PROGRESS Cohesin in cancer: chromosome segregation and beyond Ana Losada
Abstract | Cohesin is an evolutionarily conserved, four-subunit complex that entraps DNA fibres within its ring-shaped structure. It was originally identified and named for its role in mediating sister chromatid cohesion, which is essential for chromosome segregation and DNA repair. Increasing evidence indicates that cohesin participates in other processes that involve DNA looping, most importantly, transcriptional regulation. Mutations in genes encoding cohesin subunits and other regulators of the complex have recently been identified in several types of tumours. Whether aneuploidy that results from chromosome missegregation is the major contribution of cohesin mutations to cancer progression is under debate. Cohesin subunits were originally identified in yeast as mutants that displayed premature separation of sister chromatids and, soon after this identification, they were shown to form a complex that is required for sister chromatid cohesion in Xenopus laevis egg extracts and mammalian cells. Sister chromatid cohesion ensures accurate chromosome segregation and promotes faithful DNA repair by homologous recombination. Thus, cohesin is essential for genome stability. Cohesin is also a major contributor to interphase chromatin organization through the formation of chromatin loops. In this way, cohesin regulates gene expression, organizes DNA replication factories and facilitates locus rearrangement by recombination1. This plethora of functions makes it difficult to discern which of them may explain the pathological consequences of cohesin mutations that are associated with human disease. Somatic mutations in cohesin genes have been recently described in bladder cancer, acute myeloid leukaemia (AML) and some other cancer types2–13. The relevance of these mutations in cancer initiation and/or progression is unclear. In addition, germline mutations in cohesin and its regulators — most importantly, nipped-B‑like protein (NIPBL), a protein that mediates the loading of cohesin on chromatin — are at
the origin of Cornelia de Lange Syndrome. The affected patients display growth and mental retardation, limb defects and typical facial features, but an increased incidence of cancer has not been reported14. This Progress article briefly reviews our current knowledge of the composition, regulation and functions of the cohesin complex and discusses how its malfunction might affect tumorigenesis. The dynamic behaviour of cohesin Cohesin consists of four subunits that are arranged in a ring-shaped structure: that is, structural maintenance of chromosomes (SMC) proteins SMC1 and SMC3, the kleisin subunit RAD21 (sister chromatid cohesion protein 1 (Scc1) in yeast) and stromal antigen (SA; also known as STAG) (Scc3 in yeast) (FIG. 1). The integrity of this ring is crucial for the association of cohesin with chromatin, which is thought to be topological15. This means that chromatin fibres are encircled by cohesin but there is no direct binding to DNA and therefore no recognition of a specific sequence, unlike the interaction mode of transcription factors. With the exception of SMC3, all of the subunits have at least two versions, with one or more of them being meiosisspecific (FIG. 1). Two cohesin complexes co-occur in somatic vertebrate cells: each
NATURE REVIEWS | CANCER
has SMC1A, SMC3 and RAD21, and either SA1 or SA2. The relative abundance of the two complexes is likely to vary among cell types and/or developmental stages, and their specific functions are only beginning to emerge (see below). Cohesin associates with chromatin in the G1 phase of the cell cycle in a process that requires the presence of the NIPBL–MAU2 heterodimer (Scc2–Scc4 in yeast) and ATP hydrolysis (FIG. 2). Recent biochemical reconstitution of the loading reaction onto naked DNA indicates that cohesin has an intrinsic ability to load topologically on DNA but the process is inefficient unless NIPBL is present 16. The contribution of MAU2 to cohesin loading in vitro is negligible, but it is essential in vivo. Two factors, named wings apart-like protein homologue (WAPL) and PDS5 (either PDS5A or PDS5B in vertebrates), associate with each other and with chromatin-bound cohesin and promote cohesin unloading 17. Thus, the fraction of cohesin that is present on chromatin is the result of the opposing actions of NIPBL–MAU2 and PDS5–WAPL. During G1 phase, cohesin entraps one single chromatid. After passage of the DNA replication fork, cohesin rings encircle the two sister chromatids and become cohesive. The establishment of cohesion requires acetylation of two lysine residues in the SMC3 head domain by the cohesin acetyltransferases (CoATs) ESCO1 and ESCO2 (Eco1 in yeast), as well as the binding of a protein named sororin to PDS5 (REF. 18) (FIG. 2). The binding of sororin to PDS5 has been proposed to displace WAPL, thereby preventing its unloading action. Although the residence time of chromatin-bound cohesin in G1 phase, measured by fluorescence recovery after photobleaching (FRAP), is around 15 minutes, it increases to several hours for cohesive complexes19. Importantly, even in G2 phase, there is a population of dynamic cohesin that still dissociates from chromatin in a WAPL-dependent manner. This population probably corresponds either to complexes that were present on chromatin when the DNA replication fork passed but that were not acetylated and/or bound by sororin or to complexes that were loaded by NIPBL–MAU2 after DNA replication. VOLUME 14 | JUNE 2014 | 389
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS At the onset of mitosis, most cohesin is released from chromatin to allow proper sister chromatid resolution and efficient segregation during anaphase (FIG. 2). This process is known as the prophase dissociation pathway and requires the action of three protein kinases — that is, cyclin-dependent kinase 1 (CDK1), aurora kinase B (AURKB) and polo-like kinase 1 (PLK1). CDK1 and AURKB phosphory late sororin to drive its dissociation from PDS5 and, in this way, PDS5–WAPL can unload cohesin17,20. PLK1 phosphory lates the SA subunit and further facilitates cohesin release. A small proportion of cohesin, mostly enriched at centromeres, is protected from this dissociation by the Hinge SMC3
SMC1A (SMC1B)
Coiled-coil ATPase head PDS5A or PDS5B
N
RAD21 (REC8, RAD21 or RAD21L) C SA1 or SA2 (SA3)
WAPL Sororin
Figure 1 | Architecture of the cohesin complex. Nature Reviews of | Cancer In somatic cells, cohesin is composed structural maintenance of chromosomes protein 1A (SMC1A), SMC3, RAD21 and either stromal antigen 1 (SA1) or SA2, shown in bold. Additional versions of SMC1, RAD21 and SA occur in germ cells (in parentheses). SMC proteins are long polypeptides that fold back on themselves to form a rodlike structure (the coiled-coil domain) with a hinge domain at one end and an ATPase domain at the other. SMC1 and SMC3 form a V‑shaped heterodimer and interact through their hinge domains. The amino- and carboxy-terminal regions of RAD21 interact with the head domains of SMC3 and SMC1, respectively, thereby forming a tripartite ring that entraps the DNA fibre. The central region of RAD21 binds to SA. Although it is not required for maintaining the integrity of the ring, the SA subunit is essential for cohesin loading onto chromatin and for the proper function of the complex. Three factors (wings apart-like protein homologue (WAPL), PDS5A or PDS5B, and sororin) bind to chromatinbound cohesin and modulate its association with chromatin. Sororin is not present in yeast. WAPL and sororin compete for binding to PDS5, although WAPL has additional interacting sites in cohesin. RAD21L, RAD21‑like protein. Adapted from Current Opinion in Cell Biology, 25, Remeseiro, S. & Losada, A., Cohesin, a chromatin engagement ring, 63–71, Copyright (2013), with permission from Elsevier1.
