Control of Shugoshin function during fission-yeast meiosis

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.     

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.     

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.     

Regulated Separation of Sister Centromeres depends on the Spindle Assembly Checkpoint but not on the Anaphase Promoting Complex/Cyclosome

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.

Supplemental Figures     

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.     

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.     

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.     

Roles and regulation of the Drosophila centromere cohesion protein MEI-S332 family

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of 相似文献   

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.     

Shugoshin prevents dissociation of cohesin from centromeres during mitosis in vertebrate cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's alpha kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its alpha kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its alpha kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.     

Uncoordinated loss of chromatid cohesion is a common outcome of extended metaphase arrest

Chromosome segregation requires coordinated separation of sister chromatids following biorientation of all chromosomes on the mitotic spindle. Chromatid separation at the metaphase-to-anaphase transition is accomplished by cleavage of the cohesin complex that holds chromatids together. Here we show using live-cell imaging that extending the metaphase bioriented state using five independent perturbations (expression of non-degradable Cyclin B, expression of a Spindly point mutant that prevents spindle checkpoint silencing, depletion of the anaphase inducer Cdc20, treatment with a proteasome inhibitor, or treatment with an inhibitor of the mitotic kinesin CENP-E) leads to eventual scattering of chromosomes on the spindle. This scattering phenotype is characterized by uncoordinated loss of cohesion between some, but not all sister chromatids and subsequent spindle defects that include centriole separation. Cells with scattered chromosomes persist long-term in a mitotic state and eventually die or exit. Partial cohesion loss-associated scattering is observed in both transformed cells and in karyotypically normal human cells, albeit at lower penetrance. Suppressing microtubule dynamics reduces scattering, suggesting that cohesion at centromeres is unable to resist dynamic microtubule-dependent pulling forces on the kinetochores. Consistent with this view, strengthening cohesion by inhibiting the two pathways responsible for its removal significantly inhibits scattering. These results establish that chromosome scattering due to uncoordinated partial loss of chromatid cohesion is a common outcome following extended arrest with bioriented chromosomes in human cells. These findings have important implications for analysis of mitotic phenotypes in human cells and for development of anti-mitotic chemotherapeutic approaches in the treatment of cancer.     

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 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.     

Shugoshin: a centromeric guardian senses tension

To ensure accurate chromosome segregation during mitosis, the spindle checkpoint monitors chromosome alignment on the mitotic spindle. Indjeian and colleagues have investigated the precise role of the shugoshin 1 protein (Sgo1p) in this process in budding yeast. The Sgo proteins were originally identified as highly conserved proteins that protect cohesion at centromeres during the first meiotic division. Together with other recent findings, the study highlighted here has identified Sgo1 as a component that informs the mitotic spindle checkpoint when spindle tension is perturbed. This discovery has provided a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore-microtubule interface.     

The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis.     

HCP-4/CENP-C promotes the prophase timing of centromere resolution by enabling the centromere association of HCP-6 in Caenorhabditis elegans

Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.     

Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells

Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.     

MCAK facilitates chromosome movement by promoting kinetochore microtubule turnover

Mitotic centromere-associated kinesin (MCAK)/Kif2C is the most potent microtubule (MT)-destabilizing enzyme identified thus far. However, MCAK's function at the centromere has remained mechanistically elusive because of interference from cytoplasmic MCAK's global regulation of MT dynamics. In this study, we present MCAK chimeras and mutants designed to target centromere-associated MCAK for mechanistic analysis. Live imaging reveals that depletion of centromere-associated MCAK considerably decreases the directional coordination between sister kinetochores. Sister centromere directional antagonism results in decreased movement speed and increased tension. Sister centromeres appear unable to detach from kinetochore MTs efficiently in response to directional switching cues during oscillatory movement. These effects are reversed by anchoring ectopic MCAK to the centromere. We propose that MCAK increases the turnover of kinetochore MTs at all centromeres to coordinate directional switching between sister centromeres and facilitate smooth translocation. This may contribute to error correction during chromosome segregation either directly via slow MT turnover or indirectly by mechanical release of MTs during facilitated movement.     

Sumoylated BubR1 plays an important role in chromosome segregation and mitotic timing

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.Key words: BubR1, sumoylation, kinetochores, centromeric cohesion, spindle checkpoint, Sgo1     

Shugoshin 2 regulates localization of the chromosomal passenger proteins in fission yeast mitosis

Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Although Sgo1 has clearly been established as a protector of centromere cohesion in meiosis I, the roles of Sgo2 remain elusive. Here we show that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged spindle checkpoint arrest. Consistent with this, Sgo2 is essential for maintaining the Passenger proteins on centromeres upon checkpoint activation. Interestingly, lack of Sgo2 has a more penetrant effect on the localization of Survivin than on the two other Passenger proteins INCENP and Aurora B, and the Survivin-INCENP complex but not the INCENP-Aurora B complex is destabilized in the absence of Sgo2. Finally we show that the conserved C-terminus of Sgo2 is crucial to maintain Sgo2 and Passenger proteins localization on centromeres upon prolonged checkpoint activation. Taken together, our results demonstrate that Sgo2 is important for chromosome biorientation and that it controls docking of the Passenger proteins on chromosomes in early mitotic cells.