A conserved motif at the C terminus of sororin is required for sister chromatid cohesion

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.     

A model for ATP hydrolysis-dependent binding of cohesin to DNA

BACKGROUND: Cohesion between sister chromatids is promoted by the chromosomal cohesin complex that forms a proteinaceous ring, large enough in principle to embrace two sister strands. The mechanism by which cohesin binds to DNA, and how sister chromatid cohesion is established, is unknown. RESULTS: Biochemical studies of cohesin have largely been limited to protein isolated from soluble cellular fractions. Here, we characterize cohesin purified from budding yeast chromatin, suggesting that chromosomal cohesin is sufficiently described by its known distinctive ring structure. We present evidence that the two Smc subunits of cohesin by themselves form a ring, closed at interacting ATPase head domains. A motif in the Smc1 subunit implicated in ATP hydrolysis is essential for loading cohesin onto DNA. In addition to functional ATPase heads, an intact cohesin ring structure is indispensable for DNA binding, suggesting that ATP hydrolysis may be coupled to DNA transport into the cohesin ring. DNA is released in anaphase when separase cleaves cohesin's Scc1 subunit. We show that a cleavage fragment of Scc1 disrupts the interaction between the two Smc heads, thereby opening the ring. CONCLUSIONS: We present a model for cohesin binding to chromatin by ATP hydrolysis-dependent transport of DNA into the cohesin ring. After DNA replication, two DNA strands may be trapped to promote sister chromatid cohesion. In anaphase, Scc1 cleavage opens the ring to release sister chromatids.     

Sororin, a substrate of the anaphase-promoting complex, is required for sister chromatid cohesion in vertebrates

We have identified a regulator of sister chromatid cohesion in a screen for cell cycle-controlled proteins. This 35 kDa protein is degraded through anaphase-promoting complex (APC)-dependent ubiquitination in G1. The protein is nuclear in interphase cells, dispersed from the chromatin in mitosis, and interacts with the cohesin complex. In Xenopus embryos, overexpression of the protein causes failure to resolve and segregate sister chromatids in mitosis and an increase in the level of cohesin associated with metaphase chromosomes. In cultured cells, depletion of the protein causes mitotic arrest and complete failure of sister chromatid cohesion. This protein is thus an essential, cell cycle-dependent mediator of sister chromatid cohesion. Based on sequence analysis, this protein has no apparent orthologs outside of the vertebrates. We speculate that the protein, which we have named sororin, regulates the ability of the cohesin complex to mediate sister chromatid cohesion, perhaps by altering the nature of the interaction of cohesin with the chromosomes.     

Wapl controls the dynamic association of cohesin with chromatin

Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.     

The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2‐7 subcomplex of the replicative Cdc45‐MCM‐GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL. Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.     

Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts

The cohesin complex is a central player in sister chromatid cohesion, a process that ensures the faithful segregation of chromosomes in mitosis and meiosis. Previous genetic studies in yeast show that Scc2/Mis4, a HEAT-repeat-containing protein, is required for the loading of cohesin onto chromatin. In this study, we have identified two isoforms of Scc2 in humans and Xenopus (termed Scc2A and Scc2B), which are encoded by a single gene but have different carboxyl termini created by alternative splicing. Both Scc2A and Scc2B bind to chromatin concomitant with cohesin during DNA replication in Xenopus egg extracts. Simultaneous immunodepletion of Scc2A and Scc2B from the extracts impairs the association of cohesin with chromatin, leading to severe defects in sister chromatid pairing in the subsequent mitosis. The loading of Scc2 onto chromatin is inhibited in extracts treated with geminin but not with p21(CIP1), suggesting that this step depends on replication licensing but not on the initiation of DNA replication. Upon mitotic entry, Scc2 is removed from chromatin through a mechanism that requires cdc2 but not aurora B or polo-like kinase. Our results suggest that vertebrate Scc2 couples replication licensing to sister chromatid cohesion by facilitating the loading of cohesin onto chromatin.     

