Faithful anaphase is ensured by Mis4, a sister chromatid cohesion molecule required in S phase and not destroyed in G1 phase

The loss of sister chromatid cohesion triggers anaphase spindle movement. The budding yeast Mcd1/Scc1 protein, called cohesin, is required for associating chromatids, and proteins homologous to it exist in a variety of eukaryotes. Mcd1/Scc1 is removed from chromosomes in anaphase and degrades in G₁. We show that the fission yeast protein, Mis4, which is required for equal sister chromatid separation in anaphase is a different chromatid cohesion molecule that behaves independent of cohesin and is conserved from yeast to human. Its inactivation in G₁ results in cell lethality in S phase and subsequent premature sister chromatid separation. Inactivation in G₂ leads to cell death in subsequent metaphase–anaphase progression but missegregation occurs only in the next round of mitosis. Mis4 is not essential for condensation, nor does it degrade in G₁. Rather, it associates with chromosomes in a punctate fashion throughout the cell cycle. mis4 mutants are hypersensitive to hydroxyurea (HU) and UV irradiation but retain the ability to restrain cell cycle progression when damaged or sustaining a block to replication. The mis4 mutation results in synthetic lethality with a DNA ligase mutant. Mis4 may form a stable link between chromatids in S phase that is split rather than removed in anaphase.     

Sister chromatid cohesion: the cohesin cleavage model does not ring true

Sister chromatid cohesion is important for high fidelity chromosome segregation during anaphase. Gene products that provide structural components (cohesin complex or cohesin) and regulatory components responsible for cohesion are conserved through eukaryotes. A simple model where cohesion establishment occurs by replication through static cohesin rings and cohesion dissolution occurs by Esp1p/separase mediated cleavage of the cohesin rings (Mcd1p/Rad21p/Scc1p sub-unit cleavage) has become widespread. A growing body of evidence is inconsistent with this ring cleavage model. This review will summarize the evidence showing that cohesin complex is not static but is regulated at multiple cell cycle stages before anaphase in a separase independent manner. Separase is indeed required at anaphase for complete chromosome segregation. However, multiple mechanisms for cohesion dissolution appear to act concurrently during anaphase. Separase is only one such mechanism and its importance varies from organism to organism. The idea that cohesin is a dynamic complex subjected to regulation at various cell cycle stages by multiple mechanisms makes sense in light of the myriad functions in which it has been implicated, such as DNA damage repair, gene silencing and chromosome condensation.     

Rec8-containing cohesin maintains bivalents without turnover during the growing phase of mouse oocytes

During female meiosis, bivalent chromosomes are thought to be held together from birth until ovulation by sister chromatid cohesion mediated by cohesin complexes whose ring structure depends on kleisin subunits, either Rec8 or Scc1. Because cohesion is established at DNA replication in the embryo, its maintenance for such a long time may require cohesin turnover. To address whether Rec8- or Scc1-containing cohesin holds bivalents together and whether it turns over, we created mice whose kleisin subunits can be cleaved by TEV protease. We show by microinjection experiments and confocal live-cell imaging that Rec8 cleavage triggers chiasmata resolution during meiosis I and sister centromere disjunction during meiosis II, while Scc1 cleavage triggers sister chromatid disjunction in the first embryonic mitosis, demonstrating a dramatic transition from Rec8- to Scc1-containing cohesin at fertilization. Crucially, activation of an ectopic Rec8 transgene during the growing phase of Rec8(TEV)(/TEV) oocytes does not prevent TEV-mediated bivalent destruction, implying little or no cohesin turnover for ≥2 wk during oocyte growth. We suggest that the inability of oocytes to regenerate cohesion may contribute to age-related meiosis I errors.     

Cell cycle mechanisms of sister chromatid separation; roles of Cut1/separin and Cut2/securin

The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called 'anaphase'. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW approximately 180 kDa), the C-terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N-terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.     

Yeast Cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase→anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a ‘Cohesin’ complex that binds chromosomes from late G₁ until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin’s association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G₂ and M phases.     

Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl

DNA double-strand breaks (DSBs) fuel cancer-driving chromosome translocations. Two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, promote DSB repair through sister chromatid homologous recombination (SCR). Here we show that the Smc5/6 subunit Mms21 sumoylates multiple lysines of the cohesin subunit Scc1. Mms21 promotes cohesin-dependent small ubiquitin-like modifier (SUMO) accumulation at laser-induced DNA damage sites in S/G2 human cells. Cells expressing the nonsumoylatable Scc1 mutant (15KR) maintain sister chromatid cohesion during mitosis but are defective in SCR and sensitive to ionizing radiation (IR). Scc1 15KR is recruited to DNA damage sites. Depletion of Wapl, a negative cohesin regulator, rescues SCR defects of Mms21-deficient or Scc1 15KR-expressing cells. Expression of the acetylation-mimicking Smc3 mutant does not bypass the requirement for Mms21 in SCR. We propose that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after cohesin loading at DSBs and in a way not solely dependent on Smc3 acetylation.     

