Garcia-Beccariaa,*, Paula Martineza, Marta Cañamerob, Francisca Muleroc, Maria Blascoa
a
Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Research
Centre (CNIO),
b
Histopathology Unit, Biotechnology Program, Spanish National Cancer Research Centre (CNIO)
c
Molecular Imaging Unit, Biotechnology Program, Spanish National Cancer Research Centre (CNIO)
Lung cancer is the leading cause of cancer related deaths worldwide. In spite of some success of
targeted therapies, new therapeutic strategies are needed. Telomeres are considered potential anticancer targets, as telomere maintenance above a minimum length is necessary for cancer cell growth.
Indeed, inhibition of telomerase is being currently tested in various human cancer clinical trials.
Telomerase inhibition, however, has the potential shortcoming of telomere length heterogeneity
within tumors, which may result in selection of cancer cells with long telomeres. In addition,
telomerase abrogation in mouse cancer models decreases tumor growth only after several mouse
generations when telomeres reach a critically short length. Here, we address the induction of acute
telomere uncapping by abrogation of the shelterin component TRF1 as an alternative strategy to target
telomeres in cancer, independently of telomere length. To this end, we used the well-established KRasG12V lung cancer mouse model, shown to recapitulate the initiation and progression steps of
human lung carcinogenesis. TRF1 abrogation reduced the size and malignancy of K-RasG12V-induced
lung tumors, as well as increased mouse survival, compared to wild-type controls. The anti-tumorigenic
effect of Trf1 deletion was concomitant with induction of telomeric DNA damage and apoptosis in the
tumors. TRF1-abrogated tumors also showed less proliferation, G2-arrest, and signs of
endoreduplication. These results demonstrate that Trf1 deletion impairs the growth and progression of
tumors associated to increased oncogenic stress by induction of DNA damage and severe mitotic
defects. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target
for lung cancer.
Presented by: Garcia-Beccaria, Maria
119
Poster 48
Interactions between the Elg1 clamp-unloader and the DNA damage checkpoint kinase Dun1
Inbal Gazy, Martin Kupiec
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University Ramat Aviv, 69978,
Israel
Elg1 is a protein with homology to Rfc1 that forms an RFC-like complex whose precise molecular
function is still unkonwn. The Elg1 RFC-like complex interacts with the replication clamp PCNA,
particularly when it becomes SUMOylated. It has been proposed that the role of Elg1 is to unload PCNA
from stalled replication forks and from Okazaki fragments to allow their maturation. Deletion of ELG1
leads to a variety of genomic instability phenotypes, including, among others, hyper-recombination,
chromosome loss and increased telomeric silencing. In addition, Elg1 plays a role in telomere
regulation, and its deletion leads to elongated telomeres.
In order to better understand Elg1’s role we investigated the synthetic interaction of ELG1 and several
other genes known to play a role in telomere length regulation.
We found that deletion of the gene encoding the protein kinase Dun1 supresses Elg1’s telomeric length
phenotype. This kinase is under regulation of the DNA damage response and controls the level of
dNTPs in the cells and deletion of DUN1 results in a reduction of the dNTP cellular pool. Accordingly,
we found that elevation of the dNTP pools abolishes dun1 supression of elg1 elongated telomeres.
We are currently investigating the mechanisms by which Dun1 and dNTPs affect Elg1's activity.
Presented by: Gazy, Inbal
120
Poster 49
Telomeric Protein TRF2 Is an Angiogenic Target of WT1 That Regulates the Expression of PDFGRβ
Mounir El Mai, Kay Wagner, Jean-François Michiel, Valerie Renault, Nicole Wagner, Eric Gilson
Institut for Research on Cancer and Aging, Nice (IRCAN), University of Nice Sophia-Antipolis, CNRS
UMR7284/INSERM U1081, Faculty of Medicine, Nice, France
We provide evidence that TRF2 is expressed in the vasculature of most human cancer types, where it
co-localizes with the Wilms’ tumor suppressor WT1. We further show that TRF2 is a direct target of
WT1 and is required for proliferation, migration, and tube formation of endothelial cells. These
angiogenic effects of TRF2 are uncoupled from its function in telomere capping. In fact, TRF2 regulates
the expression of the angiogenic tyrosine kinase PDGFRβ. These findings demonstrate a role of TRF2 in
neoangiogenesis and delineate a distinct function of TRF2 as a regulator of angiogenic factor
expression.
Presented by: Gilson, Eric
121
Poster 50
Keeping telomere under wraps to avoid DNA damage: a job for TRF2
Delphine Bennaroch-Popivkera, Sabrina Pisanoa, Alexandre Gaya, Aaron Mendez-Bermudeza, Chrysa
Latricka, Bei Peia, Simona Mironb, Marie-Hélène Le Dub, Eric Gilsona, Marie-Josèphe Giraud-Panisa,*
a
Institute for the Research on Cancer and Aging, Nice, UMR 7284 CNRS, U1081 INSERM, Faculté de
Médecine, Université de Nice-Sophia Antipolis, Nice, France
b
DSV/IBi Tec-S/SB2SM/LBSR, Commissariat à l'Energie Atomique Saclay, Gif sur Yvette, France
The human shelterin protein TRF2 is capable of condensing DNA in vitro, a property that is thought to
be important for telomeres folding and during telomeres replication (Amiard et al, NSMB, 2007, Ye et
al, Cell, 2010, Poulet et al, NAR, 2012). Using Atomic Force Microscopy, we have discovered that this
topological activity of TRF2 is based on the wrapping of 100bp of DNA around the protein. Protein
footprinting performed on the TRF2-DNA complex show that this wrapping involves residues in the
homodimerization (TRFH) domain organized as a “DNA path” around this domain. Mutations of several
of these residues gave rise to a mutant that we dubbed Top-less since it exhibits a topology activity
largely reduced as seen by AFM, topology assays and single strand invasion assays, while it is still
capable of binding telomeric DNA. Amazingly, expression of this mutant does not rescue the activation
of the DNA damage response triggered at telomeres by TRF2 downregulation. We propose that TRF2dependent DNA wrapping is necessary for telomere protection against unsuited activation of the DNA
damage response.
Presented by: Giraud-Panis, Marie-Josèphe
122
Poster 51
RTEL1 mutations cause Hoyeraal-Hreidarsson syndrome by disrupting an essential function of RTEL1
in telomerase-dependent telomere maintenance
Galina Glouskera,*, Zhong Dengb, Arturo Londoño-Vallejoc, Paul M. Liebermanb, Yehuda Tzfatid
a
Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of
Jerusalem, Givat Ram, Jerusalem, 91904, Israel
b
Gene Expression and Regulation Group, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA
19104, USA.
c
Telomeres & Cancer Laboratory, Institut Curie, 26 rue d'Ulm, 75248 Paris, France; UPMC Univ. Paris
06, F-75005 Paris, France. “E uipe Labellis e Ligue Contre le Cancer”
d
Department of Genetics, The Silberman Institute of Life Sciences, The Hebrew University of
Jerusalem, Givat Ram, Jerusalem, 91904, Israel.
Hoyeraal-Hreidarsson Syndrome (HHS), a severe form of the inherited telomeropathy dyskeratosis
congenita (DC), is characterized by bone-marrow failure, immunodeficiency and developmental
defects. HHS-causing mutations were found in DKC, hTERT, TINF2, and recently also in RTEL1, encoding
a DNA helicase. Rtel1 was identified in mouse to be genetically associated with telomere length and
RTEL1 deficiency in HHS patients was associated with severely short telomeres in blood leukocytes,
implicating RTEL1 in telomere length maintenance. How RTEL1 regulates telomere length remains
unclear. RTEL1-deficient cells display shortening of the telomeric 3’ overhang and telomere fragility1.
An increase in telomere (T)- circles was detected by a rolling-circle DNA polymerization assay2 but not
by two-dimensional gel electrophoresis1. However, telomere deletions caused by breaks or excision of
T-circles do not readily explain the shortening of telomeres in telomerase-positive RTEL1-deficient cells
and the normal telomere length in telomerase negative primary fibroblasts, while both telomerase
positive and negative cells display telomere dysfunction-induced foci and telomere fragility. We are
using lymphoblastoid cells and fibroblasts carrying single and compound heterozygous RTEL1
mutations, and a tetracycline-inducible system for the regulated expression of each of the RTEL1 splice
variants. This system enables studying the different phenotypes associated with each variant, and with
the disease-causing mutations. Our results suggest that RTEL1 is essential for telomerase action at
telomeres; mutations in RTEL1 compromise the ability of telomerase to elongate telomeres, resulting
in severe telomere shortening, which is the primary cause for HHS.
1Deng et al. (2013) PNAS 110:e3408-16
2Ballew et al. (2013) PLoS Genetics 9:e1003695
Presented by: Glousker, Galina
123
Poster 52
Development of a high-content, automated platform for rapid analysis of alternative lengthening of
telomeres (ALT)-associated promyelocytic leukemia nuclear bodies (APBs) in human cancer cells
David Halvorsen, Thomas Hunt, Manali Aggrawal, Haroldo Silva
SENS Research Foundation
Most cancers prevent telomere loss, senescence and apoptosis by expressing telomerase. Yet, about
10-15% of cancers employ a telomerase-free, homologous recombination-based mechanism known as
alternative lengthening of telomeres (ALT). Unlike the telomerase-based pathway, no ALT-specific
targets or therapeutics are yet available due in part to a lack of robust ALT assays. Here we develop
and validate a high-speed, high-content version of the established ALT-associated promyelocytic
leukemia (PML) nuclear body (APB) assay, wherein co-localization of PML with telomeric chromatin
(e.g., TRF2) occurs prominently in ALT cells. Currently, the APB assay is impractical for high-throughput
applications as it requires complex three-dimensional (3-D) confocal image acquisition, reconstruction
and analysis using a supercomputer for robust statistical results. Here we use the Molecular Devices
ImageXpress Micro Widefield High Content Screening System and MetaXpress imaging and analysis
software running on a regular desktop computer for automated imaging and analysis of APBs in human
ALT cells in multi-well plates. We validated our platform by treating cells with agents previously
reported to modulate APB levels. During image acquisition, a maximum pixel intensity projection
algorithm collapses Z-stacks into a two-dimensional (2-D) image. This platform reproduces key
published data (e.g., average number of APBs per nuclei) in a less data-intensive format (2-D vs. 3-D)
and therefore it is about 5-6 times faster than previous methods while keeping similar statistical
power. Our platform provides useful parameters for the standardization of the APB assay and a
powerful tool to screen drugs and genetic targets for functional impact on ALT activity.
Presented by: Halvorsen, David
124
Poster 53
PARP1 and ATRX are Required for Proper Telomere Length Maintenance
Adam Harveya,*, Duncan Bairdb, Eric Hendricksona
a
b
University of Minnesota
Cardif University
Telomeres are the repetitive DNA:protein elements that protect the ends of linear chromosomes.
Here, I describe the distinct roles of two genes not attributed to canonical telomere maintenance per
se: PARP1 and ATRX, using genetic knock-out approaches in human somatic cells. PARP1 is known for
its role in DNA break sensing and for its synthetic lethality with BRCA1/2 gene mutations in inherited
breast and ovarian cancers. Unexpectedly, however, I have uncovered an underappreciated role for
PARP1 in maintaining telomere length and integrity in human cells. Additionally, in collaboration with
the laboratory of Duncan Baird, we have preliminary data suggesting that cells lacking PARP1 rarely
escape telomere crisis induced by gradual telomere loss.
The second accessory factor in my studies, ATRX, is a histone chaperone for the histone H3 variant
H3.3. Mutations in ATRX have been strongly implicated in the genesis of alternative lengthening of
telomeres-positive (ALT+) tumors. To experimentally test whether the absence of ATRX is necessary
and/or sufficient for the genesis of ALT in the context of different cellular settings, I have functionally
inactivated ATRX in 2 different cell types: i) a telomerase-positive, transformed and immortalized
cancer cell line {HCT116} and ii) a telomerase-negative, transformed, but not immortalized cell line
(JFCF). The inactivation of ATRX in HCT116 cell line did not lead to the formation of ALT. In contrast,
when JFCF cells were subjected to gene targeting, 11 independent clones immortalized, were correctly
gene targeted, lacked ATRX expression, and all have an ALT-phenotype.
Presented by: Harvey, Adam
125
Poster 54
Local telomerase over-expression promotes cancer in a tissue-dependent manner
Catarina M. Henriques, Miguel Godinho Ferreira
Instituto Gulbenkian de Ciência, Portugal
Ageing is characterized by an overall decline in tissue homeostasis, which sets a fertile ground for
disease. In humans, progressive telomere dysfunction is a good candidate to explain tissue
degeneration with age. Telomeres shorten during our lifespan. Critically short telomeres can impact
tissue homeostasis both by cell autonomous (as a barrier to cell proliferation) and non-cell
autonomous (by accumulation of senescent cells) mechanisms. Consistently, telomere dysfunction
leads to premature ageing and disease in humans. However, it is currently not known whether
functional telomeres are the limiting factor safeguarding from ageing.
We have established zebrafish as a key model to address this question. We have shown that zebrafish,
like humans; require telomerase for health and lifespan. In this model, telomere dysfunction leads to
premature ageing, triggering systemic tissue degeneration in a time and tissue-dependent manner. We
hypothesize that rescuing telomere dysfunction in key tissues may prevent age-associated
degeneration.
Our data suggest that local telomerase expression is sufficient to partially rescue telomerase mutant
phenotypes, including both gut and muscle degeneration. This effect is likely to be dose and/or tissue
dependent. Remarkably, telomerase over-expressing cells do not directly outcompete tert-/- cells in
tissue-regeneration assays, suggesting they are not transformed/malignant. However, localized
telomerase over-expression in otherwise tert-/- mutants (displaying old age phenotypes) appears to
lead to hyperplasia and cell transformation. Thus, the “aged” microenvironment may itself promote
the transformation of cells with un-restricted telomerase and, consequently, lead to cancer. These
results impart caution for telomerase-activating therapies for tissue regeneration in old individuals.
Presented by: Henriques, Catarina M.
126
Poster 55
Which domains are critical for TRF2-REST interaction?
Milan Hluchý, Jan Paleček, Ctirad Hofr
Chromatin Molecular Complexes, CEITEC and Faculty of Science, Masaryk University, Brno, CZ-62500,
Czech Republic
TRF2, one of the six shelterin subunits, is a key player in protection of telomeres from DNA-damage
response pathways. Recently discovered interaction between TRF2 and protein REST showed new nontelomeric function of TRF2. REST is a master repressor in neuronal progenitor cells. TRF2 protects REST
from proteasome degradation and helps to stall the neuronal progenitors in undifferentiated phase.
This work aims to characterise the interacting domains on both proteins and determine the specificity
of TRF2-REST interaction. Both TRF2 and REST were divided to three truncated protein variants
according to their functional domains. REST proteins expressed in E. coli cells were used as bait in pulldown assay together with radioactively labelled TRF2 prey. Fragments with candidate interacting
domains were used for further in vitro studies with surface plasmon and fluorescence spectroscopy.
Both proteins interact physically with each other in vitro. The interacting domain in REST protein was
found in the linker region covering amino acid residues 438 to 859. A candidate REST-interacting
domain on TRF2 is in the TRFH dimerisation domain. Further investigation of these unexpected
interacting partners can help to understand the dual role of REST in different cancer types where it can
act both as a tumour suppressor or oncogene. This work was supported by CEITEC
(CZ.1.05/1.1.00/02.0068), the European Social Fund (CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550),
and KONTAKT II (LH13054).
Presented by: Hluchý, Milan
127
Poster 56
A rabbit ear mechanism by which human Rap1 modulates TRF2 attraction to telomeric DNA
Eliška Janoušková, Ivona Nečasová, Michal Zimmermann, Milan Hluchý, Ctirad Hofr
Chromatin Molecular Complexes, CEITEC and Faculty of Science, Masaryk University, Brno, CZ-62500,
Czech Republic
Regardless of a successful genetic research that has identified and assign main biological functions of
shelterin proteins that safeguard telomeres, a molecular mechanism of how each protein subunit
contributes to the protecting function of the whole shelterin complex remains elusive.
Effects of full-length Rap1 protein on TRF2 binding to double-stranded DNA were investigated by
quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA binding affinity
of TRF2. Despite the reduced DNA affinity in Rap1 presence, the binding selectivity of TRF2 to telomeric
DNA is increased significantly. The lower salt dependence of the dissociation constant for TRF2 binding
to DNA in presence of Rap1 indicates the neutralization of the total net charge of Rap1-TRF2 complex.
The less pronounced electrostatic attraction between TRF2 and DNA in Rap1 presence explained the
improved selectivity of TRF2 to telomeric DNA. Thus, Rap1 prompts more accurate and selective
recognition of telomeric DNA by TRF2.
Based on our quantitative measurements and known structural data, we speculate that Rap1, after
binding to TRF2, might shield the positively charged basic domain on the N-terminus of TRF2 from
mainly non-specific electrostatic interaction with DNA. An interaction model which resembles a happy
and sad rabbit ear will be presented. The results of these quantitative studies contribute to the
understanding of the selective recognition of telomeric DNA by the whole shelterin.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), the European Social Fund
(CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550), and KONTAKT II (LH13054).
Presented by: Hofr, Ctirad
128
Poster 57
Telomere transcripts without subtelomere sequences improve targeting of telomerase in cancer cells
Sandra Sampl, Christian Stern, Doris Mejri, Klaus Holzmann
Division of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical
University Vienna, Austria
Transcripts from telomeres called TERRA were identified to bind and block telomerase activity (TA).
TERRA can further function to guide the telomerase complex to specific chromosome ends. Therefore,
TERRA without subtelomere sequences may act as decoy molecule independent of direct TA inhibition.
We expressed decoy TERRA transcripts in cell culture cells to study in vitro cell growth and viability
directly and in combination with telomerase inhibitors.
Adeno- and lentivirus constructs (AV and LV) were established for transient and stable recombinant
expression of approximately 120 units of UUAGGG repeats in sense (S) or anti-sense (AS) orientation
under control of hH1 or CMV promoter. Telomere length (TL) and expression was analyzed by qPCR, TA
by TRAP assay. Telomerase inhibitors and AVs expressing dominant-negative telomerase and shRNA
against hTERT were used in MTT cell viability and clonogenicity assays.
Moderate 1.5-1.7 fold elevation of TERRA-S compared to endogenous transcripts caused reduction of
TA to 23-38% in tumor cell lines. In contrast, TERRA-AS expression reduced 3-8 fold endogenous TERRA
without reduction of TA. TL, Population doubling time, cell viability and clonogenicity were not affected
by TERRA-S/AS expression. Significant improvement of telomerase inhibition was identified as IC-50
values and clone formation capacities decreased 2.0-2.6 and 1.3–1.7 fold, respectively, in LV cell clones
expressing moderate TERRA-S compared to TERRA-AS and controls. Fibroblasts showed 15-fold
increased TERRA expression compared to tumor cells and were not affected by treatments applied.
Our results suggest TERRA oligonucleotides as candidates for in vivo application in combination with
telomerase targeting approaches.
Presented by: Holzmann, Klaus
129
Poster 58
Telomerase modulates cyclinD1 gene in concert with NOL1
Juyeong Honga,*, Jihoon Leeb, In Kwon Chungc
a
Department of Integrated Omics for Biomedical Science, Yonsei University, Seoul, 120-749, Korea.
Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University,
Seoul 120-749, Korea.
c
Department of Integrated Omics for Biomedical Science and Department of Systems Biology, Yonsei
University, Seoul, 120-749, Korea.
b
Telomerase is upregulated in many human cancers. Telomerase, which is composed of telomerase
reverse transcriptase (TERT) and the associated RNA component (TERC), adds telomeric repeats, thus
allowing cells to keep dividing. Although recent studies have uncovered that extra-telomeric TERT
functions as a transcription factor in oncogenic signaling pathway beyond de novo synthesis of
telomere, regulatory mechanisms are still less understood. Here, we identify NOL1 as a transcription
factor that regulates cyclinD1 gene expression by association with TERT, independently of telomerase
activity. We find that NOL1 interacts with TERT using TERC and directs to TCF binding element (TBE)
site of the cyclin D1 gene promoter. NOL1 does not affect the subcellular localization of TERT,
telomerase activity, and telomere dysfunction induced foci (TIF) formation. Furthermore, Chromatin
immunoprecipitation and luciferase analyses show that depletion of NOL1 prevents TERT from binding
and activating CyclinD1 genes, which delays G1/S transition, subsequently leading to cell-cycle arrest.
Together, these findings suggest a novel mechanism that contributes to understanding how TERT plays
a role as a transcription factor in cellular proliferation.