action of shugoshin 1 (SGO1) that is bound to the protein phosphatase 2A (PP2A). SGO1–PP2A recognizes cohesin-bound sororin and antagonizes its phosphory lation21. This centromeric cohesin allows chromosome alignment in the metaphase plate. Activation of the anaphase promoting complex/cyclosome (APC/C) at the onset of anaphase leads to degradation of securin and activation of separase. Separase cleaves the RAD21 subunit of the remaining chromatin-bound cohesin, thereby destroying the integrity of the ring and allowing separation of the sister chromatids. Cohesion-dependent functions of cohesin As explained above, cohesin embraces the two sister chromatids from S phase to anaphase. Studies in mouse and human cells have shown that cohesin complexes that include SA1 (cohesin–SA1) and SA2 (cohesin–SA2) mediate sister chromatid cohesion at telomeres and centromeres, respectively, and the two complexes mediate cohesion along chromosome arms22,23. PDS5A and PDS5B, which can bind to either cohesin–SA1 or cohesin–SA2, both contribute to telomere and arm cohesion, whereas PDS5B is specifically required at centromeres24. The molecular mechanisms underlying these specificities remain to be identified. During S phase and G2, cohesin promotes restart of replication forks that stall at regions that are difficult to replicate, such as telomeres, and facilitates repair of double-strand breaks by homologous recombination23,25 (FIG. 3a). Thus, in the absence of cohesin, one might expect an increase in replication fork collapse and unrepaired DNA breaks that lead to genomic and chromosomal instability upon passage through mitosis. Indeed, mouse embryonic fibroblasts (MEFs) that lack cohesin–SA1 show robust centromere cohesion but their defect in telomere cohesion results in faulty telomere replication that leads to chromosome missegregation and aneuploidy 23. During mitosis, cohesion contributes to the proper orientation of sister kinetochores. Cohesion also prevents the premature separation of sister chromatids under the pulling forces of spindle microtubules while chromosomes try to align at the metaphase plate and become attached to both spindle poles. Centromeric cohesion that is carried out by cohesin–SA2 complexes bound to PDS5B–sororin is, in principle, most crucial for these tasks. MEFs that are deficient in PDS5B are indeed aneuploid24, and SA2 inactivation in some human cell
390 | JUNE 2014 | VOLUME 14
lines also changes the modal chromosome number 2,3,10, but this is not always the case9. In bladder tumour samples, disparate results have been reported regarding the association of mutations in STAG2 (encoding SA2) with aneuploidy 7–10. It is possible that different cell types have a different balance of SA1 and SA2, or that a less efficient prophase dissociation pathway leaves more cohesin along chromosome arms that is capable of holding sister chromatids together in the absence of robust centromeric cohesion, or even that the functional specificity of cohesin–SA1 and cohesin–SA2 that has been described for MEFs and HeLa cells is not universal. Moreover, cohesion depends not only on cohesin but also on catenation, which results from DNA replication and physically interlocks DNA along the sister chromatids. This catenation must be resolved by topoisomerase 2 (TOP2) before anaphase, and the efficiency of this process may be variable among cell types. The presence of cohesin hinders decatenation by TOP2, so that a less efficient prophase dissociation of cohesin leads to worse decatenation and increased rates of chromosome missegregation17,26. Cohesin seems to be required for keeping mother and daughter centrioles together in an orthogonal arrangement, from centriole duplication in S phase to mitotic exit 27, albeit that the mechanism underlying this function is unclear. Premature centriole separation in the absence of cohesin might also contribute to abnormal cell division and the generation of aneuploidy. Cohesion-independent functions Cohesin is present in non-cycling cells in which there are no sister chromatids to hold together. The likely reason for this is that, together with CCCTC-binding factor (CTCF), cohesin contributes to the topological organization of the genome28–30. This organization is based on a hierarchy of chromatin loops and underlies most aspects of genome function, including DNA replication and long-range transcriptional regulation (FIG. 3b). Consistent with this function as architectural organizers, cohesin and CTCF largely colocalize along the human and mouse genomes31,32. In the absence of CTCF, cohesin can no longer be found at CTCF sites, but the amount of cohesin on chromatin is unaffected31. Thus, cohesin localization to specific sites, but not its association with chromatin, probably depends on its interaction with proteins that recognize specific DNA sequences, such as CTCF and other transcription factors33,34. Cohesin might also www.nature.com/reviews/cancer
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS ESCO1 or ESCO2
Establishment of cohesion
Cohesin Ac
Ac DNA
PDS5
WAPL
Sororin
Cohesin unloading Cohesin loading
S G1
Ac
Ac
G2 M
NIPBL
• PLK1 • AURKB • CDK1
Recycling MAU2
Ac HDAC8
Prophase dissociation
Ac
Ac
SGO1 PP2A
Ac Anaphase dissociation
Ac P
P
Separase
Figure 2 | Cell cycle regulation of cohesin. Cohesin is loaded onto chromatin in early G1 phase, and this loading is assisted by the nipped-B‑like protein (NIPBL)–MAU2 heterodimer. This heterodimer makes contact with all four cohesin subunits and may function as a molecular ‘shaft’ to convey energy from the ATP hydrolysis that takes place in the head domains of structural maintenance of chromosomes protein 1A (SMC1A) and SMC3 to the hinge region, which is thought to transiently dissociate to allow entry of the DNA16. Subsequent binding of PDS5 and wings apart-like protein homologue (WAPL) to cohesin, through RAD21 and stromal antigen (SA), promotes its unloading. The DNA fibre exits the complex through the interface created by the SMC3 head domain and the amino‑terminal region of RAD21. During DNA replication, the cohesin acetyltransferases (CoATs) ESCO1 and ESCO2 acetylate (Ac) K105 and K106 in the N‑terminal domain of SMC3, and sororin is recruited to PDS5, which displaces WAPL, although it remains bound to cohesin. Sororin-bound acetylated cohesin complexes
recognize a genomic feature (for example, a chromatin loop or a nucleosome-free region). The extent to which the distribution of the cohesin loader NIPBL–MAU2 dictates the distribution of cohesin in mammalian genomes is unclear. In mouse embryonic stem cells, cohesin and NIPBL colocalize at the enhancer and promoter regions of actively transcribed genes, whereas NIPBL is absent from cohesin–CTCF sites35. The loops that are stabilized by cohesin can have different purposes. They can facilitate enhancer–promoter interactions
encircling the two sister chromatids are stably bound to chromatin. In prophase, most cohesin dissociates from chromatin when polo-like kinase 1 (PLK1) phosphorylates (P) the SA subunit, and sororin is released from Nature Reviews | Cancer cohesin after being phosphorylated by aurora kinase B (AURKB) and cyclindependent kinase 1 (CDK1). Concomitantly, shugoshin 1 (SGO1) and its partner protein phosphatase 2A (PP2A) accumulate at centromeres to counteract the above-mentioned phosphorylation events and prevent cohesin dissociation. Centromeric cohesin remains on chromatin until anaphase, when cleavage of RAD21 by separase destroys the integrity of the cohesin ring. The cohesin complexes that are released during mitosis can be reused in the ensuing G1 phase after a cohesin deacetylase (histone deacetylase 8 (HDAC8) in human cells) removes acetyl groups from SMC3 (REF. 45). Adapted from Current Opinion in Cell Biology, 25, Remeseiro, S. & Losada, A., Cohesin, a chromatin engagement ring, 63–71, Copyright (2013), with permission from Elsevier1.
