2018 Wellcome Centre for Cell Biology 2018 Historical Background

Text The expansion of research in cell biology was planned in 1992 as a result of the vision of Professor Sir Kenneth Murray, who was at the time Biogen Professor at the Institute of Cell and Molecular Biology. A seed contribution of £2.5 million from the Darwin Trust was followed by financial commitments from The Wolfson Foundation, the University and the Wellcome Trust, allowing construction of the Michael Swann Building. The majority of research space was earmarked for Wellcome Trust-funded research. Recruitment, based on research excellence at all levels in the area of cell biology, began in earnest in 1993, mostly but not exclusively, through the award of Research Fellowships from the Wellcome Trust. The Swann Building was first occupied by new arrivals in January 1996 and became “The Wellcome Trust Centre for Cell Biology” from October 2001. Core funding for the Centre from the Wellcome Trust was renewed in 2006 and 2011.

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Director’s Report 04 Robin Allshire 06 A. Jeyaprakash Arulanandam 08 Jean Beggs 10 Adrian Bird 12 Atlanta Cook 14 Bill Earnshaw 16 Patrick Heun 18 Tony Ly 20 Adele Marston 22 Hiro Ohkura 24 Juri Rappsilber 26 Kenneth E. Sawin 28 Eric Schirmer 30 David Tollervey 32 Philipp Voigt 34 Malcolm Walkinshaw 36 Julie Welburn 38 Public Engagement 40 List of Groups 42 Centre Publications 2016 - 2018 48 International Scientific Advisory Board 56

3 Director’s Report

The Wellcome Centre for Cell Biology (WCB) is one of This brochure presents a very brief overview of our research fifteen UK-based, Wellcome-funded research centres. The and highlights some of the exciting results obtained seventeen research groups that currently comprise the WCB, during the past year. I am delighted that the WCB has and our associated research facilities, occupy the Michael again made excellent progress with every group reporting Swann Building on the King’s Buildings Campus of the innovative, world-class research, as reflected in our many University of Edinburgh. Constructed in the mid 1990s, the notable publications. Centre staff have also delivered an Swann Building was designed as a centre for research in outstanding programme of public engagement; my thanks molecular cell biology. go to everyone who gave up their time and brought their enthusiasm to share our work with the broader community Cell biology is a broad discipline that encompasses the in Edinburgh. study of all that it takes to make a cell – from molecules to biological pathways and complex structures. Astonishingly, I would like to take this opportunity to congratulate WCB our bodies are made up of some 50 trillion human cells, staff for some notable successes during the past year. In every one of which contains an entire copy of the genome particular, A. Jeyaprakash Arulanandam, Atlanta Cook, and and all the machinery needed to duplicate itself. The many Julie Wellburn, who joined us as junior Research Career biochemical reactions taking place in those cells form Development Fellows, have now been awarded highly pathways that are highly organised; physically, in space competitive Wellcome Senior Research Fellowships. Their and in time. The key goal of the WCB is to gain new insights success in achieving this key step in the progression of into cell structure and function at levels from molecular their scientific careers is both a tribute to their insight and interactions to complex systems. dedication and a positive indication of the support and Research in the Centre is particularly strong in the broad field mentoring provided by their WCB colleagues. I am also of cellular epigenetic mechanisms. The term epigenetics very pleased to welcome the newest member of WCB, was coined by Conrad Waddington, the former Professor Tony Ly, who joined us in September 2017. Tony obtained a of Genetics in Edinburgh, as “the branch of biology which prestigious Sir Henry Dale Fellowship from the Royal Society studies the causal interactions between genes and their and Wellcome to establish his WCB research group, which products which bring the phenotype into being”. This focuses on the use of mass spectrometry to follow proteomic original definition embraces all systems controlling gene changes during the cell cycle. We also congratulate Dhanya expression in eukaryotes and encompasses key topics of Cheerambathur and Marcus Wilson, who are recent our research. By bringing together the major themes of recipients of Sir Henry Dale Fellowships. Dhanya, who will nuclear organisation, genome packaging and transmission, shortly join us from the University of California, San Diego, chromatin states and RNA biology, we aim to chart key aims to understand the specialised microtubule architecture interconnections between these processes and identify their of neuronal cells during nervous system development. mechanisms, regulation and role(s) in human disease. Marcus will be moving to Edinburgh from the Francis Crick

4 Institute, to determine the mechanisms that underpin establishment and maintenance of DNA methylation, a key epigenetic mark, and its functional consequences. The recruitment and progression of these outstanding younger researchers bodes well for the long-term success of WCB. More senior WCB researchers have also been honoured recently, with the award of the Charles Rudolphe Brupbacher Prize to Sir Adrian Bird, the Mendel Medal to David Tollervey and the RNA Society Lifetime Achievement Award to Jean Beggs.

Let me end by reiterating my deepest thanks to all of our talented and dedicated researchers and support staff and congratulating them again on their excellent work over the year. Their efforts underpin all of the continuing successes of the WCB.

David Tollervey

5 Epigenetic inheritance: establishment and transmission of specialized chromatin domains

Chromosomal DNA is wrapped around nucleosomes containing core histones (H3/H4/H2A/ H2B). However, at centromeres a specific histone H3 variant, CENP-A, replaces histone H3 to form specialized CENP-A nucleosomes. CENP-A chromatin is critical for assembly of the chromosome segregation machinery – kinetochores – at these specific chromosomal locations and is flanked by histone H3 lysine 9 methylated heterochromatin. Our goal is to decipher conserved mechanisms that establish, maintain and regulate the Robin Allshire assembly of heterochromatin and CENP-A chromatin domains. Heterochromatin is required for the establishment of CENP-A chromatin on centromere DNA. One objective is to provide further Co-workers: insight into mechanisms that promote heterochromatin formation on pericentromeric repeats. Heterochromatin might also silence genes throughout the genome, we therefore also investigate Tatsiana Auchynnikava how heterochromatin formation is regulated and whether such mechanisms influence Roberta Carloni phenotype. We endeavour to determine how heterochromatin, spatial nuclear organisation and non-coding RNAPII transcription combine to mediate CENP-A incorporation at centromeres. Tadhg Devlin Our main questions are: Lorenza Di Pompeo 1. How do DNA, RNA and chromatin signatures instigate the assembly of specialized chromatin domains? Max Fitz-James 2. How does chromatin architecture and subnuclear compartmentalization affect specialized chromatin domains? Alison Pidoux 3. How does heterochromatin influence gene expression? Desislava Staneva Long-read sequencing allowed the de novo assembly of the genomes of two fission yeast Manu Shukla species that are evolutionarily distinct from Schizosaccharomyces pombe. Our assemblies are contiguous across all three centromeres, and other heterochromatin regions, of both Puneet Singh species and permits comparison of centromere organization between these divergent species (Figure a). Centromeres from all three species retain an overall structural resemblance, Pin Tong however, no sequence similarity is detected between repetitive elements and central Jesus Torres-Garcia regions of even the closest two species, apart from the presence of similarly ordered tRNA genes (Figure b). Interspecies functional tests reveal that non-homologous S. octosporus Gabor Varga centromere DNA is recognized and promotes centromere assembly in S. pombe. We surmise that conserved processes recognize features associated with non-conserved sequences Sharon White allowing preservation of centromere identity and location over evolutionary time. Principal Weifang Wu Component Analysis detect a distinct pattern of all possible nucleotide pentamers enriched in CENP-A-associated DNA from all three species (Figure c). Conserved processes, such as Imtiyaz Yaseen transcription, may promote recognition of these centromeric DNAs and the replacement of histone H3 chromatin with CENP-A chromatin. Consistent with this, other analyses indicate that H3 nucleosomes turnover at a high rate on centromere DNA and de novo CENP-A assembly requires H2A.Z deposition by the Swr1C chromatin remodeling complex.

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Figure. a. Centromere organisation is conserved, sequence is not. CENP-A chromatin (Purple) and H3K9me2 heterochromatin (Orange) domains mapped by ChIP-seq to S. octosporus and S. cryophilus centromere 3. b. tDNA order is conserved but intervening sequence is not. Pairwise dotplot comparison of S. octosporus and S. cryophilus outer repeat regions encoding DVAIR tDNAs. c. PCA analysis of pentamer frequencies in 12 kb sliding windows (4.5 kb intervals) across the entire genomes of S. pombe, S. octosporus and S. cryophilus (Grey) reveals that a statistically significant distinct pattern of pentamers resides in CENP-A chromatin regions.

Selected Publications: *Subramanian, L., *Medina-Pritchard, B., Barton, R., Spiller, Yadav, R.K., Jablonowski, C.M., Fernandez, A.G., Lowe, B.R., F., Kulasegaran-Shylini, R., Radaviciute, G., Allshire, R.C., and Henry, R.A., Finkelstein, D., Barnum, K.J., Pidoux, A.L., Kuo, Y.M., Jeyaprakash A. A. (2016). Centromere Localization and Function Huang, J., O'Connell, M.J., Andrews, A.J., Onar-Thomas, A., of Mis18 Requires 'Yippee-like' Domain-Mediated Oligomerization. Allshire, R.C., Partridge, J.F. (2017). Histone H3G34R mutation EMBO Rep 17, 496-507. * joint first authors causes replication stress, homologous recombination defects and genomic instability in S. pombe. Elife 6, pii: e27406. Ard, R., and Allshire, R.C. (2016). Transcription-coupled changes to chromatin underpin gene silencing by transcriptional interference. Nucleic Acids Res. 44, 10619–10630. 7 Structural Biology of Cell Division

Cell division is an essential biological process that ensures genome integrity by equally and identically distributing chromosomes to the daughter cells. 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 A. Jeyaprakash kinetochores to spindle microtubules and successful completion of cytokinesis. These Arulanandam cellular events are regulated by a number of mitotic molecular assemblies (including the Chromosomal Passenger Complex (CPC), KMN (Knl1-Mis12-Ndc80) network, the Ska Co-workers: complex, Spindle Assembly Checkpoint and the Anaphase Promoting Complex) involving an extensive network of protein-protein interactions. Maria Alba Abad Although much is known about the basic mechanisms of cell division, structural level Fernandaz mechanistic details of pathways regulating error free chromosome segregation are still Asma Al-Murtadha emerging. In particular, a high-resolution understanding of centromere inheritance and how kinetochores employ dynamic protein interaction to harness the forces generated by Lana Buzuk spindle microtubules to drive chromosome segregation is yet to be obtained. To address Ignacio Jiménez these important questions requires an approach that integrates structural and functional methods capable of dissecting and probing individual roles of protein interactions Bethan Medina- mediated at varying timescale. We use molecular biology and biochemical approaches Pritchard to characterize protein interactions in vitro, X-ray crystallography, Cross-linking/Mass Frances Spiller spectrometry, Small Angle X-ray Scattering (SAXS) and Electron Microscopy for structural analysis and a combination of in vitro and cell-based in vivo functional assays using Reshma Thamkachy structure-guided mutations for functional characterization. The specific questions that we aim to address currently are i) What is the molecular basis for the establishment and maintenance of CENP-A nucleosomes at centromeres? ii) How do the outer kinetochore microtubule binding components cooperate to facilitate spindle driven chromosome segregation? and iii) How CPC, a key player required for eliminating incorrect kinetochore-microtubule attachment is targeted to the kinetochore? We address these question by characterizing protein complexes involved in centromere maintenance (Mis18 and Mis18-associated), physically coupling chromosomes to kinetochores (the Ska complexes and other outer kinetochore microtubule binding factors) and error-correction (CPC and its centromere/kinetochore receptors). The structural and functional insights from these studies will also provide new avenues for targeting specific protein-interactions to fight mitosis related human health disorders.

