The Wellcome Trust Centre for Cell Biology

The Wellcome Trust
Centre for Cell Biology
2015
1
The Wellcome Trust Centre
for Cell Biology
2015
1
Historical Background
The expansion of research in cell biology was planned
in 1992 as a result of the vision of Professor Sir Kenneth
Murray, who was at the time Biogen Professor at
the Institute of Cell and Molecular Biology. A seed
contribution of £2.5 million from the Darwin Trust was
followed by financial commitments from The Wolfson
Foundation, the University and the Wellcome Trust,
allowing construction of the Michael Swann Building.
The majority of research space was earmarked for
Wellcome Trust-funded research. Recruitment, based
on research excellence at all levels in the area of cell
biology, began in earnest in 1993, mostly but not
exclusively, through the award of Research Fellowships
from the Wellcome Trust. The Swann Building was first
occupied by new arrivals in January 1996 and became
“The Wellcome Trust Centre for Cell Biology” from
October 2001. Core funding for the Centre from the
Wellcome Trust was renewed in 2006 and 2011.
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Content
Page
Director’s Report
4
Robin Allshire
6
A. Jeyaprakash Arulanandam
8
Jean Beggs
10
Adrian Bird
12
Atlanta Cook
14
William C. Earnshaw
16
Kevin G. Hardwick
18
Patrick Heun
20
Adele Marston
22
Gracjan Michlewski
24
Hiro Ohkura
26
Juri Rappsilber
28
Kenneth E. Sawin
30
Eric Schirmer
32
Irina Stancheva
34
David Tollervey
36
Philipp Voigt
38
Malcolm Walkinshaw
40
Julie Welburn
42
Outreach and Public Engagement
44
List of Groups
46
Centre Publications 2013 - 2015
52
International Scientific Advisory Board
60
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Wellcome Trust Centre for Cell Biology Director’s Report
Professor David Tollervey
The Wellcome Trust Centre for Cell Biology (WTCCB) is
one of eight UK-based Wellcome Trust funded Centres,
two of which are in Scotland. The 19 research groups
that currently comprise the WTCCB occupy the Michael
Swann Building on the King’s Buildings Campus of
the University of Edinburgh. The Swann Building was
constructed in the mid 1990s as a purpose built centre for
research in molecular cell biology.
The discipline of Cell Biology aims to generate
understanding of the basic building block of all complex
organisms – the cell. Each human contains around 50
trillion cells that, although very small, are immensely
complex and intricately organised. The goal of the
WTCCB is to gain new insights into how cells function at
levels from molecular interactions to complex systems.
During 2014 the WTCCB has made outstanding progress,
with all research groups reporting exciting and innovative
research. The work undertaken in the WTCCB is driven
by the imagination and creativity of the Centre group
leaders and their dedicated teams of researchers.
However, there are major focal themes to our research,
based around the control and mechanism of cell division
and the regulation of gene expression, particularly by
epigenetic mechanisms.
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The chromosomes package and organise the DNA
molecules that hold the genetic information, and are
key structures in both cell division and gene expression.
The Rappsilber group developed a novel protocol
for the purification of DNA-protein complexes from
chromosomes and by combining this with a machine
learning approach they defined the protein composition
of interphase chromosomes in a probabilistic way.
This is a fundamentally new concept in describing the
composition of organelles and sub-cellular structures.
Within each chromosome a specialised region forms
the centromere, which acts as an attachment point
for machinery – termed the mitotic spindle - that will
pull the chromosomes into the daughter cells during
division. A new fission yeast protein, Eic1, which plays an
important role in timing the loading of key factors onto
the centromeres, was identified by the Allshire group.
The mitotic spindle is made up of long structures called
microtubules and structural analyses by the Arulanandam
group elegantly demonstrated the structural basis for
the ability of the Ska complex to bind microtubules in
a conformation independent manner, a property that
may be crucial for mitotic spindle driven chromosome
segregation. The Welburn lab reported that the spindle
shape scales anisotropically with spindle length. This
underlies a universal rule governing spindle shape
during spindle formation, a key feature of cell division.
Through research on the microtubule cytoskeleton, the
Sawin lab uncovered a crucial role for an Mto1/2 complex
in regulating microtubule polymerisation and gained
insights into its architecture and assembly.
Prior to separation, the chromosomes become more
tightly packed and the Hardwick lab identified a novel
proteasome receptor for ubiquitinylated substrates
(Dss1) and a Protein Phosphatase 1 associated complex
with major roles in chromosomal condensation. The
Ohkura lab achieved mechanistic insights, reporting
that the NuRD nucleosome remodelling complex and
the protein kinase NHK-1 are required for chromosome
condensation in oocytes. Exciting work from the
Earnshaw group revealed that a famous proliferation
marker protein, Ki-67, is required for the assembly of the
mitotic chromosome periphery. A subnuclear region,
the nucleolus, is responsible for ribosome synthesis. The
chromosome periphery is rich in nucleolar components
and Ki-67 is required for efficient reactivation of nucleolar
function, and resumption of ribosome synthesis, following
each cell division. A specialised form of cell division,
termed meiosis, is needed to generate gametes with
half the number of chromosomes in the parent cells. The
Marston lab made important and widely reported findings
on meiosis, including showing how a protein called
shugoshin recruits the chromosome organising complex,
condensin, and helps to signal when chromosome
separation has been completed.
A second major research topic involves the posttranscriptional regulation of gene expression. Most
mRNAs are spliced with the removal of internal
intervening sequences. The Beggs group demonstrated
the existence of a checkpoint in budding yeast that
acts to pause the transcribing polymerase to facilitate
co-transcriptional assembly of the splicing complex.
The concept of a splicing-dependent, transcriptional
elongation checkpoint is novel and has wider implications
for control of splicing in more complex organisms.
The microRNAs form an important class of very small,
regulatory RNAs and the Michlewski group reported
that RNA-dependent protein ubiquitination controls the
stability of microRNA precursors. This paradigm-shifting
observation suggests that such RNA-Binding-ProteinModifying enzymes may regulate the assembly and
function of many RNA-protein complexes. RNA-protein
crosslinking coupled to high-throughput sequencing
gave the Tollervey group insights into the maturation
of pre-ribosomes, and mapped the genome-wide
distribution of RNA-DNA duplexes (R-loops), which are
implicated in human disease and genome instability.
A major advance in understanding the function of
a developmentally important RNA binding protein,
nuclear factor 90 (NF90) was made by the Cook lab. They
showed that NF90 adopts a similar RNA recognition
mode to ADAR2, suggesting a role in the regulation
of RNA editing. RNA undergoes several types of posttranscriptional modification, collectively referred to as the
epitranscriptome, which forms an important new field.
At the interface between chromosome structure, RNA
biology and the regulation of gene expression lies the
important, and very topical, field of epigenetics. Two group
leaders joined the Centre during 2014; Patrick Heun, who
was awarded a Wellcome Trust SRF, and Philipp Voigt,
who obtained a Wellcome Trust/Royal Society Sir Henry
Dale Fellowship. Both of these outstanding recruits will
further strengthen our expertise in epigenetics. A direct
link between the epigenome and DNA sequence was
established by the Bird lab who used synthetic CpG islandlike DNA sequences to demonstrate that the function of
CpG island promoters depends on both base composition
(G+C richness) and the density of CpG motifs. In particular,
they demonstrated a causal link between low G+C levels
and DNA methylation. In the past year the Stancheva lab
showed that cellular immortality, but not transformation
with cooperating oncogenes, promotes DNA methylation
at gene promoters and dramatic changes in the
transcriptional output of the genome. The 3D location
of genes within the nucleus is linked to their expression
levels and interactions with the lamina proteins, which
line the inside of the nuclear envelope, are generally
associated with repression. Cells from Emery-Dreifuss
muscular dystrophy patients, which have mutations in the
lamin protein, Emerin, were analysed by the Schirmer lab
who showed that greater growth and differentiation is
associated with the defective lamin. The Walkinshaw lab
has focussed on understanding the structure and allosteric
mechanisms of enzymes in the glycolytic pathway.
Structural insights helped the design of novel inhibitors
of the enzyme phosphofructokinase from Trypanosoma
brucei, the causative agent of sleeping sickness.
I have highlighted only a fraction of the recent
contributions of our researchers to the world-wide
effort to understand cell function, an endeavour that will
ultimately underpin revolutions in the understanding and
treatment of disease. To conclude, let me thank all of our
dedicated researchers and support staff and congratulate
them on their excellent work, which has played the major
role in the continuing success of the WTCCB.
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Robin Allshire
Co-workers: Ryan Ard, Tatsiana Auchynnikava, Pauline Audergon, Emilie Castonguay,
Sandra Catania, Max Fitz-James, Raghavendran Kulasegaran Shylini, Alison Pidoux,
Manu Shukla, Puneet Singh, Lakxmi Subramanian, Nick Toda, Pin Tong, Sharon White
How centromeres are specified: the interplay between heterochromatin, CENP-A
chromatin, and kinetochore assembly.
Most chromosomal DNA is wrapped around nucleosomes
containing core histones H3, H4, H2A and H2B. However,
at centromeres a specific histone H3 variant, known
as CENP-A, replaces histone H3 to form specialized
CENP-A nucleosomes. CENP-A chromatin is critical for
kinetochore assembly; chromosome segregation is
aberrant in cells with defective CENP-A. What determines
where on a chromosome CENP-A is assembled rather
than histone H3? Primary DNA sequence is not absolute
in dictating where active regional centromeres are
formed. Instead, ‘epigenetic’ features provide cues that
promote the assembly of CENP-A chromatin and thereby
provide the foundations to build kinetochores. Our
objective is to understand the nature of these features
and how they mediate the replacement of histone H3
with CENP-A to form active centromeres.
We utilize fission yeast, Schizosaccharomyces pombe,
as a model organism. As in human cells, S. pombe
centromeres are regional, with repetitive elements
packaged in H3 lysine 9 methylation-dependent
(H3K9me) heterochromatin flanking CENP-A chromatin.
Our observations suggest that it is not the DNA sequence
per se that is important for attracting CENP-A, but rather,
the particular environment that the centromere DNA
sequence creates. RNA polymerase (RNAPII) appears to
stall while transcribing centromere DNA. We find that
Selected Publications: Ard, R., Tong, P., Allshire, R.C. (2014) Long noncoding RNA-mediated transcriptional interference of a permease gene
confers drug tolerance in fission yeast. Nature Comms. 5: 5576.
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Cantania, S., Pidoux, A.L., Allshire, R.C. (2015) Sequence features and
stalled transcription within centromere DNA promote establishment of
CENP-A chromatin. PLOS Genetics 11, e1004986
CENP-A is more easily deposited on centromere DNA
when RNAPII stalling is increased. We propose that
persistent RNAPII stalling on centromere sequences
attracts factors that mediate CENP-A incorporation.
Thus centromeric DNA itself may create a permissive
environment for CENP-A chromatin assembly (Figure 1).
Post-translational modifications on histones are frequently
referred to as ‘epigenetic marks’, however, it has not
been definitively demonstrated that H3K9me-dependent
heterochromatin exhibits epigenetic heritability. Fission
yeast has a single H3K9 methyltransferase, Clr4, required
to form all heterochromatin. We investigated if the
H3K9me mark could be maintained on a chromosomal
region where it is not normally found. To artificially H3K9
methylate chromatin we fused Clr4 methyltransferase to
a DNA binding domain that binds within the test region.
As expected, the presence of the resulting H3K9me mark
and heterochromatin turned embedded genes off. When
we removed the Clr4 fusion protein from the region we
found that H3K9me and heterochromatin was maintained
in subsequent generations in cells lacking the putative
Epe1 demethylase. Thus, since H3K9 methylation can
be copied and transmitted through cell division it is a
heritable epigenetic mark (Figure 2).
Audergon, P.N.C.B., Catania, S., Kagansky, A., Tong, P., Shukla, M.,
Pidoux, A., and Allshire, R.C. (2015) Restricted epigenetic inheritance of
H3K9 methylation. Science 348, 132-135.
Figure 1
CENP-A and
deposition factors
Figure 2
Figure 1: Transcriptional stalling promotes CENP-A chromatin assembly. Central domain centromere DNA is transcribed but
when introduced into wild-type cells H3, not CENP-A, chromatin assembles. In TFIIS and Ubp3 null cells high levels of RNAPII,
but low levels of transcript, are detected and CENP-A chromatin is efficiently assembled.
Figure 2: H3K9 methylation can be copied and transmitted through cell divisions, and transgenerationally, in the absence of
a putative counteracting demethylase, Epe1. Tetracycline releases TetR-Clr4 fusion protein from tetO sites at a test locus in
epe1D cells that also express wild-type Clr4 H3K9 methyltransferase.
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A. Jeyaprakash Arulanandam
Co-workers: Bethan Medina, Maria Alba Abad Fernandaz, Tanmay Gupta, Frances Spiller,
Lisse Bausier
Structural Biology of Cell Division
Cell division is a fundamental molecular process of life
that ensures accurate transfer of genetic information
through generations. Errors in cell division often result
in daughter cells with inappropriate chromosome
numbers, a condition associated with cancers and birth
defects. Key events that determine the accuracy of cell
division include centromere specification, kinetochore
assembly, physical attachment of kinetochores to spindle
microtubules and successful completion of cytokinesis.
These cellular events are regulated by a number of
mitotic molecular machines (including Chromosomal
Passenger Complex (CPC), KMN (Knl1-Mis12-Ndc80)
network, Ska complex, spindle checkpoint and the
anaphase promoting complex) involving an extensive
network of protein-protein interactions. We are interested in understanding how specific proteinprotein interactions involved in kinetochore assembly
and function are translated into the regulation of cell
division. Our current focus is on the protein complexes
involved in centromere specification (Mis18 and Mis18associated), the Chromosomal Passenger Complex (CPC),
a key orchestrator of cell division and the Ska complex, an
essential protein assembly required for efficient coupling
of chromosomes to the spindle microtubules. The
specific questions that we aim to address currently
are i) What is molecular basis for the establishment of
Selected Publications: Abad, M. A, Medina, B., Santamaria, A., Zou,
J., Plasberg-Hill, C., Madhumalar, A, Jayachandran, U., Redli, P. M.,
Rappsilber, J., Nigg, E. A. and Jeyaprakash. A. A. (2014). Structural
basis for microtubule recognition by the human kinetochore Ska
complex. Nat Commun 5, 2964. doi: 10.1038/ncomms3964. 8
Jeyaprakash, A. A., Santamaria, A., Jayachandran, U., Chan, Y, W.,
CENP-A nucleosomes at the centromere? ii) What is
the structural/molecular basis for the centromere and
midbody localization of the CPC? and iii) How does
the Ska complex cooperate with the Ndc80 complex to
establish stable kinetochore-microtubule attachments?
