The regulation of sister chromatid cohesion

Biochimica et Biophysica Acta 1786 (2008) 41−48

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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b b a c a n

Review

The regulation of sister chromatid cohesion Ana Losada ⁎ Chromosome Dynamics Group, Spanish National Cancer Research Centre, Melchor Fernández Almagro 3, Madrid, E-28029, Spain

a r t i c l e

i n f o

Article history: Received 18 December 2007 Received in revised form 6 March 2008 Accepted 8 April 2008 Available online 24 April 2008 Keywords: Cohesin Chromosome segregation Topoisomerase II Mouse models

a b s t r a c t Sister chromatid cohesion is a major feature of the eukaryotic chromosome. It entails the formation of a physical linkage between the two copies of a chromosome that result from the duplication process. This linkage must be maintained until chromosome segregation takes place in order to ensure the accurate distribution of the genomic information. Cohesin, a multiprotein complex conserved from yeast to humans, is largely responsible for sister chromatid cohesion. Other cohesion factors regulate the interaction of cohesin with chromatin as well as the establishment and dissolution of cohesion. In addition, the presence of cohesin throughout the genome appears to influence processes other than chromosome segregation, such as transcription and DNA repair. In this review I summarize recent advances in our understanding of cohesin function and regulation in mitosis, and discuss the consequences of impairing the cohesion process at the level of the whole organism. © 2008 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . 2. Cohesin architecture and the topology of cohesion . . . . 3. Cohesin loading and redistribution . . . . . . . . . . . 4. Cohesion establishment during S phase . . . . . . . . . 5. Cohesion maintenance . . . . . . . . . . . . . . . . . 6. Removal of cohesin in mitosis, step 1 . . . . . . . . . . 7. Removal of cohesin in mitosis, step 2 . . . . . . . . . . 8. Cohesin-independent cohesion: removal of catenations by 9. Cohesin and DNA repair . . . . . . . . . . . . . . . . 10. Cohesin and gene expression . . . . . . . . . . . . . . 11. Animal models for cohesion factors . . . . . . . . . . . 12. Concluding remarks . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction The genomes of eukaryotic organisms are organized in chromosomes, individual DNA molecules folded into complex structures. Before cell division, each of these DNA molecules is faithfully copied by the DNA replication machinery. It is at this time that a physical linkage is established between the two sister DNA molecules (or sister chromatids). This pair-wise organization allows the cell to know how

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to distribute the chromatids during cell division so the daughter cells receive a full complement of chromosomes. The inter-sister linkage has two components: the DNA catenations arisen from the replication process itself and a multiprotein complex known as cohesin. Cohesin consists of a heterodimer of Structural Maintenance of Chromosomes (SMC) subunits, SMC1 and SMC3, the kleisin subunit Scc1 (also known as Mcd1/Rad21), and Scc3 or SA (Fig. 1A). SMC proteins are conserved from bacteria to human, and in eukaryotes they form three different complexes that participate in many important aspects of chromosome biology [1]. Although it has been proposed that cohesin forms a ring that embraces the sister DNA molecules, other models are possible [2]. Moreover, cohesin may bind to chromatin and function in different

A. Losada / Biochimica et Biophysica Acta 1786 (2008) 41−48

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Fig 1.

