Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Science.gov (United States)

Zheng, Yupeng; John, Sam; Pesavento, James J.; Schultz-Norton, Jennifer R.; Schiltz, R. Louis; Baek, Sonjoon; Nardulli, Ann M.; Hager, Gordon L.; Kelleher, Neil L.

2010-01-01

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants. PMID:20439994

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

Science.gov (United States)

Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

2016-09-01

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

Directory of Open Access Journals (Sweden)

Serena Barral

2014-01-01

Full Text Available Phosphorylation of the histone H2AX (γH2AX form is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU, we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ and the granule cell precursors (cerebellum. Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS degenerative diseases.

Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

Energy Technology Data Exchange (ETDEWEB)

Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M. [NIH; (UNC); (Columbia)

2014-08-06

Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

Science.gov (United States)

Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

2013-01-01

Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons

DEFF Research Database (Denmark)

Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada

2016-01-01

Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol...... of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation...... of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K...

Increased histone H3 phosphorylation in neurons in specific brain structures after induction of status epilepticus in mice.

Directory of Open Access Journals (Sweden)

Tetsuji Mori

Full Text Available Status epilepticus (SE induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3(+ (PH3(+ neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3(+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3(+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation.

Phosphorylation of histone H2AX as an indicator of received dose of gamma radiation after whole-body irradiation of rats

Directory of Open Access Journals (Sweden)

Radim Havelek

2011-01-01

Full Text Available The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats. Wistar rats were irradiated by 1-10 Gy of gamma radiation by 60Co source. Value LD50/60 was 7.37 (4.68-8.05 Gy. Histone H2AX is phosphorylated by ATM kinase on serine 139 (γH2AX quickly after the irradiation. It forms microscopically visible foci in the site of double strand breaks of DNA. Flow-cytometric method was used for quantitative detection. This study is the first one that evaluated dose-dependency of H2AX phosphorylation in peripheral lymphocytes of rats irradiated by whole-body dose 1-10 Gy. Our data show a dose-dependent increase in γH2AX in rat peripheral blood lymphocytes 1 h after whole-body irradiation by the dose of 1-10 Gy. We proved that phosphorylation of histone H2AX is a prompt and reliable indicator of the received radiation dose suitable for rapid measurement before the number of lymphocytes in peripheral blood starts to decrease. It can be used already 1 h after the irradiation for an estimation of the received dose of radiation. Blood samples can be stored in 4 °C for 23 h without significantly affecting the result.

Synthesis and phosphorylation of histones and nonhistone proteins in the cycloheximide-synchronized hepatocytes after the effect of radiation and serotonin

International Nuclear Information System (INIS)

Aslamova, L.I.; Blyum, Ya.B.; Tsudzevich, B.A.; Kucherenko, N.E.

1984-01-01

Phosphorylation and synthesis of histones and nonhistone proteins were studied after the inhibition of translation by sublethal cycloheximide doses. Activation of the chromatin protein phosphorylation was noted: (1) at the stage of recovery and stimulation of the protein synthesis (18-24 h), and (2) at the stage of activation of the replicative DNA synthesis (30-60 h). Phosphorylation and synthesis of the chromatin poteins depended upon the individual or combined effect of X-radiation and serotonin. The possible role of the chromatin protein phosphorylation in the response of the nuclear apparatus to the effect of radiation and serotonin the latter being used as a radioprotective agent is discussed

Histone deacetylase 1 phosphorylation at S421 and S423 is constitutive in vivo, but dispensable in vitro

International Nuclear Information System (INIS)

Karwowska-Desaulniers, Paulina; Ketko, Anastasia; Kamath, Nayana; Pflum, Mary Kay H.

2007-01-01

Histone Deacetylase 1 (HDAC1) is a transcriptional regulator associated with proliferation, apoptosis, and tumorigenesis, although its precise cellular role is unclear. HDAC1 was previously characterized as a phosphoprotein where mutation of phosphorylated S421 and S423 resulted in a loss of deacetylase activity and protein association. Here, the role of phosphorylation in regulating HDAC1 function was examined using phospho-specific antibodies. The antibody studies revealed that phosphorylation at S421 and S423 is constant during the cell cycle, under stress conditions, or in the presence of kinase or phosphatase inhibitors. Further, phosphorylation is dispensable for catalysis or protein association in vitro, as revealed by phosphatase studies. Truncation mutants of HDAC1 demonstrated that binding to Sin3A is promoted by S421 and S423 phosphorylation, while interaction with RbAp48 is not. Taken together, the data are consistent with constitutive phosphorylation of HDAC1 at S421 and S423 in vivo, which is dispensable for activity in vitro

Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex.

Science.gov (United States)

van de Werken, C; Avo Santos, M; Laven, J S E; Eleveld, C; Fauser, B C J M; Lens, S M A; Baart, E B

2015-10-01

Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos? Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote. Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described. In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways. The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins. Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the

Targeting post-translational modifications of histones for cancer therapy.

Science.gov (United States)

Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H

2015-10-30

Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.

Retinol-induced changes in the phosphorylation levels of histones and high mobility group proteins from Sertoli cells

Directory of Open Access Journals (Sweden)

Moreira J.C.F.

2000-01-01

Full Text Available Chromatin proteins play a role in the organization and functions of DNA. Covalent modifications of nuclear proteins modulate their interactions with DNA sequences and are probably one of the multiple factors involved in the process of switch on/off transcriptionally active regions of DNA. Histones and high mobility group proteins (HMG are subject to many covalent modifications that may modulate their capacity to bind to DNA. We investigated the changes induced in the phosphorylation pattern of cultured Wistar rat Sertoli cell histones and high mobility group protein subfamilies exposed to 7 µM retinol for up to 48 h. In each experiment, 6 h before the end of the retinol treatment each culture flask received 370 KBq/ml [32P]-phosphate. The histone and HMGs were isolated as previously described [Moreira et al. Medical Science Research (1994 22: 783-784]. The total protein obtained by either method was quantified and electrophoresed as described by Spiker [Analytical Biochemistry (1980 108: 263-265]. The gels were stained with Coomassie brilliant blue R-250 and the stained bands were cut and dissolved in 0.5 ml 30% H2O2 at 60oC for 12 h. The vials were chilled and 5.0 ml scintillation liquid was added. The radioactivity in each vial was determined with a liquid scintillation counter. Retinol treatment significantly changed the pattern of each subfamily of histone and high mobility group proteins.

Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons.

Science.gov (United States)

Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada; Hu, Xiaochen; Hellysaz, Arash; Falconi, Anastasia; Gomes, Ana-Luisa; Broberger, Christian; Hansen, Klaus; Fisone, Gilberto

2016-08-01

Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol, a typical antipsychotic drug, increase the phosphorylation of H3 at S28 and that this effect occurs in the context of H3K27me3. The increases in H3K27me3S28p occur in distinct populations of projection neurons located in the striatum, the major component of the basal ganglia. Genetic inactivation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K27me3S28p as a potential mediator of the effects exerted by amphetamine and haloperidol, and suggest that these drugs may act by re-activating PcG repressed target genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Histone H1 interphase phosphorylation becomes largely established in G1 or early S phase and differs in G1 between T-lymphoblastoid cells and normal T cells

Directory of Open Access Journals (Sweden)

Gréen Anna

2011-08-01

Full Text Available Abstract Background Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G1, S and G2/M populations. Results We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G1 or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. Conclusion Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G1 may be affecting the G1/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.

Stress-induced brain histone H3 phosphorylation: contribution of the intensity of stressors and length of exposure.

Science.gov (United States)

Rotllant, David; Pastor-Ciurana, Jordi; Armario, Antonio

2013-05-01

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. © 2013 International Society for Neurochemistry.

cAMP Signaling Regulates Histone H3 Phosphorylation and Mitotic Entry Through a Disruption of G2 Progression

OpenAIRE

Rodriguez-Collazo, Pedro; Snyder, Sara K.; Chiffer, Rebecca C.; Bressler, Erin A.; Voss, Ty C.; Anderson, Eric P.; Genieser, Hans-Gottfried; Smith, Catharine L.

2008-01-01

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in ...

Distribution pattern of histone H3 phosphorylation at serine 10 ...

Indian Academy of Sciences (India)

2013-08-06

Aug 6, 2013 ... tant consequences for chromatin packing due to change in histone load ... Minas Gerais, Brazil), in B. brizantha (cultivar Marandu, ... (2005), who state that the ..... Mitotic microtubule development and histone H3 phosphoryla-.

Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

International Nuclear Information System (INIS)

Jo, Hye-Ryeong; Kim, Yong-Seok; Son, Hyeon

2016-01-01

Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

Erythropoietin and carbamylated erythropoietin promote histone deacetylase 5 phosphorylation and nuclear export in rat hippocampal neurons

Energy Technology Data Exchange (ETDEWEB)

Jo, Hye-Ryeong [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Kim, Yong-Seok [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of); Son, Hyeon, E-mail: hyeonson@hanyang.ac.kr [Department of Biomedical Sciences, Graduate School of Biomedical Science and Engineering (Korea, Republic of); Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791 (Korea, Republic of)

2016-01-29

Erythropoietin (EPO) produces neurotrophic effects in animal model of neurodegeneration. However, clinical use of EPO is limited due to thrombotic risk. Carbamylated EPO (cEPO), devoid of thrombotic risk, has been proposed as a novel neuroprotective and neurotrophic agent although the molecular mechanisms of cEPO remain incomplete. Here, we show a previously unidentified role of histone deacetylase 5 (HDAC5) in the actions of EPO and cEPO. EPO and cEPO regulate the HDAC5 phosphorylation at two critical sites, Ser259 and Ser498 through a protein kinase D (PKD) dependent pathway. In addition, EPO and cEPO rapidly stimulates nuclear export of HDAC5 in rat hippocampal neurons which expressing HDAC5-GFP. Consequently, EPO and cEPO enhanced the myocyte enhancer factor-2 (MEF2) target gene expression. Taken together, our results reveal that EPO and cEPO mediate MEF2 target gene expression via the regulation of HDAC5 phosphorylation at Ser259/498, and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of EPO and cEPO.

A brief histone in time: understanding the combinatorial functions of histone PTMs in the nucleosome context.

Science.gov (United States)

Ng, Marlee K; Cheung, Peter

2016-02-01

It has been over 50 years since Allfrey et al. proposed that histone acetylation regulates RNA synthesis, and the study of histone modifications has progressed at an extraordinary pace for the past two decades. In this review, we provide a perspective on some key events and advances in our understanding of histone modifications. We also highlight reagents and tools from past to present that facilitated progress in this research field. Using histone H3 phosphorylation as an underlying thread, we review the rationale that led to the proposal of the histone code hypothesis, as well as examples that illustrate the concepts of combinatorial histone modifications and cross-talk pathways. We further highlight the importance of investigating these mechanisms in the context of nucleosomes rather than just at the histone level and present current and developing approaches for such studies. Overall, research on histone modifications has yielded great mechanistic insights into the regulation of genomic functions, and extending these studies using nucleosomes will further elucidate the complexity of these pathways in a more physiologically relevant context.

Helicobacter pylori infection-induced H3Ser10 phosphorylation in stepwise gastric carcinogenesis and its clinical implications.

Science.gov (United States)

Yang, Tao-Tao; Cao, Na; Zhang, Hai-Hui; Wei, Jian-Bo; Song, Xiao-Xia; Yi, Dong-Min; Chao, Shuai-Heng; Zhang, Li-Da; Kong, Ling-Fei; Han, Shuang-Yin; Yang, Yu-Xiu; Ding, Song-Ze

2018-04-15

Our previous works have demonstrated that Helicobacter pylori (Hp) infection can alter histone H3 serine 10 phosphorylation status in gastric epithelial cells. However, whether Helicobacter pylori-induced histone H3 serine 10 phosphorylation participates in gastric carcinogenesis is unknown. We investigate the expression of histone H3 serine 10 phosphorylation in various stages of gastric disease and explore its clinical implication. Stomach biopsy samples from 129 patients were collected and stained with histone H3 serine 10 phosphorylation, Ki67, and Helicobacter pylori by immunohistochemistry staining, expressed as labeling index. They were categorized into nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia, and intestinal-type gastric cancer groups. Helicobacter pylori infection was determined by either 13 C-urea breath test or immunohistochemistry staining. In Helicobacter pylori-negative patients, labeling index of histone H3 serine 10 phosphorylation was gradually increased in nonatrophic gastritis, chronic atrophic gastritis, intestinal metaplasia groups, peaked at low-grade intraepithelial neoplasia, and declined in high-grade intraepithelial neoplasia and gastric cancer groups. In Helicobacter pylori-infected patients, labeling index of histone H3 serine 10 phosphorylation followed the similar pattern as above, with increased expression over the corresponding Helicobacter pylori-negative controls except in nonatrophic gastritis patient whose labeling index was decreased when compared with Helicobacter pylori-negative control. Labeling index of Ki67 in Helicobacter pylori-negative groups was higher in gastric cancer than chronic atrophic gastritis and low-grade intraepithelial neoplasia groups, and higher in intestinal metaplasia group compared with chronic atrophic gastritis group. In Helicobacter pylori-positive groups, Ki67 labeling index was increased

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

2014-01-01

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

KAUST Repository

Bigeard, Jean

2014-07-10

In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

Ketamine induces brain-derived neurotrophic factor expression via phosphorylation of histone deacetylase 5 in rats.

Science.gov (United States)

Choi, Miyeon; Lee, Seung Hoon; Park, Min Hyeop; Kim, Yong-Seok; Son, Hyeon

2017-08-05

Ketamine shows promise as a therapeutic agent for the treatment of depression. The increased expression of brain-derived neurotrophic factor (BDNF) has been associated with the antidepressant-like effects of ketamine, but the mechanism of BDNF induction is not well understood. In the current study, we demonstrate that the treatment of rats with ketamine results in the dose-dependent rapid upregulation of Bdnf promoter IV activity and expression of Bdnf exon IV mRNAs in rat hippocampal neurons. Transfection of histone deacetylase 5 (HDAC5) into rat hippocampal neurons similarly induces Bdnf mRNA expression in response to ketamine, whereas transfection of a HDAC5 phosphorylation-defective mutant (Ser259 and Ser498 replaced by Ala259 and Ala498), results in the suppression of ketamine-mediated BDNF promoter IV transcriptional activity. Viral-mediated hippocampal knockdown of HDAC5 induces Bdnf mRNA and protein expression, and blocks the enhancing effects of ketamine on BDNF expression in both unstressed and stressed rats, and thereby providing evidence for the role of HDAC5 in the regulation of Bdnf expression. Taken together, our findings implicate HDAC5 in the ketamine-induced transcriptional regulation of Bdnf, and suggest that the phosphorylation of HDAC5 regulates the therapeutic actions of ketamine. Copyright © 2017 Elsevier Inc. All rights reserved.

Dynamic acetylation of all lysine 4-methylated histone H3 in the mouse nucleus: analysis at c-fos and c-jun.

Directory of Open Access Journals (Sweden)

Catherine A Hazzalin

2005-12-01

Full Text Available A major focus of current research into gene induction relates to chromatin and nucleosomal regulation, especially the significance of multiple histone modifications such as phosphorylation, acetylation, and methylation during this process. We have discovered a novel physiological characteristic of all lysine 4 (K4-methylated histone H3 in the mouse nucleus, distinguishing it from lysine 9-methylated H3. K4-methylated histone H3 is subject to continuous dynamic turnover of acetylation, whereas lysine 9-methylated H3 is not. We have previously reported dynamic histone H3 phosphorylation and acetylation as a key characteristic of the inducible proto-oncogenes c-fos and c-jun. We show here that dynamically acetylated histone H3 at these genes is also K4-methylated. Although all three modifications are proven to co-exist on the same nucleosome at these genes, phosphorylation and acetylation appear transiently during gene induction, whereas K4 methylation remains detectable throughout this process. Finally, we address the functional significance of the turnover of histone acetylation on the process of gene induction. We find that inhibition of turnover, despite causing enhanced histone acetylation at these genes, produces immediate inhibition of gene induction. These data show that all K4-methylated histone H3 is subject to the continuous action of HATs and HDACs, and indicates that at c-fos and c-jun, contrary to the predominant model, turnover and not stably enhanced acetylation is relevant for efficient gene induction.

Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

DEFF Research Database (Denmark)

Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

2007-01-01

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

Inhibition of mitotic-specific histone phophorylation by sodium arsenite

Energy Technology Data Exchange (ETDEWEB)

Cobo, J.M. [Universidad de Alcala de Henares, Madrid (Spain); Valdez, J.G.; Gurley, L.R. [Los Alamos National Lab., NM (United States)

1994-10-01

Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

The Role of Histone Protein Modifications and Mutations in Histone Modifiers in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia

Science.gov (United States)

Janczar, Szymon; Janczar, Karolina; Pastorczak, Agata; Harb, Hani; Paige, Adam J. W.; Zalewska-Szewczyk, Beata; Danilewicz, Marian; Mlynarski, Wojciech

2017-01-01

While cancer has been long recognized as a disease of the genome, the importance of epigenetic mechanisms in neoplasia was acknowledged more recently. The most active epigenetic marks are DNA methylation and histone protein modifications and they are involved in basic biological phenomena in every cell. Their role in tumorigenesis is stressed by recent unbiased large-scale studies providing evidence that several epigenetic modifiers are recurrently mutated or frequently dysregulated in multiple cancers. The interest in epigenetic marks is especially due to the fact that they are potentially reversible and thus druggable. In B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) there is a relative paucity of reports on the role of histone protein modifications (acetylation, methylation, phosphorylation) as compared to acute myeloid leukemia, T-cell ALL, or other hematologic cancers, and in this setting chromatin modifications are relatively less well studied and reviewed than DNA methylation. In this paper, we discuss the biomarker associations and evidence for a driver role of dysregulated global and loci-specific histone marks, as well as mutations in epigenetic modifiers in BCP-ALL. Examples of chromatin modifiers recurrently mutated/disrupted in BCP-ALL and associated with disease outcomes include MLL1, CREBBP, NSD2, and SETD2. Altered histone marks and histone modifiers and readers may play a particular role in disease chemoresistance and relapse. We also suggest that epigenetic regulation of B-cell differentiation may have parallel roles in leukemogenesis. PMID:28054944

Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

Science.gov (United States)

Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

2015-01-01

Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

2013-04-26

Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

Implication of Posttranslational Histone Modifications in Nucleotide Excision Repair

Directory of Open Access Journals (Sweden)

Shisheng Li

2012-09-01

Full Text Available Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4 or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN.

Science.gov (United States)

Kumar, Santhosh V R; Kulkarni, Onkar P; Mulay, Shrikant R; Darisipudi, Murthy N; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen; Anders, Hans-Joachim

2015-10-01

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN. Copyright © 2015 by the American Society of Nephrology.

The Role of Dietary Histone Deacetylases (HDACs Inhibitors in Health and Disease

Directory of Open Access Journals (Sweden)

Shalome A. Bassett

2014-10-01

Full Text Available Modification of the histone proteins associated with DNA is an important process in the epigenetic regulation of DNA structure and function. There are several known modifications to histones, including methylation, acetylation, and phosphorylation, and a range of factors influence each of these. Histone deacetylases (HDACs remove the acetyl group from lysine residues within a range of proteins, including transcription factors and histones. Whilst this means that their influence on cellular processes is more complex and far-reaching than histone modifications alone, their predominant function appears to relate to histones; through deacetylation of lysine residues they can influence expression of genes encoded by DNA linked to the histone molecule. HDAC inhibitors in turn regulate the activity of HDACs, and have been widely used as therapeutics in psychiatry and neurology, in which a number of adverse outcomes are associated with aberrant HDAC function. More recently, dietary HDAC inhibitors have been shown to have a regulatory effect similar to that of pharmacological HDAC inhibitors without the possible side-effects. Here, we discuss a number of dietary HDAC inhibitors, and how they may have therapeutic potential in the context of a whole food.

Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

Science.gov (United States)

Hazzalin, Catherine A; Mahadevan, Louis C

2017-01-01

Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

Biological Significance of the Suppression of Oxidative Phosphorylation in Induced Pluripotent Stem Cells

Directory of Open Access Journals (Sweden)

Cheng Zhang

2017-11-01

Full Text Available We discovered that induced pluripotent stem cell (iPSC clones generated from aged tissue donors (A-iPSCs fail to suppress oxidative phosphorylation. Compared to embryonic stem cells (ESCs and iPSCs generated from young donors (Y-iPSCs, A-iPSCs show poor expression of the pluripotent stem cell-specific glucose transporter 3 (GLUT3 and impaired glucose uptake, making them unable to support the high glucose demands of glycolysis. Persistent oxidative phosphorylation in A-iPSCs generates higher levels of reactive oxygen species (ROS, which leads to excessive elevation of glutathione (a ROS-scavenging metabolite and a blunted DNA damage response. These phenotypes were recapitulated in Y-iPSCs by inhibiting pyruvate dehydrogenase kinase (PDK or supplying citrate to activate oxidative phosphorylation. In addition, oxidative phosphorylation in A-iPSC clones depletes citrate, a nuclear source of acetyl group donors for histone acetylation; this consequently alters histone acetylation status. Expression of GLUT3 in A-iPSCs recovers the metabolic defect, DNA damage response, and histone acetylation status.

Phosphorylation of Histone H2A.X in Peripheral Blood Mononuclear Cells May Be a Useful Marker for Monitoring Cardiometabolic Risk in Nondiabetic Individuals

Directory of Open Access Journals (Sweden)

So Ra Yoon

2017-01-01

Full Text Available Phosphorylation of H2A.X (serine 139 in the histone H2A family located in the downstream of the DNA damage kinase signaling cascade is an important indicator of DNA damage. Recently, phosphorylation of H2A.X was proposed as a sensitive biomarker of aging. This study investigated if phosphorylation of H2A.X in peripheral blood mononuclear cells (PBMCs is associated with cardiometabolic risk in nondiabetic individuals. Basic parameters and oxidative stress/inflammatory markers were measured in nondiabetic healthy Koreans (n=119. Phosphorylation of H2A.X was measured randomly among the study subjects using a flow cytometer. According to the number of metabolic syndrome risk factor (MetS-RF, the study subjects were subdivided into “super healthy” (MetS−RF=0, n=71 and “MetS-risk” (MetS−RF≥1, n=48 groups. Phosphorylation of H2A.X in PBMCs (percentages and mean fluorescence intensity was significantly higher in the MetS-risk group than in the super healthy group after adjusting for age, sex, cigarette smoking, and alcohol consumption. Phosphorylated H2A.X was positively correlated with the number of MetS-RF as well as waist circumference, blood pressures, triglyceride, HbA1C, oxidized LDL, high sensitivity C-reactive protein, tumor necrosis factor-alpha, and alanine aminotransferase after the adjustment. The present study suggested that phosphorylated H2A.X in circulating PBMCs measured by flow cytometer may be a useful marker for monitoring cardiometabolic risk in nondiabetic individuals.

Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes

Czech Academy of Sciences Publication Activity Database

Neumann, Pavel; Schubert, V.; Vrbová, Iva; Manning, Jasper Eugene; Houben, A.; Macas, Jiří

2016-01-01

Roč. 7, č. 234 (2016) ISSN 1664-462X R&D Projects: GA ČR(CZ) GAP501/11/1843 Institutional support: RVO:60077344 Keywords : Centromere structure * epigenetic modifications * histone phosphorylation * histone methylation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.298, year: 2016

Epigenetic regulation of the NR4A orphan nuclear receptor NOR1 by histone acetylation.

Science.gov (United States)

Zhao, Yue; Nomiyama, Takashi; Findeisen, Hannes M; Qing, Hua; Aono, Jun; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

2014-12-20

The nuclear receptor NOR1 is an immediate-early response gene implicated in the transcriptional control of proliferation. Since the expression level of NOR1 is rapidly induced through cAMP response element binding (CREB) protein-dependent promoter activation, we investigated the contribution of histone acetylation to this transient induction. We demonstrate that NOR1 transcription is induced by histone deacetylase (HDAC) inhibition and by depletion of HDAC1 and HDAC3. HDAC inhibition activated the NOR1 promoter, increased histone acetylation and augmented the recruitment of phosphorylated CREB to the promoter. Furthermore, HDAC inhibition increased Ser133 phosphorylation of CREB and augmented NOR1 protein stability. These data outline previously unrecognized mechanisms of NOR1 regulation and illustrate a key role for histone acetylation in the rapid induction of NOR1. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Vitamin K3 (menadione)-induced oncosis associated with keratin 8 phosphorylation and histone H3 arylation.

Science.gov (United States)

Scott, Gary K; Atsriku, Christian; Kaminker, Patrick; Held, Jason; Gibson, Brad; Baldwin, Michael A; Benz, Christopher C

2005-09-01

The vitamin K analog menadione (K3), capable of both redox cycling and arylating nucleophilic substrates by Michael addition, has been extensively studied as a model stress-inducing quinone in both cell culture and animal model systems. Exposure of keratin 8 (k-8) expressing human breast cancer cells (MCF7, T47D, SKBr3) to K3 (50-100 microM) induced rapid, sustained, and site-specific k-8 serine phosphorylation (pSer73) dependent on signaling by a single mitogen activated protein kinase (MAPK) pathway, MEK1/2. Normal nuclear morphology and k-8 immunofluorescence coupled with the lack of DNA laddering or other features of apoptosis indicated that K3-induced cytotoxicity, evident within 4 h of treatment and delayed but not prevented by MEK1/2 inhibition, was due to a form of stress-activated cell death known as oncosis. Independent of MAPK signaling was the progressive appearance of K3-induced cellular fluorescence, principally nuclear in origin and suggested by in vitro fluorimetry to have been caused by K3 thiol arylation. Imaging by UV transillumination of protein gels containing nuclear extracts from K3-treated cells revealed a prominent 17-kDa band shown to be histone H3 by immunoblotting and mass spectrometry (MS). K3 arylation of histones in vitro followed by electrospray ionization-tandem MS analyses identified the unique Cys110 residue within H3, exposed only in the open chromatin of transcriptionally active genes, as a K3 arylation target. These findings delineate new pathways associated with K3-induced stress and suggest a potentially novel role for H3 Cys110 as a nuclear stress sensor.

Development of a new rapid HPLC method for the fractionation of histones

International Nuclear Information System (INIS)

Gurley, L.R.; Valdez, J.G.; Prentice, D.A.; Spall, W.D.

1983-01-01

To study histone functions, it is necessary to fractionate the histones into their five classes (H1, H2A, H2B, H3 and H4) and then to subfractionate these classes into variants having slightly different primary structures and into different phosphorylated and acetylated forms. With the advent of high-performance liquid chromatography (HPLC), it was hoped that laborious and time-consuming conventional methods could be replaced by a simple, rapid, high-resolving HPLC method for fractionating histones. However, problems of irreversible adsorption of the histones to HPLC column packings discouraged this development. Our laboratory has now determined that the strong adsorption of histones to HPLC columns results from two different forces: (1) polar interactions between the histones and the silanol groups of silica-based HPLC column packing, and (2) hydrophobic interactions between the histones and the bound organic phase of the column packings. By minimizing these forces, we have succeeded in developing an HPLC method suitable for histone studies

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage

OpenAIRE

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-01-01

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly ...

Distribution pattern of histone H3 phosphorylation at serine 10

Indian Academy of Sciences (India)

We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated ...

Developmentally Regulated Post-translational Modification of Nucleoplasmin Controls Histone Sequestration and Deposition

Directory of Open Access Journals (Sweden)

Takashi Onikubo

2015-03-01

Full Text Available Nucleoplasmin (Npm is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here, we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity toward the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction, leading to histone sequestration in the egg.

Effect of SPL (Spent Pot Liner) and its main components on root growth, mitotic activity and phosphorylation of Histone H3 in Lactuca sativa L.

Science.gov (United States)

Freitas, Aline Silva; Fontes Cunha, Isabela Martinez; Andrade-Vieira, Larissa Fonseca; Techio, Vânia Helena

2016-02-01

Spent Pot Liner (SPL) is a solid waste from the aluminum industry frequently disposed of in industrial landfills; it can be leached and contaminate the soil, sources of drinking water and plantations, and thus may pose a risk to human health and to ecosystems. Its composition is high variable, including cyanide, fluoride and aluminum salts, which are highly toxic and environmental pollutants. This study evaluated the effect of SPL and its main components on root growth and the mitosis of Lactuca sativa, by investigating the mechanisms of cellular and chromosomal alterations with the aid of immunolocalization. To this end, newly emerged roots of L. sativa were exposed to SPL and its main components (solutions of cyanide, fluoride and aluminum) and to calcium chloride (control) for 48h. After this, root length was measured and cell cycle was examined by means of conventional cytogenetics and immunolocalization. Root growth was inhibited in the treatments with SPL and aluminum; chromosomal and nuclear alterations were observed in all treatments. The immunolocalization evidenced normal dividing cells with regular temporal and spatial distribution of histone H3 phosphorylation at serine 10 (H3S10ph). However, SPL and its main components inhibited the phosphorylation of histone H3 at serine 10, inactivated pericentromeric regions and affected the cohesion of sister chromatids, thus affecting the arrangement of chromosomes in the metaphase plate and separation of chromatids in anaphase. In addition, these substances induced breaks in pericentromeric regions, characterized as fragile sites. Copyright © 2015 Elsevier Inc. All rights reserved.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

Science.gov (United States)

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-05-19

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Trichostatin-A induces differential changes in histone protein dynamics and expression in HeLa cells

International Nuclear Information System (INIS)

Rao, Jyothsna; Bhattacharya, Dipanjan; Banerjee, Bidisha; Sarin, Apurva; Shivashankar, G.V.