Spo13 regulates cohesin cleavage

A key aspect of meiotic chromosome segregation is that cohesin, the protein complex that holds sister chromatids together, dissociates from chromosome arms during meiosis I and from centromeric regions during meiosis II. The budding yeast protein Spo13 plays a key role in preventing centromeric cohesin from being lost during meiosis I. We have determined the molecular basis for the metaphase arrest obtained when SPO13 is overexpressed during the mitotic cell cycle. Overexpression of SPO13 inhibits anaphase onset by at least two mechanisms. First, Spo13 causes a transient delay in degradation of the anaphase inhibitor Pds1. Second, Spo13 inhibits cleavage of the cohesin subunit Scc1/Mcd1 or its meiosis-specific homolog, Rec8, by the separase Esp1. The finding that Spo13 did not prevent cleavage of another Esp1 substrate, Slk19, suggests that overexpression of SPO13 is sufficient to prevent cohesin cleavage by protecting specific substrates from separase activity.     

Many functions of the meiotic cohesin

Sister chromatids are held together from the time of their formation in S phase until they segregate in anaphase by the cohesin complex. In meiosis of most organisms, the mitotic Mcd1/Scc1/Rad21 subunit of the cohesin complex is largely replaced by its paralog named Rec8. This article reviews the specialized functions of Rec8 that are crucial for diverse aspects of chromosome dynamics in meiosis, and presents some speculations relating to meiotic chromosome organization.     

Cohesin release is required for sister chromatid resolution,but not for condensin-mediated compaction,at the onset of mitosis

The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction.     

Localization of human SMC1 protein at kinetochores

Proper cohesion of sister chromatids is prerequisite for correct segregation of chromosomes during cell division. The cohesin multiprotein complex, conserved in eukaryotes, is required for sister chromatid cohesion. Human cohesin is composed of a stable heterodimer of the structural maintenance of chromosomes (SMC) family proteins, hSMC1 and hSMC3, and non-SMC components, hRAD21 and SA1 (or SA2). In yeast, cohesin associates with chromosomes from late G1 to metaphase and is required for the establishment and maintenance of both chromosome arm and centromeric cohesion. However, in human cells, the majority of cohesin dissociates from chromosomes before mitosis. Although it was recently shown that a small amount of hRAD21 localizes to the centromeres during metaphase, the presence of other cohesin components at the centromere has not been demonstrated in human cells. Here we report the mitosis-specific localization of hSMC1 to the kinetochores. hSMC1 is targeted to the kinetochore region during prophase concomitant with kinetochore assembly and remains through anaphase. Importantly, hSMC1 is targeted only to the active centromere on dicentric chromosomes. These results suggest that hSMC1 is an integral component of the functional kinetochore structure during mitosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Scc2/Scc4 cohesin loader determines the distribution of cohesin on budding yeast chromosomes

Cohesins mediate sister chromatid cohesion and DNA repair and also function in gene regulation. Chromosomal cohesins are distributed nonrandomly, and their deposition requires the heterodimeric Scc2/Scc4 loader. Whether Scc2/Scc4 establishes nonrandom cohesin distributions on chromosomes is poorly characterized, however. To better understand the spatial regulation of cohesin association, we mapped budding yeast Scc2 and Scc4 chromosomal distributions. We find that Scc2/Scc4 resides at previously mapped cohesin-associated regions (CARs) in pericentromeric and arm regions, and that Scc2/Scc4–cohesin colocalization persists after the initial deposition of cohesins in G1/S phase. Pericentromeric Scc2/Scc4 enrichment is kinetochore-dependent, and both Scc2/Scc4 and cohesin associations are coordinately reduced in these regions following chromosome biorientation. Thus, these characteristics of Scc2/Scc4 binding closely recapitulate those of cohesin. Although present in G1, Scc2/Scc4 initially has a poor affinity for CARs, but its affinity increases as cells traverse the cell cycle. Scc2/Scc4 association with CARs is independent of cohesin, however. Taken together, these observations are inconsistent with a previous suggestion that cohesins are relocated by translocating RNA polymerases from separate loading sites to intergenic regions between convergently transcribed genes. Rather, our findings suggest that budding yeast cohesins are targeted to CARs largely by Scc2/Scc4 loader association at these locations.     

