The Wellcome Trust Centre for Cell Biology
Transcription
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. 2 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 3 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. 4 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. 5 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. 6 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. 7 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. 10 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. 11 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. 12 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. 13 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 14 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. 54 Earnshaw, W.C., Allshire, R.C., Black, B.E., Bloom, K., Brinkley, B.R., Brown, W., Cheeseman, I.M., Choo, K.H., Copenhaver, G.P., Deluca, J.G., Desai, A., Diekmann, S., Erhardt, S., FitzgeraldHayes, M., Foltz, D., Fukagawa, T., Gassmann, R., Gerlich, D.W., Glover, D.M., Gorbsky, G.J., Harrison, S.C., Heun, P., Hirota, T., Jansen, L.E., Karpen, G., Kops, G.J., Lampson, M.A., Lens, S.M., Losada, A., Luger, K., Maiato, H., Maddox, P.S., Margolis, R.L., Masumoto, H., McAinsh, A.D., Mellone, B.G., Meraldi, P., Musacchio, A., Oegema, K., O’Neill, R.J., Salmon, E.D., Scott, K.C., Straight, A.F., Stukenberg, P.T., Sullivan, B.A., Sullivan, K.F., Sunkel, C.E., Swedlow, J.R., Walczak, C.E., Warburton, P.E., Westermann, S., Willard, H.F., Wordeman, L., Yanagida, M., Yen, T.J., Yoda, K., and Cleveland, D.W. (2013). Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21, 101-106. Earnshaw, W.C., and Cleveland, D.W. (2013). CENP-A and the CENP nomenclature: response to Talbert and Henikoff. Trends in Genetics 29, 500-502. Ebert, D.H., Gabel, H.W., Robinson, N.D., Kastan, N.R., Hu, L.S., Cohen, S., Navarro, A.J., Lyst, M.J., Ekiert, R., Bird, A.P., and Greenberg, M.E. (2013). Activity-dependent phosphorylation of MECP2 threonine 308 regulates interaction with NcoR. Nature 499, 341-345. 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 Genet 10, e1004716. Ernst, A., Avvakumov, G., Tong, J., Fan, Y., Zhao, Y., Alberts, P., Persaud, A., Walker, J.R., Neculai, A.M., Neculai, D., Vorobyov, A., Garg, P., Beatty, L., Chan, P.K., Juang, Y.C., Landry, M.C., Yeh, C., Zeqiraj, E., Karamboulas, K., Allali-Hassani, A., Vedadi, M., Tyers, M., Moffat, J., Sicheri, F., Pelletier, L., Durocher, D., Raught, B., Rotin, D., Yang, J., Moran, M.F., Dhe-Paganon, S., and Sidhu, S.S. (2013). A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590-595. Fernius, J., Nerusheva, O.O., Galander, S., Alves Fde, L., Rappsilber, J., and Marston, A.L. (2013). Cohesin-dependent association of scc2/4 with the centromere initiates pericentromeric cohesion establishment. Curr Biol 23, 599-606. Fischer, L., Chen, Z.A., and Rappsilber, J. (2013). Quantitative cross-linking/mass spectrometry using isotope-labelled crosslinkers. J Proteomics 88, 120-128. Folco, H.D., Campbell, C.S., May, K.M., Espinoza, C.A., Oegema, K., Hardwick, K.G., Grewal, S.I., and Desai, A. (2015). The CENP-A N-tail confers epigenetic stability to centromeres via the CENP-T branch of the CCAN in fission yeast. Curr Biol 25, 348-356. Fraser, J.A., Worrall, E.G., Lin, Y., Landre, V., Pettersson, S., Blackburn, E., Walkinshaw, M., Muller, P., Vojtesek, B., Ball, K., and Hupp, T.R. (2015). Phosphomimetic Mutation of the N-Terminal Lid of MDM2 Enhances the Polyubiquitination of p53 through Stimulation of E2-Ubiquitin Thioester Hydrolysis. Journal of Molecular Biology 427, 1728-1747. Garcia-Gomez, J.J., Fernandez-Pevida, A., Lebaron, S., Rosado, I.V., Tollervey, D., Kressler, D., and