Absence of mouse REC8 cohesin promotes synapsis of sister chromatids in meiosis

REC8 is a key component of the meiotic cohesin complex. During meiosis, cohesin is required for the establishment and maintenance of sister-chromatid cohesion, for the formation of the synaptonemal complex, and for recombination between homologous chromosomes. We show that REC8 has an essential role in mammalian meiosis, in that Rec8 null mice of both sexes have germ cell failure and are sterile. In the absence of REC8, early chromosome pairing events appear normal, but synapsis occurs in a novel fashion: between sister chromatids. This implies that a major role for REC8 in mammalian meiosis is to limit synapsis to between homologous chromosomes. In all other eukaryotic species studied to date, REC8 phenotypes have been restricted to meiosis. Unexpectedly, Rec8 null mice are born in sub-Mendelian frequencies and fail to thrive. These findings illuminate hitherto unknown REC8 functions in chromosome dynamics during mammalian meiosis and possibly in somatic development.     

Separase is required for chromosome segregation during meiosis I in Caenorhabditis elegans.

BACKGROUND: Chromosome segregation during mitosis and meiosis is triggered by dissolution of sister chromatid cohesion, which is mediated by the cohesin complex. Mitotic sister chromatid disjunction requires that cohesion be lost along the entire length of chromosomes, whereas homolog segregation at meiosis I only requires loss of cohesion along chromosome arms. During animal cell mitosis, cohesin is lost in two steps. A nonproteolytic mechanism removes cohesin along chromosome arms during prophase, while the proteolytic cleavage of cohesin's Scc1 subunit by separase removes centromeric cohesin at anaphase. In Saccharomyces cerevisiae and Caenorhabditis elegans, meiotic sister chromatid cohesion is mediated by Rec8, a meiosis-specific variant of cohesin's Scc1 subunit. Homolog segregation in S. cerevisiae is triggered by separase-mediated cleavage of Rec8 along chromosome arms. In principle, chiasmata could be resolved proteolytically by separase or nonproteolytically using a mechanism similar to the mitotic "prophase pathway." RESULTS: Inactivation of separase in C. elegans has little or no effect on homolog alignment on the meiosis I spindle but prevents their timely disjunction. It also interferes with chromatid separation during subsequent embryonic mitotic divisions but does not directly affect cytokinesis. Surprisingly, separase inactivation also causes osmosensitive embryos, possibly due to a defect in the extraembryonic structures, referred to as the "eggshell." CONCLUSIONS: Separase is essential for homologous chromosome disjunction during meiosis I. Proteolytic cleavage, presumably of Rec8, might be a common trigger for the first meiotic division in eukaryotic cells. Cleavage of proteins other than REC-8 might be necessary to render the eggshell impermeable to solutes.     

The cohesin REC8 prevents illegitimate inter‐sister synaptonemal complex assembly

During meiosis, a specialized chromosome structure is assembled to promote pairing/synapsis of homologous chromosomes and meiotic recombination, a process yielding chiasmata between homologs to ensure accurate segregation. Meiosis‐specific cohesin complexes mediating sister chromatid cohesion play pivotal roles in almost all these events, including synaptonemal complex (SC) formation. In this issue of EMBO Reports, Agostinho and colleagues have examined chromosome axes and SC structures by taking advantage of a hypomorphic Stag3 mutant in which the levels of the cohesin subunit REC8 are partly reduced 6 . Using super‐resolution microscopy, the authors illuminate previously unforeseen chromosome axis structures, showing locally separated axes in regions where REC8 is absent, regardless of RAD21L or RAD21 cohesin localization. Furthermore, they assessed the relationship between sister chromatid cohesion and inter‐sister SC formation, demonstrating that “axial opening” in the REC8‐free region is accompanied by illegitimate SC formation between sister chromatids. This study highlights the physiological importance of REC8 in sister chromatid cohesion and proper SC formation during meiosis, suggesting a new model in which a high density of REC8 deposition along the chromosome prevents illegitimate inter‐sister SC formation.     

The DIF1 gene of Arabidopsis is required for meiotic chromosome segregation and belongs to the REC8/RAD21 cohesin gene family

Cohesins are a group of conserved proteins responsible for cohesion between replicated sister chromatids during mitosis and meiosis and which are implicated in double-strand break repair and meiotic recombination. We describe here the identification and characterisation of an Arabidopsis gene - DETERMINATE, INFERTILE1 (DIF1), which is a homolog of the Schizosaccharomyces pombe REC8/RAD21 cohesin genes, and is essential for meiotic chromosome segregation. Five independent alleles of the DIF1 gene were isolated by transposon mutagenesis, and the mutants show complete male and female sterility. Pollen mother cells (PMCs) of dif1 mutants show multiple meiotic defects which are represented by univalent chromosomes and chromosome fragmentation at metaphase I, and acentric fragments and chromatin bridges in meiosis I and II. Consequently, chromosome segregation is strongly affected, resulting in meiotic products of uneven size, shape and of variable ploidy. The similarities in phenotype, and the sequence homology between DIF1 and the REC8/RAD21 cohesins suggests that cohesin function is largely conserved between eukaryotes and highlights the essential role cohesins play in plant meiosis.     