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Figure. a. CENP-A deposition model. b. Oligomerisation of s.pombe Mis18 is critical for its centromere association and centromere maintenance (Subramanian et al., 2016) c. Human Mis18 complex forms a hetero-octameric structure and the timing of the assembly is controlled by Cdk1 phosphorylation on Mis18BP1 to achieve cell-cylce controlled CENP-A deposition (Spiller et al., 2017).

Selected Publications: Spiller, F*., Medina-Pritchard, M*., Abad, M. A*., Wear, M. A., Jeyaprakash A. A. (2016) Centromere Localization and Function of Molina, O., Earnshaw, W. C and Jeyaprakash, A. A. (2017) Mis18 Requires ‘Yippee-Like’ Domain-Mediated Oligomerization. EMBO Molecular Basis for Cdk1 Regulated Timing of Mis18 Complex Rep 17, 496-507. Doi:10.15252/embr.201541520. (* equal contribution) Assembly and CENP-A Deposition. EMBO Rep 18, 894-905. Abad, M. A*., Medina, B*., Santamaria, A*., Zou, J., Plasberg-Hill, C., Doi:10.15252/embr.201643564 Madhumalar, A., Jayachandran, U., Redli, P. M., Rappsilber, J., Nigg, Subramanian, L*., Medina-Pritchard, B*., Barton, R., Spiller, E. A. and Jeyaprakash. A. A. (2014). Structural Basis for Microtubule F., Kulasegaran-Shylini, R., Radaviciute, G, Allshire, R. C and Recognition by the Human Kinetochore Ska Complex. Nat Commun 5, 2964. doi:10.1038/ncomms3964. (* equal contribution) 9 Regulation of splicing and functional links between splicing, transcription and chromatin

Transcription and RNA splicing are at the centre of gene expression in eukaryotes, controlling gene expression levels and removing introns from 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 Jean Beggs molecular studies. Our current focus is on links between RNA splicing and other metabolic processes, especially transcription and chromatin. Our approaches include: quantitative RT- Co-workers: PCR, ChIP-seq, RNA-seq, biochemical analyses and molecular genetics.

Vahid Aslanzadeh In Barrass et al. (2015) we describe the use of metabolic labelling with 4-thio-uracil for very David Barrass short times to isolate newly synthesised precursor RNAs and perform RNA-seq. In this way we compared the relative speed of splicing of different pre-mRNAs, observing that, Jim Brodie on average, ribosomal protein gene transcripts are spliced faster than most other intron- Susana De Lucas containing transcripts. Moreover, splicing is faster for introns with secondary structures that are predicted to be less stable. In Wallace and Beggs (2017) we compared data from Eve Hartswood several sources, finding that ribosomal transcripts are also spliced more efficiently (i.e. more Bella Maudlin pre-mRNA gets spliced) and more co-transcriptionality (more splicing happens while the RNA is still associated with polymerase) compared to other intron-containing transcripts. Gonzalo Mendoza-Ochoa To investigate how speed of transcription elongation affects splicing, we measured the Ema Sani efficiency, the co-transcriptionality and the fidelity (accuracy of correct splice site use) Edward Wallace of splicing in yeast strains with wild-type (WT) RNA polymerase II (Pol II), or with mutant Pol II that elongates faster or slower. We show that slow Pol II elongation increases both co-transcriptional splicing and splicing efficiency and that faster elongation reduces co- transcriptional splicing and splicing efficiency in budding yeast, suggesting that splicing is more efficient when co-transcriptional. Moreover, we demonstrate that altering the Pol II elongation rate in either direction compromises splicing fidelity (e.g. Figure 1). These effects are notably stronger for the highly expressed ribosomal protein coding transcripts, which are spliced with much higher fidelity than other intron-containing transcripts (Figure 2). We propose that transcription by RNA polymerase II is tuned to optimize the efficiency and accuracy of ribosomal protein gene expression, while allowing flexibility in splice site choice with the non-ribosomal protein transcripts.

photo © Antonia Reeve [email protected] 10 Figure 1. Sashimi plots with examples of splicing errors and how they are affected by faster or slower rates of transcription. Numbers are sequence reads across splice junctions. SEF (log2) SEF

Figure 2. Ribosomal protein transcripts are spliced with higher fidelity. Upper: Diagram illustrating how Splicing Error Frequency (SEF) was measured, using an alternative upstream 3'SS event as an example. Below: Distribution of the SEF in RP (pink) and non-RP (grey) intron-containing transcripts in the WT, slow and fast Pol II strains. (Aslanzadeh et al., 2018).

Selected Publications: Barrass, J.D., Reid, J.E.A., Huang, Y., Hector, R.D., Sanguinetti, G., Aslanzadeh, V., Huang, Y., Sanguinetti, G. and Beggs, J.D. (2018) Beggs, J.D. and Granneman, S. (2015) Transcriptome-wide RNA Transcription Rate Strongly Affects Splicing Fidelity and Co- processing kinetics revealed using extremely short 4tU labeling. transcriptionality in Budding Yeast. Genome Res. 28, 203-213. Genome Biol 16, 282. Wallace, E. and Beggs, J.D. (2017) Extremely fast and incredibly close: co-transcriptional splicing in budding yeast. RNA 23, doi: 10.1261/rna.060830. 11 MeCP2 and Rett syndrome

In 2017 we significantly advanced our understanding of the function of the chromosomal protein MeCP2. This protein, which we discovered in 1992, has been the subject of intense study since the finding that mutations within it cause the profound neurological disorder Rett syndrome. Despite these efforts, its precise role has remained uncertain, with several independent hypotheses in circulation. We originally proposed that a primary Adrian Bird role is to target sites of DNA methylation and the evidence in favour of this from several research groups has strengthened significantly during the year. A prominent cluster of Co-workers: Rett-causing missense mutations co-localises with the methyl-CpG binding domain and Christian Belton causes its inactivation. We showed that in addition to the canonical binding site mCG, MeCP2 also targets the trinucleotide mCAC, which is abundant in neurons. In addition to Beatrice-Alexander methylated DNA, MeCP2 has been linked with numerous protein partners. Of particular Howden interest, a discrete region that binds to the TBL1/R1 subunits of the NCoR and SMRT co- Kashyapp Chhatbar repressor complexes coincides with a cluster of Rett syndrome mutations. Using X-ray crystallography in a collaboration with Atlanta Cook, we found that the four amino acids Justyna Cholewa-Waclaw mutated in Rett syndrome make intimate contact with the TBL1/R1 surface. This finding John Connelly makes it highly likely that loss of this specific interaction is a root cause of the disorder. Dina De Sousa These results add to the weight of evidence supporting the “bridge hypothesis”, whereby MeCP2 recruits the corepressor complexes based on the density of methylated sites in Laura Fitzpatrick the genome. Re-analysis of chromatin immunoprecipitation data confirms the predictions Jacky Guy of this model and, further, provides evidence that transcriptional inhibition at gene loci is proportional to the density of methylated binding sites. Since the great majority of Matthew Lyst Rett syndrome mutations inactivate either the DNA binding domain or the corepressor Raphael Pantier interaction domain, we speculated that these domains alone, which amount to only Katy Paton 32% of the full-length protein, would be sufficient to fulfil key functions of MeCP2. The results strongly support this hypothesis, as the Mecp2 “minigene” prevents Rett-like Jim Selfridge phenotypes in mice. Interestingly, injection of the minigene in an adeno-associated virus Konstantina Skourti- vector rescued mice that lacked endogenous MeCP2. This raises the possibility that the Stathaki truncated MeCP2 might be used therapeutically for gene therapy. With our collaborator Stuart Cobb, also at Edinburgh University, we are exploring this possibility further. Christine Struthers

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Figure 1. Functional domains of MeCP2. a. Molecular structure of the Methyl-CpG Binding Domain (MBD) of MeCP2 bound to DNA. Grey balls denote the methyl groups on cytosine that are essential for DNA binding. b. A close-up of the molecular interaction between the NCoR Interaction Domain (NID) and the NCoR subunit TBLR1. The yellow backbone corresponds to the NID, with key amino acids (single letter code) denoted by orange numbers. The TBLR1 backbone is in blue, with important amino acids highlighted by black numbers. Stabilizing contacts between the two proteins are shown as dotted green lines. c. Top: MeCP2 shown as a grey bar with the MBD (blue), NID (pink), Enhanced Green Fluorescent Protein (EGFP) tag and Nuclear Localisation Signal (NLS) all highlighted. Radically truncated versions of MeCP2 that were introduced into mice to test their ability to prevent or reverse Rett-like phenotypes. Modified forms were missing the N-terminal domain (∆N), N- and C-terminal domains (∆NC) or N- and C-termini plus the region between the MBD and the NID (∆NIC). Each of these proteins retained wildtype or near-wildtype function suggesting that domains other than the MBD and the NID are dispensable.

Selected Publications: Tillotson R, Selfridge J, Koerner MV, Gadalla KKE, Guy J, De MD, and Bird A. MeCP2 recognizes cytosine methylated tri- Sousa D, Hector RD, Cobb SR, and Bird A. Radically truncated nucleotide and di-nucleotide sequences to tune transcription in the MeCP2 rescues Rett syndrome-like neurological defects. Nature. mammalian brain. PLoS Genet. 2017;13(5):e1006793. 2017;550(7676):398-401. Kruusvee V, Lyst MJ, Taylor C, Tarnauskaite Z, Bird AP, and Cook Lagger S, Connelly JC, Schweikert G, Webb S, Selfridge J, AG. Structure of the MeCP2-TBLR1 complex reveals a molecular Ramsahoye BH, Yu M, He C, Sanguinetti G, Sowers LC, Walkinshaw basis for Rett syndrome and related disorders. Proc Natl Acad Sci U S A. 2017;114(16):E3243-E50. 13 Structural biology of macromolecular complexes involved in RNA metabolism and transcriptional silencing

The expression of individual genes is controlled at the levels of mRNA transcription and also post-transcriptionally, by processes such as splicing, localization, modification or editing, and degradation. To gain a mechanistic understanding of these processes it is important to understand the interactions between the individual players, including both protein and nucleic acid components, at the molecular level. We have used structural Atlanta Cook approaches to tackle mechanistic questions about how protein-RNA interactions can control RNA maturation and RNA editing and how transcriptional repressors are recruited Co-workers: to methylated DNA. By combining structural studies with biochemical, biophysical Uma Jayachandran and cell-based functional assays we can gain powerful insights into these molecular processes. Valdeko Kruusvee Previously, we focused on proteins that control RNA metabolism during eukaryotic Aleksandra Kasprowicz ribosome biogenesis and RNA turnover. By solving the structure of yeast Tsr1, an essential Alexander Will ribosome biogenesis factor, we were able to model it into low-resolution maps of immature 40S ribosomal. This gave a key insight into how this protein controls the timing of events during maturation of the ribosomal small subunit.

We also solved several structures of an essential vertebrate protein complex that is thought to be an RNA chaperone on many types of transcript including rRNA. This complex, made up of nuclear factors 90 and 45 (NF90/NF45) specifically recognizes stretches of dsRNA. We have shown that NF90 has an evolutionary relationship to proteins involved in adenosine-to-inosine editing. In future work we hope to better understand how the full-length form of this protein recognizes dsRNA and how this may impact on RNA editing in cells.

