Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-delta on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles.     

Sister chromatid cohesion and recombination in meiosis

Sister chromatids are associated from their formation until their disjunction. Cohesion between sister chromatids is provided by protein complexes, of which some components are conserved across the kingdoms and between the mitotic and meiotic cell cycles. Sister chromatid cohesion is intimately linked to other aspects of chromosome behaviour and metabolism, in particular chromosome condensation, recombination and segregation. Recombination, sister chromatid cohesion and the relation between the two processes must be regulated differently in mitosis and meiosis. In meiosis, cohesion and recombination are modified in such a way that reciprocal exchange and reductional segregation of homologous chromosomes are ensured. Received: 11 October 1999; in revised form: 3 December 1999 / Accepted: 6 December 1999     

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.     

Distinct roles of the Drosophila ninaC kinase and myosin domains revealed by systematic mutagenesis

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.     

Meiotic cohesion requires accumulation of ORD on chromosomes before condensation

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei of Drosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable in ord(null) spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.     

Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells

A new method is described to visualize chromosome damage in interphase cells immediately after exposure to mutagenic agents. This method involves the fusion of treated interphase cells with untreated mitotic cells which results in the induction of premature chromosome condensation (PCC). Chinese hamster ovary (CHO) cells were treated with X-rays and chromosome aberrations were scored in G2-PCC and the mitotic chromosomes. The incidence of aberrations was significantly higher in PCC than that observed in the mitotic chromosomes of the treated cells. Post-irradiation incubation for I h before fusion allowed the repair of some of the chromosome damage. Data are also presented which indicate that the extent of radiation damage visualized in PCC is inversely proportional to the degree of chromosome condensation. These results indicate that the PCC method has a greater senstivity in the detection of induced chromosome damage than the standard method of scoring metaphase chromosomes.     

Premature chromosome condensation. II. The nature of chromosome gaps produced by alkylating agents and ultraviolet light

Chinese hamster ovary (CHO) cells were treated with ultraviolet radiation or the alkylating agents, nitrogen mustard or trenimon, and chromosome damage to G2 phase cells were scored by the premature chromosome condensation (PCC) method or the metotic chromosome method. Treatment with these agents produced gaps but not chromatid breaks or exchanges. After UV treatment, the gap frequency observed in G2-PCC was higher than in the mitotic chromosomes, while the reverse trend was observed after treatment with nitrogen mustard or trenimon. These results suggest that two types of chromosome gaps exist, both of which are observable in mitotic chromosomes while only one type is observable in PCC due to differences in the stages of condensation between PCC and mitotic chromosomes.     

Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.     

Biochemical heterogeneity and developmental varieties in T-cell leukemia

During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes – the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint “helicase”) displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.     

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.     

Functional characterization of individual genomic condensin-binding sites in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using minichromosome mitotic segregation stability assay

Proper chromatin compaction in mitosis (condensation) is required for equal chromosome distribution and the precise inheritance of genetic information. A protein complex called condensin is responsible for mitotic chromosome condensation, chromosome individualization, the timely separation of sister chromatids in mitosis, and proper tension in the mitotic spindle. The mitotic function of condensin depends on the recognition of specific binding sites in the chromosome. The mechanism for binding condensin to individual sites of mitotic chromosomes, as well as the molecular anatomy of these sites, remains to be elucidated. Even less is known about the process that translates condensin binding to individual sites into the segregation of chromosomes in anaphase. In the present work, by using minichromosome assay, we analyze seven individual condensin-binding sites in S. cerevisiae identified in the whole-genome ChIP-on-chip screening. This approach allowed us to estimate the individual contribution of condensin-binding sites to the segregation fidelity of minichromosomes.     

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.     

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.     

Relationship between histone phosphorylation and premature chromosome condensation

The relationship between histone phosphorylation and chromosome condensation was investigated by determining changes in phosphorylation levels of histones H1 and H3 following fusion between mitotic and interphase cells and subsequent premature chromosome condensation. We detected significant increases in the levels of phosphorylation of H1 and H3 from interphase chromatin in which a majority of nuclei had undergone premature chromosome condensation. In addition, we noted significant decreases in the phosphate content of the highly phosphorylated mitotic H1 and H3, presumably resulting from phosphatase activity contributed by the interphase component of mitotic/interphase fused cells. These observations further strengthen the correlation between histone phosphorylation and the changes in chromosome condensation associated with the initiation of mitosis. This study also suggests that maintenance of the mitotic chromosomes in a highly condensed state does not require the continued presence of histones in a highly phosphorylated form.     

Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants

BACKGROUND: The accurate duplication and packaging of the genome is an absolute prerequisite to the segregation of chromosomes in mitosis. To understand the process of cell-cycle chromosome dynamics further, we have performed the first detailed characterization of a mutation affecting mitotic chromosome condensation in a metazoan. Our combined genetic and cytological approaches in Drosophila complement and extend existing work employing yeast genetics and Xenopus in vitro extract systems to characterize higher-order chromosome structure and function. RESULTS: Two alleles of the ORC2 gene were found to cause death late in larval development, with defects in cell-cycle progression (delays in S-phase entry and metaphase exit) and chromosome condensation in mitosis. During S-phase progression in wild-type cells, euchromatin replicates early and heterochromatin replicates late. Both alleles disrupted the normal pattern of chromosomal replication, with some euchromatic regions replicating even later than heterochromatin. Mitotic chromosomes were irregularly condensed, with the abnormally late replicating regions of euchromatin exhibiting the greatest problems in mitotic condensation. CONCLUSIONS: The results not only reveal novel functions for ORC2 in chromosome architecture in metazoans, they also suggest that the correct timing of DNA replication may be essential for the assembly of chromatin that is fully competent to undergo mitotic condensation.     

Chromosome assembly in vitro: topoisomerase II is required for condensation.

The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion. HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control. Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II. Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation. In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.     

Phosphorylation of the Scc2 cohesin deposition complex subunit regulates chromosome condensation through cohesin integrity

The cohesion of replicated sister chromatids promotes chromosome biorientation, gene regulation, DNA repair, and chromosome condensation. Cohesion is mediated by cohesin, which is deposited on chromosomes by a separate conserved loading complex composed of Scc2 and Scc4 in Saccharomyces cerevisiae. Although it is known to be required, the role of Scc2/Scc4 in cohesin deposition remains enigmatic. Scc2 is a phosphoprotein, although the functions of phosphorylation in deposition are unknown. We identified 11 phosphorylated residues in Scc2 by mass spectrometry. Mutants of SCC2 with substitutions that mimic constitutive phosphorylation retain normal Scc2–Scc4 interactions and chromatin association but exhibit decreased viability, sensitivity to genotoxic agents, and decreased stability of the Mcd1 cohesin subunit in mitotic cells. Cohesin association on chromosome arms, but not pericentromeric regions, is reduced in the phosphomimetic mutants but remains above a key threshold, as cohesion is only modestly perturbed. However, these scc2 phosphomimetic mutants exhibit dramatic chromosome condensation defects that are likely responsible for their high inviability. From these data, we conclude that normal Scc2 function requires modulation of its phosphorylation state and suggest that scc2 phosphomimetic mutants cause an increased incidence of abortive cohesin deposition events that result in compromised cohesin complex integrity and Mcd1 turnover.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.