Cohesin Rec8 Research Articles

Functional roles of the interaction of Moa1 with CENP-C and Rec8 in meiosis of Schizosaccharomyces pombe.

The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.

Canonical and noncanonical roles of Hop1 are crucial for meiotic prophase in the fungus Sordaria macrospora.

We show here that in the fungus Sordaria macrospora, the meiosis-specific HORMA-domain protein Hop1 is not essential for the basic early events of chromosome axis development, recombination initiation, or recombination-mediated homolog coalignment/pairing. In striking contrast, Hop1 plays a critical role at the leptotene/zygotene transition which is defined by transition from pairing to synaptonemal complex (SC) formation. During this transition, Hop1 is required for maintenance of normal axis structure, formation of SC from telomere to telomere, and development of recombination foci. These hop1Δ mutant defects are DSB dependent and require Sme4/Zip1-mediated progression of the interhomolog interaction program, potentially via a pre-SC role. The same phenotype occurs not only in hop1Δ but also in absence of the cohesin Rec8 and in spo76-1, a non-null mutant of cohesin-associated Spo76/Pds5. Thus, Hop1 and cohesins collaborate at this crucial step of meiotic prophase. In addition, analysis of 4 non-null mutants that lack this transition defect reveals that Hop1 also plays important roles in modulation of axis length, homolog-axis juxtaposition, interlock resolution, and spreading of the crossover interference signal. Finally, unexpected variations in crossover density point to the existence of effects that both enhance and limit crossover formation. Links to previously described roles of the protein in other organisms are discussed.

Phosphorylation of Rec8 cohesin complexes regulates mono-orientation of kinetochores in meiosis I.

In meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase I. The conserved meiosis-specific kinetochore protein meikin (Moa1 in fission yeast) associates with polo-like kinase: Plo1 and regulates both mono-orientation and cohesion protection. Although the phosphorylation of Rec8-S450 by Plo1 associated with Moa1 plays a key role in cohesion protection, how Moa1-Plo1 regulates mono-orientation remains elusive. Here, we identify Plo1 phosphorylation sites in the cohesin subunits, Rec8 and Psm3. The non-phosphorylatable mutations at these sites showed specific defects in mono-orientation. These results enabled the genetic dissection of meikin functions at the centromeres.

Separase and Roads to Disengage Sister Chromatids during Anaphase.

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Analysis of absolute amounts of two meiotic cohesin subunits, RAD21L and REC8, in mouse spermatocytes.

RAD2lL and REC8, meiosis-specific paralogs of the canonical cohesin subunit RAD21, are essential for proper formation of axial/lateral elements of the synaptonemal complex, synapsis of homologous chromosomes, and crossover recombination in mammalian meiosis. However, how many meiotic cohesins are present in germ cells has not been investigated because of the lack of an appropriate method of analysis. In the present study, to examine the intracellular amount of meiotic cohesins, we generated two strains of knock-in (KI) mice that expressed a 3×FLAG-tag at the C-terminus of RAD21L or REC8 protein using the CRISPR/Cas9 genome editing system. Both KI mice were fertile. Western blot analyses and immunocytochemical studies revealed that expression levels and localization patterns of both RAD21L-3×FLAG and REC8-3×FLAG in KI mice were similar to those in wild-type mice. After confirming that tagging of endogenous RAD21L and REC8 with 3×FLAG did not affect their expression profiles, we evaluated the levels of RAD21L-3×FLAG and REC8-3×FLAG in the testes of 2-week-old mice in which only RAD21L and REC8 but little RAD21 are expressed in the meiocytes. By comparing the band intensities of testicular RAD21L-3×FLAG and REC8-3×FLAG with 3×FLAG-tagged recombinant proteins of known concentrations in western blot analysis, we found that there were approximately 413,000 RAD21L and 453,000 REC8 molecules per spermatocyte in the early stages of prophase I. These findings provide new insights into the role played by cohesins in the process of meiotic chromosome organization in mammalian germ cells.

Mapping separase-mediated cleavage in situ.