More recently, in collaboration with Adrian Bird’s laboratory, we gained new insights into the molecular basis for a genetic autism spectrum disorder known as Rett syndrome (RTT) (Figure 1). Mutations to the methylated DNA binding protein MeCP2 cause RTT and fall in to two clusters, one in the DNA binding domain and one in a region that recruits the transcriptional co-repressor complex called NCoR/SMRT. We mapped the MeCP2 binding to the C-terminal WD40 domain TBLR1, a tetrameric core protein of NCoR/SMRT. The crystal structure of a complex between TBLR1 and a fragment of MeCP2 reveals that the only residues that make extensive contacts to TBLR1 are exactly those mutated in RTT. This suggests that a functional interaction between this region of MeCP2 and TBLR1 is required for the development of a healthy brain. 14 a c

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Figure 1. a. MeCP2 provides a molecular bridge between methylated CpG sequences in DNA and a large multicomponent co-repressor complex called NCoR/SMRT. This complex contains HDAC3 which deacetylates histones to maintain transcriptional repression. b. The NCoR interaction domain (NID) of MeCP2 contains a cluster of amino acids that are known to be mutated in RTT syndrome. Some common mutations are shown under the wild-type sequence. This segment of MeCP2 was used in co-crystallisation studies with the C-terminal domain of TBLR1 but only the darker region was visible. c. An overview of the MeCP2-TBLR1 complex and a zoomed-in view showing that the key interactions are made by residues commonly mutated in RTT.

Selected Publications: Kruusvee, V., Lyst M.J., Taylor, C., Tarnauskaite, Z. and Bird A.P. ribosome biogenesis factor Tsr1 is an inactive structural mimic of (2017) Structure of the MeCP2-TBLR1 complex reveals a molecular translational GTPases. Nat Commun 7, 11789. basis for Rett syndrome and related disorders. Jayachandran, U., Grey, H., and Cook, A.G. (2016). Nuclear factor Proc Natl Acad Sci USA 114, E3243-E3250 90 uses an ADAR2-like binding mode to recognize specific bases in McCaughan, U.M., Jayachandran, U., Shchepachev, V., Chen, dsRNA. Nucleic Acids Res 44, 1924-1936. Z.A., Rappsilber, J., Tollervey, D., and Cook, A.G. (2016). Pre-40S

15 The role of non-histone proteins in chromosome structure and function during mitosis

Our research focuses on three main aims. 1. Making mitotic chromosomes: How do mitotic chromosomes condense, and what is the role of histones and non-histone proteins in shaping them? 2. Segregating the chromosomes: How are centromere specification and kinetochore Bill Earnshaw assembly controlled epigenetically? 3. Controlling the process: How does the chromosomal passenger complex (CPC) Co-workers: regulate chromosome segregation? This year, a collaboration exploiting Hi-C technology (with Job Dekker at U. Mass. Medical Mar Carmena School in Worcester) with mathematical modelling (with Leonid Mirny at M.I.T.) allowed us to Fernanda Cisneros propose a new model for the pathway of mitotic chromosome formation. Our lab did all the cell biology and imaging. The resulting model integrates all previous major models of chromosome Oscar Molina structure, showing that chromosomes are built of nested dynamic loops emanating from a Lidija Pavlovich spiral scaffold structure composed of condensin II. The key advance enabling this study was development of a chemical biology protocol yielding an almost perfectly synchronous entry of Emma Peat DT40 cells into mitosis. We also completed a study showing that rapid depletion of condensin leads to novel and much more dramatic phenotypes than seen previously. Earlier this year, we Elisa Pesenti published a study strongly suggesting that histone posttranslational modifications may be a Melponemi Platani key factor driving mitotic chromatin compaction. Lucy Remnant Studies of chromosome segregation focused on using human synthetic artificial chromosomes to probe the role of epigenetics and mitotic transcription in centromere stability and function. Jan Ruppert We expanded our approach to removing histone marks from centromeres, showing that we Kumiko Samejima can target multiple competing activities simultaneously to perform what are essentially in situ epistasis studies. This led to a hypothesis that centromeric H3K9ac defends centromeres Itaru Samejima against invasion by surrounding heterochromatin. We also began work on a new generation of synthetic human chromosomes containing separate heterochromatin and centromeric arrays Giulia Vargiu that will allow us to systematically probe interactions between these two centromeric domains. Alisa Zhiteneva Our work on the CPC revealed that interactions between heterochromatin protein HP1 and the CPC play a key role in targeting and activation of this important mitotic kinase complex during mitotic entry. Other studies probed the role of nucleoporin Seh1 in targeting regulators of Tor kinase signaling to mitotic chromosomes and examined the mitotic phenotypes resulting from treatment of cells with p53-activating inhibitors of the enzyme DHODH in one study and inhibitors of telomerase in a second. Our work is supported by a Wellcome Principal Research Fellowship and by the Centre for Mammalian Synthetic Biology. 16 Figure. Simulation of nested loops on a prometaphase chromosome formed by the action of condensin I and II superimposed on a micrograph showing the synchronous entry of a DT40 CDK1as cell line into mitosis. Simulation by Anton Golobdorodko. Micrograph by Kumiko Samejima.

Selected Publications: Vargiu, G., A.A. Makarov, J. Allan, T. Fukagawa, D.G. Booth and compaction in vitro. OPEN BIOL. 7, pii: 170076. PMID: 28903997; W.C. Earnshaw. (2017) Stepwise unfolding supports a subunit PMC5627050; DOI: 10.1098/rsob.170076. model for vertebrate kinetochores. PROC. NATL. ACAD. SCI. U.S.A. Gibcus, J.H., K. Samejima, A. Goloborodko, I. Samejima, N. 114: 3133-3138. PMID: 28265097; DOI: 10.1073/pnas.1614145114. Naumova, J. Nuebler, M. Kanemaki, L. Xie, J.R. Paulson, W.C. Zhiteneva, A., J.J. Bonfiglio, A. Makarov, T. Colby, P. Vagnarelli, Earnshaw, L.A. Mirny, J. Dekker. (2018). A pathway for mitotic E.C. Schirmer, I. Matic and W.C Earnshaw. (2017). Mitotic post- chromosome formation. SCIENCE 9:359. PMID: 29348367; DOI: translational modifications of histones promote chromatin 10.1126/science.aao6135.

17 Epigenetic inheritance and organization of centromeres

Our lab is interested in the epigenetic inheritance and organization of centromeres. Epigenetic transmission of centromere identity through many cell generations is required 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: Patrick Heun How is the epigenetic identity of centromeres propagated? Centromeres are found at the primary constriction of chromosomes in mitosis where they Co-workers: remain connected before cell division. This structure is essential for an equal distribution of Eduard Anselm chromosomes to the daughter cells.

cenH3 Georg Bobkov The centromere specific histone H3-variant CENP-A is essential for kinetochore formation and centromere function. We have recently established a biosynthetic approach to target Emily Fowler dCENP-AcenH3 to specific non-centromeric sequences such as the Lac Operator and follow the formation of functional neocentromeres. Using this approach we were able to directly Eftychia Kyriacou demonstrate that a dCENP-AcenH3 -LacI fusion is sufficient to induce centromere formation Vasiliki Lazou as well as self-propagation and inheritance of the epigenetic centromere mark (Figure 1a and b); Using the LacO/LacI tethering system, we are interested in dissecting the function Manuela Marescotti of centromere factors in Drosophila and human cells for propagation of CENP-AcenH3. This Virginie Roure approach has been successfully introduced into a heterologous system comprised of human centromere factors expressed in Drosophila Schneider S2 cells (Logsdon et al., 2015).. We are currently trying to reconstitute the loading and self-propagation of either human or Drosophila CENP-A at the ectopic LacO locus (Figure 1c).

What is the role of transcription at the centromere? Loading of CENP-A at the centromere occurs outside of S-phase and requires the removal of H3 placeholder" nucleosomes. Transcription at centromeres has been linked to the deposition of new CENP-A, although the molecular mechanism is not understood. Interestingly, transcription is able to evict nucleosomes, which can be recycled by the histone chaperone Spt6. We find that centromeric localization of Spt6, RNAPII and centromere-associated transcripts temporally coincides with dCENP-A loading from mitosis to G1. Using fast acting transcriptional inhibitors in combination with a newly developed CENP-A loading system, we demonstrate that centromeric transcription is required for dCENP-A loading by evicting placeholder nucleosomes and promoting dCENP-A transition from chromatin association to nucleosome incorporation. In contrast, loss of parental CENP-A in Spt6 depleted cells underlines the importance of CENP-A maintenance during transcription. Thus, co-operated actions of transcription and Spt6-mediated nucleosome recycling are essential for the stability of the epigenetic centromere mark dCENP-A. 18 Human centromere proteins Drosophila S2 cells Heterologous system Drosophila centromere proteins human cells Heterologous system

Heterolog.CENP’s Epigenetic LacO/LacI system : or Inheritance? GFP

LacI GFP Heterolog.CENP’s LacI LacO heterolog. CENP-A

Figure 1: Heterologous system to dissect the epigenetic inheritance of centromere histone CENP-A. Figurenew 1. Heterologous system to dissect the epigenetic inheritance of centromere histone CENP-A.

A MitosisMitosis new A Mitosis TranscriptionTranscription H3 H3Spt6 Spt6 Spt6 A H3 centromeres old Centromeres Spt6 Spt6 Figure 2.H3 Model for the role of transcription at centromeres: Transcriptioncentromeres remodels centromere chromatin A andFigure evicts 2: H3-nucleosomes Model for the role (green) of transcription to allow new at centromeres:CENP-A (red) loading.Transcription Evicted remodels old CENP-A is recycled by transcriptioncentromere elongation chromatin factor and evicts Spt6. H3-nucleosomes (green) to allow new CENP-A (red) old loading. Evicted old CENP-A is recycled by transcription elongation factor Spt6. Selected Publications: Barth, T.K., Schade, G.O., Schmidt, A., Vetter, I., Wirth, M., Heun, Barrey, EJ and Heun, P. (2017). Artificial Chromosomes and P.*, Thomae, A.W.*, and Imhof, A*. (2014). Identification of novel Strategies to Initiate Epigenetic Centromere Establishment. Prog Drosophila centromere-associated proteins. Proteomics 14, 2167- Mol Subcell Biol. 56, 193-212 2178 (*corresponding authors). 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 Figure 2: Model for the role ofare required transcription for centromere establishment. at J Cell centromeres: Biol 208, 521-531. Transcription remodels centromere chromatin and evicts H3-nucleosomes (green) to allow new CENP-A (red)19 loading. Evicted old CENP-A is recycled by transcription elongation factor Spt6. Cell state transitions during cell growth and division

Our research aim is to characterise the proteomic changes that accompany and control cell state transitions during cell growth and division in human cells. To achieve this aim, we use a combination of state of the art techniques, including fluorescence-activated cell sorting (FACS) and quantitative mass spectrometry (MS)-based proteomics.

We will focus initially on cell state transitions that occur during the mitotic cell division cycle. Tony Ly This cycle can be separated into four major phases (G1, S, G2, and M) that are largely defined by two major processes: DNA replication (S phase) and chromosome segregation (Mitosis). Co-worker: Cells can also enter a reversible quiescent state, called G0.

Van Kelly Mitosis can be further resolved into discrete subphases based on changes in cellular David Lewis architecture that can be visualized by light microscopy, or by immunostaining for specific molecular signaling events, including phosphorylation of histone H3 and degradation of cyclin Aymen al-Rawi proteins (e.g. cyclin A and cyclin B). Unlike in mitosis, it is less clear if gap phases are similarly structured as a linear sequence of state transitions. Evidence in support of a linear progression model during gap phases is the proposed existence of an ‘antephase’ during G2. Antephase is a short time window late in G2 that precedes nuclear envelope breakdown and chromatin condensation that is characterized by an increased sensitivity towards DNA damage. On the other hand, recent studies suggest gap phases of the cell cycle are characterized by bifurcations in cell fate trajectories, leading to heterogeneous, temporally aligned cell states.

Comprehensive, molecular definitions of cell state and identity can be obtained using quantitative mass spectrometry-based proteomics (Ly et al. eLife 2014). Recent developments in mass spectrometry (MS) enable the high throughput identification and quantitation of thousands of proteins in a single analysis. Multidimensional analysis of the proteome is now possible. Static and dynamic parameters of proteins can be measured, including protein copy numbers, post translational modifications, protein-protein interactions, and protein half-life.