modes at specific chromosomal regions [3]. Cohesin is deposited on chromatin before S phase and it has to cope with other processes that take place in the cell nucleus like DNA replication or transcription. In metazoan organisms, most cohesin dissociates from chromatin at the onset of mitosis to allow proper resolution of the sister chromatids. A small fraction that persists, enriched around centromeres, is sufficient to allow proper chromosome alignment and timely segregation. Total dissolution of cohesion in anaphase requires removal of this cohesin fraction, which occurs by proteolytic cleavage of the Scc1 subunit, and the action of topoisomerase II on the remaining catenations. A number of factors contribute to the regulation of the cohesion process (Fig. 2). In this review I describe the major aspects of this regulation in somatic cells. 2. Cohesin architecture and the topology of cohesion Although cohesin is widely conserved, different versions of several subunits have arisen during evolution. For example, meiosis-specific forms of Scc1 and Scc3/SA are present in most organisms [4]. The specific functions of some variants remain unclear, as in the case of vertebrate SA1 and SA2 that coexist in mitotically dividing cells [5]. Despite this variety, it is likely that most complexes display a similar (if not identical) structural organization. Under the electron microscope, cohesin displays a ring shape [6] (Fig. 1B). Biochemical mapping of subunit–subunit interactions suggests that cohesin forms a tripartite ring with a 35-nm diameter in which the open V structure of the SMC heterodimer is closed by the simultaneous binding of the N- and C-terminal regions of Scc1 to the globular heads of SMC3 and SMC1, respectively [7] (see schematic in Fig. 1A). The results of recent FRET experiments indicate, however, that the Scc1 C-terminus lies between the SMC heads, which would be always in contact with each other [8]. It has been proposed that cohesin may perform its function by topologically trapping the sister chromatids within its ring. In favor of the model, cohesin dissociates from chromatin upon disruption of the integrity of the ring by cleavage of one of its forming subunits or, in the case of circular minichromosomes, upon linearization of the DNA [9,10]. Nevertheless, growing experimental evidence hints at the existence of different modes of cohesin binding to DNA in specialized chromosomal regions. For example, in the mating type locus of Saccharomyces cerevisiae, each cohesin ring apparently entraps a single chromatid and interacts with silencing factors in the opposite sister in order to achieve cohesion [11]. 3. Cohesin loading and redistribution Loading of cohesin on chromatin requires a heterodimeric complex composed of Scc2 and Scc4 in S. cerevisiae [12]. Orthologs of Scc2 have been easily identified in other eukaryotes, including humans [13,14]. In contrast, Scc4 has proven much less conserved, but it has been recently found in Schizosaccharomyces pombe and humans [15–17]. In all cases,

elimination of Scc2 or Scc4 function results in cohesion defects. Scc2 is a HEAT-repeat protein that interacts with cohesin and has been proposed to stimulate ATP hydrolysis by the SMC heads [18]. In Xenopus egg extracts, but not in budding yeast, loading of Scc2 on chromatin depends on pre-replication complex assembly [19,20]. Whether this is a feature of the rapid embryonic cycles remains to be assessed. The actual mechanism of cohesin loading by Scc2–Scc4 is unclear. ATP hydrolysis by the SMC proteins appears to be important, maybe to weaken the interaction between the SMC1–SMC3 heads [21] or to promote a transient disengagement of Scc1 [18]. More recent studies have highlighted the importance of the hinge domain in this process and even proposed that opening of the ring may occur at the hinge instead of the head domains [22,23]. In contrast, extensive biochemical analyses of Bacillus subtilis SMC led to the proposal that hinge–DNA interaction stimulates ATP hydrolysis thereby promoting disengagement of SMC heads to allow passage of the DNA between them [24]. Regardless of the final mechanism, the prominent role of the hinge in cohesin dynamics implies that it could be the target of regulatory factors. Indeed, this seems to be the case of Pds5, a protein that modulates the dynamic association of cohesin with chromatin [8] (see below). Mapping the genome-wide distribution of cohesin and Scc2 in yeast by chromatin immunoprecipitation (ChIP)-on-chip analyses has shown that: (1) cohesin binds preferentially to sites of convergent transcription, and (2) cohesin and Scc2 do not colocalize. These results prompted the idea that cohesin interacts with Scc2–Scc4 at the initial loading sites but then it slides towards the ends of genes pushed by the transcription machinery [25,26]. Unlike yeast, Drosophila Scc2 (Nipped-B) and cohesin bind to the same sites throughout the nonrepetitive genome of the fruit fly, and these sites are preferentially located in actively transcribed regions [27]. The authors of this study hypothesize that transcription may facilitate cohesin loading by providing a 10-nm fiber that fits the diameter of the cohesin ring. In human cells, 35% of the 8811 cohesin-binding sites identified throughout the non-repetitive genome in a recent study are found in introns [28]. Thus, it is likely that transcription dictates or modifies cohesin localization in metazoan organisms. Loading of cohesin may not always depend on Scc2–Scc4. A recent ChIP-on-chip study in budding yeast suggests that transcript elongation into cohesin association sites results in local dissociation of cohesin, an alternative to the cohesin sliding model previously proposed. Once transcription is halted, cohesin can bind to its original sites independently of Scc2, although it is no longer functional for cohesion [29]. Also, reassociation of cohesin during “centromere breathing”, the transient splitting of centromeric DNA under the pulling forces of spindle microtubule in metaphase, can also occur in the absence of Scc2 [30]. Recent experiments in Drosophila suggest that Scc2 is not required for cohesin loading at the centromeres, at least in meiotic chromosomes [31]. Additional factors contribute to cohesin loading at particular chromosomal regions. The centromeric histone H3 variant Cse4 and other kinetochore proteins are required for pericentromeric cohesin enrichment in S. cerevisiae [32]. Similarly, the S. pombe heterochromatin protein 1 homologue Swi6 bound to methylated Lysine9 on histone H3 enhances cohesin recruitment to centromeric repeats [33,34]. 4. Cohesion establishment during S phase Association of cohesin with chromatin and establishment of cohesion are two distinct processes that can be temporally separated. In human cells, loading of cohesin occurs already in early G1 while establishment can only happen during or after DNA replication. Among the proteins required for formation of cohesive structures, but not to deposit cohesin on DNA, is Eco1/Ctf7 [35,36]. This protein associates with the DNA polymerase processivity factor PCNA and with components of the clamp loader replication factor C (RF-C)