de la Cruz, J. (2014). Final pre40S maturation depends on the functional integrity of the 60S subunit ribosomal protein L3. PLoS Genet 10, e1004205. Gardano, L., Pucci, F., Christian, L., Le Bihan, T., and Harrington, L. (2013). Telomeres, a busy platform for cell signaling. Front Oncol 3, 146. Garg, S.K., Lioy, D.T., Cheval, H., McGann, J.C., Bissonnette, J.M., Murtha, M.J., Foust, K.D., Kaspar, B.K., Bird, A., and Mandel, G. (2013). Systemic delivery of MeCP2 rescues behavioral and cellular deficits in female mouse models of Rett syndrome. The Journal of Neuroscience: the official journal of the Society for Neuroscience 33, 13612-13620. Gautam, A., Grainger, R.J., Vilardell, J., Barrass, J.D., and Beggs, J.D. (2015). Cwc21p promotes the second step conformation of the spliceosome and modulates 3’ splice site selection. Nucleic Acids Res. doi: 10.1093/nar/gkv159. Gordon, K., Clouaire, T., Bao, X.X., Kemp, S.E., Xenophontos, M., de Las Heras, J.I., and Stancheva, I. (2013). Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression. Nucleic Acids Res. 42, 3529-3541. 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. Nucleic Acids Res 42, 1213812154. Helwak, A., Kudla, G., Dudnakova, T., and Tollervey, D. (2013). Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153, 654-665. Helwak, A., and Tollervey, D. (2014). Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH). Nature Protocols 9, 711-728. stabilization of a low-affinity interface with ubiquitin. Nature Chemical Biology 10, 156-163. Illingworth, R.S., Gruenewald-Schneider, U., De Sousa, D., Webb, S., Merusi, C., Kerr, A.R., James, K.D., Smith, C., Walker, R., Andrews, R., and Bird, A.P. (2015). Inter-individual variability contrasts with regional homogeneity in the human brain DNA methylome. Nucleic Acids Res. 43(2), 732-744. Jacob, Y., Bergamin, E., Donoghue, M.T., Mongeon, V., LeBlanc, C., Voigt, P., Underwood, C.J., Brunzelle, J.S., Michaels, S.D., Reinberg, D., Couture, J.F., and Martienssen, R.A. (2014). Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343, 1249-1253. Keszei, A.F., Tang, X., McCormick, C., Zeqiraj, E., Rohde, J.R., Tyers, M., and Sicheri, F. (2014). Structure of an SspH1-PKN1 complex reveals the basis for host substrate recognition and mechanism of activation for a bacterial E3 ubiquitin ligase. Molecular and Cellular Biology 34, 362-373. Kim, H.S., Mukhopadhyay, R., Rothbart, S.B., Silva, A.C., Vanoosthuyse, V., Radovani, E., Kislinger, T., Roguev, A., Ryan, C.J., Xu, J., Jahari, H., Hardwick, K.G., Greenblatt, J.F., Krogan, N.J., Fillingham, J.S., Strahl, B.D., Bouhassira, E.E., Edelmann, W., and Keogh, M.C. (2014). Identification of a BET Family Bromodomain/ Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function. Cell Reports 6, 892-905. Kim, J.H., Zhang, T., Wong, N.C., Davidson, N., Maksimovic, J., Oshlack, A., Earnshaw, W.C., Kalitsis, P., and Hudson, D.F. (2013). Condensin I associates with structural and gene regulatory regions in vertebrate chromosomes. Nat Commun 4, 2537. Kononenko, A.V., Lee, N.C., Earnshaw, W.C., Kouprina, N., and Larionov, V. (2013). Re-engineering an alphoid(tetO)-HAC-based vector to enable high-throughput analyses of gene function. Nucleic Acids Res 41, e107. Kustatscher, G., Hegarat, N., Wills, K.L., Furlan, C., Bukowski-Wills, J.C., Hochegger, H., and Rappsilber, J. (2014). Proteomics of a fuzzy organelle: interphase chromatin. EMBO J 33, 648-664. Houston, D.R., and Walkinshaw, M.D. (2013). Consensus docking: improving the reliability of docking in a virtual screening context. J Chem Inf Model 53, 384-390. Lebaron, S., Segerstolpe, A., French, S.L., Dudnakova, T., de Lima