We developed a method combining FACS and MS to measure protein changes during mitosis proteome-wide in an asynchronous culture of human leukemia cells (Fig. 1A).

Using this method, we aim to dissect cell state transitions in the mitotic cell division cycle using quantitative, multidimensional proteomics as comprehensive readouts of cellular state (Fig. 1B).

20 a

b

Figure 1. a. Four mitotic subpopulations are isolated from an asynchronous population of human cells by FACS and intracellular immunostaining of mitotic markers. b. FACS-isolated mitotic subpopulations are then subjected to proteome-wide analysis to compare differences in protein abundance, protein-protein interactions, and post translational modifications (PTMs).

Selected Publications: *Ly, T., Whigham, A., Clarke, R., Brenes-Murillo, A.J., Estes, B., Ly, T., Ahmad Y., Shlien, A., Soroka, D., Mills, A., Emanuele, M.J., Madhessian, D., Lundberg, E., Wadsworth, P., *Lamond, A.I. (2017) Stratton, M.R., Lamond A.I. (2014) A proteomic chronology of gene Proteomic analysis of cell cycle progression in asynchronous expression through the cell cycle in human myeloid leukemia cells. cultures, including mitotic subphases, using PRIMMUS. eLife 2017, eLife 2014, 3, e01630. 6, e27574. *Co-corresponding authors Ly, T., Endo, A., Lamond, A.I. (2015) Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells. eLife 4, e04534. 21 Orienting Chromosomes during Mitosis and Meiosis

Our goal is to understand the molecular mechanisms that ensure the accurate transmission of chromosomes to daughter cells during cell division. Errors in chromosome segregation generate cells with the wrong number of chromosomes, known as aneuploidy. Aneuploid somatic cells, arising as a result of errors in mitosis, are associated with cancer. Aneuploid gametes are generated from erroneous meiosis and are causative for miscarriages, infertility Adele Marston and birth defects. We aim to uncover conserved and fundamental mechanisms in both mitosis and meiosis by employing yeast cells and frog oocytes as models, together with a wide range Co-workers: of cell biological and biochemical methodologies. Rachael Barton A central theme of current work in our laboratory is non-canonical roles of the kinetochore. The kinetochore is a complex molecular machine that assembles on the centromere and is best Julie Blyth known for its role in coupling chromosomes to microtubules, thereby mediating the movement Weronika Borek of chromosomes. Our work has uncovered key regulatory and structural functions of the kinetochore that impinge on various aspects of chromosome segregation: Stefan Galander 1. Chromosome organisation We showed that the kinetochore targets cohesin loading Bethany Harker to the centromere through a dedicated pathway that enriches cohesin throughout the surrounding chromosomal region (the pericentromere). We uncovered the mechanism of Katarina Jönsson this targeted cohesin loading (Figure 1) and demonstrated its importance for chromosomal Vasso Makrantoni organisation in this region (Figure 2). Flora Paldi 2. Cell cycle regulation Shugoshin is a pericentromeric adaptor protein that performs multiple distinct functions in chromosome segregation during mitosis and meiosis. We showed Meg Peyton-Jones that shugoshin delocalizes from the pericentromere to indicate that chromosomes are properly attached to microtubules, called biorientation. Our recent work has uncovered a regulatory Rebecca Plowman pathway that inactivates shugoshin, to allow cell cycle progression once biorientation has been Ola Pompa achieved. Xue (Bessie) Su 3. Adaptations for meiosis Meiosis is a modified cell division that produces gametes through two consecutive rounds of chromosome segregation. During the first meiotic division, Menglu (Lily) Wang uniquely, the maternal and paternal chromosomes or homologs are segregated. This requires several adaptations to the way in which chromosomes are segregating including the way in which chromosomes attach to the microtubules that will pull them apart, and the way in which linkages between them are lost. Our recent work has revealed how a master meiosis I-specific regulator establishes these modifications, essentially converting mitosis into meiosis. In addition, we have uncovered aspects of kinetochore assembly and function that are critical for meiosis, but not mitosis. Our future focus is to gain a thorough understanding of how kinetochores are adapted for meiosis both in yeast and vertebrates. 22 cohesin 1 84 Scc2--Scc4 phosphorylated Ctf19 cohesin loader cohesinP Ctf19 P 1 84 * * * * *Ctf19 * * * * Scc2--Scc4 phosphorylated 1 T4 S5 T7 T8 S10 T13 S14 S16 S19 20 cohesin loader PP Ctf19 9A centromere * * * * * * * * * 1 T4 S5 T7 T8 S10 T13 S14 S16 S19 20 Chromosome V 9A wild type Cohesin ChIP-seqcentromere

Chromosome V wildctf19-9 typeA Cohesin ChIP-seq

ctf19-9A Figure 1 Targeted cohesin loading at the centromere. Schematics top left depict mechanism of kinetochore-driven Figurecohesin 1. Targeted loading cohesin and relevant loading phosphorylation at the centromere. sites in the Schematics Ctf19 protein, depictrespectively mechanism with cohesin of kinetochore-driven ChIP-seq in wild type cohesinand loading cells (ctf19-9A (top left)) where and kinetochore-driven phosphorylation cohesin sites loading in the isCtf19 abolished. protein that are required for targeted cohesin loadingFigure (top 1 right). Targeted Cohesin cohesin ChIP-Seq loading at performed the centromere. in wild Schematics type cells top and left cells depict lacking mechanism Ctf19 of phosphorylation kinetochore-driven sites cohesin loading and relevant phosphorylation sites in the Ctf19 protein, respectively with cohesin ChIP-seq80 in wild type

(ctf19-9A) is shown below. Inter- 70 >1-4 μm ** and cells (ctf19-9A) where kinetochore-driven cohesin loading is abolished. ** centromere 60 distance 8050

Inter- 70 >1-4 μm **

40 **

centromere % cells 6030 distance 5020 4010 % cells 300 centromere 15 20 inter GFP 10 Δ spindle pole bodies CTF19ctf19 distance wild0 type ctf19-9A Figure 2 Targeted cohesin loading at the centromere ensures robustcentromere centromeric 15 cohesion. Image shows metaphase-Δ arrested cells with a single centromere and spindle pole bodies labeled with GFP and tdinterTomato GFP, respectively. Schematic spindle pole bodies CTF19ctf19 and measurements of inter-centromere distance are shown to the right. distance wild type ctf19-9A Figure 2 Targeted cohesin loading at the centromere ensures robust centromeric cohesion. Image shows metaphase- arrested cells with a single centromere and spindle pole bodies labeled with GFP and tdTomato, respectively. Schematic Figureand 2. measurements Targeted cohesin of inter-centromere loading at thedistance centromere are shown ensures to the right. robust centromeric cohesion. Image shows metaphase-arrested cells with a single centromere and spindle pole bodies labeled with GFP and tdTomato, respectively. Schematic and measurements of inter-centromere distance are shown to the right.

Selected Publications: Blyth J, Makrantoni V, Barton RE, Spanos C, Rappsilber J and Fox C, Zou J, Rappsilber J and Marston AL. (2017) Cdc14 Marston AL. (2018) Genes Important for Schizosaccharomyces phosphatase directs centrosome re-duplication at the meiosis I pombe Meiosis Identified Through a Functional Genomics Screen. to meiosis II transition in budding yeast. Wellcome Open Res. doi: Genetics. 208, 589-603. 10.12688/wellcomeopenres.10507.1. Hinshaw SM, Makrantoni V, Harrison SC and Marston AL (2017) The Kinetochore Receptor for the Cohesin Loading Complex. Cell 171, 72-84 23 The meiotic spindle and chromosomes in oocytes

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 Hiro Ohkura 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. Co-workers: Defining these molecular pathways is crucial to understand error-prone chromosome Mariana Costa segregation in human oocytes. Furthermore, evidence indicates that these apparent oocyte-specific pathways also operate in mitosis, although less prominently, to ensure the Fiona Cullen accuracy of chromosome segregation. Therefore uncovering the molecular basis of these Alex McDonnell pathways is also important to understand how somatic cells avoid chromosome instability, Jule Nieken a contributing factor for cancer development. To understand the molecular pathways which set up the chromosome segregation Charlotte Repton machinery in oocytes, we take advantage of Drosophila oocytes as a "discovery platform" Pierre Romé because of their similarity to mammalian oocytes and suitability for a genetics-led Pedro Silva mechanistic analysis. In Drosophila oocytes, as in human oocytes, meiotic chromosomes form a compact cluster called the karyosome within the nucleus. Later, meiotic Verdiana Steccanella chromosomes assemble a bipolar spindle without centrosomes in the large volume of the cytoplasm, and establish bipolar attachment. We have identified a number of genes defective in chromosome organisation and/or spindle formation in oocytes.

From studying the karyosome, we found the histone demethylase Kdm5/Lid plays an important role forming the karyosome and stable synaptonemal complex, independently of its catalytic activity. In addition, we uncovered a novel regulatory loop which controls interaction between the nuclear pore and chromatin in oocytes and somatic cells.

For bipolar spindle formation, we found that the phospho-docking 14-3-3 protein is crucial for spatial regulation of a spindle protein. It suppresses microtubule binding of the kinesin-14 Ncd in the large cytoplasm of oocytes, and this suppression is locally removed by the Aurora B kinase that acts as a chromosomal signal. We also showed that Sentin- EB1 actively prevents microtubule plus ends from forming stable kinetochore attachments during spindle formation to facilitate bipolar attachment of homologous chromosomes in Drosophila oocytes. 24 a b # # $ $ # # $ $

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Figure. a. Meiotic spindles from control and 14-3-3ε RNAi oocytes. b. The non-motor region of the kinesin-14 Ncd has two phosphorylation sites (S94, S96) and interacts with microtubules and the phospho-docking protein 14-3-3. c. Two phosphorylations on Ncd differentially regulate its microtubule binding in the presence of 14-3-3. S96 phosphorylation (by PDK2) promotes 14-3-3 binding which inhibits microtubule interaction. Further phosphorylation at S94 by Aurora B releases Ncd from 14-3-3 to allow microtubule binding. d. A model of spatial regulation of Ncd in oocytes. The phospho-docking 14-3-3 protein suppresses microtubule binding of the kinesin-14 Ncd in the large cytoplasm of oocytes, and the Aurora B kinase acting as a chromosomal signal locally removes this suppression. All mages are reproduced from Beaven et al (2017).