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Fig. 2.

[37,38]. Eco1/Ctf7 is a putative acetyl-transferase that can acetylate cohesin subunits in vitro [39]. However, this enzymatic activity may not be required for cohesion establishment during S phase [40]. An Eco1/Ctf7 ortholog, Deco, and a distinct acetyl-transferase named San are required in Drosophila to prevent precocious sister separation [41]. Both are conserved in human cells and appear to have non-redundant functions in the cohesion process [42,43]. While the human Eco1 orthologs (EFO1/ESCO1 and EFO2/ESCO2) are chromatin-bound proteins, human San is only present in the cytoplasm. Finding the substrates of these enzymes in vivo will be crucial to understand their relevance in cohesion establishment. Ctf4 and Ctf18 provide another link between cohesion and DNA replication [44,45]. Unlike Eco1, they are not essential in yeast, but cohesion establishment is clearly compromised in their absence. Ctf4 associates with the GINS complex and DNA polymerase α/primase in a putative replisome progression complex [46]. Ctf18 is part of a specific RF-C complex that loads PCNA onto DNA [47]. Less severe defects in sister chromatid cohesion are observed upon deletion or mutation of other replication-related factors (the DNA helicase Chl1, the replication fork-associated checkpoint proteins Tof1 and Csm3, DNA polymerase, the RF-C core subunit Rfc4, or the Orc5 subunit of the origin recognition complex) further supporting the existence of a mechanistic link between replication and cohesion establishment [45,48–53]. Indeed, ChIP experiments show that Eco1, Ctf4 and Ctf18 can be detected at replication forks during replication arrest in S phase, and at least Ctf4 travels along chromosomes together with the replication machinery during undisturbed S phase [54]. It is therefore likely that the establishment of cohesion takes place in the context of the replication fork. Importantly, it does not require additional loading of cohesin in S phase. Thus, one possibility is that cohesin dissociates from DNA during fork passage, but it is maintained relatively close to forks by some of the aforementioned cohesion factors. An alternative model proposes that the replication machinery slides through the cohesin ring, and this requires an appropriate geometry of the replication fork, to which those factors would contribute. In vitro reconstitution of a replication-coupled cohesion reaction will be important to fully understand cohesion establishment. 5. Cohesion maintenance Several lines of evidence suggest that the cohesin pool actively engaged in holding sister chromatids together exchanges very little