2007-01-01

Trichostatin-A (TSA), a histone deacetylase (HDAC) inhibitor, results in enhanced acetylation of core histones thereby disrupting chromatin organization within living cells. We report on changes in chromatin organization and the resultant alteration in nuclear architecture following treatment with TSA using fluorescence imaging. TSA triggers an expected increase in the euchromatin fraction which is accompanied by a significant increase in nuclear volume and alterations in chromatin compaction mapped using fluorescence anisotropy imaging. We observe differential changes in the mobility of core and linker histones as measured by fluorescence recovery after photo-bleaching (FRAP) and fluorescence correlation spectroscopy (FCS) methods. Further TSA induces a differential increase in linker histone transcription and increased phosphorylation of linker histone proteins accompanying an expected increase in core histone acetylation patterns. Thus subtle feedback responses triggered by changes in chromatin configurations impinge selectively on linker histone mobility and its expression. These observations have implications for understanding the role of HDAC in the dynamic maintenance of chromatin organization

Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

Science.gov (United States)

Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia

2015-01-15

Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to

Histone Lysine Methylation and Neurodevelopmental Disorders

Directory of Open Access Journals (Sweden)

Jeong-Hoon Kim

2017-06-01

Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

A positive feedback loop links opposing functions of P-TEFb/Cdk9 and histone H2B ubiquitylation to regulate transcript elongation in fission yeast.

Directory of Open Access Journals (Sweden)

Miriam Sansó

Full Text Available Transcript elongation by RNA polymerase II (RNAPII is accompanied by conserved patterns of histone modification. Whereas histone modifications have established roles in transcription initiation, their functions during elongation are not understood. Mono-ubiquitylation of histone H2B (H2Bub1 plays a key role in coordinating co-transcriptional histone modification by promoting site-specific methylation of histone H3. H2Bub1 also regulates gene expression through an unidentified, methylation-independent mechanism. Here we reveal bidirectional communication between H2Bub1 and Cdk9, the ortholog of metazoan positive transcription elongation factor b (P-TEFb, in the fission yeast Schizosaccharomyces pombe. Chemical and classical genetic analyses indicate that lowering Cdk9 activity or preventing phosphorylation of its substrate, the transcription processivity factor Spt5, reduces H2Bub1 in vivo. Conversely, mutations in the H2Bub1 pathway impair Cdk9 recruitment to chromatin and decrease Spt5 phosphorylation. Moreover, an Spt5 phosphorylation-site mutation, combined with deletion of the histone H3 Lys4 methyltransferase Set1, phenocopies morphologic and growth defects due to H2Bub1 loss, suggesting independent, partially redundant roles for Cdk9 and Set1 downstream of H2Bub1. Surprisingly, mutation of the histone H2B ubiquitin-acceptor residue relaxes the Cdk9 activity requirement in vivo, and cdk9 mutations suppress cell-morphology defects in H2Bub1-deficient strains. Genome-wide analyses by chromatin immunoprecipitation also demonstrate opposing effects of Cdk9 and H2Bub1 on distribution of transcribing RNAPII. Therefore, whereas mutual dependence of H2Bub1 and Spt5 phosphorylation indicates positive feedback, mutual suppression by cdk9 and H2Bub1-pathway mutations suggests antagonistic functions that must be kept in balance to regulate elongation. Loss of H2Bub1 disrupts that balance and leads to deranged gene expression and aberrant cell

Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus

Science.gov (United States)

Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene

2016-01-01

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees’ CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

Regulation of protein phosphorylation in oat mitochondria

International Nuclear Information System (INIS)

Pike, C.; Kopeck, K.; Sceppa, E.

1989-01-01

We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with 32 P-γ-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of 35 S-adenosine thiotriphosphate

Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

Science.gov (United States)

Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

2016-08-17

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity

International Nuclear Information System (INIS)

Di Renzo, Francesca; Cappelletti, Graziella; Broccia, Maria L.; Giavini, Erminio; Menegola, Elena

2007-01-01

Histone deacetylases (HDAC) control gene expression by changing histonic as well as non histonic protein conformation. HDAC inhibitors (HDACi) are considered to be among the most promising drugs for epigenetic treatment for cancer. Recently a strict relationship between histone hyperacetylation in specific tissues of mouse embryos exposed to two HDACi (valproic acid and trichostatin A) and specific axial skeleton malformations has been demonstrated. The aim of this study is to verify if boric acid (BA), that induces in rodents malformations similar to those valproic acid and trichostatin A-related, acts through similar mechanisms: HDAC inhibition and histone hyperacetylation. Pregnant mice were treated intraperitoneally with a teratogenic dose of BA (1000 mg/kg, day 8 of gestation). Western blot analysis and immunostaining were performed with anti hyperacetylated histone 4 (H4) antibody on embryos explanted 1, 3 or 4 h after treatment and revealed H4 hyperacetylation at the level of somites. HDAC enzyme assay was performed on embryonic nuclear extracts. A significant HDAC inhibition activity (compatible with a mixed type partial inhibition mechanism) was evident with BA. Kinetic analyses indicate that BA modifies substrate affinity by a factor α = 0.51 and maximum velocity by a factor β = 0.70. This work provides the first evidence for HDAC inhibition by BA and suggests such a molecular mechanism for the induction of BA-related malformations

Histone H2AX in DNA repair

International Nuclear Information System (INIS)

Lewandowska, H.; Szumiel, I.

2002-01-01

The paper reviews the recent reports on the role of the phosphorylated histone H2AX (γ-H2AX). The modification of this histone is an important part of the cellular response to the induction of DNA double strand brakes (DSB) by ionising radiation and other DSB-generating factors. In irradiated cells the modification is carried out mainly by ATM (ataxia-telangiectasia mutated) kinase, the enzyme that starts the alarm signalling upon induction of DSB.γ-H2AX molecules are formed within 1-3 min after irradiation and form foci at the sites of DSB. This seems to be necessary for the recruitment of repair factors that are later present in foci of damaged nuclei. Modification of a constant percentage of H2AX molecules per DSB takes place, corresponding to chromatin domains of megabase of DNA. (author)

MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

KAUST Repository

Latrasse, David; Jé gu, Teddy; Li, Huchen; Zé licourt, Axel de; Raynaud, Cé cile; Legras, Sté phanie; Gust, Andrea; Samajova, Olga; Veluchamy, Alaguraj; Rayapuram, Naganand; Ramirez Prado, Juan Sebastian; Kulikova, Olga; Colcombet, Jean; Bigeard, Jean; Genot, Baptiste; Bisseling, Ton; Benhamed, Moussa; Hirt, Heribert

2017-01-01

Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

MAPK-triggered chromatin reprogramming by histone deacetylase in plant innate immunity

KAUST Repository

Latrasse, David

2017-07-06

Microbial-associated molecular patterns activate several MAP kinases, which are major regulators of the innate immune response in Arabidopsis thaliana that induce large-scale changes in gene expression. Here, we determine whether microbial-associated molecular pattern-triggered gene expression involves modifications at the chromatin level.Histone acetylation and deacetylation are major regulators of microbial-associated molecular pattern-triggered gene expression and implicate the histone deacetylase HD2B in the reprogramming of defence gene expression and innate immunity. The MAP kinase MPK3 directly interacts with and phosphorylates HD2B, thereby regulating the intra-nuclear compartmentalization and function of the histone deacetylase.By studying a number of gene loci that undergo microbial-associated molecular pattern-dependent activation or repression, our data reveal a mechanistic model for how protein kinase signaling directly impacts chromatin reprogramming in plant defense.

Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1.

Science.gov (United States)

Kobayashi, Junya; Tauchi, Hiroshi; Chen, Benjamin; Burma, Sandeep; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J; Komatsu, Kenshi

2009-03-20

Phosphorylated histone H2AX (gamma-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with gamma-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether gamma-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a gamma-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-gamma-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, gamma-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.

Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1

International Nuclear Information System (INIS)

Kobayashi, Junya; Tauchi, Hiroshi; Chen, Benjamin; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J.; Komatsu, Kenshi

2009-01-01

Phosphorylated histone H2AX (γ-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with γ-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether γ-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a γ-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-γ-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, γ-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.

Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability.

Science.gov (United States)

Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Jesús Cañal, Maria; Rodríguez, Roberto

2010-03-15

Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs. Copyright 2009 Elsevier GmbH. All rights reserved.

Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns

International Nuclear Information System (INIS)

Gurley, L.R.; Prentice, D.A.; Valdez, J.G.; Spall, W.D.

1983-01-01

Previous work described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C 18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a μBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C 18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [ 3 H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species

Effects of Forced Swimming Stress on ERK and Histone H3 Phosphorylation in Limbic Areas of Roman High- and Low-Avoidance Rats.

Science.gov (United States)

Morello, Noemi; Plicato, Ornella; Piludu, Maria Antonietta; Poddighe, Laura; Serra, Maria Pina; Quartu, Marina; Corda, Maria Giuseppa; Giorgi, Osvaldo; Giustetto, Maurizio

2017-01-01

Stressful events evoke molecular adaptations of neural circuits through chromatin remodeling and regulation of gene expression. However, the identity of the molecular pathways activated by stress in experimental models of depression is not fully understood. We investigated the effect of acute forced swimming (FS) on the phosphorylation of the extracellular signal-regulated kinase (ERK)1/2 (pERK) and histone H3 (pH3) in limbic brain areas of genetic models of vulnerability (RLA, Roman low-avoidance rats) and resistance (RHA, Roman high-avoidance rats) to stress-induced depression-like behavior. We demonstrate that FS markedly increased the density of pERK-positive neurons in the infralimbic (ILCx) and the prelimbic area (PrLCx) of the prefrontal cortex (PFCx), the nucleus accumbens, and the dorsal blade of the hippocampal dentate gyrus to the same extent in RLA and RHA rats. In addition, FS induced a significant increase in the intensity of pERK immunoreactivity (IR) in neurons of the PFCx in both rat lines. However, RHA rats showed stronger pERK-IR than RLA rats in the ILCx both under basal and stressed conditions. Moreover, the density of pH3-positive neurons was equally increased by FS in the PFCx of both rat lines. Interestingly, pH3-IR was higher in RHA than RLA rats in PrLCx and ILCx, either under basal conditions or upon FS. Finally, colocalization analysis showed that in the PFCx of both rat lines, almost all pERK-positive cells express pH3, whereas only 50% of the pH3-positive neurons is also pERK-positive. Moreover, FS increased the percentage of neurons that express exclusively pH3, but reduced the percentage of cells expressing exclusively pERK. These results suggest that (i) the distinctive patterns of FS-induced ERK and H3 phosphorylation in the PFCx of RHA and RLA rats may represent molecular signatures of the behavioural traits that distinguish the two lines and (ii) FS-induced H3 phosphorylation is, at least in part, ERK-independent.

Histone deacetylase inhibitors reverse age-related increases in side effects of haloperidol in mice.

Science.gov (United States)

Montalvo-Ortiz, Janitza L; Fisher, Daniel W; Rodríguez, Guadalupe; Fang, Deyu; Csernansky, John G; Dong, Hongxin

2017-08-01

Older patients can be especially susceptible to antipsychotic-induced side effects, and the pharmacodynamic mechanism underlying this phenomenon remains unclear. We hypothesized that age-related epigenetic alterations lead to decreased expression and functionality of the dopamine D2 receptor (D2R), contributing to this susceptibility. In this study, we treated young (2-3 months old) and aged (22-24 months old) C57BL/6 mice with the D2R antagonist haloperidol (HAL) once a day for 14 days to evaluate HAL-induced motor side effects. In addition, we pretreated separate groups of young and aged mice with histone deacetylase (HDAC) inhibitors valproic acid (VPA) or entinostat (MS-275) and then administered HAL. Our results show that the motor side effects of HAL are exaggerated in aged mice as compared to young mice and that HDAC inhibitors are able to reverse the severity of these deficits. HAL-induced motor deficits in aged mice are associated with an age- and drug-dependent decrease in striatal D2R protein levels and functionality. Further, histone acetylation was reduced while histone tri-methylation was increased at specific lysine residues of H3 and H4 within the Drd2 promoter in the striatum of aged mice. HDAC inhibitors, particularly VPA, restored striatal D2R protein levels and functionality and reversed age- and drug-related histone modifications at the Drd2 promoter. These results suggest that epigenetic changes at the striatal Drd2 promoter drive age-related increases in antipsychotic side effect susceptibility, and HDAC inhibitors may be an effective adjunct treatment strategy to reduce side effects in aged populations.

Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

DEFF Research Database (Denmark)

Gotfryd, Kamil; Skladchikova, Galina; Lepekhin, Eugene E

2010-01-01

lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3). Results: VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1......ABSTRACT: BACKGROUND: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods: The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell....../2 phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK...

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

Science.gov (United States)

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

Science.gov (United States)

Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

2011-01-01

Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

Aging and radiation induced alternations in liver histones

International Nuclear Information System (INIS)

Kozurkova, M.; Misurova, E.; Kropacova, K.

1994-01-01

Age-related changes in histones in the liver of normal rats and in rats irradiated with 5.7 Gy gamma rays were examined. Quantitative histone changes in growing and aging rats (from 1 to 28 months of age) were found to be mild only. As they paralleled the DNA changes, the histone /DNA ratio remained stable with age. In total extracted histones there was a decrease in the H1 proportion in older groups with preceding increase in the H1 grad proportion. Thirty minutes after irradiation the amount of histones was reduced with age, probably due to an impaired extractability of histones. As the quantitative DNA changes were milder, the histone?DNA ratio decreased in aging liver after irradiation. Similar patterns of changes in proportion of the H1 fraction and H1 grad sub-fraction were observed in irradiated and nonirradiated animals in the former with an earlier onset. Irradiation, therefore, accelerated spontaneous age-related alternations. (author)

Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.

Science.gov (United States)

Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang

2016-01-21

Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

Science.gov (United States)

Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

2012-09-21

Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Deficit in DNA content relative to histones in X-irradiated HeLa cells

International Nuclear Information System (INIS)

Bases, R.; Mendez, F.; Neubort, S.

1976-01-01

The DNA and histone content of HeLa S-3 cell cultures was measured by direct mass assays 21 hours after 1000 rad of X-irradiation, when the cells were arrested in G2 phase. The nuclear DNA content of such cultures was found to be deficient (73 per cent of control values). In contrast, the synthesis of nuclear histones persisted, and the total histone content was close to 100 per cent of control values. When synchronously-growing cultures were irradiated in mid-S phase and examined 3.5 hours later in G2 phase, both DNA and histone content were equal to control values. (author)

Histone Lysine Methylation in Diabetic Nephropathy

Directory of Open Access Journals (Sweden)

Guang-dong Sun

2014-01-01

Full Text Available Diabetic nephropathy (DN belongs to debilitating microvascular complications of diabetes and is the leading cause of end-stage renal diseases worldwide. Furthermore, outcomes from the DCCT/EDIC study showed that DN often persists and progresses despite intensive glucose control in many diabetes patients, possibly as a result of prior episode of hyperglycemia, which is called “metabolic memory.” The underlying mechanisms responsible for the development and progression of DN remain poorly understood. Activation of multiple signaling pathways and key transcription factors can lead to aberrant expression of DN-related pathologic genes in target renal cells. Increasing evidence suggests that epigenetic mechanisms in chromatin such as DNA methylation, histone acetylation, and methylation can influence the pathophysiology of DN and metabolic memory. Exciting researches from cell culture and experimental animals have shown that key histone methylation patterns and the related histone methyltransferases and histone demethylases can play important roles in the regulation of inflammatory and profibrotic genes in renal cells under diabetic conditions. Because histone methylation is dynamic and potentially reversible, it can provide a window of opportunity for the development of much-needed novel therapeutic potential for DN in the future. In this minireview, we discuss recent advances in the field of histone methylation and its roles in the pathogenesis and progression of DN.

Patients with HBV-related acute-on-chronic liver failure have increased concentrations of extracellular histones aggravating cellular damage and systemic inflammation.

Science.gov (United States)

Li, X; Gou, C; Yao, L; Lei, Z; Gu, T; Ren, F; Wen, T

2017-01-01

Acute-on-chronic liver failure (ACLF) is the most common type of liver failure and associated with grave consequences. Systemic inflammation has been linked to its pathogenesis and outcome, but the identifiable triggers are absent. Recently, extracellular histones, especially H4, have been recognized as important mediators of cell damage in various inflammatory conditions. This study aimed to investigate whether extracellular histones have clinical implications in patients with hepatitis B virus (HBV)-related ACLF. One hundred and twelve patients with HBV-related ACLF, 90 patients with chronic hepatitis B, 88 patients with HBV-related liver cirrhosis and 40 healthy volunteers were entered into this study. Plasma histone H4 levels, cytokine profile and clinical data were obtained. Besides, patient's sera were incubated overnight with human L02 hepatocytes or monocytic U937 cells in the presence or absence of antihistone H4 antibody, and cellular damage and cytokine production were evaluated. We found that plasma histone H4 levels were greatly increased in patients with ACLF as compared with chronic hepatitis B, liver cirrhosis and healthy control subjects and were significantly associated with disease severity, systemic inflammation and outcome. Notably, ACLF patients' sera incubation decreased cultured L02 cell integrity and induced profound cytokine production in the supernatant of U937 cells. Antihistone H4 antibody treatment abrogated these adverse effects, thus confirming a cause-effect relationship between extracellular histones and organ injury/dysfunction. The data support the hypothesis that the increased extracellular histone levels in ACLF patients may aggravate disease severity by inducing cellular injury and systemic inflammation. Histone-targeted therapies may have potentially interventional value in clinical practice. © 2016 John Wiley & Sons Ltd.

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation.

Science.gov (United States)

Kurat, Christoph F; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

2014-09-30

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase-specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APC(Cdh1)) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation.

Cell cycle-regulated oscillator coordinates core histone gene transcription through histone acetylation

Science.gov (United States)

Kurat, Christoph F.; Lambert, Jean-Philippe; Petschnigg, Julia; Friesen, Helena; Pawson, Tony; Rosebrock, Adam; Gingras, Anne-Claude; Fillingham, Jeffrey; Andrews, Brenda

2014-01-01

DNA replication occurs during the synthetic (S) phase of the eukaryotic cell cycle and features a dramatic induction of histone gene expression for concomitant chromatin assembly. Ectopic production of core histones outside of S phase is toxic, underscoring the critical importance of regulatory pathways that ensure proper expression of histone genes. Several regulators of histone gene expression in the budding yeast Saccharomyces cerevisiae are known, yet the key oscillator responsible for restricting gene expression to S phase has remained elusive. Here, we show that suppressor of Ty (Spt)10, a putative histone acetyltransferase, and its binding partner Spt21 are key determinants of S-phase–specific histone gene expression. We show that Spt21 abundance is restricted to S phase in part by anaphase promoting complex Cdc20-homologue 1 (APCCdh1) and that it is recruited to histone gene promoters in S phase by Spt10. There, Spt21-Spt10 enables the recruitment of a cascade of regulators, including histone chaperones and the histone-acetyltransferase general control nonderepressible (Gcn) 5, which we hypothesize lead to histone acetylation and consequent transcription activation. PMID:25228766

Histone Acetylation in Fungal Pathogens of Plants

Directory of Open Access Journals (Sweden)

Junhyun Jeon

2014-03-01

Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

Rtt109-dependent histone H3 K56 acetylation and gene activity are essential for the biological control potential of Beauveria bassiana.

Science.gov (United States)

Cai, Qing; Wang, Juan-Juan; Shao, Wei; Ying, Sheng-Hua; Feng, Ming-Guang

2018-04-27

Rtt109 is a histone acetyltransferase that catalyzes histone H3K56 acetylation required for genomic stability, DNA damage repair and virulence-related gene activity in yeast-like human pathogens but remains functionally unknown in fungal insect pathogens. This study seeks to elucidate catalytic activity of Rtt109 orthologue and its possible role in sustaining biological control potential of Beauveria bassiana, a fungal entomopathogen. Deletion of rtt109 in B. bassiana abolished histone H3K56 acetylation and triggered histone H2A-S129 phosphorylation. Consequently, the deletion mutant showed increased sensitivities to the stresses of DNA damage, oxidation, cell wall perturbation, high osmolarity and heat shock during colony growth, severe conidiation defects under normal culture conditions, reduced conidial hydrophobicity, decreased conidial UV-B resistance, and attenuated virulence through normal cuticle infection. These phenotypic changes correlated well with reduced transcript levels of many genes, which encode the families of H2A-S129 dephosphorylation-related protein phosphotases, DNA damage-repairing factors, antioxidant enzymes, heat-shock proteins, key developmental activators, hydrophobins and cuticle-degrading Pr1 proteases respectively. Rtt109 can acetylate H3K56 and dephosphorylate H2A-S129 in direct and indirect manners respectively, and hence plays an essential role in sustaining genomic stability and global gene activity required for conidiation capacity, environmental fitness and pest-control potential in B. bassiana. This article is protected by copyright. All rights reserved.

Histones bundle F-actin filaments and affect actin structure.

Science.gov (United States)

Blotnick, Edna; Sol, Asaf; Muhlrad, Andras

2017-01-01

Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

Postmortem Changes in Pork Muscle Protein Phosphorylation in Relation to the RN Genotype

DEFF Research Database (Denmark)

Lametsch, René; Larsen, Martin Røssel; Essén-Gustavsson, Birgitta

2011-01-01

Postmortem changes in pork muscle protein phosphorylation in relation to the RN(-) genotype were investigated using one-dimensional gel electrophoresis and a phosphor specific staining. The phosphorylation levels of several protein bands were found to be affected by the RN(-) genotype and to change...... of phosphorylation of these key enzymes during the postmortem metabolism. The results illustrate that the protein phosphorylation level of the muscle proteins could be interpreted as a global metabolic fingerprint containing information about the activity status of the enzymes in the postmortem metabolism....... during postmortem development. Glycogen phosphorylase, phosphofructokinase, and pyruvate kinase were found in protein bands affected by the RN(-) genotype, and the phosphorylation profile indicates that part of the increased rate and extended pH decline of the RN(-) genotype could be a consequence...

Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes.

Science.gov (United States)

Maity, Suman Kumar; Jbara, Muhammad; Mann, Guy; Kamnesky, Guy; Brik, Ashraf

2017-11-01

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl] 2 and PdCl 2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within ∼20-30 d.

Replication stress interferes with histone recycling and predeposition marking of new histones

DEFF Research Database (Denmark)

Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine

2010-01-01

To restore chromatin on new DNA during replication, recycling of histones evicted ahead of the fork is combined with new histone deposition. The Asf1 histone chaperone, which buffers excess histones under stress, is a key player in this process. Yet how histones handled by human Asf1 are modified...... remains unclear. Here we identify marks on histones H3-H4 bound to Asf1 and changes induced upon replication stress. In S phase, distinct cytosolic and nuclear Asf1b complexes show ubiquitous H4K5K12diAc and heterogeneous H3 marks, including K9me1, K14ac, K18ac, and K56ac. Upon acute replication arrest......, the predeposition mark H3K9me1 and modifications typical of chromatin accumulate in Asf1 complexes. In parallel, ssDNA is generated at replication sites, consistent with evicted histones being trapped with Asf1. During recovery, histones stored with Asf1 are rapidly used as replication resumes. This shows...

Involvement of histone H3 phosphorylation via the activation of p38 MAPK pathway and intracellular redox status in cytotoxicity of HL-60 cells induced by Vitex agnus-castus fruit extract.

Science.gov (United States)

Kikuchi, Hidetomo; Yuan, Bo; Yuhara, Eisuke; Imai, Masahiko; Furutani, Ryota; Fukushima, Shin; Hazama, Shingo; Hirobe, Chieko; Ohyama, Kunio; Takagi, Norio; Toyoda, Hiroo

2014-08-01

We have demonstrated that an extract from the ripe fruit of Vitex angus-castus (Vitex), might be a promising anticancer candidate. In order to further provide a molecular rationale for clinical development in anticancer therapy, a detailed mechanism underlying the efficacy of Vitex against HL-60 cells was investigated. Vitex induced a dose- and time-dependent decrease in cell viability associated with induction of apoptosis and G(2)/M cell cycle arrest, both of which were suppressed by the addition of SB203580, an inhibitor for p38 MAPK. Furthermore, SB203580 significantly suppressed Vitex-induced phosphorylation of histone H3, a downstream molecule of p38 MAPK known to be involved in apoptosis induction in tumor cells. Notably, Vitex induced upregulation of intracellular ATP, known to bind its binding pocket inside activated p38 MAPK and to be required for the activation of p38 MAPK pathway. These results, thus, suggest that upregulation of intracellular ATP and phosphorylation of histone H3 are closely associated with the activation of p38 MAPK pathway, consequently contributing to Vitex-mediated cytotoxicity. Intriguingly, a significant decrease of intracellular ROS levels and downregulation of expression level of gp91(phox), an important component of NADPH oxidase, were observed in Vitex-treated cells. A greater decline in ROS levels along with enhanced apoptosis was observed after treatment with Vitex in combination with SnPP, an inhibitor specific for HO-1. Since NADPH oxidase and HO-1 are closely correlated to redox status associated with intracellular ROS levels, the two enzymes are suggested to be implicated in Vitex-mediated cytotoxicity in HL-60 cells by regulating ROS generation. We also suggest that activation of the p38 MAPK pathway may be dependent on the alterations of intracellular ATP levels, rather than that of intracellular ROS levels. These results may have important implications for appropriate clinical uses of Vitex and provide novel insights

Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

Science.gov (United States)

Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

2011-01-01

To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories. PMID:21508930

Histones bundle F-actin filaments and affect actin structure.

Directory of Open Access Journals (Sweden)

Edna Blotnick

Full Text Available Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

Energy Technology Data Exchange (ETDEWEB)

Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

2014-03-28

Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

Quantitative proteomic analysis of post-translational modifications of human histones

DEFF Research Database (Denmark)

Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune

2006-01-01

, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational...

Rapid purification of recombinant histones.

Science.gov (United States)

Klinker, Henrike; Haas, Caroline; Harrer, Nadine; Becker, Peter B; Mueller-Planitz, Felix

2014-01-01

The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

International Nuclear Information System (INIS)

Bases, R.; Mendez, F.

1971-01-01

Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal

Organ distribution of histones after intravenous infusion of FITC histones or after sepsis.

Science.gov (United States)

Fattahi, Fatemeh; Grailer, Jamison J; Jajou, Lawrence; Zetoune, Firas S; Andjelkovic, Anuska V; Ward, Peter A

2015-03-01

Histones appear in plasma during infectious or non-infectious sepsis and are associated with multiorgan injury. In the current studies, intravenous infusion of histones resulted in their localization in major organs. In vitro exposure of mouse macrophages to histones caused a buildup of histones on cell membranes followed by localization into cytosol and into the nucleus. After polymicrobial sepsis (cecal ligation and puncture), histones appeared in plasma as well as in a multiorgan pattern, peaking at 8 h followed by decline. In lungs, histones and neutrophils appeared together, with evidence for formation of neutrophil extracellular traps (NETs), which represent an innate immune response to trap and kill bacteria and other infectious agents. In liver, there was intense NET formation, featuring linear patterns containing histones and strands of DNA. When neutrophils were activated in vitro with C5a or phorbol myristate acetate, NET formation ensued. While formation of NETs represents entrapment and killing of infectious agents, the simultaneous release from neutrophils of histones often results in tissue/organ damage.

Organ Distribution of Histones after Intravenous Infusion of FITC-Histones or after Sepsis

Science.gov (United States)

Fattahi, Fatemeh; Grailer, Jamison J.; Jajou, Lawrence; Zetoune, Firas S.; Andjelkovic, Anuska V.; Ward, Peter A.

2015-01-01

Histones appear in plasma during infectious or non-infectious sepsis and are associated with multiorgan injury. In the current studies, intravenous infusion of histones resulted in their localization in major organs. In vitro exposure of mouse macrophages to histones caused a buildup of histones on cell membranes followed by localization into cytosol and into the nucleus. After polymicrobial sepsis (cecal ligation and puncture, CLP), histones appeared in plasma as well as in a multiorgan pattern, peaking at 8 hr followed by decline. In lungs, histones and neutrophils appeared together, with evidence for formation of neutrophil extracellular traps (NETs), which represent an innate immune response to trap and kill bacteria and other infectious agents. In liver, there was intense NET formation, featuring linear patterns containing histones and strands of DNA. When neutrophils were activated in vitro with C5a or phorbol myristate acetate, NET formation ensued. While formation of NETs represents entrapment and killing of infectious agents, the simultaneous release from neutrophils of histones often results in tissue/organ damage. PMID:25680340

Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

International Nuclear Information System (INIS)

Ke Qingdong; Ellen, Thomas P.; Costa, Max

2008-01-01

Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

MS_HistoneDB, a manually curated resource for proteomic analysis of human and mouse histones.