Selected Publications: Beaven, R., Bastos, R. N., Spanos, C., Rome, P., Cullen, C. F., Głuszek, A. A., Cullen, C. F., Li, W., Battaglia, R. A., Radford, S. J., Rappsilber, J., Giet, R., Goshima, G. and Ohkura, H. (2017) 14-3-3 Costa, M. F., McKim, K. S., Goshima, G., and Ohkura, H. (2015). regulation of Ncd reveals a new mechanism for targeting proteins to The microtubule catastrophe promoter Sentin delays stable the spindle in oocytes. J. Cell Biol. 216, 3029-3039. kinetochore-microtubule attachment in oocytes. J. Cell Biol. 211, 1113-1120. Breuer, M., and Ohkura, H. (2015). A negative regulatory loop within the nuclear pore complex controls global chromatin organization. Genes Dev. 29, 1789-1794. 25 3D proteomics

Genes are not randomly distributed in the genome. In humans, 10% of protein-coding genes are transcribed from bidirectional promoters and many more are organised in larger clusters. Intriguingly, neighbouring genes are frequently coexpressed but rarely functionally related. We could show recently that coexpression of bidirectional gene pairs, and closeby genes in general, is buffered at the protein level (Kustatscher et al., 2017). Taking into Juri Rappsilber account the 3D architecture of the genome, we found that co-regulation of spatially close, functionally unrelated genes is pervasive at the transcriptome level, but does not extend to Co-workers: the proteome. Non-functional mRNA coexpression in human cells appears to arise from Giulia Bartolomucci stochastic chromatin fluctuations and direct regulatory interference between spatially close genes. Protein-level buffering likely reflects a lack of coordination of post-transcriptional Adam Belsom regulation of functionally unrelated genes. Grouping human genes together along the Colin Combe genome sequence, or through long-range chromosome folding, is associated with reduced expression noise. Our results support the hypothesis that the selection for noise reduction is Therese Dau a major driver of the evolution of genome organisation. The large presence of non-functional Lutz Fischer coexpression of genes at the transcript but not protein level suggests that proteomics data should surpass transcriptomics data when screening for functional links between genes. We Martin Graham decided to follow up on this by collating protein expression datasets and mining them for Lars Kolbowski functional protein associations with machine-learning. The annotation of protein function is a longstanding challenge of cell biology that suffers Georg Kustatscher from the sheer magnitude of the task. We therefore developed ProteomeHD, which Christos Spanos documents the response of 10,323 human proteins to 294 biological perturbations, measured by isotope-labelling mass spectrometry. Using this data matrix and robust Juan Zou machine learning we create a co-regulation map of the cell that reflects functional associations between human proteins and that outperforms predictions done by STRING based on the NCBI GEO repository currently holding mRNA expression profiling data from more than one million human samples. Our map identifies a functional context for many uncharacterized proteins, including microproteins that are difficult to study with traditional methods. Co-regulation also captures relationships between proteins which do not physically interact or co-localize. For example, co-regulation of the peroxisomal membrane protein PEX11 with mitochondrial respiration factors led us to discover a novel organelle interface between peroxisomes and mitochondria in mammalian cells. The co- regulation map can be explored at www.proteomeHD.net. Our lab is also continuing its development of cross-linking/mass spectrometry as a tool to investigate in cells structures of proteins and their complexes. 26 Figure. Housekeeping genes are clustered in the human genome, which minimizes stochastic silencing but leads to partial co-expression of thousands of functionally unrelated genes. This non-functional mRNA co-expression is buffered at the protein level. • Genes that are spatially proximal in sequence or 3D structure of the human genome are often co-regulated at the mRNA abundance level. • The co-expression of spatially proximal, functionally unrelated genes is buffered at the protein level. • The co-expression of neighbouring genes at the mRNA level is driven by chromatin fluctuations and direct regulatory interference. • Regulatory interference is likely buffered by a neutral mechanism

Selected Publications: Kustatscher, G., Grabowski, P., and Rappsilber, J. (2017). Pervasive Schneider, M., Belsom, A., and Rappsilber, J. (2018). Protein coexpression of spatially proximal genes is buffered at the protein Tertiary Structure by Crosslinking/Mass Spectrometry. Trends level. Mol. Syst. Biol. 13, 937. Biochem. Sci. 43, 157–169. Kolbowski, L., Mendes, M.L., and Rappsilber, J. (2017). Optimizing the Parameters Governing the Fragmentation of Cross-Linked Peptides in a Tribrid Mass Spectrometer. Anal. Chem. 89, 5311– 5318. 27 Microtubule nucleation, cytoskeletal organisation, and cell polarity

Our laboratory is interested in two main areas related to cellular and cytoskeletal organisation: 1) the molecular mechanisms underlying microtubule nucleation; and 2) the regulation of cell polarity, in a systems context, under both normal and stress conditions. In both areas we use the fission yeast Schizosaccharomyces pombe as a model eukaryotic organism. We combine classical and molecular genetic analysis with live-cell Kenneth E. Sawin fluorescence microscopy, biochemistry, proteomics and computational modeling.

Co-workers: Microtubule nucleation in all eukaryotic cells depends on the γ-tubulin complex (γ-TuC), a multi-protein complex enriched at microtubule organizing centres such as the Sanju Ashraf centrosome. The spatial and temporal regulation of the γ-TuC remains largely a mystery. Xun Bao We discovered the fission yeast proteins Mto1 and Mto2, which form an oligomeric complex (the Mto1/2 complex) that targets the γ-TuC to different sites in the cell and also Hayley Johnson activates γ-TuC during the cell cycle. Mutations in the human homolog of Mto1 lead to the Su Ling Leong brain disease microcephaly. Our current work involves understanding the mechanism of γ-TuC activation by the Mto1/2 complex, through genetic approaches in yeast, and through Ana Rodriguez expression, purification and characterization of recombinant multi-protein complexes in Ye Dee Tay insect cells, in vitro functional reconstitution, and structural biology analysis, including X-ray crystallography. We are also using new methods to investigate how the Mto1/2 Harish Thakur complex is localized to different subcellular structures. Regulation of cell polarity in fission yeast is particularly interesting because it involves multiple internal cues that cooperate and compete with each other. The Rho-family GTPase Cdc42 and its associated regulators and effectors control the actin cytoskeleton and exocytosis. Microtubules provide an additional level of control in regulating site-selection for polarised growth, through the microtubule plus-tip-associated protein Tea1, the membrane

protein Mod5, and their interactors. We are currently studying how the Cdc42 system and the microtubule-based system “talk to each other" under different environmental stimuli and under stress, using a combination of mutational analysis, proteomics, FRAP microscopy, and mathematical modeling. This work has led to the discovery of new cell-polarity regulators, outside of the Cdc42- and microtubule-based systems. An important component of our work involves developing new tools in genetics, microscopy, 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 applying to global analysis of protein phosphorylation in cell polarity, and new methods for interrogating protein-protein interactions in complex “solid-phase” organelles. 28 Figure 1 Figure 2

1 2 wild-type + DMSO wild-type + LatA sty1∆ + LatA Multiple stresses Actin Cdc42-GTP Actin Cdc42-GTP Actin Cdc42-GTP (hypertonic, oxidative, etc.) 0 min

3 MAPKKK Win1, Wis4 30

MAPKK Wis1 60

MAPK Sty1 90

120 X Y Pyp1,Pyp2 Actin polymerised Actin depolymerised Actin depolymerised Cdc42 polarised Cdc42 depolarised Cdc42 remains polarised Figure 3 3 : = 1:1 Mto1[bonsai] dimer γ-TuSC Mto1/2 Mto2 complex oligomer

γ-TuRC-like

Figure 1. Stress-activated MAP kinase (SAPK) pathway in fission yeast. Figure 2. The Sty1 SAPK pathway is required for cell depolarisation after actin depolymerisation by latrunculin A (LatA). Arrows show Cdc42-GTP dispersal from cell tips. In sty1∆ cells, actin is depolymerised, but Cdc42-GTP remains polarised. Figure 3. Model for activation of the γ-tubulin complex by Mto1/2 complex, together with crystals of an Mto1 domain.

Selected Publications: Mutavchiev, D. R., Leda, M., & Sawin, K. E. (2016). Remodeling Lynch, E.M., Groocock, L.M., Borek, W.E., and Sawin, K.E. (2014) of the Fission Yeast Cdc42 Cell-Polarity Module via the Sty1 p38 Activation of the γ-tubulin complex by the Mto1/2 complex. Curr Biol Stress-Activated Protein Kinase Pathway. Curr Biol 26, 2921-2928. 24, 896-903. Borek, W.E., Groocock, L.M., Samejima, I., Zou, J., de Lima Alves, F., Rappsilber, J., and Sawin, K.E. (2015). Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis. Nat Commun 6, 7929. 29 Nuclear envelope transmembrane protein regulation of tissue- specific genome organisation in differentiation and disease

Mutations in widely expressed nuclear envelope (NE) proteins cause many distinct diseases with tissue-specific pathologies including muscular dystrophies, lipodystrophies, neuropathy, dermopathy, and premature-aging syndromes. This raised the question: how could mutations in the same ubiquitous protein cause distinct diseases affecting different tissues? Hypothesizing that tissue-specific partners mediate the tissue-specific Eric C. Schirmer pathologies, we identified candidate partners with proteomics. The NE connects on the inside to chromatin and genome organisation is disrupted in patient cells. If our Co-workers: hypothesis is correct, it follows that these tissue-specific NETs might direct tissue-specific Dario Barreiros patterns of genome organisation with consequences for gene expression and we have found this to be the case. Rafal Czapiewski We found three muscle-specific NETs that re-position genes to the NE that are needed Jose I. de las Heras early in myogenesis, but subsequently become inhibitory and must be tightly shut down. Their combined knockdown blocks myogenesis. Thus, NE gene recruitment enables Charles R. Dixon tighter regulatory control. Importantly, we found mutations in these muscle NETs in Alexandr Makarov unlinked Emery-Dreifuss muscular dystrophy patients, further arguing the importance of this novel regulatory mechanism. We have found similar effects with a fat-specific NET Maria Manunta in adipogenesis and found that mice lacking this protein have difficulty producing fat, Andrea Rizzotto become insensitive to insulin, have metabolic dysfunction and a general lipodystrophy phenotype (Fig. 1). Aishwarya Sivakumar It appears that NE connections can also influence gene activities in the nuclear interior as during lymphocyte activation we found that released genes that were flanked by unchanging NE-associated regions remained within <0.8 µm from the NE, presumably because the flanking contacts restrict their diffusion and thus promote their association in chromosome compartments in what we call the "constrained diffusion" hypothesis. We showed that several genes and an enhancer up to 14 Mb away from one another are all released upon lymphocyte activation and associate in A2 sub-compartments. This type of regulation could contribute temporal control to lymphocyte activation. Other lines of investigation include: 1) Studying the structure of intermediate filament lamins with the Rappsilber lab. 2) Investigating NET effects on nuclear size changes in several cancer types and screening for small molecules targeting this with the Auer and Tyers labs. Nuclear size changes mark increased disease severity and this is also tissue- specific. 3) Testing how another NET contributes to signaling of innate immune responses. 4) Investigating how herpesviruses escape through the NE, finding that vesicle fusion proteins in the NE are needed for efficient virus nuclear egress. 30 a c

b

Figure 1. Phenotype of a mouse with knockout (KO) of fat-specific NET Tmem120A. a. KO and wild-type (WT) mice are similar on a healthy diet, but KO mice fail to gain weight like WT mice on a high-fat diet. b. KO mice cannot clear glucose after insulin injection. c. Respiratory exchange rate (RER) is similar between WT and KO mice on regular diet, but KO mice show a clear metabolic defect on high-fat diet.

Selected Publications: Robson, M. I., de las Heras, J. I., Czapiewski, R., Sivakumar, A., alter genome organization and regulation even in a heterologous Kerr, A. R. W., and Schirmer, E. C. (2017). Constrained release of system. Nucleus 8, 81-97. lamina-associated enhancers and genes from the nuclear envelope Robson, M. I., de las Heras, J. I., Czapiewski, R., Le Thanh, P., during T-cell activation facilitates their association in chromosome Booth, D. G., Kelly, D. A., Webb, S., Kerr, A. R. W., and Schirmer, E. compartments. Genome Res 27, 1126-1138. C. (2016). Tissue-specific gene repositioning by muscle nuclear de las Heras, J. I., Zuleger, N., Batrakou, D. G., Czapiewski, R., membrane proteins enhances repression of critical developmental Kerr, A. R. W., and Schirmer, E. C. (2017). Tissue-specific NETs genes during myogenesis. Mol. Cell 62, 834-847. 31 Nuclear RNA Processing and Surveillance

We aim to understand the nuclear pathways that synthesise and process newly transcribed RNAs, the assembly of RNA-protein complexes and the surveillance activities that monitor their fidelity.