or regulate the expression of genes within clusters or domains by allowing or preventing certain chromatin contacts35,36. The genome-wide distribution of cohesin–SA1 and cohesin–SA2 is very similar and overlaps with the distribution of CTCF. However, in SA1‑deficient MEFs, a proportion of cohesin–SA2 relocates away from promoters and CTCF sites, and the transcriptome is altered32. From this result, it is tempting to conclude that cohesin–SA1 is more important than cohesin–SA2 for transcriptional regulation. Consistent with
NATURE REVIEWS | CANCER
this possibility, no substantial changes were found when comparing the transcriptional profiles of paired human glioblastoma cell lines with and without SA2 expression3. Nevertheless, further analyses are required to ascertain the specific contributions of cohesin–SA1 and cohesin–SA2 to genome organization and transcriptional regulation. Cohesin mutations in cancer Recent exome sequencing of 4,742 human cancer samples across 21 cancer types has identified STAG2 as one of 12 genes that VOLUME 14 | JUNE 2014 | 391
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS a Cohesion-dependent functions
b Cohesion-independent functions
DNA replication
Genome compartmentalization Cohesin
Prevent collapse and aid restart
TAD
Fork stalling Homologous recombination-mediated repair DSB
Transcription regulation
DNA damage
Enhancer
Promoter
CTCF
Transcription factor
Chromosome biorientation Cohesin
DNA replication Replicon
Centrosome
Kinetochore
Replication origin
Simultaneous origin firing
Figure 3 | Cohesin functions. a | Cohesion-dependent functions of cohesin. In interphase, cohesin Nature Reviews | Cancer is important for stabilizing stalled DNA replication forks and promoting their restart. This is particularly important for regions that are difficult to replicate, such as telomeres. Cohesion also facilitates the use of the sister chromatid as a template to repair double-strand breaks (DSBs) through homologous recombination-mediated repair, thus preventing both inaccurate repair and recombination between homologous chromosomes that would lead to loss of heterozygosity. In mitosis, cohesion ensures faithful chromosome segregation. It promotes the back‑to‑back orientation of the sister kinetochores to facilitate their attachment to microtubules from opposite spindle poles and prevents sister chromatid separation until all chromosomes achieve bipolar attachment (also known as biorientation). b | Cohesion-independent functions of cohesin are related to genome organization. The genome is partitioned into discrete units known as topologically associating domains (TADs) that range from 100 kb to 1 Mb in mammals. Both CCCTC-binding factor (CTCF) and cohesin (not shown in the top panel) contribute to this organization, probably through the formation of chromatin loops. TADs confine regulatory activities (for example, enhancers) to a specific domain. Within a domain, cohesin can promote transcription by facilitating the interaction between an enhancer and a promoter35 or contribute to the transcriptional regulation of gene clusters36. Cohesin has also been proposed to organize chromatin loops at replication factories, thereby facilitating simultaneous firing of the clustered origins39.
are mutated at substantial frequencies in at least four tumour types12. Mutations in genes encoding cohesin subunits and NIPBL were initially identified in colorectal cancer 2, and mutations in STAG2 were later found in glioblastoma, Ewing’s sarcoma and melanoma3. However, it is in urothelial bladder cancer that mutations in STAG2 are the most common, with rates of 10–15% in aggressive tumours and up to 30% in low-grade tumours7–10,13. Other cohesin subunits are not as frequently mutated in this type of cancer. By contrast, similar mutation rates across most cohesin subunits have been described in AML4,11, Down syndrome related acute megakaryocitic leukaemia5 and other
myeloid neoplasms6. Overall, mutations in STAG2 are often truncating, whereas missense mutations are more frequent in other cohesin genes. The consequence of many missense mutations in protein function is unclear. Although most identified mutations are heterozygous, the SMC1A and STAG2 genes are located on the X chromosome, which makes the corresponding mutations functionally homozygous, at least in males. It is unlikely that cells can proliferate in the absence of cohesin. The higher mutation rates of STAG2 in most tumours might be explained by the fact that a single hit is sufficient for the loss of SA2 function, and cohesin–SA1 complexes might partially
392 | JUNE 2014 | VOLUME 14
compensate for this loss. Downregulation of SA2 is less detrimental for chromosome segregation than downregulation of SMC1A or SMC3 (REF. 2). Regarding SMC1A, only one mutation among those reported in the recent studies leads to the expression of a nonfunctional protein that lacks its carboxy‑ terminal third8. All other mutations result in amino acid changes whose importance for protein function remains to be tested. It is unclear how the cells in the tumour with a truncated SMC1A8, which is from a male patient, survive without a functional cohesin complex. As suggested above, cohesin dysfunction could affect tumorigenesis by increasing genome instability due to faulty DNA replication and/or repair and chromosome missegregation37. Although aneuploidy and genome instability are detrimental to cell survival, they can accelerate tumour evolution and adaptability 38. An association between aneuploidy and cohesin mutations in cancer has been reported in some studies8,10 but not in others7,9,11. Reduced sister chromatid cohesion might also favour loss of heterozygosity by promoting recombination with the homologous chromosome instead of using the sister chromatid as a template for homologous recombination‑mediated repair. The role of cohesin in genome organization could also underlie the tumour-promoting consequences of cohesin mutations. The most obvious effect would be gene expression changes of crucial oncogenes or tumour suppressors. However, other possibilities should be considered. Altered organization of replication factories may slow replication and increase replicative stress39,40. A local alteration of chromosomal domain organization could alter the replication timing of the domain and thereby affect its mutation rate, its epigenetic modifications or the frequency of structural rearrangements41. Reduced cohesion, together with domain decompaction and an increased number of interdomain chromatin contacts30 may favour chromosomal translocations. A study in yeast showed that reducing cohesin levels to 30% of wild type compromises homologous recombination‑mediated repair, but a deleterious effect on chromosome segregation requires a reduction below 13% (REF. 25). Although these numbers can be substantially different in mammalian cells, this study is important to make us aware of the different sensitivity of diverse cohesin functions to the levels of cohesin in the cell. Moreover, the distinct versions of cohesin that carry SA1 or SA2, PDS5A or PDS5B may also show different sensitivities www.nature.com/reviews/cancer