The structural and functional insights that we would
obtain from these studies will also allow us to explore
possibilities of targeting specific protein-interactions in
fighting cancer and other mitosis related health disorders. To achieve our goals, we combine structural, biochemical
and cell biological methods. We use molecular biology
and biochemical methods to characterize protein
interactions in vitro, X-ray crystallography, SAXS and
negative staining EM for structural analysis and a
combination of in vitro and in vivo functional assays using
structure based mutants for functional characterization.
Our recent structural and biochemical work on the
microtubule-binding domain of the Ska complex in
combination with crosslinking mass spectrometry
(in collaboration with Juri Rappsibler) and siRNA
rescue experiments (in collaboration with Erich Nigg,
Biozentrum, Basel) elegantly provided the structural basis
for its ability to bind dynamic microtubule structures (both
polymerizing/’straight’ and depolymerizing/’curved’), a
property that may be critical for its role in driving accurate
chromosome segregation (Abad et al., 2014). Benda, C., Nigg, E. A., and Conti, E. (2012). Structural and functional
organization of the Ska complex, a key component of the kinetochoremicrotubule interface. Mol Cell 46, 274-286.
Chan, Y. W., Jeyaprakash, A. A., Nigg, E. A., and Santamaria, A. (2012).
Aurora B controls kinetochore-microtubule attachments by inhibiting Ska
complex-KMN network interaction. J Cell Biol 196, 563-571.
Figure 1. Molecular basis for the establishment and regulation of KT-MT attachments. Cartoon summarizing the MT-binding
modes of the Ska and Ndc80 complexes and their implications in establishing stable, yet dynamic KT-MT attachments (left)
and molecular mechanisms regulating the KT-localization and function of the CPC (Auroro-B-INCENP-Borealin-Survivin) (right).
9
Jean Beggs
Co-workers: Vahid Aslanzadeh, David Barrass, Jim Brodie, Susana De Lucas, Eve Hartswood,
Gonzalo Mendoza-Ochoa, Jane Reid, Ema Sani
Regulation of splicing and functional links between splicing and transcription
Transcription and RNA splicing are at the centre of gene
expression in eukaryotes, controlling gene expression
levels and removing introns from the primary transcripts.
The mechanisms and machineries involved in both
transcription and RNA splicing are highly conserved
throughout eukaryotes, and the budding yeast
Saccharomyces cerevisiae makes an excellent model
system, permitting the application of genetic approaches
in parallel with molecular studies. In addition to
investigating the functions and molecular interactions of
yeast splicing factors, we are interested in links between
RNA splicing and other metabolic processes, especially
transcription. Our approaches include: quantitative
RT-PCR, ChIP-seq, RNA-seq, in vivo RNA-protein crosslinking, biochemical analyses of splicing and molecular
genetics.
To investigate links between transcription and splicing,
we performed high resolution kinetic ChIP experiments
to follow the recruitment of RNA polymerase II (Pol II)
and splicing factors to inducible reporter genes. We
found that, soon after the initiation of transcription, Pol II
pauses transiently near the 3’ end of an intron in response
to splicing. The carboxy-terminal domain (CTD) of the
paused Pol II large subunit is hyper-phosphorylated on
serine 5 and on serine 2, suggesting regulation through
phosphorylation (Alexander et al., 2010). We propose that
Selected Publications: Alexander, R., Innocente, S., Barrass, J.D. and
Beggs, J.D. (2010). Splicing causes RNA polymerase pausing in yeast.
Mol Cell 40, 582-593.
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Hahn, D., Kudla, G., Tollervey, D. and Beggs, J.D. (2012). Brr2-mediated
conformational rearrangements in the spliceosome during
Image of Jean: © Antonia Reeve [email protected]
this represents a novel splicing–dependent transcriptional
checkpoint that may be associated with the quality
control activities of splicing ATPases, such as Prp5
(Figure 1A). This hypothesis is supported by the finding
that mutations (e.g. prp5-1) that block pre-spliceosome
formation cause a transcription defect, with pSer5 Pol II
accumulating on introns (Figure 1B). The U2-associated
Cus2p remains in the defective splicing complex, but
deletion of CUS2 suppresses the transcription defect. We
propose that Cus2p is a checkpoint factor that signals the
status of pre-spliceosome formation to the transcription
machinery (Chathoth et al., 2014). In future work we will
characterise these factors and investigate the mechanism
by which splicing affects transcription and how coupling
these processes benefits gene expression.
To facilitate studies of how other processes affect splicing,
we have developed a procedure for labelling RNA in
vivo with 4-thio-uracil for very short periods, followed
by biotinylation of the thiolated RNA and its affinity
purification. Reverse transcription then produces cDNA of
the newly synthesised transcripts, which can be analysed
by deep sequencing. In this way, short-lived precursor
RNAs can be sequenced during a brief time course of
labelling. Comparing intron and exon reads for each
transcript allows a comparison of the rate of splicing of
different pre-mRNAs (Figure 2).
spliceosome activation and substrate repositioning. Genes Dev. 26,
2408-2421.
Chathoth, K., Barrass, J.D., Webb, S. and Beggs, J.D. (2014) A splicingdependent transcriptional checkpoint associated with pre-spliceosome
formation. Mol Cell 53, 779-790.
Figure 1
A
5ʼ
B
checkpoint
Pol II
sasfied
CTD paused
p
p
pSer5
pSer2
p
p
OK to go
p
p Pol II
intron
Cus2
Prp5
ATPase
pSer5 p
Pol II stalled
p
3ʼ
5ʼ
pre-spliceosome
formed
p Pol
P II
Cus2
Xr 5 1
prp
prp5-1
X
pre-spliceosome 3ʼ
(ATPase mutant)
Figure 1.
Figure 2
Extremely short in vivo labelling with 4-thio-uracil: unparalleled resoluon of splicing kinecs
fast splicing
transcripts
Intron RPKM / Exon RPKM: High
Low
slow splicing
transcripts
1.5 min
2.5 min
5.0 min
steady
state
Figure 2.
Figure 1. Model for a splicing-coupled transcriptional checkpoint associated with pre-spliceosome formation in wild-type cells
(A) or in a prp5-1 mutant (B) (Alexander et al., 2010; Chathoth et al., 2014).
Figure 2. RNA-seq analysis of splicing kinetics. RNA labelled in vivo for 1.5, 2.5 or 5.0 min with 4-thio-U was affinity-purified,
reverse transcribed and sequenced along with steady state RNA. Data clustered as intron RPKM (reads per kilobase per million
reads)/exon RPKM. With time, the intron reads relative to exon reads decline towards the steady state level. Faster splicing
transcripts reach the steady state level faster than slower splicing transcripts.
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Adrian Bird
Co-workers: Beatrice Alexander-Howden, Kyla Brown, Justyna Cholewa-Waclaw,
John Connelly, Dina De Sousa, Jacky Guy, Martha Koerner, Sabine Lagger, Matthew Lyst,
Cara Merusi, Timo Quante, Jim Selfridge, Ruth Shah, Christine Struthers, Rebekah Tillotson
CpG as a genomic signalling module
We study the influence of the short DNA sequence motif
5’CG (also known as CpG) on chromatin structure and
gene expression. The frequency of this two-base pair
sequence is highly variable, being under-represented
in the great majority of the genome, but abundant in
“CpG islands”, which mark the regulatory regions of most
human genes. Our recent work indicates that CpG islands
function as landing pads for proteins that recognise
CpG in the unmethylated state and facilitate chromatin
modification of the N-terminal tails of core histones.
Using synthetic CpG island sequences lacking the ability
to drive transcription, we find that CpG frequency is
indeed the key motif responsible for recruitment of
these chromatin marks. Other CpG island-like features
are also required, however, as a high frequency of CpGs
fails to induce characteristic chromatin modifications if
embedded in DNA that is rich in the bases A and T. In
fact A+T-rich DNA reliably induces DNA methylation,
which blocks addition of other histone marks. This work
establishes that the two prominent shared features of all
mammalian CpG islands – G+C-richness and a high CpG
frequency – are both required for their function.
Methylation of CpG islands, for example on the inactive
X chromosome or at imprinted genes, excludes CpG
binding proteins and allows access by proteins that
specifically interact with methylated CpG. Such methyl-
Selected Publications: Illingworth RS, Gruenewald-Schneider U, De
Sousa D, Webb S, Merusi C, Kerr AR, James KD, Smith C, Walker R,
Andrews R, Bird AP. Inter-individual variability contrasts with regional
homogeneity in the human brain DNA methylome. Nucleic acids
research. 2015.
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CpG binding proteins often associate with transcriptional
corepressor complexes that facilitate the formation
of silent chromatin. CpG islands therefore have the
potential to participate in transcriptional switching
during development. To map their methylation in the
human brain, we used a biochemical method to retrieve
densely methylated DNA from regions of the human
brain followed by high throughput DNA sequencing. We
were surprised to find that most brain regions display
remarkably similar DNA methylation patterns, even when
derived from different cell lineages. The conspicuous
exception is cerebellum, which exhibits many
differentially methylated regions compared with the rest
of the brain. Interestingly these differences conform to a
pattern, as cerebellar DNA with higher or lower levels of
this DNA modification differ sharply in base composition.
These results again suggest that DNA base composition
– long considered to be a passive consequence of
constraints on chromatin structure and metabolism – may
act as a biological signal that affects the structure of the
epigenome. We currently seek mediators of these effects,
which may provide a “missing link” in our understanding
of the function of DNA methylation.
Wachter E, Quante T, Merusi C, Arczewska A, Stewart F, Webb S, Bird A.
Synthetic CpG islands reveal DNA sequence determinants of chromatin
structure. eLife. 2014; 3.
Lyst MJ, Bird A. Rett syndrome: a complex disorder with simple roots.
Nature Reviews Genetics. 2015.
Figure 1
Figure 3
Figure 2
Figure 1. CpG islands (blue dots) are distinct from the rest of the genome (grey dots) in both G+C content (%G+C) and their
observed-over-expected frequency of CpG (CG[o/e]).
Figure 2. Examples of CpG island promoters. CpGs are represented by vertical strokes and red boxes show the first exon only
of the genes concerned.
Figure 3. Base composition of differentially methylated regions (DMRs) in the human cerebellum in comparison with other
brain regions. Blue bars: hypo-methylated; red bars: hyper-methylated.
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Atlanta Cook
Co-workers: Urszula McCaughan, Uma Jayachandran, Valdeko Kruusvee
Structural biology of macromolecular complexes involved in RNA metabolism
Translation is the central process in biology during which
genetic information encoded on mRNAs is read by the
ribosome and polypeptides are synthesized. Extensive
biochemical studies on prokaryotic ribosomes have given
insights into the assembly of this machine, while structural
studies have illuminated its workings during translation.
In eukaryotic ribosomes, two areas where we lack a good
mechanistic understanding are in ribosome biogenesis
and translational control.
Eukaryotic ribosome biogenesis is vastly more
complex than in prokaryotes, requiring more than 200
additional factors and proceeding through multiple
cellular compartments. The process centres around
the transcription of a long ribosomal RNA transcript in
the nucleolus. Chemical modification of bases and the
association of small subunit ribosomal proteins occur at
this early stage. A series of RNA cleavage events then
separates the pre-40S and pre-60S particles, which exit
the nucleus as almost fully assembled pre-ribosomal
particles. These particles associate with late-acting
biogenesis factors that also act as transport adaptors,
mediating interactions with the nucleocytoplasmic
transport machinery. Final maturation is completed
in the cytoplasm where late-acting biogenesis factors
are removed and the mature ribosomal particles can
associate on mRNAs. I am interested in understanding the
mechanistic roles that late-acting ribosomal biogenesis
Selected Publications: Hector R.D., Burlacu E., Aitken S., Le Bihan T.,
Tuijtel M., Zaplatina A., Cook, A.G. and Granneman S. (2014) Snapshots
of pre-rRNA structural flexibility reveal eukaryotic 40S assembly
dynamics at nucleotide resolution Nucl. Acids Res. 42(19):12138-54
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factors play in final maturation in the cytoplasm. I use
structural biology as a tool to understand interactions
between ribosome biogenesis proteins and their
interactions with rRNA.
In addition to factors associated with ribosome
biogenesis, I have a further interest in proteins that
are implicated in post-transcriptional control of gene
expression. The rate of translation of a given mRNA
depends on both its stability and its availability for
translation. The association of regulatory protein
complexes with distinct elements in the 3’UTRs of
cognate mRNAs can have a major impact on the fate
of these mRNPs. Nuclear factors (NF)90 and NF45 are
thought to associate with various mRNAs and to affect
their subsequent translation. I am currently trying to
understand what is the basis of RNA recognition by NF90/
NF45 using structural biology and in vivo approaches.
Wolkowicz UM, Cook AG. (2012) NF45 dimerizes with NF90, Zfr
and SPNR via a conserved domain that has a nucleotidyltransferase
fold. Nucleic Acids Res. 40:9356-68. A
B
Figure 1. (A) Nuclear factors 90 and 45 form a dimeric complex via their N-terminal domains. In addition, NF90 has two dsRNA
binding domains that are connected through a long, unstructured linker. (B) NF45 uses the same binding interface to interact
with two developmentally important proteins that are related to NF90: spermatid perinuclear RNA binding protein (SPNR) and
zinc-finger domain protein interacting with RNA (Zfr). All three of these proteins are likely to be present in neurones but have
different sub-cellular localisation.
15
William C. Earnshaw
Co-workers: Daniel Booth, Mar Carmena, Florence Gohard, Nuno Martins, Oscar Molina,
Diana Papini, Melpi Platani, Jan Ruppert, Itaru Samejima, Kumiko Samejima, Giulia Vargiu,
Laura Wood, Alisa Zhiteneva
The role of non-histone proteins in chromosome structure and function during mitosis
Our studies aim to answer the following three questions:
How do mitotic chromosomes form and segregate
in mitosis? What is the chromatin environment of the
centromere that provides an epigenetic landscape
permissive for kinetochore assembly? How does the
chromosomal passenger complex (CPC) regulate mitotic
events?
