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Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses. [ABSTRACT FROM AUTHOR]

Background and Aims Dogroses (Rosa sect. Caninae) are mostly pentaploid, bearing 2 n  = 5 x  = 35 chromosomes in somatic cells. They evolved a unique form of asymmetrical meiosis characterized by two types of chromosomes: (1) chromosomes forming bivalents and distributed in the normal sexual way; and (2) chromosomes occurring as univalents and transferred by a female gamete only. In the mature pollen of pentaploid species, seven bivalent-derived chromosomes are transmitted to offspring, and 21 unpaired univalent chromosomes are eliminated during microsporogenesis. To discriminate between bivalent- and univalent-forming chromosomes, we studied histone H3 phosphorylation patterns regulating meiotic chromosome condensation and segregation. Methods We analysed histone modification patterns during male canina meiosis in two representative dogrose species, 5 x Rosa canina and 5 x Rosa rubiginosa , by immunohistochemical and molecular cytogenetics approaches. Immunostaining of meiotic cells included α-tubulin, histone H3 phosphorylation (H3S10p, H3S28p and H3T3p) and methylation (H3K4me3 and H3K27me3) marks. In addition, fluorescent in situ hybridization was carried out with an 18S rDNA probe. Key Results In the first meiotic division, univalent chromosomes underwent equational division into chromatids, while homologues in bivalents were segregated as regular dyads. In diakinesis, bivalent chromosomes displayed strong H3 phosphorylation signals in proximal regions, spreading to the rest of the chromosome. In contrast, in univalents, the H3 phosphorylation signals were weaker, occurring mostly outside proximal regions largely overlapping with the H3K4me3 signals. Reduced phosphorylation was associated with relative under-condensation of the univalent chromosomes, particularly at early diakinesis. Conclusions We hypothesize that the absence of pairing and/or recombination in univalent chromosomes negatively affects the histone H3 phosphorylation of their chromatin and perhaps the loading of meiotic-specific cohesins. This apparently destabilizes cohesion of sister chromatids, leading to their premature split in the first meiotic division. [ABSTRACT FROM AUTHOR]

Reversible protein phosphorylation regulates various cellular mechanisms in eukaryotes by altering the conformation, activity, localization, and stability of substrate proteins. In Arabidopsis thaliana root meristems, histone post-translational modifications are crucial for proper cell division, and they are also involved in oxidative stress signaling. To investigate the link between reactive oxygen species (ROS) and mitosis, we treated various Arabidopsis genotypes, including wild-types and mutants showing dysfunctional PP2A, with the ROS-inducing herbicide diquat (DQ). Studying the c3c4 double catalytic subunit mutant and fass regulatory subunit mutants of PP2A provided insights into phosphorylation-dependent mitotic processes. DQ treatment reduced mitotic activity in all genotypes and caused early mitotic arrest in PP2A mutants, likely due to oxidative stress-induced damage to essential mitotic processes. DQ had a minimal effect on reversible histone H3 phosphorylation in wild-type plants but significantly decreased phospho-histone H3 levels in PP2A mutants. Following drug treatment, the phosphatase activity decreased only in the stronger phenotype mutant plants (fass-5 and c3c4). Our findings demonstrate that (i) the studied PP2A loss-of-function mutants are more sensitive to increased intracellular ROS and (ii) DQ has indirect altering effects of mitotic activities and histone H3 phosphorylation. All these findings underscore the importance of PP2A in stress responses.