Presented by: Hong, Juyeong
130
Poster 59
Human Rap1 Induces TRF2 Release from Telomeric DNA
Eliška Janoušková, Ivona Nečasová, Michal Zimmermann, Milan Hluchý, Ctirad Hofr
Chromatin Molecular Complexes, CEITEC and Faculty of Science, Masaryk University, Brno, CZ-62500,
Czech Republic
Telomeric proteins TRF2 and Rap1 are components of a multi-subunit human protein complex called
shelterin. Shelterin alters the spatial arrangement of telomeres by formation of specific structures,
thereby affects telomerase and other enzymes access to DNA. In this way, shelterin proteins
participate in the processes of aging and tumor cell transformation. Here we investigate the effect of
protein Rap1 on TRF2 binding to double-stranded telomeric DNA. The quantitative analysis using
fluorescence anisotropy, surface plasmon resonance, isothermal titration calorimetry and gel
electrophoresis revealed that the binding affinity of TRF2 to Rap1 is comparable with the binding
affinity of TRF2 to DNA. The presence of Rap1 reduces the overall DNA binding affinity of TRF2,
however, increases the binding selectivity of TRF2 to telomeric DNA. Our results demonstrate that
Rap1 induces a partial release of TRF2 from a preformed TRF2-DNA complex. In Rap1 presence, TRF2
affinity to DNA depends less on salt concentration, which shows that the specific TRF2-DNA interaction
prevail; TRF2 binds telomeric DNA more selectively. The observed Rap1 induced charge neutralization
might contribute to TRF2 release from DNA. Thus, Rap1 improves significantly the accuracy of the
selective recognition of telomeric DNA by TRF2 and hence the whole shelterin complex.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), the European Social Fund
(CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550), and KONTAKT II (LH13054).
Presented by: Janoušková, Eliška
131
Poster 60
Influence of environmental factors on telomerase-independent survivor formation in fission yeast
Kristi Jensena,*, Baumann Peterb
a
b
HHMI and Stowers Institute for Medical Research
HHMI and Stowers Intitute for Medical Research; Dept. of Mol and Int. Physiology, KUMC
Whereas most human somatic cells do not express the catalytic subunit of telomerase; reactivation of
telomerase is a signature of many human cancers. This lends clinical significance to finding a
telomerase inhibitor that can be used as a chemotherapeutic drug. However, a sub-set of cancers lack
active telomerase and chromosome ends are maintained by a telomerase independent process called
alternative lengthening of telomeres (ALT). As we are learning more about the mechanisms underlying
telomerase-independent telomere maintenance, it will be important to understand how readily cells
can switch between different pathways to replenish telomeric sequences.
In fission yeast, loss of telomerase results in a population of cells that initially experience chromosome
instability and crisis after which telomerase-independent survivors arise. Three classes of survivors
have been described: (i) so called circular survivors in which all three chromosomes have undergone
circularization and chromosome ends are absent; (ii) linear survivors with long heterogeneous
telomeres maintained through recombination reminiscent of human ALT cells; and (iii) HAATI survivors
that accumulate large amounts of repetitive heterochromatic sequence at chromosome ends. How
environmental conditions affect the choice of pathway that maintains telomeres in the absence of
functional telomerase and whether cells can switch readily between pathways is poorly understood.
We will present results on factors and conditions that affect survival in the absence of the RNA subunit
of telomerase.
Presented by: Jensen, Kristi
132
Poster 61
Sequence variants of the telomerase reverse transcriptase (TERT) gene in Nicotiana tabacum
Jana Jureckovaa,*, Eva Sykorovab, Miloslava Fojtovac
a
Faculty of Science, Masaryk University Brno, Czech Republic
Institute of Biophysics, ASCR, Czech Republic
c
CEITEC, Masaryk University Brno, Czech Republic
b
Study of telomeres evolution includes the analysis of arrangement of telomere and subtelomere
regions and characterisation of the telomerase in different model organisms.
Telomerase is a ribonucleoprotein enzyme complex providing the synthesis of telomeric repeats at the
ends of eukaryotic chromosomes. Telomerase RNA (TR) serves as the teplate for elongation of the
telomere repeat motif. The gene coding for catalytic subunit of telomerase - telomerase reverse
transcriptase (TERT) is developmentally regulated and its transcription reflects the activity of
telomerase in the tissue.
Nicotiana tabacum (tobacco), an allotetraploid model plant, is the only plant representative in which
more sequence variants of the TERT gene were identified (Sýkorová et al., 2012). Two NtTERT variants
(NtTERT-C/s and NtTERT-D) were inherited from the tobacco‘s diploid progenitor Nicotiana sylvestris
and one NtTERT variant (NtTERT-C/t) from Nicotiana tomentosiformis. The aim of our study was to
analyse copy number of NtTERT-C/s, NtTERT-C/t and NtTERT-D variants in N. tabacum genome using
qPCR and to determinethe transcription of these variants in tissues with different telomerase activity
like seedlings, leaves, buds and roots by qRT-PCR.
Sykorova E, Fulneckova J, Mokros P, Fajkus J, Fojtova M, Peska V. Three TERT genes in Nicotiana
tabacum. Chromosome Res. (2012).
The research is supported by Masaryk University Brno (MUNI/C/0979/2013), by Czech Science
Foundation (13-06943S) and by the project “CEITEC - Central European Institute of Technology”
(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.
Presented by: Jureckova, Jana
133
Poster 62
Fission yeast without subtelomeres
Sanki Tashiro, Yuki Nishihara, Junko Kanoh
Institute for Protein Research, Osaka University
Recent studies have uncovered the functions of telomere, such as the maintenance of telomere DNA
length, the protection of chromosomal ends, and the regulation of chromosome movements in mitosis
and meiosis. However, little is known about the physiological roles of subtelomeres. S. pombe is an
ideal organism for the analyses of subtelomere because it has only three chromosomes. The
subtelomeres on chromosomes I and II consist of approximately 50 kb-long highly homologous DNA
sequences, and the subtelomere on chromosome III contains the partial DNA sequence (approximately
15 kb-long) homologous to the other subtelomeres. To investigate the physiological roles of
subtelomere, the all subtelomeres were deleted from the S. pombe chromosomes. The SD
(subtelomere-deleted) strain was viable with the similar growth rate to that of the wild-type. It also
showed the normal telomere DNA length, indicating that the subtelomeres are dispensable for the cell
growth and the telomere DNA maintenance. Interestingly, the subtelomere deletion led to the invasion
of heterochromatin to the subtelomere-adjacent regions (approximately 50 kb-long), which resulted in
the silencing of genes located at the heterochromatin-invaded regions. These data suggested that the
subtelomeres serve as buffer zones against the heterochromatin invasion to maintain gene
expressions.
Presented by: Kanoh, Junko
134
Poster 63
Multiple interactions within a primer influences Saccharomyces castellii telomerase activity in vitro
Cecilia Gustafsson, Ahu Karademir, Roopesh Krishnankutty, Marita Cohn
Department of Biology, Genetics group, Lund University, Lund, Sweden
The telomerase ribonucleoprotein enzyme maintains genome integrity by elongating the telomeres.
Telomerase adds tandem repeats on the telomeric DNAs via using its intrinsic RNA template.
Translocation of the DNA 3´end on the RNA template provides telomerase to add several repeats on
the substrate. The budding yeast Saccharomyces castellii synthesizes several 8-mer repeats
(CTGGGTGT) onto the primer in vitro. In this work, we determined the the primer extension
mechanisms of S. castellii telomerase by using an in vitro primer direct assay. We showed that a six
nucleotide long primer and a single-stranded 3’ overhang of four nucleotides are efficiently extended.
Moreover, we showed that multiple interactions within a primer influences the telomerase activity of
S. castellii in vitro. While S. castellii telomerase is not able to utilize the non-telomeric 3’ ends of short
primers, the interaction with the 5’ end of long primers can stimulate the extension of non-telomeric 3’
ends. Our results reveal intriguing details about the S. castellii telomerase activity.
Presented by: Karademir, Ahu
135
Poster 64
Dissecting the telomeric and non-telomeric roles of human CTC1 through gene disruption
Christopher Kasbek, Jason Stewart, Mary Chaiken, Caitlyn Goodwin, Carolyn Price
University of Cincinnati
Human CST (CTC1-STN1-TEN1) functions in DNA replication at telomeres and other genomic sites in
response to replication stress. Mutations in the CTC1 subunit cause the disease Coats plus and can lead
to dyskeratosis congenita. STN1 depletion revealed that CST functions in telomere duplex replication,
C-strand fill in, and genome-wide replication rescue after hydoxyurea treatment. TEN1 depletion
suggested additional roles as telomere loss and anaphase bridge formation were more pronounced
than with STN1 depletion. However depletion of CTC1 with shRNA resulted in variable phenotypes. To
clarify CTC1 function, we generated a conditional gene disruption in human cancer cells. Although
CTC1 mRNA is undetectable 2 days after gene disruption, phenotypes due to CTC1 deletion are not
observed until day 4. Non-telomeric defects arise earlier than telomeric defects. Initial phenotypes
include decreased ability to rescue replication after fork stalling and increased anaphase bridge
formation in the absence of telomere fusions, however growth rate remains normal. By days 5-7, Goverhang length is increased and chromosome ends lacking telomere signal become apparent. The
growth rate decreases slightly. Telomere loss then increases steadily, chromosome fusions appear and
DNA damage signaling rises. By three weeks after CTC1 disruption the cells cease to divide. These
results support the idea that CST functions in response to multiple types of replication stress. They also
suggest that the severity of symptoms in Coats plus patients stem from both telomeric and genome
wide deficiencies in DNA replication.
Presented by: Kasbek, Christopher
136
Poster 65
Interfacial inhibition of human telomerase reconstitution
Guillaume Kellermanna,*, Markus Kaiserb, Florent Dinglic, Olivier Lahunad, Florence Mahuteau-Betzere,
Damarys Loewdc, Evelyne Ségal-Bendirdjiana, Marie-Paule Teulade-Fichoue, Sophie Bombarda
a
INSERM U1007 / Université Paris Descartes
Universität Duisburg-Essen
c
Institut Curie / laboratoire de spectrométrie de masse protéomique, France.
d
INSERM U-1016, Institut Cochin
e
Institut Curie / CNRS UMR 176, Orsay, France
b
Telomerase is an almost universal cancer target lacking of specific and efficient inhibitors. We present
herein a method to produce high amount of the catalytic subunit of human telomerase and use it to
identify small-molecules interfering with telomerase assembly. This test extends the identification of
telomerase inhibitors to a new class of compounds, not detectable by conventionnal assays.
Using this method, we identified a new molecule, TAI1, that can completely inhibit telomerase activity
when added prior assembly, at a concentration which has no effect on an already assembled
telomerase. Direct assay shows that TAI1 prevents the DNA polymerase activity of telomerase, and
does not interfere with repeat addition processivity.
EMSA showed that TAI1 only slightly reduces the binding of hTERT to hTR, in conditions where
telomerase activity is completely inhibited. This means that TAI1 promotes the formation of inactive
telomerase complexes. Surprisingly, these inactive complexes are irreversibly inhibited, since their 100fold dilution failed to restore any activity, even after several hours of incubation. These observations
suggest that TAI1 is irreversibly trapped inside the telomerase complex, after assembly, in a manner
reminiscent of interfacial inhibitors. Structure-activity relationships support a model where TAI1 binds
both to hTERT and hTR, thus producing a ternary complexe.
In conclusion, our data suggest that potent telomerase inhibitors have escaped detection during all
previous screens performed with telomerase already assembled from cancer cells, and these
observations therefore recommand the future use of the present method that can both detect
assembly and catalytic inhibitors.
Presented by: Kellermann, Guillaume
137
Poster 66
Therapeutic agents influence the TRF2-TIN2 interactions and their cellular distribution: study in
human cancer cell lines
Patrycja Klos, Ctirad Hofr, Ivona Necasova, Jiri Fajkus
Central European Institute of Technology, Masaryk University, Molecular Complexes of Chromatin
Group
Among anti-cancer therapies, the use of chemicals interfering with the expression/function of
telosome components has aroused recently much interest. In this study we examined the interactions
between TRF2 and TIN2 shelterin proteins in human cancer cells with varied telomere length, treated
with therapeutic chemicals that are known to interact with the DNA, which in turn might cause TRF2
redistribution accross the DNA, and/or influence its stability and interacting properties. To compare
the number of TRF2-TIN2 interactions between the different cell lines we employed proximity ligation
assay (PLA). Immunofluorescence colocalization study was performed to check the distribution pattern
of the studied proteins in the cells. The expression level of TRF2 after the drug treatment was
measured by qRT-PCR and Western blotting. Significant changes in the number of TRF2-TIN2
interaction spots, their distribution accross the chromatin, as well as TRF2 expression level were
observed after treating the cells with the chosen chemicals. Interestingly, the obtained data varied in
different studied cell lines. Altogether our results demonstrate a strong impact of the chosen
therapeutics on cancer cell telosome, which in turn may have further implications in anti-cancer
therapy.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), the European Social Fund
(CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550), and KONTAKT II (LH13054).
Presented by: Klos, Patrycja
138
Poster 67
Telomerase RNP complex assembly in disease background
Elzbieta Kowalska, Georgeta Zemora, Michael Wildauer, Christina Waldsich
Max F. Perutz Laboratories, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
RNA structure is relevant in telomerase ribonucleoprotein complex (RNP) formation, as specific
nucleotide polymorphisms in the human telomerase RNA (hTR) have been implicated in disease
pathologies. The main protein components of the human telomerase RNP complex are hTERT, a
reverse transcriptase, and Dyskerin (DKC1), a protein important for telomerase biogenesis. As for hTR,
there are known hot spots of single nucleotide polymorphisms in both of these proteins that are linked
to dyskeratosis congenita and/or pulmonary fibrosis. To elucidate the structural basis of disease-linked
mutations, I introduced single amino acid changes in these regions of the proteins. For hTERT, the
motif B and the linker motif were mutated. In the case of DKC1, I targeted the PUA domain that
contacts the hTR. The hTERT and DKC1 constructs containing the disease-related point mutations were
expressed by transient transfection together with hTR in HEK293 cells. Employing the SHAPE chemistry,
the structure of hTR can be investigated in living cells. After chemical modification, the cells were
harvested and total RNA was extracted for reverse transcription. At modified nucleotides the reverse
transcriptase will stall or fall off. The different cDNA fragments were separated on a denaturing
polyacrylamide gel (PAGE) and intensities of bands were compared between hTR alone and hTR in the
presence of the wildtype or point mutated hTERT or DKC1 proteins. The changes in structure of hTR in
the presence of wildtype protein or the proteins with disease-related point mutations will provide
novel insights into the architecture of the telomerase RNP complex.
Presented by: Kowalska, Elzbieta
139
Poster 68
Alternative lengthening of telomeres detected in telomerase-positive canine histiocytic sarcoma
Theresa Kreilmeiera,*, Sandra Samplb, Marlene Hauckc, Ingrid Walterd, Klaus Holzmannb, Miriam Kleitera
a
Department for Companion Animals and Horses, University of Veterinary Medicine Vienna,
Veterinärplatz 1, 1210 Vienna, Austria
b
Division of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical
University Vienna, Austria
c
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 1060
William Moore Drive, Raleigh, NC 27607, USA
d
Vet Core Facility, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria
The telomere maintenance mechanism (TMM) used in the absence of telomerase activity (TA) is called
alternative lengthening of telomeres (ALT). ALT is found more often in human tumors of mesenchymal
than of epithelial origin. Our aim was to screen a cohort of canine sarcomas for prevalence of TMM
used.
Tumor tissue from 17 spontaneous occurring canine soft tissue sarcomas were analysed for telomere
length, TMM, and immunohistochemical expression of ATRX and DAXX. Telomere length was
determined by qPCR, telo-qFISH hybridisation and TRF analyses including CHEF gelelectrophoresis. TA
and ALT were measured by TRAP assay and the presence of C-circles, respectively.
One histiocytic sarcoma showed elevated C-circle levels and long telomeres with heterogeneous
lengths similar in appearance as in human cells with ALT. In contrast, TA was detected in 15 of 16
sarcomas including the case with ALT activity. In one sarcoma neither ALT nor telomerase activity was
found. The case using two TMMs showed on tissue sections loss in ATRX but not DAXX expression,
proteins known to localise at PML bodies. Further, colocalisation of DAXX at telomeres was elevated
compared to cases without ALT.
In conclusion, ALT exists in canine sarcomas with presumably less prevalence compared to human. Our
data indicate some tumor cells use both ALT and TA, but this remains to be confirmed. TA seems to be
the dominant TMM and even non-determined TMM might play a role. Therefore, all mechanisms have
to be considered for therapeutic approaches.
Presented by: Kreilmeier, Theresa
140
Poster 69
Telomere Length Kinetics Assay (TELKA) sorts the Telomere Length Maintenance (tlm) mutants into
functional groups
Martin Kupiec, Linda Rubinstein, Yaniv Harari, Vera Babin
Department of Molecular Micro and Biotechnology, Tel Aviv University, Ramat Aviv 69978, Israel.
Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length
maintenance network, or TLM), encompassing more than 400 genes, which acts coordinatively to
maintain telomere length. Identifying the genes was an important first stage; the next challenge is to
decipher their mechanism of action and to organize then into functional groups or pathways. Here we
present a new telomere-length measuring program, TelQuant, and a novel assay, TELKA (Telomere
Length Kinetic Analysis), and use them to organize tlm mutants into functional classes. Our results
show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as
mutants defective for the Ribonucleotide Reductase subunit Rnr1, in charge of the limiting step in
dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the
epistatic relationship between the mutants, and show that physical interactions exist between Rnr1
and Met7. We also show that Met7 and the Ku heterodimer affects dNTP formation, and play a role in
non-homologous end-joining (NHEJ). Thus, our telomere kinetics assay uncovers new functional
groups, as well as complex genetic interactions between tlm mutants.
Presented by: Kupiec, Martin
141
Poster 70
Involvement of SRSF11 in cell cycle-specific recruitment of active telomerase to telomeres at nuclear
speckles
Jihoon Leea,*, Sun Ah Jeongb, Prabhat Khadkaa, Juyeong Hongb, In Kwon Chungc
a
Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University,
Seoul 120-749, Korea.
b
Department of Integrated Omics for Biomedical Science, Yonsei University, Seoul, 120-749, Korea.
c
Department of Integrated Omics for Biomedical Science and Department of Systems Biology, Yonsei
University, Seoul, 120-749, Korea.
Telomerase, a unique ribonucleoprotein (RNP) complex that contains telomerase reverse transcriptase
(TERT) and the telomerase RNA component (TERC), is required for continued cell proliferation in cancer
and stem cells. By adding short telomere repeats to the 3’ ends of chromosomes, telomerase
compensates for the progressive loss of telomeric DNA produced by incomplete replication of
chromosome ends. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear
speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11
associates with active telomerase enzyme within Cajal bodies and directs it to nuclear speckles
specifically during S phase of the cell cycle. On the other hand, telomeres are shown to be
constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear
speckles may be the nuclear sites for telomerase recruitment to telomeres. Depletion of SRSF11 by
RNA interference prevents telomerase from associating with nuclear speckles and disrupts telomerase
recruitment to telomeres, thereby abrogating telomere elongation by telomerase. SRSF11 also
associates with telomeres through the interaction with TRF2, which facilitates translocation of
telomerase to telomeres. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor
that plays a major role in the telomerase association with telomeres through the interactions with
TERC and TRF2, and provide a new target for modulating telomerase activity in cancer.
Presented by: Lee, Jihoon
142
Poster 71
Anticancer mechanism of TMPyP4 in breast cancer cell lines MCF7 and MDA-MB-231
Natalia Lipinskaa,*, Hanna Holysza, Aleksandra Romaniuka, Mariusz Kaczmarekb, Blazej Rubisa
a
Department of Clinical Chemistry and Molecular Diagnostics,
Poznan University of Medical Sciences, Przybyszewskiego 49 St., 60-355 Poznan, Poland
b
Department of Immunology, Chair of Clinical Immunology, Poznan University of Medical Sciences,
Rokietnicka 5D St., 60-806 Poznan, Poland
Telomeres, the guanine-rich sequences, play a crucial role in human genome stabilization. Thus, they
became a promising target in cancer fighting strategy. One of the potential effective tool in blocking
the access of telomerase to telomeres, leading to a limited cancer cell divisions, is application of the
telomeric ends (G-quadruplex) stabilizers. It was revealed that a cationic porphyrin, TMPyP4 is an
efficient G-quadruplex stabilizing agent and hence might inhibit telomerase activity in human cancer
cells.
We analyzed the influence of clinically relevant TMPyP4 concentrations on morphology, cell cycle,
proliferation, migration and adhesion of MCF7 and MDA-MB-231 breast cancer cells.
It was shown that treatment with TMPyP4 caused a time (24, 48, 72h) and concentration (0.5µM 15µM) dependant proliferation decrease of breast cancer cells. Short-term treatment resulted in a
significant telomere shortening, while longer times of treatment (up to 20 passages) induced
restoration of the original length of telomeres (MDA-MB-231) or even telomere lengthening (MCF7).
Surprisingly it was not associated with any significant changes in cell cycle.
We suggest that this effect might be related to the mechanism of multidrug resistance since treatment
with 0.5µM TMPyP4 caused a significant increase of ABCB1 gene expression after 24h. Interestingly,
this effect was abolished after longer incubation time. Additionally, it was revealed that TMPyP4
treatment might affect adhesion and migration properties of breast cancer cells through influence on
FAK, p-FAK, β1-integrin and paxillin expression.
The work was supported by 2011/03/B/NZ7/00512 project.