In an ongoing collaboration with Juri Rappsilber, we
use Multi-Classifier Combinatorial Proteomics (MCCP)
to determine how depleting key members of important
protein complexes affects the mitotic chromosome
proteome. Our recent studies have focused on the
important nucleolar protein Ki-67. Ki-67 is a very widely
used marker for cell proliferation (over 18,000 PubMed
citations), but its function is unknown. We have confirmed
that Ki-67 is a protein phosphatase 1 targeting subunit,
with the prominent nucleolar protein nucleophosmin/
B23 as one of its substrates. Surprisingly, we found that
Ki-67 is essential for establishment of the chromosome
periphery compartment during mitosis. If Ki-67 is
depleted from cells, the assembly of this entire group of
proteins appears to be defective and nucleolar proteins
aggregate in the cytoplasm instead. We have shown
that the chromosome periphery is not essential for
mitotic chromosome assembly, structure or segregation.
It is, however essential for the efficient reactivation of
nucleolus organising regions during mitotic exit. In the
absence of Ki-67, cells have a single, smaller, nucleolus
and seem to be starved for ribosomal components. As
a result, the cells are smaller, and they appear to have
difficulties in subsequent mitoses.
16
We are using a human synthetic artificial chromosome
to map histone modifications that render chromatin
permissive for kinetochore assembly and function. Our
current studies focus on the roles of both deep (e.g.
mediated by H3 lysine 9 trimethylation) and facultative
(e.g. mediated by the Polycomb system) heterochromatin
at kinetochores. These studies reveal that centromere
chromatin is surprisingly plastic, tolerating the removal
of heterochromatin on the one hand and insertion
of facultative heterochromatin on the other. A critical
balance of transcription is apparently essential for
kinetochore maintenance.
Other studies of mitotic control by the chromosomal
passenger complex have focused on the mysterious
passenger-like protein TD-60 (telophase disk 60 kDa).
Using a synthetic gene, we have been able for the first
time to express this protein in amounts suitable for
biochemistry. These experiments reveal that TD-60 is
a Guanine Exchange factor (GEF) that activates a small
Ras-family GTPase. This activation is required for normal
targeting of the chromosomal passenger complex to
kinetochores and for robust microtubule-kinetochore
attachments to form during metaphase.
Figure 1. Structurel model of the SMC2/SMC4 core of the condensin complex from chicken prepared by template-based
structure prediction constrained by chemical cross-linking coupled with mass spectrometry (CLMS) analysis. SMC2 is shown
in green. SMC4 is shown in blue. Red lines show inter-molecular cross-links used to position the SMC2/SMC4 anti-parallel
coiled-coils relative to one another. The inset shows the length distribution of 105 cross-links represented in this structure,
which includes 1111 residues (85 %) from SMC4 and 1096 residues (92 %) of SMC2. Cross-linking mass spectrometry
by Helena Barysz in collaboration with Juri Rappsilber. Structure prediction by Dietlind Gerloff (Foundation For Applied
Molecular Evolution, Ft Lauderdale, Florida).
Selected Publications: Ribeiro, S.A., P. Vagnarelli and W.C. Earnshaw.
(2014) DNA content of a functioning chicken kinetochore.
CHROMOSOME RES. 22:7-13
Fukagawa. (2014) Histone H4 Lys 20 mono-methylation of the CENP-A
nucleosome is essential for kinetochore assembly. DEV. CELL 29:740749.
Booth, D.G., M. Takagi, L. Sanchez-Pulido, E. Petfalski, G. Vargiu, K.
Samejima, N. Imamoto, C.P. Ponting, D. Tollervey, W.C. Earnshaw* and P.
Vagnarelli*. (2014). Ki-67 is a PP1-interacting protein that organises the
mitotic chromosome periphery. ELIFE 3:e01641.
Carmena, M., M.O. Lombardia, H. Ogawa and W.C. Earnshaw. (2014).
Polo kinase regulates the localization and activity of the CPC in meiosis
and mitosis in Drosophila melanogaster. OPEN. BIOL. 4:140162. PMID:
25376909; DOI: 10.1098/rsob.140162.
Hori, T. 7, W.-H. Shang, 7, A. Toyoda, S. Misu, N. Monma, K. Ikeo, O.
Molina, G. Vargiu, A. Fujiyama, Hi. Kimura, W.C. Earnshaw, and T.
17
Kevin G. Hardwick
Co-workers: Ioanna Leontiou, Karen May, Kostas Paraskevopoulos, Onur Sen, Ivan Yuan
Functional dissection of the spindle checkpoint
We study how the cell biology of mitosis is co-ordinated
with cell cycle progression. The spindle checkpoint
normally prevents cells with spindle or kinetochore
defects from initiating chromosome segregation.
Mutations in the mad (mitotic arrest defective) or bub
(budding uninhibited by benomyl) genes inactivate
the checkpoint and allow cells with defective spindles
to proceed through mitosis. Such cell divisions lead to
inaccurate chromosome segregation, aneuploidy and
death.
A single unattached kinetochore is sufficient to activate
the spindle checkpoint, and we are particularly interested
in how such kinetochores generate signals that delay
anaphase onset throughout the mitotic apparatus. Mad1
recruits and activates Mad2 at kinetochores, and Bub1
recruits Mad3 (Fig.1A). Once activated, Mad3 and Mad2
interact with Cdc20 to form the mitotic checkpoint
complex (MCC) which is crucial for inhibition of the
anaphase-promoting complex (APC/C), and thereby
delays anaphase onset.
A major focus of our recent research was to identify
relevant substrates of the Mph1, Bub1 and Aurora (Ark1)
kinases. Mph1 and Ark1 kinase activities are critical for
Selected Publications: Paraskevopoulos K, Kriegenburg F, Tatham MH,
Rösner HI, Medina B, Larsen IB, Brandstrup R, Hardwick KG, Hay RT,
Kragelund BB, Hartmann-Petersen R, Gordon C. (2014). Dss1 is a 26S
proteasome ubiquitin receptor. Molecular Cell, 56, 453-461.
Shepperd, L.A., Meadows, J.C., Sochaj, A.M., Lancaster, T.C., Zou, J.,
Buttrick, G.J., Rappsilber, J., Hardwick, K.G. and Millar, J.B.A. (2012).
Phosphodependent recruitment of Bub1 and Bub3 to Spc7/KNL1 by
18
spindle checkpoint arrest and we have identified several
important substrates. The kinetochore protein Spc7/KNL1
is phosphorylated by both kinases, and when conserved
MELT motifs are phosphorylated by Mph1 they become
a binding site for the Bub3-Bub1 checkpoint complex
(Fig.1A).
Mph1 kinase also phosphorylates Mad2 and Mad3, and
their modification is necessary to form stable MCCAPC/C complexes and maintain spindle checkpoint
arrests (Fig.1B/C). We employ cross-linking proteomics
to analyse kinetochore, MCC and APC/C complexes to
identify and define novel protein-protein interactions for
functional studies. We have identified two small APC/C
sub-units (Apc15 and Apc14) as key links between MCC
and APC/C. Apc15 is required for stable MCC binding
and checkpoint arrest whereas Apc14 aids efficient MCC
release during checkpoint silencing and mitotic exit.
We are currently employing synthetic biology approaches
to assemble an artificial MCC generator and thereby a
delay in anaphase onset (Fig. 1D).
Mph1 kinase maintains the spindle checkpoint. Current Biology, 22, 891199.
Zich, J., Sochaj, A.M., Syred, H.M., Milne, L., Cook, A.G., Ohkura, H.,
Rappsilber, J., and Hardwick, K.G. (2012). Kinase activity of fission yeast
Mps1 is required for Mad2 and Mad3 to stably bind the anaphase
promoting complex. Current Biology, 22, 296-301.
A
B
Figure 1A. Model for spindle checkpoint
protein recruitment to kinetochores.
C
D
Figure 1B. Mph1 kinase activity is required
for stable binding of Mad2 and Mad3 to
the APC/C. Cells were released into mitosis
(from a G2 block) and then challenged with
an anti-microtubule drug (carbendazim).
The APC/C was purified from extracts
(using Apc4-TAP) and analysed for
associated checkpoint proteins by
immunoblotting.
Figure 1C. Imaging of mitotic fission
yeast cells (septa in black). Strains were
pre-synchronised in G2, released into
mitosis and then challenged with the
anti-microtubule drug carbendazim. This
data demonstrates that the mad2,mad3
phospho-mutants fail to checkpoint arrest,
with a similar phenotype to the mps1-kd
cells, consistent with them being important
Mps1 substrates.
Figure 1D. Synthetic biology approaches
to assemble the mitotic checkpoint
complex. Checkpoint scaffold proteins
(e.g. Spc7, Mad1) are tethered to an array
of tet operators, and MCC is generated
after co-recruitment of activating kinases.
19
Patrick Heun
Co-workers: Vasilik Lazou, Virginie Roure, Georg Schade, Eftychia Kyriacou, Evelyne Barrey,
Eduard Anselm, Thomas van Emden
Epigenetic regulation of chromosome and nuclear organization
All inheritable chromosome conditions not encoded
by the DNA sequence itself are called epigenetic,
including gene expression and for most eukaryotes
also centromere and telomere identity. The epigenetic
transmission of these states through many cell
generations is highly relevant for proper genome
regulation and when perturbed can lead to genome
instability and cellular malfunction. We use the fruit fly
Drosophila melanogaster and human cells as a model
organism to address the following questions:
How is the epigenetic identity of centromeres regulated?
Centromeres are found at the primary constriction of
chromosomes in mitosis where they remain connected
before cell division. This structure is essential for an
equivalent chromosomes distribution to the daughter cells.
The centromere specific histone H3-variant CENP-AcenH3
is essential for kinetochore formation and centromere
function. We have recently established a biosynthetic
approach to target dCENP-AcenH3 to specific noncentromeric sequences such as the Lac Operator and
follow the formation of functional neocentromeres. Using
this approach we were able to directly demonstrate
that a dCENP-AcenH3 -LacI fusion is sufficient to induce
centromere formation as well as self-propagation
and inheritance of the epigenetic centromere mark
(Mendiburo et al., 2011). Using the LacO/LacI tethering
system, we are now interested dissecting the function
of dCENP-AcenH3 in Drosophila (also known as CID) and
human cells for its centromere targeting, kinetochore
formation and self-propagation properties (Logsdon et
al., 2015; Figure 1).
20
How are centromeres organised in the nucleus?
The compartmentalization of the eukaryotic cell helps
regulating proper genome function and gene expression.
In D. melanogaster centromeres and the pericentric
heterochromatin are not randomly positioned in the
nucleus. Centromeres cluster together and are tethered
to the periphery of the nucleolus. We are currently
investigating the factors involved in this particular
organization. We could recently show that the protein
NLP (Nucleoplasmin Like Protein) plays a major role for
centromere positioning (Padeken et al., 2013). It binds
specifically to centromeres and causes their clustering
near the nucleolus (Figure 2). Elimination of NLP leads
to declustering of centromeres, dissociation from the
nucleolus, loss of silencing of repetitive DNA elements,
DNA damage and mitotic defects.
Our aim is to determine how NLP and its binding factors
contribute to higher-order genome organization and
maintenance of genome integrity.
Figure 1
Figure 2
Figure 1. CENP-A-LacI is efficiently targeted to (a) integrated lacO arrays or (b) lacO plasmids, where it induces de novo
centromere formation. Using this biosynthetic approach we aim to dissect CENP-A function and gain insight into the
epigenetic inheritance of centromeres.
Figure 2: Centromere clustering is mediated by a network of proteins around NLP. (a) Centromeres form clusters at the
periphery of the nucleolus in Drosophila Schneider S2 cells. (b) NLP (nucleoplasmin) and dCTCF contribute to centromere
clustering, while dNucleolin, serves as a nucleolus anchor. (Padeken et al., Mol. Cell 2013)
Selected Publications: Logsdon, G.A., Barrey, E.J., Bassett, E.A.,
DeNizio, J.E., Guo, L.Y., Panchenko, T., Dawicki-McKenna, J.M., Heun, P.,
and Black, B.E. (2015). Both tails and the centromere targeting domain
of CENP-A are required for centromere establishment. J Cell Biol 208,
521-531.
Mendiburo, M.J., Padeken, J., Fulop, S., Schepers, A., and Heun, P.
(2011). Drosophila CENH3 is sufficient for centromere formation.
Science 334, 686-690.
Padeken, J., Mendiburo, M.J., Chlamydas, S., Schwarz, H.J., Kremmer,
E., and Heun, P. (2013). The nucleoplasmin homolog NLP mediates
centromere clustering and anchoring to the nucleolus. Molecular cell 50,
236-249.
21
Adele Marston
Co-workers: Bonnie Alver, Rachael Barton, Julie Blyth, Colette Connor, Eris Duro,
Stefan Galander, Vasso Makrantoni, Claudia Schaffner, Xue (Bessie) Su, Kitty Verzjilbergen,
Nadine Vincenten
The Role of the Pericentromere during Mitosis and Meiosis
We study the molecular mechanisms that ensure the
accurate segregation of chromosomes during cell
division. Errors in chromosome segregation generate
daughter cells with the wrong number of chromosomes,
known as aneuploidy, and this is associated with cancer,
birth defects and infertility. To uncover conserved and
fundamental mechanisms we employ budding yeast
as a model system together with a wide range of cell
biological and biochemical techniques.
During mitosis, chromosomes are segregated
because kinetochores of sister chromatids, which
assemble on the centromere of each chromosome,
attach to microtubules from opposite poles. A protein
complex known as cohesin resists the pulling forces
of microtubules. Cohesin is most highly enriched in a
large domain surrounding the centromere, known as the
pericentromere, which assembles a regulatory platform
for chromosome segregation.
Once all chromosomes are properly attached to
microtubules cohesin is promptly destroyed, triggering
chromosome segregation.
Our work focuses on the establishment and functions of
the pericentromere as well as the changes that occur to
the kinetochore to bring out the specialized chromosome
segregation pattern which occurs during meiosis.
22
Specifically, we aim to address three broad questions:
1. How is cohesin established in the pericentromere?
We demonstrated that inner kinetochore proteins
and the Scc2/4 cohesin loader collaborate to target
cohesin loading to the centromere to build the cohesinrich pericentromere. Ongoing work is addressing the
mechanism of this targeting as well as defining the
boundaries of the pericentromere.
2. How does the pericentromere regulate chromosome
segregation?
Many of the regulatory functions of the pericentromere
are conferred by the pericentromeric adaptor protein,
shugoshin. We showed that shugoshin recruits multiple
protein complexes to the pericentromere to ensure
accurate chromosome segregation. We are now
investigating the role of these interactions during meiosis
and deciphering mechanisms of shugoshin inactivation.