Science.gov (United States)

El Kennani, Sara; Adrait, Annie; Shaytan, Alexey K; Khochbin, Saadi; Bruley, Christophe; Panchenko, Anna R; Landsman, David; Pflieger, Delphine; Govin, Jérôme

2017-01-01

Histones and histone variants are essential components of the nuclear chromatin. While mass spectrometry has opened a large window to their characterization and functional studies, their identification from proteomic data remains challenging. Indeed, the current interpretation of mass spectrometry data relies on public databases which are either not exhaustive (Swiss-Prot) or contain many redundant entries (UniProtKB or NCBI). Currently, no protein database is ideally suited for the analysis of histones and the complex array of mammalian histone variants. We propose two proteomics-oriented manually curated databases for mouse and human histone variants. We manually curated >1700 gene, transcript and protein entries to produce a non-redundant list of 83 mouse and 85 human histones. These entries were annotated in accordance with the current nomenclature and unified with the "HistoneDB2.0 with Variants" database. This resource is provided in a format that can be directly read by programs used for mass spectrometry data interpretation. In addition, it was used to interpret mass spectrometry data acquired on histones extracted from mouse testis. Several histone variants, which had so far only been inferred by homology or detected at the RNA level, were detected by mass spectrometry, confirming the existence of their protein form. Mouse and human histone entries were collected from different databases and subsequently curated to produce a non-redundant protein-centric resource, MS_HistoneDB. It is dedicated to the proteomic study of histones in mouse and human and will hopefully facilitate the identification and functional study of histone variants.

Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

DEFF Research Database (Denmark)

Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

2008-01-01

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated...

Bub1 autophosphorylation feeds back to regulate kinetochore docking and promote localized substrate phosphorylation.

Science.gov (United States)

Asghar, Adeel; Lajeunesse, Audrey; Dulla, Kalyan; Combes, Guillaume; Thebault, Philippe; Nigg, Erich A; Elowe, Sabine

2015-09-24

During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.

Histone deacetylase regulates insulin signaling via two pathways in pancreatic β cells.

Directory of Open Access Journals (Sweden)

Yukina Kawada

Full Text Available Recent studies demonstrated that insulin signaling plays important roles in the regulation of pancreatic β cell mass, the reduction of which is known to be involved in the development of diabetes. However, the mechanism underlying the alteration of insulin signaling in pancreatic β cells remains unclear. The involvement of epigenetic control in the onset of diabetes has also been reported. Thus, we analyzed the epigenetic control of insulin receptor substrate 2 (IRS2 expression in the MIN6 mouse insulinoma cell line. We found concomitant IRS2 up-regulation and enhanced insulin signaling in MIN6 cells, which resulted in an increase in cell proliferation. The H3K9 acetylation status of the Irs2 promoter was positively associated with IRS2 expression. Treatment of MIN6 cells with histone deacetylase inhibitors led to increased IRS2 expression, but this occurred in concert with low insulin signaling. We observed increased IRS2 lysine acetylation as a consequence of histone deacetylase inhibition, a modification that was coupled with a decrease in IRS2 tyrosine phosphorylation. These results suggest that insulin signaling in pancreatic β cells is regulated by histone deacetylases through two novel pathways affecting IRS2: the epigenetic control of IRS2 expression by H3K9 promoter acetylation, and the regulation of IRS2 activity through protein modification. The identification of the histone deacetylase isoform(s involved in these mechanisms would be a valuable approach for the treatment of type 2 diabetes.

Different effects of histone deacetylase inhibitors nicotinamide and trichostatin A (TSA) in C17.2 neural stem cells.

Science.gov (United States)

Wang, Haifeng; Cheng, Hua; Wang, Kai; Wen, Tieqiao

2012-11-01

Histone deacetylase inhibitors are involved in proliferation, apoptosis, cell cycle, mRNA transcription, and protein expression in various cells. However, the molecular mechanism underlying such functions is still not fully clear. In this study, we used C17.2 neural stem cell (NSC) line as a model to evaluate the effects of nicotinamide and trichostatin A (TSA) on cell characteristics. Results show that nicotinamide and TSA greatly inhibit cell growth, lead to cell morphology changes, and effectively induce cell apoptosis in a dose-dependent manner. Western blot analyses confirmed that nicotinamide significantly decreases the expression of bcl-2 and p38. Further insight into the molecular mechanisms shows the suppression of phosphorylation in eukaryotic initiation factor 4E-binding protein 1 (4EBP1) by nicotinamide, whereas, an increased expression of bcl-2 and p38 and phosphorylation of 4EBP1 by TSA. However, both nicotinamide and TSA significantly increase the expression of cytochrome c (cyt c). These results strongly suggest that bcl-2, p38, cyt c, and p-4EBP1 could suppress proliferation and induce apoptosis of C17.2 NSCs mediated by histone deacetylase inhibitors, nicotinamide and TSA, involving different molecular mechanisms.

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

Science.gov (United States)

Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

2007-02-01

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

Amino acid analysis and cell cycle dependent phosphorylation of an H1-like, butyrate-enhanced protein (BEP; H10; IP25) from Chinese hamster cells

International Nuclear Information System (INIS)

D'Anna, J.A.; Gurley, L.R.; Becker, R.R.; Barham, S.S.; Tobey, R.A.; Walters, R.A.

1980-01-01

A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approx. 20 amino acids so that the large fragment seen in sodium dodecyl sulfate-acrylamide gels has a molecular weight of approx. 20,000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1 0 , rat H1 0 , and IP 25 , a protein enhanced in differentiated Friend erythroleukemia cells. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP

Histone turnover within nonproliferating cells

International Nuclear Information System (INIS)

Commerford, S.L.; Carsten, A.L.; Cronkite, E.P.

1982-01-01

The turnover of DNA and histones in the livers and brains of mice has been determined. These mice had been exposed to constant levels of tritiated water from conception until they were 8 months old. At this point, exposure to tritium was discontinued, and the tritium remaining in DNA and histones was measured at various intervals afterward. The half-lives calculated for these components (with 95% confidence limits given in parentheses) were 117 (85 to 188) days for liver histone, 318 (241 to 466) days for liver DNA, 159 (129 to 208) days for brain histone and 593 (376 to 1406) days for brain DNA. The difference between histone and DNA turnover is statistically significant for both tissues and indicates that histone turnover within tissues cannot be solely accounted for by cell turnover within the tissue but also must include histone turnover within living cells. The half-life of histone within cells is estimated to be 117 (88 to 178) days in liver and 223 (187 to 277) days in brain

Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

Directory of Open Access Journals (Sweden)

Yonggang Wang

2014-01-01

Full Text Available Cardiovascular disease (CVD remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors which contribute to CVD is required in order to develop more effective treatment options. Dysregulation of epigenetic posttranscriptional modifications of histones in chromatin is thought to be associated with the pathology of many disease models, including CVD. Histone acetyltransferases (HATs and deacetylases (HDACs are regulators of histone lysine acetylation. Recent studies have implicated a fundamental role of reversible protein acetylation in the regulation of CVDs such as hypertension, pulmonary hypertension, diabetic cardiomyopathy, coronary artery disease, arrhythmia, and heart failure. This reversible acetylation is governed by enzymes that HATs add or HDACs remove acetyl groups respectively. New evidence has revealed that histone acetylation regulators blunt cardiovascular and related disease states in certain cellular processes including myocyte hypertrophy, apoptosis, fibrosis, oxidative stress, and inflammation. The accumulating evidence of the detrimental role of histone acetylation in cardiac disease combined with the cardioprotective role of histone acetylation regulators suggests that the use of histone acetylation regulators may serve as a novel approach to treating the millions of patients afflicted by cardiac diseases worldwide.

Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

Directory of Open Access Journals (Sweden)

Adi Ben Yehuda

Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

Histone H2A mobility is regulated by its tails and acetylation of core histone tails

International Nuclear Information System (INIS)

Higashi, Tsunehito; Matsunaga, Sachihiro; Isobe, Keisuke; Morimoto, Akihiro; Shimada, Tomoko; Kataoka, Shogo; Watanabe, Wataru; Uchiyama, Susumu; Itoh, Kazuyoshi; Fukui, Kiichi

2007-01-01

Histone tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions

Total levels of hippocampal histone acetylation predict normal variability in mouse behavior.

Directory of Open Access Journals (Sweden)

Addie May I Nesbitt

Full Text Available Genetic, pharmacological, and environmental interventions that alter total levels of histone acetylation in specific brain regions can modulate behaviors and treatment responses. Efforts have been made to identify specific genes that are affected by alterations in total histone acetylation and to propose that such gene specific modulation could explain the effects of total histone acetylation levels on behavior - the implication being that under naturalistic conditions variability in histone acetylation occurs primarily around the promoters of specific genes.Here we challenge this hypothesis by demonstrating with a novel flow cytometry based technique that normal variability in open field exploration, a hippocampus-related behavior, was associated with total levels of histone acetylation in the hippocampus but not in other brain regions.Results suggest that modulation of total levels of histone acetylation may play a role in regulating biological processes. We speculate in the discussion that endogenous regulation of total levels of histone acetylation may be a mechanism through which organisms regulate cellular plasticity. Flow cytometry provides a useful approach to measure total levels of histone acetylation at the single cell level. Relating such information to behavioral measures and treatment responses could inform drug delivery strategies to target histone deacetylase inhibitors and other chromatin modulators to places where they may be of benefit while avoiding areas where correction is not needed and could be harmful.

Histone Acetylome-wide Association Study of Autism Spectrum Disorder.

Science.gov (United States)

Sun, Wenjie; Poschmann, Jeremie; Cruz-Herrera Del Rosario, Ricardo; Parikshak, Neelroop N; Hajan, Hajira Shreen; Kumar, Vibhor; Ramasamy, Ramalakshmi; Belgard, T Grant; Elanggovan, Bavani; Wong, Chloe Chung Yi; Mill, Jonathan; Geschwind, Daniel H; Prabhakar, Shyam

2016-11-17

The association of histone modification changes with autism spectrum disorder (ASD) has not been systematically examined. We conducted a histone acetylome-wide association study (HAWAS) by performing H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) on 257 postmortem samples from ASD and matched control brains. Despite etiological heterogeneity, ≥68% of syndromic and idiopathic ASD cases shared a common acetylome signature at >5,000 cis-regulatory elements in prefrontal and temporal cortex. Similarly, multiple genes associated with rare genetic mutations in ASD showed common "epimutations." Acetylome aberrations in ASD were not attributable to genetic differentiation at cis-SNPs and highlighted genes involved in synaptic transmission, ion transport, epilepsy, behavioral abnormality, chemokinesis, histone deacetylation, and immunity. By correlating histone acetylation with genotype, we discovered >2,000 histone acetylation quantitative trait loci (haQTLs) in human brain regions, including four candidate causal variants for psychiatric diseases. Due to the relative stability of histone modifications postmortem, we anticipate that the HAWAS approach will be applicable to multiple diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Radiation damage to histones

International Nuclear Information System (INIS)

Mee, L.K.; Adelstein, S.J.

1985-01-01

The damage to histones irradiated in isolation is being elaborated to aid the identification of the crosslinking sites in radiation-induced DNA-histone adducts. Histones are being examined by amino acid analysis to determine the destruction of residues and by polyacrylamide gel electrophoresis to delineate changes in conformation. For the slightly lysine-rich histone, H2A, a specific attack on selective residues has been established, the aromatic residues, tyrosine and phenylalanine, and the heterocyclic residue, histidine, being significantly destroyed. In addition, a significant increase in aspartic acid was found; this may represent a radiation product from scission of the ring in the histidine residues. The similarity of the effects on residues in nitrous oxide-saturated and nitrogen-saturated solutions suggests that OH . and e/sub aq//sup -/ are equally efficient and selective in their attack. On gel electrophoresis degradation of the histone H2A was found to be greatest for irradiations in nitrous oxide-saturated solutions, suggesting CH . is the most effective radical for producing changes in conformation; O/sub 2//sup -/ was essentially ineffective. Other histones are being examined for changes in amino acid composition, conformation, and for the formation of radiation products

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

International Nuclear Information System (INIS)

Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.

1989-01-01

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

Defects in Histone H3.3 Phosphorylation and ATRX Recruitment to Misaligned Chromosomes during Mitosis Contribute to the Development of Pediatric Glioblastomas

Science.gov (United States)

2015-09-01

aneuploidy. 2. Keywords: aneuploidy, ATRX, cell cycle, chromosome missegregation, CRISPR /Cas9, DAXX, glioblastoma, histone H3.3, microinjection, mitosis...histone H3.3 with mutant constructs. We have switched from shRNA hairpins to CRISPR /Cas9 gene editing to silence both alleles of H3.3 (and an H3.3...plasmids against H3F3B. Both plasmids had the Cas9 gene and a soluble GFP reporter. The CRISPR guide sequence in one of these plasmids was 100% match

Histone variants and lipid metabolism

NARCIS (Netherlands)

Borghesan, Michela; Mazzoccoli, Gianluigi; Sheedfar, Fareeba; Oben, Jude; Pazienza, Valerio; Vinciguerra, Manlio

2014-01-01

Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis

Histone Deacetylase Inhibitor Alleviates the Neurodegenerative Phenotypes and Histone Dysregulation in Presenilins-Deficient Mice

Directory of Open Access Journals (Sweden)

Ting Cao

2018-05-01

Full Text Available Histone acetylation has been shown to play a crucial role in memory formation, and histone deacetylase (HDAC inhibitor sodium butyrate (NaB has been demonstrated to improve memory performance and rescue the neurodegeneration of several Alzheimer’s Disease (AD mouse models. The forebrain presenilin-1 and presenilin-2 conditional double knockout (cDKO mice showed memory impairment, forebrain degeneration, tau hyperphosphorylation and inflammation that closely mimics AD-like phenotypes. In this article, we have investigated the effects of systemic administration of NaB on neurodegenerative phenotypes in cDKO mice. We found that chronic NaB treatment significantly restored contextual memory but did not alter cued memory in cDKO mice while such an effect was not permanent after treatment withdrawal. We further revealed that NaB treatment did not rescue reduced synaptic numbers and cortical shrinkage in cDKO mice, but significantly increased the neurogenesis in subgranular zone of dentate gyrus (DG. We also observed that tau hyperphosphorylation and inflammation related protein glial fibrillary acidic protein (GFAP level were decreased in cDKO mice by NaB. Furthermore, GO and pathway analysis for the RNA-Seq data demonstrated that NaB treatment induced enrichment of transcripts associated with inflammation/immune processes and cytokine-cytokine receptor interactions. RT-PCR confirmed that NaB treatment inhibited the expression of inflammation related genes such as S100a9 and Ccl4 found upregulated in the brain of cDKO mice. Surprisingly, the level of brain histone acetylation in cDKO mice was dramatically increased and was decreased by the administration of NaB, which may reflect dysregulation of histone acetylation underlying memory impairment in cDKO mice. These results shed some lights on the possible molecular mechanisms of HDAC inhibitor in alleviating the neurodegenerative phenotypes of cDKO mice and provide a promising target for treating AD.

Biological significance of lysine mono-, di- and trimethylation on histone and non-histone proteins

International Nuclear Information System (INIS)

Perez-Burgos, L.

2006-01-01

Histones are the proteins that compact DNA into the repeating unit of chromatin known as the nucleosome. The N-termini of histones are subject to a series of post-translational modifications, one of which is methylation. This modification is termed 'epigenetic' because it extends the information encoded in the genome. Lysines can be mono-, di- or tri-methylated at different positions on histones H1, H3 and H4. In order to study the biological role of histone lysine methylation, antibodies were generated against mono-, di- and trimethylated H3-K9 and H3-27. Indeed, different chromatin domains in the mouse nucleus are enriched in distinct forms of histone lysine methylation, such as pericentric heterochromatin and the inactive X chromosome. Interestingly, heterochromatin in Arabidopsis thaliana is enriched in the mono- and di-, but not the trimethylated form of H3-K9. Furthermore, there exists a hierarchy of epigenetic modifications in which H3-K9 trimethylation is found to be upstream of DNA methylation on mouse major satellites. Histone lysine methylation is also involved in gene regulation upon development. One example is the chicken 61538;-globin locus, a region of facultative chromatin that undergoes a loss of di- and trimethylated H3-K27 in mature red blood cells, concomitant with expression of the 61538;-globin genes. SET-domain proteins are enzymes that methylate histones, but some of them are also able to methylate non-histone substrates. In particular, p53 is methylated by Set9 on lysine 372, G9a and Glp-1 on lysine 373 and by Smyd2 on lysine 370. Smyd2 transcript levels are greatly increased upon irradiation and dimethylated p53-370 specifically binds to 53BP1, a protein involved in recognizing DNA double-stranded breaks upon ionizing radiation. These results argue for a novel role of p53-K370 methylation in the biology of DNA damage. In summary, lysine methylation is a post-translational modification that can occur both on histone and non-histone proteins

The Mechanism of Action of the Histone Deacetylase Inhibitor Vorinostat Involves Interaction with the Insulin-Like Growth Factor Signaling Pathway

Science.gov (United States)

Sarfstein, Rive; Bruchim, Ilan; Fishman, Ami; Werner, Haim

2011-01-01

A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat. PMID:21931726

The mechanism of action of the histone deacetylase inhibitor vorinostat involves interaction with the insulin-like growth factor signaling pathway.

Directory of Open Access Journals (Sweden)

Rive Sarfstein

Full Text Available A correlation between components of the insulin-like growth factor (IGF system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa and uterine serous papillary (Type II, USPC-2 endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat.

Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases.

Science.gov (United States)

Mengel, Alexander; Ageeva, Alexandra; Georgii, Elisabeth; Bernhardt, Jörg; Wu, Keqiang; Durner, Jörg; Lindermayr, Christian

2017-02-01

Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-dl-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes. © 2017 American Society of Plant Biologists. All Rights Reserved.

Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Gardner, Nicole M., E-mail: nicolegardner@creighton.edu [Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178 (United States); Riley, Ronald T.; Showker, Jency L.; Voss, Kenneth A. [USDA-ARS, Toxicology and Mycotoxin Research Unit, Athens, GA 30605 (United States); Sachs, Andrew J.; Maddox, Joyce R.; Gelineau-van Waes, Janee B. [Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178 (United States)

2016-05-01

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs. - Highlights: • FB1 treatment results in accumulation of Sa1P primarily in the nucleus of MEFs. • FB1 treatment and elevated nuclear Sa1P are associated with HDAC inhibition. • Sphk2 inhibition alone

Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

International Nuclear Information System (INIS)

Gardner, Nicole M.; Riley, Ronald T.; Showker, Jency L.; Voss, Kenneth A.; Sachs, Andrew J.; Maddox, Joyce R.; Gelineau-van Waes, Janee B.

2016-01-01

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs. - Highlights: • FB1 treatment results in accumulation of Sa1P primarily in the nucleus of MEFs. • FB1 treatment and elevated nuclear Sa1P are associated with HDAC inhibition. • Sphk2 inhibition alone

Chemical and semisynthesis of modified histones.

Science.gov (United States)

Maity, Suman Kumar; Jbara, Muhammad; Brik, Ashraf

2016-05-01

Post-translational modifications (PTMs) of histones play critical roles in the epigenetic regulation of eukaryotic genome by directly altering the biophysical properties of chromatin or by recruiting effector proteins. The large number of PTMs and the inherent complexity in their population and signaling processes make it highly challenging to understand epigenetics-related processes. To address these challenges, accesses to homogeneously modified histones are obligatory. Over the last decade, synthetic protein chemists have been devising novel synthetic tools and applying state-of-the-art chemoselective ligation strategies to prepare precious materials useful in answering fundamental questions in this area. In this short review, we cover some of the recent breakthroughs in these directions in particular the synthesis and semi-synthesis of modified histones and their use to unravel the mysteries of epigenetics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Histone deacetylases (HDACs and brain function

Directory of Open Access Journals (Sweden)

Claude-Henry Volmar

2015-01-01

Full Text Available Modulation of gene expression is a constant and necessary event for mammalian brain function. An important way of regulating gene expression is through the remodeling of chromatin, the complex of DNA, and histone proteins around which DNA wraps. The “histone code hypothesis” places histone post-translational modifications as a significant part of chromatin remodeling to regulate transcriptional activity. Acetylation of histones by histone acetyl transferases and deacetylation by histone deacetylases (HDACs at lysine residues are the most studied histone post-translational modifications in cognition and neuropsychiatric diseases. Here, we review the literature regarding the role of HDACs in brain function. Among the roles of HDACs in the brain, studies show that they participate in glial lineage development, learning and memory, neuropsychiatric diseases, and even rare neurologic diseases. Most HDACs can be targeted with small molecules. However, additional brain-penetrant specific inhibitors with high central nervous system exposure are needed to determine the cause-and-effect relationship between individual HDACs and brain-associated diseases.

Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

Directory of Open Access Journals (Sweden)

Hao Zhang

Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

Effect of X-irradiation on soluble nucleohistone of Pinus pinea

International Nuclear Information System (INIS)

Berkofsky, J.; Roy, R.M.

1977-01-01

Exposure of isolated nuclei from Pinus pinea cotyledons to 5 kR of 260 kVp X-rays resulted in no significant alteration of viscosity, thermal denaturation or spectral characteristics of soluble nucleohistone nor was the relative amount of DNA, RNA, histone and acidic protein affected. Pine histones, resolved into 8 sub-fractions by polyacrylamide gel electrophoresis, appeared to be unaffected with respect to mobility or relative abundance following irradiation of cotyledons, isolated nuclei, soluble nucleohistone or histones. However, phosphorylation of histones at AT 32 P following irradiation of isolated nuclei with 5 kR was significantly inhibited. Although histone F 1 phosphorylation was inhibited to the greatest extent as previously reported for mammalian tissues, significant inhibition of phosphorylation of histone F 2 a2 and F 2 b was observed. Exposure of cotyledons at the 14th day of germination to 1.0 kR resulted in an apparent G 2 mitotic block within 1 hr post irradiation. The normal increase in histone content of soluble nucleohistone associated with the termination of cell division and onset of cell elongation in the cotyledon at this time was inhibited. Mitotic activity remained arrested for approximately 2 days. Recovery of mitotic activity in the irradiated cotyledons was correlated with resumption of deposition of histone into chromatin preceding the onset of cell elongation. (author)

Histones induce rapid and profound thrombocytopenia in mice

Science.gov (United States)

Bhandari, Ashish A.

2011-01-01

Histones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbβ3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbβ3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage. PMID:21700775

Social isolation stress induces ATF-7 phosphorylation and impairs silencing of the 5-HT 5B receptor gene

Science.gov (United States)

Maekawa, Toshio; Kim, Seungjoon; Nakai, Daisuke; Makino, Chieko; Takagi, Tsuyoshi; Ogura, Hiroo; Yamada, Kazuyuki; Chatton, Bruno; Ishii, Shunsuke

2010-01-01

Many symptoms induced by isolation rearing of rodents may be relevant to neuropsychiatric disorders, including depression. However, identities of transcription factors that regulate gene expression in response to chronic social isolation stress remain elusive. The transcription factor ATF-7 is structurally related to ATF-2, which is activated by various stresses, including inflammatory cytokines. Here, we report that Atf-7-deficient mice exhibit abnormal behaviours and increased 5-HT receptor 5B (Htr5b) mRNA levels in the dorsal raphe nuclei. ATF-7 silences the transcription of Htr5B by directly binding to its 5′-regulatory region, and mediates histone H3-K9 trimethylation via interaction with the ESET histone methyltransferase. Isolation-reared wild-type (WT) mice exhibit abnormal behaviours that resemble those of Atf-7-deficient mice. Upon social isolation stress, ATF-7 in the dorsal raphe nucleus is phosphorylated via p38 and is released from the Htr5b promoter, leading to the upregulation of Htr5b. Thus, ATF-7 may have a critical role in gene expression induced by social isolation stress. PMID:19893493

Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

Science.gov (United States)

Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

2017-10-16

Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Histone chaperone networks shaping chromatin function

DEFF Research Database (Denmark)

Hammond, Colin; Strømme, Caroline Bianchi; Huang, Hongda

2017-01-01

and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone...... chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin....

Epinephrine and glucose modulate training-related CREB phosphorylation in old rats: relationships to age-related memory impairments.

Science.gov (United States)

Morris, Ken A; Gold, Paul E

2013-02-01

Epinephrine enhances memory in young adult rats, in part, by increasing blood glucose levels needed to modulate memory. In old rats, epinephrine is deficient at raising blood glucose levels and thus is only moderately effective at enhancing memory. In contrast, systemic glucose injections improve memory in old rats, with resulting memory performance equal to that of young rats. The diminished response of glucose to training in old rats may blunt downstream neurochemical and molecular mechanisms needed to upregulate memory processes. In the first experiment, young adult and old rats were trained on an inhibitory avoidance task with immediate post-training injections of aCSF or glucose into the dorsal hippocampus. Old rats had significant memory impairments compared to young rats 7 days after training. Intrahippocampal injections of glucose reversed age-related deficits, improving memory scores in old rats to values seen in young rats. A second experiment examined age-related changes in activation of the transcription factor CREB, which is widely implicated in memory formation and may act downstream of hormonal and metabolic signals. Activation was assessed in response to training with systemic injections of epinephrine and glucose at doses known to enhance memory. Young adult and old rats were trained on inhibitory avoidance with immediate post-training systemic injections of saline, epinephrine, or glucose. After training, old rats had significant impairments in CREB phosphorylation in area CA1 and the dentate gyrus region of the hippocampus, and in the basolateral and lateral amygdala. Epinephrine and glucose attenuated age-related deficits in CREB phosphorylation, but were more effective in the amygdala and hippocampus, respectively. Together, these results support the view that age-related changes in blood glucose responses to epinephrine contribute to memory impairments, which may be related to alterations in regional patterns of CREB phosphorylation. Copyright

Age-related changes in the synthesis and phosphorylation of proteins

International Nuclear Information System (INIS)

Butler, J.A.; Heydari, A.; Richardson, A.

1986-01-01

It is well documented that the protein synthetic activity of liver tissue decreases significantly with age. However, very little information is available on the effect of age on the synthesis or phosphorylation of individual proteins. Hepatocytes were isolated from 5- to 30-month-old male Fischer F344 rats, and proteins were labeled with either [ 3 H]-valine or [ 32 P]-phosphate. Two-dimensional polyacrylamide gel electrophoresis was used to monitor the synthesis and phosphorylation of a wide variety of proteins. A dramatic increase or decrease in the synthesis of approximately 2 to 3% of the proteins was observed. Most of the proteins whose synthesis increased with age were found to be plasma proteins, e.g., acute phase proteins, synthesized by the liver. In general, the synthesis of most proteins decreased 20 to 40% with age. The phosphorylation of most proteins (over 200) did not appear to change with age. However the phosphorylation of two acidic proteins (molecular weights of 148 Kd and 130 Kd and pIs of 5.4 and 5.36, respectively) decreased with age while the phosphorylation of a basic protein (molecular weight of 57 Kd and pI of 8.09) increased with age

Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

KAUST Repository

Chauhan, Sakshi

2016-09-02

Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

International Nuclear Information System (INIS)

Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay

2015-01-01

Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

Energy Technology Data Exchange (ETDEWEB)

Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

2015-03-15

Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

Nitric Oxide Modulates Histone Acetylation at Stress Genes by Inhibition of Histone Deacetylases1[OPEN

Science.gov (United States)

Mengel, Alexander; Ageeva, Alexandra; Durner, Jörg

2017-01-01

Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-dl-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes. PMID:27980017

Extracting histones for the specific purpose of label-free MS.

Science.gov (United States)

Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter

2016-12-01

Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel method for isolation of histones from serum and its implications in therapeutics and prognosis of solid tumours.

Science.gov (United States)

Reddy, Divya; Khade, Bharat; Pandya, Riddhi; Gupta, Sanjay

2017-01-01

Dysregulation in post-translational modifications of histones and their modifiers are now well-recognized as a hallmark of cancer and can be used as biomarkers and potential therapeutic targets for disease progression and prognosis. In most solid tumours, a biopsy is challenging, costly, painful or potentially risky for the patient. Therefore, non-invasive methods like 'liquid biopsy' for analysis of histone modifications and their modifiers if possible will be helpful in the better clinical management of cancer patients. Here, we have developed a cost-effective and time-efficient protocol for isolation of circulating histones from serum of solid tumor, HCC, called Dual Acid Extraction (DAE) protocol and have confirmed by mass spectrometry. Also, we measured the activity of HDACs and HATs in serum samples. The serum purified histones were profiled for changes in histone PTMs and have shown a comparable pattern of modifications like acetylation (H4K16Ac), methylation (H4K20Me3, H3K27Me3, H3K9Me3) and phosphorylation (γ-H2AX and H3S10P) to paired cancer tissues. Profiling for the histone PTM changes in various other organs of normal and tumor bearing animal suggests that the changes in the histone PTMs observed in the tumor serum is indeed due to changes in the tumor tissue only. Further, we demonstrate that the observed hypo-acetylation of histone H4 in tissue and serum samples of tumor bearing animals corroborated with the elevated HDAC activity in both samples compared to normal. Interestingly, human normal and tumor serum samples also showed elevated HDAC activity with no significant changes in HAT activity. Our study provides the first evidence in the context of histone PTMs and modifiers that liquid biopsy is a valuable predictive tool for monitoring disease progression. Importantly, with the advent of drugs that target specific enzymes involved in the epigenetic regulation of gene expression, liquid biopsy-based 'real time' monitoring will be useful for

Sepsis and ARDS: The Dark Side of Histones

Science.gov (United States)

Xu, Zhiheng; Huang, Yongbo; Mao, Pu; Zhang, Jianrong; Li, Yimin

2015-01-01

Despite advances in management over the last several decades, sepsis and acute respiratory distress syndrome (ARDS) still remain major clinical challenges and the leading causes of death for patients in intensive care units (ICUs) due to insufficient understanding of the pathophysiological mechanisms of these diseases. However, recent studies have shown that histones, also known as chromatin-basic structure proteins, could be released into the extracellular space during severe stress and physical challenges to the body (e.g., sepsis and ARDS). Due to their cytotoxic and proinflammatory effects, extracellular histones can lead to excessive and overwhelming cell damage and death, thus contributing to the pathogenesis of both sepsis and ARDS. In addition, antihistone-based treatments (e.g., neutralizing antibodies, activated protein C, and heparin) have shown protective effects and have significantly improved the outcomes of mice suffering from sepsis and ARDS. Here, we review researches related to the pathological role of histone in context of sepsis and ARDS and evaluate the potential value of histones as biomarkers and therapeutic targets of these diseases. PMID:26609197

Histone deacetylase inhibition abolishes stress-induced spatial memory impairment.

