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Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.11402.001, eLife digest In every one of our cells, our DNA is wrapped together with histone proteins to make a structure called chromatin. When a cell divides, each newly formed daughter cell must receive an identical set of chromatin. As part of this process, the chromatin is copied and then compacted, which causes a characteristic “X”-shaped chromosome to form. This “X” shape is actually made up of two identical parts, or chromatids, that are joined together until a specific time during cell division. If chromosomes separate too early or too late, the DNA will not distribute evenly to daughter cells, which could lead to diseases including cancer. Histone modifications are small chemical changes on the histone proteins that the DNA wraps around. Previous research identified a new histone modification that is located at an important contact point between the DNA and a particular histone protein. However, the role of this modification in living cells was not clear. Wike et al. have now determined that in animal cells, this histone modification occurs immediately before the chromatids separate and at specific locations along the chromosomes. The amount of this histone modification is very important: in cells with too much of the modification, the chromosomes compacted incorrectly and the chromatids separated too soon. As a result, the chromosomes were incorrectly distributed among the daughter cells. Wike et al. also show that an enzyme called Aurora-A kinase is responsible for making this histone modification. The next challenge will be to understand how the Aurora-A kinase knows when and where to add the histone modification to the chromosome. This will help us to understand how the overproduction of Aurora-A causes cancer. DOI: http://dx.doi.org/10.7554/eLife.11402.002

Kaposi’s sarcoma herpesvirus (KSHV) causes Kaposi’s sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication., Author Summary Herpesviruses are known to wake up and reactivate in response to different kinds of stress. Our study now highlights the key molecular host cell events that KSHV has evolved to utilize for efficient viral lytic replication: the activation of p53 and upregulation of p21, which slows down the cell cycle, but promotes viral replication and transcription of viral lytic genes. Mutations in TP53 gene are rarely found in KSHV-associated malignancies. Therefore, our work now provides a mechanistic explanation as to why the virus has evolved to retain p53.

Summary Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

Aurora-like kinases play key roles in chromosome segregation and cytokinesis in yeast, plant, and animal systems. Here, we characterize three Arabidopsis thaliana protein kinases, designated AtAurora1, AtAurora2, and AtAurora3, which share high amino acid identities with the Ser/Thr kinase domain of yeast Ipl1 and animal Auroras. Structure and expression of AtAurora1 and AtAurora2 suggest that these genes arose by a recent gene duplication, whereas the diversification of plant α and β Aurora kinases predates the origin of land plants. The transcripts and proteins of all three kinases are most abundant in tissues containing dividing cells. Intracellular localization of green fluorescent protein–tagged AtAuroras revealed an AtAurora-type specific association mainly with dynamic mitotic structures, such as microtubule spindles and centromeres, and with the emerging cell plate of dividing tobacco (Nicotiana tabacum) BY-2 cells. Immunolabeling using AtAurora antibodies yielded specific signals at the centromeres that are coincident with histone H3 that is phosphorylated at Ser position10 during mitosis. An in vitro kinase assay demonstrated that AtAurora1 preferentially phosphorylates histone H3 at Ser 10 but not at Ser 28 or Thr 3, 11, and 32. The phylogenetic analysis of available Aurora sequences from different eukaryotic origins suggests that, although a plant Aurora gene has been duplicated early in the evolution of plants, the paralogs nevertheless maintained a role in cell cycle–related signal transduction pathways.

For successful eukaryotic mitosis, sister chromatid pairs remain linked after replication until their kinetochores have been attached to opposite spindle poles by microtubules. This linkage is broken at the metaphase–anaphase transition and the sisters separate. In budding yeast, this sister chromatid cohesion requires a multi-protein complex called cohesin. A key component of cohesin is Scc1/Mcd1 (Rad21 in fission yeast). Disruption of the chicken orthologue of Scc1 by gene targeting in DT40 cells causes premature sister chromatid separation. Cohesion between sister chromatids is likely to provide a substrate for post-replicative DNA repair by homologous recombination. In keeping with this role of cohesion, Scc1 mutants also show defects in the repair of spontaneous and induced DNA damage. Scc1-deficient cells frequently fail to complete metaphase chromosome alignment and show chromosome segregation defects, suggesting aberrant kinetochore function. Consistent with this, the chromosomal passenger protein, INCENP (inner centromere protein) fails to localize to centromeres. Survivin, another passenger protein and one which interacts with INCENP, also fails to localize to centromeres in Scc1-deficient cells. These results show that cohesin maintains genomic stability by ensuring appropriate DNA repair and equal chromosome segregation at mitosis.

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.

Udgivelsesdato: 2002-Dec-1 The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIalpha) as a potential Aurora B substrate. Purified recombinant human topo IIalpha was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.

The proinflammatory cytokine Tumour Necrosis Factor (TNF)-alpha is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with b-agonists modulates the TNF-alpha-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1) and beta 2 adrenoreceptors (beta 2-ARs). TNF-alpha activated the canonical Nuclear Factor-kappa B (NF-kappa B) pathway and Mitogen-Activated Protein Kinases (MAPKs), culminating in potent induction of NF-kappa B-dependent proinflammatory genes. Cotreatment with the beta-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6) and several chemokines. The enhanced production of chemotactic factors upon TNF-alpha/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and beta 2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-alpha/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-kappa B p65 subunit, cAMP-response element-binding protein (CREB), CREB-binding protein (CBP) and RNA polymerase II. In summary, we show that b-agonists potentiate TNF-alpha action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of beta-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

Dynamic histone acetylation, catalyzed by lysine acetyltransferases and HDACs, is critical to IEG expression. Expression of IEGs, such as FOSL1, is induced by several signal transduction pathways resulting in activation of the protein kinase MSK and phosphorylation of histone H3 at serine 10 of nucleosomes (the nucleosome response) at the upstream promoter and regulatory region of target genes. HDAC inhibitors prevent FOSL1 gene induction and the association of HDAC1, 2 and 3 with the gene body. However, HDAC inhibitors did not prevent the nucleosome response. Thus HDAC inhibitors perturb events downstream of the nucleosome response required for FOSL1 transcription initiation.