Fission yeast Bub1 is essential in setting up the meiotic pattern of chromosome segregation

In meiosis, sister-chromatids move to the same spindle pole during the first division (MI) and to opposite poles during the second division (MII). This requires that MI sister kinetochores are co-orientated and form an apparent single functional unit that only interacts with microtubules from one pole, and that sister-chromatids remain associated through their centromeres until anaphase II. Here we investigate the function of Bub1 and Mad2, which are components of the mitotic-spindle checkpoint, on chromosome segregation during meiosis. Both proteins are required to prevent the occurrence of non-disjunction events in MI, which is consistent with recent findings that components of the mitotic-spindle checkpoint also operate during meiosis. However, Bub1 has several functions that are not shared with Mad2. When the bub1 gene is deleted, sister chromatids often move to opposite spindle poles during MI, indicating that sister kinetochores are disunited. Furthermore, the cohesin Rec8 is never retained at centromeres at anaphase I and sister-chromatid cohesion is lost. Our results show that Bub1, besides its functions in monitoring chromosome attachment, is essential for two other significant aspects of MI - unification of sister kinetochores and retention of centromeric cohesion.     

Two fission yeast homologs of Drosophila Mei-S332 are required for chromosome segregation during meiosis I and II

BACKGROUND: Meiosis produces haploid gametes from diploid progenitor cells. This reduction is achieved by two successive nuclear divisions after one round of DNA replication. Correct chromosome segregation during the first division depends on sister kinetochores being oriented toward the same spindle pole while homologous kinetochores must face opposite poles. Segregation during the second division depends on retention of sister chromatid cohesion between centromeres until the onset of anaphase II, which in Drosophila melanogaster depends on a protein called Mei-S332 that binds to centromeres. RESULTS: We report the identification of two homologs of Mei-S332 in fission yeast using a knockout screen. Together with their fly ortholog they define a protein family conserved from fungi to mammals. The two identified genes, sgo1 and sgo2, are required for retention of sister centromere cohesion between meiotic divisions and kinetochore orientation during meiosis I, respectively. The amount of meiotic cohesin's Rec8 subunit retained at centromeres after meiosis I is reduced in Deltasgo1, but not in Deltasgo2, cells, and Sgo1 appears to regulate cleavage of Rec8 by separase. Both Sgo1 and Sgo2 proteins localize to centromere regions. The abundance of Sgo1 protein normally declines after the first meiotic division, but extending its expression by altering its 3'UTR sequences does not greatly affect meiosis II. Its mere presence within the cell might therefore be insufficient to protect centromeric cohesion. CONCLUSIONS: A conserved protein family based on Mei-S332 has been identified. The two fission yeast homologs are implicated in meiosis I kinetochore orientation and retention of centromeric sister chromatid cohesion until meiosis II.     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.Key words: meiosis, chromosome segregation, recombination, kinetochore, Sgo1, fission yeast     

Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.     

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.     

Bub1 is essential for assembly of the functional inner centromere

During mitosis, the inner centromeric region (ICR) recruits protein complexes that regulate sister chromatid cohesion, monitor tension, and modulate microtubule attachment. Biochemical pathways that govern formation of the inner centromere remain elusive. The kinetochore protein Bub1 was shown to promote assembly of the outer kinetochore components, such as BubR1 and CENP-F, on centromeres. Bub1 was also implicated in targeting of Shugoshin (Sgo) to the ICR. We show that Bub1 works as a master organizer of the ICR. Depletion of Bub1 from Xenopus laevis egg extract or from HeLa cells resulted in both destabilization and displacement of chromosomal passenger complex (CPC) from the ICR. Moreover, soluble Bub1 controls the binding of Sgo to chromatin, whereas the CPC restricts loading of Sgo specifically onto centromeres. We further provide evidence that Bub1 kinase activity is pivotal for recruitment of all of these components. Together, our findings demonstrate that Bub1 acts at multiple points to assure the correct kinetochore formation.     

PP2A is required for centromeric localization of Sgo1 and proper chromosome segregation

Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.     

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.     

Bub1 targeting to centromeres is sufficient for Sgo1 recruitment in the absence of kinetochores

Centromeric chromatin containing the histone H3 variant centromere protein A (CENP-A) directs kinetochore assembly through a hierarchical binding of CENPs, starting with CENP-C and CENP-T. Centromeres are also the chromosomal regions where cohesion, mediated by cohesin, is most prominently maintained in mitosis. While most cohesin dissociates from chromosome arms in prophase, Shugoshin 1 (Sgo1) prevents this process at centromeres. Centromeric localization of Sgo1 depends on histone H2A phosphorylation by the kinase Bub1, but whether additional interactions with kinetochore components are required for Sgo1 recruitment is unclear. Using the Xenopus egg cell-free system, we here show that both CENP-C and CENP-T can independently drive centromeric accumulation of Sgo1 through recruitment of Bub1 to the KNL1, MIS12, NDC80 (KMN) network. The spindle assembly checkpoint (SAC) kinase Mps1 is also required for this pathway even in the absence of checkpoint signaling. Sgo1 recruitment is abolished in chromosomes lacking kinetochore components other than CENP-A. However, forced targeting of Bub1 to centromeres is sufficient to restore Sgo1 localization under this condition.     