Science.gov (United States)

Vargas-López, Viviana; Lamprea, Marisol R; Múnera, Alejandro

2016-10-01

Acute stress induced before spatial training impairs memory consolidation. Although non-epigenetic underpinning of such effect has been described, the epigenetic mechanisms involved have not yet been studied. Since spatial training and intense stress have opposite effects on histone acetylation balance, it is conceivable that disruption of such balance may underlie acute stress-induced spatial memory consolidation impairment and that inhibiting histone deacetylases prevents such effect. Trichostatin-A (TSA, a histone deacetylase inhibitor) was used to test its effectiveness in preventing stress' deleterious effect on memory. Male Wistar rats were trained in a spatial task in the Barnes maze; 1-h movement restraint was applied to half of them before training. Immediately after training, stressed and non-stressed animals were randomly assigned to receive either TSA (1mg/kg) or vehicle intraperitoneal injection. Twenty-four hours after training, long-term spatial memory was tested; plasma and brain tissue were collected immediately after the memory test to evaluate corticosterone levels and histone H3 acetylation in several brain areas. Stressed animals receiving vehicle displayed memory impairment, increased plasma corticosterone levels and markedly reduced histone H3 acetylation in prelimbic cortex and hippocampus. Such effects did not occur in stressed animals treated with TSA. The aforementioned results support the hypothesis that acute stress induced-memory impairment is related to histone deacetylation. Copyright © 2016 Elsevier Inc. All rights reserved.

Simplified Method for Rapid Purification of Soluble Histones

Directory of Open Access Journals (Sweden)

Nives Ivić

2016-06-01

Full Text Available Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H42 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research. This work is licensed under a Creative Commons Attribution 4.0 International License.

The effect of hepatoprotective preparations thioctacid and flavobion on histones in intact and regenerating lever in irradiated rats

International Nuclear Information System (INIS)

Kozhurkova, M.; Kropachova, E.; Mishurova, R.; Reksa, R.

1992-01-01

The changes in concentration, total content of histones and relative proportion of individual histone fractions in intact and regenerating liver were followed in rats after administration of hepatoprotective agents flavobion and thioctacid and after whole-body gamma irradiation with a dose 5.7 Gy. Thioctacid alone caused an increase in histone concentration in intact liver whereas flavobion alone did not produce significant quantitative changes. Irradiation alone decreased markedly the concentration and total content of histones in intact as well as regenerating liver of unprotected rats. Administration of thioctacid or flavobion protected from these quantitative histone changes or alleviated them consideradly. In relative proportion of individual histone fractions, the most profound changes were found in H1 histone after flavobion application

Extracellular histones induce erythrocyte fragility and anemia.

Science.gov (United States)

Kordbacheh, Farzaneh; O'Meara, Connor H; Coupland, Lucy A; Lelliott, Patrick M; Parish, Christopher R

2017-12-28

Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients. © 2017 by The American Society of Hematology.

Toxic effects of extracellular histones and their neutralization by vitreous in retinal detachment.

Science.gov (United States)

Kawano, Hiroki; Ito, Takashi; Yamada, Shingo; Hashiguchi, Teruto; Maruyama, Ikuro; Hisatomi, Toshio; Nakamura, Makoto; Sakamoto, Taiji

2014-05-01

Histones are DNA-binding proteins and are involved in chromatin remodeling and regulation of gene expression. Histones can be released after tissue injuries, and the extracellular histones cause cellular damage and organ dysfunction. Regardless of their clinical significance, the role and relevance of histones in ocular diseases are unknown. We studied the role of histones in eyes with retinal detachment (RD). Vitreous samples were collected during vitrectomy, and the concentration of histone H3 was measured by enzyme-linked immunosorbent assay. The location of the histones and related molecules was examined in a rat RD model. The release of histones and their effects on rat retinal progenitor cells R28 and ARPE-19 were evaluated in vitro. In addition, the protective role of the vitreous body against histones was tested. The intravitreal concentration of histones was higher in eyes with RD (mean, 30.9 ± 9.8 ng/ml) than in control eyes (below the limit of detection, Phistone H3 was observed on the outer side of the detached retina and was associated with photoreceptor death. Histone H3 was released from cultured R28 by oxidative stress. Histones at a concentration 10 μg/ml induced the production of interleukin-8 in ARPE-19 cells (2.5-fold increase, PHistones were toxic to cells at concentrations of ≥ 20 μg/ml. Vitreous body or hyaluronan decreased toxicity of histones by inhibiting diffusion of histones. These results indicate that histones are released from retinas with RD and may modulate the subretinal microenvironment by functioning as damage-associated molecular pattern molecules, thereby inducing proinflammatory cytokines or cell toxicity. In addition, the important role of the vitreous body and hyaluronan in protecting the retina from these toxic effects is suggested.

CSR-1 RNAi pathway positively regulates histone expression in C. elegans.

Science.gov (United States)

Avgousti, Daphne C; Palani, Santhosh; Sherman, Yekaterina; Grishok, Alla

2012-10-03

Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3'UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2'-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.

HiHiMap: single-cell quantitation of histones and histone posttranslational modifications across the cell cycle by high-throughput imaging.

Science.gov (United States)

Zane, Linda; Chapus, Fleur; Pegoraro, Gianluca; Misteli, Tom

2017-08-15

We describe Hi gh-throughput Hi stone Map ping (HiHiMap), a high-throughput imaging method to measure histones and histone posttranslational modifications (PTMs) in single cells. HiHiMap uses imaging-based quantification of DNA and cyclin A to stage individual cells in the cell cycle to determine the levels of histones or histone PTMs in each stage of the cell cycle. As proof of principle, we apply HiHiMap to measure the level of 21 core histones, histone variants, and PTMs in primary, immortalized, and transformed cells. We identify several histone modifications associated with oncogenic transformation. HiHiMap allows the rapid, high-throughput study of histones and histone PTMs across the cell cycle and the study of subpopulations of cells. © 2017 Zane et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Brain pattern of histone H3 phosphorylation after acute amphetamine administration: its relationship to brain c-fos induction is strongly dependent on the particular brain area.

Science.gov (United States)

Rotllant, David; Armario, Antonio

2012-02-01

Recent evidence strongly suggests a critical role of chromatin remodelling in the acute and chronic effects of addictive drugs. We reasoned that Immunohistochemical detection of certain histone modifications may be a more specific tool than induction of immediate early genes (i.e. c-fos) to detect brain areas and neurons that are critical for the action of addictive drugs. Thus, in the present work we studied in adult male rats the effects of a high dose of amphetamine on brain pattern of histone H3 phosphorylation in serine 10 (pH3S(10)) and c-fos expression. We firstly observed that amphetamine-induced an increase in the number of pH3S(10) positive neurons in a restricted number of brain areas, with maximum levels at 30 min after the drug administration that declined at 90 min in most areas. In a second experiment we studied colocalization of pH3S(10) immunoreactivity (pH3S(10)-IR) and c-fos expression. Amphetamine increased c-fos expression in medial prefrontal cortex (mPFC), dorsal striatum, nucleus accumbens (Acb), major Island of Calleja (ICjM), central amygdala (CeA), bed nucleus of stria terminalis lateral dorsal (BSTld) and paraventricular nucleus of the hypothalamus (PVN). Whereas no evidence for increase in pH3S(10) positive neurons was found in the mPFC and the PVN, in the striatum and the Acb basically all pH3S(10) positive neurons showed colocalization with c-fos. In ICjM, CeA and BSTld a notable degree of colocalization was found, but an important number of neurons expressing c-fos were negative for pH3S(10). The present results give support to the hypothesis that amphetamine-induced pH3S(10)-IR showed a more restricted pattern than brain c-fos induction, being this difference strongly dependent on the particular brain area studied. It is likely that those nuclei and neurons showing pH3S(10)-IR are more specifically associated to important effects of the drug, including neural plasticity. This article is part of a Special Issue entitled 'Post

Overview of the Classical Histone Deacetylase Enzymes and Histone Deacetylase Inhibitors

OpenAIRE

Ververis, Katherine; Karagiannis, Tom C.

2012-01-01

The important role of histone deacetylase enzymes in regulating gene expression, cellular proliferation, and survival has made them attractive targets for the development of histone deacetylase inhibitors as anticancer drugs. Suberoylanilide hydroxamic acid (Vorinostat, Zolinza), a structural analogue of the prototypical Trichostatin A, was approved by the US Food and Drug Administration for the treatment of advanced cutaneous T-cell lymphoma in 2006. This was followed by approval of the cycl...

Identification and Interrogation of Combinatorial Histone Modifications

Directory of Open Access Journals (Sweden)

Kelly R Karch

2013-12-01

Full Text Available Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs. Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.

Molecular mechanisms and potential functions of histone demethylases

DEFF Research Database (Denmark)

Kooistra, Susanne Marije; Helin, Kristian

2012-01-01

of two families of enzymes that can demethylate histones has changed this notion. The biochemical activities of these histone demethylases towards specific Lys residues on histones, and in some cases non-histone substrates, have highlighted their importance in developmental control, cell-fate decisions...

The histone codes for meiosis.

Science.gov (United States)

Wang, Lina; Xu, Zhiliang; Khawar, Muhammad Babar; Liu, Chao; Li, Wei

2017-09-01

Meiosis is a specialized process that produces haploid gametes from diploid cells by a single round of DNA replication followed by two successive cell divisions. It contains many special events, such as programmed DNA double-strand break (DSB) formation, homologous recombination, crossover formation and resolution. These events are associated with dynamically regulated chromosomal structures, the dynamic transcriptional regulation and chromatin remodeling are mainly modulated by histone modifications, termed 'histone codes'. The purpose of this review is to summarize the histone codes that are required for meiosis during spermatogenesis and oogenesis, involving meiosis resumption, meiotic asymmetric division and other cellular processes. We not only systematically review the functional roles of histone codes in meiosis but also discuss future trends and perspectives in this field. © 2017 Society for Reproduction and Fertility.

A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism

DEFF Research Database (Denmark)

Feng, Dan; Liu, Tao; Sun, Zheng

2011-01-01

Disruption of the circadian clock exacerbates metabolic diseases, including obesity and diabetes. We show that histone deacetylase 3 (HDAC3) recruitment to the genome displays a circadian rhythm in mouse liver. Histone acetylation is inversely related to HDAC3 binding, and this rhythm is lost whe...

Rapid divergence of histones in Hydrozoa (Cnidaria) and evolution of a novel histone involved in DNA damage response in hydra.

Science.gov (United States)

Reddy, Puli Chandramouli; Ubhe, Suyog; Sirwani, Neha; Lohokare, Rasika; Galande, Sanjeev

2017-08-01

Histones are fundamental components of chromatin in all eukaryotes. Hydra, an emerging model system belonging to the basal metazoan phylum Cnidaria, provides an ideal platform to understand the evolution of core histone components at the base of eumetazoan phyla. Hydra exhibits peculiar properties such as tremendous regenerative capacity, lack of organismal senescence and rarity of malignancy. In light of the role of histone modifications and histone variants in these processes it is important to understand the nature of histones themselves and their variants in hydra. Here, we report identification of the complete repertoire of histone-coding genes in the Hydra magnipapillata genome. Hydra histones were classified based on their copy numbers, gene structure and other characteristic features. Genomic organization of canonical histone genes revealed the presence of H2A-H2B and H3-H4 paired clusters in high frequency and also a cluster with all core histones along with H1. Phylogenetic analysis of identified members of H2A and H2B histones suggested rapid expansion of these groups in Hydrozoa resulting in the appearance of unique subtypes. Amino acid sequence level comparisons of H2A and H2B forms with bilaterian counterparts suggest the possibility of a highly mobile nature of nucleosomes in hydra. Absolute quantitation of transcripts confirmed the high copy number of histones and supported the canonical nature of H2A. Furthermore, functional characterization of H2A.X.1 and a unique variant H2A.X.2 in the gastric region suggest their role in the maintenance of genome integrity and differentiation processes. These findings provide insights into the evolution of histones and their variants in hydra. Copyright © 2017 Elsevier GmbH. All rights reserved.

Gallic Acid Decreases Inflammatory Cytokine Secretion Through Histone Acetyltransferase/Histone Deacetylase Regulation in High Glucose-Induced Human Monocytes.

Science.gov (United States)

Lee, Wooje; Lee, Sang Yeol; Son, Young-Jin; Yun, Jung-Mi

2015-07-01

Hyperglycemia contributes to diabetes and several diabetes-related complications. Gallic acid is a polyhydroxy phenolic compound found in various natural products. In this study, we investigated the effects and mechanism of gallic acid on proinflammatory cytokine secretion in high glucose-induced human monocytes (THP-1 cells). THP-1 cells were cultured under normoglycemic or hyperglycemic conditions, in the absence or presence of gallic acid. Hyperglycemic conditions significantly induced histone acetylation, nuclear factor-κB (NF-κB) activation, and proinflammatory cytokine release from THP-1 cells, whereas gallic acid suppressed NF-κB activity and cytokine release. It also significantly reduced CREB-binding protein/p300 (CBP/p300, a NF-κB coactivator) gene expression, acetylation levels, and CBP/p300 histone acetyltransferase (HAT) activity. In addition, histone deacetylase 2 (HDAC2) expression was significantly induced. These results suggest that gallic acid inhibits hyperglycemic-induced cytokine production in monocytes through epigenetic changes involving NF-κB. Therefore, gallic acid may have potential for the treatment and prevention of diabetes and its complications.

Structure and Functions of Linker Histones.

Science.gov (United States)

Lyubitelev, A V; Nikitin, D V; Shaytan, A K; Studitsky, V M; Kirpichnikov, M P

2016-03-01

Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity.

Directory of Open Access Journals (Sweden)

Kanae Ando

2016-03-01

Full Text Available Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD. β-amyloid (Aβ lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing

NARCIS (Netherlands)

A. Lagarou (Anna); A.B. Mohd Sarip; Y.M. Moshkin (Yuri); G.E. Chalkley (Gillian); K. Bezstarosti (Karel); J.A.A. Demmers (Jeroen); C.P. Verrijzer (Peter)

2008-01-01

textabstractTranscription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was

Post-Translational Modifications of Histones in Human Sperm.

Science.gov (United States)

Krejčí, Jana; Stixová, Lenka; Pagáčová, Eva; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, Gabriela; Zdráhal, Zbyněk; Sehnalová, Petra; Dabravolski, Siarhei; Hejátko, Jan; Bártová, Eva

2015-10-01

We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. © 2015 Wiley Periodicals, Inc.

Evidence for gene-specific rather than transcription rate-dependent histone H3 exchange in yeast coding regions.

Science.gov (United States)

Gat-Viks, Irit; Vingron, Martin

2009-02-01

In eukaryotic organisms, histones are dynamically exchanged independently of DNA replication. Recent reports show that different coding regions differ in their amount of replication-independent histone H3 exchange. The current paradigm is that this histone exchange variability among coding regions is a consequence of transcription rate. Here we put forward the idea that this variability might be also modulated in a gene-specific manner independently of transcription rate. To that end, we study transcription rate-independent replication-independent coding region histone H3 exchange. We term such events relative exchange. Our genome-wide analysis shows conclusively that in yeast, relative exchange is a novel consistent feature of coding regions. Outside of replication, each coding region has a characteristic pattern of histone H3 exchange that is either higher or lower than what was expected by its RNAPII transcription rate alone. Histone H3 exchange in coding regions might be a way to add or remove certain histone modifications that are important for transcription elongation. Therefore, our results that gene-specific coding region histone H3 exchange is decoupled from transcription rate might hint at a new epigenetic mechanism of transcription regulation.

Histone modifications influence mediator interactions with chromatin

Science.gov (United States)

Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun; Liu, Zhongle; Quan, Amy; Costanzo, Michael; Dávila López, Marcela; Westholm, Jakub Orzechowski; Ronne, Hans; Boone, Charles; Gustafsson, Claes M.; Myers, Lawrence C.

2011-01-01

The Mediator complex transmits activation signals from DNA bound transcription factors to the core transcription machinery. Genome wide localization studies have demonstrated that Mediator occupancy not only correlates with high levels of transcription, but that the complex also is present at transcriptionally silenced locations. We provide evidence that Mediator localization is guided by an interaction with histone tails, and that this interaction is regulated by their post-translational modifications. A quantitative, high-density genetic interaction map revealed links between Mediator components and factors affecting chromatin structure, especially histone deacetylases. Peptide binding assays demonstrated that pure wild-type Mediator forms stable complexes with the tails of Histone H3 and H4. These binding assays also showed Mediator—histone H4 peptide interactions are specifically inhibited by acetylation of the histone H4 lysine 16, a residue critical in transcriptional silencing. Finally, these findings were validated by tiling array analysis that revealed a broad correlation between Mediator and nucleosome occupancy in vivo, but a negative correlation between Mediator and nucleosomes acetylated at histone H4 lysine 16. Our studies show that chromatin structure and the acetylation state of histones are intimately connected to Mediator localization. PMID:21742760

Regulation of human histone gene expression: transcriptional and posttranscriptional control in the coupling of histone messenger RNA stability with DNA replication

International Nuclear Information System (INIS)

Baumbach, L.L.; Stein, G.S.; Stein, J.L.

1987-01-01

The extent to which transcriptional and posttranscriptional regulation contributes to the coupling of histone gene expression and DNA replication was examined during the cell cycle in synchronized HeLa S3 cells. Rates of transcription were determined in vitro in isolated nuclei. A 3-5-fold increase in cell cycle dependent histone gene transcription was observed in early S phase, prior to the peak of DNA synthesis. This result is consistent with a previous determination of histone mRNA synthesis in intact cells. The transcription of these genes did not change appreciably after inhibition of DNA replication by hydroxyurea treatment, although Northern blot analysis indicated that cellular levels of histone mRNA decreased rapidly in the presence of the drug. Total cellular levels of histone mRNA closely parallel the rate of DNA synthesis as a function of cell cycle progression, reaching a maximal 20-fold increase as compared with non S phase levels. This DNA synthesis dependent accumulation of histone mRNA occurs predominantly in the cytoplasm and appears to be mediated primarily by control of histone mRNA stability. Changes in nuclear histone mRNA levels were less pronounced. These combined observations suggest that both transcriptional regulation and posttranscriptional regulation contribute toward control of the cell cycle dependent accumulation of histone mRNA during S phase, while the stability of histone mRNA throughout S phase and the selective turnover of histone mRNAs, either at the natural termination of S phase or following inhibition of DNA synthesis, are posttranscriptionally regulated

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

Directory of Open Access Journals (Sweden)

Ye Seul Kim

Full Text Available Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1 is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF, histone H3, and the cAMP response element (CRE-binding protein (CREB. In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

Extracellular histones in tissue injury and inflammation.

Science.gov (United States)

Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

2014-05-01

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

International Nuclear Information System (INIS)

Waterborg, J.H.; Robertson, A.J.; Tatar, D.L.; Borza, C.M.; Davie, J.R.

1995-01-01

Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

Role of extracellular histones in the cardiomyopathy of sepsis.

Science.gov (United States)

Kalbitz, Miriam; Grailer, Jamison J; Fattahi, Fatemeh; Jajou, Lawrence; Herron, Todd J; Campbell, Katherine F; Zetoune, Firas S; Bosmann, Markus; Sarma, J Vidya; Huber-Lang, Markus; Gebhard, Florian; Loaiza, Randall; Valdivia, Hector H; Jalife, José; Russell, Mark W; Ward, Peter A

2015-05-01

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction. © FASEB.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

International Nuclear Information System (INIS)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

2013-01-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression

Histones as mediators of host defense, inflammation and thrombosis.

Science.gov (United States)

Hoeksema, Marloes; van Eijk, Martin; Haagsman, Henk P; Hartshorn, Kevan L

2016-01-01

Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of animal hosts. In addition, histones can trigger inflammatory responses in some cases acting through Toll-like receptors or inflammasome pathways. Extracellular histones mediate organ injury (lung, liver), sepsis physiology, thrombocytopenia and thrombin generation and some proteins can bind histones and reduce these potentially harmful effects.

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

DEFF Research Database (Denmark)

Lohse, Brian; Helgstrand, Charlotte; Andersson, Jan Legaard

2013-01-01

mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc...... toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide...

Histone Deacetylase 3 Suppresses Erk Phosphorylation and Matrix Metalloproteinase (Mmp)-13 Activity in Chondrocytes

Science.gov (United States)

Carpio, Lomeli R.; Bradley, Elizabeth W.; Westendorf, Jennifer J.

2017-01-01

Histone deacetylase inhibitors are emerging therapies for many diseases including cancers and neurological disorders; however, these drugs are teratogens to the developing skeleton. Hdac3 is essential for proper endochondral ossification as its deletion in chondrocytes increases cytokine signaling and the expression of matrix remodeling enzymes. Here we explored the mechanism by which Hdac3 controls Mmp13 expression in chondrocytes. In Hdac3-depleted chondrocytes, Erk1/2 as well as its downstream substrate, Runx2, were hyperphosphorylated as a result of decreased expression and activity of the Erk1/2 specific phosphatase, Dusp6. Erk1/2 kinase inhibitors and Dusp6 adenoviruses reduced Mmp13 expression and partially rescued matrix production in Hdac3-deficient chondrocytes. Postnatal chondrocyte-specific deletion of Hdac3 with an inducible Col2a1-Cre caused premature production of pErk1/2 and Mmp13 in the growth plate. Thus, Hdac3 controls the temporal and spatial expression of tissue-remodeling genes in chondrocytes to ensure proper endochondral ossification during development. PMID:27662443

Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new Histones H3 and H4

International Nuclear Information System (INIS)

Jackson, V.

1987-01-01

The authors have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposits as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution of density gradients

Opposite replication polarities of transcribed and nontranscribed histone H5 genes

International Nuclear Information System (INIS)

Trempe, J.P.; Lindstrom, Y.I.; Leffak, M.

1988-01-01

The authors used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocyctes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, the authors conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction

Histone displacement during nucleotide excision repair

DEFF Research Database (Denmark)

Dinant, C.; Bartek, J.; Bekker-Jensen, S.

2012-01-01

Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....

The tumor suppressor Rb and its related Rbl2 genes are regulated by Utx histone demethylase

Energy Technology Data Exchange (ETDEWEB)

Terashima, Minoru; Ishimura, Akihiko; Yoshida, Masakazu [Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Ishikawa (Japan); Suzuki, Yutaka; Sugano, Sumio [Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8561, Chiba (Japan); Suzuki, Takeshi, E-mail: suzuki-t@staff.kanazawa-u.ac.jp [Division of Functional Genomics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Ishikawa (Japan)

2010-08-20

Research highlights: {yields} Utx increases expression of Rb and Rbl2 genes through its demethylase activity. {yields} Utx changes histone H3 methylation on the Rb and Rbl2 promoters. {yields} Utx induces decreased cell proliferation of mammalian primary cells. -- Abstract: Utx is a candidate tumor suppressor gene that encodes histone H3 lysine 27 (H3K27) demethylase. In this study, we found that ectopic expression of Utx enhanced the expression of retinoblastoma tumor suppressor gene Rb and its related gene Rbl2. This activation was dependent on the demethylase activity of Utx, and was suggested to contribute to the decreased cell proliferation induced by Utx. A chromatin immunoprecipitation assay showed that over-expressed Utx was associated with the promoter regions of Rb and Rbl2 resulting in the removal of repressive H3K27 tri-methylation and the increase in active H3K4 tri-methylation. Furthermore, siRNA-mediated knockdown of Utx revealed the recruitment of endogenous Utx protein on the promoters of Rb and Rbl2 genes. These results indicate that Rb and Rbl2 are downstream target genes of Utx and may play important roles in Utx-mediated cell growth control.

Hepatic radiofrequency ablation causes an increase of circulating histones in patients with hepatocellular carcinoma.

Science.gov (United States)

Gu, Tao; Ge, Yang; Song, Yuezhang; Fu, Zhanzhao; Zhang, Yunjie; Wang, Guangxia; Shao, Shasha; Wen, Tao

2015-11-01

Radiofrequency ablation (RFA) has been increasingly accepted for the treatment of hepatocellular carcinoma (HCC). However, RFA has been associated with an obvious systemic inflammatory response, but little is known about the underlying mechanisms. Circulating histones are recently identified as pivotal inflammatory mediators. Hence, we investigated whether circulating histones are involved in RFA-related inflammation. Serial blood samples were collected from 42 HCC patients undergoing RFA at 3 time points: pre-RFA, post-RFA (within 24 h), and in 4-week follow up after RFA. Plasma histones, myeloperoxidase (MPO), inflammatory cytokines (IL-1β, IL-6, IL-10, TNF-α), liver damage parameters (ALT, AST), and creatinine were measured. Compared to pre-RFA (0.837 μg/ml), there was a significant increase in the levels of circulating histones within 24 h post-RFA (4.576 μg/ml, p histones decreased to pre-RFA levels in 4-week follow up after RFA. Meanwhile, MPO, IL-6, and IL-10 were elevated remarkably within 24 h post-RFA, indicative of an occurrence of the inflammatory response. Notably, histone levels correlated well with MPO (r = 0.5678), IL-6 (r = 0.4851), and IL-10 (r = 0.3574), respectively. In addition, there was a significant damage of liver function in patients within 24 h post-RFA, evidenced by the increased levels of ALT and AST. No changes in creatinine levels were observed. These data demonstrate that circulating histones are excessively released in HCC patients treated with RFA, which may lead to systemic inflammation by stimulating neutrophil activation and promoting cytokine production. Circulating histones may act as a novel marker to indicate the extent of inflammation related to RFA.

Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

International Nuclear Information System (INIS)

Gupta, Chanchal; Kaur, Jasmine; Tikoo, Kulbhushan

2014-01-01

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. - Highlights: • High glucose induces phosphorylation of histone H3 and dephosphorylation of GSK-3β. • Moreover, hyperglycemia also leads to increased DNA methylation in MDA-MB-231 cells. • Inhibition of GSK-3β prevented histone H3 phosphorylation and reduced DNMT1 levels. • Interplay exists between GSK-3β, histone H3 phosphorylation and DNA methylation

Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

Energy Technology Data Exchange (ETDEWEB)

Gupta, Chanchal; Kaur, Jasmine; Tikoo, Kulbhushan, E-mail: tikoo.k@gmail.com

2014-05-15

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. - Highlights: • High glucose induces phosphorylation of histone H3 and dephosphorylation of GSK-3β. • Moreover, hyperglycemia also leads to increased DNA methylation in MDA-MB-231 cells. • Inhibition of GSK-3β prevented histone H3 phosphorylation and reduced DNMT1 levels. • Interplay exists between GSK-3β, histone H3 phosphorylation and DNA methylation.

Co-regulation of histone-modifying enzymes in cancer.

Directory of Open Access Journals (Sweden)

Abul B M M K Islam

Full Text Available Cancer is characterized by aberrant patterns of expression of multiple genes. These major shifts in gene expression are believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including histone modifications. However, it is unclear whether the binding of histone-modifying proteins to genomic regions and the placing of histone modifications efficiently discriminates corresponding genes from the rest of the genes in the human genome. We performed gene expression analysis of histone demethylases (HDMs and histone methyltransferases (HMTs, their target genes and genes with relevant histone modifications in normal and tumor tissues. Surprisingly, this analysis revealed the existence of correlations in the expression levels of different HDMs and HMTs. The observed HDM/HMT gene expression signature was specific to particular normal and cancer cell types and highly correlated with target gene expression and the expression of genes with histone modifications. Notably, we observed that trimethylation at lysine 4 and lysine 27 separated preferentially expressed and underexpressed genes, which was strikingly different in cancer cells compared to normal cells. We conclude that changes in coordinated regulation of enzymes executing histone modifications may underlie global epigenetic changes occurring in cancer.

Phosphorylation of mitogen-activated protein kinase (MAPK) is required for cytokinesis and progression of cell cycle in tobacco BY-2 cells.

Science.gov (United States)

Ma, Zhaowu; Yu, Guanghui

2010-02-15

The role of mitogen-activated protein kinase (MAPK) in plant cytokinesis remains largely uncharacterized. To elucidate its role, tobacco Bright Yellow-2 (BY-2) cells have been synchronized using a two-step procedure, and the different phases of the cell cycle identified by Histone 4 gene expression and the mitotic index. MAPK expression was analyzed by semi-quantitative (SQ) RT-PCR and protein gel blot analysis for phosphorylated MAPK during cell cycle progression. The SQ RT-PCR analysis indicated that MAPK expression is lower in mitosis than in interphase (G1, G2 and S). However, the amount of phosphorylated MAPK remained stable throughout the cell cycle, indicating that MAPK activity is predominantly regulated at the post-translational level and that phosphorylation of MAPK plays an important role in mitosis. Application of the specific MAPK phosphorylation inhibitor U0126 revealed that while U0126 treatment decreases the phosphorylation of MAPK and the progression from telophase to early cytokinesis is significantly inhibited. The formation of the phragmoplast is also negatively affected at this stage. These results demonstrate that MAPK phosphorylation is involved in the formation of the cell plate within the phragmoplast during cytokinesis and that MAPK predominantly functions during the cytokinesis stage of the cell cycle in tobacco BY-2 cells. Copyright 2009 Elsevier GmbH. All rights reserved.