3. How is the pericentromere modified for meiosis?
Meiosis is a modified cell division that produces gametes
through two consecutive rounds of chromosome
segregation. During the first meiotic division, uniquely,
the maternal and paternal chromosomes, known as
homologs, are segregated. We are investigating how
kinetochores are adapted to bring about this specialized
pattern of chromosome segregation.
Figure 1
Figure 2
Figure 1. Electron micrograph showing the ultrastructure of the yeast spindle pole body in meiosis II. Inset shows light
microscopy image of a cell at a similar stage. Spindle pole bodies are shown in red, and DNA is shown in blue.
Figure 2. Snapshot from a movie of live yeast cells undergoing meiosis I. Kinetochores are shown in pink and Rec8 cohesin is
shown in green.
Selected Publications: Sarangapani K, Duro E, Deng Y, de Lima Alves F,
Ye Q, Opoku KN, Ceto S, Rappsilber J, Corbett KD, Biggins S, Marston
AL and Asbury C (2014) Sister kinetochores are mechanically fused
during meiosis I in yeast. Science 346, 248-251.
Nerusheva OO, Galander, S, Fernius J and Marston AL (2014) Tensiondependent removal of pericentromeric shugoshin is an indicator of
sister chromosome biorientation. Genes and Development 28, 12911309.
Verzijlbergen KF, Nerusheva OO, Kelly D, Kerr A, Clift D, de Lima Alves
F, Rappsilber J and Marston AL (2014) Shugoshin biases chromosomes
for biorientation through condensin recruitment to the pericentromere.
eLife 3, e01374.
23
Gracjan Michlewski
Co-workers: Nila Roy Choudhury, Jakub Stanislaw Nowak, Santosh Kumar
Regulation of microRNA processing and function
MicroRNAs (miRNAs) are conserved non-coding RNAs
that regulate gene expression by targeting partially
complementary sequences in the mRNAs. Each miRNA
potentially regulates hundreds of mRNA targets, thus
controlling a variety of biological processes, including
mammalian cellular differentiation and development. In
spite of widespread efforts to understand the roles of
individual miRNAs little progress has been made towards
unravelling the regulation of their biogenesis.
RNA-binding proteins control gene expression in all living
cells by regulating all aspects of RNA biology. Because
RNA binding motives are short and abundant, a typical
RNA-binding protein has thousands of RNA targets.
Strikingly, many RNA-binding proteins are multifunctional
and, depending on their substrates, can have positive,
negative or passive effects on the related molecular
process. Although considerable progress has been made
in cataloguing RNA-Protein (RNP) complexes, it is very
hard to predict their molecular and physiological function
only based on their binary interaction.
My group has focused on elucidating the cis and transacting factors of tissue-specific miRNA biogenesis in
mammalian cells. We have identified proteins that
regulate the production of brain-enriched and brainspecific miRNAs, as well as novel RNA-binding protein,
responsible for the selective uridylation and degradation
of miRNA precursors in embryonic cells.
Selected Publications: Choudhury, N.R., Nowak, J.S., Zou, J.,
Rappsilber, J., Spoel, S.H., and Michlewski, G. (2014). Trim25 Is an RNASpecific Activator of Lin28a/TuT4-Mediated Uridylation. Cell reports 9,
1265-1272.
24
Nowak, J.S., Choudhury, N.R., de Lima Alves, F., Rappsilber, J., and
We have demonstrated that the expression profile of
brain-enriched miRNA-7, which is processed from a
ubiquitous pre-mRNA transcript coding for hnRNP K
protein, is achieved by inhibition of its biogenesis in nonbrain cells (Choudhury et al., 2013). By identifying MSI2
and HuR proteins as inhibitors of miRNA-7 maturation
in non-brain cells we provided the first insight into the
regulation of brain-enriched miRNA processing by
defined tissue-specific factors. Furthermore, we showed
that brain-specific miRNA-9 is regulated transcriptionally
and post-transcriptionally during neuronal differentiation
(Nowak et al., 2014). We revealed that Lin28a, an RNAbinding protein progressively switched off during
differentiation, inhibits the processing of brain-specific
miRNA-9 by inducing the degradation of its precursor
transcript during early stages of neuronal differentiation.
Finally, we have identified the E3 ubiquitin ligase
Trim25 as a RNA-dependent co-factor for Lin28a/TuT4mediated uridylation of let-7 precursors (Choudhury
et al., 2014). This demonstrated for the first time that a
protein-modifying enzyme, recently shown to bind RNA,
can guide the function of RNA-protein complexes in cis.
Our findings have far reaching consequences for our
understanding of how RNA-binding proteins commit to
a specific molecular function and how, through targeting
miRNA biogenesis pathway, they contribute to control of
gene expression mammalian cells.
Michlewski, G. (2014). Lin28a regulates neuronal differentiation and
controls miR-9 production. Nature communications 5, 3687.
Choudhury, N.R., de Lima Alves, F., de Andres-Aguayo, L., Graf, T., Caceres,
J.F., Rappsilber, J., and Michlewski, G. (2013). Tissue-specific control of brainenriched miR-7 biogenesis. Genes & development 27, 24-38.
Figure 1. Structural and sequence context of the GGAG motif determine Lin28a binding and functionality. (A) Schematic of
the secondary structure of wild-type and conserved terminal loop (CTL) mutants of pri-let-7a-1. The mutated nucleotides are
in green and the GGAG motif is in red. (B) Western blot analysis of Lin28a and DHX9 proteins in RNA pull-downs from day 0
(d0) P19 teratocarcinoma embryonic cell extract using wild-type pre-let-7 or its CTL mutants. (C) In vitro processing uridylation
assays performed with internally radiolabeled pre-let-7a transcripts in the presence of d0 P19 cell extract. (−) represents
an untreated control. Reactions were supplemented with 0.25 mM UTP. The products were analyzed on an 8% denaturing
polyacrylamide gel.
25
Hiro Ohkura
Co-workers: Robin Beaven, Manuel Breuer, Mariana Costa, Fiona Cullen, Pierre Romé,
Liudmila Zhaunova
Meiotic cell division and microtubule regulation
Accurate segregation of chromosomal DNA is essential
for life. A failure or error in this process during somatic
divisions could result in cell death or aneuploidy.
Furthermore, chromosome segregation in oocytes is
error prone in humans, and mis-segregation is a major
cause of infertility, miscarriages and birth defects. The
chromosome segregation machinery in oocytes shares
many similarities with these in somatic divisions, but
also has notable differences. In spite of its importance
for human health, little is known about the molecular
pathways which set up the chromosome segregation
machinery in oocytes. Defining these molecular pathways
is crucial to understand error-prone chromosome
segregation in human oocytes. Furthermore, evidence
indicates that these apparent oocyte-specific pathways
also operate in mitosis, although less prominently,
to ensure the accuracy of chromosome segregation.
Therefore uncovering the molecular basis of these
pathways is also important to understand how somatic
cells avoid chromosome instability, a contributing factor
for cancer development.
To understand the molecular pathways which set
up the chromosome segregation machinery in
oocytes, we take advantage of Drosophila oocytes as
a “discovery platform” because of their similarity to
mammalian oocytes and suitability to a genetics-led
mechanistic analysis. In Drosophila oocytes, like in
human oocytes, meiotic chromosomes form a compact
cluster called the karyosome within the nucleus. Later,
meiotic chromosomes assemble the spindle without
centrosomes, establish bipolar attachment and congress
within the spindle. We have identified a number of genes
26
defective in chromosome organisation and/or spindle
formation in oocytes.
We found that the gamma-tubulin recruiting complex,
Augmin, stably associates with spindle poles and has
a more restricted role in spindle microtubule assembly
in oocytes than mitotic cells. In contrast, Grip71WD, the
mediator of the gamma-tubulin and Augmin complexes,
is required for bulk spindle microtubule assembly in both
oocytes and mitotic cells. This suggests that oocytes have
an Augmin-independent pathway for spindle microtubule
assembly. This oocyte-specific microtubule assembly
pathway, together with pole association of Augmin, may
be important to compensate for a lack of centrosomes in
oocytes.
We have identified the Drosophila homologue of a
tumour suppressor protein, KANK as a protein that binds
to the crucial microtubule regulator EB1. We found that it
specifically localises to muscle-tendon attachment sites. In
parallel, we apply systematic approaches to understand
how EB1 binds its partners. We have identified a series
of peptides (aptamers) that bind EB1 homologues in
humans and Drosophila. Their sequences define distinct
EB1- and EB3-binding motifs. They include aptamers
that successfully interfere with microtubule dynamics in
developing flies.
Figure 1
Figure 2
FigureFigure
1
1
FigureFigure
2
2
FRAP FRAP
Dgt2-GFP
Dgt2-GFP
wild-type
wild-type
mitosismitosis
100
100
100
FRAP
Dgt2-GFP
%
%
%
wild-type meiosis
wild-type mitosis
GFP-Dgt2
GFP-Dgt2
Rcc1-mCherry
Rcc1-mCherry
merge merge
Figure 1
GFP-Dgt2
Rcc1-mCherry
merge
Wac-GFP
Rcc1-mCherry
merge
Wac-GFP
Rcc1-mCherry
Wac-GFP
Rcc1-mCherry
Figure
2 wild-type
wild-type
meiosismeiosis
100
%
merge50
merge
Augmin Augmin
chromoosomes
chromoosomes
Augmin
Figure
Figure
3 chromoosomes
3
Figure 3
Figure 3
100
%
100
%
50
total
50
total
total
0
30
50
slow
slow
fast
slow
50
fast
0 60 3090 60120 90150 120
s fast
150 s
Slow 85%
Slowt1/285%
= 5 min
t1/2= 5 min
15%
Fastt1/2
= 8120
sec
t1/2150
= 8 ssec
60
9015%
0 Fast
30
total
total
50
0
total
30
0 60 30 90 s 60
90 s
Slow 15%
Slowt1/215%
= ∞ t1/2 = ∞
Fast0 85%
Fast
85%
=15 90
sec
t1/2
s =15 sec
30 t1/260
Slow 85% t1/2= 5 min
Slow 15% t1/2 = ∞
Augmin localises
Augmin
localises
transiently
Augmin
amplifies
Centrosomes
Centrosomes
generategenerate
Fast 15%
t1/2= transiently
8 sec Augmin amplifies
Fast 85%
t1/2 =15 sec
uniformly
and uniformly
to the spindle
to the spindle
microtubules
microtubules
microtubules
microtubules
from poles
fromand
poles
Centrosomes generate
microtubules from poles
mitosis
mitosis
Augmin localises transiently
and uniformly to the spindle
Augmin amplifies
microtubules
mitosis
meiosis
meiosis
Oocytes Oocytes
lack centrosomes
lack centrosomes
Augmin stably
Augmin
associates
stably associates
Augmin generates
Augmin generates
meiosis
with the spindle
with thepoles
spindle poles
microtubules
microtubules
from the from
polesthe poles
to
congress
to
congress
chromosomes
chromosomes
Oocytes lack centrosomes
Augmin stably associates Augmin generates
with the spindle poles
microtubules from the poles
to congress chromosomes
Figure 1. Augmin subunits, Dgt2, and Wac, accumulate at spindle poles in oocytes.
Figure 2. Augmin turns over rapidly in mitosis, but much more slowly in meiosis.
Figure 3. A model for Augmin function in oocytes in contrast to in mitosis.
Selected Publications: H. Ohkura. (2015). Meiosis: An overview of key
differences from mitosis. Cold Spring Harb Perspect Biol. a015859.
Nikalayevich, N. and Ohkura, H. (2015). The NuRD nucleosome
remodelling complex and NHK-1 kinase are required for chromosome
condensation in oocytes. J. Cell Sci. 128, 566-575.
Lesniewska, K., Warbrick, E., and Ohkura, H. (2014). Peptide aptamers
define distinct EB1- and EB3-binding motifs and interfere with
microtubule dynamics. Mol. Biol. Cell. 25, 1025-1036.
27
Juri Rappsilber
Co-workers:
Adam Belsom, Zhuo Angel Chen, Colin Combe, Lutz Fischer, Sven Giese, Piotr Grabowski,
Georg Kustatscher, Christos Spanos, Juan Zou
3D proteomics
We aim at understanding how proteins fold, interact and
associate in larger structures in their native environments.
We interrogate proteins by two approaches that we
developed in our lab.
1) We are one of the key labs driving the transition of
cross-linking/mass spectrometry (CLMS) into a powerful
and useful addition to the structural biology toolbox.
Advancements have included the development of
algorithms, protocols and mass spectrometric methods.
We thoroughly tested and then exploited these tools
in numerous biological applications in our own lab and
in collaboration with world-leading scientists in their
respective disciplines. These efforts included the first
analysis of a large multi-protein complex using crosslinking/mass spectrometry (RNA Pol II-TFIIF) (Chen et al.
EMBO J. 2010) and the efforts in conducting quantitative
analyses (Fischer et al. J. Proteomics 2013). As the next
step we want to increase the resolution of CLMS such that
protein modelling becomes feasible. We have provided
data to CASP11 (11th Community Wide Experiment
on the Critical Assessment of Techniques for Protein
Structure Prediction), to support modelling by data. This
was the first time for CASP to include experimental data.
Selected Publications: Combe CW, Fischer L, Rappsilber J. xiNET: crosslink network maps with residue resolution. Mol Cell Proteomics. 2015 Feb 3.
pii: mcp.O114.042259. [Epub ahead of print] PubMed PMID: 25648531.
28
Kustatscher G, Wills KL, Furlan C, Rappsilber J. Chromatin enrichment
for proteomics. Nat Protoc. 2014 Sep;9(9):2090-9. doi: 10.1038/
nprot.2014.142. Epub 2014 Aug 7. PubMed PMID: 25101823; PubMed
Central PMCID: PMC4300392.
2) We propose a fundamentally new approach to protein
function analysis. While trying to establish a definitive
list of proteins that associate with mitotic chromosomes
in vertebrates we realised shortcomings in standard
concepts of organellar proteomics (that the composition
of an organelle can be defined through biochemical
purification). We developed a different approach, in
collaboration with Bill Earnshaw’s lab, by analysing the
composition of mitotic chromosome preps obtained in
multiple different ways and integrating this by machine
learning (Ohta, Bukowski-Wills et al. Cell 2010). We
expanded this conceptually in several ways and proposed
a view of interphase chromatin (Kustatscher et al. EMBO
J. 2014) that helped reveal proteins involved in DNA
replication and chromatin maturation (Alabert et al. Nat.