Abnormal histone modifications have been recognized as an important contributing factor to the initiation and progression of osteosarcoma. Sirtuin 1 (Sirt1) up-regulation has been discovered in osteosarcoma cells. This study tested the influence of Sirt1 on histone H3 phosphorylation at threonine 3 (H3T3ph) in osteosarcoma cells, along with Sirt1-H3T3ph axis on osteosarcoma cell autophagy. Plasmids or si-RNAs transfection was carried out to alter Sirt1 or H3T3ph expressions. Co-immunoprecipitation analysis and GST pull-down assay were done to probe the relationship between Sirt1 and H3T3ph. Phosphoryltransferase activity of Sirt1 was tested by in vitro kinase activity assay. Cell autophagy was measured by a number of autophagosome, conversion of LC3-I to LC3-II, degradation of long-lived protein and ATG protein expressions. We found that Sirt1 directly interacted with H3 and phosphorylate H3T3 at threonine 3 in osteosarcoma cells. Moreover, Sirt1 facilitated osteosarcoma cell autophagy under starvation condition. H3T3ph took part in the Sirt1-facilitated osteosarcoma cell autophagy under starvation condition. Besides, Sirt1-H3T3ph axis facilitated osteosarcoma cell autophagy might be achieved through transcriptional activation of ATG genes. Sirt1 promoted osteosarcoma initiation and progression might be via phosphorylate H3T3 and then facilitate osteosarcoma cell autophagy through activating ATG genes transcription under starvation condition.

Summary: Casein kinase I (CK1), a ubiquitous Ser/Thr protein kinase in eukaryotes, plays a critical role in higher plant flowering. Arabidopsis CK1 family member MUT9‐LIKE KINASEs, such as MLK1 and MLK3, have been shown to phosphorylate histone H3 at threonine 3 (H3T3), an evolutionarily conserved residue, and the modification is associated with the transcriptional repression of euchromatic and heterochromatic loci. This study demonstrates that mlk4‐3, a T‐DNA insertion mutant of MLK4, flowered late, and that overexpression of MLK4 caused early flowering. The nuclear protein MLK4 phosphorylated histone H3T3 both in vitro and in vivo, and this catalytic activity required the conserved lysine residue K175. mutation of MLK4 at K175 failed to restore the level of phosphorylated H3T3 (H3T3ph) or to complement the phenotypic defects of mlk4‐3. The FLC/MAF‐clade genes, including FLC, MAF4 and MAF5, were significantly upregulated in mlk4‐3. The double mutant mlk4‐3 flc‐3 flowered earlier than mlk4‐3, suggesting that functional FLC is crucial for flowering repression in mlk4‐3. Chromatin immunoprecipitation assays showed that MLK4 bound to FLC/MAF chromatin and that H3T3ph occupancy at the promoter of FLC/MAF was negatively associated with its transcriptional level. In accordance, H3T3ph accumulated at FLC/MAF in 35S::MLK4/mlk4‐3 but diminished in 35S::MLK4(K175R)/mlk4‐3 plants. Moreover, the amount of RNA Pol II deposited at FLC/MAF was clearly enriched in mlk4‐3 relative to the wild type. Therefore, MLK4‐dependent phosphorylation of H3T3 contributes to accelerating flowering by repressing the transcription of negative flowering regulator FLC/MAF. This study sheds light on the delicate control of flowering by the plant‐specific CK1, MLK4, via post‐translational modification of histone H3. Significance Statement: Histone H3T3 is one of the most conserved residues phosphorylated by casein kinase I (CK1) in eukaryotes. This study investigated the role of H3T3 phosphorylation (H3T3ph) in flowering and demonstrated that MLK4 phosphorylates H3T3, and that elimination of the modification by mutating the enzyme de‐represses the transcription of FLC‐clade genes, causing late flowering. The findings provide an insight into MLK4‐mediated H3T3ph as a repressive epigenetic mark in flowering regulation via the transcriptional silencing of flowering repressor FLC/MAF. [ABSTRACT FROM AUTHOR]