Shugoshin 1 Plays a Central Role in Kinetochore Assembly and is Required for Kinetochore Targeting of Plk1

Physical connection between the sister chromatids is mediated by the cohesin protein complex. During prophase, cohesin is removed from the chromosome arms while the centromeres remain united. Shugoshin1 (Sgo1) is required for maintenance of centromeric cohesion from prophase to the metaphase-anaphase transition. Furthermore, Sgo1 has been proposed to regulate kinetochore microtubule stability and sense interkinetochore tension, two tasks which are tightly coupled with the function of the Chromosomal Passenger Complex (CPC) and Polo-like kinase 1 (Plk1). Here we show that depletion or chemical inhibition of Aurora B kinase (AurB), the catalytic subunit of the CPC, disrupts accumulation of Sgo1 on the kinetochores in HeLa cells and causes Sgo1 to localize on the chromosome arms. RNAi assays show that depletion of Sgo1 did not affect AurB localization but diminished Plk1 kinetochore binding. Furthermore, we demonstrate that vertebrate Sgo1 is phosphorylated by both AurB and Plk1 in vitro. The data presented here includes an extensive analysis of kinetochore targeting interdependencies of mitotic proteins that propose a novel branch in kinetochore assembly where Sgo1 and Plk1 have central roles. Furthermore our studies implicate Sgo1 in the tension sensing mechanism of the spindle checkpoint by regulating Plk1 kinetochore affinity.     

Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis

BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated. RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20. CONCLUSION: Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin.     

Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I

Aurora-B kinases are important regulators of mitotic chromosome segregation, where they are required for the faithful bi-orientation of sister chromatids. In contrast to mitosis, sister chromatids have to be oriented toward the same spindle pole in meiosis-I, while homologous chromosomes are bi-oriented. We find that the fission yeast Aurora kinase Ark1 is required for the faithful bi-orientation of sister chromatids in mitosis and of homologous chromosomes in meiosis-I. Unexpectedly, Ark1 is also necessary for the faithful mono-orientation of sister chromatids in meiosis-I, even though the canonical mono-orientation pathway, which depends on Moa1 and Rec8, seems intact. Our data suggest that Ark1 prevents unified sister kinetochores during metaphase-I from merotelic attachment to both spindle poles and thus from being torn apart during anaphase-I, revealing a novel mechanism promoting monopolar attachment. Furthermore, our results provide an explanation for the previously enigmatic observation that fission yeast Shugoshin Sgo2, which assists in loading Aurora to centromeres, and its regulator Bub1 are required for the mono-orientation of sister chromatids in meiosis-I.     

Sister chromatid cohesion along arms and at centromeres

There is an obvious difference between the regulation of sister chromatid cohesion at centromeres and along chromosome arms during meiosis, because centromeric cohesion, but not arm cohesion, persists throughout anaphase of the first meiotic division. This regional difference of sister chromatid cohesion is also observed during mitosis; the cohesion is much more robust at the centromere at metaphase, where it antagonizes the pulling force of spindle microtubules that attach to the kinetochores from opposite poles. Recent studies have illuminated the underlying molecular mechanisms that strengthen and protect centromeric cohesion in mitosis and meiosis, and the central role of a conserved protein, shugoshin.     

Maintenance of cohesin at centromeres after meiosis I in budding yeast requires a kinetochore-associated protein related to MEI-S332

BACKGROUND: The halving of chromosome number that occurs during meiosis depends on three factors. First, homologs must pair and recombine. Second, sister centromeres must attach to microtubules that emanate from the same spindle pole, which ensures that homologous maternal and paternal pairs can be pulled in opposite directions (called homolog biorientation). Third, cohesion between sister centromeres must persist after the first meiotic division to enable their biorientation at the second. RESULTS: A screen performed in fission yeast to identify meiotic chromosome missegregation mutants has identified a conserved protein called Sgo1 that is required to maintain sister chromatid cohesion after the first meiotic division. We describe here an orthologous protein in the budding yeast S. cerevisiae (Sc), which has not only meiotic but also mitotic chromosome segregation functions. Deletion of Sc SGO1 not only causes frequent homolog nondisjunction at meiosis I but also random segregation of sister centromeres at meiosis II. Meiotic cohesion fails to persist at centromeres after the first meiotic division, and sister centromeres frequently separate precociously. Sgo1 is a kinetochore-associated protein whose abundance declines at anaphase I but, nevertheless, persists on chromatin until anaphase II. CONCLUSIONS: The finding that Sgo1 is localized to the centromere at the time of the first division suggests that it may play a direct role in preventing the removal of centromeric cohesin. The similarity in sequence composition, chromosomal location, and mutant phenotypes of sgo1 mutants in two distant yeasts with that of MEI-S332 in Drosophila suggests that these proteins define an orthologous family conserved in most eukaryotic lineages.     