The Cohesin Subunit Rad21 Is Required for Synaptonemal Complex Maintenance,but Not Sister Chromatid Cohesion,during Drosophila Female Meiosis

Replicated sister chromatids are held in close association from the time of their synthesis until their separation during the next mitosis. This association is mediated by the ring-shaped cohesin complex that appears to embrace the sister chromatids. Upon proteolytic cleavage of the α-kleisin cohesin subunit at the metaphase-to-anaphase transition by separase, sister chromatids are separated and segregated onto the daughter nuclei. The more complex segregation of chromosomes during meiosis is thought to depend on the replacement of the mitotic α-kleisin cohesin subunit Rad21/Scc1/Mcd1 by the meiotic paralog Rec8. In Drosophila, however, no clear Rec8 homolog has been identified so far. Therefore, we have analyzed the role of the mitotic Drosophila α-kleisin Rad21 during female meiosis. Inactivation of an engineered Rad21 variant by premature, ectopic cleavage during oogenesis results not only in loss of cohesin from meiotic chromatin, but also in precocious disassembly of the synaptonemal complex (SC). We demonstrate that the lateral SC component C(2)M can interact directly with Rad21, potentially explaining why Rad21 is required for SC maintenance. Intriguingly, the experimentally induced premature Rad21 elimination, as well as the expression of a Rad21 variant with destroyed separase consensus cleavage sites, do not interfere with chromosome segregation during meiosis, while successful mitotic divisions are completely prevented. Thus, chromatid cohesion during female meiosis does not depend on Rad21-containing cohesin.     

Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization

Shugoshin (Sgo) proteins constitute a conserved protein family defined as centromeric protectors of Rec8-containing cohesin complexes in meiosis . In vertebrate mitosis, Scc1/Rad21-containing cohesin complexes are also protected at centromeres because arm cohesin, but not centromeric cohesin, is largely dissociated in pro- and prometaphase . The dissociation process is dependent on the activity of polo-like kinase (Plk1) and partly dependent on Aurora B . Recently, it has been demonstrated that vertebrate shugoshin is required for preserving centromeric cohesion during mitosis ; however, it was not addressed whether human shugoshin protects cohesin itself. Here, we show that the persistence of human Scc1 at centromeres in mitosis is indeed dependent on human Sgo1. In fission yeast, Sgo localization depends on Bub1, a conserved spindle checkpoint protein, which is enigmatically also required for chromosome congression during prometaphase in vertebrate cells. We demonstrate that human Sgo1 fails to localize at centromeres in Bub1-repressed cells, and centromeric cohesion is significantly loosened. Remarkably, in these cells, Sgo1 relocates to chromosomes all along their length and provokes ectopic protection from dissociation of Scc1 on chromosome arms. These results reveal a hitherto concealed role for human Bub1 in defining the persistent cohesion site of mitotic chromosomes.     

Identification and molecular characterization of the mammalian α-kleisin RAD21L

Meiosis is a fundamental process that generates new combinations between maternal and paternal genomes and haploid gametes from diploid progenitors. Many of the meiosis-specific events stem from the behavior of the cohesin complex (CC), a proteinaceous ring structure that entraps sister chromatids until the onset of anaphase. CCs ensure chromosome segregation, participate in DNA repair, regulate gene expression, and also contribute to synaptonemal complex (SC) formation at meiosis by keeping long-range distant DNA interactions through its conserved structure. Studies from yeast to humans have led to the assumption that Scc1/RAD21 is the α-kleisin that closes the tripartite CC that entraps two DNA molecules in mitosis, while its paralog REC8 is essential for meiosis. Here we describe the identification of RAD21L, a novel mammalian CC subunit with homology to the RAD21/REC8 α-kleisin subfamily, which is expressed in mouse testis. RAD21L interacts with other cohesin subunits such as SMC1α, SMC1b, SMC3 and with the meiosis-specific STAG3 protein. Thus, our results demonstrate the existence of a new meiotic-specific CC constituted by this α-kleisin and expand the view of REC8 as the only specific meiotic α-kleisin.     

Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.     

Meiotic telomere clustering requires actin for its formation and cohesin for its resolution

In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster-SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8delta meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization-dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.     

Cohesin loading factor Nipbl localizes to chromosome axes during mammalian meiotic prophase

Background

Sister chromatid cohesion mediated by the cohesin complex is essential for accurate chromosome segregation during mitosis and meiosis. Loading of cohesin onto chromosomes is dependent on another protein complex called kollerin, containing Nipbl/Scc2 and Mau2/Scc4. Nipbl is an evolutionarily conserved large protein whose haploinsufficiency in humans causes a developmental disorder called Cornelia de Lange syndrome. Although the function of Nipbl homologues for chromosome cohesion in meiotic cells of non-vertebrate models has been elucidated, Nipbl has not been characterized so far in mammalian spermatocytes or oocytes.

