Protein kinase A-mediated serine 35 phosphorylation dissociates histone H1.4 from mitotic chromosome

Global histone H1 phosphorylation correlates with cell cycle progression. However, the function of site-specific H1 variant phosphorylation remains unclear. Our mass spectrometry analysis revealed a novel N-terminal phosphorylation of the major H1 variant H1.4 at serine 35 (H1.4S35ph), which accumulates at mitosis immediately after H3 phosphorylation at serine 10. Protein kinase A (PKA) was found to be a kinase for H1.4S35. Importantly, Ser-35-phosphorylated H1.4 dissociates from mitotic chromatin. Moreover, H1.4S35A substitution mutant cannot efficiently rescue the mitotic defect following H1.4 depletion, and inhibition of PKA activity increases the mitotic chromatin compaction depending on H1.4. Our results not only indicate that PKA-mediated H1.4S35 phosphorylation dissociates H1.4 from mitotic chromatin but also suggest that this phosphorylation is necessary for specific mitotic functions.     

Middle‐Down MS is Ready to Answer Complex Questions in Chromatin Biology

Histones are the most abundant protein family in the cells of complex organisms such as mammals and, together with DNA, they define the backbone of chromatin. Histone PTMs are key players of chromatin biology, as they function as anchors for proteins that bind and modulate chromatin readout, including gene expression. Middle‐down mass spectrometry (MS) has been optimized for about 10 years to study histone N‐terminal tails, but it has been rarely applied to identify the role of coexisting histone marks in biology. In this work, Jiang et al. used middle‐down MS to study the dynamics of coexisting PTMs on histone H4 in two breast cancer cell lines. 1 They found that overall serine 1 phosphorylation (S1ph) is mildly regulated during the cell cycle, but S1ph coexistence frequency with acetylations and methylations on the lysine residues of the N‐terminal tail is remarkably tuned during S phase and G2/M phase. Together, the team placed another benchmark proving that MS analysis of combinatorial histone PTMs provides a more comprehensive view on chromatin state than studying individual marks. We should then constantly question ourselves regarding the limitations of analyzing single PTMs when we attempt to define their effect on protein functions.     

Middle‐Down Characterization of the Cell Cycle Dependence of Histone H4 Posttranslational Modifications and Proteoforms

Post‐translational modifications (PTMs) of histones are important epigenetic regulatory mechanisms that are often dysregulated in cancer. We employ middle‐down proteomics to investigate the PTMs and proteoforms of histone H4 during cell cycle progression. We use pH gradient weak cation exchange‐hydrophilic interaction liquid chromatography (WCX‐HILIC) for on‐line liquid chromatography‐mass spectrometry analysis to separate and analyze the proteoforms of histone H4. This procedure provides enhanced separation of proteoforms, including positional isomers, and simplifies downstream data analysis. We use ultrahigh mass accuracy and resolution Fourier transform‐ion cyclotron resonance (FT‐ICR) mass spectrometer to unambiguously distinguish between acetylation and tri‐methylation (?m = 0.036 Da). In total, we identify and quantify 233 proteoforms of histone H4 in two breast cancer cell lines. We observe significant increases in S1 phosphorylation during mitosis, implicating an important role in mitotic chromatin condensation. A decrease of K20 unmodified proteoforms is observed as the cell cycle progresses, corresponding to an increase of K20 mono‐ and di‐methylation. Acetylation at K5, K8, K12, and K16 declines as cells traverse from S phase to mitosis, suggesting cell cycle–dependence and an important role during chromatin replication and condensation. These new insights into the epigenetics of the cell cycle may provide new diagnostic and prognostic biomarkers.     

The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells

We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM- 26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26- induced G2 arrest of the cell.     

M Phase-Specific Phosphorylation of Histone H1.5 at Threonine 10 by GSK-3

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.     

The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-004-0281-9Communicated by G. Almouzni     

Histone H1 of Trypanosoma cruzi is concentrated in the nucleolus region and disperses upon phosphorylation during progression to mitosis

Phosphorylation of histone H1 is intimately related to the cell cycle progression in higher eukaryotes, reaching maximum levels during mitosis. We have previously shown that in the flagellated protozoan Trypanosoma cruzi, which does not condense chromatin during mitosis, histone H1 is phosphorylated at a single cyclin-dependent kinase site. By using an antibody that recognizes specifically the phosphorylated T. cruzi histone H1 site, we have now confirmed that T. cruzi histone H1 is also phosphorylated in a cell cycle-dependent manner. Differently from core histones, the bulk of nonphosphorylated histone H1 in G(1) and S phases of the cell cycle is concentrated in the central regions of the nucleus, which contains the nucleolus and less densely packed chromatin. When cells pass G(2), histone H1 becomes phosphorylated and starts to diffuse. At the onset of mitosis, histone H1 phosphorylation is maximal and found in the entire nuclear space. As permeabilized parasites preferentially lose phosphorylated histone H1, we conclude that this modification promotes its release from less condensed and nucleolar chromatin after G(2).     