Histone tail-independent chromatin binding activity of recombinant cohesin holocomplex

Cohesin, an SMC (structural maintenance of chromosomes) protein-containing complex, governs several important aspects of chromatin dynamics, including the essential chromosomal process of sister chromatid cohesion. The exact mechanism by which cohesin achieves the bridging of sister chromatids is not known. To elucidate this mechanism, we reconstituted a recombinant cohesin complex and investigated its binding to DNA fragments corresponding to natural chromosomal sites with high and low cohesin occupancy in vivo. Cohesin displayed uniform but nonspecific binding activity with all DNA fragments tested. Interestingly, DNA fragments with high occupancy by cohesin in vivo showed strong nucleosome positioning in vitro. We therefore utilized a defined model chromatin fragment (purified reconstituted dinucleosome) as a substrate to analyze cohesin interaction with chromatin. The four-subunit cohesin holocomplex showed a distinct chromatin binding activity in vitro, whereas the Smc1p-Smc3p dimer was unable to bind chromatin. Histone tails and ATP are dispensable for cohesin binding to chromatin in this reaction. A model for cohesin association with chromatin is proposed.     

Sororin is required for stable binding of cohesin to chromatin and for sister chromatid cohesion in interphase

Sister chromatid cohesion depends on cohesin [1-3]. Cohesin associates with chromatin dynamically throughout interphase [4]. During DNA replication, cohesin establishes cohesion [5], and this process coincides with the generation of a cohesin subpopulation that is more stably bound to chromatin [4]. In mitosis, cohesin is removed from chromosomes, enabling sister chromatid separation [6]. How cohesin associates with chromatin and establishes cohesion is poorly understood. By searching for proteins that are associated with chromatin-bound cohesin, we have identified sororin, a protein that was known to be required for cohesion [7]. To obtain further insight into sororin's function, we have addressed when during the cell cycle sororin is required for cohesion. We show that sororin is dispensable for the association of cohesin with chromatin but that sororin is essential for proper cohesion during G2 phase. Like cohesin, sororin is also needed for efficient repair of DNA double-strand breaks in G2. Finally, sororin is required for the presence of normal amounts of the stably chromatin-bound cohesin population in G2. Our data indicate that sororin interacts with chromatin-bound cohesin and functions during the establishment or maintenance of cohesion in S or G2 phase, respectively.     

Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

A wapl a day keeps the sisters apart: wapl and cohesin dynamics

Sister chromatids are held together by the cohesin complex from the time they are made until cell division. In recent articles published in Cell and Current Biology, the characterization of Wapl, a newly identified cohesin-interacting protein, suggests that a dynamic interaction between the cohesin complex and chromatin is important for normal regulation of sister chromatid cohesion.     

Centrosomal Aki1 and cohesin function in separase-regulated centriole disengagement

Sister chromatid separation at anaphase is triggered by cleavage of the cohesin subunit Scc1, which is mediated by separase. Centriole disengagement also requires separase. This dual role of separase permits concurrent control of these events for accurate metaphase to anaphase transition. Although the molecular mechanism underlying sister chromatid cohesion has been clarified, that of centriole cohesion is poorly understood. In this study, we show that Akt kinase–interacting protein 1 (Aki1) localizes to centrosomes and regulates centriole cohesion. Aki1 depletion causes formation of multipolar spindles accompanied by centriole splitting, which is separase dependent. We also show that cohesin subunits localize to centrosomes and that centrosomal Scc1 is cleaved by separase coincidentally with chromatin Scc1, suggesting a role of Scc1 as a connector of centrioles as well as sister chromatids. Interestingly, Scc1 depletion strongly induces centriole splitting. Furthermore, Aki1 interacts with cohesin in centrosomes, and this interaction is required for centriole cohesion. We demonstrate that centrosome-associated Aki1 and cohesin play pivotal roles in preventing premature cleavage in centriole cohesion.     

A multi-step pathway for the establishment of sister chromatid cohesion

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.     

Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.     

Targeted sister chromatid cohesion by Sir2

The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase.     

Identification of cohesin association sites at centromeres and along chromosome arms.