Separase is a protease that performs critical functions in the maintenance of genetic homeostasis. Among them, the cleavage of the meiotic cohesin during meiosis is a key step in producing gametes in eukaryotes. However, the exact chromosomal localization of this proteolytic cleavage was not addressed due to the lack of experimental tools. To this end, we developed a method based on monoclonal antibodies capable of recognizing the predicted neo-epitopes produced by separase-mediated proteolysis in the RAD21 and REC8 cohesin subunits. To validate the epigenomic strategy of mapping cohesin proteolysis, anti-RAD21 neo-epitopes antibodies were used in ChIP-On-ChEPseq analysis of human cells undergoing mitotic anaphase. Second, a similar analysis applied for mapping of REC8 cleavage in germline cells in Macaque showed a correlation with a subset of alpha-satellites and other repeats, directly demonstrating that the site-specific mei-cohesin proteolysis hotspots are coincident but not identical with centromeres. The sequences for the corresponding immunoglobulin genes show a convergence of antibodies with close specificity. This approach could be potentially used to investigate cohesin ring opening events in other chromosomal locations, if applied to single cells.

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination.

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.

Rec8 Cohesin: A Structural Platform for Shaping the Meiotic Chromosomes.

Meiosis is critically different from mitosis in that during meiosis, pairing and segregation of homologous chromosomes occur. During meiosis, the morphology of sister chromatids changes drastically, forming a prominent axial structure in the synaptonemal complex. The meiosis-specific cohesin complex plays a central role in the regulation of the processes required for recombination. In particular, the Rec8 subunit of the meiotic cohesin complex, which is conserved in a wide range of eukaryotes, has been analyzed for its function in modulating chromosomal architecture during the pairing and recombination of homologous chromosomes in meiosis. Here, we review the current understanding of Rec8 cohesin as a structural platform for meiotic chromosomes.

Loss of sister kinetochore co-orientation and peri-centromeric cohesin protection after meiosis I depends on cleavage of centromeric REC8.

SummaryProtection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required

Erratum: Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I.

Erratum: Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I.

Meikin synergizes with shugoshin to protect cohesin Rec8 during meiosis I.

The conserved meiosis-specific kinetochore regulator, meikin (Moa1 in fission yeast) plays a central role in establishing meiosis-specific kinetochore function. However, the underlying molecular mechanisms remain elusive. Here, we show how Moa1 regulates centromeric cohesion protection, a function that has been previously attributed to shugoshin (Sgo1). Moa1 is known to associate with Plo1 kinase. We explore Plo1-dependent Rec8 phosphorylation and identify a key phosphorylation site required for cohesion protection. The phosphorylation of Rec8 by Moa1-Plo1 potentiates the activity of PP2A associated with Sgo1. This leads to dephosphorylation of Rec8 at another site, which thereby prevents cleavage of Rec8 by separase.

Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8.

Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the double strand break-forming topoisomerase Spo11 and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells lacking the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly1; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodeling factors mediate centromere dismantlement by Spo11 and Rec8. Remarkably, ectopic expression of either protein in proliferating cells leads to mitotic kinetochore loss in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability, and may jeopardize kinetochores in the increasing number of cancers known to express meiotic proteins.

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin‐PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C‐dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.

Quantitative cytogenetics reveals molecular stoichiometry and longitudinal organization of meiotic chromosome axes and loops.

During meiosis, chromosomes adopt a specialized organization involving assembly of a cohesin-based axis along their lengths, with DNA loops emanating from this axis. We applied novel, quantitative, and widely applicable cytogenetic strategies to elucidate the molecular bases of this organization using Caenorhabditis elegans. Analyses of wild-type (WT) chromosomes and de novo circular minichromosomes revealed that meiosis-specific HORMA-domain proteins assemble into cohorts in defined numbers and co-organize the axis together with 2 functionally distinct cohesin complexes (REC-8 and COH-3/4) in defined stoichiometry. We further found that REC-8 cohesins, which load during S phase and mediate sister-chromatid cohesion, usually occur as individual complexes, supporting a model wherein sister cohesion is mediated locally by a single cohesin ring. REC-8 complexes are interspersed in an alternating pattern with cohorts of axis-organizing COH-3/4 complexes (averaging 3 per cohort), which are insufficient to confer cohesion but can bind to individual chromatids, suggesting a mechanism to enable formation of asymmetric sister-chromatid loops. Indeed, immunofluorescence/fluorescence in situ hybridization (immuno-FISH) assays demonstrate frequent asymmetry in genomic content between the loops formed on sister chromatids. We discuss how features of chromosome axis/loop architecture inferred from our data can help to explain enigmatic, yet essential, aspects of the meiotic program.

