Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.     

Human chromokinesin KIF4A functions in chromosome condensation and segregation

Accurate chromosome alignment at metaphase and subsequent segregation of condensed chromosomes is a complex process involving elaborate and only partially characterized molecular machinery. Although several spindle associated molecular motors have been shown to be essential for mitotic function, only a few chromosome arm--associated motors have been described. Here, we show that human chromokinesin human HKIF4A (HKIF4A) is an essential chromosome-associated molecular motor involved in faithful chromosome segregation. HKIF4A localizes in the nucleoplasm during interphase and on condensed chromosome arms during mitosis. It accumulates in the mid-zone from late anaphase and localizes to the cytokinetic ring during cytokinesis. RNA interference--mediated depletion of HKIF4A in human cells results in defective prometaphase organization, chromosome mis-alignment at metaphase, spindle defects, and chromosome mis-segregation. HKIF4A interacts with the condensin I and II complexes and HKIF4A depletion results in chromosome hypercondensation, suggesting that HKIF4A is required for maintaining normal chromosome architecture. Our results provide functional evidence that human KIF4A is a novel component of the chromosome condensation and segregation machinery functioning in multiple steps of mitotic division.     

Flanking sequence preference modulates de novo DNA methylation in the mouse genome

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.     

Analysis of the role of Aurora B on the chromosomal targeting of condensin I

During mitosis, chromosome condensation takes place, which entails the conversion of interphase chromatin into compacted mitotic chromosomes. Condensin I is a five-subunit protein complex that plays a central role in this process. Condensin I is targeted to chromosomes in a mitosis-specific manner, which is regulated by phosphorylation by mitotic kinases. Phosphorylation of histone H3at serine 10 (Ser10) occurs during mitosis and its physiological role is a longstanding question. We examined the function of Aurora B, a kinase that phosphorylates Ser10, in the chromosomal binding of condensin I and mitotic chromosome condensation, using an in vitro system derived from Xenopus egg extract. Aurora B depletion from a mitotic egg extract resulted in the loss of H3 phosphorylation, accompanied with a 50% reduction of chromosomal targeting of condensin I. Alternatively, a portion of condensin I was bound to sperm chromatin, and chromosome-like structures were assembled when okadaic acid (OA) was supplemented in an interphase extract that lacks Cdc2 activity. However, chromosomal targeting of condensin I was abolished when Aurora B was depleted from the OA-treated interphase extract. From these results, it is suggested that Aurora B-dependent and Cdc2-independent pathways of the chromosomal targeting of condensin I are present.     

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIα

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic “X” shape. Here we report results of a detailed morphological analysis, which revealed that chromokinesin KIF4 cooperated in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the “intrinsic structure” of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIα contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology.     

Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos

DNA methyltransferases (DNMTs) comprise a family of proteins involved in the establishment and maintenance of DNA methylation patterns in the mammalian genome. DNA methylation involves the transfer of the methyl group of the coenzyme S-adenosyl-L-methionine to the 5 position of cytosine residues within CpG dinucleotides. DNA methylation is implicated in the control of imprinted genes expression, X chromosome silencing, development of certain types of cancer, and embryonic development. DNA methylation is also believed to protect the genome from parasitic elements such as transposons, retrotransposons, and viruses. The aim of this study was to analyze the expression patterns of DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L genes in rhesus macaque (Macaca mulatta) oocytes and preimplantation stage embryos from fertilization to the hatched blastocyst stage, and to compare these results with the expression profiles in the mouse and other mammalian species. We describe species-dependent differences as well as similarities in expression patterns of DNMT genes among mammals.     

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.     

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.     

Epstein-Barr virus BGLF4 kinase induces premature chromosome condensation through activation of condensin and topoisomerase II

Previous studies of Epstein-Barr virus (EBV) replication focused mainly on the viral and cellular factors involved in replication compartment assembly and controlling the cell cycle. However, little is known about how EBV reorganizes nuclear architecture and the chromatin territories. In EBV-positive nasopharyngeal carcinoma NA cells or Akata cells, we noticed that cellular chromatin becomes highly condensed upon EBV reactivation. In searching for the possible mechanisms involved, we found that transient expression of EBV BGLF4 kinase induces unscheduled chromosome condensation, nuclear lamina disassembly, and stress fiber rearrangements, independently of cellular DNA replication and Cdc2 activity. BGLF4 interacts with condensin complexes, the major components in mitotic chromosome assembly, and induces condensin phosphorylation at Cdc2 consensus motifs. BGLF4 also stimulates the decatenation activity of topoisomerase II, suggesting that it may induce chromosome condensation through condensin and topoisomerase II activation. The ability to induce chromosome condensation is conserved in another gammaherpesvirus kinase, murine herpesvirus 68 ORF36. Together, these findings suggest a novel mechanism by which gammaherpesvirus kinases may induce multiple premature mitotic events to provide more extrachromosomal space for viral DNA replication and successful egress of nucleocapsid from the nucleus.     

Three-step model for condensin activation during mitotic chromosome condensation

Chromosomes undergo a major structural reorganization during mitosis. The first step in this reorganization is the compaction of interphase chromatin into highly condensed mitotic chromosomes. An evolutionarily conserved multi-subunit ATPase, the condensin complex, plays a critical role in establishing chromosome architecture and promoting chromosome condensation in mitosis. How does condensin promote chromosome condensation and how, in turn, is the cell cycle machinery activating or restraining condensin activity during the cell cycle are fundamental questions for cell biology. In this review, we examine the role of post-translational modifications, and in particular multi-site phosphorylation, in the regulation of condensin activity during the cell cycle. Remarkably, inspection of phosphorylation sites identified through multiple proteome-wide mass spectrometry analyses reveals that the phosphorylation landscape of condensin is highly conserved evolutionarily and that several kinases regulate condensin in vivo. This analysis leads us to propose the ultrasensitive-kinase switch model, whereby the phosphorylation of condensin by multiple kinases allows the process of chromosome condensation to be maintained and even increased under fluctuating levels of cyclin-CDK activity during mitosis. Our model reconciles how chromosome condensation might be highly sensitive to low levels of CDK activity in early mitosis and subsequently insensitive to the declining levels CDK activity in late mitosis.     

Distinct but overlapping domains of AKAP95 are implicated in chromosome condensation and condensin targeting

A-kinase (or PKA)-anchoring protein AKAP95 is a zinc-finger protein implicated in mitotic chromosome condensation by acting as a targeting molecule for the condensin complex. We have identified determinants of chromatin-binding, condensin-targeting and chromosome-condensation activities of AKAP95. Binding of AKAP95 to chromatin is conferred by residues 387–450 and requires zinc finger ZF1. Residues 525–569 are essential for condensation of AKAP95-free chromatin and condensin recruitment to chromosomes. Mutation of either zinc finger of AKAP95 abolishes condensation. However, ZF1 is dispensable for condensin targeting, whereas the C-terminal ZF2 is required. AKAP95 interacts with Xenopus XCAP-H condensin subunit in vitro and in vivo but not with the human hCAP-D2 subunit. The data illustrate the involvement of overlapping, but distinct, domains of AKAP95 for condensin recruitment and chromosome condensation and argue for a key role of ZF1 in chromosome condensation and ZF2 in condensin targeting. Moreover, condensin recruitment to chromatin is not sufficient to promote condensation.