Presented by: Lipinska, Natalia
143
Poster 72
The Mechanism of Epigenetic Behavior at the Mre11/Rad50 Interface
Danielle Tatum, In-Joon Baek, Heewon Park, Arthur Lustig, et al.
Tulane University Medical Center
We have identified an unusual characteristic that, to date, has only been observed in the mre11A470T
allele that, based on crystallography, lies in one of the alpha helical residues present at the HLH
domain of Mre11. We find that the pattern of recessive and dominant alleles in these mutants and the
outcome is dependent upon the pre-existing chromatin present at the telomere. We have found
identical results in CEN plasmid studies and ectopic integration studies of the effect. The pattern of
heritability is stable to the genotype that is the recipient of the mutant gene and vise versa. We have
ruled out possible copy number artifacts and differential turnover of Mre11 and Mre11A470T. We are
finally determining whether this is truly an epigenetic state by determining the The DNA sequence of
the mutant strain.
We have begun on our next test in determining the mechanism of action of the putative epigenetic
effect We have introduced one of the rap1 alleles, rap1-5, into both wild type and mutant chromatin
states to determine if disrupted Rap1-based chromatin contributes to or removes the epigenetic
heritable state. In this regard, our studies have shown an inability to heal broken chromosomes
containing seed sequences while still stably maintaining a shorter telomere size. The inability of allele
to act in the healing is unlikely to be due to the abundance of telomerase itself given parallel successful
healing experiments with strains with similar sized telomeres e,g, the tel1 allele. As a result we are
analyzing only replication at the telomere
Presented by: Lustig, Arthur
144
Poster 73
Identification of the subtelomeric sequence of the long non-coding RNA TERRA unveils its role at
telomeres
Isabel López de Silanes, Osvaldo Graña, Maria Luigia De Bonis, Orlando Domínguez, David G. Pisano,
Maria Antonia Blasco
Spanish National Cancer Research Centre
TERRAs are telomeric repeat-containing RNAs that are transcribed from the subtelomere towards the
telomere. However, TERRA’s full se uence remains unknown to date. By using a whole-genome RNAsequencing approach, we have identified novel mouse transcripts arising from the subtelomere of
chromosome 18 resembling TERRA in many key features. Namely, they are heterogeneous in size and
are bound by TERRA RNA-binding proteins. These transcripts co-localize with TERRA and are found at
the telomeres in interphase and metaphases, as well as are regulated in a similar manner to TERRA in
response to stress and with induction of pluripotency. siRNA depletion of either these transcripts or
TERRA causes an increase in telomere dysfunction-induced foci (TIF). We have also identified the
promoter region responsible for their transcription. The identification of the subtelomeric sequence of
TERRA will facilitate the design of molecular tools to understand the functional significance of TERRA
transcripts in telomere biology and telomere-related diseases.
Presented by: López de Silanes, Isabel
145
Poster 74
Epigenetic landscape of terminal and internal telomeric DNA using different plant models
Eva Majerova, Terezie Mandakova, Miloslava Fojtova, Jiri Fajkus
Department of Functional Genomics and Proteomics, Faculty of Science and Central European Institute
of Technology, Masaryk University, CZ-62500 Brno, Czech Republic
The epigenetic state of telomeres has been animatedly discussed but has not been clearly described
yet. The most studied model plant, Arabidopsis thaliana, shows distinct epigenetic marks in
dependence to the used methodology what correlates with the fact that considerable portion of
telomeric repeats is found at intrachromosomal positions. One of the postulated hypotheses says that
interstitial telomeric repeats (ITR) are heterochromatin while genuine telomeres show rather
euchromatic character. Intriguingly, transcription of telomeres is found mostly at ITR with only small
portion of TERRA and ARRET transcripts originating from terminal telomeres. Here, we present data on
epigenetic landscape of telomeric repeats introducing two plant models – (i) Ballantinia antipoda with
large blocks of ITR; and (ii) Nicotiana tabacum, plant with long telomeres and no detectable ITR.
Our results show that the distribution of 5mC along telomeres is not uniform but proximal parts of
telomeres are less densely methylated. Long tobacco telomeres have ambiguous character; we found
marks for constitutive heterochromatin (H3K9me2) as well as for active or developmentally silenced
genes (H3K4me3, H3K27me3). Moreover, telomeres are transcribed from both strands, which indicates
that tobacco telomeres must include an active transcription start site. As for ITR, we found sparsely as
well as densely methylated region, and both of them showed the same heterochromatic pattern (only
H3K9me2 was found). Intriguingly, with no respect to different DNA methylation levels, both regions
are transcribed to the same extent and from both strands. In addition, transcription of telomeric
repeats is tissue-specific in both plants.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068) and by the European Social Fund
(CZ.1.07/2.3.00/20.0043).
Presented by: Majerova, Eva
146
Poster 75
Unraveling the changes in telomere composition during senescence, immortalization, and
tumorigenic conversion of normal human fibroblasts
Jana Majerská, Joachim Lingner
Swiss Institute for Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne
(EPFL), Lausanne, 1015, Switzerland
The dynamic composition of telomere-associated proteins allows the cell to address the special
challenges of telomere protection and maintenance. Perturbations of telomere function play an
essential role in the induction of cellular senescence, genomic instability and cancer. However, the
critical protein factors contributing to these processes have remained elusive. To address this question,
we have employed a genetically defined cell culture model of tumorigenesis, developed by Hahn et al.
[1, 2], which allows us to study telomeric states during different stages of the transformation process.
Normal human lung fibroblasts were converted into malignant cells in a step-by-step fashion using
serial introduction of genes encoding (a) the catalytic subunit of telomerase, (b) the SV40 large T and
small T antigens, and (c) the H-RasV12 oncogene. We will discuss our progress in identifying differences
in the telomeric protein composition between these cell lines, using the Quantitative telomeric
chromatin isolation protocol (Q-TIP) [3] recently developed in our lab.
1. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW and Weinberg RA (1999). Nature
400:464-8.
2. Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM, DeCaprio JA and Weinberg RA
(2002). Mol Cell Biol 22:2111-23.
3. Grolimund L, Aeby E, Hamelin R, Armand F, Chiappe D, Moniatte M and Lingner J (2013). Nat
Commun 4:2848.
Presented by: Majerská, Jana
147
Poster 76
Differential processing of double strand breaks and telomeres at the nuclear envelope in S.
cerevisiae
Isabella Marcomini, Chihiro Horigome, Susan M. Gasser
Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland
Telomeres, in yeast as in some higher eukaryotes, are anchored at the nuclear envelope, and this
anchorage is necessary to prevent ectopic recombination and genomic instability. The anchorage
pathway in S phase, dependent on yKu80-Est1 interaction, requires the SUN domain protein Mps3.
When they become critically short in absence of telomerase, telomeres have been shown to colocalize
with nuclear pores.
Our lab has shown that in S.cerevisiae persistent double strand breaks (DSBs) are also relocated to the
nuclear periphery. Two anchorage sites have been identified: nuclear pores throughout the cell cycle,
and Mps3 in S phase in a resection-dependent way.
It is remarkable that DSBs and telomeres share common anchorage sites considering that their fate in
the cell must be very different: the activation of DNA repair pathways at telomeres could cause
telomere-telomere fusions and senescence, while persistent DSBs lead to loss of somatic material and
cell death. We propose that the flanking sequence near the DSB influences its binding site at the
nuclear envelope, which in turn may determine the outcome of repair. We are testing this hypothesis
with a construct made of a DSB site flanked by TG telomeric-like repeats of different lengths,
integrated on a chromosome arm. The presence of long TG repeats (TG250) at a break is already
known to have an anticheckpoint effect dependent on Rif1 and Rif2. The construct allows us to analyze
DSB and telomeric processes at once and hopefully identify whether subnuclear positioning
contributes to the identity of telomeres versus DSBs.
Presented by: Marcomini, Isabella
148
Poster 77
Bridging ageing and metabolism through RAP1?
Paula Martinez, David Pisano, Maria A. Blasco
Spanish National Cancer Research Center (CNIO), Madrid, Spain
RAP1 is one of the components of the shelterin complex that protects the telomeres. RAP1 binds to
both telomeres and non-telomeric sites throughout the whole genome exerting a transcriptional
regulatory role. Mice lacking RAP1 gain weight dramatically faster than wild type mice, show symptoms
of diabetes type II and hepatic steatosis. Gene expression profile analyses in liver of adult mice show
that in the absence of RAP1, several metabolic pathways are remarkably affected. Telomere length is
highly dynamic and does vary among different cell types and along ageing. It is therefore conciveable
that telomere length would affect the ratio between telomere-bound RAP1 and genomic DNA-bound
RAP1 that is available to perform its transcriptional regulatory function. To test this hypothesis we
have made used of the telomerase deficient mouse model. Succesive generations of Terc-/- mouse
show a progressive telomere shortening that is accompanied by a proportional declive in regenerative
capacity of the tissues that leads to an acceletared ageing. We have generated the Rap1-/- Terc-/compound mouse. We are currently analyzing whether Rap1-deletion has an impact on body weight
and survival compared to (G1-G3) Rap1+/+ Terc-/-. Telomere length dynamics in these mouse cohorts
is also under study to address whether RAP1 could have a role in telomere length homeostasis. Chipseq analysis in Rap1+/+ Terc+/+, G1 Rap1+/+ Terc-/- and G3 Rap1+/+ Terc-/- cells will shed light on
whether Rap1 occupancy at telomeric-DNA versus genomic-DNA does change depending on telomere
length. In this meeting we will report our results regarding the abovementioned studies.
Presented by: Martinez, Paula
149
Poster 78
Telomere shortening induces skeletal and cardiac muscle failure in Duchenne Muscular Dystrophy
Foteini Mourkiotia,*, Alex Changb, Alan Meekerc, Helen Blaub
a
Instructor
Baxter Laboratory for Stem Cell Biology,
Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative
Medicine, Clinical Sciences Research Center, 269 Campus Drive, Stanford, CA 94305-5175, USA
b
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for
Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, 269 Campus Drive,
Stanford, CA 94305-5175, USA
c
Department of Pathology, Department of Oncology, Johns Hopkins Medical Institution, Baltimore, MD
21231, USA.
Duchenne muscular dystrophy (DMD) is the most common inherited myopathy of childhood that leads
to early death, generally before the age of 30. DMD is characterized by severe progressive muscle
wasting due to mutations in dystrophin, which links the inner cytoskeleton with the extracellular
matrix and therefore plays a key role in plasma membrane integrity in both skeletal and cardiac
muscles. Paradoxically, mdx mice, which share the same dystrophin deficiency as DMD patients, exhibit
only mild muscle weakness and minimal cardiac dysfunction in contrast to patients. We reasoned that
telomere length differences between mice and humans could account for this discrepancy and
developed a new mdx mouse model with shortened telomeres (mdx/mTR) (Mourkioti et. al., Nature
Cell Biology, 2013 and Sacco, Mourkioti et. al., Cell, 2010). This model is the first dystrophic mouse that
exhibits all the pathological hallmarks of human DMD: severe loss of muscle force, kyphosis, muscle
membrane disruption, progressive wasting of limb and diaphragm muscles, and dilated
cardiomyopathy, culminating in early death. Most importantly, we demonstrated 45% cardiomyocytespecific telomere shortening in both mice and DMD patients. Here, we will present new data on
telomere shortening during DMD progression that will provide novel mechanistic insight into the role
of telomeres in the etiology of disease.
Presented by: Mourkioti, Foteini
150
Poster 79
HOMOLOGY-DEPENDENT REPAIR IS INVOLVED IN 45S rDNA LOSS IN FAS MUTANTS
Veronika Muchovaa,*, Simon Amiardb, Iva Mozgováa, Martina Dvořáčkovác, Maria E. Gallegob, Charles
Whiteb, Jiří Fajkusa
a
Mendel Centre for Plant Genomics and Proteomics, CEITEC, and Faculty of Science, Masaryk
University, Kamenice 5, CZ-62500 Brno, Czech Republic
b
Génétique, Reproduction et Développement, UMR CNRS 6293 - Clermont Université - INSERM U1103,
Campus des Cézeaux, Université Blaise Pascal, Clermont Ferrand, France
c
Institute of Biophysics ASCR, Královopolská 135, CZ-61265 Brno, Czech Republic
In our previous studies we reported the progressive loss of 45S rDNA sequences and severe telomere
shortening in A. thaliana CAF-1-deficient (fas1 or fas2 mutant) plants (Mozgova et al., 2010). Our
results further demonstrate that the truncation of both repetitions is mitotic. Since fas mutants were
reported to have increased levels of HR, we decided to investigate the possible involvement of this
pathway in advertised phenomenon. For this purpose the double mutants in FAS and AtRAD51B
(essential for mitotic HDR, paralogue of the Rad51 recombinase) genes were created. Here we
demonstrate the significant role of AtRAD51B in 45S rDNA maintenance mechanism in fas mutants
while the influence of this knock out in telomeres loss is negligible – suggesting different mechanisms
of the loss of the two kinds of repeats at the ends of chromosomes. The involvement of imprecise DNA
damage repair in rDNA dynamics in fas mutants is further supported by accumulation of double-strand
breaks (measured as γ-H2AX foci) in 45S rDNA which are not associated with rDNA replication and are
ATR-dependent. Our data also suggest that the repair of DSB present in the transcribed fraction of
rDNA (in the nucleolus) is RAD51B-dependent and contributes to rDNA loss, in CAF-1 (FAS) plant
mutants, presumably via single strand annealing pathway.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), GACR (P501/11/0289) and by the
European Social Fund (CZ.1.07/2.3.00/20.0043).
Presented by: Muchova, Veronika
151
Poster 80
The effects of lifestyle factors on leukocyte telomere length dynamics: Results from the ESTHER
Cohort
Aysel Muezzinler, Aida Karina Dieffenbach, Katja Butterbach, Hermann Brenner
Division of Clinical Epidemiology and Ageing Research, German Cancer Research Center (DKFZ), Im
Neuenheimer Feld 581, 69120 Heidelberg, Germany
In recent years, the influence of lifestyle factors on leukocyte telomere length (LTL) has drawn
considerable attention. However, there is limited and contradictive evidence in the literature.
Therefore, we aim to assess the association between BMI, smoking and LTL in both cross-sectional and
longitudinal analyses.
LTL of 3600 samples of an elderly population were measured by quantitative PCR method. 8-year
follow-up LTL of 1000 samples were also measured for the longitudinal analyses. Terminal Restriction
Fragment analysis was additionally performed in a sub-sample to obtain absolute TL in base pairs.
Multivariate linear regression models were used to estimate the regression coefficients.
Age was inversely associated with LTL (r=-0.098, p<0.0001), but not with yearly LTL change (r=0.005,
p=0.882). Women had longer LTL than men (p<0.0001). Mean LTL was longer in never smokers
(p=0.002), but the rate of yearly LTL change showed no difference. No difference in LTL and yearly LTL
change was observed for intensity of smoking, different categories of BMI or duration of obesity.
The analysed lifestyle factors point to a very limited role on LTL. Only some significant associations
between smoking and BMI-related variables and LTL and yearly LTL change were observed. The power
of the longitudinal analysis could be limited due to sample size. LTL dynamics differ in different age
groups. Therefore, using a composite index for lifestyle to evaluate the value of LTL as a biological
marker of ageing could be a more appropriate approach than focusing on individual factors.
Presented by: Muezzinler, Aysel
152
Poster 81
Expression of ATRX represses the Alternative Lengthening of Telomeres phenotype
Christine Napier, Jane Noble, Roger Reddel
Cancer Research Unit, Children’s Medical Research Institute, Westmead, NSW, Australia
Normal mortal fibroblasts contain repressors of the ALT mechanism, as demonstrated by analyses of
somatic cell hybrids of ALT cells with normal mortal fibroblasts (1). Previous studies revealed that many
ALT-positive tumours and cell lines have mutations in one or other member of the ATRX/DAXX
chromatin remodelling complex (2,3). We have shown that knockdown of ATRX was not sufficient to
activate ALT activity, but that it significantly decreased the length of time in crisis prior to activation of
ALT and immortalisation. We also expressed exogenous ATRX in ALT cell lines that lack ATRX
expression. Western blotting showed that the ATRX protein level peaked at day 2 and was
undetectable by day 8 post-transfection. The C-circle levels decreased by up to 50% after 2 days of
ATRX expression, accompanied by a significant decrease in the number of ALT-associated PML bodies
in ATRX-positive nuclei. Cells transfected with ATRX were selected with G418 and resistant clones
isolated. More than 80 clones were obtained from two ALT cell lines but only one clone expressed
ATRX, suggesting a strong selection pressure against ATRX expression in ALT cells. These data are
consistent with ATRX being an ALT repressor.
1. Perrem, K, et al., Oncogene 18: 3383-3390, 1999.
2. Heaphy, CM, et al., Science 333: 425, 2011.
3. Lovejoy, CA, et al., PLoS Genet. 8: e1002772, 2012.
Presented by: Napier, Christine
153
Poster 82
In vitro assessment of the protective role of Cdc13 against degradation of telomeric single-stranded
3’ overhangs
Saishyam Narayanan, Cecilia Gustafsson, Marita Cohn
Department of Biology, Genetics group, Lund University
Telomeres are nucleoprotein structures in eukaryotic chromosomes which protect the chromosomal
ends from fusion and degradation by exonucleases. The telosome of budding yeast Saccharomyces
castellii includes the Cdc13 protein, which binds to specific sequences on the TG-rich single-stranded 3’
overhangs of the telomere. Cdc13 plays a dual role in regulating the telomere lengths by recruiting
telomerase by interaction with EST1 and by forming a protective cap (as Cdc13-Stn1-Ten1(CST)
complex) at telomeric ends, making it inaccessible to telomerase. Remarkable fluctuations in the levels
of Cdc13 along the cell cycle indicate that Cdc13/CST complex may not be essentially forming a
protective cap in all the phases of cell cycle. Thus, Cdc13 binding and its functional roles are thought to
be tightly regulated.
We analyzed the end protection delimitations of Cdc13 using an in vitro Telomere End Protection Assay
(TEPA). We found that Cdc13 on its own was able to provide protection to the single-stranded 3’
overhangs against degradation by various exonucleases, when analyzed on a short overhang of 20 nt.
The distance of the Cdc13 minimum binding site on the single-strand from the ds-ss junction
influenced the protection. Interestingly, Cdc13 bound at the minimum binding site conferred
protection to the nucleotides beyond the minimum binding site in the 3’ overhang. Our studies proved
that TEPA could be used as an effective method to determine the protective ability of Cdc13 at
telomeres with different 3’ overhang lengths and 5’ end permutations.
Presented by: Narayanan, Saishyam
154
Poster 83
TRF2 Overexpression Induces Telomeric Ultrafine Anaphase Bridges
Bernadette Nera, Hui-Shun Huang, Thao Lai, Lifeng Xu
University of California, Davis, Microbiology and Molecular Genetics, Davis, CA, 95616
Overexpression of full-length TRF2 is known to cause rapid telomere shortening. Here we report that
TRF2 overexpression inhibits telomere replication and induces the formation of telomeric ultrafine
anaphase bridges whose resolution leads to drastic telomere shortening.
Ultra-fine anaphase bridges (UFBs) derive from sister chromatids that are held together at anaphase
due to DNA catenation or incomplete replication. The BLM helicase protein is reported to align along
UFBs. We found that elevated levels of TRF2 caused replication fork stalling at telomeres and induced
telomeric UFBs. Depletion of BLM exacerbates TRF2-induced telomeric UFB formation. In contrast to
the canonical UFBs, only a proportion of such telomeric UFBs are aligned by the BLM protein,
suggesting that they represent a novel class of UFBs. The frequency of TRF2-induced telomeric UFBs
correlates with the average telomere length of cells. Comparable levels of TRF2 expression can induce
significant amount of telomeric UFBs in cells with pre-extended telomeres, but not in cells with very
short average telomere lengths. We also found that the formation of telomeric UFBs correlate with the
TRF2-induced rapid telomere shortening phenotype.
Presented by: Nera, Bernadette
155
Poster 84
How specific is TRF2 binding to telomeric DNA?
Ivona Nečasová, Michal Zimmermann, Ctirad Hofr
Chromatin Molecular Complexes, CEITEC and Faculty of Science, Masaryk University, Brno, CZ-62500,
Czech Republic
Telomeres form caps at the very ends of linear chromosomes. Any disturbance in integrity of telomeres
causes chromosomal instabilities leading to unfavorable chromosomal rearrangements or even to
carcinogenesis.
In mammals, telomere-associated proteins form a shelterin complex which protects chromosome ends
from DNA repair machinery. Shelterin serves as a negative regulator of telomerase, a
ribonucleoprotein enzyme that adds telomeric DNA to chromosomal ends to ensure complete genome
replication.
TRF2 plays a central role within shelterin. TRF2 binds to double-stranded telomeric DNA via a Cterminal Myb domain. It has been proposed that a N-terminal basic domain of TRF2 might be involved
in DNA binding as well.
Our results obtained by fluorescence anisotropy and isothermal titration calorimetry quantify the
contribution of the basic domain to the overall TRF2 binding to telomeric DNA. Additionally, our results
suggest the importance of the basic domain for long-range electrostatic interactions between TRF2 and
DNA. Furthermore, NMR measurements indicate structural changes associated with the binding of
TRF2 basic domain to DNA.