with the soluble pool [55]. FRAP analyses of GFP-tagged cohesin in rat cells have identified two distinct binding modes of cohesin to chromatin: about one-third of nuclear cohesin is bound very stably to chromosomes after replication while a second pool is dynamically exchanged on and off chromatin during interphase. The first pool, a part of which persists until anaphase, could represent those complexes actively involved in cohesion [56]. If this is the case, two thirds of chromatin-bound cohesin have a function distinct from cohesion, maybe related to the overall organization of the interphase nucleus, or even regulation of gene expression (see below). There are at least three proteins that interact closely with cohesin and modulate its chromatin association dynamics, although none of them is required for cohesin loading: Pds5, Wapl and Sororin (see Fig.1A). The three factors co-immunoprecipitate with cohesin from HeLa nuclear extracts, and their own association with chromatin depends on cohesin [57–61]. Pds5 is a HEAT-repeat protein conserved from fungi to vertebrates that plays a major role in cohesion maintenance [62–65]. Yeast Pds5 also interacts with Eco1, and in this way may contribute to cohesion establishment [66,67]. FRAP experiments in HeLa cells reveal that depletion of Sororin decreases the fraction of stably chromatinbound cohesin, thereby causing cohesion defects already in interphase [68]. In contrast, Wapl depletion prolongs the residence time of, at least, the dynamically exchanged population of cohesin on interphase chromatin, and is required for proper dissociation of cohesin in prophase [60]. Wapl, but not Sororin, is conserved in yeast where it also contributes to destabilize cohesin binding to chromatin [69]. 6. Removal of cohesin in mitosis, step 1 In metazoa, most cohesin dissociates from chromatin at prophase and only a small population remains on chromosomes by metaphase, enriched in the pericentromeric region [5,70]. Prophase dissociation requires phosphorylation of the SA cohesin subunits by Polo and phosphorylation of some other target by Aurora B [71–75]. The action of Wapl is also crucial either to “unlock” cohesin [60] or to prevent reassociation of cohesin following its release [61]. Centromeric cohesin is protected from dissociation by shugoshin (Sgo), which concentrates at centromeres in early mitosis and acts in concert with PP2A [76–80]. The phosphatase counteracts presumably cohesin phosphorylation by Polo thereby preventing its release. In addition to its role as cohesin protector, Sgo may function as a sensor of tension

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[81–83]. Proper localization of Sgo at centromeres requires both the spindle checkpoint protein Bub1 and Aurora B [84–87]. Moreover, the centromeric localization of these two kinases depends on one another [88]. Interestingly, recent results in mouse fibroblasts from Bub1 knock out animals indicate that Bub1 protection of centromeric cohesion is due to its role on the activation of the spindle checkpoint, not on Sgo localization [89]. Additional factors modulate the prophase dissociation pathway. Depletion of human Haspin, a histone H3 kinase on Threonine 3, results in a premature separation of sister chromatids (PSSC) phenotype, and its overexpression rescues the cohesion defects observed in the absence of Sgo [90]. Whether this histone phosphorylation event renders chromatin refractory to cohesin dissociation or affects the binding of some other regulator of cohesin remains to be discovered. Depletion of Prohibitin 2 (PHB2) also causes PSSC in mitosis without affecting Sgo localization, a phenotype rescued by co-depletion of Polo [91]. Writing up the connectivity map between all the modulators of the prophase pathway awaits further experimentation. What it is clear from the recent publications is that the map is more complex than previously thought. 7. Removal of cohesin in mitosis, step 2 Total dissolution of cohesion takes place at anaphase, once all chromosomes are properly bi-oriented in the metaphase plate so that the spindle checkpoint is satisfied. Cohesin release at this time results from site-specific cleavage of the Scc1 subunit by a cysteine protease called separase [92]. Scc1 phosphorylation by Polo is not essential but enhances cleavage by separase, at least in vitro [72,93,94]. The protease itself has a complex regulation that includes inhibitory binding to securin and Cdk1/cyclin B, and less understood mechanisms involving autocleavage, interaction with PP2A, or the microtubule and kinetochore protein astrin [95–97]. Separase cleaves any cohesin left on chromosomes, either at arms or at centromeres, even in cells lacking Wapl or expressing a non-phosphorylatable version of SA2 [60,72]. It is likely that Sgo has to be inactivated for separase to access and cleave cohesin in anaphase. In fact, Sgo disappears from the chromosomes in anaphase in most organisms. In Drosophila, delocalization of Sgo/Meis332 from centromeres involves both the separase pathway and phosphorylation by Polo [98,99] and Sgo is a substrate of the APC/C in vertebrates [80]. However, it is still unclear whether removal or degradation of Sgo is required for sister chromatid separation. 8. Cohesin-independent cohesion: removal of catenations by topoisomerase II Catenation of the sister chromatids that result from DNA replication is an additional mechanism of cohesion. Most catenations are removed by topoisomerase II (topo II) in interphase and the early stages of mitosis, thereby contributing to resolution of the sister chromatids. Inhibition or depletion of topo II rescues the cohesion defects of cohesin-depleted vertebrate cells [100,101]. Some catenations apparently persist by the time chromosomes align at the metaphase plate and must be resolved in anaphase to allow sister chromatid segregation [102,103]. Recent support for this idea comes from the identification of PICH, a centromere-associated protein of the SNF2 family of ATPases that decorates very thin threads connecting separating sister kinetochores in anaphase human cells [104]. These threads consist of alphoid centromeric DNA and their resolution requires the action of topo II [105,106]. Several lines of evidence suggest that topo II function in anaphase could be regulated by sumoylation (e.g, [107]. PIAS gamma, a member of the E3 family of SUMO ligases, sumoylates topo II and other centromere-enriched chromosomal factors in mitotic Xenopus extracts [108]. Inhibition of PIAS gamma prevents anaphase sister