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS to the reduction of their levels. MEFs that are heterozygous for Rad21 or Stag1 show increased mitotic defects and aneuploidy compared with wild-type MEFs and, in the case of Rad21 deficiency, reduced homologous recombination‑mediated repair has also been reported23,42. Postmitotic murine cells that were progressively depleted of RAD21 showed a concomitant loss of chromatin contacts and transcriptional dysregulation at several loci when RAD21 levels decreased below 50% of wild type30. In cells that are derived from heterozygous Nipbl mice, which recapitulate several phenotypes of Cornelia de Lange Syndrome, the overall levels of cohesin on chromatin are normal and cohesion defects are not observed; however, transcription is disrupted at several loci43,44. Thus, transcriptional regulation of specific genes could be most sensitive to a reduction in the levels of functional cohesin available in the cell. Conclusions and perspectives The recent identification of cohesin mutations in several tumour types, most notably bladder cancer and myeloid neoplasms, raises the question of how cohesin dysfunction affects tumorigenesis. Answering this question is complicated by the fact that cohesin has essential roles in both genome stability and transcriptional regulation. Mutations in cohesin genes that have been identified in cancer genomes remain poorly characterized in terms of how they affect the protein itself (folding and stability), the formation of the cohesin complex, its association to chromatin or its interaction with other proteins. Heterozygous mutations may reduce the amount of functional cohesin in the cell, but they can also produce a truncated protein with a dominantnegative effect. Distinct aspects of cohesin regulation and function could be tissuespecific and thereby explain why mutations in cohesin are more prominent in certain types of tumours. So far, synergy with mutations in additional pathways is also unclear. Our growing knowledge of the basic biology of cohesin and the generation of cell and animal models deficient for cohesin or carrying the mutations identified in cancer will help us to understand the functional importance of such mutations and hopefully contribute to improve the diagnosis and treatment of patients. Ana Losada is at the Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. e‑mail: [email protected] doi:10.1038/nrc3743
Remeseiro, S. & Losada, A. Cohesin, a chromatin engagement ring. Curr. Opin. Cell Biol. 25, 63–71 (2013). 2. Barber, T. D. et al. Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers. Proc. Natl Acad. Sci. USA 105, 3443–3448 (2008). 3. Solomon, D. A. et al. Mutational inactivation of STAG2 causes aneuploidy in human cancer. Science 333, 1039–1043 (2011). 4. Welch, J. S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278 (2012). 5. Yoshida, K. et al. The landscape of somatic mutations in Down syndrome-related myeloid disorders. Nature Genet. 45, 1293–1299 (2013). 6. Kon, A. et al. Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms. Nature Genet. 45, 1232–1237 (2013). 7. Taylor, C. F., Platt, F. M., Hurst, C. D., Thygesen, H. H. & Knowles, M. A. Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes. Hum. Mol. Genet. 23, 1964–1974 (2013). 8. Guo, G. et al. Whole-genome and whole-exome sequencing of bladder cancer identifies frequent alterations in genes involved in sister chromatid cohesion and segregation. Nature Genet. 45, 1459–1463 (2013). 9. Balbas-Martinez, C. et al. Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy. Nature Genet. 45, 1464–1469 (2013). 10. Solomon, D. A. et al. Frequent truncating mutations of STAG2 in bladder cancer. Nature Genet. 45, 1428–1430 (2013). 11. Thol, F. et al. Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications. Blood 123, 914–920 (2013). 12. Lawrence, M. S. et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014). 13. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507, 315–322. (2014). 14. Liu, J. & Krantz, I. D. Cornelia de Lange syndrome, cohesin, and beyond. Clin. Genet. 76, 303–314 (2009). 15. Haering, C. H., Lowe, J., Hochwagen, A. & Nasmyth, K. Molecular architecture of SMC proteins and the yeast cohesin complex. Mol. Cell 9, 773–788 (2002). 16. Murayama, Y. & Uhlmann, F. Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505, 367–371 (2014). 17. Tedeschi, A. et al. Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature 501, 564–568 (2013). 18. Nishiyama, T. et al. Sororin mediates sister chromatid cohesion by antagonizing Wapl. Cell 143, 737–749 (2010). 19. Gerlich, D., Koch, B., Dupeux, F., Peters, J. M. & Ellenberg, J. Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication. Curr. Biol. 16, 1571–1578 (2006). 20. Nishiyama, T., Sykora, M. M., Huis in ‘t Veld, P. J., Mechtler, K. & Peters, J. M. Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin. Proc. Natl Acad. Sci. USA 110, 13404–13409 (2013). 21. Liu, H., Rankin, S. & Yu, H. Phosphorylation-enabled binding of SGO1‑PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nature Cell Biol. 15, 40–49 (2013). 22. Canudas, S. & Smith, S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells. J. Cell Biol. 187, 165–173 (2009). 23. Remeseiro, S. et al. Cohesin‑SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres. EMBO J. 31, 2076–2089 (2012). 24. Carretero, M., Ruiz-Torres, M., Rodriguez-Corsino, M., Barthelemy, I. & Losada, A. Pds5B is required for cohesion establishment and Aurora B 1.
NATURE REVIEWS | CANCER
accumulation at centromeres. EMBO J. 32, 2938–2949 (2013). 25. Heidinger-Pauli, J. M., Mert, O., Davenport, C., Guacci, V. & Koshland, D. Systematic reduction of cohesin differentially affects chromosome segregation, condensation, and DNA repair. Curr. Biol. 20, 957–963 (2010). 26. Wang, L. H., Mayer, B., Stemmann, O. & Nigg, E. A. Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division. J. Cell Sci. 123, 806–813 (2010). 27. Schockel, L., Mockel, M., Mayer, B., Boos, D. & Stemmann, O. Cleavage of cohesin rings coordinates the separation of centrioles and chromatids. Nature Cell Biol. 13, 966–972 (2011). 28. Gibcus, J. H. & Dekker, J. The hierarchy of the 3D genome. Mol. Cell 49, 773–782 (2013). 29. Zuin, J. et al. Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc. Natl Acad. Sci. USA 111, 996–1001 (2014). 30. Sofueva, S. et al. Cohesin-mediated interactions organize chromosomal domain architecture. EMBO J. 32, 3119–3129 (2013). 31. Wendt, K. S. et al. Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451, 796–801 (2008). 32. Remeseiro, S., Cuadrado, A., Gómez-López, G., Pisano, D. G. & Losada, A. A unique role of cohesin‑SA1 in gene regulation and development. EMBO J. 31, 2090–2102 (2012). 33. Rubio, E. D. et al. CTCF physically links cohesin to chromatin. Proc. Natl Acad. Sci. USA 105, 8309–8314 (2008). 34. Schmidt, D. et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res. 20, 578–588 (2010). 35. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010). 36. Monahan, K. et al. Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression. Proc. Natl Acad. Sci. USA 109, 9125–9130 (2012). 37. Duijf, P. H. & Benezra, R. The cancer biology of wholechromosome instability. Oncogene 32, 4727–4736 (2013). 38. Holland, A. J. & Cleveland, D. W. Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep. 13, 501–514 (2012). 39. Guillou, E. et al. Cohesin organizes chromatin loops at DNA replication factories. Genes Dev. 24, 2812–2822 (2010). 40. Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492–496 (2013). 41. Stamatoyannopoulos, J. A. et al. Human mutation rate associated with DNA replication timing. Nature Genet. 41, 393–395 (2009). 42. Xu, H. et al. Rad21‑cohesin haploinsufficiency impedes DNA repair and enhances gastrointestinal radiosensitivity in mice. PLoS ONE 5, e12112 (2010). 43. Chien, R. et al. Cohesin mediates chromatin interactions that regulate mammalian β-globin expression. J. Biol. Chem. 286, 17870–17878 (2011). 44. Remeseiro, S. et al. Reduction of Nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of Cornelia de Lange syndrome. Biochim. Biophys. Acta 1832, 2097–2102 (2013). 45. Deardorff, M. A. et al. HDAC8 mutations in Cornelia de Lange Syndrome affect the cohesin acetylation cycle. Nature 489, 313–317 (2012).
Acknowledgements
The author apologizes to colleagues whose relevant work on cohesin is not cited here. She thanks the group of F. X. Real (Spanish National Cancer Research Centre (CNIO), Madrid, Spain), as well as members of her own group, for discussions. The author’s research is currently funded by the Spanish Ministry of Economy and Competitiveness (MINECO), grant SAF‑2010‑21517.
Competing interests statement
The author declares no competing interests.