Alves, F., Granneman, S., Rappsilber, J., Beyer, A.L., Wieslander, L., and Tollervey, D. (2013). Rrp5 binding at multiple sites coordinates pre-rRNA processing and assembly. Molecular Cell 52, 707-719. Huang, H., Ceccarelli, D.F., Orlicky, S., St-Cyr, D.J., Ziemba, A., Garg, P., Plamondon, S., Auer, M., Sidhu, S., Marinier, A., Kleiger, G., Tyers, M., and Sicheri, F. (2014). E2 enzyme inhibition by Lee, H.S., Lee, N.C., Grimes, B.R., Samoshkin, A., Kononenko, A.V., Bansal, R., Masumoto, H., Earnshaw, W.C., Kouprina, N., and Larionov, V. (2013). A new assay for measuring chromosome 55 instability (CIN) and identification of drugs that elevate CIN in cancer cells. BMC cancer 13, 252. Lee, N.C., Kononenko, A.V., Lee, H.S., Tolkunova, E.N., Liskovykh, M.A., Masumoto, H., Earnshaw, W.C., Tomilin, A.N., Larionov, V., and Kouprina, N. (2013). Protecting a transgene expression from the HAC-based vector by different chromatin insulators. Cellular and molecular life sciences : CMLS 70, 3723-3737. Marston, A.L. (2015). Shugoshins: tension-sensitive pericentromeric adaptors safeguarding chromosome segregation. Molecular and Cellular Biology 35, 634-648. 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. Matsuo, Y., Granneman, S., Thoms, M., Manikas, R.G., Tollervey, D., and Hurt, E. (2014). Coupled GTPase and remodelling ATPase activities form a checkpoint for ribosome export. Nature 505, 112-116. Leung, E., Schneider, C., Yan, F., Mohi-El-Din, H., Kudla, G., Tuck, A., Wlotzka, W., Doronina, V.A., Bartley, R., Watkins, N.J., Tollervey, D., and Brown, J.D. (2014). Integrity of SRP RNA is ensured by La and the nuclear RNA quality control machinery. Nucleic Acids Res 42, 10698-10710. Mayer, M.C., Kaden, D., Schauenburg, L., Hancock, M.A., Voigt, P., Roeser, D., Barucker, C., Than, M.E., Schaefer, M., and Multhaup, G. (2014). Novel zinc-binding site in the E2 domain regulates amyloid precursor-like protein 1 (APLP1) oligomerization. J Biol Chem 289, 19019-19030. Liz, J., Portela, A., Soler, M., Gomez, A., Ling, H., Michlewski, G., Calin, G.A., Guil, S., and Esteller, M. (2014). Regulation of primiRNA processing by a long noncoding RNA transcribed from an ultraconserved region. Molecular cell 55, 138-147. McLeod, F., Ganley, R., Williams, L., Selfridge, J., Bird, A., and Cobb, S.R. (2013). Reduced seizure threshold and altered network oscillatory properties in a mouse model of Rett syndrome. Neuroscience 231, 195-205. Loc’h, J., Blaud, M., Rety, S., Lebaron, S., Deschamps, P., Bareille, J., Jombart, J., Robert-Paganin, J., Delbos, L., Chardon, F., Zhang, E., Charenton, C., Tollervey, D., and Leulliot, N. (2014). RNA mimicry by the fap7 adenylate kinase in ribosome biogenesis. PLoS Biol 12, e1001860. Meinke, P., Makarov, A.A., Lê Thành, P., Sadurska, D., and Schirmer, E.C. (2015). Nucleoskeleton dynamics and functions in health and disease. . Cell Health and Cytoskeleton 7, 55-69. 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. The Journal of Cell Biology 208, 521-531. Lynch, E.M., Groocock, L.M., Borek, W.E., and Sawin, K.E. (2014). Activation of the γ-tubulin complex by the Mto1/2 complex. Curr Biol 10.1016/j.cub.2014.03.006. Lyst, M.J., and Bird, A. (2015). Rett syndrome: a complex disorder with simple roots. Nature Reviews Genetics 10.1038/nrg3897. Lyst, M.J., Ekiert, R., Ebert, D.H., Merusi, C., Nowak, J., Selfridge, J., Guy, J., Kastan, N.R., Robinson, N.D., de Lima Alves, F., Rappsilber, J., Greenberg, M.E., and Bird, A. (2013). Rett syndrome mutations abolish the interaction of MeCP2 with the NCoR/SMRT co-repressor. Nat Neurosci 16, 898-902. 