Our studies provided the first detailed quantitative and thermodynamic description of the contribution
of the basic domain to the DNA affinity of human TRF2. The obtained findings contribute to address
interactions of shelterin proteins that are essential for the regulation of telomere length and thus
maintenance of the whole genome stability.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068), the European Social Fund
(CZ.1.07/2.3.00/30.0019), GACR (P205/12/0550), and KONTAKT II (LH13054).
Presented by: Nečasová, Ivona
156
Poster 85
Selective Bioluminogenic HDAC Activity Assays for Profiling HDAC Inhibitors
Andrew L. Niles1, Nathan J. Evans1, Kevin R. Kupcho1, Thomas A. Kirkland2, and Dan F. Lazar1
1
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711
Promega Biosciences LLC, 277 Granada Drive, San Luis Obispo, CA 93401
2
Histone deacetylases (HDACs) play critical roles in the regulation of gene transcription and cell
signaling events by deacetylating histones and other important non-histone substrates. Aberrant
increases in HDAC enzyme activities are therefore implicated in a number of human infirmities,
including cancers, metabolic disease and neurodegeneration. Fortunately, HDAC enzymes represent
attractive pharmacological targets because they are readily tractable with small molecule inhibitors. In
fact, several HDAC inhibitors (HDACi) have recently proceeded through (or are near) the FDA approval
process for the treatment of hematologic malignancies. However, the promise of clinical HDACi
therapy has been hampered by significant dose-limiting toxicities. These off-target effects have led to a
renewed focus on basic HDAC biology and the development of isoenzyme-specific HDAC inhibitors
which could avoid off-target effects. To help facilitate the discovery of compounds with better defined
selectivity profiles, we have developed lysine deacetylase assays that selectively measure specific
isoenzyme activities in cells, extracts, or purified recombinant preparations. These assays are based on
substrates that are selective due to a combination of extended peptide sequence and novel chemical
modifications. Deacetylase activity is measured by delivering a single, pro-luminogenic, homogeneous
assay reagent to assay wells, resulting in luminescence proportional to HDAC activity. In addition to
being isoenzyme selective, these novel substrates are cell permeable allowing for lytic and non-lytic
cell-based HDAC assays. Lastly, these assays are also fully compatible with fluorescent viability and/or
cytotoxicity assays. This provides additional flexibility for multiplexed formats which examine not only
selective HDAC inhibition, but the functional consequences they exert on cell health.
Presented by: Niles, Andrew L.
157
Poster 86
IDENTIFICATION OF NEW TERT GENE MUTATIONS IN FAMILIAL STUDIES OF PATIENTS PRESENTING
WITH APLASTIC ANEMIA/HYPOCELLULAR MYELODYSPLASTIC SYNDROME
Valeria Nofrinia,*, Valentina Pierinia, Francesco Berardinellib, Caterina Matteuccia, Valeria Di Battistaa,
Antonella Sgurab, Tamara Iannottia, Alessandro Brozzic, Roberta La Starzaa, Cristina Mecuccia
a
Haematology and Bone Marrow Transplantation Unit, University of Perugia, Perugia, Italy
Department of Science, University ‘Roma Tre’, Rome, Italy
c
Department of Medicine, Section of Gerontology and Geriatrics, University of Perugia, Perugia, Italy
b
We describe new familial TERT gene mutations in 3 patients with diagnosis of severe aplastic anemia
(AA) or hypocellular Myelodysplastic Syndrome (h-MDS). Pedigrees were obtained in all cases. A family
history of telomere syndrome was documented in 2 cases. Telomere length was measured by Q-FISH
comparing each proband with at least 3 age- and sex-matched healthy controls.
First patient (female, 53) had severe AA, idiopathic pulmonary fibrosis, liver cirrhosis and died 41
months after diagnosis. She bore a missense mutation (c. 2093 G>A p. R698Q at exon 5) defined as
damaging by in silico analysis (SIFT and PolyPhen-2 databases). Her brother (42 years) bore the same
mutation but clinical effects were significantly different since he presented slight macrocytic anemia
and mild pulmonary fibrosis. Q-FISH revealed, in the brother, significant telomere shortening (mean
T/C%: 10.9 vs 17.4, p<0.05).
Second patient (male, 18 years) presented severe AA and bore a new missense mutation (c. 2020 G>A
p. G674S) at exon 5 defined as benign (SIFT and PolyPhen-2). Q-FISH revealed telomere shortening
(mean T/C%: 18.6 vs 24.6, p<0.05). The patient’s mother, 1 sister and 1 brother had the same exon 5
mutation without phenotype. In these cases telomere length studies are ongoing.
Third patient (female, 53 years) with family background had a stable (+136 months) h-MDS and an
acquired 47,XX,+8 karyotype. Phenotype included idiopathic pulmonary fibrosis, skin hyperpigmentation, osteoarthritis. As compound heterozygous, she bore two germline mutations (nonsense
mutation c.1209C>A p.C403*, exon 2; missense mutation c.2455C>T p.R819C, exon 8). Telomere
length: mean T/C% 9.3 vs. 18.1 (p<0.05). Her healthy 23 year old son bore the exon 2 mutation and
presented telomere shortening (mean T/C% 12.3 vs. 24.1, p<0.05).
All new mutations had a striking different penetrance.
Presented by: Nofrini, Valeria
158
Poster 87
Androgenetic fish as models in studies concerning healing of chromosome breaks by de novo
telomere addition
Konrad Ocalewicz
Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, Poland
Data concerning telomere healing of the chromosome breaks usually derive from the studies using cell
lines exposed to the clastogenic agents including ionizing radiation that damage chromosomal DNA by
inducing double strand breaks (DSBs). Ionizing radiation is also applied to inactivate nuclear DNA in fish
eggs during induced artificial androgenesis in order to produce individuals possessing exclusively
paternal chromosomes. Although X and gamma rays are thought to eliminate entire maternal nuclear
DNA from the subsequent zygotes, we have found many radiation-induced fragments of the maternal
chromosomes in the androgenetic rainbow trout and brook trout embryonic and adult cells.
Distribution patterns of the telomeric hybridization signals on such chromosome fragments enabled to
discriminate their several groups: 1) linear chromosome fragments with telomeric DNA sequences at
both ends, 2) ring chromosomes with fused broken chromosome arms, 3) fragments with intact
telomeres from one arm and fused unprotected sister chromatids of another broken arm and 4)
acentric telomeric fragments incorporated into paternal chromosomes. Our observations proved, the
some of the broken ends of the fish chromosomes could have been repaired with the telomeric DNA
repeats synthesized de novo by telomerase or another mechanism capable of de novo telomere
addition. Easy access to many fish species and availability of the techniques enabling study of the fish
telomeres and telomerase make these organisms to be promising models in researches concerning
healing of the broken chromosomal ends in the embryonic and adult cells.
Presented by: Ocalewicz, Konrad
159
Poster 88
A new role for histone deacetylase 5 in the maintenance of long telomeres
Catherine Polese1,3, Clara Lopes Novo1,2, Nicolas Matheus*, Anabelle Decottignies†,
Arturo Londono-Vallejo‡,§, Vincent Castronovo*, and Denis Mottet3
*University of Liege Sart-Tilman, GIGA-Cancer, Metastasis Research Laboratory, Pathology Institute
B23, Liege, Belgium
†Genetic and Epigenetic Alterations of the Genome, de Duve Institute, Faculty of Pharmacy and
Biomedical Sciences, Catholic University of Louvain, Brussels, Belgium
‡Telomeres and Cancer Laboratory, Labellis Ligue Contre le Cancer Institut Curie, Paris, France
§Université Pierre et Marie Curie (UPMC), University of Paris 06, Paris, France
1These authors contributed equally to this work
2Current Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge,
UK
3Laboratory of Protein Signaling and Interactions, GIGA-Research, B34, Liege, Belgium
Telomeres are major regulators of genome stability and cell proliferation. A detailed understanding of
the mechanisms involved in their maintenance is of foremost importance. Of those, telomere
chromatin remodeling is probably the least studied; thus, we intended to explore the role of a specific
histone deacetylase on telomere maintenance. We uncovered a new role for histone deacetylase 5
(HDAC5) in telomere biology. We report that HDAC5 is recruited to the long telomeres of
osteosarcoma- and fibrosarcoma-derived cell lines, where it ensures proper maintenance of these
repetitive regions. Indeed, depletion of HDAC5 by RNAi resulted in the shortening of longer telomeres
and homogenization of telomere length in cells that use either telomerase or an alternative
mechanism of telomere maintenance. Furthermore, we present evidence for the activation of
telomere recombination on depletion of HDAC5 in fibrosarcoma telomerase-positive cancer cells. Of
potential importance, we also found that depletion of HDAC5 sensitizes cancer cells with long
telomeres to chemotherapeutic drugs. Cells with shorter telomeres were used to control the specificity
of HDAC5 role in the maintenance of long telomeres. HDAC5 is essential for the length maintenance of
long telomeres and its depletion is required for sensitization of cancer cells with long telomeres to
chemotherapy.
Presented by: Polese, Catherine
160
Poster 89
Unusual telomeres in plants
Vratislav Peska, Jiri Fajkus
CEITEC MU
Telomeres are physical ends of eukaryotic chromosomes that (i) compensate the loss of DNA due to
incomplete replication, (ii) differ chromosomal ends from dsDNA breaks, (iii) prevent inappropriate
recombination and end-fusions. These features are conditioned by the presence of telomere proteins
interacting in a complex called shelterin. Telomerase - reverse transcriptase - plays major role in
telomere length maintenance. Thanks to telomerase activity, telomeres consist of minisatelites with
repetitive units type 5'-(TxAyGz)n-3 '. But more and more exceptions has been found with altered or
unknown terminal DNA sequence, where telomerase activity is replaced by mechanism of alternative
lengthening of telomeres (ALT) (e.g., Diptera, some laboratory yeast, some tumors and plants).
Dicotyledonous plants of the Solanaceae family (Vestia, Sessea and Cestrum) and the
monocotyledonous of the family Alliaceae (e.g. A. cepa and A. sativum) are an excellent opportunity to
study how the eukaryotic cell cope with loss of telomerase system without loss of integrity and
evolutionary perspective.
Alternative telomeric sequence in plants remains unknown despite its importance. We have sequenced
two Allium species to compare their repetitive DNA and to obtain telomeric candidates.
This work was supported by CEITEC (CZ.1.05/1.1.00/02.0068) and by the European Social Fund
(CZ.1.07/2.3.00/30.0019).
Presented by: Peska, Vratislav
161
Poster 90
A study of telomere-related biomarkers in an arsenic-exposed Bangladeshi population
Brandon Pierce, Shantanu Roy, Farzana Jasmine, Muhammad Kibriya, Habibul Ahsan
University of Chicago
Chronic exposure to inorganic arsenic through contaminated drinking water affects >100 million
people worldwide and can lead to increased risk for various cancers, including lung, bladder, liver, skin,
and kidney. The mechanisms by which arsenic contributes to carcinogenesis are not entirely
understood, but telomere dysfunction has been hypothesized as one potential carcinogenic
mechanism. Supporting this notion, recent experimental and observational research suggests that
arsenic exposure may influence telomere length and telomere maintenance. Our group is currently
conducting a large population-based study of arsenic-exposed individuals in Bangladeshi in order to
characterize the relationships among arsenic exposure (measured in water and urine), various
telomere-related biomarkers, and arsenic-related health outcomes. We are using a case-cohort design
to assess the association of telomere length (measured prospectively in baseline blood samples) with
incident cases of arsenic-induced skin lesions (n=1000), non-melanoma skin cancer (n=250), and overall
mortality (n=500), as compared to control cohort members. Preliminary data emerging from this work
suggests that arsenic exposure is associated with longer telomeres, consistent with a recent
publication on this topic. Analysis of gene expression data for telomere-related genes suggests that
telomere lengthening may occur through the “alternative lengthening of telomeres” (ALT) pathway,
although lengthening via telomerase is also a possibility. As a part of this study, we also plan to
measure several reported biomarkers of telomere dysfunction (CRMAP and chitinase) and assess
associations with arsenic exposure and arsenic-related health outcomes. This ongoing study is a critical
step towards understanding the mechanisms of arsenic toxicity in humans.
Presented by: Pierce, Brandon
162
Poster 91
Long-term particulate air pollution in association with the mitochondrial-telomere axis of ageing
Nicky Pietersa,*, Bram G. Janssena, Karen Smeetsa, Harrie Dewitteb, Bianca Coxa, Michelle Plusquina, Ann
Cuypersa, Tim S. Nawrota
a
b
Hasselt University
Primary Health Care Center Genk
Background: Telomere length and mitochondrial DNA content are markers of ageing and ageingassociated diseases including cardiovascular morbidity. Airborne particulate matter (PM) is associated
with adverse effects on the cardiovascular system, but its effects on markers of ageing is currently
inconclusive. Sirtuin1 down-regulation has been associated with the development of age-related
diseases and might be an underlying mechanism in the etiology of PM-induced health effects.
Aim: The present study examines the association between long-term PM exposure and leukocyte
telomere length and mitochondrial DNA content in elderly. In addition, we examined the intermediateterm effects of Sirtuin1 on this association.
Methods: Among 182 non-smoking elderly, leukocyte telomere length (LTL) was measured using a
quantitative real-time polymerase chain assay (qPCR). Mitochondrial DNA content and gene expression
of Sirtuin1 were measured using qPCR. Annual PM2.5 exposure was calculated for each participant’s
home address using a Kriging interpolation model.
Results: Annual PM2.5 concentrations ranged from 15 to 23 ug/m3. Independent of gender, age, BMI,
socio-economic status and statin use, an annual 5 µg/m3 increase in PM2.5 was associated with a
relative decrease of 23% (95% CI: -43% to -3%) in LTL and a relative decrease of 31% (95% CI: -50% to 12%) in mitochondrial DNA content. Mediation analysis showed Sirtuin1 as an intermediate
mechanism for PM-related effects on markers of ageing.
Conclusion: We found significant effects of PM2.5 exposure on the telomere-mitochondrial axis of
ageing, mediated through a decreased expression of Sirtuin1. The studied targets may have important
roles in the chronic health effects of PM2.5.
Presented by: Pieters, Nicky
163
Poster 92
Mitochondria dysfunction in telomerase zebrafish mutants
Inês Pimenta de Castro, Madalena Carneiro, Miguel Godinho Ferreira
Instituto Gulbenkian de Ciência, Oeiras, Portugal
Different biological processes cooperate progressively to diminish organ function with age. There are
two main molecular theories of ageing: one proposes that short telomeres trigger cell senescence
leading to tissue functional decline; the second postulates that cellular ageing results from the
progressive loss of mitochondrial metabolism.
Zebrafish have restricted telomerase expression and human-size telomeres. We recently showed that
first generation telomerase mutant zebrafish die prematurely with shorter telomeres and displaying
several ageing phenotypes.
We now report that telomerase loss leads to intrinsic mitochondrial abnormalities in proliferative
tissues such as the head kidney and gut. These include increased aberrant mitochondria that are not
capable of respiring, producing ATP or generating ROS. Additionally, we observe a significant activation
of the Akt pathway that results in the inactivation of the FOXO family of transcription factors and
subsequent autophagy impairment.
These data support a model in which telomere shortening leads to mitochondrial dysfunction through
the activation of the Akt/FOXO pathway, thus orchestrating old age phenotypes. We anticipate that
modulation of this pathway may attenuate the consequences of telomere shortening that naturally
occurs with age.
Presented by: Pimenta de Castro, Inês
164
Poster 93
Preclinical assessment of the telomerase inhibitor Imetelstat combined with radiotherapy in an
orthotopic model of human glioblastoma
Sylvain Ferrandona, Céline Mallevalb, Priscillia Battiston-Montagnea, Badia El Hamdania, Radu Bolbosc,
Patrick Manasd, Sergei M Gryaznove, Claire Rodriguez-Lafrassea, Jérôme Honnoratb, Delphine Ponceta,*,
et al.
a
EMR3738, Cellular and Molecular Radiobiology Laboratory, South Lyon Charles Mérieux Medicine
Faculty, F-69921 Oullins, France
b
Team « neuro-oncology and neuro-inflammation », Lyon Neuroscience Research Center, INSERM
U1028/CNRS UMR 5292, Laennec Medicine Faculty, 69372 LYON cedex 08, France
c
CERMEP-imagerie du vivant, Groupement Hospitalier Est, 59 Bd Pinel, 69677 Bron, France
d
UMS 3444 gerland CNRS P.B.E.S, Ecole Normale Supérieure Lyon, BP 7000, 69342 Lyon Cedex 07,
France
e
Geron Corporation, 149 Commonwealth, Menlo Park, CA 94025, USA
Glioblastoma (GBM) is the most frequent and aggressive adult brain tumor. Development of therapies
in GBM faces two obstacles: a high tumoral molecular heterogeneity and the blood brain barrier (BBB)
passage. The presence of a telomerase activity (TA) in most GBM and its level are predictive of a poor
prognosis in GBM. Inhibiting the TA is thus a promising option to treat GBM. TA inhibition also
increases the response to radiotherapy (RT) in other tumors. We thus tested an oligonucleotide
targeting the telomerase (Imetelstat) alone and combined with RT to treat GBM. In vitro, after a shortterm pre-treatment by Imetelstat of the U87MG cell line, we observed an increase in telomeric
damage and in cell death, both potentialized by RT. In vivo, using a murine orthotopic model of human
GBM, µMRI imaging and a protocol easily transferable into clinical practice, we demonstrated that
Imetelstat significantly : (i) inhibits the TA in the center of the tumor, (ii) reduces tumor volume; (iii)
increases tumor response to RT, in terms of tumor volume regression and survival increase. Imetelstat
has proven is efficiency in clinical trials in numerous cancers. These data support its clinical evaluation
combined with RT in human GBM.
Presented by: Poncet, Delphine
165
Poster 94
Human RTEL1 is required for the maintenance of long telomeres
Rosa Maria Porreca, Christian Naucke, Mike Schertzer, Mylène Perderiset, Irena Draskovic, Arturo
Londoño-Vallejo
Telomeres & Cancer laboratory, UMR3244- UPMC, Institut Curie, 26 rue d’Ulm, 75005 Paris
Rtel1, regulator of telomere elongation helicase 1, was initially discovered to be an essential factor for
telomere length maintenance in mice. Further experiments showed that mouse cells lacking Rtel1
display telomeric loss and chromosome aberrations, caused by abnormal replication and inappropriate
resolution of T-loops. It has been also suggested that Rtel1 is implicated in genome replication,
recombination and DNA repair. Interestingly, recent germline mutations in RTEL1 have been found in
patients with Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenital.
However, the precise mechanism of action of this protein in human cells remains largely unknown.
To investigate the role of RTEL1 in human telomere metabolism we perform loss of function
experiments with specific siRNAs and we make use of quantitative-FISH to measure telomere length
after depletion of RTEL1 in a panel of cell lines. We show that down-regulation of RTEL1 induces
telomere shortening exclusively in cells with very long telomeres (HT1080-ST and 1301) whereas the
telomere length distributions in cells with short telomeres (HeLa and HT1080) remain unchanged.
However, we find that chromosome aberrations were present regardless of the telomere length.
Specifically, we observe chromatid/chromosome breaks, fusions and triradial or quadriradial
structures, with activation of the DNA damage response through ATM-CHK2 and γH2AX signaling. Our
recent experiments further demonstrate that upon depletion of RTEL1 there is a different
stoichiometry of sheltering proteins at telomeres. Based on these results we are currently testing the
hypothesis as to whether RTEL1 might be required for the overhang stability.
Presented by: Porreca, Rosa Maria
166
Poster 95
Telomere Repeat Binding (TRB) proteins serve as functional components of telomeres and interact
with telomerase (TERT)
Petra Prochazkova Schrumpfovaa,*, Ivona Vychodilovaa, Martina Dvorackovab, Jana Majerskac, Ladislav
Dokladalb, Sarka Schorovaa, Jiri Fajkusa
a
Mendel Centre for Plant Genomics and Proteomics, CEITEC and Department of Functional Genomics
and Proteomics, NCBR, Faculty of Science, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech
Republic
b
Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ61265 Brno, Czech Republic
c
Current address: Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique
Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland
Although telomeric proteins across the species share many similarities, in vivo data indicating the
existence of a structure similar to mammalian shelterin complex in plants are still very poor. Partial
characterization of a number of candidate proteins has not identified true components of plant
shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from
Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro,
and have been shown to interact with POT1b (Protection of telomeres 1). We discover co-localization
of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction
using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant
telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of
the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic (TERT)
subunits. These findings suggest that TRB proteins are part of the telomeric interactome of A. thaliana.
The research was supported by the Czech Science Foundation (13-06943S), by project CEITEC
(CZ.1.05/1.1.00/02.0068) of the European Regional Development Fund, and project
(CZ.1.07/2.3.00/20.0043) co-financed from European Social Fund.