A multisubunit cohesin complex holds sister chromatids together after DNA replication. Using chromatin immunoprecipitation, we detected cohesin association with centromeres and with discrete sites along chromosome arms from S phase until metaphase in S. cerevisiae. Short DNA sequences (130-280 bp) are sufficient to confer cohesin association. Cohesin association with a centromere depends on Mif2p, the centromere binding factor CBF3, and a centromere-specific histone variant, Cse4p. Because only active centromeres confer cohesin association with centromeric DNA, we suggest that cohesin is recruited by the same chromatin structure that confers the attachment of microtubules. Propagation of this structure might be partly epigenetic. Finally, cohesion associated with "minimal" centromeres is insufficient to resist the splitting force exerted by microtubules and appears to be reinforced by cohesion provided by their flanking DNA sequences.     

Cohesin Acetylation Promotes Sister Chromatid Cohesion Only in Association with the Replication Machinery

Acetylation of the Smc3 subunit of cohesin is essential to establish functional cohesion between sister chromatids. Smc3 acetylation is catalyzed by members of the Eco family of acetyltransferases, although the mechanism by which acetylation is regulated and how it promotes cohesion are largely unknown. In vertebrates, the cohesin complex binds to chromatin during mitotic exit and is converted to a functional form during or shortly after DNA replication. The conserved proliferating cell nuclear antigen-interacting protein box motif in yeast Eco1 is required for function, and cohesin is acetylated during the S phase. This has led to the notion that acetylation of cohesin is stimulated by interaction of Eco1 with the replication machinery. Here we show that in vertebrates Smc3 acetylation occurs independently of DNA replication. Smc3 is readily acetylated before replication is initiated and after DNA replication is complete. However, we also show that functional acetylation occurs only in association with the replication machinery: disruption of the interaction between XEco2 and proliferating cell nuclear antigen prevents cohesion establishment while having little impact on the overall levels of Smc3 acetylation. These results demonstrate that Smc3 acetylation can occur throughout interphase but that only acetylation in association with the replication fork promotes sister chromatid cohesion. These data reveal how the generation of cohesion is limited to the appropriate time and place during the cell cycle and provide insight into the mechanism by which acetylation ensures cohesion.     

C-terminus of Sororin interacts with SA2 and regulates sister chromatid cohesion

Sororin is a conserved protein required for accurate separation of sister chromatids in each cell cycle. Sororin is recruited to chromatin during DNA replication, protects sister chromatid cohesion in S and G2 phase, and regulates the resolution of sister chromatid cohesion in mitosis. Sororin binds to cohesin complex, but how Sororin and cohesin subunits interact remains unclear. Here we report that the C-terminus of Sororin, especially the last 12 amino acid (aa) residues, is important for Sororin to bind cohesin core subunit SA2. Deletion of the last 12aa residues not only inhibits the interactions between Sororin and SA2 but also causes precocious chromosome separation. Our data suggest that the C-terminus of Sororin functions as an anchor binding to SA2, which facilitates other conserved motifs on Sororin to interact with other proteins to regulate sister chromatid cohesion and separation.     

Live-cell imaging reveals a stable cohesin-chromatin interaction after but not before DNA replication

Cohesin is a multisubunit protein complex that links sister chromatids from replication until segregation. The lack of obvious cohesin-targeting-specific sequences on DNA, as well as cohesin's molecular arrangement as a large ring, has led to the working hypothesis that cohesin acts as a direct topological linker. To preserve the identity of sister chromatids, such a linkage would need to stably persist throughout the entire S and G2 phases of the cell cycle. Unexpectedly, cohesin binds chromatin already in telophase, and a large fraction dissociates from chromosomes during prophase in a phosphorylation-dependent manner, whereas initiation of anaphase requires proteolytic cleavage of only a small fraction of cohesin. These observations raised the question of how and when cohesin interacts with chromatin during the cell cycle. Here, we report a cell-cycle dependence in the stability of cohesin binding to chromatin. Using photobleaching and quantitative live-cell imaging, we identified several cohesin pools with different chromatin binding stabilities. Although all chromatin bound cohesin dissociated after a mean residence time of less than 25 min before replication, about one-third of cohesin was bound much more stably after S phase and persisted until metaphase, consistent with long-lived links mediating cohesion between sister chromatids.