Cohesin component dynamics during meiotic prophase I in mammalian oocytes

Cohesins are chromosomal proteins that form complexes involved in the maintenance of sister chromatid cohesion during division of somatic and germ cells. Three meiosis-specific cohesin subunits have been reported in mammals, REC8, STAG3 and SMC1 beta; their expression in mouse spermatocytes has also been described. Here we studied the localization of different meiotic and mitotic cohesin components during prophase I in human and murine female germ cells. In normal and atretic human fetal oocytes, from leptotene to diplotene stages, REC8 and STAG3 colocalize in fibers. In murine oocytes, SMC1beta, SMC3 and STAG3 are localized along fibers that correspond first to the chromosome axis and then to the synaptonemal complex in pachytene. Mitotic cohesin subunit RAD21 is also found in fibers that decorate the SC during prophase I in mouse oocytes, suggesting a role for this cohesin in mammalian sister chromatid cohesion in female meiosis. We observed that, unlike human oocytes, murine synaptonemal complex protein SYCP3 localizes to nucleoli throughout prophase I stages, and centromeres cluster in discrete locations from leptotene to dictyate. At difference from meiosis in male mice, the cohesin axis is progressively lost during the first week after birth in females with a parallel destruction of the axial elements at dictyate arrest, demonstrating sexual dimorphism in sister chromatid cohesion in meiosis.     

The cohesin complex in mammalian meiosis

Cohesin is an evolutionary conserved multi‐protein complex that plays a pivotal role in chromosome dynamics. It plays a role both in sister chromatid cohesion and in establishing higher order chromosome architecture, in somatic and germ cells. Notably, the cohesin complex in meiosis differs from that in mitosis. In mammalian meiosis, distinct types of cohesin complexes are produced by altering the combination of meiosis‐specific subunits. The meiosis‐specific subunits endow the cohesin complex with specific functions for numerous meiosis‐associated chromosomal events, such as chromosome axis formation, homologue association, meiotic recombination and centromeric cohesion for sister kinetochore geometry. This review mainly focuses on the cohesin complex in mammalian meiosis, pointing out the differences in its roles from those in mitosis. Further, common and divergent aspects of the meiosis‐specific cohesin complex between mammals and other organisms are discussed.     

Cohesion proteins are present in centromere protein bodies associated with avian lampbrush chromosomes

Proteins of sister chromatid cohesion are important for maintenance of meiotic chromosome structure and retention of homologous chromosomes in bivalents during diplotene. Localization of the cohesion proteins within nuclei of growing oocytes merits special attention, particularly in avian oocytes, in which diplotene chromosomes assume the form of lampbrush chromosomes (LBCs). We performed indirect immunostaining using antibodies against cohesins SMC1α, SMC1β, SMC3, Rad21, and the SA/STAG family on chaffinch, pigeon and duck LBCs spreads, and frozen ovary sections. On LBCs spreads, antibodies to the majority of cohesins showed punctate staining on chromosome axes. LBC lateral loops, where sister chromatids are separated, did not show cohesin components. The spherical entities attached to the LBCs centromeres in avian germinal vesicles, the so-called protein bodies (PBs), were enriched in SMC1α, SMC3, Rad21, STAG1 and STAG2. The synaptonemal complex component SYCP3, which also participates in cohesion, was detected in the axes of avian lampbrush bivalents and, to a greater degree, in the PBs. In vitellogenic oocytes, cohesion proteins persist in the PBs associated with condensing bivalents when they concentrate into the karyosphere. These results indicate that cohesion proteins accumulate in centromere PBs in avian oocytes and are involved into structural maintenance of lampbrush chromosome axes.     

Sororin is enriched at the central region of synapsed meiotic chromosomes

During meiotic prophase, cohesin complexes mediate cohesion between sister chromatids and promote pairing and synapsis of homologous chromosomes. Precisely how the activity of cohesin is controlled to promote these events is not fully understood. In metazoans, cohesion establishment between sister chromatids during mitotic divisions is accompanied by recruitment of the cohesion-stabilizing protein Sororin. During somatic cell division cycles, Sororin is recruited in response to DNA replication-dependent modification of the cohesin complex by ESCO acetyltransferases. How Sororin is recruited and acts in meiosis is less clear. Here, we have surveyed the chromosomal localization of Sororin and its relationship to the meiotic cohesins and other chromatin modifiers with the objective of determining how Sororin contributes to meiotic chromosome dynamics. We show that Sororin localizes to the cores of meiotic chromosomes in a manner that is dependent on synapsis and the synaptonemal complex protein SYCP1. In contrast, cohesin, with which Sororin interacts in mitotic cells, shows axial enrichment on meiotic chromosomes even in the absence of synapsis between homologs. Using high-resolution microscopy, we show that Sororin is localized to the central region of the synaptonemal complex. These results indicate that Sororin regulation during meiosis is distinct from its regulation in mitotic cells and may suggest that it interacts with a distinctly different partner to ensure proper chromosome dynamics in meiosis.     