Procházková Schrumpfová P., Vychodilová I., Dvořáčková M., Majerská J., Dokládal L., Schořová Š. and
Fajkus J. (2014) Telomere Repeat Binding proteins are functional components of Arabidopsis telomeres
and interact with telomerase. The Plant Journal (2014) 77, 770–781
Presented by: Prochazkova Schrumpfova, Petra
167
Poster 96
In vivo selection for new telomerase RNA template regions in S. pombe
Margaret Pruitt1,2, Richard Dannebaum1, Peter Baumann1,2
1
HHMI and Stowers Institute for Medical Research
2
Dept. of Mol. and Int. Physiology, KUMC
The most common telomeric repeat sequence is GGTTAG, present in all vertebrates, and some
invertebrates, fungi, and plant species. This predominance suggests that response to selective
pressures drove many systems to this sequence to protect chromosome ends and solve the end
replication problem. Yet, despite the apparent success of this repeat, different sequence repeats are
found in diverse genera of algae, fungi, and ciliates. Perhaps the most substantial repeat diversity is
found among fungi ranging from perfect 25 nucleotide repeats in Kluyveromyces lactis to highly
heterogeneous repeats in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Whereas
perfect repeat sequences result from faithful reverse transcription of the RNA template, the synthesis
of heterogeneous repeats requires stuttering, slippage or poorly defined boundary elements in the
telomerase RNA subunit. Telomeric DNA sequence diversity is limited by the need for telomerase to
reverse transcribe the sequence in a processive manner, the binding specificity of proteins that bind
single- and double- stranded parts of the telomere, and possibly the requirement to form Gquadruplexes. The diversity of telomeric sequences found among fungi is surprising in light of these
multiple constraints that favor a unique and constant repeat sequence.
We aim to characterize the level of diversity of telomeric sequences that can exist in S. pombe. We
replaced the TER1 template sequence with all 16,384 possible nucleotide combinations and allowed
cells containing this library to grow competitively in liquid culture. Using Illumina sequencing
technology, we followed dynamic changes in the abundance of different templates. Additionally, we
are monitoring the length and sequence of telomeric DNA, the ability of Pot1 and Taz1 to bind and the
extent to which alternative repeats can maintain the full spectrum of telomeric functions. This study
will inform our understanding of the process and functional significance of the co-evolution of the Grich telomere and sequence-specific, telomere-associated proteins.
Presented by: Pruitt, Margaret
168
Poster 97
Effects of dyskeratosis congenita patient mutations in hTERT on telomerase dimerization and activity
Alix Christen, Sophie Redon, Sascha Feuerhahn, Joachim Lingner
EPFL-ISREC
Biochemical experiments and structural analysis by electron microscopy indicated that human
telomerase forms dimers and that it contains two active sites (Sauerwald et al. 2013). Heterozygous
mutations in hTERT cause dyskeratosis congenita. Whether hTERT mutations cause disease because of
haploinsufficiency or whether hTERT mutations may have dominant negative effects has remained
elusive. We now tested whether disease mutations in hTERT impinge on the ability of telomerase to
multimerize and determined whether disease mutant hTERT interferes with wild type hTERT to
catalyze telomere extension when co-assembled into heterodimers. We demonstrate that the
sedimentation coefficient of endogenous telomerase in HEK293T cells is the same as that of
“supertelomerase” that was assembled upon co-overexpression of hTERT and hTR in these cells. By
performing co-immunoprecipitation assays with active sucrose gradient fractions we confirm that
active telomerase is dimeric. We demonstrate that the tested dyskeratosis congenita hTERT mutations
do not impair with the ability of telomerase to multimerize. We purify wild type/disease mutant
telomerase heterodimers and characterize telomerase activity and processivity. Our data indicate that
wild type/mutant telomerase heterodimers have a strongly reduced catalytic activity in comparison to
wild type hTERT homodimers. Thus, hTERT mutations found in dyskeratosis congenita are dominant
negative in heteromeric complexes interfering with wild type hTERT activity.
Sauerwald, A., Sandin, S., Cristofari, G., Scheres, S.H., Lingner, J., and Rhodes, D. (2013). Structure of
active dimeric human telomerase. Nature structural & molecular biology 20, 454-460.
Presented by: Redon, Sophie
169
Poster 98
Quantile regression for flow-FISH telomere length measurement as a diagnostic tool for telomere
diseases
Fernanda Gutierrez Rodrigues, Priscila Santos Scheucher, Edson Zangiacomi Martinez, Bárbara Amélia
Aparecida Santana, Rodrigo Tocantins Calado
Ribeirão Preto Medical School, University of São Paulo
Telomere diseases are a variety of disorders that may clinically manifest as bone marrow failure (BMF),
dyskeratosis congenita, hepatic cirrhosis, and idiopathic pulmonary fibrosis (IPF). Telomere length
measurement has important implications for diagnosis and therapy in telomere diseases. Methods
capable of accurately measuring telomere length contribute to the improvement of diagnostic services.
The goals of this study were evaluate the flow-FISH performance for telomere length measurement
and assess the differential diagnosis contribution of this technique in patients with BMF syndromes
and IPF. For this purpose, telomere length was assessed in peripheral blood leukocytes from a cohort
of 180 healthy Brazilian subjects ranging in age from zero to 92 years. The data obtained fitted a thirdorder polynomial curve (R2 = 0.6099) and showed that telomeres decline with aging (rate of loss = 47
bp/year). The 1st, 10th, 50th, 90th, and 99th percentiles of healthy controls were adjusted to the curve
applying quantile regression. Telomere length was also evaluated in 37 BMF patients and 17 IPF
patients. Thirty-seven percent of individuals with BMF, 7% of sporadic IPF and 66% of familial IPF
patients had short telomeres (<10th percentile of controls). The Bland-Altman agreement analysis
between these methods was high. The mean difference was - 0,172 and the limits of agreement varied
from -2,207 - 1,862. Therefore, for the first time, we were able to measure telomere length of
Brazilians healthy controls and patients with BMF and IPF by flow-FISH. Our results reveal that applying
quantile regression analysis for flow-FISH telomere length measurement is an accurate technique for
the diagnosis of telomere diseases.
Presented by: Rodrigues, Fernanda Gutierrez
170
Poster 99
MTV, a telomeric ssDNA-binding complex, protects Drosophila telomeres and recruits retrotransposon to chromosome ends
yi zhanga, liang zhanga, guanjun gaob, yikang ronga,*
a
b
lbmb, nci, nih
tsinghua university, china
Telomeres are protected by multiple protein complexes. In telomerase-maintained organisms, the
Shelterin complex occupies primarily the duplex region of the telomere while the CST complex binds
the single stranded region. Drosophila uses a transposon-based mechanism for end protection. We
showed that the HipHop-HOAP complex occupies the duplex region. The revelation by the Gatti group
that the Ver protein shares significant sequence similarities with Stn1 suggests that a similar ssDNAbinding complex exists in Drosophila. Using yeast 2-hybrid and recombinant proteins, we identified a
trimeric complex consisting of Ver and Moi, two known capping proteins, as well as TEA, a novel
capping protein that we identified. The Moi-TEA-Ver (MTV) complex purified in vitro binds and protects
ssDNA in a sequence-independent manner. In vivo, MTV components are specifically enriched at
chromosome ends where they prevent telomere fusion. Their telomeric localization is interdependent, and point mutations disrupting MTV interaction in yeast 2-hybrid assays behave as nulls in
Drosophila, consistent with these proteins functioning as a complex in vivo. Interestingly, MTV as a
complex may regulate end-elongation by participating in the recruitment of telomeric transposon RNPs
to chromosome ends. MTV thus shares functional similarities with both the CST and the Tpp1-Pot1
complex in telomerase-maintained organisms, highlighting a possible conserved feature between
telomerase-based and transposon-based telomere protecting mechanisms.
Presented by: rong, yikang
171
Poster 100
Telomere length dynamics and hematopoietic differentiation in human DKC1-mutant iPSCs
Flavia Sacilotto Donairesa,*, Thomas Winklerb, Cynthia Dunbarb, Rodrigo Tocantins Caladoa
a
Department of Genetics, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto, São
Paulo, Brazil.
CTC – Center for Cell-based Therapy, University of São Paulo at Ribeirão Preto Medical School, Ribeirão
Preto, São Paulo, Brazil.
b
Hematology Branch, National Heart Lung and Blood Institute (NHLBI), NIH, Bethesda, Maryland, USA.
X-linked DC is caused by mutations in dyskerin (encoded by DKC1 gene), a protein that provides
stability to the telomerase complex. Induced pluripotent stem cells (iPSCs) have been established as
patient-specific disease models, as patients’ somatic cells can be reprogrammed to a pluripotent state,
maintaining genotypic characteristics. Moreover, the pluripotent capacity of iPSCs enables
differentiation into virtually any cell type. In an attempt to elucidate telomere dynamics in DC, we
derived iPSCs from dermal fibroblasts of a DC patient carrying a DKC1 mutation (A353V substitution) by
forced expression of OCT4, SOX2, KLF4, and MYC using an excisable polycistronic lentiviral vector
(STEMCCA). We analyzed telomere length of three independent iPSC clones up to 40 passages, and
observed telomere shortening during the first 20 passages, followed by length stabilization at a shorter
state than in parental fibroblasts. We differentiated the three DKC1-mutant iPSCs clones towards the
hematopoietic lineage, and observed a marked defect in generation of CD34+/CD45+ hematopoietic
lineage cells. One clone had a shift towards hemogenic endothelial precursors (CD34+/CD31+). Our
findings suggest that the control of telomere length in DKC1-mutant iPSCs is complex, with shortening
as expected in early passages post reprogramming, followed by stabilization over time, suggesting an
alternative pathway to maintain telomere length, and in contrast to findings reported in some previous
studies. Telomere length was not completely recovered after reprogramming, suggesting that full
dyskerin function is necessary for actual telomere elongation during reprogramming. Finally, our
results also suggest that normal dyskerin function is necessary for adequate HSC differentiation.
Presented by: Sacilotto Donaires, Flavia
172
Poster 101
Induced pluripotent stem cells as a model for telomeric abnormalities in ICF type I syndrome
Shira Sagiea,*, Erika Ellrana, Hagar Katzira, Rony Shakeda, Alaa Ghanayimb, Maty Tzukermana, Sara Seliga
a
Laboratory of Molecular Medicine, Rambam Health Care Campus and Rappaport Faculty of Medicine
and Research
Institute, Technion, Haifa 31096, Israel
b
Computer Science Department, Technion, Haifa
32000, Israel
Human telomeric regions are packaged as constitutive heterochromatin, characterized by extensive
subtelomeric DNA methylation and specific histone modifications. ICF (Immunodeficiency, Centromeric
instability, Facial anomalies) type I patients carry mutations in DNA methyltransferase 3B (DNMT3B)
that methylates de novo repetitive sequences during early embryonic development. ICF type I patientfibroblasts display hypomethylated subtelomeres, abnormally short telomeres and premature
senescence. In order to study the molecular mechanism by which the failure to de novo methylate
subtelomeres results in accelerated telomere shortening, we generated induced pluripotent stem cells
(iPSCs) from three ICF type I patients. Telomeres were elongated in ICF-iPSCs during reprogramming,
and the senescence phenotype was abolished despite sustained subtelomeric-hypomethylation and
high TERRA levels. Fibroblast-like cells (FLs) isolated from differentiated ICF-iPSCs maintained
abnormally high TERRA levels, and telomeres in these cells shortened at an accelerated rate, leading to
early senescence, thus recapitulating the telomeric phenotype of the parental fibroblasts. These
findings demonstrate that the abnormal telomere phenotype associated with subtelomeric
hypomethylation is overridden in cells expressing telomerase, therefore excluding telomerase
inhibition by TERRA as a central mechanism responsible for telomere shortening in ICF syndrome. The
data in this study lend support to the use of ICF-iPSCs for modeling of phenotypic and molecular
defects in ICF syndrome and for unraveling the mechanism whereby subtelomeric hypomethylation is
linked to accelerated telomeric loss in this syndrome.
Presented by: Sagie, Shira
173
Poster 102
Studies of G quadruplex-hRPA interactions in the telomere maintenance context
Layal Safaa, Jean-François Rioub, Carole Saintoméb,*
a
Structure et instabilit des g nomes, INSERM U565, CNRS UMR 7196, Mus um National d’Histoire
Naturelle,
43 rue cuvier, 75005, Paris, France
b
UMR 7196, Mus um National d’Histoire Naturelle,
43 rue cuvier, 75005, Paris, France
Telomeres are the terminal protein-DNA complexes of linear eukaryotic chromosomes. They play an
important role in critical processes underlying genome stability, cancer, and aging. Human telomeric
DNA consists of a duplex region composed of TTAGGG repeats, which terminates in a 100–200
nucleotide G-rich single-stranded overhang (G-overhang) able to adopt secondary structures such as tloop and G-quadruplex.
The G-overhang could be elongated in cancer cells through a reverse transcriptase named telomerase
or by alternative lengthening of telomeres. Human Replication Protein A (hRPA) is able to bind and
unfold G-quadruplexes in vitro. A binding model has been proposed suggesting the requirement of a
single-stranded region created at one extremity of the G-quadruplex and due to the equilibrium
between different conformations. Furthermore hRPA can modulate telomerase activity.
In order to validate the previously described G-quadruplex-hRPA binding model, we have analyzed the
interactions of hRPA with different sequences able to form G-quadruplex, using specific small molecule
ligands (G4 ligands) that bind and stabilize the G-quadruplex conformation. We have also realized
photocrosslinking experiments using the 4-thiothymine as probe to determine the nature of subunits
implicated in G-quadruplex hRPA interactions.
In this study, we showed that G4 ligands were able to efficiently inhibit hRPA binding to telomeric Gquadruplex. However, the addition of polyT tails with different length at either the 3’ or the 5’
extremity of the G-quadruplex sequence promotes hRPA binding. The presence of G4 ligands on these
extended G-quadruplex coupled with photocrosslinking experiments permitted to reveal the binding
and opening polarity of RPA.
Presented by: Saintomé, Carole
174
Poster 103
The inhibition of protein-protein interactions within telomeres: a route to cancer chemotherapy?
Twana Salih, Weng Chan, Lodewijk Dekker, Charles Laughton
Division of Medicinal Chemistry and Structural Biology, School of Pharmacy and Centre for
Biomolecular Sciences, University of Nottingham, NG7 2RD
Stable telomeres are critical to the survival of cancer cells; therefore a number of different cancer
chemotherapeutic pipelines have been developed in order to disrupt or destabilize telomeres or
telomerase. One of the newest approaches is the disruption of key protein-protein interactions in the
telomere, such as that between shelterin components TRF1 and TIN2. The goal of this project is to
synthesize and evaluate novel peptide-like molecules, analogues of a key interacting region of TIN2
that can compete effectively for the binding sites on TRF1 and so lead to the destabilization of
telomere structure.
We will report results from a combination of computational and experimental investigations. We will
discuss the application of the MM-GBSA molecular modeling method to predict short TIN2-like
peptides with optimal TRF1-binding affinities. We will describe the synthesis of selected peptides
bearing a fluorescent tag designed to avoid compromising binding affinity. We will discuss the
expression and purification of full-length hTRF1 protein and the development of an in vitro
(fluorescence polarization) assay, with the aim of identifying a lead compound.
Presented by: Salih, Twana
175
Poster 104
Uncapped telomeres as determinant for cell sensitivity to G-quadruplex ligands
Erica Salvati, Angela Rizzo, Sara Iachettini, Pasquale Zizza, Chiara Cingolani, Carmen D'Angelo, Manuela
Porru, Annamaria Biroccio
Experimental Chemotherapy Laboratory, Regina Elena National Cancer Institute, Rome, Italy
Over the past decade, many chemical classes of G-quadruplex (G4) ligands have been described for
their ability to target and damage telomeres, leading to detrimental effects on tumor cells.
The apparent selectivity of G4 ligands towards transformed cells, led us to ascertain the presence of
differences in telomere structures and functions in normal vs transformed cells in a cell model system
of genetically controlled transformation. Among the different comparative evaluations, the analysis of
the telomere status, in interphasic and metaphasic nuclei, revealed a progressive increase in the
number of telomere dysfunction (identified as TIFs) from normal to transformed cells. The majority of
TIFs in interphasic nuclei exhibited the presence of gammaH2AX but not of 53BP1 foci, indicating that
these telomere damages were not-processed by the DNA damage repair pathways. In agreement with
this, gammaH2AX foci were detectable also in metaphase telomeres (meta-TIFs) in transformed cells
and the presence of a basal telomere dysfunction correlated with the drug-induced telomere damage
and cell death. The functional relevance of this observation was directly assessed by showing that
fibroblasts with artificially uncapped telomeres, obtained by depleting components of the telomeric
complex, became sensitive to G4 stabilization. On the other hand, telomeres of normal cells were not
completely resistant to G4 stabilization, resulting damaged upon exposure to higher dosage of G4
ligands as well, and leading to cell death. Anyway the different degree of sensitivity offers a
therapeutic window for a new class of molecules with anti-tumoral features.
Presented by: Salvati, Erica
176
Poster 105
Telomere maintenance mechanisms without telomerase are of clinical relevance in colorectal
carcinoma
Sandra Sampla,*, Stefan Stättnerb, Fabienne Bastianc, Friedrich Wrbad, Brigitte Wolfe, Jeremy Hensonf,
Loretta Laug, Roger Reddelh, Klaus Holzmanna
a
Division of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical
University Vienna, Austria
b
Department of Surgery, Paracelsus Medical University, Salzburg, Austria
c
Department of Surgery, Kaiser Franz Josef-Spital/SMZ-Süd, Vienna, Austria
d
Department of Pathology, University Hospital, Vienna, Austria
e
Department of General Surgery, Medical University Vienna, Austria
f
Prince of Wales Clinical School, Faculty of Medicine, UNSW Australia
g
The Children’s Hospital at Westmead, Sydney, NSW, Australia
h
Children's Medical Research Institute, Sydney, NSW, Australia
Most colorectal carcinomas (CRCs) use telomerase as telomere maintenance mechanism (TMM). The
estimated 10% without telomerase activity (TA) may use alternative lengthening of telomeres (ALT) or
not defined TMM (NDTMM), with unknown clinical importance. This led us to study the potential
association between TMM and outcome for CRC patients.
Tumor and adjacent non-tumor tissues from 68 patients were analyzed for TA and ALT by qPCR-TRAP
and C-circle assay, respectively. Negative qPCR-TRAP results were validated by IP-TRAP. TP53, ATRX,
DAXX and TERT expression and ALT-associated PML bodies were investigated by
immunohistochemistry and TP53 mutations by exon-sequencing.
TA was detected in 90% (61/68) of tumors and 34% (23/67) of non-tumor tissues, with means of 31
and 1.8 total product generated (TPG) units, respectively. ALT and NDTMM were each identified in
6.0% (4/68) of tumors. All four ALT tumors showed either mutated TP53 or loss of ATRX expression.
One ALT tumor additionally showed TA and heterogeneous TERT expression on tumor sections. TP53
was expressed in all cells of this tumor. In contrast, tumors with either TA or ALT showed TERT
expression in all or none of the cells, respectively. Outcome was best when tumors used ALT compared
to TA (p=0.0393) or NDTMM (p<0.0001), with median survival time of >116, 68.3 and 18.4 months,
respectively. Screening a second cohort of 262 patients identified 8 further ALT cases with favorable
outcome.
TA and ALT tumorcells might coexist in CRC patient’s tumors. Our data suggest clinical relevance of
TMM without telomerase as prognostic marker for CRC.
Presented by: Sampl, Sandra
177
Poster 106
Telomere Structure and Maintenance in Trypanosoma brucei
Ranjodh Sandhu, Bibo Li
1Cleveland State University, Center for Gene Regulation in Health and Disease, Dept. of Biological,
Geological, and Environmental Sciences, Cleveland, OH 44115
Telomere recombination is particularly important for Trypanosoma brucei, a protozoan parasite and
the causative agent of human African trypanosomiasis. T. brucei evades the host’s immune response
by regularly changing its variant surface glycoproteins (VSG), and homologous recombination is one of
several important mechanisms for VSG switching. Therefore, abnormal telomere recombination may
affect VSG switching efficiency. So far, the telomere terminal structure in T. brucei is poorly
understood. We have recently adopted adaptor ligation, ligation-mediated primer extension, and
ligation-mediated PCR assays. These assays allowed us to determine the terminal nucleotide on both
strands and the length of the telomere G-overhang. We observed that the majority of T. brucei
telomeres have very short G-overhangs that end in 5’-TTAGGG-3’. In addition, terminal nucleotide of
the C-rich strand is tightly regulated and mostly ends in 5’-ACC. We also identified the RNA component
of T. brucei telomerase, TbTR, and provide phylogenetic and in vivo evidences for TbTR’s native folding
and activity. TbTR is processed through trans-splicing and is a capped transcript that interacts and copurifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of
3–5 bp/PD, which can be rescued by ectopic expression of a wild type allele of TbTR in an apparent
dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted
in corresponding mutant telomere sequences. In addition, we found that the 5’-TTAGGG-3’ ending
telomere G-overhangs are greatly diminished in cells lacking either telomerase or TbKU80, indicating
that T. brucei telomerase contributes greatly to the normal telomere G-overhang structure.