Cell Biol. 2014). Our approach is large-scale data-driven
and could complement annotation databases such as
Gene Ontology with an urgently needed quantitative
dimension. It infers protein function from co-regulation
in vivo rather than co-purification, and could thus
become a cornerstone in characterizing the proteome of
biochemically intractable cellular structures.
Barysz H, Kim JH, Chen ZA, Hudson DF, Rappsilber* J, Gerloff* DL,
Earnshaw* WC. Three-dimensional topology of the SMC2/SMC4
subcomplex from chicken condensin I revealed by cross-linking and
molecular modelling. Open Biol. 2015 Feb;5(2):150005. doi: 10.1098/
rsob.150005. PubMed PMID: 25716199; PubMed Central PMCID:
PMC4345284. [*shared commun. authors]
A
B
C
Cross-linking for Human serum albumin (HSA, shown in gray) A. Standard cross-linking under ideal conditions (left, 0.15 links/
residue, 85 links, purple). B. Our photo-cross-linking in the natural environment (right, 2.54 links/residue, 1485 links, red).
C.The first protein structure determined with 3DProteomics: domain A of HSA, computed using 320 cross links, in comparison
to the x-ray structure (gray)
29
Kenneth E. Sawin
Co-workers: Weronika Borek, Su Ling Leong, Ye Dee Tay, Harish Thakur, Sanju Ashraf, Xun Bao,
Delyan Mutavchiev
Microtubule nucleation, cytoskeletal organisation, and cell polarity
Research interests in the laboratory are focused on
cellular organisation and currently fall into two main
areas: 1) molecular mechanisms of microtubule
nucleation and organisation; and 2) understanding
regulation of cell polarity in a dynamic systems context. In
both areas we use the fission yeast Schizosaccharomyces
pombe as a model eukaryotic organism, combining
classical and molecular genetics with microscopy,
biochemistry and proteomics.
Microtubule nucleation depends on the g-tubulin
complex (g-TuC), a multi-protein complex enriched
at microtubule organizing centres such as the
centrosome. The mechanisms that target the g-TuC to
specific subcellular locales and regulate g-TuC activity
throughout the cell cycle remain largely unknown.
In fission yeast, the proteins Mto1 and Mto2 form an
oligomeric complex (Mto1/2 complex) that binds the
g-TuC and targets it to different sites during the cell cycle.
Mutations in the human homolog of Mto1 lead to the
brain disease microcephaly. Our recent work has shown
that Mto1/2 not only localizes the g-TuC to specific sites
but also activates the g-TuC. Current work is focused on
understanding the mechanism of activation, through
expression and purification of recombinant multi-protein
complexes in insect cells, in vitro functional reconstitution,
and structural biology approaches, including X-ray
crystallography. In addition, we have shown that the
30
molecular architecture of the Mto1/2 complex is
regulated by phosphorylation during the cell cycle, and
this contributes to cell cycle-dependent regulation of
microtubule nucleation.
Regulation of cell polarity in fission yeast is particularly
interesting because it involves multiple internal cues.
The Rho-family GTPase Cdc42 and its associated
regulators and effectors control the actin cytoskeleton
and exocytosis. In addition, however, microtubules
provide additional control of site-selection for polarised
growth, through the microtubule plus-tip-associated
protein Tea1, the cell-tip membrane protein Mod5, and
their interactors. In past work we have focused on the
regulation and dynamics of the Tea1-Mod5 system. We
are currently studying how the Cdc42 system and the
Tea1-Mod5 system “talk to each other” under different
environmental stimuli, using a combination of genetics,
proteomics, FRAP microscopy, and mathematical
modeling.
An important component of our research involves
developing new tools for fission yeast genetics,
microscopy, biochemistry and proteomics. This includes a
robust platform for differential proteomics in fission yeast,
using Stable Isotope Labeling by Amino Acids in Culture
(SILAC), which we are now applying to the analysis of
protein phosphorylation.
Selected Publications: Lynch, E.M., Groocock, L.M., Borek, W.E., and
Sawin, K.E. (2014) Activation of the g-tubulin complex by the Mto1/2
complex. Curr Biol 24, 896-903.
Bicho, C.C., Kelly, D.A., Snaith, H.A., Goryachev, A.B., and Sawin, K.E.
(2010). A catalytic role for Mod5 in the formation of the Tea1 cell polarity
landmark. Curr Biol 20, 1752-1757.
Anders, A., and Sawin, K.E. (2011). Microtubule stabilization in vivo by
nucleation-incompetent g-tubulin complex. J Cell Sci 124, 1207-1213.
Bicho, C.C., de Lima Alves, F., Chen, Z.A., Rappsilber, J., and Sawin,
K.E. (2010). A genetic-engineering solution to the “arginine-conversion
problem” in SILAC. Mol Cell Proteomics 9, 1567-1577.
Figure 1
Figure 2
Figure 2
kDa
200 -
m
to
2G
FP
mto1∆
m
to
2+
wild-type
m
to
2G
FP
anti-GFP
immunoprecipitation
Input
m
to
2+
Figure 1
Mto1
116 97 -
Mto2-GFP
66 45 -
31 -
22-
Mto1[bonsai]-GFP
Figure 3
Alp4-tdT
merge
alp16+
alp16∆
Figure 1: Cell morphology of wild-type and mto1∆ fission yeast.
Figure 3
Figure 2: Immunoprecipitation of GFP-tagged Mto2 reveals Mto1 and Mto2 as the major constituents of the Mto1/2 complex.
Figure 3: A “minimal” truncated form of Mto1, Mto1[bonsai] (GFP-tagged; green), forms puncta that recruit the g-tubulin small
complex (Alp4-tdT; red) and support microtubule nucleation. Recruitment of g-tubulin small complex occurs independently of
other g-tubulin complex proteins such as Alp16 (yeast homolog of human GCP6).
31
Eric C. Schirmer
Co-workers: Dzmitry G. Batrakou, Rafal Czapiewski, Jose de las Hera, Alexandr Makarov,
Peter Meinke, Andrea Rizzotto, Michael I. Robson, Natalia Saiz Ros, Phu Le Thanh, Sylvain Tollis
Nuclear envelope transmembrane protein regulation of tissue-specific genome and
cytoskeletal organization
Mutations in ubiquitous nuclear envelope (NE) proteins
cause a range of diseases with tissue-specific pathologies
including muscular dystrophies, lipodystrophies,
neuropathy, and premature-aging syndromes. To explain
how mutations in ubiquitous proteins can yield tissuespecific pathologies we postulated that tissue-specific
partners mediate pathology and identified candidate
partners with proteomics. Strikingly, we found that the
majority of NE transmembrane proteins (NETs) are tissuespecific and screening these NETs has identified distinct
sets with functions in cytoskeletal organisation, cell cycle
progression, nuclear size regulation, differentiation and
genome organization, perhaps explaining the NE-linked
diseases.
The laboratory currently has five focus areas, three deriving
from our screens. For cytoskeletal organisation we study
nucleo-cytoskeletal interactions in muscle contraction as
well as cell migration. We are also using a crosslinkingmass spectrometry approach in collaboration with the
Rappsilber lab to study intermediate filament architecture.
For nuclear size regulation we are screening for small
molecule effectors in collaboration with the Auer and Tyers
labs because nuclear size changes relate to increased
metastasis in cancer. For spatial genome organisation and
differentiation we are studying the mechanism underlying
NETs we found affect chromosome and gene positioning
Selected Publications: Meinke, P., Schneiderat, P., Srsen, V., Korfali, N., Le
Thanh, P., Cowan, G., Cavanagh, D. R., Wehnert, M., Schirmer, E. C., and
Walter, M. C. (2015) Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked EmeryDreifuss muscular dystrophy. Neuromuscul. Disord. 25, 127-136.
32
Zuleger, N., Boyle, S., Kelly, D. A, de las Heras, J., Lazou, V., Korfali, N.,
Batrakou, D. G., Randles, K. N., Morris, G. E., Harrison, D. J.,
in liver, blood, muscle and fat. We are manipulating these
NETs to determine the contribution of positioning at the
nuclear periphery to regulation of gene expression by
comparing global changes in genes at the periphery with
global changes in gene expression and confirming results
with fluorescence in situ hybridization (FISH). For example,
when the liver-specific NET47 is knocked out in mice the
FMO3 gene locus moves away from the nuclear periphery
with a concomitant 20-fold increase in expression. Tellingly,
NET29, which is mostly expressed in fat, when knocked
down in cultured adipocytes yields de-repression of
several muscle specific genes. As fat and muscle come
from the same progenitors, these effects could explain the
pathology of NE-linked lipodystrophies and potentially
also indicate a role in diabetes and obesity.
The lab is also testing a novel approach to identifying
orphan and genetically variable disease alleles by
applying iterative exome, genome and focused deep
sequencing approaches to Emery-Dreifuss muscular
dystrophy. This disease is currently linked to 5 different
NE proteins, but 53% of patients remain unlinked and our
approach has identified a dozen new high probability
candidate genes. Finally, we are investigating the
interaction of herpesviruses with the NE, predicting that
NET interactions will be crucial to this poorly studied
phase of the virus life cycle.
Bickmore, W. A., and Schirmer, E. C. (2013) Specific nuclear envelope
transmembrane proteins can promote the location of chromosomes to
and from the nuclear periphery. Genome Biol. 14(2), R14. de las Heras, J. I., Meinke, P., Batrakou, D. G. Srsen, V., Zuleger, N., Kerr,
A. R. W., and Schirmer, E. C. (2013) Tissue specificity in the nuclear
envelope supports its functional complexity. Nucleus 4(6). 460-477.
A. Lamin A induction during superantigen stimulation of lymphocytes causes repositioning of the IL-2 locus away from the
nuclear periphery. Lymphocytes expressing GFP, GFP-Lamin A or GFP-Progerin (a mutant version of lamin A that causes the
premature ageing disease Progeria used as a non-functional control) were stimulated with antigen presenting cells (Raji)
exposed to superantigen (SEE). The IL-2 locus moves from the periphery only in the cells previously expressing GFP-Lamin
A with quantification of 100 cells graphed on the bottom for three repeats. B. This repositioning is associated with a >10fold increase in IL-2 expression compared to the increases observed for GFP and GFP-Progerin expressing cells. C. DamID
determination of the global profile of genes at the nuclear periphery as they change during myogenesis. The area marked in
the box changes from not being at the periphery (red lines) to being at the periphery (blue lines). The gene encoded in this
region, Nid1, normally is repressed during myogenesis and this repression is greatly reduced by depletion of NET39, a NET
that directs muscle-specific patterns of radial chromosome positioning in myogenesis.
33
Irina Stancheva
Co-workers: Ilaria Amendola, Christian Belton, Burak Ozkan, Natalia Torrea, Simon Verzandeh
Epigenetic Regulation of Gene Expression
The main focus of our research is to gain mechanistic
insights into how DNA methylation and silencing histone
modifications regulate gene expression in mammalian
cells during differentiation and development. In
particular, we study the molecular mechanisms that
guide the establishment of DNA methylation at specific
loci and genome-wide as well as the crosstalk between
DNA methylation and histone modifications during the
establishment and propagation of silenced chromatin
states.
role in DNA methylation, the LSH ATPase is essential for
efficient repair of DNA double-strand breaks in somatic
cells (Burrage et al, 2012) and for cell proliferation. We
are currently using mouse models (Figure 1), generated
in our lab, and a combination of genomic, proteomic
and biochemical approaches to unravel the mechanistic
details of how the chromatin remodelling by LSH
promotes the epigenetic gene silencing, DNA repair and
cellular proliferation during embryonic development and
differentiation of specific cell lineages.
In early mammalian development, lineage-specific
DNA methylation patterns are established in the postimplantation embryo by de novo DNA methyltransferase
enzymes DNMT3A and DNMT3B. Unlike DNA methylation
maintenance enzyme DNMT1, which functions during
DNA replication on DNA largely free of nucleosomes, the
substrate for DNMT3A/3B-dependent methylation is DNA
packaged into chromatin. We are interested in proteins
that facilitate the action of DNMT enzymes either globally
or in a locus-specific manner. Recently, we found that, in
the absence of chromatin remodelling ATPase LSH/HELLS
DNA methylation patterns at specific loci cannot be
correctly established in embryonic lineage cells leading
to missexpression of a large number of genes (Myant et
al, 2011). We also discovered that, independently of its
We also study the mode of action of other regulators
of DNA methylation in mammalian cells, in particular,
the complex of histone H3 lysine 9 methylases G9a and
GLP. Our recent data has shown that G9a/GLP complex
not only cooperates with LSH in stable silencing of
pluripotency genes during differentiation of embryonic
stem cells (Myant et al, 2011), but is also required for
maintenance of DNA methylation at imprinting control
regions. Our current research aims to address why and
how the action of LSH and G9a/GLP complex is restricted
to specific loci in the genome, how is the complex
recruited to DNA/chromatin and what are the essential
players that enable de novo establishment of H3 K9
dimethylation.
Selected Publications: Myant, K., Termanis, A., Sundaram, A. Y., Boe,
T., Li, C., Merusi, C., Burrage, J., de Las Heras, J. I., and Stancheva, I.
(2011). LSH and G9a/GLP complex are required for developmentally
programmed DNA methylation. Genome Res 21, 83-94.
34
Burrage, J., Termanis, A., Geissner, A., Myant, K., Gordon, K.
and Stancheva, I. (2012) The SNF2 family ATPase LSH promotes
phosphorylation of H2AX and efficient repair of DNA double-strand
breaks in mammalian cells. J Cell Sci 125, 5524-5534
Gendrel, A.V., Apedaile, A., Coker, H., Termanis, A., Zvetkova, I., Godwin,
J., Tang, A., Huntley, D., Montana, G., Taylor, S., Giannoulatou, E., Heard,
E., Stancheva, I. and Brockdorff, N. (2012) Smchd1 dependent and
independent pathways determine developmental dynamics of CpG
island methylation on the inactive X chromosome, Dev Cell 23, 265-279
Western blots
Lsh +/off
+/
+
of
f/
+/ off
+
+/
of
+/ f
o
+/ ff
o
+/ ff
o
of ff
f/
+/ off
+
+/
+
+/
on
of
f/o
on n
/o
n
embryos E12.5
Lsh+/+
LSH
DNMT1
off/off
Lsh
DNMT3A
DNMT3B
Total 5mC in embryos (HPLC)
Bisulfite sequencing: Rhox 6 promoter
+/off embryo E12.5
off/off embryo E12.5
% 5mC
100
50
0
+/+ +/off off/off on/on
Figure1: The Lshoff/off mice, carrying conditionally-reversible knockout alleles of Lsh, display growth retardation and reduced
DNA methylation, but normal expression of DNMTs. These mice are also sterile, develop neurological problems and die within
24 weeks. The Lshon/on mice are phenotypically normal.