56 Marston, A.L. (2014). Chromosome segregation in budding yeast: sister chromatid cohesion and related mechanisms. Genetics 196, 31-63. Malik, P., Zuleger, N., de las Heras, J.I., Ros, N., Makarov, A.A., Lazou, V., Meinke, P., Waterfall, M., Kelly, D.A., and Schirmer, E.C. (2014). NET23/STING promotes chromatin compaction from the nuclear envelope. PLoS One 9(11). Meinke, P., Schneiderat, P., Srsen, V., Korfali, N., Le Thanh, P., Cowan, G.J., 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 Emery-Dreifuss muscular dystrophy. Neuromuscular disorders. NMD 25, 127-136. Miell, M.D., Fuller, C.J., Guse, A., Barysz, H.M., Downes, A., Owen-Hughes, T., Rappsilber, J., Straight, A.F., and Allshire, R.C. (2013). CENP-A confers a reduction in height on octameric nucleosomes. Nat Struct Mol Biol 20, 763-765. Miell, M.D., Straight, A.F., and Allshire, R.C. (2014). Reply to “CENP-A octamers do not confer a reduction in nucleosome height by AFM”. Nat Struct Mol Biol 21, 5-8. Monnier, P., Martinet, C., Pontis, J., Stancheva, I., Ait-Si-Ali, S., and Dandolo, L. (2013). H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1. Proc Natl Acad Sci USA 110, 20693-20698. Morgan, H.P., O’Reilly, F.J., Wear, M.A., O’Neill, J.R., FothergillGilmore, L.A., Hupp, T., and Walkinshaw, M.D. (2013). M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation. Proc Natl Acad Sci USA 110, 5881-5886. Morgan, H.P., Zhong, W., McNae, I.W., Michels, P.A.M., FothergillGilmore, L.A., and Walkinshaw, M.D. (2014). Structures of pyruvate kinases display evolutionarily divergent allosteric strategies. Royal Society Open Science 1(1), 140120. Nerusheva, O.O., Galander, S., Fernius, J., Kelly, D., and Marston, A.L. (2014). Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation. Genes Dev 28, 1291-1309. Nicholson, J., Scherl, A., Way, L., Blackburn, E.A., Walkinshaw, M.D., Ball, K.L., and Hupp, T.R. (2014). A systems wide mass spectrometric based linear motif screen to identify dominant invivo interacting proteins for the ubiquitin ligase MDM2. Cellular Signalling 26, 1243-1257. 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. Pai, C.C., Deegan, R.S., Subramanian, L., Gal, C., Sarkar, S., Blaikley, E.J., Walker, C., Hulme, L., Bernhard, E., Codlin, S., Bahler, J., Allshire, R., Whitehall, S., and Humphrey, T.C. (2014). A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice. Nat Commun 5, 4091. Paraskevopoulos, K., Kriegenburg, F., Tatham, M.H., Rosner, H.I., Medina, B., Larsen, I.B., Brandstrup, R., Hardwick, K.G., Hay, R.T., Kragelund, B.B., Hartmann-Petersen, R., and Gordon, C. (2014). Dss1 is a 26S proteasome ubiquitin receptor. Molecular Cell 56, 453-461. Patel, H., Zich, J., Serrels, B., Rickman, C., Hardwick, K.G., Frame, M.C., and Brunton, V.G. (2013). Kindlin-1 regulates mitotic spindle formation by interacting with integrins and Plk-1. Nat Commun 4, 2056. Patton, E.E., and Harrington, L. (2013). Cancer: Trouble upstream. Nature 495, 320-321. Nowak, J., Choudhury, N.R., de Lima Alves, F., Rappsilber, J., and Michlewski, G. (2014). Lin28a regulates neuronal differentiation and inhibits miR-9 production. Nat Commun 5, 3687. Pucci, F., Gardano, L., and Harrington, L. (2013). Short telomeres in ESCs lead to unstable differentiation. Cell Stem Cell 12, 479486. Nowak, J.S., and Michlewski, G. (2013). miRNAs in development and pathogenesis of the nervous system. Biochemical Society Transactions 41, 815-820. Ribeiro, S.A., Vagnarelli, P., and Earnshaw, W.C. (2014). DNA content of a functioning chicken kinetochore. Chromosome Res 10.1007/s10577-014-9410-3. Ohkura, H. (2015). Meiosis: An Overview of Key Differences from Mitosis. Cold Spring Harbor Perspectives in Biology. a015859. Robson, M.I., Le Thanh, P., and Schirmer, E.C. (2014). NETs