Translesion synthesis polymerases contribute to meiotic chromosome segregation and cohesin dynamics in Schizosaccharomycespombe.

Translesion synthesis polymerases (TLSPs) are non-essential error-prone enzymes that ensure cell survival by facilitating DNA replication in the presence of DNA damage. In addition to their role in bypassing lesions, TLSPs have been implicated in meiotic double-strand break repair in several systems. Here, we examine the joint contribution of four TLSPs to meiotic progression in the fission yeast Schizosaccharomyces pombe. We observed a dramatic loss of spore viability in fission yeast lacking all four TLSPs, which is accompanied by disruptions in chromosome segregation during meiosis I and II. Rec8 cohesin dynamics are altered in the absence of the TLSPs. These data suggest that the TLSPs contribute to multiple aspects of meiotic chromosome dynamics.

Synaptonemal Complex Central Region Proteins Promote Localization of Pro-crossover Factors to Recombination Events During Caenorhabditis elegans Meiosis.

Crossovers (COs) between homologous chromosomes are critical for meiotic chromosome segregation and form in the context of the synaptonemal complex (SC), a meiosis-specific structure that assembles between aligned homologs. During Caenorhabditis elegans meiosis, central region components of the SC (SYP proteins) are essential to repair double-strand DNA breaks (DSBs) as COs. Here, we investigate the relationships between the SYP proteins and conserved pro-CO factors by examining the immunolocalization of these proteins in meiotic mutants where SYP proteins are absent, reduced, or mislocalized. Although COs do not form in syp null mutants, pro-CO factors COSA-1, MSH-5, and ZHP-3 nevertheless colocalize at DSB-dependent sites during late prophase, reflecting an inherent affinity of these factors for DSB repair sites. In contrast, in mutants where SYP proteins are present but form aggregates or display abnormal synapsis, pro-CO factors consistently track with SYP-1 localization. Further, pro-CO factors usually localize to a single site per SYP-1 structure, even in SYP aggregates or in mutants where the SC forms between sister chromatids, suggesting that CO regulation occurs within these aberrant SC structures. Moreover, we find that the meiotic cohesin REC-8 is required to ensure that SC formation occurs between homologs and not sister chromatids. Taken together, our findings support a model in which SYP proteins promote CO formation by promoting the localization of pro-CO factors to recombination events within an SC compartment, thereby ensuring that pro-CO factors identify a recombination event within an SC structure and that CO maturation occurs only between properly aligned homologous chromosomes.

Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat

BackgroundMeiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition.ResultsWe cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses.ConclusionsThe Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.

EWSR1 Regulates PRDM9-Dependent Histone 3 Methylation and Links Recombination Hotspots With the Chromosomal Axis

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 lysine-4 and lysine-36 methyltransferase PRDM9 to ensure successful double-strand break initiation and repair. Here we show that EWSR1, a protein with a previously uncharacterized role in meiosis, binds to PRDM9 and increases the efficiency of trimethylation at hotspots in vivo. Additionally, EWSR1 links PRDM9-activated hotspots and the nascent chromosomal axis through the phosphorylated meiotic-specific cohesin REC8. Together, these two functions of EWSR1 direct the choice of which hotspots will be used for double-strand break formation and repair, assuring that a sufficient number of crossovers are properly positioned along the centromere-telomere axis. These results show that successful recombination and meiosis require proper association of activated hotspots with the chromosomal axis mediated by interactions between EWSR1, PRDM9, and pREC8.

Meikin-associated polo-like kinase specifies Bub1 distribution in meiosis I.

In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono-orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP-C during meiosis I, recruits Plo1 (polo-like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis-specific chromosome segregation. We provide evidence that the meiosis-specific Bub1 regulation is conserved in mouse.