Findings

Here we describe our analyses on the expression and localization of Nipbl in nuclei of mouse spermatocytes and oocytes at different stages of meiotic prophase. In both spermatocytes and oocytes we found that Nipbl is associated with the axial/lateral element of the synaptonemal complex (AE/LE) to which cohesin also localizes. Interestingly, Nipbl in spermatocytes, but not in oocytes, dissociates from the AE/LE at mid-pachytene stage coincident with completion of DNA double-strand break repair.

Conclusions

Our data propose that cohesin loading activity is maintained during early stages of meiotic prophase in mammalian spermatocytes and oocytes.

     

Novel genes required for meiotic chromosome segregation are identified by a high-throughput knockout screen in fission yeast

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.     

Rec8 cleavage by separase is required for meiotic nuclear divisions in fission yeast

Sister chromatid cohesion in meiosis is established by cohesin complexes, including the Rec8 subunit. During meiosis I, sister chromatid cohesion is destroyed along the chromosome arms to release connections of recombined homologous chromosomes (homologues), whereas centromeric cohesion persists until it is finally destroyed at anaphase II. In fission yeast, as in mammals, distinct cohesin complexes are used depending on the chromosomal region; Rec8 forms a complex with Rec11 (equivalent to SA3) mainly along chromosome arms, while Psc3 (equivalent to SA1 and SA2) forms a complex mainly in the vicinity of the centromeres. Here we show that separase activation and resultant Rec8 cleavage are required for meiotic chromosome segregation in fission yeast. A non-cleavable form of Rec8 blocks disjunction of homologues at meiosis I. However, displacing non-cleavable Rec8 restrictively from the chromosome arm by genetically depleting Rec11 alleviated the blockage of homologue segregation, but not of sister segregation. We propose that the segregation of homologues at meiosis I and of sisters at meiosis II requires the cleavage of Rec8 along chromosome arms and at the centromeres, respectively.     

Shugoshin protects cohesin complexes at centromeres

The different regulation of sister chromatid cohesion at centromeres and along chromosome arms is obvious during meiosis, because centromeric cohesion, but not arm cohesion, persists throughout anaphase of the first division. A protein required to protect centromeric cohesin Rec8 from separase cleavage has been identified and named shugoshin (or Sgo1) after shugoshin ("guardian spirit" in Japanese). It has become apparent that shugoshin shows marginal homology with Drosophila Mei-S332 and several uncharacterized proteins in other eukaryotic organisms. Because Mei-S332 is a protein previously shown to be required for centromeric cohesion in meiosis, it is now established that shugoshin represents a conserved protein family defined as a centromeric protector of Rec8 cohesin complexes in meiosis. The regional difference of sister chromatid cohesion is also observed during mitosis in vertebrates; the cohesion is much more robust at the centromere at metaphase, where it antagonizes the pulling force of spindle microtubules that attach the kinetochores from opposite poles. The human shugoshin homologue (hSgo1) is required to protect the centromeric localization of the mitotic cohesin, Scc1, until metaphase. Bub1 plays a crucial role in the localization of shugoshin to centromeres in both fission yeast and humans.     

Together until separin do us part

Loss of sister-chromatid cohesion triggers chromosome segregation. Several recent reports show that the protease Esp1 cleaves the cohesin subunit Scc1/Mcd1 to induce sister-chromatid segregation in yeast and vertebrates. This finding indicates that cohesin cleavage may control sister-chromatid separation in all vertebrates.     

Expression of Epitope-Tagged SYN3 Cohesin Proteins Can Disrupt Meiosis in Arabidopsis

a-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes encoding four a-kleisins. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCCI orthologs and function in mitosis. SYN3 is enriched in the nucleolus of meiotic and mitotic cells and is essential for megagametogenesis. It was recently shown that expression of SYN3-RNAi constructs in buds cause changes in meiotic gene expression that result in meiotic alterations. In this report we show that expression of SYN3 from the 35S promoter with either a c-terminal Myc or FAST tag causes a reduction in SYN1 mRNA levels that results in al- terations in sister chromatid cohesion, homologous chromosome synapsis female meiosis. and synaptonemal complex formation during both male and     

The Multiple Roles of Cohesin in Meiotic Chromosome Morphogenesis and Pairing

Sister chromatid cohesion, mediated by cohesin complexes, is laid down during DNA replication and is essential for the accurate segregation of chromosomes. Previous studies indicated that, in addition to their cohesion function, cohesins are essential for completion of recombination, pairing, meiotic chromosome axis formation, and assembly of the synaptonemal complex (SC). Using mutants in the cohesin subunit Rec8, in which phosphorylated residues were mutated to alanines, we show that cohesin phosphorylation is not only important for cohesin removal, but that cohesin's meiotic prophase functions are distinct from each other. We find pairing and SC formation to be dependent on Rec8, but independent of the presence of a sister chromatid and hence sister chromatid cohesion. We identified mutations in REC8 that differentially affect Rec8's cohesion, pairing, recombination, chromosome axis and SC assembly function. These findings define Rec8 as a key determinant of meiotic chromosome morphogenesis and a central player in multiple meiotic events.