Histone demethylases in development and disease

DEFF Research Database (Denmark)

Pedersen, Marianne Terndrup; Helin, Kristian

2010-01-01

Histone modifications serve as regulatory marks that are instrumental for the control of transcription and chromatin architecture. Strict regulation of gene expression patterns is crucial during development and differentiation, where diverse cell types evolve from common predecessors. Since...... the first histone lysine demethylase was discovered in 2004, a number of demethylases have been identified and implicated in the control of gene expression programmes and cell fate decisions. Histone demethylases are now emerging as important players in developmental processes and have been linked to human...

Flexible histone tails in a new mesoscopic oligonucleosome model.

Science.gov (United States)

Arya, Gaurav; Zhang, Qing; Schlick, Tamar

2006-07-01

We describe a new mesoscopic model of oligonucleosomes that incorporates flexible histone tails. The nucleosome cores are modeled using the discrete surface-charge optimization model, which treats the nucleosome as an electrostatic surface represented by hundreds of point charges; the linker DNAs are treated using a discrete elastic chain model; and the histone tails are modeled using a bead/chain hydrodynamic approach as chains of connected beads where each bead represents five protein residues. Appropriate charges and force fields are assigned to each histone chain so as to reproduce the electrostatic potential, structure, and dynamics of the corresponding atomistic histone tails at different salt conditions. The dynamics of resulting oligonucleosomes at different sizes and varying salt concentrations are simulated by Brownian dynamics with complete hydrodynamic interactions. The analyses demonstrate that the new mesoscopic model reproduces experimental results better than its predecessors, which modeled histone tails as rigid entities. In particular, our model with flexible histone tails: correctly accounts for salt-dependent conformational changes in the histone tails; yields the experimentally obtained values of histone-tail mediated core/core attraction energies; and considers the partial shielding of electrostatic repulsion between DNA linkers as a result of the spatial distribution of histone tails. These effects are crucial for regulating chromatin structure but are absent or improperly treated in models with rigid histone tails. The development of this model of oligonucleosomes thus opens new avenues for studying the role of histone tails and their variants in mediating gene expression through modulation of chromatin structure.

Potential non-oncological applications of histone deacetylase inhibitors.

Science.gov (United States)

Ververis, Katherine; Karagiannis, Tom C

2011-01-01

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutic drugs. Their clinical utility in oncology stems from their intrinsic cytotoxic properties and combinatorial effects with other conventional cancer therapies. To date, the histone deacetylase inhibitors suberoylanilide hydroxamic acid (Vorinostat, Zolinza®) and depsipeptide (Romidepsin, Istodax®) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Further, there are currently over 100 clinical trials involving the use of histone deacetylase inhibitors in a wide range of solid and hematological malignancies. The therapeutic potential of histone deacetylase inhibitors has also been investigated for numerous other diseases. For example, the cytotoxic properties of histone deacetylase inhibitors are currently being harnessed as a potential treatment for malaria, whereas the efficacy of these compounds for HIV relies on de-silencing latent virus. The anti-inflammatory properties of histone deacetylase inhibitors are the predominant mechanisms for other diseases, such as hepatitis, systemic lupus erythematosus and a wide range of neurodegenerative conditions. Additionally, histone deacetylase inhibitors have been shown to be efficacious in animal models of cardiac hypertrophy and asthma. Broad-spectrum histone deacetylase inhibitors are clinically available and have been used almost exclusively in preclinical systems to date. However, it is emerging that class- or isoform-specific compounds, which are becoming more readily available, may be more efficacious particularly for non-oncological applications. The aim of this review is to provide an overview of the effects and clinical potential of histone deacetylase inhibitors in various diseases. Apart from applications in oncology, the discussion is focused on the potential efficacy of histone deacetylase inhibitors for the treatment of neurodegenerative diseases, cardiac

SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

Energy Technology Data Exchange (ETDEWEB)

JOHN C WALKER

2011-11-01

Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

Further evidence for poly-ADP-ribosylated histones as DNA suppressors

International Nuclear Information System (INIS)

Yu, F.L.; Geronimo, I.H.; Bender, W.; Meginniss, K.E.

1986-01-01

For many years histones have been considered to be the gene suppressors in eukaryotic cells. Recently, the authors have found strong evidence indicating that poly-ADP-ribosylated histones, rather than histones, are the potent inhibitors of DNA-dependent RNA synthesis. They now report additional evidence for this concept: 1) using histone inhibitor isolated directly from nuclei, the authors are able to confirm their earlier findings that the inhibitor substances are sensitive to pronase, snake venom phosphodiesterase digestion and 0.1N KOH hydrolysis, and are resistant to DNase I and RNase A digestion, 2) the O.D. 260/O.D.280 ratio of the histone inhibitor is between pure protein and nuclei acid, suggesting the inhibitor substance is a nucleoprotein hybrid. This result directly supports the fact that the isolated histone inhibitor is radioactive poly (ADP-ribose) labeled, 3) commercial histones show big differences in inhibitor activity. The authors believe this reflects the variation in poly-ADP-ribosylation among commercial histones, and 4) 0.1N KOH hydrolysis eliminates the poly (ADP-ribose) radioactivity from the acceptor proteins as well as histone inhibitor activity. Yet, on gel, the inhibitor shows identical histone bands and stain intensity before and after hydrolysis, indicating the histones per se are qualitatively and quantitatively unaffected by alkaline treatment. This result strongly suggests that histones themselves are not capable of inhibiting DNA-dependent RNA synthesis

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones.

Science.gov (United States)

Liokatis, Stamatios

2017-03-26

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications (PTMs). They are newly identified species with unknown means of establishment and functional implications. Current analytical methods are inadequate to detect the copy-specific occurrence of PTMs on the nucleosomal sister histones. This protocol presents a biochemical method for the in vitro reconstitution of nucleosomes containing differentially isotope-labeled sister histones. The generated complex can be also asymmetrically modified, after including a premodified histone pool during refolding of histone subcomplexes. These asymmetric nucleosome preparations can be readily reacted with histone-modifying enzymes to study modification cross-talk mechanisms imposed by the asymmetrically pre-incorporated PTM using nuclear magnetic resonance (NMR) spectroscopy. Particularly, the modification reactions in real-time can be mapped independently on the two sister histones by performing different types of NMR correlation experiments, tailored for the respective isotope type. This methodology provides the means to study crosstalk mechanisms that contribute to the formation and propagation of asymmetric PTM patterns on nucleosomal complexes.

Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism.

Directory of Open Access Journals (Sweden)

Mayumi Nakahara

Full Text Available INTRODUCTION: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM. rTM has been approved for the treatment of disseminated intravascular coagulation (DIC in Japan, and is currently undergoing a phase III clinical trial in the United States. METHODS: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. RESULTS: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. CONCLUSIONS: Extracellular histones cause massive

Histone modifications: Cycling with chromosomal replication

DEFF Research Database (Denmark)

Thon, Genevieve

2008-01-01

Histone modifications tend to be lost during chromosome duplication. Several recent studies suggest that the RNA interference pathway becomes active during the weakened transcriptional repression occurring at centromeres in S phase, resulting in the re-establishment of histone modifications...

Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

Science.gov (United States)

Zhang, Qian; Giebler, Holli A; Isaacson, Marisa K; Nyborg, Jennifer K

2015-01-01

In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo. These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust

Presence of Cytotoxic Extracellular Histones in Machine Perfusate of Donation After Circulatory Death Kidneys.

Science.gov (United States)

van Smaalen, Tim C; Beurskens, Daniëlle M H; Hoogland, E R Pieter; Winkens, Bjorn; Christiaans, Maarten H L; Reutelingsperger, Chris P; van Heurn, L W Ernest; Nicolaes, Gerry A F

2017-04-01

Extracellular histones are cytotoxic molecules that are related to cell stress and death. They have been shown to play a crucial role in multiple pathophysiologic processes like sepsis, inflammation, vascular dysfunction, and thrombosis. Their role in organ donation and graft function and survival is still unknown. The aim of this study was to assess whether an association exists between the presence of extracellular histones in machine perfusates and deceased donor kidney viability. Machine perfusates of 390 donations after circulatory death kidneys were analyzed for histone concentration, and corresponding graft function and survival were assessed. Extracellular histone concentrations were significantly higher in perfusates of kidneys with posttransplant graft dysfunction (primary nonfunction and delayed graft function) and were an independent risk factor for delayed graft function (odds ratio, 2.152; 95% confidence interval [95% CI], 1.199-3.863) and 1 year graft failure (hazard ratio, 1.386; 95% CI, 1.037-1.853), but not for primary nonfunction (odds ratio, 1.342; 95% CI, 0.900-2.002). One year graft survival was 12% higher in the group with low histone concentrations (P = 0.008) as compared with the group that contained higher histone concentrations. This study warrants future studies to probe for a possible role of cytotoxic extracellular histones in organ viability and suggests that quantitation of extracellular histones might contribute to assessment of posttransplant graft function and survival.

Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Rakesh Kumar Singh

Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones.

Science.gov (United States)

Baranova, Svetlana V; Buneva, Valentina N; Nevinsky, Georgy A

2016-08-01

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Here, using ELISA it was shown that sera of HIV-infected patients and healthy donors contain autoantibodies against histones. Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether antibodies from sera of HIV-infected patients can hydrolyze human histones. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of HIV-infected patients by chromatography on several affinity sorbents. We present first evidence showing that 100% of IgGs purified from the sera of 32 HIV-infected patients efficiently hydrolyze from one to five human histones. Several rigid criteria have been applied to show that the histone-hydrolyzing activity is an intrinsic property of IgGs of HIV-infected patients. The relative efficiency of hydrolysis of histones (H1, H2a, H2b, H3, and H4) significantly varied for IgGs of different patients. IgGs from the sera of 40% of healthy donors also hydrolyze histones but with an average efficiency approximately 16-fold lower than that of HIV-infected patients. Similar to proteolytic abzymes from the sera of patients with several autoimmune diseases, histone-hydrolyzing IgGs from HIV-infected patients were inhibited by specific inhibitors of serine and of metal-dependent proteases, but an unexpected significant inhibition of the activity by specific inhibitor of thiol-like proteases was also observed. Because IgGs can efficiently hydrolyze histones, a negative role of abzymes in development of acquired immune deficiency syndrome cannot be

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

Science.gov (United States)

Ashar, Harshini K; Mueller, Nathan C; Rudd, Jennifer M; Snider, Timothy A; Achanta, Mallika; Prasanthi, Maram; Pulavendran, Sivasami; Thomas, Paul G; Ramachandran, Akhilesh; Malayer, Jerry R; Ritchey, Jerry W; Rajasekhar, Rachakatla; Chow, Vincent T K; Esmon, Charles T; Teluguakula, Narasaraju

2018-01-01

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Thiophene-derivatized Fluorescent Benzamides as Possible Probes for Histone Deacetylases

International Nuclear Information System (INIS)

Seo, Young Jun

2013-01-01

We have synthesized a series of novel fluorescent benzamides inhibitors possessing intrinsic fluorescence properties. Most of these benzamide fluorophores exhibit high quantum yields, making them suitable for use in imaging studies, with colors ranging from blue to green; a couple of them were also water-soluble. Notably, TB1 and TB2 display a high quantum yield and TB1 exhibits high binding affinity to HDAC enzymes. We believe that these new fluorescent benzamide inhibitors might be useful diagnostic tools for in vitro studies of HDACs. Histone deacetylases (HDACs) are crucial gene regulating enzymes that control the expression of histones-epigenetic targets in research related to developing new therapies for cancer, central nervous system disorders, and heart disease. The deacetylation of histones is a vital repression process in transcriptional gene expression; it also affects apoptosis, cell-cycle arrest, and angiogenesis

Thiophene-derivatized Fluorescent Benzamides as Possible Probes for Histone Deacetylases

Energy Technology Data Exchange (ETDEWEB)

Seo, Young Jun [Chonbuk National Univ., Jeonju (Korea, Republic of)

2013-08-15

We have synthesized a series of novel fluorescent benzamides inhibitors possessing intrinsic fluorescence properties. Most of these benzamide fluorophores exhibit high quantum yields, making them suitable for use in imaging studies, with colors ranging from blue to green; a couple of them were also water-soluble. Notably, TB1 and TB2 display a high quantum yield and TB1 exhibits high binding affinity to HDAC enzymes. We believe that these new fluorescent benzamide inhibitors might be useful diagnostic tools for in vitro studies of HDACs. Histone deacetylases (HDACs) are crucial gene regulating enzymes that control the expression of histones-epigenetic targets in research related to developing new therapies for cancer, central nervous system disorders, and heart disease. The deacetylation of histones is a vital repression process in transcriptional gene expression; it also affects apoptosis, cell-cycle arrest, and angiogenesis.

Histone methylation and aging: Lessons learned from model systems

Science.gov (United States)

McCauley, Brenna S.; Dang, Weiwei

2014-01-01

Aging induces myriad cellular and, ultimately, physiological changes that cause a decline in an organism's functional capabilities. Although the aging process and pathways that regulate it have been extensively studied, only in the last decade have we begun to appreciate that dynamic histone methylation may contribute to this process. In this review, we discuss recent work implicating histone methylation in aging. Loss of certain histone methyltransferases and demethylases changes lifespan in invertebrates, and alterations in histone methylation in aged organisms regulate lifespan and aging phenotypes, including oxidative stress-induced hormesis in yeast, insulin signaling in Caenorhabiditis elegans and mammals, and the senescence-associated secretory phenotype in mammals. In all cases where histone methylation has been shown to impact aging and aging phenotypes, it does so by regulating transcription, suggesting that this is a major mechanism of its action in this context. Histone methylation additionally regulates or is regulated by other cellular pathways that contribute to or combat aging. Given the numerous processes that regulate aging and histone methylation, and are in turn regulated by them, the role of histone methylation in aging is almost certainly underappreciated. PMID:24859460

Dynamics of Histone Tails within Chromatin

Science.gov (United States)

Bernier, Morgan; North, Justin; Page, Michael; Jaroniec, Christopher; Hammel, Christopher; Poirier, Michael

2012-02-01

Genetic information in humans is encoded within DNA molecules that is wrapped around histone octamer proteins and compacted into a highly conserved structural polymer, chromatin. The physical and material properties of chromatin appear to influence gene expression by altering the accessibility of proteins to the DNA. The tails of the histones are flexible domains that are thought to play a role in regulating DNA accessibility and compaction; however the molecular mechanisms for these phenomena are not understood. I will present CW-EPR studies on site directed spin labeled nucleosomes that probe the structure and dynamics of these histone tails within nucleosomes.

Histone deacetylase inhibitors: can we consider potent anti-neoplastic agents for the treatment of asthma?

Science.gov (United States)

Royce, Simon G; Ververis, Katherine; Karagiannis, Tom C

2012-01-01

Histone deacetylase inhibitors have emerged as a new class of anti-cancer therapeutics due to their potent anti-proliferative and apoptotic effects in malignant cells. Accumulating evidence is indicating that histone deacetylase inhibitors may also have potential clinical utility in non-oncological applications, including asthma. However, the potential of histone deacetylase inhibitors in asthma remains controversial. For example, the mechanisms of action of the broad-spectrum histone deacetylase inhibitor, Trichostatin A, in animal models of allergic airways disease are conflicting. Further, there is evidence suggesting potential problems associated with histone deacetylase 2 inhibition and conventional glucocorticosteroid therapy. Similarly, disparate findings are emerging following modulation of the class III, sirtuin 1 enzyme. Indeed, it is becoming apparent that the mechanism of action may not be related to histone deacetylase inhibition activity per se. Further, there is only limited evidence that these compounds possess anti-inflammatory effects in models of asthma. In this review, we provide an overview of the biology of the metal-dependent and sirtuin deacetylases in the context of asthma. The controversies surrounding the potential use of histone deacetylase inhibitors in asthma are discussed and future directions involving the investigation of more specific analogues are explored.

The histones of the endosymbiont alga of Peridinium balticum (Dinophyceae).

Science.gov (United States)

Rizzo, P J; Morris, R L; Zweidler, A

1988-01-01

The histones of the endosymbiont nucleus of the binucleate dinoflagellate Peridinium balticum were characterized by amino acid analysis and peptide mapping, and compared to calf thymus histones. Using these and various other criteria we have identified two H1-like histones as well as the highly conserved histones H3 and H4. A 13,000 dalton component in sodium dodecyl sulphate (SDS) gels can be separated into two components in Triton-containing gels. We suggest that these histones (HPb1 and HPb2) correspond to the vertebrate histones H2A and H2B, respectively.

Regulation of replication fork progression through histone supply and demand

DEFF Research Database (Denmark)

Groth, Anja; Corpet, Armelle; Cook, Adam J L

2007-01-01

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone c...... progression and histone supply and demand.......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...

Histones and their modifications in ovarian cancer - drivers of disease and therapeutic targets.

Science.gov (United States)

Marsh, Deborah J; Shah, Jaynish S; Cole, Alexander J

2014-01-01

Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC) is the most common subtype, with the majority of women presenting with advanced disease where 5-year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation, and monoubiquitination, with involvement of enzymes including histone methyltransferases, histone acetyltransferases/deacetylases, and ubiquitin ligases/deubiquitinases, respectively. Complexes such as the Polycomb repressive complex also play roles in the control of histone modifications and more recently roles for long non-coding RNA and microRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here, we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for women with ovarian cancer.

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

Science.gov (United States)

Permyakov, Serge E; Pershikova, Irina V; Khokhlova, Tatyana I; Uversky, Vladimir N; Permyakov, Eugene A

2004-05-18

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.

Structural Mechanisms of Nucleosome Recognition by Linker Histones.

Science.gov (United States)

Zhou, Bing-Rui; Jiang, Jiansheng; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T Sam; Bai, Yawen

2015-08-20

Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures. Copyright © 2015 Elsevier Inc. All rights reserved.

Histone deacetylase inhibitors promote the tumoricidal effect of HAMLET.

Science.gov (United States)

Brest, Patrick; Gustafsson, Mattias; Mossberg, Ann-Kristin; Gustafsson, Lotta; Duringer, Caroline; Hamiche, Ali; Svanborg, Catharina

2007-12-01

Histone deacetylase inhibitors (HDIs) and HAMLET (human alpha-lactalbumin made lethal to tumor cells) interact with histones, modify the structure of chromatin, and trigger tumor cell death. This study investigated how the combination of HDIs and HAMLET influences cell viability, histone acetylation, and DNA integrity. The pretreatment of tumor cells with HDIs was shown to enhance the lethal effect of HAMLET and the histone hyperacetylation response to HDIs increased even further after HAMLET treatment. HDIs and HAMLET were shown to target different histone domains as HAMLET bound tailless core histones, whereas HDIs modify the acetylation of the histone tail. DNA damage in response to HAMLET was increased by HDIs. The DNA repair response (p21WAFI expression) was induced by both agonists but abolished when the two agonists were combined. The results suggest that the synergy of HDIs and HAMLET is based on different but converging death pathways, both involving chromatin alterations. We speculate that HAMLET and HDIs might be combined to promote tumor cell death in vivo.

Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

Directory of Open Access Journals (Sweden)

Buro Lauren J

2010-09-01

Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

Histone acetylation regulates the time of replication origin firing.

Science.gov (United States)

Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

2002-11-01

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.

OpenAIRE

Hays, Shan M; Swanson, Johanna; Selker, Eric U

2002-01-01

We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged ...

Antibodies to H2a and H2b histones from the sera of HIV-infected patients catalyze site-specific degradation of these histones.

Science.gov (United States)

Baranova, Svetlana V; Dmitrienok, Pavel S; Ivanisenko, Nikita V; Buneva, Valentina N; Nevinsky, Georgy A

2017-06-01

Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecules when they are released into the extracellular space. Administration of histones to animals leads to systemic inflammatory and toxic responses. Autoantibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical enzymes were isolated from the sera of HIV-infected patients by chromatography on several affinity sorbents including anti-histone Sepharose. In contrast to canonical proteases (trypsin, chymotrypsin, proteinase K), IgGs from HIV-infected patients specifically hydrolyzed only histones but not many other tested globular proteins. Using MALDI mass spectrometry the sites of H2a and H2b histone cleavage by anti-histone IgGs were determined for the first time. One cluster of H2a hydrolysis contains two major (↕) and four moderate (↓) cleavage sites: 31-H↓R↓L↓L↓R↕K G↕N-38. One major and two moderate sites of cleavage were revealed in the second cluster: 14-A↕KSRS↓SRA↓G-22. The third cluster corresponding to the H2a C-terminal part contains only five minor (†) sites of cleavage: 82-H†LQLAIRNDEELN†KLLG†RV†T†I-102. It was shown that two major and four moderate sites of cleavage were present in the main cluster of H2b hydrolysis: 46-K↕QvhpD↓TgiS↓SkA↓M↕GiM↓N-63. Two moderate sites of cleavage correspond to a relatively short 6-mer cluster: 12-K↓GskK↓A-17. The third relatively long 9-mer cluster contains one major and two minor sites of H2b cleavage: 80-L↕AHYN†KRS†T-88. In the nucleosome core particle, most of the major and moderate cleavage sites are located at the H2a/H2b interaction interface. Minor cleavage sites of H2a are involved in binding with H3 in the nucleosome core. Two moderate cleavage sites of H2b and one

Quantitative mass spectrometry of histones H3.2 and H3.3 in Suz12-deficient mouse embryonic stem cells reveals distinct, dynamic post-translational modifications at Lys-27 and Lys-36

DEFF Research Database (Denmark)

Jung, Hye Ryung; Pasini, Diego; Helin, Kristian

2010-01-01

distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides....... The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27......me3 and an increase of H3K27ac, thereby uncovering an antagonistic methyl/acetyl switch at H3K27. The reduction in H3K27 methylation and increase in H3K27 acetylation was accompanied by H3K36 acetylation and methylation. Estimation of the global isoform percentage of unmodified and modified histone...

Histones as mediators of host defense, inflammation and thrombosis

OpenAIRE

Hoeksema, Marloes; van Eijk, Martin; Haagsman, Henk P; Hartshorn, Kevan L

2016-01-01

Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that contribute to bacterial killing but also to inflammatory injury. Histones can act as antimicrobial peptides and directly kill bacteria, fungi, parasites and viruses, in vitro and in a variety of ani...

Effects of protein phosphorylation on color stability of ground meat.

Science.gov (United States)

Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

2017-03-15

The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Heparin defends against the toxicity of circulating histones in sepsis.

Science.gov (United States)

Wang, Feifei; Zhang, Naipu; Li, Biru; Liu, Lanbo; Ding, Lei; Wang, Ying; Zhu, Yimin; Mo, Xi; Cao, Qing

2015-06-01

Although circulating histones were demonstrated as major mediators of death in septic mice models, their roles in septic patients are not clarified. The present study sought to evaluate the clinical relevance of the circulating histone levels in septic children, and the antagonizing effects of heparin on circulating histones. Histone levels in the plasma of septic children were significantly higher than healthy controls, and positively correlated with disease severity. Histone treatment could activate NF-κB pathway of the endothelial cells and induce the secretion of large amount of cytokines that further amplify inflammation, subsequently leading to organ damage. Co-injection of low dose heparin with lethal dose histones could protect mouse from organ damage and death by antagonizing circulating histones, and similar effects were also observed in other septic models. Collectively, these findings indicated that circulating histones might serve as key factors in the pathogenesis of sepsis and their levels in plasma might be a marker for disease progression and prognosis. Furthermore, low dose heparin might be an effective therapy to hamper sepsis progression and reduce the mortality.

Potentials and limitations of histone repeat sequences for phylogenetic reconstruction of Sophophora.

Science.gov (United States)

Baldo, A M; Les, D H; Strausbaugh, L D

1999-11-01

Simplified DNA sequence acquisition has provided many new data sets that are useful for phylogenetic reconstruction, including single- and multiple-copy nuclear and organellar genes. Although transcribed regions receive much attention, nontranscribed regions have recently been added to the repertoire of sequences suitable for phylogenetic studies, especially for closely related taxa. We evaluated the efficacy of a small portion of the histone repeat for phylogenetic reconstruction among Drosophila species. Histone repeats in invertebrates offer distinct advantages similar to those of widely used ribosomal repeats. First, the units are tandemly repeated and undergo concerted evolution. Second, histone repeats include both highly conserved coding and variable intergenic regions. This composition facilitates application of "universal" primers spanning potentially informative sites. We examined a small region of the histone repeat, including the intergenic spacer segments of coding regions from the divergently transcribed H2A and H2B histone genes. The spacer (about 230 bp) exists as a mosaic with highly conserved functional motifs interspersed with rapidly diverging regions; the former aid in alignment of the spacer. There are no ambiguities in alignment of coding regions. Coding and noncoding regions were analyzed together and separately for phylogenetic information. Parsimony, distance, and maximum-likelihood methods successfully retrieve the corroborated phylogeny for the taxa examined. This study demonstrates the resolving power of a small histone region which may now be added to the growing collection of phylogenetically useful DNA sequences.

Histones as mediators of host defense, inflammation and thrombosis

NARCIS (Netherlands)

Hoeksema, Marloes; Eijk, Martin van; Haagsman, Henk P; Hartshorn, Kevan L

2016-01-01

Histones are known for their ability to bind to and regulate expression of DNA. However, histones are also present in cytoplasm and extracellular fluids where they serve host defense functions and promote inflammatory responses. Histones are a major component of neutrophil extracellular traps that

Stromal serine protein kinase activity in spinach chloroplasts

International Nuclear Information System (INIS)

Cortez, N.; Lucero, H.A.; Vallejos, R.H.

1987-01-01

At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

Identification of distinct SET/TAF-Ibeta domains required for core histone binding and quantitative characterisation of the interaction.

Science.gov (United States)

Karetsou, Zoe; Emmanouilidou, Anastasia; Sanidas, Ioannis; Liokatis, Stamatis; Nikolakaki, Eleni; Politou, Anastasia S; Papamarcaki, Thomais

2009-04-09

The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Ibeta belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Ibeta, we designed several SET/TAF-Ibeta truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. Wild type SET/TAF-Ibeta binds to histones H2B and H3 with Kd values of 2.87 and 0.15 microM, respectively. The preferential binding of SET/TAF-Ibeta to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Ibeta, as well as the H3 amino-terminal tail, are dispensable for this interaction. This type of analysis allowed us to assess the relative affinities of SET/TAF-Ibeta for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Ibeta and can be valuable to understand the role of SET/TAF-Ibeta in chromatin function.

Novel chemokine-like activities of histones in tumor metastasis.

Science.gov (United States)

Chen, Ruochan; Xie, Yangchun; Zhong, Xiao; Fu, Yongmin; Huang, Yan; Zhen, Yixiang; Pan, Pinhua; Wang, Haichao; Bartlett, David L; Billiar, Timothy R; Lotze, Michael T; Zeh, Herbert J; Fan, Xue-Gong; Tang, Daolin; Kang, Rui

2016-09-20

Histones are intracellular nucleosomal components and extracellular damage-associated molecular pattern molecules that modulate chromatin remodeling, as well as the immune response. However, their extracellular roles in cell migration and invasion remain undefined. Here, we demonstrate that histones are novel regulators of tumor metastasis with chemokine-like activities. Indeed, exogenous histones promote both hepatocellular carcinoma (HCC) cell migration and invasion through toll-like receptor (TLR)4, but not TLR2 or the receptor for advanced glycosylation end product. TLR4-mediated activation of nuclear factor-κB (NF-κB) by extracellular signal-regulated kinase (ERK) is required for histone-induced chemokine (e.g., C-C motif ligand 9/10) production. Pharmacological and genetic inhibition of TLR4-ERK-NF-κB signaling impairs histone-induced chemokine production and HCC cell migration. Additionally, TLR4 depletion (by using TLR4-/- mice and TLR4-shRNA) or inhibition of histone release/activity (by administration of heparin and H3 neutralizing antibody) attenuates lung metastasis of HCC cells injected via the tail vein of mice. Thus, histones promote tumor metastasis of HCC cells through the TLR4-NF-κB pathway and represent novel targets for treating patients with HCC.

Genome-wide analysis of regions similar to promoters of histone genes

KAUST Repository

Chowdhary, Rajesh

2010-05-28

Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

Science.gov (United States)

Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

2017-08-01

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Plant Responses to Abiotic Stress Regulated by Histone Deacetylases

Directory of Open Access Journals (Sweden)

Ming Luo

2017-12-01

Full Text Available In eukaryotic cells, histone acetylation and deacetylation play an important role in the regulation of gene expression. Histone acetylation levels are modulated by histone acetyltransferases and histone deacetylases (HDACs. Recent studies indicate that HDACs play essential roles in the regulation of gene expression in plant response to environmental stress. In this review, we discussed the recent advance regarding the plant HDACs and their functions in the regulation of abiotic stress responses. The role of HDACs in autophagy was also discussed.

Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin.

Science.gov (United States)

Matsushita, Masaya; Ochiai, Hiroshi; Suzuki, Ken-Ichi T; Hayashi, Sayaka; Yamamoto, Takashi; Awazu, Akinori; Sakamoto, Naoaki

2017-12-15

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development. © 2017. Published by The Company of Biologists Ltd.

Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors

Directory of Open Access Journals (Sweden)

Sylva Brabencová

2017-12-01

Full Text Available Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0 and Wassilewskija (Ws homogenized by two techniques (grinding in a cryomill or with a mortar and pestle. We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants. Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

Variations of Histone Modification Patterns: Contributions of Inter-plant Variability and Technical Factors.

Science.gov (United States)

Brabencová, Sylva; Ihnatová, Ivana; Potěšil, David; Fojtová, Miloslava; Fajkus, Jiří; Zdráhal, Zbyněk; Lochmanová, Gabriela

2017-01-01

Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

Directory of Open Access Journals (Sweden)

Michael J Morrison

Full Text Available WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

Science.gov (United States)

Fabry, S; Müller, K; Lindauer, A; Park, P B; Cornelius, T; Schmitt, R

1995-09-01

The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II

Histone Variants and Composition in the Developing Brain: Should MeCP2 Care?

Science.gov (United States)

Zago, Valentina; Pinar-CabezaDeVaca, Cristina; Vincent, John B; Ausio, Juan

2017-01-01

Specific compositional chromatin features distinguish brain/neuronal chromatin from that of other tissues and are critical to this organ and cell type development and neuroplasticity. These features include a significant turnover of the major constitutive chromosomal proteins, including the (canonical) replication-dependent histones, the replication-independent replacement histone variants, as well as the chromatin associated transcriptional regulator MeCP2 (methyl CpG binding protein 2). Alterations of histones and MeCP2 have already been implicated in many brain disorders. Despite the relevance of histone variants to chromatin structure and function, only recently has some exciting literature started to re-emerge that directly relates them to neuron plasticity and cognition. However, the amount of information available on the functional role of these histones is still very limited. The purpose of this review is to focus attention to this important group of chromatin proteins, which, in the brain, possess overlapping structural and functional roles with the highly abundant presence of MeCP2. There is an imperative need to understand how all these proteins communicate with each other, and future research will hopefully provide us with answers.

Arginine-rich histones have strong antiviral activity for influenza A viruses.

Science.gov (United States)

Hoeksema, Marloes; Tripathi, Shweta; White, Mitchell; Qi, Li; Taubenberger, Jeffery; van Eijk, Martin; Haagsman, Henk; Hartshorn, Kevan L

2015-10-01

While histones are best known for DNA binding and transcription-regulating properties, they also have antimicrobial activity against a broad range of potentially pathogenic organisms. Histones are abundant in neutrophil extracellular traps, where they play an important role in NET-mediated antimicrobial killing. Here, we show anti-influenza activity of histones against both seasonal H3N2 and H1N1, but not pandemic H1N1. The arginine rich histones, H3 and H4, had greater neutralizing and viral aggregating activity than the lysine rich histones, H2A and H2B. Of all core histones, histone H4 is most potent in neutralizing IAV, and incubation with IAV with histone H4 results in a decrease in uptake and viral replication by epithelial cells when measured by qRT-PCR. The antiviral activity of histone H4 is mediated principally by direct effects on viral particles. Histone H4 binds to IAV as assessed by ELISA and co-sedimentation of H4 with IAV. H4 also induces aggregation, as assessed by confocal microscopy and light transmission assays. Despite strong antiviral activity against the seasonal IAV strains, H4 was inactive against pandemic H1N1. These findings indicate a possible role for histones in the innate immune response against IAV. © The Author(s) 2015.

Histones and their modifications in ovarian cancer – drivers of disease and therapeutic targets

Directory of Open Access Journals (Sweden)

Deborah Joy Marsh

2014-06-01

Full Text Available Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC is the most common subtype, with the majority of women presenting with advanced disease where 5 year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation and monoubiquitination, with involvement of enzymes including histone methyl transferases (HMTases, histone acetyltransferases/deacetylases and ubiquitin ligases/deubiquitinases respectively. Complexes such as the Polycomb Repressive Complex also play roles in the control of histone modifications and more recently roles for long non-coding (lnc RNA and microRNAs (miRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for

The role of extracellular histones in haematological disorders.

Science.gov (United States)

Alhamdi, Yasir; Toh, Cheng-Hock

2016-06-01

Over the past decades, chromosomal alterations have been extensively investigated for their pathophysiological relevance in haematological malignancies. In particular, epigenetic modifications of intra-nuclear histones are now known as key regulators of healthy cell cycles that have also evolved into novel therapeutic targets for certain blood cancers. Thus, for most haematologists, histones are DNA-chained proteins that are buried deep within chromatin. However, the plot has deepened with recent revelations on the function of histones when unchained and released extracellularly upon cell death or from activated neutrophils as part of neutrophil extracellular traps (NETs). Extracellular histones and NETs are increasingly recognized for profound cytotoxicity and pro-coagulant effects. This article highlights the importance of recognizing this new paradigm of extracellular histones as a key player in host defence through its damage-associated molecular patterns, which could translate into novel diagnostic and therapeutic biomarkers in various haematological and critical disorders. © 2016 John Wiley & Sons Ltd.

Circulating histones are mediators of trauma-associated lung injury.

Science.gov (United States)

Abrams, Simon T; Zhang, Nan; Manson, Joanna; Liu, Tingting; Dart, Caroline; Baluwa, Florence; Wang, Susan Siyu; Brohi, Karim; Kipar, Anja; Yu, Weiping; Wang, Guozheng; Toh, Cheng-Hock

2013-01-15

Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. To investigate the pathological roles of circulating histones in trauma-induced lung injury. Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause-effect relationship was studied using cells and mouse models. In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival outcomes in patients.

Circulating Histones Are Mediators of Trauma-associated Lung Injury

Science.gov (United States)

Abrams, Simon T.; Zhang, Nan; Manson, Joanna; Liu, Tingting; Dart, Caroline; Baluwa, Florence; Wang, Susan Siyu; Brohi, Karim; Kipar, Anja; Yu, Weiping

2013-01-01

Rationale: Acute lung injury is a common complication after severe trauma, which predisposes patients to multiple organ failure. This syndrome largely accounts for the late mortality that arises and despite many theories, the pathological mechanism is not fully understood. Discovery of histone-induced toxicity in mice presents a new dimension for elucidating the underlying pathophysiology. Objectives: To investigate the pathological roles of circulating histones in trauma-induced lung injury. Methods: Circulating histone levels in patients with severe trauma were determined and correlated with respiratory failure and Sequential Organ Failure Assessment (SOFA) scores. Their cause–effect relationship was studied using cells and mouse models. Measurements and Main Results: In a cohort of 52 patients with severe nonthoracic blunt trauma, circulating histones surged immediately after trauma to levels that were toxic to cultured endothelial cells. The high levels were significantly associated with the incidence of acute lung injury and SOFA scores, as well as markers of endothelial damage and coagulation activation. In in vitro systems, histones damaged endothelial cells, stimulated cytokine release, and induced neutrophil extracellular trap formation and myeloperoxidase release. Cellular toxicity resulted from their direct membrane interaction and resultant calcium influx. In mouse models, cytokines and markers for endothelial damage and coagulation activation significantly increased immediately after trauma or histone infusion. Pathological examinations showed that lungs were the predominantly affected organ with edema, hemorrhage, microvascular thrombosis, and neutrophil congestion. An anti-histone antibody could reduce these changes and protect mice from histone-induced lethality. Conclusions: This study elucidates a new mechanism for acute lung injury after severe trauma and proposes that circulating histones are viable therapeutic targets for improving survival

Characterization of the UV-crosslinked heterodimer of histones H2B and H4

International Nuclear Information System (INIS)

Johnson, E.R.; Brown, D.M.; DeLange, R.J.

1986-01-01

At relatively high salt concentrations (1.2 M), histone 2B (H2B) and histone 4 (H4) can be covalently crosslinked by irradiation with ultraviolet light to yield a mixture of the three possible dimers: H2B-H2B, H4-H4, and H2B-H4. The formation of the H2B-H4 heterodimer was found to be favored at lower histone concentrations (> 90% H2B-H4 at 0.1 mg/ml total histone protein). CNBr cleavage of the H2B-H4 dimer produced three fragments which were separated by reverse phase HPLC. These fragments were identified by amino acid compositional analysis to be H4(85-102), H2B(62-125), and the crosslinked N-terminal regions H2B(1-59)-H4(1-84). Amino acid sequence analysis of the crosslinked fragment indicated that tyrosine-40 of H2B is likely involved in the covalent crosslinkage which joins the histone monomers to form the heterodimer

17ß-Estradiol Regulates Histone Alterations Associated with Memory Consolidation and Increases "Bdnf" Promoter Acetylation in Middle-Aged Female Mice

Science.gov (United States)

Fortress, Ashley M.; Kim, Jaekyoon; Poole, Rachel L.; Gould, Thomas J.; Frick, Karyn M.

2014-01-01

Histone acetylation is essential for hippocampal memory formation in young adult rodents. Although dysfunctional histone acetylation has been associated with age-related memory decline in male rodents, little is known about whether histone acetylation is altered by aging in female rodents. In young female mice, the ability of 17ß-estradiol…

Germline-specific H1 variants: the "sexy" linker histones.

Science.gov (United States)

Pérez-Montero, Salvador; Carbonell, Albert; Azorín, Fernando

2016-03-01

The eukaryotic genome is packed into chromatin, a nucleoprotein complex mainly formed by the interaction of DNA with the abundant basic histone proteins. The fundamental structural and functional subunit of chromatin is the nucleosome core particle, which is composed by 146 bp of DNA wrapped around an octameric protein complex formed by two copies of each core histone H2A, H2B, H3, and H4. In addition, although not an intrinsic component of the nucleosome core particle, linker histone H1 directly interacts with it in a monomeric form. Histone H1 binds nucleosomes near the exit/entry sites of linker DNA, determines nucleosome repeat length and stabilizes higher-order organization of nucleosomes into the ∼30 nm chromatin fiber. In comparison to core histones, histone H1 is less well conserved through evolution. Furthermore, histone H1 composition in metazoans is generally complex with most species containing multiple variants that play redundant as well as specific functions. In this regard, a characteristic feature is the presence of specific H1 variants that replace somatic H1s in the germline and during early embryogenesis. In this review, we summarize our current knowledge about their structural and functional properties.

The in vivo phosphorylation sites of rat brain dynamin I

DEFF Research Database (Denmark)

Graham, Mark E; Anggono, Victor; Bache, Nicolai

2007-01-01

-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation...... of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results...

Histone H4 Lysine 20 methylation

DEFF Research Database (Denmark)

Jørgensen, Stine; Schotta, Gunnar; Sørensen, Claus Storgaard

2013-01-01

of histones have emerged as key regulators of genomic integrity. Intense research during the past few years has revealed histone H4 lysine 20 methylation (H4K20me) as critically important for the biological processes that ensure genome integrity, such as DNA damage repair, DNA replication and chromatin...... compaction. The distinct H4K20 methylation states are mediated by SET8/PR-Set7 that catalyses monomethylation of H4K20, whereas SUV4-20H1 and SUV4-20H2 enzymes mediate further H4K20 methylation to H4K20me2 and H4K20me3. Disruption of these H4K20-specific histone methyltransferases leads to genomic...

Identification of distinct SET/TAF-Iβ domains required for core histone binding and quantitative characterisation of the interaction

Science.gov (United States)

Karetsou, Zoe; Emmanouilidou, Anastasia; Sanidas, Ioannis; Liokatis, Stamatis; Nikolakaki, Eleni; Politou, Anastasia S; Papamarcaki, Thomais

2009-01-01

Background The assembly of nucleosomes to higher-order chromatin structures is finely tuned by the relative affinities of histones for chaperones and nucleosomal binding sites. The myeloid leukaemia protein SET/TAF-Iβ belongs to the NAP1 family of histone chaperones and participates in several chromatin-based mechanisms, such as chromatin assembly, nucleosome reorganisation and transcriptional activation. To better understand the histone chaperone function of SET/TAF-Iβ, we designed several SET/TAF-Iβ truncations, examined their structural integrity by circular Dichroism and assessed qualitatively and quantitatively the histone binding properties of wild-type protein and mutant forms using GST-pull down experiments and fluorescence spectroscopy-based binding assays. Results Wild type SET/TAF-Iβ binds to histones H2B and H3 with Kd values of 2.87 and 0.15 μM, respectively. The preferential binding of SET/TAF-Iβ to histone H3 is mediated by its central region and the globular part of H3. On the contrary, the acidic C-terminal tail and the amino-terminal dimerisation domain of SET/TAF-Iβ, as well as the H3 amino-terminal tail, are dispensable for this interaction. Conclusion This type of analysis allowed us to assess the relative affinities of SET/TAF-Iβ for different histones and identify the domains of the protein required for effective histone recognition. Our findings are consistent with recent structural studies of SET/TAF-Iβ and can be valuable to understand the role of SET/TAF-Iβ in chromatin function. PMID:19358706

Brownian dynamics simulation of the cross-talking effect among modified histones on conformations of nucleosomes

International Nuclear Information System (INIS)

Zhao-Wen, Duan; Wei, Li; Ping, Xie; Shuo-Xing, Dou; Peng-Ye, Wang

2010-01-01

Using Brownian dynamics simulation, we studied the effect of histone modifications on conformations of an array of nucleosomes in a segment of chromatin. The simulation demonstrated that the segment of chromatin shows the dynamic behaviour that its conformation can switch between a state with nearly all of the histones being wrapped by DNA and a state with nearly all of the histones being unwrapped by DNA, thus involving the “cross-talking” interactions among the histones. Each state can stay for a sufficiently long time. These conformational states are essential for gene expression or gene silence. The simulation also shows that these conformational states can be inherited by the daughter DNAs during DNA replication, giving a theoretical explanation of the epigenetic phenomenon. (cross-disciplinary physics and related areas of science and technology)

Biotinylation is a natural, albeit rare, modification of human histones

Science.gov (United States)

Kuroishi, Toshinobu; Rios-Avila, Luisa; Pestinger, Valerie; Wijeratne, Subhashinee S. K.; Zempleni, Janos

2011-01-01

Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites. PMID:21930408

O-antigen protects gram-negative bacteria from histone killing.

Directory of Open Access Journals (Sweden)

Catherine Chaput

Full Text Available Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous Escherichia coli libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens Shigella flexneri and Klebsiella pneumoniae.

Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C

DEFF Research Database (Denmark)

Leurs, Ulrike; Lohse, Brian; Rand, Kasper Dyrberg

2014-01-01

Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most...... inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide...... sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation...

Open and closed: the roles of linker histones in plants and animals.

Science.gov (United States)

Over, Ryan S; Michaels, Scott D

2014-03-01

Histones package DNA in all eukaryotes and play key roles in regulating gene expression. Approximately 150 base pairs of DNA wraps around an octamer of core histones to form the nucleosome, the basic unit of chromatin. Linker histones compact chromatin further by binding to and neutralizing the charge of the DNA between nucleosomes. It is well established that chromatin packing is regulated by a complex pattern of posttranslational modifications (PTMs) to core histones, but linker histone function is less well understood. In this review, we describe the current understanding of the many roles that linker histones play in cellular processes, including gene regulation, cell division, and development, while putting the linker histone in the context of other nuclear proteins. Although intriguing roles for plant linker histones are beginning to emerge, much of our current understanding comes from work in animal systems. Many unanswered questions remain and additional work is required to fully elucidate the complex processes mediated by linker histones in plants.

Histone deacetylase 1, 2, 6 and acetylated histone H4 in B- and T-cell lymphomas

DEFF Research Database (Denmark)

Marquard, L.; Poulsen, C.B.; Gjerdrum, L.M.

2009-01-01

AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim was to det......AIMS: Histone deacetylase (HDAC) inhibitors are novel therapeutics in the treatment of peripheral T-cell lymphoma, unspecified (PTCL) and diffuse large B-cell lymphoma (DLBCL), where, for unknown reasons, T-cell malignancies appear to be more sensitive than B-cell malignancies. The aim...... was to determine HDAC expression in DLBCL and PTCL which has not previously been investigated. METHODS AND RESULTS: The expression of HDAC1, HDAC2, HDAC6 and acetylated histone H4 was examined immunohistochemically in 31 DLBCL and 45 PTCL. All four markers showed high expression in both DLBCL and PTCL compared...

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

Energy Technology Data Exchange (ETDEWEB)

Shan, Chun-Min; Wang, Jiyong; Xu, Ke; Chen, Huijie; Yue, Jia-Xing; Andrews, Stuart; Moresco, James J.; Yates, John R.; Nagy, Peter L.; Tong, Liang; Jia, Songtao

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.

Epigenetic regulation of vascular smooth muscle cell proliferation and neointima formation by histone deacetylase inhibition.

Science.gov (United States)

Findeisen, Hannes M; Gizard, Florence; Zhao, Yue; Qing, Hua; Heywood, Elizabeth B; Jones, Karrie L; Cohn, Dianne; Bruemmer, Dennis

2011-04-01

Proliferation of smooth muscle cells (SMC) in response to vascular injury is central to neointimal vascular remodeling. There is accumulating evidence that histone acetylation constitutes a major epigenetic modification for the transcriptional control of proliferative gene expression; however, the physiological role of histone acetylation for proliferative vascular disease remains elusive. In the present study, we investigated the role of histone deacetylase (HDAC) inhibition in SMC proliferation and neointimal remodeling. We demonstrate that mitogens induce transcription of HDAC 1, 2, and 3 in SMC. Short interfering RNA-mediated knockdown of either HDAC 1, 2, or 3 and pharmacological inhibition of HDAC prevented mitogen-induced SMC proliferation. The mechanisms underlying this reduction of SMC proliferation by HDAC inhibition involve a growth arrest in the G(1) phase of the cell cycle that is due to an inhibition of retinoblastoma protein phosphorylation. HDAC inhibition resulted in a transcriptional and posttranscriptional regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip). Furthermore, HDAC inhibition repressed mitogen-induced cyclin D1 mRNA expression and cyclin D1 promoter activity. As a result of this differential cell cycle-regulatory gene expression by HDAC inhibition, the retinoblastoma protein retains a transcriptional repression of its downstream target genes required for S phase entry. Finally, we provide evidence that these observations are applicable in vivo by demonstrating that HDAC inhibition decreased neointima formation and expression of cyclin D1 in a murine model of vascular injury. These findings identify HDAC as a critical component of a transcriptional cascade regulating SMC proliferation and suggest that HDAC might play a pivotal role in the development of proliferative vascular diseases, including atherosclerosis and in-stent restenosis.

Middle-down hybrid chromatography/tandem mass spectrometry workflow for characterization of combinatorial post-translational modifications in histones.

Science.gov (United States)

Sidoli, Simone; Schwämmle, Veit; Ruminowicz, Chrystian; Hansen, Thomas A; Wu, Xudong; Helin, Kristian; Jensen, Ole N

2014-10-01

We present an integrated middle-down proteomics platform for sensitive mapping and quantification of coexisting PTMs in large polypeptides (5-7 kDa). We combined an RP trap column with subsequent weak cation exchange-hydrophilic interaction LC interfaced directly to high mass accuracy ESI MS/MS using electron transfer dissociation. This enabled automated and efficient separation and sequencing of hypermodified histone N-terminal tails for unambiguous localization of combinatorial PTMs. We present Histone Coder and IsoScale software to extract, filter, and analyze MS/MS data, including quantification of cofragmenting isobaric polypeptide species. We characterized histone tails derived from murine embryonic stem cells knockout in suppressor of zeste12 (Suz12(-/-) ) and quantified 256 combinatorial histone marks in histones H3, H4, and H2A. Furthermore, a total of 713 different combinatorial histone marks were identified in purified histone H3. We measured a seven-fold reduction of H3K27me2/me3 (where me2 and me3 are dimethylation and trimethylation, respectively) in Suz12(-) (/) (-) cells and detected significant changes of the relative abundance of 16 other single PTMs of histone H3 and other combinatorial marks. We conclude that the inactivation of Suz12 is associated with changes in the abundance of not only H3K27 methylation but also multiple other PTMs in histone H3 tails. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two distinct modes for propagation of histone PTMs across the cell cycle

DEFF Research Database (Denmark)

Alabert, Constance; Barth, Teresa K; Reverón-Gómez, Nazaret

2015-01-01

Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling...... to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence...... of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment...

Circulating Extracellular Histones Are Clinically Relevant Mediators of Multiple Organ Injury.

Science.gov (United States)

Kawai, Chihiro; Kotani, Hirokazu; Miyao, Masashi; Ishida, Tokiko; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

2016-04-01

Extracellular histones are a damage-associated molecular pattern (DAMP) involved in the pathogenesis of various diseases. The mechanisms of histone-mediated injury in certain organs have been extensively studied, but an understanding of the pathophysiological role of histone-mediated injury in multiple organ injury remains elusive. To elucidate this role, we systemically subjected C57BL/6 mice to various doses of histones and performed a chronological evaluation of the morphological and functional changes in the lungs, liver, and kidneys. Notably, histone administration ultimately led to death after a dose-dependent aggravation of multiple organ injury. In chronological studies, pulmonary and hepatic injuries occurred within 15 minutes, whereas renal injuries presented at a later phase, suggesting that susceptibility to extracellular histones varies among organs. Histones bound to pulmonary and hepatic endothelial cells immediately after administration, leading to endothelial damage, which could be ameliorated by pretreatment with heparin. Furthermore, release of another DAMP, high-mobility group protein box 1, followed the histone-induced tissue damage, and an antibody against the molecule ameliorated hepatic and renal failure in a late phase. These findings indicate that extracellular histones induce multiple organ injury in two progressive stages-direct injury to endothelial cells and the subsequent release of other DAMPs-and that combination therapies against extracellular histones and high-mobility group protein box 1 may be a promising strategy for treating multiple organ injury. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Circulating histones for predicting prognosis after cardiac surgery: a prospective study.

Science.gov (United States)

Gao, Hongxiang; Zhang, Naipu; Lu, Fangfang; Yu, Xindi; Zhu, Limin; Mo, Xi; Wang, Wei

2016-11-01

The objective of this study was to assess the perioperative changes in circulating histones and their relationships with other biomarkers and clinical outcomes after cardiac surgery with cardiopulmonary bypass (CPB) in patients. Forty-eight patients with congenital cardiac diseases undergoing corrective procedure with CPB were prospectively enrolled in this study. Circulating histones, N-terminal pro-brain natriuretic peptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) were measured preoperatively (T0) and at 0 (T1), 24 (T2), 48 (T3) and 72 (T4) h postoperatively. The relationships between biomarkers and clinical outcomes were analysed. Circulating histones, NT-proBNP, PCT and CRP increased significantly postoperatively, with histones reaching the peak value earliest at T1. Circulating histone levels were higher in patients with adverse events. Receiver operating characteristic curve analysis showed that peak histone levels had a better predictive value for adverse events postoperatively. Peak histone levels correlated with the peak level of NT-proBNP (r = 0.563, P histones reached peak levels faster than NT-proBNP, PCT and CRP. Furthermore, peak histone levels correlated with biomarkers and postoperative clinical outcomes. Circulating histones may be used as a prognostic indicator for patients after cardiac surgery with CPB. ClinicalTrials.gov (ID: NCT02325765). © The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Histone modifications in response to DNA damage

International Nuclear Information System (INIS)

Altaf, Mohammed; Saksouk, Nehme; Cote, Jacques

2007-01-01

The packaging of the eukaryotic genome into highly condensed chromatin makes it inaccessible to the factors required for gene transcription, DNA replication, recombination and repair. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Histone modifying enzymes and ATP-dependent chromatin remodeling complexes play key roles here as they regulate many nuclear processes by altering the chromatin structure. Significantly, these activities are integral to the process of DNA repair where histone modifications act as signals and landing platforms for various repair proteins. This review summarizes the recent developments in our understanding of histone modifications and their role in the maintenance of genome integrity

Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.

Science.gov (United States)

Moraes, Izabel; Yuan, Zuo-Fei; Liu, Shichong; Souza, Glaucia Mendes; Garcia, Benjamin A; Casas-Mollano, J Armando

2015-01-01

Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in

Identification of histone modifications in biomedical text for supporting epigenomic research.

Science.gov (United States)

Kolárik, Corinna; Klinger, Roman; Hofmann-Apitius, Martin

2009-01-30

Posttranslational modifications of histones influence the structure of chromatine and in such a way take part in the regulation of gene expression. Certain histone modification patterns, distributed over the genome, are connected to cell as well as tissue differentiation and to the adaption of organisms to their environment. Abnormal changes instead influence the development of disease states like cancer. The regulation mechanisms for modifying histones and its functionalities are the subject of epigenomics investigation and are still not completely understood. Text provides a rich resource of knowledge on epigenomics and modifications of histones in particular. It contains information about experimental studies, the conditions used, and results. To our knowledge, no approach has been published so far for identifying histone modifications in text. We have developed an approach for identifying histone modifications in biomedical literature with Conditional Random Fields (CRF) and for resolving the recognized histone modification term variants by term standardization. For the term identification F1 measures of 0.84 by 10-fold cross-validation on the training corpus and 0.81 on an independent test corpus have been obtained. The standardization enabled the correct transformation of 96% of the terms from training and 98% from test the corpus. Due to the lack of terminologies exhaustively covering specific histone modification types, we developed a histone modification term hierarchy for use in a semantic text retrieval system. The developed approach highly improves the retrieval of articles describing histone modifications. Since text contains context information about performed studies and experiments, the identification of histone modifications is the basis for supporting literature-based knowledge discovery and hypothesis generation to accelerate epigenomic research.

Histone Methylation and Epigenetic Silencing in Breast Cancer

National Research Council Canada - National Science Library

Simon, Jeffrey A; Lange, Carol A

2008-01-01

.... EZH2 is a histone methyltransferase which modifies lysine-27 of histone H3 an epigenetic mark which is generally linked to gene silencing and is implicated in tumor suppressor silencing during breast cancer progression...

Exercise increases hyper-acetylation of histones on the Cis-element of NRF-1 binding to the Mef2a promoter: Implications on type 2 diabetes.

Science.gov (United States)

Joseph, Jitcy S; Ayeleso, Ademola O; Mukwevho, Emmanuel

2017-04-22

Exercise brings changes on the chromatin ensuing the upregulation of many genes that confer protection from type 2 diabetes. In type-2 diabetes, critical genes are down-regulated such as those involved in glucose transport (GLUT4, MEF2A) and also oxidative phosphorylation (NRF-1 and its target genes). Recent reports have shown that NRF-1 not only regulate mitochondrial oxidative genes but also controls MEF2A, the main transcription factor for glucose transporter, GLUT4. Such dual control of the two pathways by NRF-1 place it as critical gene in the design of therapeutic modalities much needed to cure or better manage type 2 diabetes. Although it is known that NRF-1 controls these dual pathways (glucose transport and oxidative phosphorylation), the actual molecular mechanisms involved surrounding this regulation remains elusive. NRF-1 itself is regulated through posttranslational modifications (acetylation, methylation and phosphorylation) resulting in enhanced binding to its target genes. This study is therefore aimed at assessing whether CaMKII, a kinase activated by exercise brings about hyper-acetylation of histones in the vicinity of NRF-1 target gene, Mef2a. Five to six weeks old male Wistar rats were used in this study. Chromatin immunoprecipitation (ChIP) assay was used to investigate the extent through which NRF-1 is bound to the Mef2a gene and if this was associated with hyper-acetylation of histones in the region of NRF-1 binding site of the Mef2a gene. Quantitative real time PCR (qPCR) was used to determine the gene expression of MEF2A and NRF-1. Results from this study indicated that exercise-induced CaMKII activation increased hyper-acetylation of histones in the region of NRF-1 binding site on vicinity of Mef2a gene and this was associated with the increased binding of NRF-1 to Mef2a gene. Exercise also increased the expression of NRF-1 and MEF2A genes. Administration of CaMKII inhibitor (KN93) prior to exercise attenuated the observed exercise

Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation.

Science.gov (United States)

Marsman, Gerben; Zeerleder, Sacha; Luken, Brenda M

2016-12-08

In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo, to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought about by histones and dsDNA, and there appear to be some marked differences. Remarkably, little distinction between the different forms in which histones circulate has been made throughout literature. This is partly due to the limitations of existing techniques to differentiate between histones in their free or DNA-bound form. Here we review the current understanding of immunostimulation induced by extracellular histones, dsDNA and nucleosomes, and discuss the importance of techniques that in their detection differentiate between these different chromatin components.

Histone deacetylases and their roles in mineralized tissue regeneration

Directory of Open Access Journals (Sweden)

Nam Cong-Nhat Huynh

2017-12-01

Full Text Available Histone acetylation is an important epigenetic mechanism that controls expression of certain genes. It includes non-sequence-based changes of chromosomal regional structure that can alter the expression of genes. Acetylation of histones is controlled by the activity of two groups of enzymes: the histone acetyltransferases (HATs and histone deacetylases (HDACs. HDACs remove acetyl groups from the histone tail, which alters its charge and thus promotes compaction of DNA in the nucleosome. HDACs render the chromatin structure into a more compact form of heterochromatin, which makes the genes inaccessible for transcription. By altering the transcriptional activity of bone-associated genes, HDACs control both osteogenesis and osteoclastogenesis. This review presents an overview of the function of HDACs in the modulation of bone formation. Special attention is paid to the use of HDAC inhibitors in mineralized tissue regeneration from cells of dental origin.

Impact of cigarette smoking on histone (H2B) to protamine ratio in human spermatozoa and its relation to sperm parameters.

