Histone hyperacetylation affects meiotic recombination and chromosome segregation in Arabidopsis

In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5‐related histone N‐acetyltransferase, is described in Arabidopsis. Analysis of the over‐expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over‐expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast

The accurate segregation of chromosomes at mitosis requires that all pairs of chromatids bind correctly to microtubules prior to the dissolution of sister cohesion and the initiation of anaphase. By analyzing the motion of GFP-tagged S. cerevisiae chromosomes, we show that kinetochore-microtubule attachments impose sufficient tension on sisters during prometaphase to transiently separate centromeric chromatin toward opposite sides of the spindle. Transient separations of 2-10 min duration occur in the absence of cohesin proteolysis, are characterized by independent motion of the sisters along the spindle, and are followed by the apparent reestablishment of sister linkages. The existence of transient sister separation in yeast explains the unusual bilobed localization of kinetochore proteins and supports an alternative model for spindle structure. By analogy with animal cells, we propose that yeast centromeric chromatin acts as a tensiometer.     

Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae

Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.     

Cold-sensitive and caffeine-supersensitive mutants of the Schizosaccharomyces pombe dis genes implicated in sister chromatid separation during mitosis

We isolated novel classes of Schizosaccharomyces pombe cold-sensitive dis mutants that block mitotic chromosome separation (nine mapped in the dis1 gene and one each in the dis2 and dis3 genes). Defective phenotype at restrictive temperature is similar among the mutants; the chromosomes condense and anomalously move to the cell ends in the absence of their disjoining so that they are unequally distributed at the two cell ends. Synchronous culture analyses indicate that the cells can enter into mitosis at normal timing but become lethal during mitosis. In comparison with the wild-type mitosis, defects are found in the early spindle structure, the mitotic chromosome structure, the poleward chromosome movement by the spindle elongation and the telophase spindle degradation. The dis mutants lose at permissive temperature an artificial minichromosome at higher rates than occur in the wild type. We found that all the dis mutants isolated are supersensitive to caffeine at permissive temperature. Furthermore, the mutant cells in the presence of caffeine produce a phenotype similar to that obtained at restrictive temperature. We suggest that the dis genes are required for the sister chromatid separation at the time of mitosis and that caffeine might affect the dis gene expression. We cloned, in addition to the dis2+ and dis3+ genes, multicopy extragenic suppressor sequences which complement dis1 and dis2 mutations. A complex regulatory system may exist for the execution of the dis+ gene functions.     

Frontier questions about sister chromatid separation in anaphase

Sister chromatid separation in anaphase is an important event in the cell's transmission of genetic information to a descendent. It has been investigated from different aspects: cell cycle regulation, spindle and chromosome dynamics within the three-dimensional cell architecture, transmission fidelity control and cellular signaling. Integrated studies directed toward unified understanding are possible using multidisciplinary methods with model organisms. Ubiquitin-dependent proteolysis, protein dephosphorylation, an unknown function by the TPR repeat proteins, chromosome transport by microtubule-based motors and DNA topological change by DNA topoisomerase II are all necessary for progression from metaphase to anaphase. Chromosome condensation, mitotic kinetochore function and spindle formation require a large number of proteins, which are prerequisites for successful sister chromatid separation. Factors that help to retain sister chromatid connection after replication and prevent premature separation remain to be determined. Although sister chromatid separation occurs in anaphase, gene functions in other cell cycle stages also ensure the progression of correct chromatid separation.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.     

Cohesin defects lead to premature sister chromatid separation,kinetochore dysfunction,and spindle-assembly checkpoint activation

Scc1/Mcd1 is a component of the cohesin complex that plays an essential role in sister chromatid cohesion in eukaryote cells. Knockout experiments of this gene have been described in budding yeast, fission yeast, and chicken cells, but no study has been reported on human Scc1 thus far. In this study, we found that an N-terminally truncated human Scc1 shows a dominant-negative effect, and we examined the phenotypes of human cells defective in Scc1 function. Scc1 defects led to failure of sister chromatid cohesion in both interphase and mitotic cells. Interestingly, four chromatids derived from two homologues occupied four distinct territories in the nucleus in chromosome painting experiments. In mitotic Scc1-defective cells, chromatids were disjoined with normal condensation, and the spindle-assembly checkpoint was activated. We also found that, although the disjoined kinetochore (half-kinetochore) in Scc1-defective cells contains CENP-A, -B, -C, and -E normally, it apparently does not establish the kinetochore-microtubule association. These results indicate that Scc1 is essential for the association of kinetochores with microtubules.     