35
David Tollervey
Co-workers: Stefan Bresson, Clémentine Delan-Forino, Hywel Dunn-Davies, Aziz El Hage,
Tatiana Dudnakova, Aleksandra Helwak, Rebecca Holmes, Laura Milligan, Elisabeth Petfalski,
Camille Sayou, Vadim Shchepachev, Tomasz Turowski, Marie-Luise Winz
Nuclear RNA Processing and Surveillance
Our aim is to understand the nuclear pathways that
process newly transcribed RNAs and assemble RNAprotein complexes, the mechanisms that regulate these
pathways and the surveillance activities that monitor their
fidelity. A prominent feature of our recent work has been
the application of in vivo RNA-protein crosslinking and
analysis of cDNA (CRAC). This provides many starting
points for detailed analyses of specific RNA-protein
complexes, as well as allowing unbiased, genome-wide
views of the targets of RNA surveillance and related
pathways.
We have a long-standing interest in the pathway of
eukaryotic ribosome synthesis, which occupies a key
place in the metabolism of all cells and is closely linked to
cell growth and division. We developed a mathematical
model for the pre-rRNA processing pathway and used
this as a practical tool in the functional analysis of the
processing pathway (Axt et al., 2014). We previously
developed the only available in vitro pre-rRNA
processing system, and we used this, together with
CRAC, to understand the last steps in the maturation
of the 40S ribosomal subunits, identifying the key
involvement of an atypical kinase Rio1 (Turkowski et
al., 2014). In collaboration with other research groups,
we characterised the roles of other factors in pre-40S
Selected Publications: Turowski, T.W., Lebaron, S., Zhang, E., Peil, L.,
Dudnakova, T., Petfalski, E., Granneman, S., Rappsilber, J. and Tollervey, D.
(2014) Rio1 mediates ATP-dependent final maturation of 40S ribosomal
subunits. Nucleic Acids Res., 42, 12189-12199. PMID: 25294836
36
Tree, J.J., Granneman, S., McAteer, S.P., Tollervey, D. and Gally,
D.L. (2014) Identification of bacteriophage-encoded anti-sRNAs in
pathogenic Escherchia coli. Mol. Cell, 55, 199-213.
maturation; components of 60S ribosomal subunits
(García-Gómez et al. 2014) and an adenylate kinase
(Loc’h et al., 2014). Other collaborations identified a novel
quality control step acting prior to ribosomal subunit
export from the nucleus (Matsuo, et al., 2014) and a relay
system that transduces chemical energy into physical
reorganisation of the pre-ribosome (Baßler, et al., 2014).
CRAC was also applied to assess the roles of small RNAs
in regulating the expression of virulence genes in the
important pathogen E.coli O157:H7, in collaboration
with David Gally (CMVM). This work identified a novel
class of prophage-encoded ncRNAs, that function by
antagonising endogenous small regulatory RNAs (sRNAs)
(Tree et al., 2014). At least one of these “anti-sRNAs” is
important for adaptation of the pathogen for growth
in the bovine host. Using a different high-throughput
technique, ChIP-seq, we identified the locations of sites
of RNA-DNA duplexes (R-loops) throughout the yeast
genome (El Hage et al., 2014). This work clarified the
roles of topoisomerase and RNase H in the avoidance
and clearance of R-loops and functional links with the
mobility of Ty retrotransposons (Figure 1).
Together, these reports increased our understanding of
the mechanism and regulation of important pathways in
RNA biology.
El Hage, A., Webb, S., Kerr, A. and Tollervey, D. (2014) Genomewide distribution of RNA-DNA hybrids identifies RNase H targets in
tRNA genes, retrotransposons and mitochondria. PLoS Gen., doi:
pgen.1004716. PMID: 25357144.
Figure 1. R-loops form in the dark by Hratch Arbach. The foreground shows the transcribing polymerase with an R-loop
forming between the nascent transcript and the DNA. In the Ty1 pre-integration complex two integrase molecules (colored in
pink) target nucleosomes upstream of tRNA genes. Rightward arrows indicate transcription direction. Leftward arrows indicate
potential interactions between the R-loop and Ty1 pre-integration complex.
37
Philipp Voigt
Co-worker: Kim Webb
Molecular Mechanisms of Epigenetic Gene Regulation
The genetic information of eukaryotic organisms is
packaged into chromatin, a complex assembly of
DNA, RNA, and proteins. The basic unit of chromatin
is the nucleosome, which is formed by DNA wrapping
around a histone octamer. Histones not only constitute
the structural backbone of chromatin but also provide
ample opportunity for regulating gene expression. They
undergo a host of posttranslational modifications, which
are thought to impact transcription either by directly
modulating chromatin structure or by recruiting effector
proteins. The overarching goal of research in our lab
is to determine the mechanistic intricacies of histone
modifications. Moreover, we aim to understand how
different systems of chromatin modifiers interact to
regulate and fine-tune gene expression.
In particular, we are interested in the repressive histone
modification histone H3 lysine 27 trimethylation
(H3K27me3), which is placed by Polycomb Repressive
Complex 2 (PRC2). Despite the well-established role of
PRC2 and H3K27me3 during development, it remains
unclear exactly how this modification functions to
repress genes in mechanistic terms. Paradoxically, in
embryonic stem cells, H3K27me3 is found at promoters
of developmental genes alongside the active mark
H3K4me3, which is catalysed by members of the SET1
and MLL methyltransferases (Figure 1A). The resulting
Selected Publications: Bonasio, R., Lecona, E., Narendra, V., Voigt, P.,
Parisi, F., Kluger, Y., and Reinberg, D. (2014). Interactions with RNA
direct the Polycomb group protein SCML2 to chromatin where it
represses target genes. eLife 2014;3, e02637.
38
Voigt, P., Tee, W.-W., and Reinberg, D. (2013). A double take on bivalent
promoters. Genes Dev. 27, 1318–1338.
bivalent domains are presumed to poise developmental
genes for activation, facilitating rapid and robust
expression upon appropriate differentiation cues while
keeping them repressed in embryonic stem cells.
However, direct evidence for this concept remains largely
elusive.
Our previous work shed light on the conformation and
establishment of bivalent domains, demonstrating that
bivalent nucleosomes are in an asymmetric conformation,
carrying H3K27me3 and H3K4me3 on different copies
of histone H3 within single nucleosomes (illustrated in
Figure 1). However, it remains unclear how the bivalent
marks are decoded and how bivalency contributes to
gene activation and plasticity during differentiation. We
are currently addressing these questions by employing
biochemical and cell-biological approaches. In addition,
we are aiming to establish quantitative, systems biologyinspired approaches to define the physiological functions
of bivalent domains. Specifically, we are testing the
hypothesis that bivalency may represent a dynamic
equilibrium between active and repressive factors that
ensures proper timing of developmental gene expression
(Figure 1B). As both temporal and spatial accuracy
of expression patterns is essential for development,
bivalency may be indispensable to proper embryonic
development.
Voigt, P., Leroy, G., Drury, W. J., Zee, B. M., Son, J., Beck, D B., Young,
N. L., Garcia, B A., and Reinberg, D. (2012). Asymmetrically Modified
Nucleosomes. Cell 151, 181–193.
Figure 1. A. Interactions between active and repressive factors control the establishment of bivalent domains in embryonic
stem cells. B. As their key function, bivalent domains may ensure proper timing of expression of developmental genes.
39
Malcolm Walkinshaw
Co-workers: Yiyuan Chen, Jaqueline Dornan, Peter Fernandes, Charis Georgiou,
James Kinkead, Divya Malik, Iain McNae, Paul. Michels, Jia Ning, Giulia Romanelli,
Andromachi Xipnitou, Li-Hsuan Yen, Meng Yuan
EPPF staff: Liz Blackburn, Sandra Bruce, Matthew Nowicki, Paul Taylor, Martin Wear
Molecular Recognition in Biological Systems
Communication within and between living cells
depends on molecular interactions. We are interested in
understanding the factors that determine how proteins
and small molecule ligands interact and regulate cellular
processes. The enzymes in the glycolytic pathway provide
an interesting model system, as the ten enzymatic
steps that break down glucose to eventually generate
pyruvate are all strictly regulated by elaborate feedback
mechanisms. The pathway has evolved over a period of
2 billion years and most of the enzymatic steps are well
conserved between bacteria, protozoans and mammals.
X-ray structural studies show that the active sites are
highly conserved. In contrast, control mechanisms have
evolved in a variety of surprisingly different ways. We
have studied isoforms of the enzyme pyruvate kinase
(PYK) in the ‘Tri Tryps’ protozoan parasites Trypanosoma
cruzi, Trypanosoma brucei and Leishmania Mexicana
which cause a number of diseases including Chagas
disease, sleeping sickness and kalazar. By trapping
the enzymes in different conformational states and
solving their crystal structures it has been possible to
understand how the enzyme activity is regulated by the
binding of small molecule metabolites and nutrients in
different allosteric pockets. The human, bacterial and
trypanosomal PYK isoforms have distinctly different
allosteric mechanisms for regulating enzyme activity
Selected Publications: Morgan,H.P.,Zhong,W.,McNae,I.W.,Michels,P
.A.M.,Fothergill-Gilmore,L.A., Walkinshaw,M.D.,(2014) Structures of
pyruvate kinases display evolutionarily divergent allosteric strategies
Royal Society Open Science 1 (1), 140120
Shave,S.,Blackburn,E.A.,Adie,J.,Houston,D.R.,Auer,M.,Webster,S.P.,Tayl
or, P. and Walkinshaw, M.D., UFSRAT: Ultra-Fast Shape Recognition
40
and use a different selection of allosteric effectors and
different ways for locking the protein conformation in an
active (R-state) or inactive (T-state) form (1) (See Figure).
These differences provide an excellent opportunity for
identifying or designing small drug-like molecules that
can activate or inhibit the enzymes in a selective, speciesspecific way. Indeed the glycolytic pathway provides a
potentially excellent drug target against trypanosomes
as they are totally dependent on this pathway for the
production of ATP and inhibiting glycolysis at any stage
in the pathway kills the parasite instantly. We have also
been developing and testing a range of software tools to
carry out small molecule database mining and docking
studies to identify novel inhibitors- particularly against
these trypanosome enzymes (2). The search for small
molecule trypanocidal inhibitors is being supported by
a Wellcome Trust Seeding Drug Discovery award and
we have now developed a number of compounds that
inhibit another allosterically regulated protein in the
pathway, phosphofructokinase (PFK). These compounds
show proof of principal by selectively killing parasites in a
matter of minutes with submicromolar EC50 values. These
are encouraging results given the very poor potency and
efficacy of the drugs currently available to treat these
diseases.
with Atom Types–The Discovery of Novel Bioactive Small Molecular
Scaffolds for FKBP12 and 11βHSD1 (2015),PloS one 10 (2), e0116570
Zhong,W.,Morgan,H.P.,Nowicki,M.N.,McNae,I.W.,Yuan,M.,Juraj,B.,Mi
chels P.A.M.,Fothergill-Gilmore,L.A.,Walkinshaw,M.D.,(2014),Pyruvate
kinases have an intrinsic and conserved decarboxylase activity. Biochem.
J.,1;458(2), 301-311.
The evolution of allosteric control of pyruvate kinase (PYK). Schematic representations of the effector induced T- to R-state
allosteric transition. This transition results in the formation of molecular bridges between chains creating a more rigid, highly
active R-state conformer. (a) The ‘inactive’ Geobacillus stearothermophilus PYK T-state tetramer. An extra domain (shown in
green) is predicted to have a hinge type movement mimicking the trypanosomatid effector loop, forming molecular bridges
between chains (as shown in panel b) and the thermally stable, highly active R-state tetramer observed in solution. (c) In
trypanosomatid PYKs F26BP binding stabilises the effector loop, resulting in the formation of a series of stabilising salt bridge
interactions across the C-C interface (as shown in panel d), generating the highly stable R-state tetramer. (e) The ‘inactive’ form
of the human M2PYK is monomeric in solution but forms stable and highly active tetramers upon F16BP binding.
41
Julie Welburn
Co-workers: Sandeep Talapatra, Sarah Young, Bethany Harker, Jovana Deretic, Agata Gluzcek
Structural and cell biology of mitosis, microtubules and motors
Microtubules are dynamic polymers made of tubulin
dimers that undergo stochastic switching between
growth and shrinkage, marked by catastrophe and
rescue events. These highly dynamic microtubule
polymers drive the formation and maintenance of the
bipolar spindle, kinetochore-microtubule attachments,
chromosome oscillations, and chromosome movement
and segregation during mitosis. Microtubules in mitosis
are tightly regulated by multiple players to allow selforganisation into the mitotic spindle and chromosome
alignment and segregation. In particular microtubule
motors and proteins that regulate the dynamic ends
of microtubules are critical to ensure these processes.
They are tightly spatially and temporally regulated
by localized mitotic kinases to fine-tune their activity
on microtubules and kinetochores. Work in the lab
focuses on the function of kinesin motors, in particular
microtubule depolymerases of the kinesin superfamily
Kinesin-13 and Kinesin-8 and microtubule stabilizing
proteins, that promote microtubule growth. Our goals
are to understand how microtubules and their regulatory
players mechanistically control the spindle and cell
division. We integrate both molecular and cellular
approaches to understand the mechanisms underlying
faithful mitosis.
Selected Publications: Analysis of microtubule dynamic regulators
highlights length-dependent anisotropic scaling of spindle shape.
Biology Open. (2014). 3:1217-1223. Young S., Besson S., Welburn JPI.
42
Syred, H.M., Welburn, J., Rappsilber, J., and Ohkura, H. (2013). Cell
cycle regulation of microtubule interactomes: multi-layered regulation
is critical for the interphase/mitosis transition. Mol Cell Proteomics 12,
3135-3147.
Recently, we have focused on the mechanism of the
kinesin-13 motor protein MCAK, a potent microtubule
depolymerase essential in many cellular processes
from mitosis to neuron formation and primary cilia
maintenance. The divergent non-motor regions flanking
the ATPase domain are critical in regulating its targeting
and activity. However, the molecular basis for the function
of the non-motor regions within the context of full-length
MCAK is unknown. Here we determine the structure of
MCAK motor domain bound to its regulatory C-terminus.