A tDNA establishes cohesion of a neighboring silent chromatin domain

DNA replication generates sister chromatid pairs that are bound to one another until anaphase onset. The process, termed sister chromatid cohesion, requires the multisubunit cohesin complex that resides at centromeres and sites where genes converge. At the HMR mating-type locus of budding yeast, cohesin associates with a heterochromatin-like structure known as silent chromatin. In this report, we show that silent chromatin is necessary but not sufficient for cohesion of the replicating locus. A tRNA gene (tDNA) that delimits the silent chromatin domain is also required, as are subunits of the TFIIIB and RSC complexes that bind the gene. Non-tDNA boundary elements do not substitute for tDNAs in cohesion, suggesting that barrier activity is not responsible for the phenomenon. The results reveal an unexpected role for tDNAs and RNA polymerase III-associated proteins in establishment of sister chromatid cohesion.     

Intersection of ChIP and FLIP, genomic methods to study the dynamics of the cohesin proteins

The evolutionarily conserved cohesin proteins Smc1, Smc3, Rad21 (Mcd1), and Scc3 function in the cohesin complex that provides the basis for chromosome cohesion and is involved in gene regulation. Understanding how these proteins link together the genome requires the use of whole-genome approaches to study the molecular mechanisms of these essential proteins. While chromatin immunoprecipitation followed by DNA microarray (ChIP-chip) studies have provided a snapshot in time of where these proteins associate with various genomes, the cohesin proteins are dynamic in their localization and interactions on chromatin. Study of the dynamic nature of these proteins requires approaches such as live cell imaging. We present evidence from fluorescence loss in photobleaching (FLIP) experiments in budding yeast that the decay constant of each cohesin subunit is ∼60–90 s in interphase. The decay constant on chromatin increases from G₁ to S phase to metaphase, consistent with the interaction with chromatin becoming more stable once chromosomes are cohered. A small population of Smc3 at a position consistent with centromeric location has a longer decay constant than bulk Smc3. The characterization of the interaction of cohesin with chromatin, in terms of both its position and its dynamics, may be key to understanding how this protein complex contributes to chromosome segregation and gene regulation.     

Releasing cohesin from chromosome arms in early mitosis: opposing actions of Wapl–Pds5 and Sgo1

The cohesin complex establishes sister chromatid cohesion during S phase. In metazoan cells, most if not all cohesin dissociates from chromatin during mitotic prophase, leading to the formation of metaphase chromosomes with two cytologically discernible chromatids. This process, known as sister chromatid resolution, is believed to be a prerequisite for synchronous separation of sister chromatids in subsequent anaphase. To dissect this process at a mechanistic level, we set up an in vitro system. Sister chromatid resolution is severely impaired upon depletion of Wapl from Xenopus egg extracts. Exogenously added human Wapl can rescue these defects and, remarkably, it can do so in a very short time window of early mitosis. A similar set of observations is made for Pds5, a factor implicated previously in the stabilization of interphase cohesion. Characteristic amino acid motifs (the FGF motifs) in Wapl coordinate its physical and functional interactions with Pds5 and cohesin subunits. We propose that Wapl and Pds5 directly modulate conformational changes of cohesin to make it competent for dissociation from chromatin during prophase. Evidence is also presented that Sgo1 plays a hitherto underappreciated role in stabilizing cohesin along chromosome arms, which is antagonized by the mitotic kinases polo-like kinsase (Plk1) and aurora B.     

Shugoshin prevents cohesin cleavage by PP2ACdc55-dependent inhibition of separase

Chromosome segregation is triggered by separase, an enzyme that cleaves cohesin, the protein complex that holds sister chromatids together. Separase activation requires the destruction of its inhibitor, securin, which occurs only upon the correct attachment of chromosomes to the spindle. However, other mechanisms restrict separase activity to the appropriate window in the cell cycle because cohesin is cleaved in a timely manner in securin-deficient cells. We investigated the mechanism by which the protector protein Shugoshin counteracts cohesin cleavage in budding yeast. We show that Shugoshin can prevent separase activation independently of securin. Instead, PP2A^Cdc55 is essential for Shugoshin-mediated inhibition of separase. Loss of both securin and Cdc55 leads to premature sister chromatid separation, resulting in aneuploidy. We propose that Cdc55 is a separase inhibitor that acts downstream from Shugoshin under conditions where sister chromatids are not under tension.     