Briefly depicted are the publications in CYTOMETRY that received the highest frequency of citations. Among them are seminal papers describing application of metachromatic fluorochrome acridine orange to differentially stain DNA versus RNA or to analyze susceptibility of DNA in situ to denaturation; both features being markers of different sections of the cell cycle including identification of noncycling quiescent cells. The papers reviewing detection of cyclins D1, E, A or B1, each in relation to cell cycle phase, were also among the highly cited ones. The highest citation rates received publications describing development of the TUNEL methodology to detect apoptotic DNA fragmentation, and more recently expression of ϒH2AX to reveal DNA damage. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Abnormal histone modifications have been recognized as an important contributing factor to the initiation and progression of osteosarcoma. Sirtuin 1 (Sirt1) up-regulation has been discovered in osteosarcoma cells. This study tested the influence of Sirt1 on histone H3 phosphorylation at threonine 3 (H3T3ph) in osteosarcoma cells, along with Sirt1-H3T3ph axis on osteosarcoma cell autophagy. Plasmids or si-RNAs transfection was carried out to alter Sirt1 or H3T3ph expressions. Co-immunoprecipitation analysis and GST pull-down assay were done to probe the relationship between Sirt1 and H3T3ph. Phosphoryltransferase activity of Sirt1 was tested by in vitro kinase activity assay. Cell autophagy was measured by a number of autophagosome, conversion of LC3-I to LC3-II, degradation of long-lived protein and ATG protein expressions. We found that Sirt1 directly interacted with H3 and phosphorylate H3T3 at threonine 3 in osteosarcoma cells. Moreover, Sirt1 facilitated osteosarcoma cell autophagy under starvation condition. H3T3ph took part in the Sirt1-facilitated osteosarcoma cell autophagy under starvation condition. Besides, Sirt1-H3T3ph axis facilitated osteosarcoma cell autophagy might be achieved through transcriptional activation of ATG genes. Sirt1 promoted osteosarcoma initiation and progression might be via phosphorylate H3T3 and then facilitate osteosarcoma cell autophagy through activating ATG genes transcription under starvation condition. [ABSTRACT FROM AUTHOR]

Abstract Overproduction of toxic cyanobacteria is a type of harmful algal blooms (HABs). The heptapeptide microcystins (MCs) are one of the most common cyanotoxins. There is increasing research concerning the effects of MCs on growth and physiology of vascular plants, however there is a lack of studies on their direct effects on aquatic macrophytes in the real environment. Here we report the occurrence of a MC producing HAB in Lake Bárdos, Hungary in 2012 with harmful effects on cytological, histological and biochemical parameters of Ceratophyllum submersum (soft hornwort) plants naturally growing at the blooming site. Blue-Green Sinapis Test (BGST) showed high toxicity of HAB samples. Cell-free water samples contained a significant amount of MCs (7.31 ± 0.17 μg L−1) while C. submersum plants contained 1.01 ± 0.21 μg g DW−1 MCs. Plants showed significant increases of protein content and decreases of anthocyanin content and carotenoid/chlorophyll ratio, indicating physiological stress- as compared to plants from the control (MC free) sampling site of the same water body. Histological and cytological studies showed (i) radial swelling and the abnormal formation of lateral buds at the shoot tip leading to abnormal development; (ii) the fragmentation of nuclei as well as accumulation of phenolics in the nucleus indicating that the HAB induced cell death and stress reactions at the nuclear level. The most relevant effect was the increase of histone H3 phosphorylation in metaphase chromosomes: since MCs are strong inhibitors of protein phosphatases, this alteration is related to the biochemical targets of these toxins. The HAB decreased peroxidase activity, but increased nuclease and protease activities, showing the decreased capacity of plants to face biotic stress and as the cytological changes, the induction of cell death. This study is one of the first to show the complex harmful changes in aquatic plants that co-exist with HABs. Graphical abstract Unlabelled Image Highlights • This is a study of cyanobacterium-plant interaction in the real environment. • Microcystis blooming affected a C. submersum (macrophyte) population in Hungary. • Microcystins were found in the cyanobacterial cells, aquatic plants and water body. • Histone H3 hyperphosphorylation in plant cells is directly related to microcystins. [ABSTRACT FROM AUTHOR]