and Cell Cycle Regulation. Advances in Experimental Medicine and Biology 773, 165-185. Orchard, S., Kerrien, S., Abbani, S., Aranda, B., Bhate, J., Bidwell, S., Bridge, A., Briganti, L., Brinkman, F.S., Cesareni, G., Chatraryamontri, A., Chautard, E., Chen, C., Dumousseau, M., Goll, J., Hancock, R.E., Hannick, L.I., Jurisica, I., Khadake, J., Lynn, D.J., Mahadevan, U., Perfetto, L., Raghunath, A., Ricard-Blum, S., Roechert, B., Salwinski, L., Stumpflen, V., Tyers, M., Uetz, P., Xenarios, I., and Hermjakob, H. (2013). Protein interaction data curation: the International Molecular Exchange (IMEx) consortium. Nat Methods 9, 345-350. Padeken, J., and Heun, P. (2013). Centromeres in nuclear architecture. Cell Cycle 12, 3455-3456. Padeken, J., and Heun, P. (2014). Nucleolus and nuclear periphery: velcro for heterochromatin. Current Opinion in Cell Biology 28, 54-60. 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. Sadowski, I., Breitkreutz, B.J., Stark, C., Su, T.C., Dahabieh, M., Raithatha, S., Bernhard, W., Oughtred, R., Dolinski, K., Barreto, K., and Tyers, M. (2013). The PhosphoGRID Saccharomyces cerevisiae protein phosphorylation site database: version 2.0 update. Database (Oxford) 2013, bat026. Sarangapani, K.K., Duro, E., Deng, Y., Alves Fde, L., Ye, Q., Opoku, K.N., Ceto, S., Rappsilber, J., Corbett, K.D., Biggins, S., Marston, A.L., and Asbury, C.L. (2014). Sister kinetochores are mechanically fused during meiosis I in yeast. Science 346, 248-251. Sardana, R., Liu, X., Granneman, S., Zhu, J., Gill, M., Papoulas, O., Marcotte, E.M., Tollervey, D., Correll, C.C., and Johnson, A.W. (2014). The DEAH-box helicase Dhr1 Dissociates U3 from the pre-rRNA to promote formation of the Central Pseudoknot. . PLoS Biol 13(2). Schneider, C., and Tollervey, D. (2013). Threading the barrel of the RNA exosome. Trends in biochemical sciences 38, 485-493. 57 Schneider, C., and Tollervey, D. (2014). Looking into the barrel of the RNA exosome. Nat Struct Mol Biol 21, 17-18. expression. Advances in experimental medicine and biology 773, 209-244. Segerstolpe, A., Granneman, S., Bjork, P., de Lima Alves, F., Rappsilber, J., Andersson, C., Hogbom, M., Tollervey, D., and Wieslander, L. (2013). Multiple RNA interactions position Mrd1 at the site of the small subunit pseudoknot within the 90S preribosome. Nucleic Acids Res 41, 1178-1190. Subramanian, L., Toda, N.R.T., Rappsilber, J., Allshire, R.C. (2014). Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly. Open Biology 4(4), 140043. 10.1098/ rsob.140043. Serrano, L., Martinez-Redondo, P., Marazuela-Duque, A., Vazquez, B.N., Dooley, S.J., Voigt, P., Beck, D.B., Kane-Goldsmith, N., Tong, Q., Rabanal, R.M., Fondevila, D., Munoz, P., Kruger, M., Tischfield, J.A., and Vaquero, A. (2013). The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation. Genes Dev 27, 639-653. Shang, W.H., Hori, T., Martins, N.M., Toyoda, A., Misu, S., Monma, N., Hiratani, I., Maeshima, K., Ikeo, K., Fujiyama, A., Kimura, H., Earnshaw, W.C., and Fukagawa, T. (2013). Chromosome engineering allows the efficient isolation of vertebrate neocentromeres. Dev Cell 24, 635-648. Sharma, A., Pohlentz, G., Bobbili, K.B., Jeyaprakash, A.A., Chandran, T., Mormann, M., Swamy, M.J., and Vijayan, M. (2013). The sequence and structure of Snake Gourd (Trichosanthes Anguina) Seed Lectin, a three-chain nontoxic homologue of type II RIP. Acta Crystallogr Sect F Struct Biol Cryst Commun D69, 1493-1503. Shave, S., Blackburn, E.A., Adie, J., Houston, D.R., Auer, M., Webster, S.P., Taylor, P., and Walkinshaw, M.D. (2015). UFSRAT: Ultra-fast Shape Recognition with Atom Types--the discovery of novel bioactive small molecular scaffolds for FKBP12 and 11betaHSD1. PLoS One 10, e0116570. Sloan, K.E., Mattijssen, S., Lebaron, S., Tollervey, D., Pruijn, G.J., and Watkins, N.J. (2013). Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing. The Journal of Cell Biology 200, 577-588. Song, C.D., Feodorova, Y., Guy, J., Peichl, L., Jost, K.L., Kimura, H., Cardoso, M.C., Bird, A., Leonhardt, H., Joffe, B., and Solovei, I. (2014). DNA methylation reader MECP2: cell type- and differentiation stage-specific protein distribution. Epigenet Chromatin 7:17 doi:10.1186/1756-8935-7-17. Spitzer, M., Wildenhain, J., Rappsilber, J., and Tyers, M. (2014). BoxPlotR: a web tool for generation of box plots. Nat Methods 11, 121-122. 58 Stancheva, I., and Schirmer, E.C. (2014). Nuclear envelope: connecting structural genome organization to regulation of gene Suzuki, M.M., Yoshinari, A., Obara, M., Takuno, S., Shigenobu, S., Sasakura, Y., Kerr, A.R., Webb, S., Bird, A., and Nakayama, A. (2013). Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells. Epigenetics & Chromatin 6, 38. 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. Molecular & Cellular Proteomics: MCP 12, 3135-3147. Tachiwana, H., Miya, Y., Shono, N., Ohzeki, J., Osakabe, A., Otake, K., Larionov, V., Earnshaw, W.C., Kimura, H., Masumoto, H., and Kurumizaka, H. (2013). Nap1 regulates proper CENP-B binding to nucleosomes. Nucleic Acids Res 41, 2869-2880. Tannock, I., Hill, R., Bristow, R.G., and Harrington, L. (2013). Basical Science of Oncology. 5 edn (New York, McGraw Hill). Thomae, A.W., Schade, G.O., Padeken, J., Borath, M., Vetter, I., Kremmer, E., Heun, P., and Imhof, A. (2013). A pair of centromeric proteins mediates reproductive isolation in Drosophila species. Dev Cell 27, 412-424. Travis, A.J., Moody, J., Helwak, A., Tollervey, D., and Kudla, G. (2014). Hyb: a bioinformatics pipeline for the analysis of CLASH (crosslinking, ligation and sequencing of hybrids) data. Methods 65, 263-273. Tree, J.J., Granneman, S., McAteer, S.P., Tollervey, D., and Gally, D.L. (2014). Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli. Molecular Cell 55, 199-213. Trubitsyna, M., Michlewski, G., Cai, Y., Elfick, A., and French, C.E. (2014). PaperClip: rapid multi-part DNA assembly from existing libraries. Nucleic Acids Res 42, e154. Tuck, A., and Tollervey, D. (2013). Functions of long non-coding RNAs in non-mammalian. Systems Molecular Biology of Long Non-coding RNAs (Khalil, A and Coller, J Eds), 137-162 (Springer). Tuck, A.C., and Tollervey, D. (2013). A transcriptome-wide atlas of RNP composition reveals diverse classes of mRNAs and lncRNAs. Cell 154, 996-1009. 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. Zhong, W., Morgan, H.P., Nowicki, M.W., McNae, I.W., Yuan, M., Bella, J., Michels, P.A., Fothergill-Gilmore, L.A., and Walkinshaw, M.D. (2014). Pyruvate kinases have an intrinsic and conserved decarboxylase activity. The Biochemical Journal 458, 301-311. Turowski, T.W., and Tollervey, D. (2015). Cotranscriptional events in eukaryotic ribosome synthesis. Wiley interdisciplinary reviews RNA 6, 129-139. Zuleger, N., Boyle, S., Kelly, D.A., de Las Heras, J.I., Lazou, V., Korfali, N., Batrakou, D.G., Randles, K.N., Morris, G.E., Harrison, D.J., 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, R14. Verzijlbergen, K.F., Nerusheva, O.O., Kelly, D., Kerr, A., Clift, D., de Lima Alves, F., Rappsilber, J., and Marston, A.L. (2014). Shugoshin biases chromosomes for biorientation through condensin recruitment to the pericentromere. eLife 3, e01374. Voigt, P., and Reinberg, D. (2013). Putting a halt on PRC2 in pediatric glioblastoma. Nature Genetics 45, 587-589. Zuleger, N., Kelly, D.A., and Schirmer, E.C. (2013). Considering