Studies of meiosis disclose distinct roles of cohesion in the core centromere and pericentromeric regions

During meiosis, a single round of genome duplication is followed by two sequential rounds of chromosome segregation. Through this process, a diploid parent cell generates gametes with a haploid set of chromosomes. A characteristic of meiotic chromosome segregation is a stepwise loss of sister chromatid cohesion along chromosomal arms and at centromeres. Whereas arm cohesion plays an important role in ensuring homologue disjunction at meiosis I, persisting cohesion at pericentromeric regions throughout meiosis I is essential for the faithful equational segregation of sisters in the following meiosis II, similar to mitosis. A widely conserved pericentromeric protein called shugoshin, which associates with protein phosphatase 2A (PP2A), plays a critical role in this protection of cohesin. Another key aspect of meiosis I is the establishment of monopolar attachment of sister kinetochores to spindle microtubules. Cohesion or physical linkage at the core centromeres, where kinetochores assemble, may conjoin sister kinetochores, leading to monopolar attachment. A meiosis-specific kinetochore factor such as fission yeast Moa1 or budding yeast monopolin contributes to this regulation. We propose that cohesion at the core centromere and pericentromeric regions plays distinct roles, especially in defining the orientation of kinetochores.     

Spo13 protects meiotic cohesin at centromeres in meiosis I

In the absence of Spo13, budding yeast cells complete a single meiotic division during which sister chromatids often separate. We investigated the function of Spo13 by following chromosomes tagged with green fluorescent protein. The occurrence of a single division in spo13Delta homozygous diploids depends on the spindle checkpoint. Eliminating the checkpoint accelerates meiosis I in spo13Delta cells and allows them to undergo two divisions in which sister chromatids often separate in meiosis I and segregate randomly in meiosis II. Overexpression of Spo13 and the meiosis-specific cohesin Rec8 in mitotic cells prevents separation of sister chromatids despite destruction of Pds1 and activation of Esp1. This phenotype depends on the combined overexpression of both proteins and mimics one aspect of meiosis I chromosome behavior. Overexpressing the mitotic cohesin, Scc1/Mcd1, does not substitute for Rec8, suggesting that the combined actions of Spo13 and Rec8 are important for preventing sister centromere separation in meiosis I.     

Isolated NIBPL missense mutations that cause Cornelia de Lange syndrome alter MAU2 interaction

Cornelia de Lange syndrome (CdLS; or Brachmann-de Lange syndrome) is a dominantly inherited congenital malformation disorder with features that include characteristic facies, cognitive delays, growth retardation and limb anomalies. Mutations in nearly 60% of CdLS patients have been identified in NIPBL, which encodes a regulator of the sister chromatid cohesion complex. NIPBL, also known as delangin, is a homolog of yeast and amphibian Scc2 and C. elegans PQN-85. Although the exact mechanism of NIPBL function in sister chromatid cohesion is unclear, in vivo yeast and C. elegans experiments and in vitro vertebrate cell experiments have demonstrated that NIPBL/Scc2 functionally interacts with the MAU2/Scc4 protein to initiate loading of cohesin onto chromatin. To test the significance of this model in the clinical setting of CdLS, we fine-mapped the NIBPL-MAU2 interaction domain and tested the functional significance of missense mutations and variants in NIPBL and MAU2 identified in these minimal domains in a cohort of patients with CdLS. We demonstrate that specific novel mutations at the N-terminus of the MAU2-interacting domain of NIBPL result in markedly reduced MAU2 binding, although we appreciate no consistent clinical difference in the small group of patients with these mutations. These data suggest that factors in addition to MAU2 are essential in determining the clinical features and severity of CdLS.     

Meiosis‐specific cohesin component,Rec8, promotes the localization of Mps3 SUN domain protein on the nuclear envelope

Proteins in the nuclear envelope (NE) play a role in the dynamics and functions of the nucleus and of chromosomes during mitosis and meiosis. Mps3, a yeast NE protein with a conserved SUN domain, predominantly localizes on a yeast centrosome equivalent, spindle pole body (SPB), in mitotic cells. During meiosis, Mps3, together with SPB, forms a distinct multiple ensemble on NE. How meiosis‐specific NE localization of Mps3 is regulated remains largely unknown. In this study, we found that a meiosis‐specific component of the protein complex essential for sister chromatid cohesion, Rec8, binds to Mps3 during meiosis and controls Mps3 localization and proper dynamics on NE. Ectopic expression of Rec8 in mitotic yeast cells induced the formation of Mps3 patches/foci on NE. This required the cohesin regulator, WAPL ortholog, Rad61/Wpl1, suggesting that a meiosis‐specific cohesin complex with Rec8 controls NE localization of Mps3. We also observed that two domains of the nucleoplasmic region of Mps3 are essential for NE localization of Mps3 in mitotic as well as meiotic cells. We speculate that the interaction of Mps3 with the meiosis‐specific cohesin in the nucleoplasm is a key determinant for NE localization/function of Mps3.