RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.     

A kinase‐dependent role for Haspin in antagonizing Wapl and protecting mitotic centromere cohesion

Sister‐chromatid cohesion mediated by the cohesin complex is fundamental for precise chromosome segregation in mitosis. Through binding the cohesin subunit Pds5, Wapl releases the bulk of cohesin from chromosome arms in prophase, whereas centromeric cohesin is protected from Wapl until anaphase onset. Strong centromere cohesion requires centromeric localization of the mitotic histone kinase Haspin, which is dependent on the interaction of its non‐catalytic N‐terminus with Pds5B. It remains unclear how Haspin fully blocks the Wapl–Pds5B interaction at centromeres. Here, we show that the C‐terminal kinase domain of Haspin (Haspin‐KD) binds and phosphorylates the YSR motif of Wapl (Wapl‐YSR), thereby directly inhibiting the YSR motif‐dependent interaction of Wapl with Pds5B. Cells expressing a Wapl‐binding‐deficient mutant of Haspin or treated with Haspin inhibitors show centromeric cohesion defects. Phospho‐mimetic mutation in Wapl‐YSR prevents Wapl from binding Pds5B and releasing cohesin. Forced targeting Haspin‐KD to centromeres partly bypasses the need for Haspin–Pds5B interaction in cohesion protection. Taken together, these results indicate a kinase‐dependent role for Haspin in antagonizing Wapl and protecting centromeric cohesion in mitosis.     

Structural insights into the regulation of cohesion establishment by Wpl1

Correct segregation of duplicated chromosomes to daughter cells during mitosis requires the action of the cohesin complex. This tripartite ring‐shaped molecule is involved in holding replicated sister chromatids together from S phase until anaphase onset. Establishment of stable cohesion involves acetylation of the Smc3 component of cohesin during replication by the Eco1 acetyltransferase. This has been proposed to antagonise the activity of another member of the cohesin complex, Wpl1. Here, we describe the X‐ray structure of the conserved Wapl domain, and demonstrate that it binds the ATPase head of the Smc3 protein. We present data that suggest that Wpl1 may be involved in regulating the ATPase activity of cohesin, and that this may be subject to the acetylation state of Smc3. In addition, we present a structure of the Wapl domain bound to a functionally relevant segment of the Smc3 ATPase.     

Human Wapl is a cohesin-binding protein that promotes sister-chromatid resolution in mitotic prophase

BACKGROUND: The linkage between duplicated chromosomes (sister chromatids) is established during S phase by the action of cohesin, a multisubunit complex conserved from yeast to humans. Most cohesin dissociates from chromosome arms when the cell enters mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process is known as sister-chromatid resolution. Although two mitotic kinases have been implicated in this process, it remains unknown exactly how the cohesin-mediated linkage is destabilized at a mechanistic level. RESULTS: The wings apart-like (Wapl) protein was originally identified as a gene product that potentially regulates heterochromatin organization in Drosophila melanogaster. We show that the human ortholog of Wapl is a cohesin-binding protein that facilitates cohesin's timely release from chromosome arms during prophase. Depletion of Wapl from HeLa cells causes transient accumulation of prometaphase-like cells with chromosomes that display poorly resolved sister chromatids with a high level of cohesin. Reduction of cohesin relieves the Wapl-depletion phenotype, and depletion of Wapl rescues premature sister separation observed in Sgo1-depleted or Esco2-depleted cells. Conversely, overexpression of Wapl causes premature separation of sister chromatids. Wapl physically associates with cohesin in HeLa-cell nuclear extracts. Remarkably, in vitro reconstitution experiments demonstrate that Wapl forms a stoichiometric, ternary complex with two regulatory subunits of cohesin, implicating its noncatalytic function in inactivating cohesin's ability to interact with chromatin. CONCLUSIONS: Wapl is a new regulator of sister chromatid resolution and promotes release of cohesin from chromosomes by directly interacting with its regulatory subunits.     

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.     