Over the past year, we discovered novel functional links between chromatin structure, David Tollervey transcription and RNA metabolism in baker’s yeast Saccharomyces cerevisiae. Reversible phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII) provides a Co-workers: flow of information from transcribing RNAPII to the RNA processing and surveillance Stefan Bresson machinery, acting on the nascent transcript. We recently reported that the catalytic RNAPII subunit is ubiquitinated close to the DNA entry path (Ref. 1). This provides a reverse flow Clémentine Delan-Forino of information linking events on the nascent transcript back to transcribing RNAPII. In Hywel Dunn-Davies particular, we proposed that splicing-associated transcriptional pausing is enforced by RNAPII ubiquitination (Figure 1). This promotes co-transcriptional splicing of the nascent Aziz El Hage pre-mRNA, which is the norm in both yeast and humans.

Tatiana Dudnakova We also discovered links between the nascent transcript and major chromatin Aleksandra Helwak modifications; methylation of histone H3 at lysine 4 (H3K4) and lysine 36 (H3K36), catalysed by the Set1 and Set2 methyltransferases, respectively. We reported that both Set1 and Set2 Laura Milligan bind nascent RNA transcripts (Ref 2). Interactions between Set1 and RNA are predominately Elisabeth Petfalski mediated by RRM2 and deletion of this region reduced the chromatin association of Set1, accompanied by reduced levels of H3K4 tri-methylation and increased di-methylation on Nic Robertson protein coding genes. Notably, a class of non-coding RNAs (ncRNAs), termed CUTs, failed Camille Sayou to bind Set1 and their genes showed high levels of H3K4 mono-methylation rather than tri-methylation that characterises protein coding genes. H3K4 mono-methylation is also a Vadim Shchepachev feature of human enhancers, which are transcribed into ncRNAs, termed eRNAs. Both yeast Tomasz Turowski CUTs and human eRNAs are highly unstable, due to rapid degradation by the exosome complex, which is potentially linked to their histone modification status.. Marie-Luise Winz Long-standing observations by the group indicated that the activity of nuclear RNA degradation by the exosome nuclease complex is responsive to nutrient availability. We have now discovered that alterations in the targeting of nuclear surveillance pathways function, together with transcriptional changes, to rapidly remodel gene expression following nutrient shift - acting both positively and negatively (Ref. 3). This identified nuclear RNA surveillance as an actively regulated step in gene expression. It seems likely that changes in nuclear RNA degradation pathways will play important roles in other situations that require large scale reprogramming of gene expression, such as developmental steps in metazoans. 32 a

b

c

d

Figure 1. Many RNA processing factors associate with the RNA polymerase, allowing information to be transmitted from the transcribing polymerase to the processing machinery on the nascent transcript. However, it was unclear whether or how information might be transmitted to the polymerase. We recently reported (Ref. 1) that ubiquitination close to the active site of RNAPII occurs in response to RNA processing events, including pre-mRNA splicing, and is linked to transcriptional pausing. This is released by de-ubiquitination catalysed by a Bre5-Ubp3 complex that associates with the nascent transcript following successful completion of splicing.

Selected Publications: Milligan, L., Sayou, C., Tuck, A., Auchynnikava, T., Reid, J.E.A., RNA binding by the histone methyltransferases Set1 and Set2. Mol. Alexander, R., de Lima Alves, F., Allshire, R., Spanos, C., Rappsilber, Cell Biol. doi: 10.1128/MCB.00165-17. PMID: PMC28483910 J., Beggs, J.D. Kudla, G. and Tollervey, D. (2017) RNA polymerase II Bresson, S., Tuck, A., Staneva, D. and Tollervey, D. (2017) Nuclear stalling at pre-mRNA splice sites is enforced by ubiquitination of the RNA decay pathways aid rapid remodeling of gene expression in catalytic subunit. eLife, 6, e27082. PMCID:PMC5673307 yeast. Mol. Cell, 65, 787-800. PMCID: PMC5344683 Sayou, C., Millán-Zambrano, G., Santos-Rosa, H., Petfalski, E., Robson, S., Houseley, J., Kouzarides, T. and Tollervey, D. (2017) 33 Molecular Mechanisms of Epigenetic Gene Regulation

The overarching goal of research in our lab is to elucidate how histone modifications regulate gene expression. We are keen to understand how different histone modifiers and readers interact to establish complex regulatory systems that control development and affect disease states. We are taking a multidisciplinary approach to tackle these questions, combining biochemistry with proteomic, genomic, cell-biological, imaging- Philipp Voigt based, and systems biology-inspired techniques.

Co-workers: We focus on the interplay between Polycomb and Trithorax group proteins at bivalent domains and poised enhancers, in order to clarify how these complexes regulate Elana Bryan expression of developmental genes in embryonic stem (ES) cells. Bivalent domains Katy McLaughlin harbour a distinctive histone modification signature featuring both the active histone H3 lysine 4 trimethylation (H3K4me3) mark and the repressive H3K27me3 mark (Figure 1A). Viktoria Major They are presumed to maintain developmental genes in a poised state, allowing for Stefania del Prete timely expression upon differentiation while maintaining repression in ES cells. Bivalent nucleosomes adopt a previously unknown asymmetric conformation, carrying the active Thomas Sheahan and repressive mark on opposite copies of histone H3 (Voigt et al., Cell, 2012). However, it remains unclear how bivalent domains function to poise genes for expression in ES cells Marie Warburton and whether they are essential for proper ES cell differentiation and development. Kim Webb To address these questions mechanistically, we performed pulldown experiments with ES cell nuclear extract and recombinant asymmetric, bivalent nucleosomes. We found that bivalent nucleosomes are unable to recruit binding proteins for H3K4me3, despite presence of the mark. In contrast, bivalent nucleosomes retain binding of H3K27me3 binders. Moreover, we further uncovered binding proteins for bivalent nucleosomes that are not recruited by either mark on its own, representing factors that might specifically read the asymmetric bivalent configuration. Our findings suggest a model by which bivalent nucleosomes mediate poising by preventing binding of activating factors while being bound by Polycomb complexes and bivalency-specific binding proteins. To explore the function of bivalent domains and associated genetic elements from a systems biology-inspired perspective, we are establishing a reporter gene system that allows us to quantify the impact of bivalent domains on gene activation and repression kinetics in order to understand how this chromatin signature acts to fine tune gene expression during development. We are testing the hypothesis that bivalent domains integrate repressive and activating signals in a dynamic fashion to fine-tune expression during development (Figure 1B). 34 a

b

Figure 1. a. Interactions between active and repressive factors control the establishment and function of bivalent domains in embryonic stem cells. b. By maintaining a plastic state, bivalent domains may ensure proper timing of expression during development.

Selected Publications: Jacob, Y., and Voigt, P. (2018). In vitro assays to measure histone Voigt, P., Leroy, G., Drury, W.J., Zee, B.M., Son, J., Beck, D.B., methyltransferase activity using different chromatin substrates. Young, N.L., Garcia, B.A., and Reinberg, D. (2012). Asymmetrically Methods Mol. Biol. 1675, 345–360. Modified Nucleosomes. Cell 151, 181–193. Brewster, R.C., Gavins, G.C., Günthardt, B., Farr, S., Webb. K.M., Voigt, P., & Hulme, A.N. (2016). Chloromethyl-triazole: a new motif for site-selective pseudo-acylation of proteins. Chem. Commun. 52, 12230–12232. 35 Drug Discovery and Molecular Recognition in Biological Systems

Organisms from bacteria to mammals have a remarkably well conserved glycolytic (and gluconeogenic) pathways that use the same ten enzymatic steps to convert glucose to pyruvate. Though the active sites have been conserved throughout 2 billion years of evolution, there is an interesting divergence in allosteric regulatory mechanisms. We have studied the enzymatic mechanisms of allosteric enzymes from mammals, trypanosomatid Malcolm Walkinshaw parasites and pathogenic bacteria including Mycobacterium tuberculosis. Co-workers: By trapping the enzymes in different conformational states it is possible to show how small molecule effector molecules including amino acids, AMP and ADP can enhance or Tsabieh Bilal inhibit enzyme activities. For example, the enzyme FBPase from the Leishmania parasite Liz Blackburn can be regulated by a simple feedback mechanism with increasing concentration of AMP (1) that traps the tetramer in an inactive twisted form (Figure A). Pyruvate kinase from Jaqueline Dornan Mycobacterium tuberculosis can act like a logical ‘OR-gate’ (3) in which two metabolites Khadar Dudekula (AMP and glucose-6-P) bind to different allosteric pockets and synergistically activate the tetramer (Figure B). More sophisticated still is the regulation of the M2 isoform of human Hannah Florance pyruvate kinase (M2PYK) where we have shown that selected amino acids (Ala, Phe, Lisa Imrie Trp) and reactive oxidation species (ROS) are potent inhibitors while other metabolites James Kinkead and amino acids (His and Ser) are activators. M2PYK interprets these multiple input signals that display the nutritional and stress state of the cell providing an appropriate Marianna Leite de Avellar output response to rebalance cellular metabolism (Figure C). This competition at a single Iain McNae allosteric site between activators and inhibitors provides a novel regulatory mechanism by which M2PYK activity is finely tuned by the relative (but not absolute) concentrations of Paul Michels activator and inhibitor amino acids which we call ‘allostatic regulation’.

Jia Ning Apart from the basic biochemical, structural and evolutionary insights gained from these Matthew Nowicki studies, the allosteric binding pockets all provide excellent targets for species-specific inhibitors. We have already identified Tb phosphofructokinase inhibitors that cure mouse Eliane Salvo-Chirnside models of African Trypanosomiasis. High throughput screens of bacterial targets have Emmaline Stotter allowed us to identify a series of hit compounds currently being modified in collaboration with the European Lead Factory. The link between cancer and glycolysis and more Paul Taylor recently as a regulator of immune response make glycolytic enzymes interesting and Martin Wear relatively unexplored therapeutic targets. We are currently screening human PFK isoforms for new families of allosteric inhibitors as anticancer therapeutics.

36 a

b c

Figure 1. The diversity of allosteric regulatory mechanisms in glycolysis. a. Leishmania mexicana fructose bisphosphatase is regulated in by its allosteric inhibitor AMP that keeps the tetramer in an inactive conformation. b. Mycobacterium tuberculosis pyruvate kinase acts like an ‘logical OR’ gate and is activated synergistically by two allosteric activators that bind at two distinct sites. c. Human M2 pyruvate kinase is allosterically regulated by amino acids and fructose 2,6-bisphosphate. Blue solid arrows show the transformation of M2PYK between active tetramer (green) and inactive tetramer (orange). Green dotted arrows show that FBP and serine stabilise M2PYK in the active tetrameric form while phenylalanine, tryptophan, valine and alanine inhibit M2PYK.

Selected Publications: Fragment-Based Drug Discovery: Identification of New Cyclophilin Yuan M, Vasquez-Valdivieso MG, McNae IW, Michels PAM, Binders. Journal of molecular biology. 2017;429:2556-70. Fothergill-Gilmore LA, Walkinshaw MD. Structures of Leishmania Fructose-1,6-Bisphosphatase Reveal Species-Specific Differences Zhong W, Cui L, Goh BC, Cai Q, Ho P, Chionh YH, et al. Allosteric in the Mechanism of Allosteric Inhibition. Journal of molecular pyruvate kinase-based "logic gate" synergistically senses biology. 2017;429:3075-89. energy and sugar levels in Mycobacterium tuberculosis. Nature communications. 2017;8:1986. Georgiou C, McNae I, Wear M, Ioannidis H, Michel J, Walkinshaw M. Pushing the Limits of Detection of Weak Binding Using 37 How microtubule motors coordinate mitosis

To maintain their genomic integrity, eukaryotic cells must replicate their DNA faithfully and distribute it equally to the daughter cells. Mitotic defects lead to aneuploidy and cancer. This indicates that the mitotic mechanisms that are in place to allow faithful division have been compromised. The segregation of chromosomes is mediated by polarized and highly dynamic filaments, termed microtubules. Microtubules depend on motor proteins Julie Welburn to assemble into a spindle and segregate chromosomes. These motors play key roles in cytoskeletal organization during cell division but also in cell migration, polarity, and axonal Co-workers: and cytoplasmic transport. However, the reductionist approach to studying these motors Ben Craske in isolation is not sufficient to understand their function in the cellular context. It remains unclear how the activities of individual motors and their interacting regulatory networks Giovanna de la Hoz cooperate to generate physiological cellular function such as chromosome segregation. Banos We aim to define how kinesin motors are modulated by their cargos to provide a specific Agata Gluszek output, and how the coordinated activities of kinesin motors are greater than the sum of their individual activities in vitro and in human cells. Thibault Legal Kinetochores and motors. CENP-E is a huge motor (312 kD), recruited to unattached Toni McHugh kinetochores. CENP-E moves kinetochores along microtubules to facilitate chromosome alignment. How CENP-E associates with the kinetochore, how human CENP-E is activated to walk on microtubules and how CENP-E motor ensembles are coordinated to move chromosomes is currently not known. We are addressing these questions.