Although the regulatory network of G2/M phase transition has been intensively studied in mammalian cell lines, the identification of morphological and molecular markers to identify G2/M phase transition in vivo remains elusive. In this study, we found no obvious morphological changes between the S phase and G2 phase in mice intestinal epithelial cells. The G2 phase could be identified by Brdu incorporation resistance, marginal and scattered foci of histone H3 phosphorylated at Ser10 (pHH3), and relatively intact Golgi ribbon. Prophase starts with nuclear transformation in situ, which was identified by a series of prophase markers including nuclear translocation of cyclinB1, fragmentation of the Golgi complex, and a significant increase in pHH3. The nucleus started to move upwards in the late prophase and finally rounded up at the apical surface. Then, metaphase was initiated as the level of pHH3 peaked. During anaphase and telophase, pHH3 sharply decreased, while Ki67 was obviously bound to chromosomes, and PCNA was distributed throughout the whole cell. Based on the aforementioned markers and Brdu pulse labeling, it was estimated to take about one hour for most crypt cells to go through the G2 phase and about two hours to go through the G2-M phase. It took much longer for crypt base columnar (CBC) stem cells to undergo G2-prophase than rapid transit amplifying cells. In summary, a series of sequentially presenting markers could be used to indicate the progress of G2/M events in intestinal epithelial cells and other epithelial systems in vivo. [ABSTRACT FROM AUTHOR]

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.

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.

Gene transcription and translation in the hippocampus is of critical importance in hippocampus-dependent memory formation, including during Morris water maze (MWM) learning. Previous work using gene deletion models has shown that the immediate-early genes (IEGs) c-Fos, Egr-1, and Arc are crucial for such learning. Recently, we reported that induction of IEGs in sparse dentate gyrus neurons requires ERK MAPK signaling and downstream formation of a distinct epigenetic histone mark (i.e., phospho-acetylated histone H3). Until now, this signaling, epigenetic and gene transcriptional pathway has not been comprehensively studied in the MWM model. Therefore, we conducted a detailed study of the phosphorylation of ERK1/2 and serine10 in histone H3 (H3S10p) and induction of IEGs in the hippocampus of MWM trained rats and matched controls. MWM training evoked consecutive waves of ERK1/2 phosphorylation and H3S10 phosphorylation, as well as c-Fos, Egr-1, and Arc induction in sparse hippocampal neurons. The observed effects were most pronounced in the dentate gyrus. A positive correlation was found between the average latency to find the platform and the number of H3S10p-positive dentate gyrus neurons. Furthermore, chromatin immuno-precipitation (ChIP) revealed a significantly increased association of phospho-acetylated histone H3 (H3K9ac-S10p) with the gene promoters of c-Fos and Egr-1, but not Arc, after MWM exposure compared with controls. Surprisingly, however, we found very little difference between IEG responses (regarding both protein and mRNA) in MWM-trained rats compared with matched swim controls. We conclude that exposure to the water maze evokes ERK MAPK activation, distinct epigenetic changes and IEG induction predominantly in sparse dentate gyrus neurons. It appears, however, that a specific role for IEGs in the learning aspect of MWM training may become apparent in downstream AP-1- and Egr-1-regulated (second wave) genes and Arc-dependent effector mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Expression of c-fos is used for the characterization of brain areas activated by stressors. Recently, some epigenetic markers associated with enhanced transcription have been identified that may be also useful to detect neuronal populations important for the processing of stressors: phosphorylation of histone H3 in serine 10 or 28 ( pH3S10 or pH3S28). Then, we compared in rats the response to stress of c-fos and these epigenetic changes. More specifically, we studied the influence of the type of stressor (novel environment vs. immobilization, IMO) and the dynamics of the response to IMO. Stress increased pH3S10 positive neurons, with a more restricted pattern than that of c-fos, both in terms of brain areas activated and number of positive neurons. Changes in pH3S10 showed a maximum at 30 min, then progressively declining in most areas in spite of the persistence of IMO. Moreover, the decline was in general more sensitive than c-fos to the termination of IMO. The pattern of pH3S28 was even more restricted that of pH3S10, but they showed co-localization. The present data demonstrate a more selective pattern of stress-induced histone H3 phosphorylation than c-fos. The factors determining such a selectivity and its biological meaning remain to be studied. [ABSTRACT FROM AUTHOR]

