PIASgamma is required for faithful chromosome segregation in human cells.
ABSTRACT: BACKGROUND:The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. PRINCIPAL FINDINGS:We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II. CONCLUSIONS:PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.
Project description:Cohesin maintains sister chromatid cohesion until its Rad21/Scc1/Mcd1 is cleaved by separase during anaphase. DNA topoisomerase II (topo II) maintains the proper topology of chromatid DNAs and is essential for chromosome segregation. Here we report direct observations of mitotic progression in individual HeLa cells after functional disruptions of hRad21, NIPBL, a loading factor for hRad21, and topo II alpha,beta by RNAi and a topo II inhibitor, ICRF-193. Mitosis is delayed in a Mad2-dependent manner after disruption of either or both cohesin and topo II. In hRad21 depletion, interphase pericentric architecture becomes aberrant, and anaphase is virtually permanently delayed as preseparated chromosomes are misaligned on the metaphase spindle. Topo II disruption perturbs centromere organization leading to intense Bub1, but no Mad2, on kinetochores and sustains a Mad2-dependent delay in anaphase onset with persisting securin. Thus topo II impinges upon centromere/kinetochore function. Disruption of topo II by RNAi or ICRF-193 overrides the mitotic delay induced by cohesin depletion: sister centromeres are aligned and anaphase spindle movements occur. The ensuing accumulation of catenations in preseparated sister chromatids may overcome the reduced tension arising from cohesin depletion, causing the override. Cohesin and topo II have distinct, yet coordinated functions in metaphase alignment.
Project description: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.
Project description:The mitotic checkpoint, also known as the spindle assembly checkpoint, delays anaphase onset until all chromosomes have reached bipolar tension on the mitotic spindle [1-3]. Once this is achieved, the protease separase is activated to cleave the chromosomal cohesin complex, thereby triggering anaphase. Cohesin cleavage releases tension between sister chromatids, but why the mitotic checkpoint now remains silent is poorly understood. Here, using budding yeast as a model, we show that loss of sister chromatid cohesion at anaphase onset would engage the mitotic checkpoint if this was not prevented by concomitant separase-dependent activation of the Cdc14 phosphatase. Cdc14, in turn, inactivates the mitotic checkpoint by dephosphorylating Sli15(INCENP), a subunit of the conserved Aurora B kinase complex that forms part of the proposed chromosomal tension sensor. Dephosphorylation-dependent relocation of Sli15(INCENP) from centromeres to the central spindle during anaphase is seen in organisms from yeast to human [4-8]. Our results suggest that Sli15(INCENP) dephosphorylation is part of an evolutionarily conserved mechanism that prevents the mitotic checkpoint from reengaging when tension between sister chromatids is lost at anaphase onset.
Project description: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.
Project description: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.
Project description:Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit.Here we show that during experimentally induced metaphase delay, spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term "cohesion fatigue." Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the "prophase pathway" of cohesin release from chromosomes, or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids.In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.
Project description:Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.
Project description:PICH (Plk1-interacting checkpoint helicase) was recently identified as an essential component of the spindle assembly checkpoint and shown to localize to kinetochores, inner centromeres, and thin threads connecting separating chromosomes even during anaphase. In this paper, we have used immuno-fiber fluorescence in situ hybridization and chromatin-immunoprecipitation to demonstrate that PICH associates with centromeric chromatin during anaphase. Furthermore, by careful analysis of PICH-positive anaphase threads through FISH as well as bromo-deoxyurdine and CREST labeling, we strengthen the evidence that these threads comprise mainly alphoid centromere deoxyribonucleic acid. Finally, by timing the addition of ICRF-193 (a specific inhibitor of topoisomerase-II alpha) to cells synchronized in anaphase, we demonstrate that topoisomerase activity is required specifically to resolve PICH-positive threads during anaphase (as opposed to being required to prevent the formation of such threads during earlier cell cycle stages). These data indicate that PICH associates with centromeres during anaphase and that most PICH-positive threads evolve from inner centromeres as these stretch in response to tension. Moreover, they show that topoisomerase activity is required during anaphase for the resolution of PICH-positive threads, implying that the complete separation of sister chromatids occurs later than previously assumed.
Project description:Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called "anaphase problem" is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore-microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear. Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/C(Cdc20) substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/C(Cdc20) activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10-15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/C(Cdc20) in late anaphase helps to keep cyclin B1 levels low.
Project description:In budding yeast, we have found that sister rDNA arrays marked with fluorescent probes can be visualized as two distinguishable strands during metaphase. Upon anaphase, these arm loci are drawn into the spindle, where they adopt a cruciform-like structure and stretch 2.5-fold as they migrate to the poles. Therefore, while sister rDNA arrays appear separated in metaphase, mechanical linkages between sister arm loci persist throughout anaphase in yeast, as shown in grasshopper spermatocytes (Paliulis and Nicklas 2004). These linkages are partially dependent on the protector of cohesin, SGO1. In anaphase, the spatially regulated dissolution of these mechanical linkages serves to prevent premature sister separation and restrain the rate of spindle elongation. Thus, sister separation is temporally controlled and linkages between sister chromatids contribute to the regulation of anaphase spindle elongation.