Mitotic motors and microtubule dynamics. Our lab has made new discoveries on the mechanism of mitotic microtubule depolymerases. While Kinesin-13 motors are major microtubule depolymerases, the role of Kinesin-8 motors in microtubule depolymerization remains controversial. We have shown using gene knockout that the Kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase (Fig 1A, B). Using in vitro reconstitution, we reveal that Kif18b is a highly processive plus end-directed motor that uses a C-terminal non-motor microtubule-binding region to accumulate at growing microtubule plus ends (Fig 1C). This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length (Fig 1D). Finally we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b utilizes its motile properties to reach microtubule ends where it regulates astral microtubule length to ensure spindle centering.

38 a b

c d

Figure 1. Kif18b is a processive plus end-directed microtubule motor that controls astral microtubule length and spindle positioning. a. Representative immunofluorescence images of control and Kif18b-KO HeLa cells, acquired using antibodies against Ndc80 and β-tubulin. b. Box and whisker plot showing quantification of the spindle displacement from the center of the cell during metaphase. Each point represents the displacement of 1 spindle over at least 30 minutes. c. Kif18b is a plus end directed motor with a run length of >7μm. Motors walk processively towards the plus end of the microtubules, pausing at the microtubule tips before dissociation. d. Schematic model of Kif18b walking towards the plus end of a microtubule. Kif18b is phosphorylated close to centrosomes and chromosomes. As Kif18b reaches the cortex, the C terminus is dephosphorylated by cytoplasmic or cortical phosphatases, which allows Kif18b to accumulate at the ends of astral microtubules to destabilize them. Kif18b and MCAK could cooperate at microtubule plus ends to destabilize microtubules.

Selected Publications: Gigant E., Stefanutti M, Laband K, Gluszek-Kustusz A, Edwards F, architecture of the Dam1 complex-microtubule interaction. (2016). Maton G,Lacroix B, Canman JC., Welburn JPI , Dumont J. Inhibition Open Biology. DOI: 10.1098. PMID: 26962051. of ectopic microtubule assembly by the kinesin-13 KLP-7MCAK Talapatra S., Harker B., Welburn JPI. The C-terminal region prevents oocyte aneuploidy. Development. (2017). 144: 1674-1686. of MCAK controls its activity and structure through a major Legal T., Zou J., Sochaj A., Rappsilber J., Welburn JPI. Molecular conformational switch. Elife (2015). 39 Public Engagement

In 2017, ninety-two of our researchers engaged with over 2200 members of the public, our support for Midlothian Science Festival (MSF) engaged a further 9500 members of the public: 11,833 people in total. The time that each engagement lasted was high, 51 minutes on average. Sarah Keer-Keer Our most successful new project in 2017, was Tattoo my Science, a project designed for adults and teenagers at festivals. We ran this at the Meadows Festival with 395 Co-workers: people and received some of the most positive feedback we have ever had. We also ran the event with families in Midlothian (it worked equally well) and presented it at Maria Fanourgiaki Engage 2017 to engagement professionals. In a survey to follow up after Engage, 94% Claire Jellema of respondents said they would or probably would use tattoos in their own engagement projects. Figs 1, 2 & 3 Laura Reed We completed many new presentations of projects that have been run successfully in previous years: two phases of Pupils in Labs with local senior pupils (in total 120 pupils spent the day with us) Figs 4 & 5, two days of Life Through a Lens at the Botanic gardens held together with the Epigenetic Voyage Figs 6 & 7, and 4 sessions of Life Through a Lens at primary schools.

New for 2017, we:

• Supported two of our researchers in developing and running their own fantastic engagement project DNA journey, which was presented to over 300 people, Fig 8,

• Produced nine days of Public Engagement using Glass training, reaching 15 researchers, including many outside of our centre: two of these researchers went on to hold an exhibition of their work,

• Produced a workshop that uses story-telling for children age 3-7,

• Produced a day in a local shopping centre engaging the shoppers (a different demographic to other venues).

We are currently focussed on our Cell Block Science project, a collaboration with St. Andrews University where we are delivering public engagement in HMP Edinburgh prison, Fig 9 shows a photo of a page from a prisoner’s lab book.

40 1

3 2

4 5 6

7 8 9 41 List of Groups

Robin Allshire Jean Beggs Wellcome Principal Research Fellow Professor of Molecular Biology

Tatsiana Auchynnikava Wellcome Research Associate Vahid Aslanzadeh Darwin Trust Graduate Student Roberta Carloni Wellcome Research Associate David Barrass Wellcome Research Associate Tadhg Devlin BBSRC EASTBIO Graduate Student Jim Brodie Wellcome Research Associate Lorenza Di Pompeo Wellcome Research Assistant Susana De Lucas Wellcome Research Associate Max Fitz-James Wellcome 4yr Graduate Student Eve Hartswood Wellcome Research Associate Alison Pidoux Wellcome Research Associate Bella Maudlin Wellcome 4yr Graduate Student Desislava Staneva Wellcome 4yr Graduate Student Gonzalo Mendoza-Ochoa CONACYT Graduate Student Manu Shukla Wellcome Research Associate Julia Neumeier Erasmus Plus visiting MSc Student Puneet Singh Darwin Trust Graduate Student Ema Sani Wellcome Research Associate Pin Tong Wellcome Research Associate Edward Wallace Marie Curie Research Fellow Jesus Torres-Garcia Darwin Trust Graduate Student Gabor Varga Wellcome Technician (part-time) Sharon White Wellcome Research Associate Adrian Bird Weifang Wu Darwin Trust Graduate Student Buchanan Professor of Genetics, Edinburgh University Imtiyaz Yaseen Wellcome Research Associate Christian Belton Graduate Student Beatrice A. Jeyaprakash Arulanandam Alexander-Howden Wellcome Research Assistant Wellcome Senior Research Fellow Kashyap Chhatbar RSRT Research Assistant Justyna Cholewa-Waclaw RSRT Research Associate Maria Alba Abad John Connelly Wellcome Research Associate Fernandaz Wellcome Research Associate Dina De Sousa Wellcome Research Assistant Asma Al-Murtadha Darwin Trust Graduate Student Laura Fitzpatrick Wellcome 4yr Graduate Student Lana Buzuk Research Technician Jacky Guy Wellcome Research Associate Ignacio Jiménez Principal’s Career Development Matthew Lyst Wellcome Research Associate Graduate Student Raphael Pantier ERC Research Associate Bethan Medina-Pritchard Wellcome Research Assistant Katy Paton SBS/SBRC cohort Graduate Student Frances Spiller Darwin Trust Graduate Student Jim Selfridge Wellcome Research Associate Reshma Thamkachy SERB Overseas Postdoctoral Fellow Konstantina Skourti-Stathaki Sir Henry Wellcome Postdoctoral Fellow Christine Struthers Personal Assistant

42 Atlanta Cook Tony Ly Wellcome Senior Research Fellow Sir Henry Dale Fellow

Uma Jayachandran Wellcome Research Assistant Van Kelly Wellcome Research Associate Valdeko Kruusvee Wellcome Research Associate David Lewis BBSRC EASTBIO Graduate Student Aleksandra Kasprowicz Wellcome Research Associate Aymen al-Rawi Darwin Trust Graduate Student Alexander Will Darwin Trust Graduate Student

Bill Earnshaw Adele Marston Wellcome Principal Research Fellow Wellcome Senior Research Fellow

Mar Carmena Wellcome Research Associate Rachael Barton Wellcome 4yr Graduate Student Fernanda Cisneros CONACYT (Mexico) Research Associate Julie Blyth Wellcome Research Assistant Oscar Molina Wellcome Research Associate Weronika Borek Wellcome Research Associate Lidija Pavlovic Principal's Career Development Stefan Galander Wellcome Research Associate Graduate Scholarship Bethany Harker Wellcome 4yr Graduate Student Emma Peat Part-time Technician Katarina Jönsson EPSRC/Agilent Graduate Student Elisa Pesenti CMSB Postdoctoral Fellow Vasso Makrantoni Wellcome Research Associate Melpomeni Platani Wellcome Research Associate Flora Paldi Wellcome 4yr Graduate Student Lucy Remnant Wellcome Research Associate Meg Peyton-Jones EASTBIO Graduate student Jan Ruppert Marie Curie Early Stage Researcher Rebecca Plowman MRC Graduate Student Itaru Samejima Wellcome Research Associate Ola Pompa Darwin Trust Graduate student Kumiko Samejima Wellcome Research Associate Xue (Bessie) Su Wellcome Research Associate Giulia Vargiu Wellcome Research Associate Menglu (Lily) Wang Wellcome Research Associate Alisa Zhiteneva Wellcome 4yr Graduate Student

Patrick Heun Hiro Ohkura Wellcome Senior Research Fellow Wellcome Investigator in Science

Eduard Anselm Wellcome Research Associate Mariana Costa Wellcome Research Associate Georg Bobkov ERC Research Associate Fiona Cullen Wellcome Research Associate Emily Fowler Graduate Student Alex McDonnell Wellcome Research Assistant Eftychia Kyriacou ERC Research Associate Jule Nieken Darwin Trust Graduate Student Vasiliki Lazou Wellcome Research Assistant Charlotte Repton BBSRC EASTBIO Graduate Student Manuela Marescotti Wellcome Research Associate Pierre Romé Wellcome Research Associate Virginie Roure ERC Research Associate Pedro Silva Darwin Trust Graduate Student Verdiana Steccanella Wellcome Research Associate

43 Juri Rappsilber David Tollervey Wellcome Senior Research Fellow Director, Wellcome Principal Research Fellow

Giulia Bartolomucci Wellcome Research Assistant Stefan Bresson Wellcome Research Associate Adam Belsom Wellcome Research Associate Clémentine Delan-Forino FEBS Fellow Colin Combe Wellcome Research Associate Hywel Dunn-Davies BBSRC Research Associate Therese Dau DFG Research Associate Aziz El Hage Wellcome Research Associate Lutz Fischer Wellcome Research Assistant Tatiana Dudnakova BBSRC Research Associate Martin Graham Wellcome Research Associate Aleksandra Helwak Wellcome Research Associate Lars Kolbowski Wellcome Research Assistant Laura Milligan Wellcome Research Associate Georg Kustatscher Wellcome Research Associate Elisabeth Petfalski Wellcome Research Associate Christos Spanos Wellcome Research Associate Nic Robertson ECAT Fellow Juan Zou Wellcome Proteomics Data Camille Sayou EMBO Fellow Analysis Manager Vadim Shchepachev SNSF Research Fellow Tomasz Turowski Polish Academy Research Fellow Marie-Luise Winz EMBO Fellow Ken Sawin Professor of Cell Biology