Shugoshin-2 is essential for the completion of meiosis but not for mitotic cell division in mice

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.     

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 structural maintenance of chromosomes (SMC) family of proteins in mammals

Structural maintenance of chromosomes (SMC) family proteins play critical roles in chromosome structural changes. SMC proteins are known to be involved in two major chromosome structural organization events required for mitotic segregation of chromosomes: mitotic chromosome condensation and sister chromatid cohesion. In eukaryotes, two separate sets of SMC heterodimers form the cores of two distinct multiprotein complexes termed condensin and cohesin, each specialized for condensation or cohesion, respectively. It is clear that both condensin and cohesin are conserved in mammals, including humans. The mammalian complexes demonstrate dynamic changes in intracellular distribution in a cell cycle-dependent manner. At any point in the cell cycle, the intracellular localization of the majority of mammalian cohesin and condensin appears to be complementary. Cohesin is associated with chromatin in interphase, while condensin is largely cytoplasmic. Similarly, in mitosis, cohesin is mostly excluded from chromosomes while condensin is distinctly bound to them. Cell cycle-dependent targeting of the two complexes appears to play a major role in regulating their cell cycle-specific activities, and how this redistribution is controlled is an area of active research. Finally, there is evidence that SMC proteins may be involved in DNA recombination and repair. This review focuses on what we have learned about SMC family proteins in humans and other mammalian species in comparison to those in lower eukaryotes. The authors present their own views with regard to some of the major outstanding questions surrounding the nature and functions of the SMC family of proteins.     

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.     

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.     

The core centromere and Sgo1 establish a 50-kb cohesin-protected domain around centromeres during meiosis I

The stepwise loss of cohesins, the complexes that hold sister chromatids together, is required for faithful meiotic chromosome segregation. Cohesins are removed from chromosome arms during meiosis I but are maintained around centromeres until meiosis II. Here we show that Sgo1, a protein required for protecting centromeric cohesins from removal during meiosis I, localizes to cohesin-associated regions (CARs) at the centromere and the 50-kb region surrounding it. Establishment of this Sgo1-binding domain requires the 120-base-pair (bp) core centromere, the kinetochore component Bub1, and the meiosis-specific factor Spo13. Interestingly, cohesins and the kinetochore proteins Iml3 and Chl4 are necessary for Sgo1 to associate with pericentric regions but less so for Sgo1 to associate with the core centromeric regions. Finally, we show that the 50-kb Sgo1-binding domain is the chromosomal region where cohesins are protected from removal during meiosis I. Our results identify the portions of chromosomes where cohesins are protected from removal during meiosis I and show that kinetochore components and cohesins themselves are required to establish this cohesin protective domain.     

The axial element protein HTP-3 promotes cohesin loading and meiotic axis assembly in C. elegans to implement the meiotic program of chromosome segregation

Faithful transmission of the genome through sexual reproduction requires reduction of genome copy number during meiosis to produce haploid sperm and eggs. Meiosis entails steps absent from mitosis to achieve this goal. When meiosis begins, sisters are held together by sister chromatid cohesion (SCC), mediated by the cohesin complex. Homologs then become linked through crossover recombination. SCC subsequently holds both sisters and homologs together. Separation of homologs and then sisters requires two successive rounds of chromosome segregation and the stepwise removal of Rec8, a meiosis-specific cohesin subunit. We show that HTP-3, a known component of the C. elegans axial element (AE), molecularly links these meiotic innovations. We identified HTP-3 in a genetic screen for factors necessary to maintain SCC until meiosis II. Our data show that interdependent loading of HTP-3 and cohesin is a principal step in assembling the meiotic chromosomal axis and in establishing SCC. HTP-3 recruits all known AE components to meiotic chromosomes and promotes cohesin loading, the first known involvement of an AE protein in this process. Furthermore, REC-8 and two paralogs, called COH-3 and COH-4, together mediate meiotic SCC, but they perform specialized functions. REC-8 alone is necessary and sufficient for the persistence of SCC after meiosis I. In htp-3 and rec-8 mutants, sister chromatids segregate away from one another in meiosis I (equational division), rather than segregating randomly, as expected if SCC were completely eliminated. AE assembly fails only when REC-8, COH-3, and COH-4 are simultaneously disrupted. Premature equational sister separation in rec8 mutants of other organisms suggests the involvement of multiple REC-8 paralogs, which may have masked a conserved requirement for cohesin in AE assembly.     

Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.     

Dynamic molecular linkers of the genome: the first decade of SMC proteins

Structural maintenance of chromosomes (SMC) proteins are chromosomal ATPases, highly conserved from bacteria to humans, that play fundamental roles in many aspects of higher-order chromosome organization and dynamics. In eukaryotes, SMC1 and SMC3 act as the core of the cohesin complexes that mediate sister chromatid cohesion, whereas SMC2 and SMC4 function as the core of the condensin complexes that are essential for chromosome assembly and segregation. Another complex containing SMC5 and SMC6 is implicated in DNA repair and checkpoint responses. The SMC complexes form unique ring- or V-shaped structures with long coiled-coil arms, and function as ATP-modulated, dynamic molecular linkers of the genome. Recent studies shed new light on the mechanistic action of these SMC machines and also expanded the repertoire of their diverse cellular functions. Dissecting this class of chromosomal ATPases is likely to be central to our understanding of the structural basis of genome organization, stability, and evolution.     

A model for chromosome structure during the mitotic and meiotic cell cycles

The chromosome scaffold model in which loops of chromatin are attached to a central, coiled chromosome core (scaffold) is the current paradigm for chromosome structure. Here we present a modified version of the chromosome scaffold model to describe chromosome structure and behavior through the mitotic and meiotic cell cycles. We suggest that a salient feature of chromosome structure is established during DNA replication when sister loops of DNA extend in opposite directions from replication sites on nuclear matrix strands. This orientation is maintained into prophase when the nuclear matrix strand is converted into two closely associated sister chromatid cores with sister DNA loops extending in opposite directions. We propose that chromatid cores are contractile and show, using a physical model, that contraction of cores during late prophase can result in coiled chromatids. Coiling accounts for the majority of chromosome shortening that is needed to separate sister chromatids within the confines of a cell. In early prophase I of meiosis, the orientation of sister DNA loops in opposite directions from axial elements assures that DNA loops interact preferentially with homologous DNA loops rather than with sister DNA loops. In this context, we propose a bar code model for homologous presynaptic chromosome alignment that involves weak paranemic interactions of homologous DNA loops. Opposite orientation of sister loops also suppresses crossing over between sister chromatids in favor of crossing over between homologous non-sister chromatids. After crossing over is completed in pachytene and the synaptonemal complex breaks down in early diplotene (= diffuse stage), new contractile cores are laid down along each chromatid. These chromatid cores are comparable to the chromatid cores in mitotic prophase chromosomes. As an aside, we propose that leptotene through early diplotene represent the missing G2 period of the premeiotic interphase. The new chromosome cores, along with sister chromatid cohesion, stabilize chiasmata. Contraction of cores in late diplotene causes chromosomes to coil in a configuration that encourages subsequent syntelic orientation of sister kinetochores and amphitelic orientation of homologous kinetochore pairs on the spindle at metaphase I.     

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.     

The enhancement of pericentromeric cohesin association by conserved kinetochore components promotes high-fidelity chromosome segregation and is sensitive to microtubule-based tension

Sister chromatid cohesion, conferred by the evolutionarily conserved cohesin complex, is essential for proper chromosome segregation. Cohesin binds to discrete sites along chromosome arms, and is especially enriched surrounding centromeres, but past studies have not clearly defined the roles of arm and pericentromeric cohesion in chromosome segregation. To address this issue, we developed a technique that specifically reduced pericentromeric cohesin association on a single chromosome without affecting arm cohesin binding. Under these conditions, we observed more extensive stretching of centromeric chromatin and elevated frequencies of chromosome loss, suggesting that pericentromeric cohesin enrichment is essential for high-fidelity chromosome transmission. The magnitude of pericentromeric cohesin association was negatively correlated with tension between sister kinetochores, with the highest levels of association in cells lacking kinetochore-microtubule attachments. Pericentromeric cohesin recruitment required evolutionarily conserved components of the inner and central kinetochore. Together, these observations suggest that pericentromeric cohesin levels reflect the balance of opposing forces: the kinetochore-mediated enhancement of cohesin binding and the disruption of binding by mechanical tension at kinetochores. The involvement of conserved kinetochore components suggests that this pathway for pericentromeric cohesin enrichment may have been retained in higher eukaryotes to promote chromosome biorientation and accurate sister chromatid segregation.     

LAB-1 antagonizes the Aurora B kinase in C. elegans

The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. Here we show that Caenorhabditis elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. We propose that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area.