Nuclear p120 catenin is a component of the perichromosomal layer and coordinates sister chromatid segregation during mitosis in lung cancer cells

Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression.Subject terms: Non-small-cell lung cancer, Cell biology     

Elevated rates of sister chromatid exchange at chromosome ends

Chromosome ends are known hotspots of meiotic recombination and double-strand breaks. We monitored mitotic sister chromatid exchange (SCE) in telomeres and subtelomeres and found that 17% of all SCE occurs in the terminal 0.1% of the chromosome. Telomeres and subtelomeres are significantly enriched for SCEs, exhibiting rates of SCE per basepair that are at least 1,600 and 160 times greater, respectively, than elsewhere in the genome.     

Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

Class I_A phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP₃). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP₃ generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.     

Dual inhibition of sister chromatid separation at metaphase.

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.     

Actin-binding proteins required for reliable chromosome segregation in mitosis

While studying mitosis in Dictyostelium mutants with deficiencies in actin-binding proteins, we found that two of these proteins, cortexillin and Aip1, are required for the precise segregation of chromosomes. Atypical spindles and nuclei with varying DNA content indicate that mutants lacking cortexillin or Aip1 are genetically unstable. These aberrations are caused by the detachment and irregular reattachment of centrosomes to the nuclear surface. Live imaging showed how coalescing mitotic complexes give rise to a multipolar spindle, and how excess centrosomes can be eliminated by mitotic cleavage between anucleate and nucleated portions of a cell. We hypothesize that mutations in regulatory proteins of the actin network might be one cause of genetic instability of malignant tumor cells.     

Okadaic acid inhibits sister chromatid separation in mammalian cells.

Mitotic HeLa cells were treated with different concentrations of okadaic acid inhibiting phosphatase 2A activity alone or in addition to phosphatase 1 activity. Phosphatase 2A inhibition alone had no visible effect on mitosis, but inhibition of both phosphatase 1 and 2A produced mitotic abnormalities, including inhibition of anaphase mimicking the effect of colchicine. Recovery experiments in okadaic acid-free medium showed formation of diplochromosomes, indicating a failure of sister chromatid separation in the treated mitotic cells. The universality of the phosphatase 1 requirement in sister chromatid separation is discussed.     

The small molecule CS1 inhibits mitosis and sister chromatid resolution in HeLa cells

Background

Mitosis, the most dramatic event in the cell cycle, involves the reorganization of virtually all cellular components. Antimitotic agents are useful for dissecting the mechanism of this reorganization. Previously, we found that the small molecule CS1 accumulates cells in G2/M phase [1], but the mechanism of its action remains unknown.

Aurora B prevents chromosome arm separation defects by promoting telomere dispersion and disjunction

The segregation of centromeres and telomeres at mitosis is coordinated at multiple levels to prevent the formation of aneuploid cells, a phenotype frequently observed in cancer. Mitotic instability arises from chromosome segregation defects, giving rise to chromatin bridges at anaphase. Most of these defects are corrected before anaphase onset by a mechanism involving Aurora B kinase, a key regulator of mitosis in a wide range of organisms. Here, we describe a new role for Aurora B in telomere dispersion and disjunction during fission yeast mitosis. Telomere dispersion initiates in metaphase, whereas disjunction takes place in anaphase. Dispersion is promoted by the dissociation of Swi6/HP1 and cohesin Rad21 from telomeres, whereas disjunction occurs at anaphase after the phosphorylation of condensin subunit Cnd2. Strikingly, we demonstrate that deletion of Ccq1, a telomeric shelterin component, rescued cell death after Aurora inhibition by promoting the loading of condensin on chromosome arms. Our findings reveal an essential role for telomeres in chromosome arm segregation.     

Kinetochore-independent mechanisms of sister chromosome separation

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.     

Relationship of spontaneous chromosome instability and sister chromatid exchanges in Fanconi's anemia

The frequency of spontaneous instability of lymphocyte chromosomes of the first 2 mitoses, the rate of sister chromatid exchanges (SCEs), and the proliferative kinetics of lymphocytes were studied in a 6-year-old girl with Fanconi's anemia (FA) and in 4 healthy donors. The frequencies of aberrant cells and the total number of chromosome breaks in the FA patient decreased with cell transition from the first to the second mitosis. The FA lymphocytes had a slower proliferative kinetics and the level of SCEs was higher as compared with control. The probability of chromatid deletions at the sites of SCEs localization and in the dark and light stained chromatids was unequal. 33.8% of chromatid breaks were associated with SCEs. The data point to the relationship between SCEs and spontaneous chromosome instability in AF cells.