Pds5 promotes cohesin acetylation and stable cohesin-chromosome interaction

Pds5 and Wpl1 act as anti-establishment factors preventing sister-chromatid cohesion until counteracted in S-phase by the cohesin acetyl-transferase Eso1. However, Pds5 is also required to maintain sister-chromatid cohesion in G2. Here, we show that Pds5 is essential for cohesin acetylation by Eso1 and ensures the maintenance of cohesion by promoting a stable cohesin interaction with replicated chromosomes. The latter requires Eso1 only in the presence of Wapl, indicating that cohesin stabilization relies on Eso1 only to neutralize the anti-establishment activity. We suggest that Eso1 requires Pds5 to counteract anti-establishment. This allows both cohesion establishment and Pds5-dependent stable cohesin binding to chromosomes.     

Cohesin acetyltransferase Esco2 is a cell viability factor and is required for cohesion in pericentric heterochromatin

Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is essential for chromosome segregation. In mammalian cells, cohesion establishment and Sororin recruitment to chromatin-bound cohesin depends on the acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a developmental disease in which mitotic chromosomes have a 'railroad' track morphology. Here, we show that Esco2 deficiency leads to termination of mouse development at pre- and post-implantation stages, indicating that Esco2 functions non-redundantly with Esco1. Esco2 is transiently expressed during S-phase when it localizes to pericentric heterochromatin (PCH). In interphase, Esco2 depletion leads to a reduction in cohesin acetylation and Sororin recruitment to chromatin. In early mitosis, Esco2 deficiency causes changes in the chromosomal localization of cohesin and its protector Sgo1. Our results suggest that Esco2 is needed for cohesin acetylation in PCH and that this modification is required for the proper distribution of cohesin on mitotic chromosomes and for centromeric cohesion.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

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.     

Cell-cycle regulation of cohesin stability along fission yeast chromosomes

Sister chromatid cohesion is mediated by cohesin, but the process of cohesion establishment during S-phase is still enigmatic. In mammalian cells, cohesin binding to chromatin is dynamic in G1, but becomes stabilized during S-phase. Whether the regulation of cohesin stability is integral to the process of cohesion establishment is unknown. Here, we provide evidence that fission yeast cohesin also displays dynamic behavior. Cohesin association with G1 chromosomes requires continued activity of the cohesin loader Mis4/Ssl3, suggesting that repeated loading cycles maintain cohesin binding. Cohesin instability in G1 depends on wpl1, the fission yeast ortholog of mammalian Wapl, suggestive of a conserved mechanism that controls cohesin stability on chromosomes. wpl1 is nonessential, indicating that a change in wpl1-dependent cohesin dynamics is dispensable for cohesion establishment. Instead, we find that cohesin stability increases at the time of S-phase in a reaction that can be uncoupled from DNA replication. Hence, cohesin stabilization might be a pre-requisite for cohesion establishment rather than its consequence.     

Shugoshin regulates cohesion by driving relocalization of PP2A in Xenopus extracts

Sister chromatid cohesion is mediated by cohesin. At the onset of mitosis, most cohesin dissociates from chromatin with the exception of a small population, present along chromosome arms and enriched at centromeres. A protein known as shugoshin (Sgo) is essential to maintain arm and centromeric cohesion until the onset of anaphase in transformed human cells, but not in other organisms like Drosophila or mouse. We have used Xenopus egg extracts to further explore this issue. Chromosomes assembled in extracts depleted of Sgo have little or no cohesin at centromeres and display centromeric cohesion defects. Unlike transformed human cells, however, arm cohesion is maintained in the absence of Sgo. Furthermore, Sgo depletion impairs the prophase dissociation of cohesin. This phenotype can be rescued by inhibition of PP2A. The protein phosphatase interacts with Sgo and accumulates at centromeres in mitosis in a Sgo-dependent manner. We propose that Sgo drives relocalization of PP2A from arms to centromeres and, in this way, coordinates release of arm cohesin with protection of centromeric cohesin in mitosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

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.     