Vertebrate shugoshin links sister centromere cohesion and kinetochore microtubule stability in mitosis

Drosophila MEI-S332 and fungal Sgo1 genes are essential for sister centromere cohesion in meiosis I. We demonstrate that the related vertebrate Sgo localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis, thus providing an explanation for the differential cohesion observed between the arms and the centromeres of mitotic sister chromatids. Sgo is degraded by the anaphase-promoting complex, allowing the separation of sister centromeres in anaphase. Intriguingly, we show that Sgo interacts strongly with microtubules in vitro and that it regulates kinetochore microtubule stability in vivo, consistent with a direct microtubule interaction. Sgo is thus critical for mitotic progression and chromosome segregation and provides an unexpected link between sister centromere cohesion and microtubule interactions at kinetochores.     

A Highlights from MBoC Selection: Ipl1/Aurora-B is necessary for kinetochore restructuring in meiosis I in Saccharomyces cerevisiae

In mitosis, the centromeres of sister chromosomes are pulled toward opposite poles of the spindle. In meiosis I, the opposite is true: the sister centromeres move together to the same pole, and the homologous chromosomes are pulled apart. This change in segregation patterns demands that between the final mitosis preceding meiosis and the first meiotic division, the kinetochores must be restructured. In budding yeast, unlike mammals, kinetochores are largely stable throughout the mitotic cycle. In contrast, previous work with budding and fission yeast showed that some outer kinetochore proteins are lost in early meiosis. We use quantitative mass spectrometry methods and imaging approaches to explore the kinetochore restructuring process that occurs in meiosis I in budding yeast. The Ndc80 outer kinetochore complex, but not other subcomplexes, is shed upon meiotic entry. This shedding is regulated by the conserved protein kinase Ipl1/Aurora-B and promotes the subsequent assembly of a kinetochore that will confer meiosis-specific segregation patterns on the chromosome.     

Shugoshin promotes sister kinetochore biorientation in Saccharomyces cerevisiae

Chromosome segregation must be executed accurately during both mitotic and meiotic cell divisions. Sgo1 plays a key role in ensuring faithful chromosome segregation in at least two ways. During meiosis this protein regulates the removal of cohesins, the proteins that hold sister chromatids together, from chromosomes. During mitosis, Sgo1 is required for sensing the absence of tension caused by sister kinetochores not being attached to microtubules emanating from opposite poles. Here we describe a differential requirement for Sgo1 in the segregation of homologous chromosomes and sister chromatids. Sgo1 plays only a minor role in segregating homologous chromosomes at meiosis I. In contrast, Sgo1 is important to bias sister kinetochores toward biorientation. We suggest that Sgo1 acts at sister kinetochores to promote their biorientation.     

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1-INCENP and HP1-Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres.     

Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B-independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B-mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.     

Bub1 kinase targets Sgo1 to ensure efficient chromosome biorientation in budding yeast mitosis

During cell division all chromosomes must be segregated accurately to each daughter cell. Errors in this process give rise to aneuploidy, which leads to birth defects and is implicated in cancer progression. The spindle checkpoint is a surveillance mechanism that ensures high fidelity of chromosome segregation by inhibiting anaphase until all kinetochores have established bipolar attachments to spindle microtubules. Bub1 kinase is a core component of the spindle checkpoint, and cells lacking Bub1 fail to arrest in response to microtubule drugs and precociously segregate their DNA. The mitotic role(s) of Bub1 kinase activity remain elusive, and it is controversial whether this C-terminal domain of Bub1p is required for spindle checkpoint arrest. Here we make a detailed analysis of budding yeast cells lacking the kinase domain (bub1ΔK). We show that despite being able to arrest in response to microtubule depolymerisation and kinetochore-microtubule attachment defects, bub1ΔK cells are sensitive to microtubule drugs. This is because bub1ΔK cells display significant chromosome mis-segregation upon release from nocodazole arrest. bub1ΔK cells mislocalise Sgo1p, and we demonstrate that both the Bub1 kinase domain and Sgo1p are required for accurate chromosome biorientation after nocodazole treatment. We propose that Bub1 kinase and Sgo1p act together to ensure efficient biorientation of sister chromatids during mitosis.