Background: Increased histone H3 phosphorylation is an essential regulatory mechanism for neoplastic cell transformation. We aimed to explore the role of histone H3 phosphorylation at serine10 (p-H3Ser10) in Epstein-Barr virus (EBV) latent membrane protein-1 (LMP1)-induced carcinogenesis of nasopharyngeal carcinoma (NPC). Methods: The expression of p-H3Ser10 was detected by the immunohistochemical analysis in NPC, chronic nasopharyngitis and normal nasopharynx tissues, and its correlation with LMP1 was analyzed in NPC tissues and cell lines. Using the small interfering RNA (siRNA)-H3 and histone H3 mutant (S10A), the effect of histone H3 Ser10 motif on LMP1-induced CNE1 cell proliferation, transformation and activator protein-1 (AP-1) activation were evaluated by CCK-8, focus-forming and reporter gene assay respectively. Mitogen- and stress-activated kinase 1 (MSK1) kinase activity and phosphorylation were detected by in vitro kinase assay and western blot. Using MSK1 inhibitor H89 or siRNA-MSK1, the regulatory role of MSK1 on histone H3 phosphorylation and AP-1 activation were analyzed. Results: Immunohistochemical analysis revealed that the expression of p-H3Ser10 was significantly higher in the poorly differentiated NPC tissues than that in chronic nasopharyngitis (p <0.05) and normal nasopharynx tissues (p <0.001). Moreover, high level of p-H3Ser10 was positively correlated with the expression of LMP1 in NPC tissues (?²=6.700, p =0.01; C=0.350) and cell lines. The knockdown and mutant (S10A) of histone H3 suppressed LMP1- induced CNE1 cell proliferation, foci formation and AP-1 activation. In addition, LMP1 could increase MSK1 kinase activity and phosphorylation. MSK1 inhibitor H89 or knockdown of MSK1 by siRNA blocked LMP1-induced phosphorylation of histone H3 at Ser10 and AP-1 activation. Conclusion: EBV-LMP1 can induce phosphorylation of histone H3 at Ser10 via MSK1. Increased phosphorylation of histone H3 at Ser10 is likely a crucial regulatory mechanism involved in LMP1-induced carcinogenesis of NPC. [ABSTRACT FROM AUTHOR]

Background and Aims Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. Methods Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H2O2 production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). Key Results Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H2O2, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. Conclusions The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus. [ABSTRACT FROM AUTHOR]

We have demonstrated that heat acclimation (AC) causes selective, long-lasting, transcriptional changes in cytoprotective and chromatin remodeling-associated genes, which maintain their AC transcriptome profile, despite the loss of the AC phenotype (Tetievsky et al. Physiol Genomics 34: 78-87, 2008). We postulated that AC memory involves upstream epigenetic information, which predisposes to rapid reacclimation (ReAC) and cytoprotective memory. Here we tested the hypothesis that posttranslational histone modifications are linked to this process. Rats subjected to AC (34°C for 2 or 30 days), deacclimation (DeAC; 24°C, 30 days), and ReAC (34°C, 2 days), and untreated controls were used. Histone H4 lysine acetylation and histone H3 acetylation and phosphorylation in the heat shock element (HSE) of the promoters of heat shock protein-70 (hsp70) and -90 (hsp90) genes were examined. Histone acetyltransferase recruitment of TIP60 (60-kDa histone acetyltransferase-interactive protein), the catalytic subunit of NuH4, was used to validate acetylation. Heat shock factor-1 (HSF-1)-HSE binding to the hsp70 and hap90 genes was measured to confirm HSF-1 binding to euchromatin. Our results indicate that, while histone H3Serl0 phosphorylation occurred during the AC 2-day phase, AC constitutively elevated histone H4 acetylation in the HSE of hsp70 and hsp90 promoters. HSF-1-HSE binding was detected in the hsp70 gene throughout AC-DeAC-ReAC. The hsp90 gene lacked HSF-1 binding during DeAC, but resumed a high binding level upon ReAC. HSP-90 is a critical cytoprotective protein, and the HSF-1-hsp90 binding profile matched levels of this protein. We conclude that, while early histone H3 phosphorylation is probably required for subsequent histone H4 acetylation, the constitutively acetylated histone H4 and the preserved euchromatin state throughout AC-DeAC-ReAC predispose to rapid cytoprotective acclimatory memory. [ABSTRACT FROM AUTHOR]