Presented by: Sandhu, Ranjodh
178
Poster 107
A Shelterin-RTEL1 interaction required for maintaining telomere integrity
Grzegorz Sarek, Stephanie Panier, Jean-Baptiste Vannier, Simon Boulton
DNA Damage Response laboratory, London Research Institute, Cancer Research UK, Clare Hall, South
Mimms, EN6 3LD, UK
Telomere homeostasis and DNA repair are fundamental processes, which are vital for genome
integrity. RTEL1 (Regulator of telomere length 1) is an essential DNA helicase that resolves telomere
loops (T-loops) and counteracts telomere fragility, thereby ensuring integrity of the telomere.
Recently, we demonstrated that RTEL1 associates with the replisome through binding to proliferating
cell nuclear antigen (PCNA) via a PCNA interacting protein (PIP) motif. Mutant cells deficient for the
RTEL1-PCNA interaction exhibited telomere replication defects, evident from the accumulation of
fragile sites at telomeres, which we attributed to a failure to unwind telomeric G4-DNA structures. In
contrast, the RTEL1–PCNA complex was dispensable for dismantling T-loops. These data raise the
possibility that RTEL1 engages other interacting partners to resolve T-loops.
We show here that RTEL1 binds directly to Shelterin and this interaction occurs predominantly during
S-phase. We identify key amino acids required for the RTEL1-Shelterin interaction, including specific
residue within Shelterin and conserved residues within RTEL1, some of which are mutated in a subset
of Dyskeratosis congenital (DC) patients. Analysis of mutant cells defective for the RTEL1-Shelterin
interaction revealed inappropriate T-loop resolution accompanied with telomere loss and telomeric
circle formation. In contrast to the RTEL1-PIP box mutant, loss of the RTEL1-Shelterin interaction had
no measurable impact on genome-wide or telomere replication. Finally, we identify a single amino-acid
substitution mutation within Shelterin that abolishes RTEL1 recruitment to telomeres and phenocopies
RTEL1 deficiency. These findings define a critical RTEL1-Shelterin interaction, which is required to
promote T-loop disassembly during S-phase and is compromised in a subset of RTEL1 DC patients.
Presented by: Sarek, Grzegorz
179
Poster 108
Associations between five-factor model of personality and leukocyte telomere length in elderly men
and women – the Helsinki Birth Cohort Study (HBCS)
Katri Savolainena,*, Johan Erikssonb, Eero Kajantieb, Anu-Katriina Pesonena, Katri Räikkönena
a
Institute of Behavioural Sciences, University of Helsinki, FINLAND
Diabetes Prevention Unit, Department of Chronic Disease Prevention, National Institution for Health
and Welfare, Helsinki, FINLAND
b
Introduction
Personality traits have been associated with longevity, and morbidity and mortality of cardiometabolic
diseases and mental disorders. The underlying mechanisms are not fully understood. Accelerated
cellular aging may play a role in this process. We studied if personality traits in later adulthood, as
defined in the five-factor model, are associated with a biomarker of cellular vitality, leukocyte telomere
length (LTL).
Methods
At a mean age of 63.4 (SD = 2.8) years, 1671 (742 men, 929 women) participants from the Helsinki
Birth Cohort Study, filled in the Neuroticism, Extraversion and Openness Personality Inventory (NEOPI). LTL was measured at a mean age of 61.5 (SD = 2.9) years by using a real-time quantitative PCR
method.
Results
None of the NEO-PI personality traits were significantly associated with the LTL in the analyses of both
sexes combined. In the sex-specific analyses, men who scored higher on agreeableness (p = .016) or
lower on neuroticism (p= .040) and women who scored lower on agreeableness (p = .016) had shorter
LTL.
Conclusions
FFM dimensions of personality were not associated with LTL in a sample of elderly individuals. Our
findings suggest that LTL, a biomarker of cellular aging, may not offer insight into the associations
between personality, longevity and health.
Presented by: Savolainen, Katri
180
Poster 109
A mouse model for ICF syndrome does not recapitulate the telomeric phenotype of human ICF
syndrome patients
Gal Laroma, Shiran Yehezkela, Robert J. Duszynskib, Lucy A. Godleyb, Claire Francastelc, Guillaume
Velascoc, Sara Seliga,*
a
Rambam Health Care Campus and
Rappaport Faculty of Medicine and Research Institute, Technion
b
Department of Medicine, Section of Hematology Oncology, The University of Chicago
c
Centre Epigénétique et Destin Cellulaire, Université Paris
Mutations in the human DNA Methyltransferase 3B (DNMT3B) gene result in autosomal-recessive ICF
(Immunodeficiency, Centromeric instability, Facial anomalies) type I syndrome. The subtelomeres in
these patients are abnormally hypomethylated and subtelomeric-hypomethylation is associated with
high levels of TERRA, accelerated telomere shortening and premature senescence. With the aim of
establishing a model system for studying the in vivo consequences of hypomethylated subtelomeres,
we characterized the telomeric phenotype in mouse embryonic fibroblasts (MEFs) carrying human ICF
type I mutations.
Mouse subtelomeric regions in ICF-MEFs were only slightly hypomethylated in comparison to wild-type
(WT) subtelomeres. Also dissimilar to human ICF cells, telomere-FISH analysis did not reveal missing
signals, TRF analysis demonstrated no significant difference in the mean telomere length and TERRA
levels were extremely low. We found normal levels of telomere sister chromatid exchange and no
colocalization of PML bodies with telomeres. 3D analysis of centromeric and telomeric-nuclear
positioning indicated that the position of telomeres, but not centromeres, differed significantly
between WT and ICF MEFs, however human WT and ICF fibroblasts demonstrated similar nuclear
positioning of these chromosomal regions.
Our studies indicate that subtelomeric methylation, which plays an important role in telomere length
and function in human cells, is a less significant player in the generation of the proper
heterochromatinization of mouse telomeres. To this end, ICF-MEFs do not exhibit the main telomeric
phenotypes observed in human ICF cells and therefore are not a good surrogate model for studying
this phenotypic aspect of human ICF syndrome.
Presented by: Selig, Sara
181
Poster 110
Dissecting Alternative Lengthening of Telomeres in the Nematode Caenorhabditis elegans
Beomseok Seoa,*, Chuna Kima, Mark Hillsb, Sung Sanghyuna, Stephane Flibottec, Donald Moermanc, Lee
Junhod
a
School of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National
University, Seoul, Korea, 151-742
b
Terry Fox Laboratory, BC Cancer Agency, Vancouver V5Z 1L3, Canada
c
Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada
d
School of Biological Sciences and Institute of Molecular Biology and Genetics, Seoul National
University, Seoul, Korea, 151-742; Department of Biophysics and Chemical Biology, Seoul National
University, Seoul, Korea 151-742
Eukaryotes have linear chromosomes, which causes the 'end-replication problem'. With consecutive
rounds of DNA replication, the ends of the chromosomes cannot be completely replicated due to the
inability to prime the lagging strand. One way to solve this problem is to use a reverse transcriptase
telomerase, which adds telomeric repeats to the end of the chromosomes de novo. However, it has
also been found that telomeres can be maintained without telomerase in yeast and some cancer cells.
This is called Alternative Lengthening of Telomere (ALT) mechanism, which is proposed to be based on
a process involving recombination although exact mechanisms are largely unknown. In the nematode
Caenorhabditis elegans, the telomerase deficient trt-1(ok410) mutant strain becomes sterile after
several generations as a result of shortening of telomeres (mortal germline phenotype). We
mutagenized trt-1 mutant worms and isolated suppressor mutants whose mortal germline phenotype
was suppressed. The transgenerational lifespan of these mutants are fully recovered. The Terminal
Restriction Fragment (TRF) assays and fluorescent in situ hybridization (FISH) revealed that telomeres
of the suppressor mutants were lengthened and had distinct properties from wild type telomeres.
Whole genome sequencing revealed that the telomeres of suppressor mutants were amplified with
variant repeats originated from either internal telomere-like repeats or subtelomeric DNA. N2 and
CB4856 wild isolates of C. elegans seem to have different hot spots for telomere recombination. We
are currently seeking candidate genes responsible for ALT mechanism in C. elegans. These results
suggest that C. elegans ALT mechanism has a new mode of template selection, which can be
differentially regulated in distinct genetic backgrounds.
Presented by: Seo, Beomseok
182
Poster 111
Subtelomeric elements of the shortest telomere are required for TERRA expression and buffer
senescence onset
Kamar al zaman Serhala,*, Marco Grafb, Pascale Joliveta, Maria Teresa Teixeiraa, Brian Lukeb
a
Institut de Biologie Physico-chimique, Laboratoire de Biologie Moléculaire et Cellulaire des
Eucaryotes, UMR8226 CNRS/UPMC
b
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches
Krebsforschungszentrum (DKFZ)-ZMBH Alliance, Heidelberg, Germany.
Despite their heterochromatic state, telomeres express telomeric repeat containing RNAs (TERRA). In
telomerase negative pre-senescent cells, TERRA RNA-DNA hybrids were shown to promote homologydirected repair (HDR) at telomeres. In a parallel study, it was determined that the shortest telomeres in
senescing cells, are more prone to undergo processing events related to HDR. Taken together, we set
out to determine whether critically short telomeres were subject to increased levels of HDR due to the
up-regulation of TERRA and hence RNA-DNA hybrids at short telomeres.
Here, we have employed the FLP-FRT system to engineer a yeast strain in which one specific telomere
is the shortest telomere in the cell. We compared two variants of an inducible Very Short Telomere
(VST), one where the endogenous subtelomere was left intact (VST-6R) and one where the
subtelomere was removed, preventing TERRA expression (VST-7L). We found that upon telomerase
deletion and shortening of the engineered telomere, the VST strain lacking subtelomeric elements
senesced at a significantly increased rate compared to the strain containing the non-modified VST
subtelomere. This shows that the subtelomeric elements of the shortest telomere delay senescence.
Strikingly, TERRA levels were significantly increased at the VST-6R, supporting the idea that
transcription at telomeres is derepressed as telomeres shorten in the absence of telomerase.
Moreover, we detect an increase of RNA-DNA hybrids at this telomere. We propose a model whereby
TERRA transcription is increased at critically short telomeres. This generates RNA-DNA hybrids, which
in turn promote HDR at the shortest telomeres, delaying the senescence onset.
Presented by: Serhal, Kamar al zaman
183
Poster 112
Telomere alterations and chromosome segregation defects induced by prolonged oxidative stress
treatment
Elisa Coluzzia, Rossella Buonsantea, Anthony J. Asmarb, Kelley L. Millerb, Daniela Ciminib, Antonella
Sguraa
a
b
University of Roma Tre, Department of Science, V.le G. Marconi, 446, 00146, Rome, Italy
Virginia Tech, Department of Biological Sciences, 1015 Life Science Circle, Blacksburg, VA, 24061, USA
Telomeres represent an important target of oxidative stress, and oxidative stress-induced damage may
produce telomere length-dependent chromosome mis-segregation. In this study, we investigated the
effects of prolonged oxidative stress on telomere length and chromosome segregation in human
primary fibroblasts (MRC-5) treated daily with 10μM H2O2 and analysed at regular intervals over a 45–
day period. Analysis of telomere length showed initial telomere shortening followed by lengthening in
H2O2-treated cells over the first 20 days of treatment. This cycle also seemed to repeat at later times,
but we focused our subsequent analyses on the first 20 days of treatment. We next investigate
potential mechanisms of telomere elongation, and first performed RTQ-TRAP assay, which did not
reveal any telomerase activity. We then asked whether telomere elongation may be induced by the
ALT mechanism. To this end, we assessed two ALT markers: telomeric-sister chromatid exchanges (TSCE) and colocalization of telomeres with PML proteins. Both these ALT markers were observed at
higher frequencies in H2O2-treated cells compared to untreated cells at the 15 day time point, which
corresponded to the time of observed telomere elongation. Finally, live- and fixed-cell experiments
showed a H2O2-induced increase chromosome bridges at 5 days of treatment and a reduction at 15
days, corresponding to the observed telomere shortening and elongation, respectively. –Taken
together, these data suggest that oxidative stress initially induces telomere shortening and anaphase
chromosome bridges in human primary fibroblasts. Under these conditions, these cells activate the ALT
mechanism, which restores telomere length and suppresses chromosome bridge formation.
Presented by: Sgura, Antonella
184
Poster 113
Development of a high-throughput, fluorescence-based, PCR-free method of C-circle detection to
screen drugs and genes targeting the alternative lengthening of telomeres (ALT) pathway in human
cancers
David Halvorsen, Haroldo Silva
SENS Research Foundation
While most cancers rely on telomerase for immortalization, a significant proportion of tumors show no
detectable telomerase activity. These cancers often use a homologous recombination-based pathway
known as alternative lengthening of telomeres (ALT). However, no ALT-specific targets or therapies are
currently available partially due to a lack of robust assays that quantitatively measure ALT hallmarks.
Here we develop and validate a fast, high-throughput version of the established C-circle assay used to
identify ALT-positive cancer cells and tumors. ALT causes abnormal telomere metabolism that
generates linear and circular extra-chromosomal telomeric repeats, including partially double-stranded
C-rich (CCCTAA)n circles or C-circles (CC). ALT activity is strongly correlated with and proportional to CC
levels, which are measured by rolling circle amplification (RCA) using phi29 DNA polymerase. Currently,
CC assays often take a few days to complete and use radioactive or chemiluminescent probes on DNAspotted membranes or complex PCR techniques that make them incompatible with high-throughput or
clinical diagnostics. Our CC assay adapts established fluorescence-based methods previously validated
to detect telomerase activity to quantitatively measure CC in human ALT cells from multi-well plates.
We validated our assay using drugs and siRNA treatments previously established to regulate ALT
activity. Unlike current CC assays, our method generates complete results in one hour post RCA or 8
hours including genomic DNA isolation. Our assay also has a broader linear response than conventional
methods without loss of sensitivity. This novel CC detection platform enables large-scale screening of
drugs and genetic targets for a functional role on the ALT mechanism.
Presented by: Silva, Haroldo
185
Poster 114
TERT Promoter Mutations Are a Major Indicator of Poor Outcome in Differentiated Thyroid
Carcinomas
Paula Soaresa,*, Miguel Melob, Adriana Gaspar da Rochaa, João Vinagrec, et al.
a
IPATIMUP and Medical Faculty, University of Porto, Portugal
Departments of Endocrinology, Diabetes, and Metabolism and Unit of Endocrinology,Faculty of
Medicine, University and Hospital Center of Coimbra
c
IPATIMUP and ICBAS, University of Porto, Portugal
b
Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid
carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers.
We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed
in five University Hospitals were included. Mean follow-up (±SD) was 7.8±5.8 years. We evaluate the
predictive value of TERT promoter mutations and other clinico-pathological and molecular features
(BRAF, NRAS) for distant metastasization, disease persistence at the end of follow-up and diseasespecific mortality.
TERT promoter mutations were found in 7.5% of papillary carcinomas (PTC), 17.1% of follicular
carcinomas (FTC), 29.0% of poorly differentiated carcinomas and 33.3% of anaplastic thyroid
carcinomas. Patients with TERT-mutated tumors were older (p<0.001) and had larger tumors
(p=0.002). In DTC, TERT promoter mutations were significantly associated with distant metastases
(p<0.001) and higher stage (p<0.001). Patients with DTC harboring TERT promoter mutations were
submitted to more radioiodine treatments (p=0.009) with higher cumulative dose (p=0.004), and to
more treatment modalities (p=0.001). At the end of follow-up, patients with TERT-mutated DTC were
more prone to have persistent disease (p=0.001). TERT promoter mutations were significantly
associated with disease-specific mortality [in the whole FCDTC (p<0.001)] in DTC (p<0.001), in PTC
(p=0.001) and in FTC (p<0.001). After adjusting for age at diagnosis and gender, the HR was 10.35
(95%CI 2.01-53.24; p=0.005) in DTC and 23.81 (95%CI 1.36-415.76; p=0.03) in PTC.
TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse
outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent
prognostic value in DTC and, notably, in PTC.
Presented by: Soares, Paula
186
Poster 115
The role of DAXX and ATRX in telomere maintenance
Zhou Songyanga,*, Quanyuan Heb, Mengfan Tangc
a
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas
77030; and SYSU-BCM Joint Research Center, School of Life Sciences, SYSU, Guangzhou, P.R. China
b
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas
77030
c
SYSU-BCM Joint Research Center, School of Life Sciences, SYSU, Guangzhou, P.R. China
Mutations in the DAXX and ATRX have been found in human pancreatic neuroendocrine tumors and
pediatric glioblastoma, and correlate with the Alternative Lengthening of Telomeres (ALT) phenotype.
DAXX has been shown to interact with ATRX and mediate histone H3.3 deposition. However, the role
of DAXX and ATRX in telomere maintenance and development of ALT-type cancers remains unclear.
We found that endogenous DAXX can localize to Cajal bodies, associate with telomerase, and regulate
telomerase assembly and targeting. Furthermore, disease mutations located in different regions of
DAXX differentially impacted its ability to interact with its binding partners, and its targeting to Cajal
bodies and telomeres. These findings support a DAXX-centric pathway for telomere regulation. To
further understand the function of DAXX and ATRX, we studied DAXX and ATRX protein complexes by
IP mass spec and performed genome-wide Chip-seq analyses. Our data suggested that DAXX and ATRX
have both overlaping and distinct functions in controling chromatin integrity, providing new insights
into cellular mechanisms that regulate telomere stability and ALT.
Presented by: Songyang, Zhou
187
Poster 116
Choice of processing dysfunctional telomeres influences stem cell aging and survival
Tobias Sperka, Satjavani Ravipati, Omid Omrani, Lenhard Rudolph
Leibniz Institute of Age Research, Fritz-Lipmann-Institute
Telomere dysfunction endangers genome integrity, engages the DNA repair machinery and evokes
checkpoint responses including the p53 network. The pool of proliferating stem and progenitor cells is
protected from genomic instability and malignant transformation by checkpoint induction and
depletion of damaged stem cells. Accumulating depletion subsequently impairs organ maintenance
contributing to tissue aging. Our previous data indicates that critically short telomeres induce cell cycle
arrest whereas telomere-fusion evokes apoptotic cell death.
Here, we want to explore the possibility of directing the processing of dysfunctional telomeres in aging
late generation mTerc-/- mice by co-deleting the DNA repair factors 53BP1, H2AX or MDC1,
respectively. The homozygous deletions of genes encoding for inhibitors of DNA end-resection (MDC1,
H2AX) leads to increased activation of p53 in telomere dysfunctional stem cells, telomere shortening,
and chromosomal fusions. The affected mice have shorter lifespan and aggravated organ atrophy. In
contrast, deletion of 53BP1 – an inhibitor of end-resection, which also mediates ligation of DNA ends –
reveals evidence that end-resection per se is not limiting stem cell function. Instead the increase in
chromosomal fusion in response to end-resection is mediating adverse effects of telomere dysfunction
on stem cell maintenance.
Presented by: Sperka, Tobias
188
Poster 117
Evidence for telomerase- and Rad52-independent sequence alterations at yeast telomeres
Clémence Claussin, Michael Chang, Sonia Stinus
European Research Institute for the Biology of Ageing, University of Groningen, University
Medical Center Groningen
In the yeast Saccharomyces cerevisiae, cells lacking telomerase senesce after 60-80 generations but a
small subset of cells can overcome senescence using recombination-mediated mechanisms to become
‘survivors’. Two main types of survivors have been described: type I and type II. Both types re uire
Rad52, a protein necessary for almost all recombination events in yeast. Type I survivors involve the
amplification of subtelomeric elements, while type II survivors resemble the majority of human ALT
cancer cells in that they both exhibit amplification of the terminal telomere repeats. Recombination
proteins are also known to be important in pre-senescent cells, before the formation of survivors.
Telomerase mutants lacking Rad52 exhibit accelerated senescence, but the shortening rate of
telomeres is unchanged. The precise function of recombination proteins in presenescent cells is
unknown. Using a telomere sequencing approach, telomere recombination events have been detected
in pre-senescent cells, although at a much lower frequency than in survivors. Surprisingly, we have
found that these events still occur in rad52Δ mutants and therefore take place in a Rad52-independent
manner. We are currently studying how these events occur as well as their significance with respect to
telomere maintenance.
Presented by: Stinus, Sonia
189
Poster 118
In vivo Interaction of Telomerase, Telomere homeostasis and the Human Papillomavirus Oncogenes
Charis Achilleos, Stella Michael, Katerina Strati
Department of Biological Sciences
University of Cyprus
Human papillomaviruses (HPVs) are responsible for the majority of cervical cancers as well as other
types of epithelial cancers. Carcinogenesis is dependent on the long-term expression of the viral
oncogenes E6 and E7. E6 has been shown to regulate the telomerase enzyme at the transcriptional and
post-transcriptional level and even bind telomeric repeats in vitro. E7 has been shown to induce
alternative lengthening of telomeres (ALT). The multiple ways of regulating in particular the telomerase
enzyme suggest that it represents a crucial target for the virus. It has been speculated that this
regulation may be important to the cancer-causing properties of the virus but also integral to its viral
lifecycle. In order to understand the in vivo implications of the interaction of the HPV oncogene and
telomere homeostasis we have employed mouse models. We are utilizing mice in which the keratin 14
promoter directs the expression of HPV16 E6 or E7 to stratified epithelial tissues of mice. These tissues
are the target of natural infection. The expression of E6 and E7 leads to aberrant differentiation,
increased and unscheduled DNA synthesis, a mobilization of epidermal stem cells, an expansion of
stemness marker expression, formation of tumors etc. We have crossed these mice on a Terc-/background and will present acute phenotypes on the epidermis and their dependence on functional
telomerase enzyme.