VOLUME 14 | JUNE 2014 | 393 © 2014 Macmillan Publishers Limited. All rights reserved
Abstract | Cohesin is an evolutionarily conserved, four-subunit complex that entraps DNA fibres within its ring-shaped structure. It was originally identified and named for its role in mediating sister chromatid cohesion, which is essential for chromosome segregation and DNA repair. Increasing evidence indicates that cohesin participates in other processes that involve DNA looping, most importantly, transcriptional regulation. Mutations in genes encoding cohesin subunits and other regulators of the complex have recently been identified in several types of tumours. Whether aneuploidy that results from chromosome missegregation is the major contribution of cohesin mutations to cancer progression is under debate. Cohesin subunits were originally identified in yeast as mutants that displayed premature separation of sister chromatids and, soon after this identification, they were shown to form a complex that is required for sister chromatid cohesion in Xenopus laevis egg extracts and mammalian cells. Sister chromatid cohesion ensures accurate chromosome segregation and promotes faithful DNA repair by homologous recombination. Thus, cohesin is essential for genome stability. Cohesin is also a major contributor to interphase chromatin organization through the formation of chromatin loops. In this way, cohesin regulates gene expression, organizes DNA replication factories and facilitates locus rearrangement by recombination1. This plethora of functions makes it difficult to discern which of them may explain the pathological consequences of cohesin mutations that are associated with human disease. Somatic mutations in cohesin genes have been recently described in bladder cancer, acute myeloid leukaemia (AML) and some other cancer types2–13. The relevance of these mutations in cancer initiation and/or progression is unclear. In addition, germline mutations in cohesin and its regulators — most importantly, nipped-B‑like protein (NIPBL), a protein that mediates the loading of cohesin on chromatin — are at
the origin of Cornelia de Lange Syndrome. The affected patients display growth and mental retardation, limb defects and typical facial features, but an increased incidence of cancer has not been reported14. This Progress article briefly reviews our current knowledge of the composition, regulation and functions of the cohesin complex and discusses how its malfunction might affect tumorigenesis. The dynamic behaviour of cohesin Cohesin consists of four subunits that are arranged in a ring-shaped structure: that is, structural maintenance of chromosomes (SMC) proteins SMC1 and SMC3, the kleisin subunit RAD21 (sister chromatid cohesion protein 1 (Scc1) in yeast) and stromal antigen (SA; also known as STAG) (Scc3 in yeast) (FIG. 1). The integrity of this ring is crucial for the association of cohesin with chromatin, which is thought to be topological15. This means that chromatin fibres are encircled by cohesin but there is no direct binding to DNA and therefore no recognition of a specific sequence, unlike the interaction mode of transcription factors. With the exception of SMC3, all of the subunits have at least two versions, with one or more of them being meiosisspecific (FIG. 1). Two cohesin complexes co-occur in somatic vertebrate cells: each
NATURE REVIEWS | CANCER
has SMC1A, SMC3 and RAD21, and either SA1 or SA2. The relative abundance of the two complexes is likely to vary among cell types and/or developmental stages, and their specific functions are only beginning to emerge (see below). Cohesin associates with chromatin in the G1 phase of the cell cycle in a process that requires the presence of the NIPBL–MAU2 heterodimer (Scc2–Scc4 in yeast) and ATP hydrolysis (FIG. 2). Recent biochemical reconstitution of the loading reaction onto naked DNA indicates that cohesin has an intrinsic ability to load topologically on DNA but the process is inefficient unless NIPBL is present 16. The contribution of MAU2 to cohesin loading in vitro is negligible, but it is essential in vivo. Two factors, named wings apart-like protein homologue (WAPL) and PDS5 (either PDS5A or PDS5B in vertebrates), associate with each other and with chromatin-bound cohesin and promote cohesin unloading 17. Thus, the fraction of cohesin that is present on chromatin is the result of the opposing actions of NIPBL–MAU2 and PDS5–WAPL. During G1 phase, cohesin entraps one single chromatid. After passage of the DNA replication fork, cohesin rings encircle the two sister chromatids and become cohesive. The establishment of cohesion requires acetylation of two lysine residues in the SMC3 head domain by the cohesin acetyltransferases (CoATs) ESCO1 and ESCO2 (Eco1 in yeast), as well as the binding of a protein named sororin to PDS5 (REF. 18) (FIG. 2). The binding of sororin to PDS5 has been proposed to displace WAPL, thereby preventing its unloading action. Although the residence time of chromatin-bound cohesin in G1 phase, measured by fluorescence recovery after photobleaching (FRAP), is around 15 minutes, it increases to several hours for cohesive complexes19. Importantly, even in G2 phase, there is a population of dynamic cohesin that still dissociates from chromatin in a WAPL-dependent manner. This population probably corresponds either to complexes that were present on chromatin when the DNA replication fork passed but that were not acetylated and/or bound by sororin or to complexes that were loaded by NIPBL–MAU2 after DNA replication. VOLUME 14 | JUNE 2014 | 389
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS At the onset of mitosis, most cohesin is released from chromatin to allow proper sister chromatid resolution and efficient segregation during anaphase (FIG. 2). This process is known as the prophase dissociation pathway and requires the action of three protein kinases — that is, cyclin-dependent kinase 1 (CDK1), aurora kinase B (AURKB) and polo-like kinase 1 (PLK1). CDK1 and AURKB phosphory late sororin to drive its dissociation from PDS5 and, in this way, PDS5–WAPL can unload cohesin17,20. PLK1 phosphory lates the SA subunit and further facilitates cohesin release. A small proportion of cohesin, mostly enriched at centromeres, is protected from this dissociation by the Hinge SMC3
SMC1A (SMC1B)
Coiled-coil ATPase head PDS5A or PDS5B
N
RAD21 (REC8, RAD21 or RAD21L) C SA1 or SA2 (SA3)
WAPL Sororin
Figure 1 | Architecture of the cohesin complex. Nature Reviews of | Cancer In somatic cells, cohesin is composed structural maintenance of chromosomes protein 1A (SMC1A), SMC3, RAD21 and either stromal antigen 1 (SA1) or SA2, shown in bold. Additional versions of SMC1, RAD21 and SA occur in germ cells (in parentheses). SMC proteins are long polypeptides that fold back on themselves to form a rodlike structure (the coiled-coil domain) with a hinge domain at one end and an ATPase domain at the other. SMC1 and SMC3 form a V‑shaped heterodimer and interact through their hinge domains. The amino- and carboxy-terminal regions of RAD21 interact with the head domains of SMC3 and SMC1, respectively, thereby forming a tripartite ring that entraps the DNA fibre. The central region of RAD21 binds to SA. Although it is not required for maintaining the integrity of the ring, the SA subunit is essential for cohesin loading onto chromatin and for the proper function of the complex. Three factors (wings apart-like protein homologue (WAPL), PDS5A or PDS5B, and sororin) bind to chromatinbound cohesin and modulate its association with chromatin. Sororin is not present in yeast. WAPL and sororin compete for binding to PDS5, although WAPL has additional interacting sites in cohesin. RAD21L, RAD21‑like protein. Adapted from Current Opinion in Cell Biology, 25, Remeseiro, S. & Losada, A., Cohesin, a chromatin engagement ring, 63–71, Copyright (2013), with permission from Elsevier1.