The IκB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-κB (NF-κB) pathway. It has been reported to function in physiologic responses, including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, the mechanism by which oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKα (pIKKα) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-κB-inducing kinase (NIK) (an upstream kinase of IKKα), pIKKα, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and pIKKα in mouse brain endothelial cells after tFCI. Nuclear factor-κB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKα after oxygen–glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKα were confirmed by coimmunoprecipitation. Treatment with IKKα small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKα, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke). [ABSTRACT FROM AUTHOR]

The enzymological properties of AtAurora1, a kinase responsible for the cell cycle-dependent phosphorylation of histone H3 at S10, and its cross-talk with other post-translational histone modifications, were determined. In vitro phosphorylation of H3S10 by AtAurora1 is strongly increased by K9 acetylation, and decreased by K14 acetylation and T11 phosphorylation. However, S10 phosphorylation activity is unaltered by mono-, di- or trimethylation of K9. An interference of H3K9 dimethylation by SUVR4 occurs by a pre-existing phosphorylation at S10. Hence, cross-talk in plants exists between phosphorylation of H3S10 and methylation, acetylation or phosphorylation of neighbouring amino acid residues. AtAurora1 undergoes autophosphorylation in vivo regardless of the presence of substrate, and forms dimers in planta. Of the three ATP-competitive Aurora inhibitors tested, Hesperadin was most effective in reducing the in vivo kinase activity of AtAurora1. Hesperadin consistently inhibited histone H3S10 phosphorylation during mitosis in Arabidopsis cells, but did not affect other H3 post-translational modifications, suggesting a specific inhibition of AtAurora in vivo. Inactivation of AtAurora also caused lagging chromosomes in a number of anaphase cells, but, unlike the situation in mammalian cells, Hesperadin did not influence the microtubule dynamics in dividing cells. [ABSTRACT FROM AUTHOR]

Animal cells react to mitogenic or stress stimuli by rapid up-regulation of immediate-early (IE) genes and a parallel increase in characteristic modifications of core histones: chromatin changes, collectively termed the nucleosomal response. With regard to plants little is known about the accompanying changes at the chromatin level. We have used tobacco BY-2 and Arabidopsis T87 cell lines to study the nucleosomal response of plant cells to high salinity, cold and exogenous abscisic acid (ABA). When in quiescent stage, both tobacco and Arabidopsis cells show the typical nucleosomal response to high salinity and cold stress, manifested by rapid transient up-regulation of histone H3 Ser-10 phosphorylation, immediately followed by transient up-regulation of H3 phosphoacetylation and histone H4 acetylation. For each of the studied stresses the observed nucleosomal response was strictly correlated with the induction of stress-type specific genes. The dynamics of histone modifications in BY-2 cells in response to exogenous ABA exhibited a more complex pattern than that evoked by the two abiotic stresses, probably due to superposition of the primary and secondary effects of ABA. A rapid increase in H3 Ser-10 phosphorylation was also observed in whole leaves subjected to high salinity; however, the rate of change in this modification was much slower than in cultured cells. Together, these results indicate that the quiescent BY-2 and T87 cell lines show a typical nucleosomal response to abiotic stresses and ABA treatment and may represent suitable models for the study of chromatin-mediated mechanisms of stress tolerance in plants. [ABSTRACT FROM AUTHOR]