discrete protein pools when measuring the dynamics of nuclear membrane proteins. Methods in Molecular Biology 1042, 275298. Voigt, P., and Reinberg, D. (2013). Epigenome editing. Nature Biotechnology 31, 1097-1099. Voigt, P., Tee, W.W., and Reinberg, D. (2013). A double take on bivalent promoters. Genes Dev 27, 1318-1338. Wachter, E., Quante, T., Merusi, C., Arczewska, A., Stewart, F., Webb, S., and Bird, A. (2014). Synthetic CpG islands reveal DNA sequence determinants of chromatin structure. eLife 3. Walkinshaw, M. (2014). Multiple chemical scaffolds inhibit a promising Leishmania drug target. IUCrJ 1, 202-203. Weber, G., Cristao, V.F., Santos, K.F., Jovin, S.M., Heroven, A.C., Holton, N., Luhrmann, R., Beggs, J.D., and Wahl, M.C. (2013). Structural basis for dual roles of Aar2p in U5 snRNP assembly. Genes Dev 27, 525-540. Welburn, J.P. (2013). The molecular basis for kinesin functional specificity during mitosis. Cytoskeleton 70, 476-493. White, S.A., Buscaino, A., Sanchez-Pulido, L., Ponting, C.P., Nowicki, M.W., and Allshire, R.C. (2014). The RFTS domain of Raf2 is required for Cul4 interaction and heterochromatin integrity in fission yeast. PLoS One 9, e104161. Wong, L.H., Unciti-Broceta, A., Spitzer, M., White, R., Tyers, M., and Harrington, L. (2013). A yeast chemical genetic screen identifies inhibitors of human telomerase. Chem Biol 20, 333-340. Wongsombat, C., Aroonsri, A., Kamchonwongpaisan, S., Morgan, H.P., Walkinshaw, M.D., Yuthavong, Y., and Shaw, P.J. (2014). Molecular characterization of Plasmodium falciparum Bruno/CELF RNA binding proteins. Molecular and Biochemical Parasitology 198, 1-10. 59 International Scientific Advisory Board Angelika Amon Center for Cancer Research Howard Hughes Medical Institute Massachusetts Institute of Technology 40 Ames Street Cambridge MA 02139 Frank Grosveld Department of Cell Biology Erasmus Medical Center Dr Molewaterplein 50 3000 Rotterdam Netherlands Michael Rout The Rockefeller University 1230 York Avenue New York, NY 10021 USA Eric Karsenti European Molecular Biology Laboratories Meyerhofstraße 1 69117 Heidelberg Germany Nick Proudfoot Sir William Dunn School of Pathology University of Oxford South Parks Road Oxford OX3 60 Margaret Fuller Department of Developmental Biology and Department of Genetics Stanford University School of Medicine 291 Campus Drive, Li Ka Shing Building Stanford, CA 94305-5101 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 Biology offers training in cell biology from outstanding researchers in a stimulating environment. Five places are available each year by competitive interview. The first year of the programme combines miniprojects with taught courses which cover a wide range of techniques important for modern cell biology including advanced microscopy, molecular biology, proteomics, bioinformatics and systems biology. At the end of the first year, the degree of MSc by Research is awarded to qualifying students, based on rotation project reports and a PhD project outline. This is followed by three further years of full time research. Funding from the Wellcome Trust for this programme includes a generous student stipend and payment of tuition fees (at the EU rate). For further information visit www.wcb.ed.ac.uk/phd Wellcome Trust Centre for Cell Biology School of Biological Sciences The University of Edinburgh Michael Swann Building Mayfield Road Edinburgh EH9 3JR Scotland, UK Telephone +44 (0)131 650 7005 Fax +44 (0)131 650 4968 Website www.wcb.ed.ac.uk Cover image: Cover image shows human neurons in culture, contributed by Justyna Cholewa-Waclaw and Adrian Bird The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336 1