Presented by: Strati, Katerina
190
Poster 119
in vivo isolation of telomeric proteins in the nematode Caenorhabditis elegans
Sanghyun Sunga,*, Beomseok Seoa, Junho Leeb
a
Department of Biological Sciences, Seoul National University, Seoul, Korea, 151-742
Department of Biological Sciences &
Department of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea, 151-742
b
Telomeres are special nucleoprotein complexes that cap the ends of the eukaryotic linear
chromosomes to maintain genomic integrity. Many interesting aspects of the telomeres of
Caenorhabditis elegans have been studied; for example, the extension of organismic lifespan by longer
telomeres and alternative lengthening of telomeres at the organismic level have been reported. It is
conceivable that the protein components of the telomeres may have critical roles in these processes as
regulators of length, stability, and yet unknown interesting mechanisms of telomeres. Because most
telomeric proteins found in higher mammals (or even yeasts) do not have homologous partners in C.
elegans, there is a strong need for biochemical approaches to identify them in C. elegans. Previously,
we used a nucleic acid affinity capture method to isolate specific proteins associated with telomeric
DNA. Although this in vitro method was useful, it had limitations for describing all proteins that may
act in the in vivo context. Therefore, in this study, we utilized the recently developed technique called
proteomics of isolated chromatin segments(PICh). PICh uses unique nucleic acid to capture the
endogenous segments of chromatin which are fixed before hybridization, so it can be a direct and
quantitative tool. Currently, we are investigating the telomere-specific proteins of wild type C. elegans.
Presented by: Sung, Sanghyun
191
Poster 120
The DNA-end-replication problem and the establishment of replicative senescence
Julien Soudeta, Emilie Falleta, Pascale Joliveta, Michael Lisbyb, Eric Gilsonc, Maria Teresa Teixeiraa,*
a
Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie PhysicoChimique, UMR8226 CNRS/UPMC
b
Department of Biology, University of Copenhagen
c
Institute for Research on Cancer and Aging, Nice (IRCAN), University of Nice Sophia-Antipolis, CNRS
UMR7284/INSERM U1081, Faculty of Medicine
In the absence of telomerase, yeast telomeres shorten 2.5-5 nt/cell division leading to replicative
senescence. However, the model for telomere shortening is currently incomplete and the exact
contribution of the telomeric 3’-overhang to the shortening rate remains unclear. By following the
dynamics of telomeres during replication at near-nucleotide resolution, we find that the leading strand
synthesis generates blunt-end intermediates before being 5’-resected and filled-in. The shortening rate
is set by positioning the last Okazaki fragments at the very ends of the chromosome. Thus, telomeres
shorten in direct proportion to the 3’-overhang lengths of 5–10 nucleotides which are present in
parental templates. Furthermore, Cdc13 coordinates leading and lagging strand syntheses. Yet, these
telomere shortening “rules” do not apply when telomeres reach a critical length. By tracking individual
telomeres, we show that telomeres are subjected to different pathways depending on their length. We
demonstrate a progressive accumulation of subtelomeric ssDNA through 5’-3’-resection as telomeres
shorten. We also uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the
DNA damage tolerance (DDT) pathway associates with native telomeres. We propose that DDT acts in
a length-independent manner whereas a HR-based repair using the sister chromatid as a template
buffers precocious 5’-3’-resection at the shortest telomeres. Taken together, our data unravel a precise
choreography of telomere replication elucidating the DNA-end-replication problem and provide a new
framework to understand the control of the cell proliferation potential.
Funded by ERC-STG-2010 D-END.
Presented by: Teixeira, Maria Teresa
192
Poster 121
DNA DAMAGE SIGNALLING IN THE RECRUITMENT OF TELOMERASE TO TELOMERES
Adrian Tonga,*, Josh Sterna, Scott Cohena, Xu-Dong Zhub, Tracy Bryana
a
b
Children's Medical Research Institute, Sydney, Australia.
McMaster University, Hamilton, Ontario, Canada.
Telomerase recruitment to telomeres is a cell-cycle regulated process important in determining
telomere length. Understanding this process is important for telomere biology and potential
therapeutic drug design in regulating tumorigenesis.
The yeast homologues of the DNA damage protein ATM is involved in telomerase recruitment, but
ATM’s involvement in telomerase recruitment to telomeres has not been demonstrated in human
cells. TRF1 has been shown to be phosphorylated by ATM, leading to telomere elongation. We
therefore directly tested the hypothesis that ATM mediates telomerase recruitment to the telomere
via phosphorylation of TRF1.
We have employed an hTR/telomere fluorescent in situ hybridisation assay that allows the direct
visualisation of endogenous telomerase to confirm that telomerase is recruited to the telomere at mid
S phase in 293T cells. ATM silencing resulted in decreased telomerase recruitment, as well as a slight
decrease in the ability of telomerase to assemble and form a functional protein complex, while TRF1
knockdown resulted in the deregulation of telomerase recruitment across the cell cycle. Co-knockdown
of TRF1 partially rescued the phenotype of diminished telomerase recruitment resulting from ATM
knockdown alone, demonstrating that these two proteins act in the same pathway. Treatment of cells
with the ATM kinase inhibitor Ku55933 established that the kinase activity of ATM is vital in
determining telomerase localisation to the telomere via TRF1.
We have provided direct evidence that ATM phosphorylation events regulate telomerase recruitment,
assembly and telomere accessibility through TRF1.
Presented by: Tong, Adrian
193
Poster 122
Effects of bilirubin on telomere integrity and dynamics
Anela Tosevskaa,*, Christine Moelzerb, Karl-Heinz Wagnera
a
b
Research Platform Active Ageing, University of Vienna, Austria
University of Vienna, Austria
Bilirubin has long been considered a redundant byproduct of heme catabolism and potentially toxic if
present in high concentrations in human serum. In contrast, mildly elevated bilirubin levels,
characteristic of Gilbert Syndrome, are considered beneficial and are inversely correlated with cancer
and CVD risk as well as the onset of metabolic syndrome. Mechanistically, this double nature of
bilirubin is attributed to its concentration-dependent pro- or anti- oxidant capacity.
Low grade hyperbilirubinemia (≈17,1µM in serum) is proposed to protect cellular macromolecules such
as DNA from damage caused by excess generation of reactive oxygen species (ROS). We hypothesize
that bilirubin also has protective effects against ROS-mediated telomere attrition. Our present study
examines its possible direct or indirect effects on telomere integrity.
To address this issue, we use a cell culture model of healthy primary human PBMCs, treated with
unconjugated bilirubin (UCB) in concentrations physiologically present in the circulation. We use qPCRbased methods to assess telomere length and FPG-sensitive (8-oxo-dG) sites in telomeres.
Furthermore, we evaluate the role of bilirubin in the recruitment of DNA repair mechanisms and
telomere length maintenance under defined stress conditions. We combine these outcomes with flowcytometric cell cycle analysis in order to get an insight of cell survival and functionality following
bilirubin treatment.
Our findings suggest that telomeres could act as mediators in the bilirubin protective machinery
against age-related and metabolic diseases.
Presented by: Tosevska, Anela
194
Poster 123
Single-molecule imaging of RTEL1 in embryonic stem cells
Evert-Jan Uringaa,*, Andrew Robinsonb, Karl Duderstadtb, Antoine Van Oijenb, Peter Lansdorpc
a
European Research Institute for the Biology of Ageing, University Medical Center Groningen,
University of Groningen, The Netherlands
b
Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, The
Netherlands
c
European Research Institute for the Biology of Ageing, University Medical Center Groningen,
University of Groningen, The Netherlands; Terry Fox Laboratory, BC Cancer Research Centre and
Department of Medicine, University of British Columbia, Vancouver, BC
Telomere maintenance and DNA repair are crucial processes that protect the genome against
instability. The essential helicase Regulator of Telomere Length 1 (RTEL1) is required for unwinding
DNA structures that arise during replication, recombination and repair. Substrates for RTEL1 include
secondary structures formed from trinucleotide repeats and G-rich DNA. In addition, RTEL1 promotes
T-loop disassembly and acts during homologous recombination by dismantling the displacement-loop.
Fluorescence imaging can provide crucial information on the behavior of RTEL1 in living cells. Since
overexpression of RTEL1 is toxic to cells, we generated mouse embryonic stem cells (ESCs) in which
endogenous RTEL1 was replaced with a fluorescently-tagged RTEL1 fusion protein (RTEL1-venus). Our
previous attempts to study RTEL1-venus in ESCs have been hampered by the very low expression levels
of endogenous RTEL1. Here we report studies of RTEL1 using single-molecule localization microscopy.
This technique has been used to study replication and repair in bacterial cells and allows studies of
molecular properties of proteins that are not accessible through more traditional microscopy methods.
Upon treatment with the topoisomerase II inhibitor etoposide, we observed a dose-dependent
increase in the number of RTEL1-venus foci. Using single-molecule localization microscopy we
determined that of these foci, 52% contain a single molecule of RTEL1-venus. To gain insight into the
dynamic behavior of RTEL1 molecules, we also analysed the recovery of photobleached foci. We found
that 50% of RTEL1 foci recover fluorescence after photobleaching, with a recovery rate similar to that
of the diffuse signal. This finding suggests that RTEL1 molecules at sites of damage have either fast or
slow exchange kinetics.
Presented by: Uringa, Evert-Jan
195
Poster 124
Insights into telomere protection by Ku heterodimer
Sona Valuchovaa,*, Eliska Janouskovab, Ctirad Hofrb, Karel Rihaa
a
b
Gregor Mendel Institute of Molecular Plant Biology GmbH, Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Central European Institute of Technology, Kamenice 753/5, 601 77 Brno, Czech Republic
Ku is a conserved protein present in all domains of life and viruses. It is involved in DNA double-strand
break repair and telomere protection. Both processes are essential for maintenance of genome
integrity and deficiencies in these processes were associated with human diseases. At first, the role of
Ku in telomere protection in eukaryotic organisms seems counterintuitive, because it mediates fusions
of unprotected telomeres. However, its action as a resection and recombination inhibitor seems to be
beneficial for natural chromosome ends. We are trying to understand the mechanism that prevents
initiation of DNA repair by Ku on telomeres while retaining its end-protective function. In Arabidopsis
thaliana, presence of Ku heterodimer is necessary for integrity of blunt ended telomeres by inhibiting
nucleolytic resection. Such resection takes place after DNA replication to create 3’ G-overhangs in
many organisms. We hypothesize that Ku physically hinders/regulates the telomeric resection by
binding to chromosome ends. To examine the importance of DNA-Ku interaction in telomere
protection, we engineered series of mutant Ku complexes with altered DNA binding properties. We
mutated conserved residues present on DNA interaction surface according to the available crystal
structure. We identified human Ku proteins with in vitro decreased affinity to DNA and increased
incidence on DNA termini. The same mutations of homologous residues had similar effect on
complexes from A. thaliana. Analysis of the mutant complexes in vivo suggests that the DNA
interacting residues are important for telomeric function(s) of the Ku complex.
Presented by: Valuchova, Sona
196
Poster 125
Depression and shortened telomeres: implication on shelterin and telomerase
Yabin Weia,*, Lena Backlundb, Lina Martinssonb, Aleksander A. Mathéc, Gregers Wegenerd, Martin
Schallinga, Catharina Lavebratta
a
Department of Molecular Medicine and Surgery, Neurogenetics Unit, Karolinska Institutet, Stockholm,
Sweden
b
Department of Clinical Neuroscience, Centre for Psychiatric Research and Education, Karolinska
Institutet, Clinic for Affective Disorders, Karolinska University Hospital,
Stockholm, Sweden
c
Department of Clinical Neuroscience, Section for Psychiatry, Karolinska Institutet, Stockholm, Sweden
d
Centre for Psychiatric Research, Aarhus University Hospital, Risskov, Denmark
Eukaryotic telomeres are protective DNA-protein complexes that form the chromosome ends. A
number of studies reported shorter leukocyte telomere length (LTL) to be associated with major
depression, but telomere protective proteins, known as shelterin, has never been investigated.
Further, how long-term lithium treatment protected against telomere shortening remains elusive. In
the hippocampus region, shelterin components, telomerase activity and telomere length (TL) were
assessed: 1) between the Flinders Sensitive Line (FSL), a genetic rat model of depression-like and its
controls, the Flinders Resistant Line (FRL); 2) the FSL rats before and after dietary lithium treatment for
3 weeks. In addition, we assessed if the single nucleotide polymorphism (SNP, rs2736100) associated
with depression or depressive episodes in a Swedish population based cohort (PART) and in bipolar
patients (BP) respectively. Finally, by using human whole saliva DNA we compared the TL between
depression patients and healthy controls, and testing the correlation of TL between DNA from whole
saliva and leukocytes. The FSL rats, compared to its counterpart, exhibited significantly downregulation
of Terf2, Rap1,Tert expression and telomerase activity, which were associated with shorter
hippocampal TL. Lithium treatment only rescued the Tert expression and telomerase activity in the FSL.
rs2736100 was associated with depression in PART cohort and increased depressive epidosdes in BP.
TL, tested in whole saliva DNA, was decreased in depression patients compared to the controls, which
positively correlated with leukocytes TL. This is the first study characterizing shelterin in depression-like
rat model and lithium’s mechanism in protecting against telomere shortening.
Presented by: Wei, Yabin
197
Poster 126
Chromosome Instability: Major Events Likely Start in the Telomeres, Stupid
Ted Weinerta,*, Tracey Beyera, Pete Vintona, Rachel Langstona, Margherita Paschinib, Vicki Lundbladb
a
b
University of Arizona
Salk Institute for Biological Studies
We have been studying how large-scale chromosome rearrangements arise. We developed a budding
yeast model system in which loss of a gene (CAN1) allows identification of loss or recombination on a
marker chromosome that is not essential (in a ChrVII disome). Surprisingly, a major event is one in
which an unstable chromosome is formed, probably a dicentric. The dicentric undergoes additional
events to form progeny cells (forming photogenic sectored colonies, if we do say so ourselves) that
contain different chromosome structures. We believe that replication fork error leads to faulty
template switch to form dicentrics. A major site of fusion was proposed to be 403 kb from the
telomere end (on a 1.1Mb chromosome); we proposed this 403 site is "fragile" and where events
initiate.
We now find that the 403 site is NOT where events likely initiate, but rather is where instability events
often resolve. Events appear to initiate, instead, in the telomere; events are up in est2 mutants (5 fold
increase compared to wildtype cels), in cdc13 hypomorphs (13 fold increase) , and in tel1 rad17 double
mutants (>300 fold increase)! (And since, as it’s widely known, at least one of us--TW--loves telomere
people, that events initiate in or near a telomere is a lovely turn of events!) We are re-interpreting a lot
of our earlier data with this telomere-centric view, and propose that replication forks stall in
telomeres, then undergo template switching to form a first dicentric, which rearranges as cells struggle
to divide.
Presented by: Weinert, Ted
198
Poster 127
Structure-Function studies on Cdc13
Sofiane Y. Mersaoui, Raymund J. Wellinger
Université de Sherbrooke
Budding yeast Cdc13p is a key player acting to assure telomere maintenance and genome stability.
Previous studies proposed an architectural organisation with five distinct domains for Cdc13p, which
include four OB-fold domains and one recruitment domain. However, many details on the in vivo roles
of those domains and their potential inter-domain interactions remained elusive. We therefore
constructed tagged versions of the protein where each new allele lacks one of the five proposed
domains. Highlights of the results show that the first 80 amino acids of the OB1 domain contribute to
stabilization of the Cdc13p. We hypothesise that this stabilization occurs after dimerization of Cdc13p.
Second, the data show that the poorly characterized third domain of the protein (putative OB2) is
essential for the viability. Also, Cdc13-ΔOB2 is unable to bind telomeric DNA in vivo and results in a
capping deficiency. We also introduced all these alleles into yeast strains that grow in the absence of
Cdc13p. Although viable, those adapted cells are sensitive to genotoxic drugs; yet an ectopic
expression of Cdc13p in these cells restores normal resistance, providing evidence for a new function
of Cdc13p in resistance to DNA damage. Those adapted cells thus allow an assessment of functions of
cdc13-alleles that lack essential domains and also permit a delineation of the domains involved in the
presumed new function of Cdc13p. We will report our findings with these experiments and integrate
them into the domain organization of Cdc13p that is pertinent to its in vivo functions.
Presented by: Wellinger, Raymund J.
199
Poster 128
The structural organization of the human telomerase
Michael Wildauer, Christina Waldsich
Max F. Perutz Laboratories, Department of Biochemistry and Cell biology, University of Vienna, Austria
Telomerase plays an essential role in the maintenance of chromosome ends. This ribonucleoprotein
complex consists of two major players: an RNA component called telomerase RNA (TR) and the
catalytic subunit termed telomerase reverse transcriptase (TERT). Together they prevent the
shortening of the telomeres during each round of cell division. This problem is also called endreplication problem and was described for the first time by Russian theorist Alexey Olovnikov in the
late 1970s. Despite on-going research for more than 3 decades the exact mechanism of the telomerase
complex is not known. Describing the architecture of human telomerase will be critical to understand
its mechanism. Our research focuses on studying the interface between the human telomerase RNA
(hTR) and the human TERT protein. So far the detailed interaction pattern of hTR and hTERT in the
functional core complex remains to be discovered. Via in vivo UV crosslinking we have been able to
identify several crosslinks within hTR, mainly in the pseudoknot domain and the CR4/CR5 region. These
structural elements have been implicated to be part of the hTERT binding site.
Presented by: Wildauer, Michael
200
Poster 129
Roles of ATRX and H3.3 to assemble a repressive chromatin state at telomeres
Maheshi Udugamaa, Fiona Changa, Lyn Chana, James McGhiea, Philippe Collasb, Jeffrey Mannc, Lee
Wonga,*
a
Monash University, Victoria, Australia
University of Oslo, Oslo, Norway
c
Murdoch Childrens Research Institute, Victoria, Australia
b
Telomeres are specialised ribonucleoprotein complexes found at chromosome ends and they are
absolutely essential for maintaining chromosome and genome stability. The telomere DNA is enriched
for typical heterochromatin marks such as hypoacetylated histone, H4K20me3 (H4 trimethylated lysine
20), H3K9me3 (H3 trimethylated lysine 9) and Heterochromatin Protein 1 (HP1). Telomere DNA is also
hypermethylated. The heterochromatic nature of telomere chromatin is important for its structure and
function. Beside these epigenetic marks, our previous studies show that Alpha Thalassemia Mental
Retardation X-linked (ATRX) and H3.3 are also key regulators of the telomeric chromatin. Deposition of
H3.3 by ATRX and its interacting partner DAXX is essential for chromatin repression at the telomere.
Function of ATRX-H3.3 in maintaining telomere chromatin repression is further demonstrated by
recent studies that show a strong association of ATRX mutation with alternative lengthening of
telomeres (ALT) in human cancers; 90% of ALT cancer cell lines suffering from inactivating mutations of
the ATRX gene and loss of ATRX function in somatic cell hybrids segregated with the ALT pathway.
Here, we will investigate the roles of ATRX and H3.3 functions in controlling DNA replication fidelity
and repressive chromatin assembly at the telomeres. The study is carried out using mouse embryonic
stem cells (ESCs) carrying conditional gene knockout of h3f3a and h3f3b (H3.3a and b genes). We show
evidence that H3.3 is required for assembly heterochromatin enriched with H3K9me3 at telomeres.
Presented by: Wong, Lee
201
Poster 130
A cryptic route to senescence reveals a constitutive protective function of telomerase
Zhou Xua,*, Emilie Falleta, Camille Paolettib, Steffen Fehrmannb, Gilles Charvinb, Maria Teresa Teixeiraa
a
Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, Institut de Biologie PhysicoChimique, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, UMR8226, 13
rue Pierre et Marie Curie 75005 Paris, France
b
Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries 67400 Illkirch Cedex,
France
In the absence of telomerase, eukaryotic cells still undergo divisions but eventually arrest. To study the
dynamics of this transition in Saccharomyces cerevisiae, we used a microfluidics-based approach and
tracked down individual cell lineages from telomerase inactivation to cell death. We found that most
lineages are characterized by a sharp on/off transition from normal cell divisions to an arrested state.
This is consistent with a single event controlling senescence onset when telomere(s) reach a critical
length due to the end-replication problem. To our surprise, we also observed the existence of a cryptic
noncanonical route leading to replicative senescence, which comprises frequent cell cycle delays early
after telomerase inactivation, signalled through Mec1 and inconsistent with gradual telomere attrition.