Sanju Ashraf Wellcome Research Associate Philipp Voigt Xun Bao Wellcome Research Associate Sir Henry Dale Fellow Hayley Johnson Wellcome 4yr Graduate Student Elana Bryan Wellcome 4yr Graduate Student Su Ling Leong Wellcome Research Associate Katy McLaughlin Postdoctoral Research Associate Ana Rodriguez Darwin Trust Graduate Student Viktoria Major Research Technician Ye Dee Tay BBSRC/Wellcome Research Associate Stefania del Prete Postdoctoral Research Associate Harish Thakur Wellcome Research Associate Thomas Sheahan Graduate Student Marie Warburton Graduate Student Eric Schirmer Kim Webb Wellcome Research Assistant Wellcome Senior Research Fellow

Dario Barreiros Ker Memorial Studentship Rafal Czapiewski Wellcome Research Associate Jose I. de las Heras Wellcome Research Associate Charles R. Dixon Wellcome 4yr Graduate Student Alexandr Makarov Principle's Scholarship Graduate Student Maria Manunta Wellcome Research Associate Andrea Rizzotto Darwin Trust Graduate Student Aishwarya Sivakumar Darwin Trust Graduate Student

44 Malcolm Walkinshaw Administration/Support Staff Chair of Structural Biochemistry, Director, Edinburgh Protein Production Facility Greg Anderson Centre Laboratory Manager Elizabeth Blackburn EPPF Research Associate Tsabieh Bilal Graduate Student Julie Blyth Centre Imaging Facility Assistant Liz Blackburn EPPF Research Associate Maria Fanourgiaki Science Communicator Jacqueline Dornan EPPF Research Associate Carolyn Fleming Centre Administrative Assistant Khadar Dudekula Research Associate Claire Jellema Midlothian Science Festival Hannah Florance Research Associate Manager/Administrator Lisa Imrie Research Technician, Edinomics Sarah Keer-Keer Centre Public Engagement Manager James Kinkead Wellcome Research Assistant David Kelly Centre Optical Instrumentation Marianna Leite de Avellar MSc student Laboratory Manager Iain McNae Wellcome Research Associate Alastair Kerr Centre Bioinformatics Core Paul Michels Visiting Professor Facility Manager Jia Ning Darwin Trust Graduate Student Colin McLaren Computing Support Matthew Nowicki EPPF Research Associate Jane Paget Application Scientist, Edinburgh Eliane Salvo-Chirnside Research Associate (Edinomics) Genome Foundry Emmaline Stotter MSc student Laura Reed Glass Technician/Artist Paul Taylor University Senior Lecturer/EPPF Daniel Robertson Bioinformatics Support Officer Research Associate Christos Spanos Proteomics Facility Manager Martin Wear Senior Lecturer/EPPF Facility Manager Christine Struthers PA to Adrian Bird/Centre Administrator Karen Traill Centre Manager/Wellcome 4yr PhD Programme Administrator Julie Welburn Shaun Webb Bioinformatician Wellcome Senior Research Fellow Juan Zou Proteomics Data Analysis Manager

Ben Craske BBSRC DTP Graduate Student Giovanna de la Hoz Banos Erasmus+ student Technical Support Agata Gluszek Postdoctoral Research Assistant Thibault Legal CRUK Research Assistant Lloyd Mitchell Toni McHugh CRUK Postdoctoral Research Associate Washing-up/Media

Denise Affleck Andrew Kerr Margaret Martin Donna Pratt

45 46 47 Centre Publications 2016 – 2018

Abad, M.A., J. Zou, B. Medina-Pritchard, E.A. Nigg, J. Rappsilber, Belsom, A., M. Schneider, O. Brock and J. Rappsilber (2016). A. Santamaria and A.A. Jeyaprakash (2016). “Ska3 Ensures Timely “Blind Evaluation of Hybrid Protein Structure Analysis Methods Mitotic Progression by Interacting Directly With Microtubules and based on Cross-Linking.” Trends Biochem Sci 41(7): 564-567. Ska1 Microtubule Binding Domain.” Sci Rep 6: 34042. Belsom, A., M. Schneider, L. Fischer, O. Brock and J. Rappsilber Agmon, N., Z. Tang, K. Yang, B. Sutter, S. Ikushima, Y. Cai, (2016). “Serum Albumin Domain Structures in Human Blood Serum K. Caravelli, J.A. Martin, X. Sun, W.J. Choi, A. Zhang, G. by Mass Spectrometry and Computational Biology.” Mol Cell Stracquadanio, H. Hao, B.P. Tu, D. Fenyo, J.S. Bader and J.D. Proteomics 15(3): 1105-1116. Boeke (2017). “Low escape-rate genome safeguards with minimal Bharathavikru, R., T. Dudnakova, S. Aitken, J. Slight, M. Artibani, P. molecular perturbation of Saccharomyces cerevisiae.” Proc Natl Hohenstein, D. Tollervey and N. Hastie (2017). “Transcription factor Acad Sci U S A 114(8): E1470-E1479. Wilms’ tumor 1 regulates developmental RNAs through 3’ UTR Allshire, R.C. and H.D. Madhani (2018). “Ten principles of interaction.” Genes Dev 31(4): 347-352. heterochromatin formation and function.” Nat Rev Mol Cell Biol Bird, A. (2017). “Genetic determinants of the epigenome in 19(4): 229-244. development and cancer.” Swiss Med Wkly 147: w14523. Antequera, F. and A. Bird (2018). “CpG Islands: A Historical Blyth, J., V. Makrantoni, R.E. Barton, C. Spanos, J. Rappsilber and Perspective.” Methods Mol Biol 1766: 3-13. A.L. Marston (2018). “Genes Important for Schizosaccharomyces Ard, R. and R.C. Allshire (2016). “Transcription-coupled changes to pombe meiosis identified through a functional genomics screen.” chromatin underpin gene silencing by transcriptional interference.” Genetics 208(2): 589-603. Nucleic Acids Res 44(22): 10619-10630. Bobkov, G., Gilbert, N., and Heun, P. (2018). Centromere Ard, R., R.C. Allshire and S. Marquardt (2017). “Emerging transcription allows CENP-A to transit from chromatin association Properties and Functional Consequences of Noncoding to stable incorporation. J. Cell Biol. 217, DOI: 10.1083/ Transcription.” Genetics 207(2): 357-367. jcb.201611087 Aslanzadeh, V., Y. Huang, G. Sanguinetti and J.D. Beggs Boeke, J.D., G. Church, A. Hessel, N.J. Kelley, A. Arkin, Y. Cai, (2018). “Transcription rate strongly affects splicing fidelity and R. Carlson, A. Chakravarti, V.W. Cornish, L. Holt, F.J. Isaacs, cotranscriptionality in budding yeast.” Genome Res 28(2): 203-213. T. Kuiken, M. Lajoie, T. Lessor, J. Lunshof, M.T. Maurano, L.A. Barrey, E.J. and P. Heun (2017). “Artificial Chromosomes and Mitchell, J. Rine, S. Rosser, N.E. Sanjana, P.A. Silver, D. Valle, H. Strategies to Initiate Epigenetic Centromere Establishment.” Prog Wang, J.C. Way and L. Yang (2016). “GENOME ENGINEERING. Mol Subcell Biol 56: 193-212. The Genome Project-Write.” Science 353(6295): 126-127. Beaven, R., R.N. Bastos, C. Spanos, P. Rome, C.F. Cullen, J. Booth, D.G., A.J. Beckett, O. Molina, I. Samejima, H. Masumoto, Rappsilber, R. Giet, G. Goshima and H. Ohkura (2017). “14-3-3 N. Kouprina, V. Larionov, I.A. Prior and W.C. Earnshaw (2016). regulation of Ncd reveals a new mechanism for targeting proteins “3D-CLEM Reveals that a major portion of mitotic chromosomes is to the spindle in oocytes.” J Cell Biol 216(10): 3029-3039. not chromatin.” Mol Cell 64(4): 790-802. Beggs, J.D. (2017). “Pre-mRNA Splicing.” In Reference Module in Booth, D.G. and W.C. Earnshaw (2017). “Ki-67 and the Life Sciences, Elsevier, chromosome periphery compartment in mitosis.” Trends Cell Biol 27(12): 906-916. 2017, ISBN: 978-0-12-809633-8, http://dx.doi.org/10.1016/B978-0- 12-809633-8.06947-8 Botte, M., N.R. Zaccai, J.L. Nijeholt, R. Martin, K. Knoops, G. Papai, J. Zou, A. Deniaud, M. Karuppasamy, Q. Jiang, A.S. Roy, Belsom, A., G. Mudd, S. Giese, M. Auer and J. Rappsilber K. Schulten, P. Schultz, J. Rappsilber, G. Zaccai, I. Berger, I. (2017). “Complementary Benzophenone Cross-Linking/Mass Collinson and C. Schaffitzel (2016). “A central cavity within the Spectrometry Photochemistry.” Anal Chem 89(10): 5319-5324. holo-translocon suggests a mechanism for membrane protein insertion.” Sci Rep 6: 38399. 48 Bresson, S. and D. Tollervey (2018). “Surveillance-ready Meldal, G. Micklem, S. Orchard and J. Rappsilber (2017). transcription: nuclear RNA decay as a default fate.” Open Biol 8(3). “ComplexViewer: visualization of curated macromolecular Bresson, S., A. Tuck, D. Staneva and D. Tollervey (2017). “Nuclear complexes.” Bioinformatics 33(22): 3673-3675. RNA Decay Pathways Aid Rapid Remodeling of Gene Expression Crozier, T.W.M., M. Tinti, R.J. Wheeler, T. Ly, M.A.J. Ferguson and in Yeast.” Mol Cell 65(5): 787-800 e785. A.I. Lamond (2018). “Proteomic analysis of the cell cycle of procylic Brewster, R.C., G.C. Gavins, B. Gunthardt, S. Farr, K.M. Webb, P. form Trypanosoma brucei.” Mol. Cell. Proteomics (in press). Voigt and A.N. Hulme (2016). “Chloromethyl-triazole: a new motif Czapiewski, R., M.I. Robson and E.C. Schirmer (2016). “Anchoring for site-selective pseudo-acylation of proteins.” Chem Commun a Leviathan: How the Nuclear Membrane Tethers the Genome.” (Camb) 52(82): 12230-12232. Front Genet 7: 82. Brown, K., J. Selfridge, S. Lagger, J. Connelly, D. De Sousa, A. Davis, M.P., C. Carrieri, H.K. Saini, S. van Dongen, T. Leonardi, G. Kerr, S. Webb, J. Guy, C. Merusi, M.V. Koerner and A. Bird (2016). Bussotti, J.M. Monahan, T. Auchynnikava, A. Bitetti, J. Rappsilber, “The molecular basis of variable phenotypic severity among R.C. Allshire, A. Shkumatava, D. O’Carroll and A.J. 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Margaret Fuller Nick Proudfoot Department of Developmental Biology Sir William Dunn School of Pathology and Department of Genetics University of Oxford Stanford University School of Medicine South Parks Road 291 Campus Drive, Li Ka Shing Building Oxford OX3 Stanford, CA 94305-5101 USA Michael Rout The Rockefeller University Frank Grosveld 1230 York Avenue Department of Cell Biology New York, NY 10021 Erasmus Medical Center USA Dr Molewaterplein 50 3000 Rotterdam Netherlands

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Iain Mattaj European Molecular Biology Laboratories Meyerhofstrasse 1 69117 Heidelberg Germany

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Cover image: Mitotic chromosomes are made up of loops (various colours) organised by a helical scaffold of condensin II (red circles). Image by Anton Golobdorodko from a simulation based on Gibcus, J.H., K. Samejima, A. Goloborodko, I. Samejima, N. Naumova, J. Nuebler, M. Kanemaki, L. Xie, J.R. Paulson, W.C. Earnshaw, L.A. Mirny, J. Dekker. (2018). A pathway for mitotic chromosome formation. SCIENCE 9:359.

The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336. Anton Golobdorodko