action of shugoshin 1 (SGO1) that is bound to the protein phosphatase 2A (PP2A). SGO1–PP2A recognizes cohesin-bound sororin and antagonizes its phosphory lation21. This centromeric cohesin allows chromosome alignment in the metaphase plate. Activation of the anaphase promoting complex/cyclosome (APC/C) at the onset of anaphase leads to degradation of securin and activation of separase. Separase cleaves the RAD21 subunit of the remaining chromatin-bound cohesin, thereby destroying the integrity of the ring and allowing separation of the sister chromatids. Cohesion-dependent functions of cohesin As explained above, cohesin embraces the two sister chromatids from S phase to anaphase. Studies in mouse and human cells have shown that cohesin complexes that include SA1 (cohesin–SA1) and SA2 (cohesin–SA2) mediate sister chromatid cohesion at telomeres and centromeres, respectively, and the two complexes mediate cohesion along chromosome arms22,23. PDS5A and PDS5B, which can bind to either cohesin–SA1 or cohesin–SA2, both contribute to telomere and arm cohesion, whereas PDS5B is specifically required at centromeres24. The molecular mechanisms underlying these specificities remain to be identified. During S phase and G2, cohesin promotes restart of replication forks that stall at regions that are difficult to replicate, such as telomeres, and facilitates repair of double-strand breaks by homologous recombination23,25 (FIG. 3a). Thus, in the absence of cohesin, one might expect an increase in replication fork collapse and unrepaired DNA breaks that lead to genomic and chromosomal instability upon passage through mitosis. Indeed, mouse embryonic fibroblasts (MEFs) that lack cohesin–SA1 show robust centromere cohesion but their defect in telomere cohesion results in faulty telomere replication that leads to chromosome missegregation and aneuploidy 23. During mitosis, cohesion contributes to the proper orientation of sister kinetochores. Cohesion also prevents the premature separation of sister chromatids under the pulling forces of spindle microtubules while chromosomes try to align at the metaphase plate and become attached to both spindle poles. Centromeric cohesion that is carried out by cohesin–SA2 complexes bound to PDS5B–sororin is, in principle, most crucial for these tasks. MEFs that are deficient in PDS5B are indeed aneuploid24, and SA2 inactivation in some human cell
390 | JUNE 2014 | VOLUME 14
lines also changes the modal chromosome number 2,3,10, but this is not always the case9. In bladder tumour samples, disparate results have been reported regarding the association of mutations in STAG2 (encoding SA2) with aneuploidy 7–10. It is possible that different cell types have a different balance of SA1 and SA2, or that a less efficient prophase dissociation pathway leaves more cohesin along chromosome arms that is capable of holding sister chromatids together in the absence of robust centromeric cohesion, or even that the functional specificity of cohesin–SA1 and cohesin–SA2 that has been described for MEFs and HeLa cells is not universal. Moreover, cohesion depends not only on cohesin but also on catenation, which results from DNA replication and physically interlocks DNA along the sister chromatids. This catenation must be resolved by topoisomerase 2 (TOP2) before anaphase, and the efficiency of this process may be variable among cell types. The presence of cohesin hinders decatenation by TOP2, so that a less efficient prophase dissociation of cohesin leads to worse decatenation and increased rates of chromosome missegregation17,26. Cohesin seems to be required for keeping mother and daughter centrioles together in an orthogonal arrangement, from centriole duplication in S phase to mitotic exit 27, albeit that the mechanism underlying this function is unclear. Premature centriole separation in the absence of cohesin might also contribute to abnormal cell division and the generation of aneuploidy. Cohesion-independent functions Cohesin is present in non-cycling cells in which there are no sister chromatids to hold together. The likely reason for this is that, together with CCCTC-binding factor (CTCF), cohesin contributes to the topological organization of the genome28–30. This organization is based on a hierarchy of chromatin loops and underlies most aspects of genome function, including DNA replication and long-range transcriptional regulation (FIG. 3b). Consistent with this function as architectural organizers, cohesin and CTCF largely colocalize along the human and mouse genomes31,32. In the absence of CTCF, cohesin can no longer be found at CTCF sites, but the amount of cohesin on chromatin is unaffected31. Thus, cohesin localization to specific sites, but not its association with chromatin, probably depends on its interaction with proteins that recognize specific DNA sequences, such as CTCF and other transcription factors33,34. Cohesin might also www.nature.com/reviews/cancer
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS ESCO1 or ESCO2
Establishment of cohesion
Cohesin Ac
Ac DNA
PDS5
WAPL
Sororin
Cohesin unloading Cohesin loading
S G1
Ac
Ac
G2 M
NIPBL
• PLK1 • AURKB • CDK1
Recycling MAU2
Ac HDAC8
Prophase dissociation
Ac
Ac
SGO1 PP2A
Ac Anaphase dissociation
Ac P
P
Separase
Figure 2 | Cell cycle regulation of cohesin. Cohesin is loaded onto chromatin in early G1 phase, and this loading is assisted by the nipped-B‑like protein (NIPBL)–MAU2 heterodimer. This heterodimer makes contact with all four cohesin subunits and may function as a molecular ‘shaft’ to convey energy from the ATP hydrolysis that takes place in the head domains of structural maintenance of chromosomes protein 1A (SMC1A) and SMC3 to the hinge region, which is thought to transiently dissociate to allow entry of the DNA16. Subsequent binding of PDS5 and wings apart-like protein homologue (WAPL) to cohesin, through RAD21 and stromal antigen (SA), promotes its unloading. The DNA fibre exits the complex through the interface created by the SMC3 head domain and the amino‑terminal region of RAD21. During DNA replication, the cohesin acetyltransferases (CoATs) ESCO1 and ESCO2 acetylate (Ac) K105 and K106 in the N‑terminal domain of SMC3, and sororin is recruited to PDS5, which displaces WAPL, although it remains bound to cohesin. Sororin-bound acetylated cohesin complexes
recognize a genomic feature (for example, a chromatin loop or a nucleosome-free region). The extent to which the distribution of the cohesin loader NIPBL–MAU2 dictates the distribution of cohesin in mammalian genomes is unclear. In mouse embryonic stem cells, cohesin and NIPBL colocalize at the enhancer and promoter regions of actively transcribed genes, whereas NIPBL is absent from cohesin–CTCF sites35. The loops that are stabilized by cohesin can have different purposes. They can facilitate enhancer–promoter interactions
encircling the two sister chromatids are stably bound to chromatin. In prophase, most cohesin dissociates from chromatin when polo-like kinase 1 (PLK1) phosphorylates (P) the SA subunit, and sororin is released from Nature Reviews | Cancer cohesin after being phosphorylated by aurora kinase B (AURKB) and cyclindependent kinase 1 (CDK1). Concomitantly, shugoshin 1 (SGO1) and its partner protein phosphatase 2A (PP2A) accumulate at centromeres to counteract the above-mentioned phosphorylation events and prevent cohesin dissociation. Centromeric cohesin remains on chromatin until anaphase, when cleavage of RAD21 by separase destroys the integrity of the cohesin ring. The cohesin complexes that are released during mitosis can be reused in the ensuing G1 phase after a cohesin deacetylase (histone deacetylase 8 (HDAC8) in human cells) removes acetyl groups from SMC3 (REF. 45). Adapted from Current Opinion in Cell Biology, 25, Remeseiro, S. & Losada, A., Cohesin, a chromatin engagement ring, 63–71, Copyright (2013), with permission from Elsevier1.