Science.gov (United States)

Hamad, M F; Shelko, N; Kartarius, S; Montenarh, M; Hammadeh, M E

2014-09-01

Smoking is strongly associated with abnormalities in histone-to-protamine transition and with alteration of protamine expression in human spermatozoa. A proper protamine to histone ratio is, however, essential for sperm chromatin maturity and DNA integrity. Alterations in these sperm nuclear proteins were observed in infertile men. The present prospective study is aimed at evaluating the possible relationship among smoking, semen quality and the histone-to-protamine transition ratio in mature spermatozoa. Histone H2B and protamine 1 (P1) and 2 (P2) were quantified using acid-urea polyacrylamide gel electrophoresis in the spermatozoa of 35 smokers and 19 non-smokers. Levels of lipid peroxidation marker malondialdehyde (MDA) were measured in seminal plasma by thiobarbituric acid assay. Cotinine concentrations were determined in seminal plasma using an enzyme-linked immunosorbent assay. Histone H2B levels in smokers (292.27 ± 58.24 ng/10(6)) were significantly higher (p = 0.001) than that of non-smokers (109.1 ± 43.70 ng/10(6)), besides, a significant difference (p > 0.0001) was found for the P1 and P2 ratio between smokers (1.71 ± 0.071) and non-smokers (1.05 ± 0.033). The H2B/(H2B+P1 + P2) ratio (0.29 ± 0.71) of smokers were significantly higher (p = sperm count, motility (p = 0.018), vitality (p = 0.009) and membrane integrity (p = 0.0001) than non-smokers. These results reveal that patients who smoke possess a higher proportion of spermatozoa with an alteration of the histone to protamine ratio than patients who do not smoke, and suggest that cigarette smoking may inversely affect male fertility. © 2014 American Society of Andrology and European Academy of Andrology.

Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

DEFF Research Database (Denmark)

Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane

2016-01-01

In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA repli...

Surface recognition and fluorescence sensing of histone by dansyl-appended cyclophane-based resorcinarene trimer.

Science.gov (United States)

Hayashida, Osamu; Ogawa, Naoyuki; Uchiyama, Masaki

2007-11-07

A cyclophane-based resorcinarene trimer (3) bearing a dansyl moiety as an environmentally sensitive fluorophore was prepared by stepwise condensation of a tetraaza[6.1.6.1]paracyclophane skeleton with a dansyl moiety and three resorcinarene derivatives having heptacarboxylic acid residues in this sequence. The dansyl-appended cyclophane exhibited the following fluorescence properties regarding solvent polarity dependency and histone surface recognition: With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 3 increased along with a concomitant blue shift of the fluorescence maximum (lambdaem). The microenvironmentally sensitive fluorescence properties of dansyl fluorophore were maintained, even when the dansyl moiety was covalently attached to a cyclophane. Most interestingly, the cyclophane-based resorcinarene trimer exhibited recognition and fluorescence sensing capabilities toward histone, a small basic protein of eukaryotic chromatins. The fluorescence intensity originating from 3 increased along with a concomitant blue shift of lambdaem upon the addition of histone, reflecting the formation of 3-histone complexes. A relatively large fluorescence polarization (P) value was obtained for the 3-histone complexes (0.15), reflecting highly restricted conformations of 3, and the obtained P value was much larger than that of 3 alone in aqueous medium (0.07). The binding constant (K) of 3 with histone (unit basis) was estimated to be 2.1 x 106 M-1. On the other hand, upon the addition of acetylated histone (Ac-histone) to an aqueous solution containing 3, the extent of change in fluorescence intensity originating from the dansyl group of 3 was almost negligible, indicating that the electrostatic interactions between 3 and Ac-histone were weak. In addition, the fluorescence spectral changes were also small or negligible upon the addition of other proteins such as albumin, ovalbumin, peanut agglutinin

Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

NARCIS (Netherlands)

T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

2004-01-01

textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

Global turnover of histone post-translational modifications and variants in human cells

Directory of Open Access Journals (Sweden)

Zee Barry M

2010-12-01

Full Text Available Abstract Background Post-translational modifications (PTMs on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement. Results In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same

HAMLET interacts with histones and chromatin in tumor cell nuclei.

Science.gov (United States)

Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

2003-10-24

HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53

Science.gov (United States)

Charvet, Céline; Wissler, Manuela; Brauns-Schubert, Prisca; Wang, Shang-Jui; Tang, Yi; Sigloch, Florian C.; Mellert, Hestia; Brandenburg, Martin; Lindner, Silke E.; Breit, Bernhard; Green, Douglas R.; McMahon, Steven B.; Borner, Christoph; Gu, Wei; Maurer, Ulrich

2011-01-01

Summary Activation of p53 by DNA damage results in either cell cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the pro-apoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60S86A mutant was less active to induce p53 K120 acetylation, Histone 4 acetylation and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86-phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53. PMID:21658600

Histone modifications and nuclear architecture: A review

Czech Academy of Sciences Publication Activity Database

Bártová, Eva; Kroupová, Jana; Harničarová, Andrea; Galiová-Šustáčková, Gabriela; Kozubek, Stanislav

2008-01-01

Roč. 56, č. 8 (2008), s. 711-721 ISSN 0722-186X R&D Projects: GA ČR(CZ) GA204/06/0978; GA MŠk(CZ) LC535 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : histones * histone modifications * nuclear architecture Subject RIV: BO - Biophysics

Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications

Science.gov (United States)

2015-01-01

Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118. PMID:25167464

Evaluation of proteomic search engines for the analysis of histone modifications.

Science.gov (United States)

Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C; Garcia, Benjamin A

2014-10-03

Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

DEFF Research Database (Denmark)

Rao, R Shyama Prasad; Møller, Ian Max

2012-01-01

in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

Pathological histone acetylation in Parkinson's disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition.

Science.gov (United States)

Harrison, Ian F; Smith, Andrew D; Dexter, David T

2018-02-14

Parkinson's disease (PD) is associated with degeneration of nigrostriatal neurons due to intracytoplasmic inclusions composed predominantly of a synaptic protein called α-synuclein. Accumulations of α-synuclein are thought to 'mask' acetylation sites on histone proteins, inhibiting the action of histone acetyltransferase (HAT) enzymes in their equilibrium with histone deacetylases (HDACs), thus deregulating the dynamic control of gene transcription. It is therefore hypothesised that the misbalance in the actions of HATs/HDACs in neurodegeneration can be rectified with the use of HDAC inhibitors, limiting the deregulation of transcription and aiding neuronal homeostasis and neuroprotection in disorders such as PD. Here we quantify histone acetylation in the Substantia Nigra pars compacta (SNpc) in the brains of control, early and late stage PD cases to determine if histone acetylation is a function of disease progression. PD development is associated with Braak-dependent increases in histone acetylation. Concurrently, we show that as expected disease progression is associated with reduced markers of dopaminergic neurons and increased markers of activated microglia. We go on to demonstrate that in vitro, degenerating dopaminergic neurons exhibit histone hypoacetylation whereas activated microglia exhibit histone hyperacetylation. This suggests that the disease-dependent increase in histone acetylation observed in human PD cases is likely a combination of the contributions of both degenerating dopaminergic neurons and infiltrating activated microglia. The HDAC SIRT 2 has become increasingly implicated as a novel target for mediation of neuroprotection in PD: the neuronal and microglial specific effects of its inhibition however remain unclear. We demonstrate that SIRT 2 expression in the SNpc of PD brains remains relatively unchanged from controls and that SIRT 2 inhibition, via AGK2 treatment of neuronal and microglial cultures, results in neuroprotection of

Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

Science.gov (United States)

Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

2014-11-01

To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Biochemical profiling of histone binding selectivity of the yeast bromodomain family.

Directory of Open Access Journals (Sweden)

Qiang Zhang

2010-01-01

Full Text Available It has been shown that molecular interactions between site-specific chemical modifications such as acetylation and methylation on DNA-packing histones and conserved structural modules present in transcriptional proteins are closely associated with chromatin structural changes and gene activation. Unlike methyl-lysine that can interact with different protein modules including chromodomains, Tudor and MBT domains, as well as PHD fingers, acetyl-lysine (Kac is known thus far to be recognized only by bromodomains. While histone lysine acetylation plays a crucial role in regulation of chromatin-mediated gene transcription, a high degree of sequence variation of the acetyl-lysine binding site in the bromodomains has limited our understanding of histone binding selectivity of the bromodomain family. Here, we report a systematic family-wide analysis of 14 yeast bromodomains binding to 32 lysine-acetylated peptides derived from known major acetylation sites in four core histones that are conserved in eukaryotes.The histone binding selectivity of purified recombinant yeast bromodomains was assessed by using the native core histones in an overlay assay, as well as N-terminally biotinylated lysine-acetylated histone peptides spotted on streptavidin-coated nitrocellulose membrane in a dot blot assay. NMR binding analysis further validated the interactions between histones and selected bromodomain. Structural models of all yeast bromodomains were built using comparative modeling to provide insights into the molecular basis of their histone binding selectivity.Our study reveals that while not all members of the bromodomain family are privileged to interact with acetylated-lysine, identifiable sequence features from those that bind histone emerge. These include an asparagine residue at the C-terminus of the third helix in the 4-helix bundle, negatively charged residues around the ZA loop, and preponderance of aromatic amino acid residues in the binding pocket

Extracellular DNA and histones: double-edged swords in immunothrombosis.

Science.gov (United States)

Gould, T J; Lysov, Z; Liaw, P C

2015-06-01

The existence of extracellular DNA in human plasma, also known as cell-free DNA (cfDNA), was first described in the 1940s. In recent years, there has been a resurgence of interest in the functional significance of cfDNA, particularly in the context of neutrophil extracellular traps (NETs). cfDNA and histones are key components of NETs that aid in the host response to infection and inflammation. However, cfDNA and histones may also exert harmful effects by triggering coagulation, inflammation, and cell death and by impairing fibrinolysis. In this article, we will review the pathologic nature of cfDNA and histones in macrovascular and microvascular thrombosis, including venous thromboembolism, cancer, sepsis, and trauma. We will also discuss the prognostic value of cfDNA and histones in these disease states. Understanding the molecular and cellular pathways regulated by cfDNA and histones may provide novel insights to prevent pathological thrombus formation and vascular occlusion. © 2015 International Society on Thrombosis and Haemostasis.

Immune activation by histones: plusses and minuses in inflammation.

Science.gov (United States)

Pisetsky, David S

2013-12-01

Histones are highly cationic proteins that are essential components of the cell nucleus, interacting with DNA to form the nucleosome and regulating transcription. Histones, however, can transit from the cell nucleus during cell death and, once in an extracellular location, can serve as danger signals and activate immune cells. An article in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 3336-3342] reports that histones can activate monocyte-derived DCs via the NRLP3 inflammasome to induce the production of IL-1β. As such, histones, which can also stimulate TLRs, may drive events in the immunopathogenesis of a wide range of acute and chronic diseases marked by sterile inflammation. While the mechanism of this stimulation is not known, the positive charge of histones may provide a structural element to promote interaction with cells and activation of downstream signaling systems. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Histones induce phosphatidylserine exposure and a procoagulant phenotype in human red blood cells.

Science.gov (United States)

Semeraro, F; Ammollo, C T; Esmon, N L; Esmon, C T

2014-10-01

Extracellular histones exert part of their prothrombotic activity through the stimulation of blood cells. Besides platelets, histones can bind to red blood cells (RBCs), which are important contributors to thrombogenesis, but little is known about the functional consequences of this interaction. To evaluate the effect of histones on the procoagulant potential of human RBCs with particular regard to the expression of surface phosphatidylserine (PS). PS exposure on human RBCs treated with a natural mixture of histones or recombinant individual histones was evaluated with fluorescein isothiocyanate-annexin-V binding and measured with flow cytometry. Calcium influx in RBCs loaded with the calcium-sensitive fluorophore Fluo-4 AM was assessed with flow cytometry. The procoagulant potential of histone-treated RBCs was evaluated with a purified prothrombinase assay and a one-stage plasma recalcification clotting test. Natural histones induced PS exposure on RBCs in a dose-dependent manner, and neutralization or cleavage of histones by heparin or activated protein C, respectively, abolished PS externalization. H4 was mainly responsible for the stimulating activity of histones, whereas the other subtypes were almost ineffective. Similarly, natural histones and H4 induced influx of calcium into RBCs, whereas the other individual histones did not. Histone-induced exposure of PS on RBCs translated into increased prothrombinase complex-mediated prothrombin activation and accelerated fibrin formation in plasma. Histones induce RBCs to express a procoagulant phenotype through the externalization of PS. This finding provides new insights into the prothrombotic activity of extracellular histones. © 2014 International Society on Thrombosis and Haemostasis.

Differential affinity of mammalian histone H1 somatic subtypes for DNA and chromatin

Directory of Open Access Journals (Sweden)

Mora Xavier

2007-05-01

Full Text Available Abstract Background Histone H1 is involved in the formation and maintenance of chromatin higher order structure. H1 has multiple isoforms; the subtypes differ in timing of expression, extent of phosphorylation and turnover rate. In vertebrates, the amino acid substitution rates differ among subtypes by almost one order of magnitude, suggesting that each subtype might have acquired a unique function. We have devised a competitive assay to estimate the relative binding affinities of histone H1 mammalian somatic subtypes H1a-e and H1° for long chromatin fragments (30–35 nucleosomes in physiological salt (0.14 M NaCl at constant stoichiometry. Results The H1 complement of native chromatin was perturbed by adding an additional amount of one of the subtypes. A certain amount of SAR (scaffold-associated region DNA was present in the mixture to avoid precipitation of chromatin by excess H1. SAR DNA also provided a set of reference relative affinities, which were needed to estimate the relative affinities of the subtypes for chromatin from the distribution of the subtypes between the SAR and the chromatin. The amounts of chromatin, SAR and additional H1 were adjusted so as to keep the stoichiometry of perturbed chromatin similar to that of native chromatin. H1 molecules freely exchanged between the chromatin and SAR binding sites. In conditions of free exchange, H1a was the subtype of lowest affinity, H1b and H1c had intermediate affinities and H1d, H1e and H1° the highest affinities. Subtype affinities for chromatin differed by up to 19-fold. The relative affinities of the subtypes for chromatin were equivalent to those estimated for a SAR DNA fragment and a pUC19 fragment of similar length. Avian H5 had an affinity ~12-fold higher than H1e for both DNA and chromatin. Conclusion H1 subtypes freely exchange in vitro between chromatin binding sites in physiological salt (0.14 M NaCl. The large differences in relative affinity of the H1 subtypes for

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

Science.gov (United States)

Earnshaw, W C; Allshire, R C; Black, B E; Bloom, K; Brinkley, B R; Brown, W; Cheeseman, I M; Choo, K H A; Copenhaver, G P; Deluca, J G; Desai, A; Diekmann, S; Erhardt, S; Fitzgerald-Hayes, M; Foltz, D; Fukagawa, T; Gassmann, R; Gerlich, D W; Glover, D M; Gorbsky, G J; Harrison, S C; Heun, P; Hirota, T; Jansen, L E T; Karpen, G; Kops, G J P L; Lampson, M A; Lens, S M; Losada, A; Luger, K; Maiato, H; Maddox, P S; Margolis, R L; Masumoto, H; McAinsh, A D; Mellone, B G; Meraldi, P; Musacchio, A; Oegema, K; O'Neill, R J; Salmon, E D; Scott, K C; Straight, A F; Stukenberg, P T; Sullivan, B A; Sullivan, K F; Sunkel, C E; Swedlow, J R; Walczak, C E; Warburton, P E; Westermann, S; Willard, H F; Wordeman, L; Yanagida, M; Yen, T J; Yoda, K; Cleveland, D W

2013-04-01

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3

OpenAIRE

Keith, Kevin C.; Baker, Richard E.; Chen, Yinhuai; Harris, Kendra; Stoler, Sam; Fitzgerald-Hayes, Molly

1999-01-01

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions o...

Genome-wide analysis of regions similar to promoters of histone genes

KAUST Repository

Chowdhary, Rajesh; Bajic, Vladimir B.; Dong, Difeng; Wong, Limsoon; Liu, Jun S

2010-01-01

of histone and histone-coregulated gene transcription initiation. While these hypotheses still remain to be verified, we believe that these form a useful resource for researchers to further explore regulation of human histone genes and human genome

Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer

OpenAIRE

O'Sullivan, Aine G.; Mulvaney, Eamon P.; Hyland, Paula B.; Kinsella, B. Therese

2015-01-01

The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TP? and TP? isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TP?/TP?-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenet...

Biophysical characterization of the association of histones with single-stranded DNA.

Science.gov (United States)

Wang, Ying; van Merwyk, Luis; Tönsing, Katja; Walhorn, Volker; Anselmetti, Dario; Fernàndez-Busquets, Xavier

2017-11-01

Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin. Copyright © 2017 Elsevier B.V. All rights reserved.

EPC1/TIP60-mediated histone acetylation facilitates spermiogenesis in mice

DEFF Research Database (Denmark)

Dong, Yixin; Isono, Kyo Ichi; Ohbo, Kazuyuki

2017-01-01

Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation- mediated histone replacement remains poorly understood. Here...

Histone acetyltransferases : challenges in targeting bi-substrate enzymes

NARCIS (Netherlands)

Wapenaar, Hannah; Dekker, Frank J

2016-01-01

Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to

Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

Science.gov (United States)

Jo-Watanabe, Airi; Ohse, Takamoto; Nishimatsu, Hiroaki; Takahashi, Masao; Ikeda, Yoichiro; Wada, Takehiko; Shirakawa, Jun-ichi; Nagai, Ryoji; Miyata, Toshio; Nagano, Tetsuo; Hirata, Yasunobu; Inagi, Reiko; Nangaku, Masaomi

2014-06-01

Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons

AMPA receptor phosphorylation and recognition memory: learning-related, time-dependent changes in the chick brain following filial imprinting.

Science.gov (United States)

Solomonia, Revaz O; Meparishvili, Maia; Mikautadze, Ekaterine; Kunelauri, Nana; Apkhazava, David; McCabe, Brian J

2013-04-01

There is strong evidence that a restricted part of the chick forebrain, the intermediate medial mesopallium (IMM), stores information acquired through the learning process of visual imprinting. We have previously demonstrated that at 1 h but not 24 h after imprinting training, a learning-specific increase in the amount of membrane Thr286-autophosphorylated α-calcium/calmodulin-dependent protein kinase II (αCaMKII), and in the proportion of total αCaMKII that is phosphorylated, occurs in the IMM but not in a control brain region, the posterior pole of the nidopallium (PPN). αCaMKII directly phosphorylates Ser831 in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. In the present study we have inquired whether the learning-related increase in αCaMKII autophosphorylation is followed by changes in the Ser831 phosphorylation of GluA1 (P-GluA1) and in the total amount of this subunit (T-GluA1). Trained chicks together with untrained control chicks were killed either 1 or 24 h after training. Tissue was removed from the IMM together with tissue from the PPN as a control. Amounts of P-GluA1 and T-GluA1 were measured. In the left IMM of the 1 h group the P-GluA1/T-GluA1 ratio increased in a learning-specific way. No learning-related changes were observed in other brain regions at 1 h or in any region 24 h after training. The results indicate that a time- and regionally-dependent, learning-specific increase in GluA1 phosphorylation occurs early in recognition memory formation.

Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

Science.gov (United States)

Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

2013-01-01

SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

Small molecule inhibitors of histone deacetylases and acetyltransferases as potential therapeutics in oncology

NARCIS (Netherlands)

van den Bosch, Thea; Leus, Niek; Timmerman, Tirza; Dekker, Frank J

2016-01-01

Uncontrolled cell proliferation and resistance to apoptosis in cancer are, among others, regulated by post-translational modifications of histone proteins. The most investigated type of histone modification is lysine acetylation. Histone acetyltransferases (HATs), acetylate histone lysine residues,

C1qTNF-related protein 1 improve insulin resistance by reducing phosphorylation of serine 1101 in insulin receptor substrate 1.

Science.gov (United States)

Xin, Yaping; Zhang, Dongming; Fu, Yanqin; Wang, Chongxian; Li, Qingju; Tian, Chenguang; Zhang, Suhe; Lyu, Xiaodong

2017-08-30

C1qTNF-related protein 1 (CTRP1) is independently associated with type 2 diabetes. However, the relationship between CTRP1 and insulin resistance is still not established. This study aimed to explore the role of CTRP1 under the situation of insulin resistance in adipose tissue. Plasma CTRP1 level was investigated in type 2 diabetic subjects (n = 35) and non-diabetic subjects (n = 35). The relationship between CTRP1 and phosphorylation of multi insulin receptor substrate 1 (IRS-1) serine (Ser) sites was further explored. Our data showed that Plasma CTRP1 was higher and negative correlation with insulin resistance in diabetic subjects (r = -0.283, p = 0.018). Glucose utilisation test revealed that the glucose utilisation rate of mature adipocytes was improved by CTRP1 in the presence of insulin. CTRP1 was not only related to IRS-1 protein, but also negatively correlated with IRS-1 Ser1101 phosphorylation (r = -0.398, p = 0.031). Furthermore, Phosphorylation levels of IRS-1 Ser1101 were significantly lower after incubation with 40 ng/mL CTRP1 in mature adipocytes than those with no intervention (p insulin resistance by reducing the phosphorylation of IRS-1 Ser1101, induced in the situation of insulin resistance as a feedback adipokine.

Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease

DEFF Research Database (Denmark)

Cloos, Paul A C; Christensen, Jesper; Agger, Karl

2008-01-01

The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone...... demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases......, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer....

Histone hypoacetylation is required to maintain late replication timing of constitutive heterochromatin.

Science.gov (United States)

Casas-Delucchi, Corella S; van Bemmel, Joke G; Haase, Sebastian; Herce, Henry D; Nowak, Danny; Meilinger, Daniela; Stear, Jeffrey H; Leonhardt, Heinrich; Cardoso, M Cristina

2012-01-01

The replication of the genome is a spatio-temporally highly organized process. Yet, its flexibility throughout development suggests that this process is not genetically regulated. However, the mechanisms and chromatin modifications controlling replication timing are still unclear. We made use of the prominent structure and defined heterochromatic landscape of pericentric regions as an example of late replicating constitutive heterochromatin. We manipulated the major chromatin markers of these regions, namely histone acetylation, DNA and histone methylation, as well as chromatin condensation and determined the effects of these altered chromatin states on replication timing. Here, we show that manipulation of DNA and histone methylation as well as acetylation levels caused large-scale heterochromatin decondensation. Histone demethylation and the concomitant decondensation, however, did not affect replication timing. In contrast, immuno-FISH and time-lapse analyses showed that lowering DNA methylation, as well as increasing histone acetylation, advanced the onset of heterochromatin replication. While dnmt1(-)(/)(-) cells showed increased histone acetylation at chromocenters, histone hyperacetylation did not induce DNA demethylation. Hence, we propose that histone hypoacetylation is required to maintain normal heterochromatin duplication dynamics. We speculate that a high histone acetylation level might increase the firing efficiency of origins and, concomitantly, advances the replication timing of distinct genomic regions.

Biochemical studies on histones of the central nervous system. 2

International Nuclear Information System (INIS)

Schmitt, M.; Matthies, H.

1979-01-01

There are no qualitative differences in the electrophoretic patterns of histones from neurones and glia. A 25% increased acetylation rate is found in neutronal histones as compared to glial histones after incubation of chopped brain in a [ 14 C]-acetate containing medium. This result probably reflects different condensation states of the chromatins of both cell types, as demonstrated by electron microscopy. (author)

Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

Science.gov (United States)

Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

2015-10-22

One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.

Science.gov (United States)

Poirier, Aurore C; Schmitt, Paulina; Rosa, Rafael D; Vanhove, Audrey S; Kieffer-Jaquinod, Sylvie; Rubio, Tristan P; Charrière, Guillaume M; Destoumieux-Garzón, Delphine

2014-09-05

Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Abnormal levels of histone methylation in the retinas of diabetic rats are reversed by minocycline treatment

DEFF Research Database (Denmark)

Wang, Wenjun; Sidoli, Simone; Zhang, Wenquan

2017-01-01

67% of these marks had their relative abundance restored to non-diabetic levels after minocycline treatment. Mono-and di-methylation states of histone H4 lysine 20 (H4K20me1/me2), markers related to DNA damage response, were found to be up-regulated in the retinas of diabetic rats and restored......In this study we quantified the alterations of retinal histone post-translational modifications (PTMs) in diabetic rats using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. Some diabetic rats were subsequently treated with minocycline, a tetracycline antibiotic, which has...... been shown to inhibit the diabetes-induced chronic inflammation in the retinas of rodents. We quantified 266 differentially modified histone peptides, including 48 out of 83 methylation marks with significantly different abundancein retinas of diabetic rats as compared to non-diabetic controls. About...

Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

Energy Technology Data Exchange (ETDEWEB)

Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

2012-11-01

Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of histones.

Science.gov (United States)

Pesavento, James J; Garcia, Benjamin A; Streeky, James A; Kelleher, Neil L; Mizzen, Craig A

2007-09-01

Recent developments in top down mass spectrometry have enabled closely related histone variants and their modified forms to be identified and quantitated with unprecedented precision, facilitating efforts to better understand how histones contribute to the epigenetic regulation of gene transcription and other nuclear processes. It is therefore crucial that intact MS profiles accurately reflect the levels of variants and modified forms present in a given cell type or cell state for the full benefit of such efforts to be realized. Here we show that partial oxidation of Met and Cys residues in histone samples prepared by conventional methods, together with oxidation that can accrue during storage or during chip-based automated nanoflow electrospray ionization, confounds MS analysis by altering the intact MS profile as well as hindering posttranslational modification localization after MS/MS. We also describe an optimized performic acid oxidation procedure that circumvents these problems without catalyzing additional oxidations or altering the levels of posttranslational modifications common in histones. MS and MS/MS of HeLa cell core histones confirmed that Met and Cys were the only residues oxidized and that complete oxidation restored true intact abundance ratios and significantly enhanced MS/MS data quality. This allowed for the unequivocal detection, at the intact molecule level, of novel combinatorially modified forms of H4 that would have been missed otherwise. Oxidation also enhanced the separation of human core histones by reverse phase chromatography and decreased the levels of salt-adducted forms observed in ESI-FTMS. This method represents a simple and easily automated means for enhancing the accuracy and sensitivity of top down analyses of combinatorially modified forms of histones that may also be of benefit for top down or bottom up analyses of other proteins.

Tyrosine phosphorylation of Grb14 by Tie2

Directory of Open Access Journals (Sweden)

Dumont Daniel J

2010-10-01

Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

Epigenetic Metabolite Acetate Inhibits Class I/II Histone Deacetylases, Promotes Histone Acetylation, and Increases HIV-1 Integration in CD4+ T Cells.

Science.gov (United States)

Bolduc, Jean-François; Hany, Laurent; Barat, Corinne; Ouellet, Michel; Tremblay, Michel J

2017-08-15

In this study, we investigated the effect of acetate, the most concentrated short-chain fatty acid (SCFA) in the gut and bloodstream, on the susceptibility of primary human CD4 + T cells to HIV-1 infection. We report that HIV-1 replication is increased in CD3/CD28-costimulated CD4 + T cells upon acetate treatment. This enhancing effect correlates with increased expression of the early activation marker CD69 and impaired class I/II histone deacetylase (HDAC) activity. In addition, acetate enhances acetylation of histones H3 and H4 and augments HIV-1 integration into the genome of CD4 + T cells. Thus, we propose that upon antigen presentation, acetate influences class I/II HDAC activity that transforms condensed chromatin into a more relaxed structure. This event leads to a higher level of viral integration and enhanced HIV-1 production. In line with previous studies showing reactivation of latent HIV-1 by SCFAs, we provide evidence that acetate can also increase the susceptibility of primary human CD4 + T cells to productive HIV-1 infection. IMPORTANCE Alterations in the fecal microbiota and intestinal epithelial damage involved in the gastrointestinal disorder associated with HIV-1 infection result in microbial translocation that leads to disease progression and virus-related comorbidities. Indeed, notably via production of short-chain fatty acids, bacteria migrating from the lumen to the intestinal mucosa could influence HIV-1 replication by epigenetic regulatory mechanisms, such as histone acetylation. We demonstrate that acetate enhances virus production in primary human CD4 + T cells. Moreover, we report that acetate impairs class I/II histone deacetylase activity and increases integration of HIV-1 DNA into the host genome. Therefore, it can be postulated that bacterial metabolites such as acetate modulate HIV-1-mediated disease progression. Copyright © 2017 American Society for Microbiology.

Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

Science.gov (United States)

Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

2012-01-01

Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.

Directory of Open Access Journals (Sweden)

Yang Shu

Full Text Available Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2 gene with Parkinson's disease (PD. Among the mutations, LRRK2 c.4883G>C (R1628P variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5. Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+ phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

Science.gov (United States)

Parkel, Sven; Lopez-Atalaya, Jose P.; Barco, Angel

2013-01-01

Recent research indicates that epigenetic mechanisms and, in particular, the post-translational modification (PTM) of histones may contribute to memory encoding and storage. Among the dozens of possible histone PTMs, the methylation/demethylation of lysines in the N-terminal tail of histone H3 exhibits particularly strong links with cognitive…

Phosphorylated nano-diamond/ Polyimide Nanocomposites

International Nuclear Information System (INIS)

Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

2014-01-01

In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

Histone deacetylases in memory and cognition.