separation in these extracts and arrests human cells in metaphase with paired sisters, even in the absence of Sgo [109]. In contrast, PIAS gamma-deficient mice appear phenotypically normal, suggesting that its function is not essential for proliferation or that there is a compensatory mechanism [110]. Whether sumoylation regulates topo II localization, its affinity for chromatin, or its enzymatic activity remains to be elucidated. 9. Cohesin and DNA repair Studies in yeast and vertebrate cells have revealed the importance of cohesin in DNA repair [111]. The complex was shown to bind to sites of laser-induced DNA damage in the S- and G2-phases of the cell cycle [112]. Later on, cohesin was found to be recruited to an extended region of ~100 kb surrounding a single double strand break (DSB) in yeast [113,114]. This recruitment is needed for repair and is under the control of Scc2 and damage repair factors such as Mre11. Cohesin enrichment around the DSB likely promotes repair by homologous recombination, using the undamaged sister as a template [115] although it could have also additional signaling functions [116]. On the other hand, recent experiments in yeast show that DSB-induced cohesin loading generates competent cohesion not only at the DSB but also in undamaged regions of the genome [117,118]. Cohesion establishment at this time has different requirements, e.g. the acetyl-transferase activity of Eco1 appears to be important for DSB-induced cohesion in G2/M but not during S phase [118]. Since DSBs can arise as a result of endogenous cellular metabolism (e.g. DNA replication) it is possible that cohesion is routinely reinforced in G2 cells through this mechanism. The advantage of “adding” cohesin at this time is that there would be less interference with the replication process. 10. Cohesin and gene expression Cohesin is a major component of interphase chromatin, but in metazoan organisms, only a small fraction that persists on chromatin by metaphase is sufficient to allow proper chromosome alignment and timely segregation. The cell may assemble such a large excess of cohesin in interphase simply to ensure proper cohesion in mitosis, but a more interesting possibility is that cohesin plays additional roles in interphase. Studies in Drosophila first revealed a connection between cohesin and transcription. Mutations in the cohesin loading factor Scc2/Nipped-B reduced the expression of certain homeotic genes whereas cohesin mutants had the opposite effect [119,120]. Similarly, Pds5 mutants defective in cohesion which still allowed cohesin binding to chromatin did not show alterations in gene expression whereas mutants affecting cohesin binding did [62]. Thus, the presence of cohesin may hinder communication between enhancers and promoters, or get in the way of transcription complexes. In other instances, cohesin enhances gene expression, as is the case of the zebrafish runx transcription factors, involved in cell fate determination of many lineages [121]. New genomewide chromatin immunoprecipitation experiments in human and mouse cells have just shown that cohesin accumulates at sites bound also by CTCF, a zinc-finger protein with boundary element activity that blocks the communication between enhancers and promoters [28,122,123]. Mutations in the human Scc2 ortholog NPBL and other cohesion factors, including cohesin, cause a human disease known as Cornelia de Lange syndrome [13,14,124,125]. This is a developmental disorder characterized by facial dysmorphia, upper extremity malformations, cardiac and gastrointestinal anomalies and growth and cognitive retardation. A related yet distinct disorder, known as Roberts syndrome/SC phocomelia, is the result of mutations in the gene encoding the cohesion establishment factor ESCO2 [126]. At least some of the birth defects of these patients could result from chromosome segregation defects leading to reduced numbers of viable cells during embryonic development. Supporting this possibility, cohesion defects have been