Microcystin-LR, a protein phosphatase inhibitor, induces alterations in mitotic chromatin and microtubule organization leading to the formation of micronuclei in Vicia faba

Background and Aims Microcystin-LR (MCY-LR) is a cyanobacterial toxin, a specific inhibitor of type 1 and 2A protein phosphatases (PP1 and PP2A) with significant impact on aquatic ecosystems. It has the potential to alter regulation of the plant cell cycle. The aim of this study was improved understanding of the mitotic alterations induced by cyanotoxin in Vicia faba, a model organism for plant cell biology studies. Methods Vicia faba seedlings were treated over the long and short term with MCY-LR purified in our laboratory. Short-term treatments were performed on root meristems synchronized with hydroxylurea. Sections of lateral root tips were labelled for chromatin, phosphorylated histone H3 and β-tubulin via histochemical and immunohistochemical methods. Mitotic activity and the occurrence of mitotic alterations were detected and analysed by fluorescence microscopy. The phosphorylation state of histone H3 was studied by Western blotting. Key Results Long-term MCY-LR exposure of lateral root tip meristems increased the percentage of either early or late mitosis in a concentration-dependent manner. We observed hypercondensed chromosomes and altered sister chromatid segregation (lagging chromosomes) leading to the formation of micronuclei, accompanied by the formation of disrupted, multipolar and monopolar spindles, disrupted phragmoplasts and the hyperphosphorylation of histone H3 at Ser10. Short-term MCY-LR treatment of synchronized cells showed that PP1 and PP2A inhibition delayed the onset of anaphase at 1 μg mL(-1) MCY-LR, accelerated cell cycle at 10 μg mL(-1) MCY-LR and induced the formation of lagging chromosomes. In this case mitotic microtubule alterations were not detected, but histone H3 was hyperphosphorylated. Conclusions MCY-LR delayed metaphase-anaphase transition. Consequently, it induced aberrant chromatid segregation and micronucleus formation that could be associated with both H3 hyperphosphorylation and altered microtubule organization. However, these two phenomena seemed to be independent. The toxin may be a useful tool in the study of plant cell cycle regulation.     

Mitotic histone H3 phosphorylation by vaccinia-related kinase 1 in mammalian cells

Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.     

H2AX: tailoring histone H2A for chromatin-dependent genomic integrity.

During the last decade, chromatin research has been focusing on the role of histone variability as a modulator of chromatin structure and function. Histone variability can be the result of either post-translational modifications or intrinsic variation at the primary structure level: histone variants. In this review, we center our attention on one of the most extensively characterized of such histone variants in recent years, histone H2AX. The molecular phylogeny of this variant seems to have run in parallel with that of the major canonical somatic H2A1 in eukaryotes. Functionally, H2AX appears to be mainly associated with maintaining the genome integrity by participating in the repair of the double-stranded DNA breaks exogenously introduced by environmental damage (ionizing radiation, chemicals) or in the process of homologous recombination during meiosis. At the structural level, these processes involve the phosphorylation of serine at the SQE motif, which is present at the very end of the C-terminal domain of H2AX, and possibly other PTMs, some of which have recently started to be defined. We discuss a model to account for how these H2AX PTMs in conjunction with chromatin remodeling complexes (such as INO80 and SWRI) can modify chromatin structure (remodeling) to support the DNA unraveling ultimately required for DNA repair.     

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.     

Extent of histone modifications and H1 content during cell cycle progression in the presence of butyrate

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.     

Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. We have used an in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts to study mitotic histone H3 phosphorylation. We identified a histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes. The histone H3 kinase was not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity could be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity was regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibited kinase activity. The presence of PP1 on chromatin suggested that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generated an H3 kinase activity that was also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, we found that stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1.     

Phosphorylation of the linker histone H1 by CDK regulates its binding to HP1alpha

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.