Our analysis reveals that the MCAK C-terminus binds to
the motor domain to stabilize MCAK conformation in
solution and is displaced allosterically upon microtubule
binding, which allows its robust accumulation at
microtubule ends. These results demonstrate that MCAK
undergoes long-range conformational changes involving
its C-terminus during the soluble to microtubule-bound
transition and that the C-terminus-motor interaction
represents a structural intermediate in the catalytic cycle
of MCAK. Together, our work reveals intrinsic molecular
mechanisms underlying the regulation of kinesin-13
activity.
Welburn, J.P. (2013). The molecular basis for kinesin functional specificity
during mitosis. Cytoskeleton 70, 476-493.
Welburn, J.P., and Cheeseman, I.M. (2012). The microtubule-binding
protein Cep170 promotes the targeting of the kinesin-13 depolymerase
Kif2b to the mitotic spindle. Mol Biol Cell 23, 4786-4795.
Structure of a human motor-CT domain MCAK complex.
A. Kinesin motor domain dimers (cyan and green) bound to the CT domain (yellow, spacefill) of MCAK. ADP is in red.
B. Overlay of the motor domain and C terminus structure (blue) with the structure of murine MCAK. The respective neck
regions containing the α0 and neck linker are in royal blue and magenta respectively and The CT domain of MCAK is drawn in
yellow.
C. Sequence alignment of the conserved CT domain of MCAK for various species alongside the Drosophila kinesin-13 Klp10A
and human Kif2a.The conserved residues are highlighted in red and the ones essential for binding to the motor are boxed in
green.
D. Graph plotting for the average microtubule binding activity of MCAK and MCAKS715E in absence of nucleotide. The dashed
and full curves correspond to subtilisin-treated and untreated microtubules respectively. Both the C-termini of MCAK and
tubulin reduce the affinity of MCAK for microtubules, to ultimately enable plus-tip targeting.
43
Sarah Keer-Keer
Co-workers: Maria Fanourgiaki, Laura Reed, Juliet Ridgway-Tait
Outreach and Public Engagement
In 2014, we ran many public engagement projects,
as always taking place with the help and support of
researchers. In 2014, we tried a number of new projects,
including taking a giant inflatable cell (zorb ball) out
to parks and gardens. We created a scale set of human
chromosomes to put inside the nucleus, and invited
people to clamber inside and talk to a researcher. Figs
A-C
Each year in November we run Life Through a Lens
public drop-in activities for four weekend afternoons of
in November, attracting over 900 visitors and getting 40
or more WTCCB staff involved with events (Fig J). The Life
Through a Lens project continues to provide workshops
and support to the local Liberton High School, and in
2014 we also supported two pupils with their Advanced
Higher projects.
Our involvement with the running and delivery of
Midlothian Science Festival continues. This festival
takes events out to community halls, libraries, schools,
parks and other local venues and engages hard to reach
audiences. Figs D-F. In the 2014, festival events were
attended by over 8000 people from all backgrounds.
This year we will repeat the most successful event - The
WTCCB Science Alive Gala day - and also add events on
cancer, aging and RNA splicing.
44
In December 2014, we won a substantial award that
allows us to employ a part-time science communicator
to help develop and run events. It also enabled us
to employ a part-time glass artist to create glass that
explains or showcases WTCCB research. This increase in
capacity should help us deliver some really good projects
from summer 2015 – Autumn 2016. We have been
piloting the use of blown glass (to explain epigenetics)
and creating more complex fused glass throughout this
year. Figs G-I
Sarah Keer-Keer won the 2014 Tam Dalyell Prize for
Excellence in Engaging the Public with Science and
performed the prize lecture in April 2015.
Planning for the future, we are working hard to ensure
that the new planned hive building will contain a
dedicated public engagement area.
Figs A-C: The giant inflatable cell
Figs D-F: Midlothian Science Festival and the WTCCB
Science Alive Gala Day
Figs G-I: Blown glass (nucleosome) and fused glass
(bacteriophages)
Fig J: Life Through a Lens
J
B
C
A
D
E
F
G
H
I
45
List of Groups
Robin Allshire
Wellcome Trust Principal Research Fellow
Jean Beggs
Royal Society Darwin Trust Professor
Ryan Ard
Darwin Trust Graduate Student
Tania Auchynnikava
Wellcome Research Associate
Pauline Audergon
Wellcome 4yr Graduate Student
Emilie Castonguay
Wellcome Research Associate
Sandra Catania
Wellcome 4yr Graduate Student
Max Fitz-James
Wellcome 4yr Graduate Student
Ragahavendran
Kulasegaran-Shylini
Engelhorn Foundation
Research Associate
Alison Pidoux
Wellcome Research Associate
Manu Shukla
EMBO Research Associate
Puneet Singh
Darwin Trust Graduate Student
Lakxmi Subramanian EC Marie Curie Research Associate
Nick Toda
Darwin Trust Graduate Student
Pin Tong
EC FP7 EpiGeneSys Research Associate
Sharon White
Wellcome Research Associate
Vahid Aslanzadeh
David Barrass
Jim Brodie
Susana De Lucas
Eve Hartswood
Gonzalo
Mendoza-Ochoa
Jane Reid
Ema Sani
A. Jeyaprakash Arulanandam
Wellcome Research Career Development Fellow
Lisse Baussier
Bethan Medina
Maria Alba Abad
Fernandaz
Tanmay Gupta
Frances Spiller
46
Research Technician
Wellcome Research Assistant
Postdoctoral Research Assistant
Darwin Trust Graduate Student
Darwin Trust Graduate Student
Darwin Trust Graduate student
Wellcome Research Associate
Wellcome Research Associate
Wellcome Research Associate
Wellcome Research Associate
Graduate Student
Wellcome 4yr Graduate Student
Wellcome Research Associate
Adrian Bird
Buchanan Professor of Genetics, Edinburgh University
Beatrice
Alexander-Howden
Wellcome Research Assistant
Kyla Brown
ECAT Graduate Student
Justyna
Cholewa-Waclaw
RSRT Research Associate
John Connelly
Wellcome Research Associate
Dina De Sousa
Wellcome Research Assistant
Jacky Guy
Wellcome Research Associate
Martha Koerner
Erwin-Schrödinger Research Associate
Sabine Lagger
EMBO Research Associate
Matthew Lyst
Wellcome Research Associate
Cara Merusi
RSRT Research Assistant
Timo Quante
Marie Curie Research Associate
Jim Selfridge
Wellcome Research Associate
Ruth Shah
Wellcome 4yr Graduate Student
Christine Struthers
Personal Assistant
Rebekah Tillotson
BBSRC EASTBIO Graduate Student
Atlanta Cook
MRC Career Development Fellow
Adele Marston
Wellcome Trust Senior Research Fellow
Uma Jayachandran
Urszula McCaughan
Valdeko Kruusvee
Bonnie Alver
Wellcome Research Associate
Rachael Barton
Wellcome 4yr Graduate Student
Julie Blyth
Wellcome Research Assistant
Colette Connor
Graduate student
Eris Duro
Sir Henry Wellcome Postdoctoral Fellow
Stefan Galander
Wellcome 4yr Graduate Student
Vasso Makrantoni
Wellcome Research Associate
Claudia Schaffner
Wellcome Research Associate
Xue (Bessie) Su
Wellcome Research Associate
Kitty Verzijlbergen
EMBO Long Term Fellow
Nadine Vincenten
Wellcome 4yr Graduate Student
MRC Research Assistant
Research Associate
Graduate Student
Bill Earnshaw
Wellcome Trust Principal Research Fellow
Dan Booth
Wellcome Research Associate
Mar Carmena
Wellcome Research Associate
Nuno Martins
FCT (Portugal) Graduate Student
Oscar Molina
EMBO Research Associate
Melpi Platani
Wellcome Research Associate
Jan Ruppert
Marie Curie Early Stage Researcher
Itaru Samejima
Wellcome Research Associate
Kumiko Samejima
Wellcome Research Associate
Giulia Vargiu
Graduate Student
Alisa Zhiteneva
Wellcome 4yr Graduate Student
Kevin Hardwick
Professor of Molecular Genetics
Ioanna Leontiou
Darwin Trust Graduate student
Karen May
Wellcome Research Associate
Kostas Paraskevopoulos Wellcome Research Associate
Onur Sen
EBSS Graduate Student
Ivan Yuan
Graduate student
Patrick Heun
Wellcome Trust Senior Research Fellow
Eduard Anselm
Evelyne Barrey
Eftychia Kyriacou
Vasiliki Lazou
Virginie Roure
Georg Schade
Thomas van Emden
Gracjan Michlewski
MRC Career Development Fellow
Nila Roy Choudhury
Jakub Nowak
Santosh Kumar
MRC Research Associate
Wellcome 4yr Graduate Student
MRC Research Assistant
Hiro Ohkura
Wellcome Trust Senior Research Fellow
Robin Beaven
Manuel Breuer
Mariana Costa
Fiona Cullen
Pierre Romé
Liudmila Zhaunova
Wellcome Research Associate
Wellcome Research Associate
Wellcome 4yr Graduate Student
Wellcome Research Associate
Wellcome Research Associate
Darwin Trust Graduate Student
Graduate Student
Graduate Student
Graduate Student
Wellcome Research Assistant
Wellcome Research Associate
Graduate Student
Wellcome Research Assistant
47
Juri Rappsilber
Wellcome Trust Senior Research Fellow
Irina Stancheva
CR-UK Senior Research Fellow
Adam Belsom Zhuo Angel Chen
Colin Combe Lutz Fischer Sven Giese Piotr Grabowski Georg
Kustatscher Christos Spanos Juan Zou Ilaria Amendola
Christian Belton
Burak Ozkan
Natalia Torrea
Simon Verzandeh
Wellcome Research Associate
Wellcome Research Associate
Wellcome Research Associate
Wellcome Research Assistant
TU Berlin Research Associate
Wellcome Research Assistant
Wellcome Research Associate
Wellcome Research Associate
Wellcome Proteomics Data Analysis Manager
Ken Sawin
Professor of Cell Biology
Sanju Ashraf
Xun Bao
Weronika Borek
Su Ling Leong
Delyan Mutavchiev
Ye Dee Tay
Harish Thakur
Principal’s Career Development Graduate Student
Darwin Trust Graduate Student
Wellcome Research Associate
Wellcome Research Associate
Wellcome 4yr Graduate Student
BBSRC Research Associate
Wellcome Research Associate
Eric Schirmer
Wellcome Trust Senior Research Fellow
48
Dzmitry Batrakou
Wellcome Research Associate
Rafal Czapiewski
Wellcome Research Associate
Jose de las Heras
Wellcome Research Associate
Alexander Makarov
Principal’s Scholarship Graduate Student
Peter Meinke
Wellcome Research Associate
Andrea Rizzotto
Darwin Trust Graduate Student
Michael I Robson
Wellcome 4yr Graduate Student
Natalia Saiz Ros
Principal’s Scholarship Graduate Student
Phu Le Thanh
MRC Graduate Student
Sylvain Tollis
Wellcome Research Associate
Wellcome 4yr Graduate Student
MRC Graduate student
Darwin Trust Graduate student
Darwin Trust Graduate student
MRC Graduate Student
David Tollervey, Director;
Wellcome Trust Principal Research Fellow
Stefan Bresson
Wellcome Research Associate
Clémentine
Delan-Forino
FEBS Research Associate
Hywel Dunn-Davies
BBSRC Research Associate
Aziz El Hage
Wellcome Research Associate
Tatiana Dudnakova
BBSRC Research Associate
Aleksandra Helwak
Wellcome Research Associate
Rebecca Holmes
Sir Henry Wellcome Research Fellow
Laura Milligan
Wellcome Research Associate
Rosie Peters Wellcome 4yr Graduate Student
Elisabeth Petfalski
Wellcome Research Associate
Camille Sayou EMBO Research Associate
Vadim Shchepachev
SNSF Research Associate
Tomasz Turowski
Polish Academy Research Associate
Marie-Luise Winz
Wellcome Research Associate
Philipp Voigt
Sir Henry Dale Fellow
Kim Webb
Wellcome Research Assistant
Malcolm Walkinshaw
Chair of Structural Biochemistry,
Director, Edinburgh Protein Production Facility
Elizabeth Blackburn
EPPF Research Associate
Sandra Bruce
EPPF/Group Laboratory Manager
Yiyuan Chen
Graduate Student
Jacqueline Dornan
Wellcome Research Associate
Charis Georgiou
BBSRC/EASTBIO Graduate Student
Peter Fernandes
ECAT Graduate Student
James Kinkead
Wellcome Research Assistant
Iain McNae
Wellcome Research Associate
Divya Malik
Wellcome Research Technician
Jia Ning
Darwin Graduate Student
Matt Nowicki
EPPF Research Associate
Paul Michels
Visiting Professor
Giulia Romanelli
MSc by Research Student
Paul Taylor
University Senior Lecturer/EPPF Research Associate
Martin Wear
EPPF Facility Manager
Andromachi Xypnitou MRS Graduate Student
Li Hsuan Yen
BBSRC Case Graduate Student
Meng Yuan
Darwin Graduate Student
Julie Welburn
CRUK Research Career Development Fellow
Jovana Deretic
Agata Gluszek-Kustusz
Bethany Harker
Sandeep Talapatra
Sarah Young
Darwin Trust Graduate Student
CRUK Research Associate
Wellcome 4yr Graduate Student
CRUK Research Associate
Marie Curie Research Assistant
Administration/Support Staff
Greg Anderson
Centre Laboratory Manager
Carolyn Fleming
Centre Administrative Assistant
Sarah Keer-Keer
Centre Public Engagement and Outreach Manager
David Kelly
Centre Optical Instrumentation Laboratory Manager
Alastair Kerr
Centre Bioinformatics Core Facility Manager
Colin McLaren
Computing Support
Stefan Mann
Chemical Biology Research Associate
Daniel Robertson
Bioinformatics Support Officer
Christos Spanos
Proteomics Facility Manager
Christine Struthers
PA to Adrian Bird/Centre Administrator
Paul Taylor
Computing Support
Karen Traill
Centre Manager/Wellcome 4yr PhD Programme Administrator
Shaun Webb
Bioinformatician
Juan Zou
Proteomics Data Analysis Manager
Technical Support Lloyd Mitchell
Alistair Wilson
Washing-up/Media
Denise Affleck
Andrew Kerr
Margaret Martin
Donna Pratt
49
50
51
Centre Publications 2013 - 2015
Abad, M.A., Medina, B., Santamaria, A., Zou, J., Plasberg-Hill,
C., Madhumalar, A., Jayachandran, U., Redli, P.M., Rappsilber,
J., Nigg, E.A., and Jeyaprakash, A.A. (2014). Structural basis for
microtubule recognition by the human kinetochore Ska complex.