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observed in the cells of some Cornelia de Lange and Roberts syndrome patients [124,126]. Alternatively, and in consonance with the abovementioned studies, the expression of certain genes crucial for development – as is the case of homeotic genes – could be very sensitive to the presence of cohesin around their promoters. In addition, two recent papers describe the contribution of cohesin to the morphogenesis of nondividing neurons in Drosophila, providing another clue to understand the mental retardation displayed by these patients [127,128]. 11. Animal models for cohesion factors In mammalian cells, the function of most cohesion factors including cohesin has been studied by RNA interference. Knock out (KO) mice would be a desirable alternative to study gene function in the more physiological setting of tissues within animals. In addition, these mice would provide the opportunity to investigate whether aneuploidy caused by chromosome segregation defects can initiate or promote tumorigenesis, as well as the etiology of the syndromes mentioned in the previous section. Remarkably, a recent survey of a panel of 132 colorectal cancer samples found 11 somatic mutations in genes encoding for cohesin subunits and Scc2/NPBL, suggesting that defective cohesion may be a major cause of chromosome instability in human cancers [129]. To date, the only mouse models deficient for a component of the cohesin complex are those of the meiosis-specific subunits Rec8 and SMC1beta, both of them sterile [130,131]. Regarding cohesin-interacting factors, a recent publication reports the generation of KO animals for one of the two genes encoding a vertebrate Pds5 protein, PDS5B. PDS5B−/− mice die soon after birth and, importantly, they recapitulate many of the congenital anomalies found in patients of the Cornelia de Lange syndrome [132]. So far, no correlation is found in the literature between this disease and a higher incidence of cancer. Mouse models for separase, securin and Bub1 have been reported recently. The study of separase-deficient mice has shown that this protease is essential for mammalian embryonic development, as well as for chromosome segregation at the onset of anaphase, both in hepatocytes in vivo and in MEFs in vitro [133,134]. In contrast, securin deficiency does not lead to any obvious phenotype. These studies do not mention whether chromosome segregation defects observed in the mutant mice correlate with cancer predisposition. However, carcinogenesis studies in zebrafish reveal an eightfold increase in the percentage of fish bearing epithelial tumors for heterozygous mutants in the gene encoding separase [135]. Consistent with this phenotype, loss of separase in Drosophila causes defects in epithelial organization and integrity [136]. Mice with reduced amounts of Bub1 protein are apparently normal despite the frequency of aneuploid cells, but they are highly susceptible to spontaneous tumors [137]. This susceptibility could result from a weakened mitotic checkpoint activity, since heterozygous mice for other components of the spindle checkpoint are also predisposed to either spontaneous or carcinogen-induced tumors (e.g. [138,139]). Two other animal models related to cohesion correspond to DNA helicases. Deficiency of Ddx11, the mouse homolog of Chl1 helicase, results in embryonic lethality, and the mutant embryos show increased frequency of chromosome missegregation and aneuploidy [140]. Mice carrying a mutant allele of the RECQL4 gene lacking the helicase domain are cancer-prone, and cells derived from these animals present a PSSC phenotype and aneuploidy [141]. It remains unclear how Recql4 contributes to sister chromatid cohesion, although it could participate in cohesion establishment (like the Chl1 DNA helicase) or collaborate with topo II in the decatenation process [142]. 12. Concluding remarks Sister chromatid cohesion contributes significantly to the high fidelity of chromosome segregation in eukaryotic cells. Many of the proteins that participate in the regulation of this process are conserved from yeast to humans. The contribution from studies carried out in

