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

The Radially Swollen 4 Separase Mutation of Arabidopsis thaliana Blocks Chromosome Disjunction and Disrupts the Radial Microtubule System in Meiocytes

The caspase-family protease, separase, is required at the onset of anaphase to cleave the cohesin complex that joins replicated sister chromatids. However, in various eukaryotes, separase has acquired additional and distinct functions. A single amino-acid substitution in separase is responsible for phenotypes of the Arabidopsis thaliana mutant, radially swollen 4 (rsw4). This is a conditional mutant, resembling the wild type at the permissive temperature (∼20°C) and expressing mutant phenotypes at the restrictive temperature (∼30°C). Root cells in rsw4 at the restrictive temperature undergo non-disjunction and other indications of the loss of separase function. To determine to what extent separase activity remains at 30°C, we examined the effect of the mutation on meiosis, where the effects of loss of separase activity through RNA interference are known; and in addition, we examined female gametophyte development. Here, we report that, at the restrictive temperature, replicated chromosomes in rsw4 meiocytes typically fail to disjoin and the cohesin complex remains at centromeres after metaphase. Meiotic spindles appear normal in rsw4 male meiocytes; however the mutation disrupts the radial microtubule system, which is replaced by asymmetric arrays. Surprisingly, female gametophyte development was relatively insensitive to loss of separase activity, through either rsw4 or RNAi. These effects confirm that phenotypes in rsw4 result from loss of separase activity and establish a role for separase in regulating cell polarization following male meiosis

Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres where it provides the cohesive counterforce to bipolar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometers. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast Saccharomyces cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, which has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is reloaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres

Cohesin Is Dispensable for Centromere Cohesion in Human Cells

BACKGROUND: Proper regulation of the cohesion at the centromeres of human chromosomes is essential for accurate genome transmission. Exactly how cohesion is maintained and is then dissolved in anaphase is not understood. PRINCIPAL FINDINGS: We have investigated the role of the cohesin complex at centromeres in human cells both by depleting cohesin subunits using RNA interference and also by expressing a non-cleavable version of the Rad21 cohesin protein. Rad21 depletion results in aberrant anaphase, during which the sister chromatids separate and segregate in an asynchronous fashion. However, centromere cohesion was maintained before anaphase in Rad21-depleted cells, and the primary constrictions at centromeres were indistinguishable from those in control cells. Expression of non-cleavable Rad21 (NC-Rad21), in which the sites normally cleaved by separase are mutated, resulted in delayed sister chromatid resolution in prophase and prometaphase, and a blockage of chromosome arm separation in anaphase, but did not impede centromere separation. CONCLUSIONS: These data indicate that cohesin complexes are dispensable for sister cohesion in early mitosis, yet play an important part in the fidelity of sister separation and segregation during anaphase. Cleavage at the separase-sensitive sites of Rad21 is important for arm separation, but not for centromere separation

PIASγ Is Required for Faithful Chromosome Segregation in Human Cells

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 PIASγ as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASγ, 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. PIASγ-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASγ and Topoisomerase II. CONCLUSIONS: PIASγ 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 PIASγ in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel

Sequential Assembly of Centromeric Proteins in Male Mouse Meiosis

The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E

SMC complexes differentially compact mitotic chromosomes according to genomic context

Structural maintenance of chromosomes (SMC) protein complexes are key determinants of chromosome conformation. Using Hi-C and polymer modelling, we study how cohesin and condensin, two deeply conserved SMC complexes, organize chromosomes in the budding yeast Saccharomyces cerevisiae. The canonical role of cohesin is to co-align sister chromatids, while condensin generally compacts mitotic chromosomes. We find strikingly different roles for the two complexes in budding yeast mitosis. First, cohesin is responsible for compacting mitotic chromosome arms, independently of sister chromatid cohesion. Polymer simulations demonstrate that this role can be fully accounted for through cis-looping of chromatin. Second, condensin is generally dispensable for compaction along chromosome arms. Instead, it plays a targeted role compacting the rDNA proximal regions and promoting resolution of peri-centromeric regions. Our results argue that the conserved mechanism of SMC complexes is to form chromatin loops and that distinct SMC-dependent looping activities are selectively deployed to appropriately compact chromosomes

Heterochromatin Protein 1 (HP1) Proteins Do Not Drive Pericentromeric Cohesin Enrichment in Human Cells

Sister chromatid cohesion mediated by cohesin is essential for accurate chromosome segregation. Classical studies suggest that heterochromatin promotes cohesion, but whether this happens through regulation of cohesin remains to be determined. Heterochromatin protein 1 (HP1) is a major component of heterochromatin. In fission yeast, the HP1 homologue Swi6 interacts with cohesin and is required for proper targeting and/or stabilization of cohesin at the centromeric region. To test whether this pathway is conserved in human cells, we have examined the behavior of cohesin in cells in which the levels of HP1 alpha, beta or gamma (the three HP1 proteins present in mammalian organisms) have been reduced by siRNA. We have also studied the consequences of treating human cells with drugs that change the histone modification profile of heterochromatin and thereby affect HP1 localization. Our results show no evidence for a requirement of HP1 proteins for either loading of bulk cohesin onto chromatin in interphase or retention of cohesin at pericentric heterochromatin in mitosis. However, depletion of HP1gamma leads to defects in mitotic progression