Nat Commun 5, 2964.
Agirre, X., Castellano, G., Pascual, M., Heath, S., Kulis, M., Segura,
V., Bergmann, A., Esteve, A., Merkel, A., Raineri, E., Agueda,
L., Blanc, J., Richardson, D., Clarke, L., Datta, A., Russinol, N.,
Queiros, A.C., Beekman, R., Rodriguez-Madoz, J.R., Jose-Eneriz,
E.S., Fang, F., Gutierrez, N.C., Garcia-Verdugo, J.M., Robson, M.I.,
Schirmer, E.C., Guruceaga, E., Martens, J.H., Gut, M., Calasanz,
M.J., Flicek, P., Siebert, R., Campo, E., Miguel, J.F., Melnick, A.,
Stunnenberg, H.G., Gut, I.G., Prosper, F., and Martin-Subero, J.I.
(2015). Whole-epigenome analysis in multiple myeloma reveals
DNA hypermethylation of B cell-specific enhancers. Genome
Research. Pii: gr180240.114. PMID: 25644835
Aitken, S., Alexander, R.D., and Beggs, J.D. (2013). A rulebased kinetic model of RNA polymerase II C-terminal domain
phosphorylation. J R Soc Interface 10, 20130438.
Alabert, C., Bukowski-Wills, J.C., Lee, S.B., Kustatscher, G.,
Nakamura, K., de Lima Alves, F., Menard, P., Mejlvang, J.,
Rappsilber, J., and Groth, A. (2014). Nascent chromatin capture
proteomics determines chromatin dynamics during DNA
replication and identifies unknown fork components. Nature Cell
Biology 16, 281-293.
Ard, R., Tong, P., and Allshire, R.C. (2014). Long non-coding
RNA-mediated transcriptional interference of a permease gene
confers drug tolerance in fission yeast. Nat Commun 5, 5576.
Axt, K., French, S.L., Beyer, A.L., and Tollervey, D. (2014). Kinetic
analysis demonstrates a requirement for the Rat1 exonuclease in
cotranscriptional pre-rRNA cleavage. PLoS One 9, e85703.
Ban, N., Beckmann, R., Cate, J.H., Dinman, J.D., Dragon, F.,
Ellis, S.R., Lafontaine, D.L., Lindahl, L., Liljas, A., Lipton, J.M.,
McAlear, M.A., Moore, P.B., Noller, H.F., Ortega, J., Panse, V.G.,
Ramakrishnan, V., Spahn, C.M., Steitz, T.A., Tchorzewski, M.,
52
Tollervey, D., Warren, A.J., Williamson, J.R., Wilson, D., Yonath,
A., and Yusupov, M. (2014). A new system for naming ribosomal
proteins. Current opinion in structural biology 24, 165-169.
Barth, T.K., Schade, G.O., Schmidt, A., Vetter, I., Wirth, M., Heun,
P., Thomae, A.W., and Imhof, A. (2014). Identification of novel
Drosophila centromere-associated proteins. Proteomics 14,
2167-2178.
Bassett, A.R., Akhtar, A., Barlow, D.P., Bird, A.P., Brockdorff, N.,
Duboule, D., Ephrussi, A., Ferguson-Smith, A.C., Gingeras, T.R.,
Haerty, W., Higgs, D.R., Miska, E.A., and Ponting, C.P. (2014).
Considerations when investigating lncRNA function in vivo. eLife
3, e03058.
Bassler, J., Paternoga, H., Holdermann, I., Thoms, M., Granneman,
S., Barrio-Garcia, C., Nyarko, A., Stier, G., Clark, S.A., Schraivogel,
D., Kallas, M., Beckmann, R., Tollervey, D., Barbar, E., Sinning, I.,
and Hurt, E. (2014). A network of assembly factors is involved in
remodeling rRNA elements during preribosome maturation. The
Journal of cell biology 207, 481-498.
Bayne, E.H., Bijos, D.A., White, S.A., de Lima Alves, F., Rappsilber,
J., and Allshire, R.C. (2014). A systematic genetic screen identifies
new factors influencing centromeric heterochromatin integrity in
fission yeast. Genome Biol 15, 481.
Beaven, R., Dzhindzhev, N.S., Qu, Y., Hahn, I., Dajas-Bailador,
F., Ohkura, H., and Prokop, A. (2015). Drosophila CLIP-190 and
mammalian CLIP-170 display reduced microtubule plus end
association in the nervous system. Mol Biol Cell. in press
Bird, A. (2013). Instant Expert 29: Epigenetics. New Scientist
No2898, i-viii.
Bird, A. (2013). Genome biology: not drowning but waving. Cell
154, 951-952.
Blackburn, E.A., Fuad, F.A., Morgan, H.P., Nowicki, M.W., Wear,
M.A., Michels, P.A., Fothergill-Gilmore, L.A., and Walkinshaw,
M.D. (2014). Trypanosomatid phosphoglycerate mutases have
multiple conformational and oligomeric states. Biochemical and
Biophysical Research Communications 450, 936-941.
Bonasio, R., Lecona, E., Narendra, V., Voigt, P., Parisi, F., Kluger,
Y., and Reinberg, D. (2014). Interactions with RNA direct the
Polycomb group protein SCML2 to chromatin where it represses
target genes. eLife 3, e02637.
Booth, D.G., Takagi, M., Sanchez-Pulido, L., Petfalski, E., Vargiu, G.,
Samejima, K., Imamoto, N., Ponting, C.P., Tollervey, D., Earnshaw,
W.C., and Vagnarelli, P. (2014). Ki-67 is a PP1-interacting protein
that organises the mitotic chromosome periphery. eLife 3,
e01641.
Brimacombe, K.R., Walsh, M.J., Liu, L., Vasquez-Valdivieso, M.G.,
Morgan, H.P., McNae, I., Fothergill-Gilmore, L.A., Michels, P.A.,
Auld, D.S., Simeonov, A., Walkinshaw, M.D., Shen, M., and Boxer,
M.B. (2014). Identification of ML251, a Potent Inhibitor of T. brucei
and T. cruzi Phosphofructokinase. ACS medicinal chemistry
letters 5, 12-17.
Brimacombe, K.R., Walsh, M.J., Liu, L., Vasquez-Valdivieso, M.G.,
Morgan, H.P., McNae, I., Fothergill-Gilmore, L.A., Michels, P.A.M.,
Auld, D.S., Simeonov, A., Walkinshaw, M.D., Shen, M., and Boxer,
M.B. (2014). Identification of ML251, a Potent Inhibitor of T. brucei
and T. cruzi Phosphofructokinase. Acs Med Chem Lett 5, 12-17.
Promote Establishment of CENP-A Chromatin. PLoS Genet 11,
e1004986.
Chathoth, K.B., J.D., Webb, S. and Beggs, J.D. (2014). A splicingdependent transcriptional checkpoint associated with prespliceosome formation. Mol Cell 53, 779-70.
Chatr-Aryamontri, A., Breitkreutz, B.J., Heinicke, S., Boucher, L.,
Winter, A., Stark, C., Nixon, J., Ramage, L., Kolas, N., O’Donnell, L.,
Reguly, T., Breitkreutz, A., Sellam, A., Chen, D., Chang, C., Rust, J.,
Livstone, M., Oughtred, R., Dolinski, K., and Tyers, M. (2013). The
BioGRID interaction database: 2013 update. Nucleic Acids Res
41, D816-823.
Chen, C.C., Dechassa, M.L., Bettini, E., Ledoux, M.B., Belisario,
C., Heun, P., Luger, K., and Mellone, B.G. (2014). CAL1 is the
Drosophila CENP-A assembly factor. The Journal of cell biology
204, 313-329.
Choi, H., Liu, G., Mellacheruvu, D., Tyers, M., Gingras, A.C., and
Nesvizhskii, A.I. (2013). Analyzing protein-protein interactions
from affinity purification-mass spectrometry data with SAINT. Curr
Protoc Bioinformatics Chapter 8, Unit8 15.
Buscaino, A., Lejeune, E., Audergon, P., Hamilton, G., Pidoux,
A., and Allshire, R.C. (2013). Distinct roles for Sir2 and RNAi
in centromeric heterochromatin nucleation, spreading and
maintenance. EMBO J 32, 1250-1264.
Choudhury, N.R., de Lima Alves, F., de Andres-Aguayo, L., Graf,
T., Caceres, J.F., Rappsilber, J., and Michlewski, G. (2013). Tissuespecific control of brain-enriched miR-7 biogenesis. Genes Dev
27, 24-38.
Carrillo, E., Ben-Ari, G., Wildenhain, J., Tyers, M., Grammentz, D.,
and Lee, T.A. (2013). Characterizing the roles of Met31 and Met32
in coordinating Met4-activated transcription in the absence of
Met30. Mol Biol Cell 23, 1928-1942.
Choudhury, N.R., Nowak, J.S., Zuo, J., Rappsilber, J., Spoel, S.H.,
and Michlewski, G. (2014). Trim25 Is an RNA-Specific Activator of
Lin28a/TuT4-Mediated Uridylation. Cell reports 9, 1265-1272.
Castillo, A.G., Pidoux, A.L., Catania, S., Durand-Dubief, M., Choi,
E.S., Hamilton, G., Ekwall, K., and Allshire, R.C. (2013). Telomeric
repeats facilitate CENP-A(Cnp1) incorporation via telomere
binding proteins. PLoS One 8, e69673.
Castonguay, E., White, S.A., Kagansky, A., St-Cyr, D.J., Castillo,
A.G., Brugger, C., White, R., Bonilla, C., Spitzer, M., Earnshaw,
W.C., Schalch, T., Ekwall, K., Tyers, M., and Allshire, R.C. (2015).
Panspecies small-molecule disruptors of heterochromatinmediated transcriptional gene silencing. Molecular and Cellular
Biology 35, 662-674.
Catania, S., and Allshire, R.C. (2014). Anarchic centromeres:
deciphering order from apparent chaos. Current Opinion in Cell
Biology 26C, 41-50.
Catania, S., Pidoux, A.L., and Allshire, R.C. (2015). Sequence
Features and Transcriptional Stalling within Centromere DNA
Clohisey, S.M., Dzhindzhev, N.S., and Ohkura, H. (2014). Kank
Is an EB1 interacting protein that localises to muscle-tendon
attachment sites in Drosophila. PLoS One 9, e106112.
Clouaire, T., Webb, S., and Bird, A. (2014). Cfp1 is required for
gene expression dependent H3K4me3 and H3K9 acetylation in
embryonic stem cells. Genome Biol 15, 451.
Colombie, N., Gluszek, A.A., Meireles, A.M., and Ohkura,
H. (2013). Meiosis-specific stable binding of augmin to
acentrosomal spindle poles promotes biased microtubule
assembly in oocytes. PLoS Genet 9, e1003562.
Cordin, O., and Beggs, J.D. (2013). RNA helicases in splicing. RNA
Biol 10, 83-95.
Cordin, O., Hahn, D., Alexander, R., Gautam, A., Saveanu, C.,
Barrass, J.D., and Beggs, J.D. (2014). Brr2p carboxy-terminal
Sec63 domain modulates Prp16 splicing RNA helicase. Nucleic
Acids Res 42, 13897-13910.
53
Davidson, L., Muniz, L., and West, S. (2014). 3’ end formation of
pre-mRNA and phosphorylation of Ser2 on the RNA polymerase
II CTD are reciprocally coupled in human cells. Genes Dev 28,
342-356.
Davidson, L., and West, S. (2013). Splicing-coupled 3’ end
formation requires a terminal splice acceptor site, but not intron
excision. Nucleic Acids Res 41, 7101-7114.
de Las Heras, J.I., Batrakou, D.G., and Schirmer, E.C. (2013).
Cancer biology and the nuclear envelope: a convoluted
relationship. Semin Cancer Biol 23, 125-137.
de Las Heras, J.I., Meinke, P., Batrakou, D.G., Srsen, V., Zuleger,
N., Kerr, A.R., and Schirmer, E.C. (2013). Tissue specificity in the
nuclear envelope supports its functional complexity. Nucleus 4,
460-477.
de Las Heras, J.I., and Schirmer, E.C. (2014). The nuclear
envelope and cancer: a diagnostic perspective and historical
overview. Advances in experimental medicine and biology 773,
5-26.
Deaton, A.M., Cook, P.C., De Sousa, D., Phythian-Adams, A.T.,
Bird, A., and Macdonald, A.S. (2014). A unique DNA methylation
signature defines a population of IFN-gamma/IL-4 doublepositive T cells during helminth infection. European Journal of
Immunology 44, 1835-1841.
Delan-Forino, C., and Tollervey, D. (2014). Lighting Up pre-mRNA
recognition. Molecular Cell 55, 649-651.
Delgoshaie, N., Tang, X., Kanshin, E.D., Williams, E.C., Rudner,
A.D., Thibault, P., Tyers, M., and Verreault, A. (2014). Regulation of
the histone deactylase Hst3 by cyclin-dependent kinases and the
ubiquitin ligase SCFCdc4. J Biol Chem in press.
Dix, C.I., Soundararajan, H.C., Dzhindzhev, N.S., Begum, F.,
Suter, B., Ohkura, H., Stephens, E., and Bullock, S.L. (2013).
Lissencephaly-1 promotes the recruitment of dynein and
dynactin to transported mRNAs. The Journal of Cell Biology 202,
479-494.
Duro, E., and Marston, A.L. (2015). From equator to pole: splitting
chromosomes in mitosis and meiosis. Genes Dev 29, 109-122.
Earnshaw, W.C. (2013). Deducing protein function by forensic
integrative cell biology. PLoS Biol 11, e1001742.
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59
International Scientific Advisory Board
Angelika Amon
Center for Cancer Research
Howard Hughes Medical Institute
Massachusetts Institute of Technology
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Frank Grosveld
Department of Cell Biology
Erasmus Medical Center
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The Rockefeller University
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Eric Karsenti
European Molecular Biology Laboratories
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Nick Proudfoot
Sir William Dunn School of Pathology
University of Oxford
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Margaret Fuller
Department of Developmental Biology
and Department of Genetics
Stanford University School of Medicine
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USA
Wellcome Trust Four-year PhD Programme
in Cell Biology
Wellcome Trust Centre for Cell Biology
University of Edinburgh
The four-year PhD programme at the Centre for Cell
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At the end of the first year, the degree of MSc by
Research is awarded to qualifying students, based
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This is followed by three further years of full time
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Cover image: Cover image shows human neurons in culture, contributed by Justyna Cholewa-Waclaw and Adrian Bird
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