or regulate the expression of genes within clusters or domains by allowing or preventing certain chromatin contacts35,36. The genome-wide distribution of cohesin–SA1 and cohesin–SA2 is very similar and overlaps with the distribution of CTCF. However, in SA1‑deficient MEFs, a proportion of cohesin–SA2 relocates away from promoters and CTCF sites, and the transcriptome is altered32. From this result, it is tempting to conclude that cohesin–SA1 is more important than cohesin–SA2 for transcriptional regulation. Consistent with
NATURE REVIEWS | CANCER
this possibility, no substantial changes were found when comparing the transcriptional profiles of paired human glioblastoma cell lines with and without SA2 expression3. Nevertheless, further analyses are required to ascertain the specific contributions of cohesin–SA1 and cohesin–SA2 to genome organization and transcriptional regulation. Cohesin mutations in cancer Recent exome sequencing of 4,742 human cancer samples across 21 cancer types has identified STAG2 as one of 12 genes that VOLUME 14 | JUNE 2014 | 391
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS a Cohesion-dependent functions
b Cohesion-independent functions
DNA replication
Genome compartmentalization Cohesin
Prevent collapse and aid restart
TAD
Fork stalling Homologous recombination-mediated repair DSB
Transcription regulation
DNA damage
Enhancer
Promoter
CTCF
Transcription factor
Chromosome biorientation Cohesin
DNA replication Replicon
Centrosome
Kinetochore
Replication origin
Simultaneous origin firing
Figure 3 | Cohesin functions. a | Cohesion-dependent functions of cohesin. In interphase, cohesin Nature Reviews | Cancer is important for stabilizing stalled DNA replication forks and promoting their restart. This is particularly important for regions that are difficult to replicate, such as telomeres. Cohesion also facilitates the use of the sister chromatid as a template to repair double-strand breaks (DSBs) through homologous recombination-mediated repair, thus preventing both inaccurate repair and recombination between homologous chromosomes that would lead to loss of heterozygosity. In mitosis, cohesion ensures faithful chromosome segregation. It promotes the back‑to‑back orientation of the sister kinetochores to facilitate their attachment to microtubules from opposite spindle poles and prevents sister chromatid separation until all chromosomes achieve bipolar attachment (also known as biorientation). b | Cohesion-independent functions of cohesin are related to genome organization. The genome is partitioned into discrete units known as topologically associating domains (TADs) that range from 100 kb to 1 Mb in mammals. Both CCCTC-binding factor (CTCF) and cohesin (not shown in the top panel) contribute to this organization, probably through the formation of chromatin loops. TADs confine regulatory activities (for example, enhancers) to a specific domain. Within a domain, cohesin can promote transcription by facilitating the interaction between an enhancer and a promoter35 or contribute to the transcriptional regulation of gene clusters36. Cohesin has also been proposed to organize chromatin loops at replication factories, thereby facilitating simultaneous firing of the clustered origins39.
are mutated at substantial frequencies in at least four tumour types12. Mutations in genes encoding cohesin subunits and NIPBL were initially identified in colorectal cancer 2, and mutations in STAG2 were later found in glioblastoma, Ewing’s sarcoma and melanoma3. However, it is in urothelial bladder cancer that mutations in STAG2 are the most common, with rates of 10–15% in aggressive tumours and up to 30% in low-grade tumours7–10,13. Other cohesin subunits are not as frequently mutated in this type of cancer. By contrast, similar mutation rates across most cohesin subunits have been described in AML4,11, Down syndrome related acute megakaryocitic leukaemia5 and other
myeloid neoplasms6. Overall, mutations in STAG2 are often truncating, whereas missense mutations are more frequent in other cohesin genes. The consequence of many missense mutations in protein function is unclear. Although most identified mutations are heterozygous, the SMC1A and STAG2 genes are located on the X chromosome, which makes the corresponding mutations functionally homozygous, at least in males. It is unlikely that cells can proliferate in the absence of cohesin. The higher mutation rates of STAG2 in most tumours might be explained by the fact that a single hit is sufficient for the loss of SA2 function, and cohesin–SA1 complexes might partially
392 | JUNE 2014 | VOLUME 14
compensate for this loss. Downregulation of SA2 is less detrimental for chromosome segregation than downregulation of SMC1A or SMC3 (REF. 2). Regarding SMC1A, only one mutation among those reported in the recent studies leads to the expression of a nonfunctional protein that lacks its carboxy‑ terminal third8. All other mutations result in amino acid changes whose importance for protein function remains to be tested. It is unclear how the cells in the tumour with a truncated SMC1A8, which is from a male patient, survive without a functional cohesin complex. As suggested above, cohesin dysfunction could affect tumorigenesis by increasing genome instability due to faulty DNA replication and/or repair and chromosome missegregation37. Although aneuploidy and genome instability are detrimental to cell survival, they can accelerate tumour evolution and adaptability 38. An association between aneuploidy and cohesin mutations in cancer has been reported in some studies8,10 but not in others7,9,11. Reduced sister chromatid cohesion might also favour loss of heterozygosity by promoting recombination with the homologous chromosome instead of using the sister chromatid as a template for homologous recombination‑mediated repair. The role of cohesin in genome organization could also underlie the tumour-promoting consequences of cohesin mutations. The most obvious effect would be gene expression changes of crucial oncogenes or tumour suppressors. However, other possibilities should be considered. Altered organization of replication factories may slow replication and increase replicative stress39,40. A local alteration of chromosomal domain organization could alter the replication timing of the domain and thereby affect its mutation rate, its epigenetic modifications or the frequency of structural rearrangements41. Reduced cohesion, together with domain decompaction and an increased number of interdomain chromatin contacts30 may favour chromosomal translocations. A study in yeast showed that reducing cohesin levels to 30% of wild type compromises homologous recombination‑mediated repair, but a deleterious effect on chromosome segregation requires a reduction below 13% (REF. 25). Although these numbers can be substantially different in mammalian cells, this study is important to make us aware of the different sensitivity of diverse cohesin functions to the levels of cohesin in the cell. Moreover, the distinct versions of cohesin that carry SA1 or SA2, PDS5A or PDS5B may also show different sensitivities www.nature.com/reviews/cancer
© 2014 Macmillan Publishers Limited. All rights reserved
