Tomoya S. Kitajima

The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis.

Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.

Shugoshin collaborates with protein phosphatase 2A to protect cohesin

Sister chromatid cohesion, mediated by a complex called cohesin, is crucial—particularly at centromeres—for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.

Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast

Fission yeast centromeres, like those of higher eukaryotes, are composed of repeated DNA structures and associated heterochromatin protein complexes, that have a critical function in the faithful segregation of chromosomes during cell division. Cohesin protein complexes, which are essential for sister-chromatid cohesion and proper chromosome segregation, are enriched at centromeric repeats. We have identified a functional and physical link between heterochromatin and cohesin. We find that the preferential localization of cohesins at the centromeric repeats is dependent on Swi6, a conserved heterochromatin protein that is required for proper kinetochore function. Cohesin is also enriched at the mating-type heterochromatic region in a manner that depends on Swi6 and is required to preserve the genomic integrity of this locus. We provide evidence that a cohesin subunit Psc3 interacts with Swi6 and its mouse homologue HP1. These data define a conserved function of Swi6/HP1 in recruitment of cohesin to heterochromatic regions, promoting the proper segregation of chromosomes.

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.

Unified mode of centromeric protection by shugoshin in mammalian oocytes and somatic cells

Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes, proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the metaphase–anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.

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.

Phosphorylation of mammalian Sgo2 by Aurora B recruits PP2A and MCAK to centromeres

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. We demonstrate that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, we identified the phosphorylation of hSgo2 by Aurora B at the N-terminal coiled-coil region and the middle region, and showed that these phosphorylations separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, our study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells.

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.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly 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. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

Bivalent separation into univalents precedes age-related meiosis I errors in oocytes

The frequency of chromosome segregation errors during meiosis I (MI) in oocytes increases with age. The two-hit model suggests that errors are caused by the combination of a first hit that creates susceptible crossover configurations and a second hit comprising an age-related reduction in chromosome cohesion. This model predicts an age-related increase in univalents, but direct evidence of this phenomenon as a major cause of segregation errors has been lacking. Here, we provide the first live analysis of single chromosomes undergoing segregation errors during MI in the oocytes of naturally aged mice. Chromosome tracking reveals that 80% of the errors are preceded by bivalent separation into univalents. The set of the univalents is biased towards balanced and unbalanced predivision of sister chromatids during MI. Moreover, we find univalents predisposed to predivision in human oocytes. This study defines premature bivalent separation into univalents as the primary defect responsible for age-related aneuploidy.