Rad51 is required in both types of lineages to maintain viability, in an age-independent manner.
Conversely, we quantified the major contribution of telomerase to cell viability when homologous
recombination is compromised, and showed a two-fold reduction in the mortality rate when
telomerase is active. This suggests that in wild-type cells, telomeres are stochastically damaged with a
measurable low frequency, regardless of the end-replication problem, and require telomerase and/or
homologous recombination for repair. This work reveals a constitutive protective role for telomerase
elongation activity, distinct from the rescue of progressively shortened telomeres.
Funded by ERC-STG-2010 D-END
Presented by: Xu, Zhou
202
Poster 131
TPP1 is a substrate for the deubiquitinating enzyme USP7
Ivo Zemp, Patrick Reichenbach, Joachim Lingner
Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne
(EPFL), Switzerland
The shelterin component TPP1 has apparently opposing roles in telomerase regulation. On one hand,
its depletion leads to telomere decapping and telomere extension, indicating that TPP1 is necessary for
telomerase inhibition. On the other hand, TPP1, and more specifically the TEL patch in its OB domain, is
required for efficient telomerase recruitment to telomeres and telomere elongation. How the opposing
roles of TPP1 are regulated remains poorly understood.
To gain further insights into the regulation of TPP1, we performed a yeast two-hybrid screen using the
OB domain of TPP1 as a bait, and identified the deubiquitinating enzyme USP7 as an interacting protein
of TPP1. We show that TPP1 is ubiquitinated at several residues in its OB fold and C-terminal region,
and USP7 deubiquitinates TPP1 both in vivo and in vitro. We observe that TPP1 protein levels are
regulated by the proteasome, but USP7 appears not to be involved in this process, raising the
intriguing possibility that TPP1 ubiquitination and its deubiquitination by USP7 regulate TPP1 functions.
We show that TPP1 ubiquitination is not required for its proper localization, since ubiquitinationdeficient mutants of TPP1 still interact with the shelterin components POT1 and TIN2, and co-localize
with the telomeric marker TRF1. We will report on whether, in contrast, ubiquitination of TPP1
interferes with its binding to shelterin components or recruitment of telomerase.
Presented by: Zemp, Ivo
203
Poster 132
SUMOylation of Tpz1 controls telomerase action in fission yeast
Mansi Garg, Sahar Mansoubi, Resham Lal Gurung, Steve Plumb, Felicity Watts, Alessandro Bianchi
Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton, UK
Previous work had indicated that SUMO controls telomerase action in fission yeast. In a systematic
search for SUMOylated proteins in S. pombe, we have identified Tpz1 as a telomeric factor that is
subject to modification by SUMO. We have identified a single lysine residue in Tpz1 that is targeted by
the modification. Mutation of this lysine leads to telomere elongation in a telomerase-dependent
manner (recombination is not involved). Genetic and phenotypic analysis indicates that Pli1 is the
SUMO E3 ligase primarily active in the modification of Tpz1 and that this single modification on the
Tpz1 protein is largely responsible for the effect of SUMO in the telomerase pathway in fission yeast.
The telomere association of several telomeric proteins in only minimally affected in the SUMOylationdefective tpz1 mutant, with the notable exception of Stn1, which shows diminished telomere binding.
Intriguingly, Stn1/Ten interact with a SUMO-Tpz1 fusion protein, and also with Tpz1 and SUMO
independently, in two-hybrid assays. Our data suggest that SUMOylation down-regulates telomerase in
fission yeast by promoting Stn1/Ten1 action at telomeres through Tpz1, likely through a direct
interaction between SUMOylated Tpz1 and Stn1. These findings uncover an evolutionary conservation
of the regulation of CST function by SUMOylation.
Presented by: Bianchi, Alessandro
204
Participant List
Eric Aeby
Swiss Institute for Experimental Cancer Research
(ISREC), Ecole Polytechnique Fédérale de Lausanne
(EPFL)
[email protected]
Julio Aguado
IFOM - the FIRC Institute of Molecular Oncology
[email protected]
Shawn Ahmed
University of North Carolina
[email protected]
Patrizia Alberti
Museum National d'Histoire Naturelle
[email protected]
Genevieve Almouzni
Institut Curie
[email protected]
Sara Anjomani Virmouni
Brunel University
[email protected]
Helene Antoine-Poirel
Cliniques Universitaires Saint-Luc , de Duve Institute,
UCL
[email protected]
Mary Armanios
Johns Hopkins Univeristy School of Medicine
[email protected]
Nausica Arnoult
The Salk Institute
[email protected]
Rajika Arora
Institute of Biochemistry / ETH Zurich
[email protected]
Steven Artandi
Stanford
[email protected]
Usha Aryal
University of Leicester
[email protected]
Claus Azzalin
ETHZ Zurich
[email protected]
Duncan Baird
Cardiff University
[email protected]
Christian Bär
CNIO
[email protected]
Aurelia Barascu
UMR CNRS 8226 Institut de Biologie
PhysicoChimique
[email protected]
Peter Baumann
HHMI, Stowers Institute for Medical Research
[email protected]
Tara Beattie
University of Calgary
[email protected]
Oliver Bechter
UZ Leuven
[email protected]
Karen Beemon
Johns Hopkins University
[email protected]
205
Petra Bencurova
Masaryk University
[email protected]
Patrick Brest
IRCAN
[email protected]
Carlos Benitez
Spanish National Cancer Research Center (CNIO)
[email protected]
Tracy Bryan
Children's Medical Research Institute
[email protected]
Maname Benyelles
ENS-Lyon, France
[email protected]
Stefano Cacchione
Sapienza University of Rome, Dept of Biology &
Biotechnology
[email protected]
Benjamin Best
Life Extension Foundation
[email protected]
Alessandro Bianchi
Genome Damage and Stability Centre, School of Life
Sciences, University of Sussex
[email protected]
Elizabeth Blackburn
University of California, San Francisco
[email protected]
Maria Blasco
Spanish National Cancer Research Centre
[email protected]
Sophie Bombard
INSERM UMR-S-1007, Universite Paris Descartes
[email protected]
Sumit Borah
University of Colorado Boulder
[email protected]
Joanna Boros
de Duve Institute / Catholic University of Louvain
[email protected]
Simon Boulton
London Research Institute, Clare Hall Laboratories
[email protected]
Maria Isabel Cano
Universidade Estadual Paulista Julio de Mesquita
Filho, UNESP, Biosciences Institute, Genetics Dept.
[email protected]
Chris Caridi
University of California, Riverside
[email protected]
Jaime Carrillo Garcia
Instituto de Investigaciones Biomedicas "Alberto
Sols"
[email protected]
Maria Antonietta Cerone
UCL Cancer Institute
[email protected]
Weihang Chai
Washington State University, USA
[email protected]
Alex Chang
Stanford
[email protected]
Michael Chang
University of Groningen
[email protected]
Razan Charif
AP-HP
[email protected]
206
Pascal Chartrand
Universite de Montreal
[email protected]
Elisa Coluzzi
University of Rome
[email protected]
Liuh-Yow Chen
Institute of Molecular Biology / Academia Sinica
[email protected]
Dimitri Conomos
Children's Medical Research Institute
[email protected]
Julian J.-L. Chen
Arizona State University
[email protected]
Julia Cooper
National Cancer Institute, NIH
[email protected]
Shantanu Chowdhury
CSIR - Institute of Genomics and Integrative Biology
[email protected]
Sandro Cosconati
Second University of Naples
[email protected]
Dmitri Churikov
Marseille Cancer Research Center
[email protected]
Stephane Coulon
CRCM
[email protected]
Alessandro Cicconi
"Sapienza" University of Rome
[email protected]
Jannie De Jong
European Research Institute for the Biology of
Ageing / Laboratory of telomeres and genome
integrity
[email protected]
Francesca Cipressa
University of Rome 'La Sapienza'
[email protected]
Kate Clark
Newcastle University
[email protected]
Veryan Codd
University of Leicester
[email protected]
Marita Cohn
Lund University / Department of Biology
[email protected]
Kathleen Collins
UC Berkeley
[email protected]
Titia de Lange
The Rockefeller Univeristy
[email protected]
Anita De Rossi
University of Padova
[email protected]
Anabelle Decottignies
de Duve Institute / Catholic University of Louvain
[email protected]
Katharina Deeg
German Cancer Research Center (DKFZ)
[email protected]
Erin Degelman
Univeristy of Calgary
[email protected]
207
Jerome Dejardin
IGH CNRS INSERM
[email protected]
Jiri Fajkus
Masaryk University and CEITEC
[email protected]
Salvatore Di Maro
University of Naples "Federico II"
[email protected]
Virginie Faure
Albert Bonniot Institute - University of Grenoble
[email protected]
Aurelie Diman
de Duve Institute / Catholic University of Louvain
[email protected]
Miguel Ferreira
Instituto Gulbenkian de Ciencia
[email protected]
Irena Draskovic
Institut Curie
[email protected]
Ignacio Flores
CNIC
[email protected]
Rachid Drissi
Cincinnati Children's Hospital Medical Center
[email protected]
Sebastian Foersch
Leibniz Institute for Age Research
[email protected]
Yimin Duan
Shanghai Institute of Biochemistry and Cell Biology
[email protected]
Miloslava Fojtova
Masaryk University
[email protected]
Katerina Duskova
University of Alcala
[email protected]
Adam Freund
Stanford
[email protected]
Charlene Emerson
Baylor College of Medicine
[email protected]
Jana Fulneckova
CEITEC MU & Inst. Of Biophysics, ASCR
[email protected]
Harikleia Episkopou
de Duve Institute / Catholic University of Louvain
[email protected]
Sarantis Gagos
Biomedical Research Foundation of the Academy of
Athens Greece (BRFAA)
[email protected]
Jose Escandell Planells
Instituto Gulbenkian de Ciencia
[email protected]
Laura Esteban Lafuente
University of Alcala
[email protected]
Roberto Galletto
Washington University School of Medicine
[email protected]
Maria Garcia Beccaria
Spanish National Cancer Research Center (CNIO)
[email protected]
208
Inbal Gazy
Tel Aviv University, Israel
[email protected]
Adam Harvey
University of Minnesota
[email protected]
Vincent Geli
CRCM Marseille
[email protected]
Makoto Hayashi
The Salk Institute
[email protected]
Eric Gilson
IRCAN
[email protected]
Sandra Henkelman
ERIBA
[email protected]
Marie-Josephe Giraud-Panis
IRCAN
[email protected]
Catarina Henriques
Instituto Gulbenkian de Ciencia
[email protected]
Galina Glousker
The Hebrew University of Jerusalem
[email protected]
Milan Hluchy
Masaryk University
[email protected]
Daniel Gomez
Universidad Nacional de Quilmes
[email protected]
Dirk Hockemeyer
UC Berkeley
[email protected]
Marco Graf
ZMBH
[email protected]
Ctirad Hofr
CEITEC - Masaryk University
[email protected]
Roger Greenberg
University of Pennsylvania
[email protected]
Klaus Holzmann
Institute of Cancer Research, Comprehensive Cancer
Center, Medical University Vienna, Austria
[email protected]
Fernanda Gutierrez
University of Sao Paulo
[email protected]
David Halvorsen
SENS Research Foundation
[email protected]
Lea Harrington
Institute for Research in Immunology and
Cancer/Univ Montreal
[email protected]
Juyeong Hong
Department of Integrated Omics for Biomedical
Science / Yonsei University
[email protected]
Jacqueline Jacobs
The Netherlands Cancer Institute
[email protected]
Eliska Janouskova
Masaryk University
[email protected]
209
Clare Jelinska
Nanyang Technological University
[email protected]
Patrycja Klos
Masaryk Univeristy
[email protected]
Kristi Jensen
Stowers Institute for Medical Research
[email protected]
Elzbieta Kowalska
Max F. Perutz Laboratories (MFPL)
[email protected]
Jana Jureckova
Masaryk University, Faculty of Science
[email protected]
Theresa Kreilmeier
Department for Companion Animals and Horses,
University of Veterinary Medicine Vienna,
Veterinarplatz 1, 1210 Vienna, Austria
[email protected]
Junko Kanoh
Institute for Protein Research, Osaka University
[email protected]
Ahu Karademir
Lund University / Department of Biology
[email protected]
Jan Karlseder
The Salk Institute for Biological Studies
[email protected]
Galina Kartasheva
LLC "Telomerase Activation Sciences"
[email protected]
Christopher Kasbek
University of Cincinnati
[email protected]
Guillaume Kellermann
Universite Paris Descartes
[email protected]
Chuna Kim
Seoul National University
[email protected]
Antonis Kirmizis
University of Cyprus
[email protected]
Martin Kupiec
Tel Aviv University, Israel
[email protected]
Mattia la Torre
Sapienza University of Rome
[email protected]
Peter Lansdorp
European Research Institute for the Biology of
Ageing
[email protected]
Eros Lazzerini Denchi
The Scripps Research Institute
[email protected]
Junho Lee
Seoul National University
[email protected]
Jihoon Lee
Department of Integrated Omics for Biomedical
Science / Yonsei University
[email protected]
Ming Lei
National Center for Protein Science, Shanghai,
Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy
of Sciences
[email protected]
210
Paul Lieberman
The Wistar Institute
[email protected]
Isabella Marcomini
Friedrich Miescher Institute
[email protected]
Joachim Lingner
EPFL
[email protected]
Dries Martens
Universiteit Hasselt
[email protected]
Natalia Lipinska
Department of Clinical Chemistry and Molecular
Diagnostics, Poznan University of Medical Sciences
[email protected]
Paula Martinez
CNIO
[email protected]
Arturo Londono-Vallejo
Institut Curie
[email protected]
Isabel Lopez de Silanes
Spanish Cancer Research Centre
[email protected]
Brian Luke
University of Heidelberg, ZMBH
[email protected]
Vicki Lundblad
The Salk Institute for Biological Studies
[email protected]
Arthur Lustig
Tulane University
[email protected]
Paulina Marzec
Institute of Human Genetics (IGH), CNRS
[email protected]
Aaron Mendez Bermudez
IRCAN
[email protected]
Javier Miralles Fuste
The Salk Institute
[email protected]
Beth Moorefield
Nature Structural & Molecular Biology
[email protected]
Foteini Mourkioti
Stanford University
[email protected]
David Lydall
Newcastle University
[email protected]
Thanchanok Muangman
Thailand Institute of Scientific and Technological
Research
[email protected]
Eva Majerova
Masaryk University, Brno, CZE
[email protected]
Veronika Muchova
CEITEC MU
[email protected]
Jana Majerska
EPFL SV ISREC
[email protected]
Aysel Muezzinler
German Cancer Research Center (DKFZ)
[email protected]
211
Toru Nakamura
University of Illinois at Chicago
[email protected]
Ozgun Ozer
University of Copenhagen
[email protected]
Christine Napier
Children's Medical Research Institute
[email protected]
Raquel Paiva
University of Sao Paulo
[email protected]
Saishyam Narayanan
Lund University / Department of Biology
[email protected]
Lili Pan
Stowers Institute for Medical Research
[email protected]
Ivona Necasova
CEITEC MU
[email protected]
Stephanie Panier
London Research Institute
[email protected]
Nya Nelson
Baylor College of Medicine
[email protected]
Omesha Perera
Children's Medical Research Institute
[email protected]
Bernadette Nera
UC Davis
[email protected]
Vratislav Peska
CEITEC - Masaryk University
[email protected]
Drew Niles
10 - Promega Madison
[email protected]
Verena Pfeiffer
EPFL
[email protected]
Valeria Nofrini
University of Perugia
[email protected]
Hilda Pickett
Children's Medical Research Institute
[email protected]
Konrad Ocalewicz
Univeristy of Warmia and Mazury in Olsztyn, Poland
[email protected]
Brandon Pierce
University of Chicago
[email protected]
Galina Orlova
LLC "Telomerase Activation Sciences"
[email protected]
Nicky Pieters
Hasselt University
[email protected]
Jessica Owens
Stowers Institute for Medical Research
[email protected]
Ines Pimenta de Castro
Instituto Gulbenkian de Ciencia
[email protected]
212
Catherine Polese
Laboratory of Protein Signaling and Interactions,
Interdisciplinary Cluster for Applied Genoproteomics
(GIGA-R), University of Liege
[email protected]
Delphine Poncet
Claude Bernard University Lyon1
[email protected]
Rosa Maria Porreca
Institut Curie
[email protected]
Florian Poulain
de Duve Institute / Catholic University of Louvain
[email protected]
Carolyn Price
University of Cincinnati
[email protected]
Petra Prochazkova Schrumpfova
Masaryk University and CEITEC
[email protected]
Margaret Pruitt
Stowers Institute for Medical Research
[email protected]
Robert Radford
The Salk Institute
[email protected]
Grazia Daniela Raffa
Sapienza Universita di Roma
[email protected]
Roger Reddel
Children's Medical Research Institute
[email protected]
Sophie Redon
ISREC / EPFL
[email protected]
Patrick Revy
Imagine Institute
[email protected]
Daniela Rhodes
Nanyang Technological University
[email protected]
Yikang Rong
National Institute of Health
[email protected]
Francesca Rossiello
IFOM - the FIRC Institute of Molecular Oncology
[email protected]
Karl Lenhard Rudolph
Leibinz Institute for Age Research/Fritz-Lipman
Institute Jena
[email protected]
Flavia Sacilotto Donaires
University of Sao Paulo
[email protected]
Mahito Sadaie
Kyoto University
[email protected]
Isabella Saggio
Sapienza University of Rome
[email protected]
Shira Sagie
Technion
[email protected]
Carole Saintome
Museum National d'Histoire Naturelle
[email protected]
Twana Salih
University of Nottingham
[email protected]
213
Erica Salvati
Regina Elena National Cancer Institute
[email protected]
Antonella Sgura
University of Rome
[email protected]
Sandra Sampl
Institut for Cancer Research / Department of
Medicine I / Medical University of Vienna
[email protected]
Yusuke Shima
Kyoto University
[email protected]
Ranjodh Singh Sandhu
Cleveland State University
[email protected]
Grzegorz Sarek
London Research Institute, Cancer Research UK
[email protected]
Sharon Savage
National Cancer Institute, NIH
[email protected]
Katri Savolainen
University of Helsinki
[email protected]
Jens Schmidt
University of Colorado Boulder
[email protected]
Sara Selig
Rambam Health Care Campus and Rappaport Faculty
of Medicine and Research Institute, Technion
[email protected]
Beomseok Seo
Seoul National University
[email protected]
David Shore
University of Geneva
[email protected]
Stefan Sigurdsson
University of Iceland
[email protected]
Haroldo Silva
SENS Research Foundation
[email protected]
Susan Smith
The Skirball Institute NYU SOM
[email protected]
Paula Soares
IPATIMUP
[email protected]
Zhou Songyang
Baylor College of Medicine
[email protected]
Tobias Sperka
Leibniz Institute for Age Research - Fritz Lipmann
Institute
[email protected]
Kamar al Zaman Serhal
Institut de Biologie Physico - Chimique / UMR 8226
[email protected]
Sonia Stinus
ERIBA - European Research Institute for the Biology
of Ageing
[email protected]
Agnel Sfeir
NYU/Skirball Institute
[email protected]
Katerina Strati
University of Cyprus
[email protected]
214
Estelle Suaud
Active Motif
[email protected]
Jean-Baptiste Vannier
Cancer Research UK
[email protected]
Sanghyun Sung
Seoul National University
[email protected]
Hartmut Vodermaier
The EMBO Journal
[email protected]
Maria Teresa Teixeira
Laboratoire de Biologie Moleculaire et Cellulaire des
Eucaryotes, Institut de Biologie Physico-Chimique,
UMR8226 CNRS/UPMC
[email protected]
Leonid Vorslov
LLC "Telomerase Activation Sciences"
[email protected]
Nico Thoma
Friedrich Miescher Institute for Biomedical Research
[email protected]
Kazunori Tomita
University College London
[email protected]
Adrian Tong
Children's Medical Research Institute
[email protected]
Anela Tosevska
University of Vienna
[email protected]
Yehuda (Dudy) Tzfati
The Hebrew University of Jerusalem
[email protected]
Evert-Jan Uringa
European Research Institute for the Biology of
Ageing, University Medical Center Groningen
[email protected]
Yabin Wei
Karolinska Institutet
[email protected]
Ted Weinert
University of Arizona
[email protected]
Mundy Wellinger
Universite de Sherbrooke
[email protected]
Brigitte Wevers
The Netherlands Cancer Institute
[email protected]
Michael Wildauer
University of Vienna, Department of Biochemistry
and Cell Biology
[email protected]
Lee Wong
Monash University
[email protected]
Woodring Wright
UT Soutwestern Med Ctr
[email protected]
Sona Valuchova
Gregor Mendel Institute of Molecular Plant Biology
GmbH
[email protected]
215
Zhou Xu
Laboratoire de Biologie Moleculaire et Cellulaire
des Eucaryotes, Institut de Biologie PhysicoChimique, Centre National de la Recherche
Scientifique, Universite Pierre et Marie Curie
UMR8226
[email protected]
Lifeng Xu
UC Davis
[email protected]
Ivo Zemp
Institute for Experimental Cancer Research, EPF
Lausanne
[email protected]
216
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