PROGRESS to the reduction of their levels. MEFs that are heterozygous for Rad21 or Stag1 show increased mitotic defects and aneuploidy compared with wild-type MEFs and, in the case of Rad21 deficiency, reduced homologous recombination‑mediated repair has also been reported23,42. Postmitotic murine cells that were progressively depleted of RAD21 showed a concomitant loss of chromatin contacts and transcriptional dysregulation at several loci when RAD21 levels decreased below 50% of wild type30. In cells that are derived from heterozygous Nipbl mice, which recapitulate several phenotypes of Cornelia de Lange Syndrome, the overall levels of cohesin on chromatin are normal and cohesion defects are not observed; however, transcription is disrupted at several loci43,44. Thus, transcriptional regulation of specific genes could be most sensitive to a reduction in the levels of functional cohesin available in the cell. Conclusions and perspectives The recent identification of cohesin mutations in several tumour types, most notably bladder cancer and myeloid neoplasms, raises the question of how cohesin dysfunction affects tumorigenesis. Answering this question is complicated by the fact that cohesin has essential roles in both genome stability and transcriptional regulation. Mutations in cohesin genes that have been identified in cancer genomes remain poorly characterized in terms of how they affect the protein itself (folding and stability), the formation of the cohesin complex, its association to chromatin or its interaction with other proteins. Heterozygous mutations may reduce the amount of functional cohesin in the cell, but they can also produce a truncated protein with a dominantnegative effect. Distinct aspects of cohesin regulation and function could be tissuespecific and thereby explain why mutations in cohesin are more prominent in certain types of tumours. So far, synergy with mutations in additional pathways is also unclear. Our growing knowledge of the basic biology of cohesin and the generation of cell and animal models deficient for cohesin or carrying the mutations identified in cancer will help us to understand the functional importance of such mutations and hopefully contribute to improve the diagnosis and treatment of patients. Ana Losada is at the Chromosome Dynamics Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. e‑mail: [email protected] doi:10.1038/nrc3743
Remeseiro, S. & Losada, A. Cohesin, a chromatin engagement ring. Curr. Opin. Cell Biol. 25, 63–71 (2013). 2. Barber, T. D. et al. Chromatid cohesion defects may underlie chromosome instability in human colorectal cancers. Proc. Natl Acad. Sci. USA 105, 3443–3448 (2008). 3. Solomon, D. A. et al. Mutational inactivation of STAG2 causes aneuploidy in human cancer. Science 333, 1039–1043 (2011). 4. Welch, J. S. et al. The origin and evolution of mutations in acute myeloid leukemia. Cell 150, 264–278 (2012). 5. Yoshida, K. et al. The landscape of somatic mutations in Down syndrome-related myeloid disorders. Nature Genet. 45, 1293–1299 (2013). 6. Kon, A. et al. Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms. Nature Genet. 45, 1232–1237 (2013). 7. Taylor, C. F., Platt, F. M., Hurst, C. D., Thygesen, H. H. & Knowles, M. A. Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes. Hum. Mol. Genet. 23, 1964–1974 (2013). 8. Guo, G. et al. Whole-genome and whole-exome sequencing of bladder cancer identifies frequent alterations in genes involved in sister chromatid cohesion and segregation. Nature Genet. 45, 1459–1463 (2013). 9. Balbas-Martinez, C. et al. Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy. Nature Genet. 45, 1464–1469 (2013). 10. Solomon, D. A. et al. Frequent truncating mutations of STAG2 in bladder cancer. Nature Genet. 45, 1428–1430 (2013). 11. Thol, F. et al. Mutations in the cohesin complex in acute myeloid leukemia: clinical and prognostic implications. Blood 123, 914–920 (2013). 12. Lawrence, M. S. et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014). 13. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507, 315–322. (2014). 14. Liu, J. & Krantz, I. D. Cornelia de Lange syndrome, cohesin, and beyond. Clin. Genet. 76, 303–314 (2009). 15. Haering, C. H., Lowe, J., Hochwagen, A. & Nasmyth, K. Molecular architecture of SMC proteins and the yeast cohesin complex. Mol. Cell 9, 773–788 (2002). 16. Murayama, Y. & Uhlmann, F. Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505, 367–371 (2014). 17. Tedeschi, A. et al. Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature 501, 564–568 (2013). 18. Nishiyama, T. et al. Sororin mediates sister chromatid cohesion by antagonizing Wapl. Cell 143, 737–749 (2010). 19. Gerlich, D., Koch, B., Dupeux, F., Peters, J. M. & Ellenberg, J. Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication. Curr. Biol. 16, 1571–1578 (2006). 20. Nishiyama, T., Sykora, M. M., Huis in ‘t Veld, P. J., Mechtler, K. & Peters, J. M. Aurora B and Cdk1 mediate Wapl activation and release of acetylated cohesin from chromosomes by phosphorylating Sororin. Proc. Natl Acad. Sci. USA 110, 13404–13409 (2013). 21. Liu, H., Rankin, S. & Yu, H. Phosphorylation-enabled binding of SGO1‑PP2A to cohesin protects sororin and centromeric cohesion during mitosis. Nature Cell Biol. 15, 40–49 (2013). 22. Canudas, S. & Smith, S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells. J. Cell Biol. 187, 165–173 (2009). 23. Remeseiro, S. et al. Cohesin‑SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres. EMBO J. 31, 2076–2089 (2012). 24. Carretero, M., Ruiz-Torres, M., Rodriguez-Corsino, M., Barthelemy, I. & Losada, A. Pds5B is required for cohesion establishment and Aurora B 1.
NATURE REVIEWS | CANCER
accumulation at centromeres. EMBO J. 32, 2938–2949 (2013). 25. Heidinger-Pauli, J. M., Mert, O., Davenport, C., Guacci, V. & Koshland, D. Systematic reduction of cohesin differentially affects chromosome segregation, condensation, and DNA repair. Curr. Biol. 20, 957–963 (2010). 26. Wang, L. H., Mayer, B., Stemmann, O. & Nigg, E. A. Centromere DNA decatenation depends on cohesin removal and is required for mammalian cell division. J. Cell Sci. 123, 806–813 (2010). 27. Schockel, L., Mockel, M., Mayer, B., Boos, D. & Stemmann, O. Cleavage of cohesin rings coordinates the separation of centrioles and chromatids. Nature Cell Biol. 13, 966–972 (2011). 28. Gibcus, J. H. & Dekker, J. The hierarchy of the 3D genome. Mol. Cell 49, 773–782 (2013). 29. Zuin, J. et al. Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells. Proc. Natl Acad. Sci. USA 111, 996–1001 (2014). 30. Sofueva, S. et al. Cohesin-mediated interactions organize chromosomal domain architecture. EMBO J. 32, 3119–3129 (2013). 31. Wendt, K. S. et al. Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451, 796–801 (2008). 32. Remeseiro, S., Cuadrado, A., Gómez-López, G., Pisano, D. G. & Losada, A. A unique role of cohesin‑SA1 in gene regulation and development. EMBO J. 31, 2090–2102 (2012). 33. Rubio, E. D. et al. CTCF physically links cohesin to chromatin. Proc. Natl Acad. Sci. USA 105, 8309–8314 (2008). 34. Schmidt, D. et al. A CTCF-independent role for cohesin in tissue-specific transcription. Genome Res. 20, 578–588 (2010). 35. Kagey, M. H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010). 36. Monahan, K. et al. Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression. Proc. Natl Acad. Sci. USA 109, 9125–9130 (2012). 37. Duijf, P. H. & Benezra, R. The cancer biology of wholechromosome instability. Oncogene 32, 4727–4736 (2013). 38. Holland, A. J. & Cleveland, D. W. Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep. 13, 501–514 (2012). 39. Guillou, E. et al. Cohesin organizes chromatin loops at DNA replication factories. Genes Dev. 24, 2812–2822 (2010). 40. Burrell, R. A. et al. Replication stress links structural and numerical cancer chromosomal instability. Nature 494, 492–496 (2013). 41. Stamatoyannopoulos, J. A. et al. Human mutation rate associated with DNA replication timing. Nature Genet. 41, 393–395 (2009). 42. Xu, H. et al. Rad21‑cohesin haploinsufficiency impedes DNA repair and enhances gastrointestinal radiosensitivity in mice. PLoS ONE 5, e12112 (2010). 43. Chien, R. et al. Cohesin mediates chromatin interactions that regulate mammalian β-globin expression. J. Biol. Chem. 286, 17870–17878 (2011). 44. Remeseiro, S. et al. Reduction of Nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of Cornelia de Lange syndrome. Biochim. Biophys. Acta 1832, 2097–2102 (2013). 45. Deardorff, M. A. et al. HDAC8 mutations in Cornelia de Lange Syndrome affect the cohesin acetylation cycle. Nature 489, 313–317 (2012).
Acknowledgements
The author apologizes to colleagues whose relevant work on cohesin is not cited here. She thanks the group of F. X. Real (Spanish National Cancer Research Centre (CNIO), Madrid, Spain), as well as members of her own group, for discussions. The author’s research is currently funded by the Spanish Ministry of Economy and Competitiveness (MINECO), grant SAF‑2010‑21517.
Competing interests statement
The author declares no competing interests.
VOLUME 14 | JUNE 2014 | 393 © 2014 Macmillan Publishers Limited. All rights reserved