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition. [ABSTRACT FROM AUTHOR]

pp32 belongs to a family of leucine-rich acidic nuclear proteins, which play important roles in many cellular processes including regulation of chromatin remodeling, transcription, RNA transport, transformation and apoptosis. pp32 is described as a new tumor suppressor. It is unknown as to how pp32 works in tumor suppression. We found that overexpression of pp32 in human Jurkat T cells inhibits cell growth, and silenced pp32 promotes growth. We first showed that hyperacetylation and hyperphosphorylation of histone H3 are required for T-cell activation. Phosphorylation of histone H3 precedes acetylation during T-cell activation. pp32 specifically binds to histone H3 and blocks its acetylation and phosphorylation. pp32 directly initiates caspase activity and also promotes granzyme A-mediated caspase-independent cell death. Taken together, pp32 plays a repressive role by inhibiting transcription and triggering apoptosis.Cell Death and Differentiation (2006) 13, 1485–1494. doi:10.1038/sj.cdd.4401825; published online 9 December 2005 [ABSTRACT FROM AUTHOR]

ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes - including those at Fos and Jun - concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails. Indeed, on recombinant histone octamers in vitro, MSK1 efficiently phosphorylates both sites on the same H3 tail. However, sequential immunoprecipitation studies show that antibodies against phosphorylated S10-H3 recover virtually all this epitope without depletion of phosphorylated S28-H3, and vice versa, indicating that the two phospho-epitopes are not located on the same H3 tail in vivo. Confocal immunocytochemistry confirms the clear physical separation of the two phospho-epitopes in the intact manse nucleus. Finally, we used transfection-based experiments to test models that might explain such differential targeting. Overexpression and delocalisation of MSK1 does not result in the breakdown of targeting in vivo despite the fact that the ectopic kinase is fully activated by external stimuli. These studies reveal a remarkable level of targeting of S10 and S28 phosphorylation to distinct H3 tails within chromatin in the interphase mouse nucleus. Possible models for such exquisite targeting are discussed. [ABSTRACT FROM AUTHOR]

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression. [ABSTRACT FROM AUTHOR]

High-resolution detection of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes for scanning electron microscopy was shown using a novel application of indirect immunogold labeling with Nanogold. This method permits localization and quantification of signals in a three-dimensional context. Because the chromosome structure is well preserved, characterization of binding sites (chromomeres, parallel matrix fibers, solenoids), currently in the realm of nanometer decades, is possible. Quantification and three-dimensional localization of labels is possible with stereoscopic analysis. Limitations of the method pertain to the challenges in preservation of chromosome ultrastructure, accessibility of immunoreactants into the fixed chromatin and unspecific labeling. The differences between silver and gold enhancement and the current status of labeling efficiency are addressed. [ABSTRACT FROM AUTHOR]

The function of the phosphorylation of histone H3 at Ser 10 in plant cell division is uncertain. The timing correlates with chromosome condensation, and studies in plant meiosis suggest that it is involved in sister chromatid cohesion. In mitosis, plant chromosomes are highly phosphorylated in the pericentromeric region only. In order to modulate H3 phosphorylation, root meristems of different plant species were treated with the protein phosphatase inhibitor cantharidin or with ice-water. Immunostaining using an antibody specific to phosphorylated H3 at Ser 10 revealed a high level of H3 phosphorylation along the whole mitotic chromosome after cantharidin treatment, which resembles the distribution seen exclusively in first meiotic division. In chromosomes that were isolated from meristems treated with ice-water, the heterochromatic regions and nucleolar organizer regions, in addition to the pericentromeric region, were highly phosphorylated at H3. Cantharidin and ice-water also affected spindle assembly and chromosome length, but these effects did not seem to be directly linked to changes in H3 phosphorylation. [ABSTRACT FROM AUTHOR]

cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation., (© 2015 Wiley Periodicals, Inc.)

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.

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.

Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.