The hsSsu72 phosphatase is a cohesin‐binding protein that regulates the resolution of sister chromatid arm cohesion

Cohesin is a multiprotein complex that establishes sister chromatid cohesion from S phase until mitosis or meiosis. In vertebrates, sister chromatid cohesion is dissolved in a stepwise manner: most cohesins are removed from the chromosome arms via a process that requires polo‐like kinase 1 (Plk1), aurora B and Wapl, whereas a minor amount of cohesin, found preferentially at the centromere, is cleaved by separase following its activation by the anaphase‐promoting complex/cyclosome. Here, we report that our budding yeast two‐hybrid assay identified hsSsu72 phosphatase as a Rad21‐binding protein. Additional experiments revealed that Ssu72 directly interacts with Rad21 and SA2 in vitro and in vivo, and associates with sister chromatids in human cells. Interestingly, depletion or mutational inactivation of Ssu72 phosphatase activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72 yielded high resistance to this resolution. Interestingly, it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting the phosphorylation of SA2. Thus, our study provides important new evidence, suggesting that Ssu72 is a novel cohesin‐binding protein capable of regulating cohesion between sister chromatid arms.     

Functional characterization of cohesin SMC3 and separase and their roles in the segregation of large and minichromosomes in Trypanosoma brucei

Minichromosomes in the nuclear genome of Trypanosoma brucei exhibit unusual patterns of mitotic segregation. To address whether differences in their mode of segregation in relation to large chromosomes are reflected at a molecular level, we characterized two different proteins that have highly conserved functions in eukaryotic chromosomes segregation: the SMC3 protein, a component of the chromatid cohesion apparatus, and the protease separase that resolves the cohesin complex at the onset of anaphase and has, in other organisms, additional functions during mitosis. Using in situ hybridization we show that RNA interference-mediated depletion of SMC3 has no visible effect on the segregation of the minichromosomal population but interferes with the faithful mitotic separation of large chromosomes. In contrast, separase depletion causes missegregation of both mini- and large chromosomes. We also show that SMC3 persists as a soluble protein throughout the cell cycle and only associates with chromatin between G1 and metaphase. Separase is present in the cell during the entire cell cycle, but is excluded from the nucleus until the metaphase–anaphase transition, thereby providing a potential control mechanism to prevent the untimely cleavage of the cohesin complex.     

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.     

Psm3 acetylation on conserved lysine residues is dispensable for viability in fission yeast but contributes to Eso1-mediated sister chromatid cohesion by antagonizing Wpl1

In budding yeast and humans, cohesion establishment during S phase requires the acetyltransferase Eco1/Esco1-2, which acetylates the cohesin subunit Smc3 on two conserved lysine residues. Whether Smc3 is the sole Eco1/Esco1-2 effector and how Smc3 acetylation promotes cohesion are unknown. In fission yeast (Schizosaccharomyces pombe), as in humans, cohesin binding to G(1) chromosomes is dynamic and the unloading reaction is stimulated by Wpl1 (human ortholog, Wapl). During S phase, a subpopulation of cohesin becomes stably bound to chromatin in an Eso1 (fission yeast Eco1/Esco1-2)-dependent manner. Cohesin stabilization occurs unevenly along chromosomes. Cohesin remains largely labile at the rDNA repeats but binds mostly in the stable mode to pericentromere regions. This pattern is largely unchanged in eso1Δ wpl1Δ cells, and cohesion is unaffected, indicating that the main Eso1 role is counteracting Wpl1. A mutant of Psm3 (fission yeast Smc3) that mimics its acetylated state renders cohesin less sensitive to Wpl1-dependent unloading and partially bypasses the Eso1 requirement but cannot generate the stable mode of cohesin binding in the absence of Eso1. Conversely, nonacetylatable Psm3 reduces the stable cohesin fraction and affects cohesion in a Wpl1-dependent manner, but cells are viable. We propose that Psm3 acetylation contributes to Eso1 counteracting of Wpl1 to secure stable cohesin interaction with postreplicative chromosomes but that it is not the sole molecular event by which this occurs.