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different experimental models has allowed a rapid advance in our understanding of cohesion. The growing knowledge of the molecular mechanisms that govern the establishment, maintenance and dissolution of cohesion in a timely manner must be complemented by studies exploring the connection of cohesion and other aspects of DNA metabolism such as repair and transcription. In the future it will be necessary to address the function of cohesin in non-proliferative tissues (e.g., postmitotic neurons). Importantly, reduced dosage of a number of cohesion factors appears to have dramatic effects in human development and cause severe syndromes such as Cornelia de Lange or Roberts phocomelia syndrome. The generation of animal models for cohesion factors will be a useful tool to understand the mechanism of pathogenesis for these syndromes as well as to probe the relevance of chromosome segregation fidelity in cancer. Acknowledgements I thank J. Méndez and members of the lab for critically reading the manuscript, and Frank Uhlmann for helpful discussions. I also acknowledge the financial support of the Spanish Ministry of Science and Education (BFU2007-66627 and CSD2007-0015), the European Union (Epigenome NoE and Marie Curie Reintegration Grant MIRGCT-2005-031126) and Fundación CajaMadrid. References [1] T. Hirano, At the heart of the chromosome: SMC proteins in action, Nat. Rev., Mol. Cell Biol. 7 (2006) 311–322. [2] K. Nasmyth, C.H. Haering, The structure and function of SMC and kleisin complexes, Annu. Rev. Biochem. 74 (2005) 595–648. [3] A. Losada, Cohesin regulation: fashionable ways to wear a ring, Chromosoma 116 (2007) 321–329. [4] A. Losada, T. Hirano, Dynamic molecular linkers of the genome: the first decade ofSMC proteins, Genes Dev. 19 (2005) 1269–1287. [5] A. Losada, T. Yokochi, R. Kobayashi, T. Hirano, Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes, J. Cell Biol. 150 (2000) 405–416. [6] D.E. Anderson, A. Losada, H.P. Erickson, T. Hirano, Condensin and cohesin display different arm conformations with characteristic hinge angles, J. Cell Biol. 156 (2002) 419–424. [7] C.H. Haering, J. Lowe, A. Hochwagen, K. Nasmyth, Molecular architecture of SMC proteins and the yeast cohesin complex, Mol. Cell 9 (2002) 773–788. [8] J. Mc Intyre, E.G. Muller, S. Weitzer, B.E. Snydsman, T.N. Davis, F. Uhlmann, In vivo analysis of cohesin architecture using FRET in the budding yeast Saccharomyces cerevisiae, EMBO J. 26 (2007) 3783–3793. [9] S. Gruber, C.H. Haering, K. Nasmyth, Chromosomal cohesin forms a ring, Cell 112 (2003) 765–777. [10] D. Ivanov, K. Nasmyth, A physical assay for sister chromatid cohesion in vitro, Mol. Cell 27 (2007) 300–310. [11] C.R. Chang, C.S. Wu, Y. Hom, M.R. Gartenberg, Targeting of cohesin by transcriptionally silent chromatin, Genes Dev. 19 (2005) 3031–3042. [12] R. Ciosk, M. Shirayama, A. Shevchenko, T. Tanaka, A. Toth, A. Shevchenko, K. Nasmyth, Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins, Mol. Cell 5 (2000) 243–254. [13] E.T. Tonkin, T.J. Wang, S. Lisgo, M.J. Bamshad, T. Strachan, NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly NippedB, is mutated in Cornelia de Lange syndrome, Nat. Genet. 36 (2004) 636–641. [14] I.D. Krantz, J. McCallum, C. DeScipio, M. Kaur, L.A. Gillis, D. Yaeger, L. Jukofsky, N. Wasserman, A. Bottani, C.A. Morris, M.J. Nowaczyk, H. Toriello, M.J. Bamshad, J.C. Carey, E. Rappaport, S. Kawauchi, A.D. Lander, A.L. Calof, H.H. Li, M. Devoto, L.G. Jackson, Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B, Nat. Genet. 36 (2004) 631–635. [15] P. Bernard, J. Drogat, J.F. Maure, S. Dheur, S. Vaur, S. Genier, J.P. Javerzat, A screen for cohesion mutants uncovers Ssl3, the fission yeast counterpart of the cohesin loading factor Scc4, Curr. Biol. 16 (2006) 875–881. [16] E. Watrin, A. Schleiffer, K. Tanaka, F. Eisenhaber, K. Nasmyth, J.M. Peters, Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression, Curr. Biol. 16 (2006) 863–874. [17] V.C. Seitan, P. Banks, S. Laval, N.A. Majid, D. Dorsett, A. Rana, J. Smith, A. Bateman, S. Krpic, A. Hostert, R.A. Rollins, H. Erdjument-Bromage, P. Tempst, C.Y. Benard, S. Hekimi, S.F. Newbury, T. Strachan, Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance, PLoS Biol. 4 (2006) E242. [18] P. Arumugam, S. Gruber, K. Tanaka, C.H. Haering, K. Mechtler, K. Nasmyth, ATP hydrolysis is required for cohesin's association with chromosomes, Curr. Biol. 13 (2003) 1941–1953. [19] P.J. Gillespie, T. Hirano, Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts, Curr. Biol. 14 (2004) 1598–1603.

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