Science.gov (United States)

Penney, Jay; Tsai, Li-Huei

2014-12-09

Over the past 30 years, lysine acetylation of histone and nonhistone proteins has become established as a key modulator of gene expression regulating numerous aspects of cell biology. Neuronal growth and plasticity are no exception; roles for lysine acetylation and deacetylation in brain function and dysfunction continue to be uncovered. Transcriptional programs coupling synaptic activity to changes in gene expression are critical to the plasticity mechanisms underlying higher brain functions. These transcriptional programs can be modulated by changes in histone acetylation, and in many cases, transcription factors and histone-modifying enzymes are recruited together to plasticity-associated genes. Lysine acetylation, catalyzed by lysine acetyltransferases (KATs), generally promotes cognitive performance, whereas the opposing process, catalyzed by histone lysine deacetylases (HDACs), appears to negatively regulate cognition in multiple brain regions. Consistently, mutation or deregulation of different KATs or HDACs contributes to neurological dysfunction and neurodegeneration. HDAC inhibitors have shown promise as a treatment to combat the cognitive decline associated with aging and neurodegenerative disease, as well as to ameliorate the symptoms of depression and posttraumatic stress disorder, among others. In this review, we discuss the evidence for the roles of HDACs in cognitive function as well as in neurological disorders and disease. In particular, we focus on HDAC2, which plays a central role in coupling lysine acetylation to synaptic plasticity and mediates many of the effects of HDAC inhibition in cognition and disease. Copyright © 2014, American Association for the Advancement of Science.

The histone H5 variant in Xenopus laevis

NARCIS (Netherlands)

Moorman, A. F.; de Boer, P. A.; Linders, M. T.; Charles, R.

1984-01-01

The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal

Transcriptional regulation by histone modifications: towards a theory of chromatin re-organization during stem cell differentiation

International Nuclear Information System (INIS)

Binder, Hans; Steiner, Lydia; Przybilla, Jens; Rohlf, Thimo; Prohaska, Sonja; Galle, Jörg

2013-01-01

Chromatin-related mechanisms, as e.g. histone modifications, are known to be involved in regulatory switches within the transcriptome. Only recently, mathematical models of these mechanisms have been established. So far they have not been applied to genome-wide data. We here introduce a mathematical model of transcriptional regulation by histone modifications and apply it to data of trimethylation of histone 3 at lysine 4 (H3K4me3) and 27 (H3K27me3) in mouse pluripotent and lineage-committed cells. The model describes binding of protein complexes to chromatin which are capable of reading and writing histone marks. Molecular interactions of the complexes with DNA and modified histones create a regulatory switch of transcriptional activity. The regulatory states of the switch depend on the activity of histone (de-) methylases, the strength of complex-DNA-binding and the number of nucleosomes capable of cooperatively contributing to complex-binding. Our model explains experimentally measured length distributions of modified chromatin regions. It suggests (i) that high CpG-density facilitates recruitment of the modifying complexes in embryonic stem cells and (ii) that re-organization of extended chromatin regions during lineage specification into neuronal progenitor cells requires targeted de-modification. Our approach represents a basic step towards multi-scale models of transcriptional control during development and lineage specification. (paper)

Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

Science.gov (United States)

Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

2016-01-01

Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

Histone methylations in heart development, congenital and adult heart diseases.

Science.gov (United States)

Zhang, Qing-Jun; Liu, Zhi-Ping

2015-01-01

Heart development comprises myocyte specification, differentiation and cardiac morphogenesis. These processes are regulated by a group of core cardiac transcription factors in a coordinated temporal and spatial manner. Histone methylation is an emerging epigenetic mechanism for regulating gene transcription. Interplay among cardiac transcription factors and histone lysine modifiers plays important role in heart development. Aberrant expression and mutation of the histone lysine modifiers during development and in adult life can cause either embryonic lethality or congenital heart diseases, and influences the response of adult hearts to pathological stresses. In this review, we describe current body of literature on the role of several common histone methylations and their modifying enzymes in heart development, congenital and adult heart diseases.

Histone H1x is highly expressed in human neuroendocrine cells and tumours

International Nuclear Information System (INIS)

Warneboldt, Julia; Haller, Florian; Horstmann, Olaf; Danner, Bernhard C; Füzesi, László; Doenecke, Detlef; Happel, Nicole

2008-01-01

Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation. Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study. We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues. We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate

Stage-specific histone modification profiles reveal global transitions in the Xenopus embryonic epigenome.

Directory of Open Access Journals (Sweden)

Tobias D Schneider

Full Text Available Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.

High-resolution structure of the native histone octamer

International Nuclear Information System (INIS)

Wood, Christopher M.; Nicholson, James M.; Lambert, Stanley J.; Chantalat, Laurent; Reynolds, Colin D.; Baldwin, John P.

2005-01-01

The high-resolution (1.90 Å) model of the native histone octamer allows structural comparisons to be made with the nucleosome-core particle, along with an identification of a likely core-histone binding site. Crystals of native histone octamers (H2A–H2B)–(H4–H3)–(H3′–H4′)–(H2B′–H2A′) from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 Å resolution, yielding a structure with an R work value of 18.7% and an R free of 22.2%. The crystal space group is P6 5 , the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A′–H3–H4 molecular cluster (also H2A–H3′–H4′), the H4–H2B′ interaction (also H4′–H2B) and the H2A′–H4 β-sheet interaction (also H2A–H4′). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle

Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes

DEFF Research Database (Denmark)

Jeppesen, A N; Hvas, A-M; Grejs, A M

2017-01-01

Background Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone...... after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus. The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. Results We found no difference...... in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P

Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

Science.gov (United States)

Ververis, Katherine; Rodd, Annabelle L; Tang, Michelle M; El-Osta, Assam; Karagiannis, Tom C

2011-12-01

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

Open and Closed: The Roles of Linker Histones in Plants and Animals

OpenAIRE

Over, Ryan S.; Michaels, Scott D.

2014-01-01

Linker histones play key roles alongside core histones in the regulation and maintenance of chromatin. Here, we illustrate our current understanding of the contributions of linker histones to the cell cycle, development, and chromatin structure in plants and animals.

Histone deacetylase inhibition reduces cardiac Connexin43 expression and gap junction communication

Directory of Open Access Journals (Sweden)

Qin eXu

2013-04-01

Full Text Available Histone deactylase (HDAC inhibitors are being investigated as novel therapies for cancer, inflammation, neurodegeneration, and heart failure. The effects of HDAC inhibitors on the functional expression of cardiac gap junctions (GJ are essentially unknown. The purpose of this study was to determine the effects of trichostatin A (TSA and vorinostat (VOR on functional GJ expression in ventricular cardiomyocytes. The effects of HDAC inhibition on connexin43 (Cx43 expression and functional GJ assembly were examined in primary cultured neonatal mouse ventricular myocytes. TSA and VOR reduced Cx43 mRNA, protein expression, and immunolocalized Cx43 GJ plaque area within ventricular myocyte monolayer cultures in a dose-dependent manner. Chromatin-immunoprecipitation experiments revealed altered protein interactions with the Cx43 promoter. VOR also altered the phosphorylation state of several key regulatory Cx43 phospho-serine sites. Patch clamp analysis revealed reduced electrical coupling between isolated ventricular myocyte pairs, altered transjunctional voltage-dependent inactivation kinetics, and steady state junctional conductance inactivation and recovery relationships. Single GJ channel conductance was reduced to 54 pS only by maximum inhibitory doses of TSA (>= 100 nM. These two hydroxamate pan-HDAC inhibitors exert multiple levels of regulation on ventricular GJ communication by altering Cx43 expression, GJ area, post-translational modifications (e.g. phosphorylation, acetylation, gating, and channel conductance. Although a 50% downregulation of Cx43 GJ communication alone may not be sufficient to slow ventricular conduction or induce arrhythmias, the development of class-selective HDAC inhibitors may help avoid the potential negative cardiovascular effects of pan-HDACI.

DNA and factor VII-activating protease protect against the cytotoxicity of histones

NARCIS (Netherlands)

Marsman, Gerben; von Richthofen, Helen; Bulder, Ingrid; Lupu, Florea; Hazelzet, Jan; Luken, Brenda M.; Zeerleder, Sacha

2017-01-01

Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize

Post-Training Intrahippocampal Inhibition of Class I Histone Deacetylases Enhances Long-Term Object-Location Memory

Science.gov (United States)

Hawk, Joshua D.; Florian, Cedrick; Abel, Ted

2011-01-01

Long-term memory formation involves covalent modification of the histone proteins that package DNA. Reducing histone acetylation by mutating histone acetyltransferases impairs long-term memory, and enhancing histone acetylation by inhibiting histone deacetylases (HDACs) improves long-term memory. Previous studies using HDAC inhibitors to enhance…

Characterization of human papillomavirus type 16 pseudovirus containing histones.

Science.gov (United States)

Kim, Hyoung Jin; Kwag, Hye-Lim; Kim, Hong-Jin

2016-08-27

Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities. The amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity. Our findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most

Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

International Nuclear Information System (INIS)

Beretta, L.; Boutterin, M.C.; Sobel, A.

1988-01-01

We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and [ 32 P] phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins

The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

Science.gov (United States)

Ravi, Maruthachalam; Kwong, Pak N; Menorca, Ron M G; Valencia, Joel T; Ramahi, Joseph S; Stewart, Jodi L; Tran, Robert K; Sundaresan, Venkatesan; Comai, Luca; Chan, Simon W-L

2010-10-01

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

Science.gov (United States)

Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

2017-08-15

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Quantitative analysis of histone modifications: formaldehyde is a source of pathological n(6-formyllysine that is refractory to histone deacetylases.

Directory of Open Access Journals (Sweden)

Bahar Edrissi

Full Text Available Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N(6-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3'-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N(6-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N(6-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N(6-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1-4 modifications per 10(4 lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10(4 lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N(6-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N(6-formyllysine, with use of [(13C,(2H2]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N(6-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10% with a peptide substrate containing the formyl adduct. These data suggest that N(6-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary

The histone demethylase Jhdm1a regulates hepatic gluconeogenesis.

Directory of Open Access Journals (Sweden)

Dongning Pan

Full Text Available Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36 demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.

The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

Science.gov (United States)

Zou, Tie; Yao, Annie Y.; Cooper, Marcus P.; Boyartchuk, Victor; Wang, Yong-Xu

2012-01-01

Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes. PMID:22719268

Histones trigger sterile inflammation by activating the NLRP3 inflammasome.

Science.gov (United States)

Allam, Ramanjaneyulu; Darisipudi, Murthy Narayana; Tschopp, Jurg; Anders, Hans-Joachim

2013-12-01

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll-like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)-1β and IL-18. Here, we report that histones released from necrotic cells induce IL-1β secretion in an NLRP3-ASC-caspase-1-dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone-induced IL-1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone-neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell-derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL-1β secretion via oxidative stress. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

International Nuclear Information System (INIS)

Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario

2006-01-01

Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes

Structure of the human histone chaperone FACT Spt16 N-terminal domain

Energy Technology Data Exchange (ETDEWEB)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk [Cancer Research UK Beatson Institute, Garscube Estate, Switchback Road, Glasgow G61 1BD, Scotland (United Kingdom)

2016-01-22

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

Structure of the human histone chaperone FACT Spt16 N-terminal domain

International Nuclear Information System (INIS)

Marcianò, G.; Huang, D. T.

2016-01-01

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding

Class IIa histone deacetylases are hormone-activated regulators of FOXO and mammalian glucose homeostasis.

Science.gov (United States)

Mihaylova, Maria M; Vasquez, Debbie S; Ravnskjaer, Kim; Denechaud, Pierre-Damien; Yu, Ruth T; Alvarez, Jacqueline G; Downes, Michael; Evans, Ronald M; Montminy, Marc; Shaw, Reuben J

2011-05-13

Class IIa histone deacetylases (HDACs) are signal-dependent modulators of transcription with established roles in muscle differentiation and neuronal survival. We show here that in liver, class IIa HDACs (HDAC4, 5, and 7) are phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, class IIa HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 recruit HDAC3, which results in the acute transcriptional induction of these genes via deacetylation and activation of FOXO family transcription factors. Loss of class IIa HDACs in murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Finally, suppression of class IIa HDACs in mouse models of type 2 diabetes ameliorates hyperglycemia, suggesting that inhibitors of class I/II HDACs may be potential therapeutics for metabolic syndrome. Copyright © 2011 Elsevier Inc. All rights reserved.

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

1988-01-01

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

Chemical mechanisms of histone lysine and arginine modifications

OpenAIRE

Smith, Brian C.; Denu, John M.

2008-01-01

Histone lysine and arginine residues are subject to a wide array of post-translational modifications including methylation, citrullination, acetylation, ubiquitination, and sumoylation. The combinatorial action of these modifications regulates critical DNA processes including replication, repair, and transcription. In addition, enzymes that modify histone lysine and arginine residues have been correlated with a variety of human diseases including arthritis, cancer, heart disease, diabetes, an...

histoneHMM: Differential analysis of histone modifications with broad genomic footprints

Czech Academy of Sciences Publication Activity Database

Heinig, M.; Colomé-Tatché, M.; Taudt, A.; Rintisch, C.; Schafer, S.; Pravenec, Michal; Hubner, N.; Vingron, M.; Johannes, F.

2015-01-01

Roč. 16, Feb 22 (2015), s. 60 ISSN 1471-2105 R&D Projects: GA MŠk(CZ) 7E10067; GA ČR(CZ) GA13-04420S Institutional support: RVO:67985823 Keywords : ChIP - seq * histone modifications * Hidden Markov model * computational biology * differential analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.435, year: 2015

UV laser-induced histone-DNA crosslinking proceeds via the N-terminal tails

International Nuclear Information System (INIS)

Stefanovski, V.; Dimitrov, S.; Angelov, D.; Keskinova, E.; Pashev, I.

1990-01-01

The covalent crosslinking of histones to DNA by UV laser irradiation is accomplished solely via the N-terminal part of the molecule. Irradiated isolated calfthymus nuclei are treated with clostripain. The crosslinked protein-DNA complexes are isolated and the presence of each core histone analyzed by dot-immunoassay using antibodies, specific to the central globular domain of the respective histone. The reaction is negative for all core histones i.e. the globular domain is absent. It means that this domain has not been crosslinked to DNA and, once cleaved by clostripain, it has been stripped from DNA during the centrigugation in CsCl. This peculiar property of the crosslinked procedure makes it particularly useful in addressing some yet unanswered questions concerning histone-DNA interactions, such as the interaction of the N-terminal tails with linker DNA, the effect of the transient postsynthetic histone acetylation on its interaction with DNA, etc. These questions are now under study. 1 fig., 6 refs

Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

Science.gov (United States)

Zhang, Menghuan; Li, Hong; He, Ying; Sun, Han; Xia, Li; Wang, Lishun; Sun, Bo; Ma, Liangxiao; Zhang, Guoqing; Li, Jing; Li, Yixue; Xie, Lu

2015-07-02

Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

Proteasome phosphorylation regulates cocaine-induced sensitization.

Science.gov (United States)

Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanism for the decrease in the FIP1L1-PDGFRalpha protein level in EoL-1 cells by histone deacetylase inhibitors.

Science.gov (United States)

Ishihara, Kenji; Kaneko, Motoko; Kitamura, Hajime; Takahashi, Aki; Hong, Jang Ja; Seyama, Toshio; Iida, Koji; Wada, Hiroshi; Hirasawa, Noriyasu; Ohuchi, Kazuo

2008-01-01

Acetylation and deacetylation of proteins occur in cells in response to various stimuli, and are reversibly catalyzed by histone acetyltransferase and histone deacetylase (HDAC), respectively. EoL-1 cells have an FIP1L1-PDGFRA fusion gene that causes transformation of eosinophilic precursor cells into leukemia cells. The HDAC inhibitors apicidin and n-butyrate suppress the proliferation of EoL-1 cells and induce differentiation into eosinophils by a decrease in the protein level of FIP1L1-PDGFRalpha without affecting the mRNA level for FIP1L1-PDGFRA. In this study, we analyzed the mechanism by which the protein level of FIP1L1-PDGFRalpha is decreased by apicidin and n-butyrate. EoL-1 cells were incubated in the presence of the HDAC inhibitors apicidin, trichostatin A or n-butyrate. The protein levels of FIP1L1-PDGFRalpha and phosphorylated eIF-2alpha were determined by Western blotting. Actinomycin D and cycloheximide were used to block RNA synthesis and protein synthesis, respectively, in the chasing experiment of the amount of FIP1L1-PDGFRalpha protein. When apicidin- and n-butyrate-treated EoL-1 cells were incubated in the presence of actinomycin D, the decrease in the protein level of FIP1L1-PDGFRalpha was significantly enhanced when compared with controls. In contrast, the protein levels were not changed by cycloheximide among these groups. Apicidin and n-butyrate induced the continuous phosphorylation of eIF-2alpha for up to 8 days. The decrease in the level of FIP1L1-PDGFRalpha protein by continuous inhibition of HDAC may be due to the decrease in the translation rate of FIP1L1-PDGFRA. Copyright 2008 S. Karger AG, Basel.

Phosphorylation of myelin basic proteins and its relevance to myelin biogenesis

Energy Technology Data Exchange (ETDEWEB)

Ulmer, J.B.

1985-01-01

Age-related differences in the in vivo incorporation of (32-P) into mouse myelin basic proteins (MBPs) of the central nervous system were observed. The resulting specific radioactivity (S.A.) of the MBPs appeared to be related to the S.A. of the acid-soluble pool of phosphates of myelin. In development, MBPs were phosphorylated in vivo prior to the onset of myelination in the brain, indicating that MBPs are phosphorylated prior to their deposition in the myelin sheath. The incorporation of (32-P) into MBPs and the turnover rates of MBP phosphates were studied in vivo in developmentally-related myelin compartments. The results suggest that there are two separate events in MBP phosphorylation and that the turnover rates of the MBP phosphates derived from these two events are different. A model for MBP phosphorylation, that could explain in these observations, is postulated and discussed in the light of existing information.

New clinical developments in histone deacetylase inhibitors for epigenetic therapy of cancer

Directory of Open Access Journals (Sweden)

Ma Yuehua

2009-06-01

Full Text Available Abstract DNA methylation and histone acetylation are two well known epigenetic chromatin modifications. Epigenetic agents leading to DNA hypomethylation and histone hyperacetylation have been approved for treatment of hematological disorders. The first histone deacetylase inhibitor, vorinostat, has been licensed for cutaneous T cell lymphoma treatment. More than 11 new epigenetic agents are in various stages of clinical development for therapy of multiple cancer types. In this review we summarize novel histone deacetylase inhibitors and new regimens from clinical trials for epigenetic therapy of cancer.

Oxidative phosphorylation revisited

DEFF Research Database (Denmark)

Nath, Sunil; Villadsen, John

2015-01-01

The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

Extracellular histones play an inflammatory role in acid aspiration-induced acute respiratory distress syndrome.

Science.gov (United States)

Zhang, Yanlin; Wen, Zongmei; Guan, Li; Jiang, Ping; Gu, Tao; Zhao, Jinyuan; Lv, Xin; Wen, Tao

2015-01-01

Systemic inflammation is a key feature in acid aspiration-induced acute respiratory distress syndrome (ARDS), but the factors that trigger inflammation are unclear. The authors hypothesize that extracellular histones, a newly identified inflammatory mediator, play important roles in the pathogenesis of ARDS. The authors used a hydrochloric acid aspiration-induced ARDS model to investigate whether extracellular histones are pathogenic and whether targeting histones are protective. Exogenous histones and antihistone antibody were administered to mice. Heparin can bind to histones, so the authors studied whether heparin could protect from ARDS using cell and mouse models. Furthermore, the authors analyzed whether extracellular histones are clinically involved in ARDS patients caused by gastric aspiration. Extracellular histones in bronchoalveolar lavage fluid of acid-treated mice were significantly higher (1.832 ± 0.698) at 3 h after injury than in sham-treated group (0.63 ± 0.153; P = 0.0252, n = 5 per group). Elevated histones may originate from damaged lung cells and neutrophil infiltration. Exogenous histones aggravated lung injury, whereas antihistone antibody markedly attenuated the intensity of ARDS. Notably, heparin provided a similar protective effect against ARDS. Analysis of plasma from ARDS patients (n = 21) showed elevated histones were significantly correlated with the degree of ARDS and were higher in nonsurvivors (2.723 ± 0.2933, n = 7) than in survivors (1.725 ± 0.1787, P = 0.006, n = 14). Extracellular histones may play a contributory role toward ARDS by promoting tissue damage and systemic inflammation and may become a novel marker reflecting disease activity. Targeting histones by neutralizing antibody or heparin shows potent protective effects, suggesting a potentially therapeutic strategy.

Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

Science.gov (United States)

Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio

2016-02-01

The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Investigation of the reactions of histone protein hydroperoxides and their role in DNA damage

International Nuclear Information System (INIS)

Luxford, C.; Dean, R.T.; Davies, M.J.

1998-01-01

Free radical attack on DNA results in base changes, cross-linking and strand cleavage leading to mutations if unrepaired. Histone proteins are intimately involved in DNA packaging and are excellent candidates for investigating DNA damage arising from protein-OOH-derived radicals. This study aimed (i) to investigate the formation of hydroperoxide on the linker histone H1 via radical reactions in the presence of O 2 ; (ii) to examine the radicals formed from transition metal ion-catalyzed breakdown of histone H1-OOH and (iii) to determine whether histone H1-OOH-derived radicals can damage DNA and free bases. (i) Histone H1 solutions were γ-irradiated ( 60 Co source) in the presence of O 2 and histone H1-OOH concentrations determined using a manual iodometric assay. Formation ( histone H1-OOH was dose-dependent and, in the absence of light or transition metal ions these hydroperoxides were found to be very stable (half life of 24 hours at 4degC ). (ii) Electron Paramagnetic Resonance (EPR) spectroscopy and spin trapping was used t investigate the Cu + -catalyzed breakdown of histone H1-OOH to form histone H1 protein side chain and -backbone carbon-centred radicals. Further EPR/spin trapping experiments showed that histone H1-OOH-derived radicals can oxidise pyrimidine bases (eg. uridine with the resultant trapping of three radical species; two pyrimidine radicals, C5-yl and Ct yl adducts (via addition of histone H1-OOH-derived radicals to the C5-C6 double bond o the pyrimidine ring) and an acyl radical adduct, whose origin is currently unknown. (iii) Damage to DNA and 2'-deoxyguanosine after reaction of histone H1-OOH-derive radicals were detected and quantified using HPLC (with EC and UV detection). We have identified 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a significant product ( histone H1-OOH-derived oxidative DNA modification. Increasing histone H1-OOH concentrations resulted in a concomitant increase in the amount of 8-oxodG formed. Our studies show

Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer

International Nuclear Information System (INIS)

Paller, Channing J; Wissing, Michel D; Mendonca, Janet; Sharma, Anup; Kim, Eugene; Kim, Hea-Soo; Kortenhorst, Madeleine S Q; Gerber, Stephanie; Rosen, Marc; Shaikh, Faraz; Zahurak, Marianna L; Rudek, Michelle A; Hammers, Hans; Rudin, Charles M; Carducci, Michael A; Kachhap, Sushant K

2014-01-01

Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA

Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

Science.gov (United States)

Swetha, Medchalmi; Ramaiah, Kolluru V A

2015-11-01

Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan; Fulvio, Mauricio Di; Gomez-Cambronero, Julian

2009-01-01

Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27 KIP1 and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y→F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y 179 and Y 511 .

Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA

DEFF Research Database (Denmark)

Luxford, C; Dean, R T; Davies, Michael Jonathan

2000-01-01

Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion......; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin...... trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give...

Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

Science.gov (United States)

Dulmage, Keely A; Todor, Horia; Schmid, Amy K

2015-09-08

In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode

Histone Deacetylase Inhibitor Therapy in Epithelial Ovarian Cancer

Directory of Open Access Journals (Sweden)

Noriyuki Takai

2010-01-01

Full Text Available Since epigenetic alterations are believed to be involved in the repression of tumor suppressor genes and promotion of tumorigenesis in ovarian cancers, novel compounds endowed with a histone deacetylase (HDAC inhibitory activity are an attractive therapeutic approach. In this review, we discuss the biologic and therapeutic effects of HDAC inhibitors (HDACIs in treating ovarian cancer. HDACIs were able to mediate inhibition of cell growth, cell cycle arrest, apoptosis, and expression of genes related to the malignant phenotype in a variety of ovarian cancer cell lines. Furthermore, HDACIs were able to induce the accumulation of acetylated histones in the chromatin of the p21WAF1 gene in human ovarian carcinoma cells. In xenograft models, some of HDACIs have demonstrated antitumor activity with only few side effects. Some clinical trials demonstrate that HDACI drugs provide an important class of new mechanism-based therapeutics for ovarian cancer. In this review, we discuss the biologic and therapeutic effects of HDACIs in treating ovarian cancer, especially focusing on preclinical studies and clinical trials.

Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism.

Science.gov (United States)

Gómez-Rodríguez, Elida Yazmín; Uresti-Rivera, Edith Elena; Patrón-Soberano, Olga Araceli; Islas-Osuna, María Auxiliadora; Flores-Martínez, Alberto; Riego-Ruiz, Lina; Rosales-Saavedra, María Teresa; Casas-Flores, Sergio

2018-01-01

Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism

Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism.

Directory of Open Access Journals (Sweden)

Elida Yazmín Gómez-Rodríguez

Full Text Available Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1, a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA, a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression

Exposure to Tumescent Solution Significantly Increases Phosphorylation of Perilipin in Adipocytes.

Science.gov (United States)

Keskin, Ilknur; Sutcu, Mustafa; Eren, Hilal; Keskin, Mustafa

2017-02-01

Lidocaine and epinephrine could potentially decrease adipocyte viability, but these effects have not been substantiated. The phosphorylation status of perilipin in adipocytes may be predictive of cell viability. Perilipin coats lipid droplets and restricts access of lipases; phospho-perilipin lacks this protective function. The authors investigated the effects of tumescent solution containing lidocaine and epinephrine on the phosphorylation status of perilipin in adipocytes. In this in vitro study, lipoaspirates were collected before and after tumescence from 15 women who underwent abdominoplasty. Fat samples were fixed, sectioned, and stained for histologic and immunohistochemical analyses. Relative phosphorylation of perilipin was inferred from pixel intensities of immunostained adipocytes observed with confocal microscopy. For adipocytes collected before tumescent infiltration, 10.08% of total perilipin was phosphorylated. In contrast, 30.62% of total perilipin was phosphorylated for adipocytes collected from tumescent tissue (P < .01). The tumescent technique increases the relative phosphorylation of perilipin in adipocytes, making these cells more vulnerable to lipolysis. Tumescent solution applied for analgesia or hemostasis of the donor site should contain the lowest possible concentrations of lidocaine and epinephrine. LEVEL OF EVIDENCE 5. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

Energy Technology Data Exchange (ETDEWEB)

Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

Effect of gamma irradiation on rat thymus arginine-rich H3 histone in vitro

International Nuclear Information System (INIS)

Patil, M.S.; Narasimhan, Saroja; Sreenivasan, A.

1977-01-01

Physicochemical properties of rat thymus H3 histone have been studied following gamma radiation (25-90 krad) in 0.2 N HCl. Polyacrylamide gel electrophoretic pattern (PGE) of H3 histone indicated that aggregates were formed in the histone fraction following gamma irradiation. The PGE pattern of the irradiated-histone fraction remained unaltered even after it was treated with 8.0 M urea to eliminate noncovalent bonding. On the other hand, the irradiated sample treated with β-mercaptoethanol exhibited the PGE pattern which was essentially similar to that of unirradiated sample. These results indicate that the aggregates seen in the PGE pattern of irradiated-H3 histone may be formed through interpolypeptide chain disulphide linkeges rather than by noncovalent bonding. This contention is also supported by the fact that irradiated-H3 histone exhibited hyperchromic shift at 240-250 nm region as well as increased disulphide content. Other results revealed that DNA-dependent RNA synthesis in vitro was inhibited to a greater extent by irradiated-H3 histone than by unirradiated-H3 histone. (author)

An atlas of histone deacetylase expression in breast cancer: fluorescence methodology for comparative semi-quantitative analysis.

Science.gov (United States)

Ververis, Katherine; Karagiannis, Tom C

2012-01-01

The histone deacetylase inhibitors, suberoylanilide hydroxamic acid (Vorinostat, Zolinza™) and depsipeptide (Romidepsin, Istodax™) have been approved by the US Food and Drug Administration for the treatment of refractory cutaneous T-cell lymphoma. Numerous histone deacetylase inhibitors are currently undergoing clinical trials, predominantly in combination with other cancer modalities, for the treatment of various haematological and solid malignancies. Most of the traditional compounds are known as broad-spectrum or pan-histone deacetylase inhibitors, possessing activity against a number of the 11 metal-dependent enzymes. One of the main questions in the field is whether class- or isoform-specific compounds would offer a therapeutic benefit compared to broad-spectrum inhibitors. Therefore, analysis of the relative expression of the different histone deacetylase enzymes in cancer cells and tissues is important to determine whether there are specific targets. We used a panel of antibodies directed against the 11 known mammalian histone deacetylases to determine expression levels in MCF7 breast cancer cells and in tissue representative of invasive ductal cell carcinoma and ductal carcinoma in situ. Firstly, we utilized a semi-quantitative method based on immunofluorescence staining to examine expression of the different histone deacetylases in MCF7 cells. Our findings indicate high expression levels of HDAC1, 3 and 6 in accordance with findings from others using RT-PCR and immunoblotting. Following validation of our approach we examined the expression of the different isoforms in representative control and breast cancer tissue. In general, our findings indicate higher expression of class I histone deacetylases compared to class II enzymes in breast cancer tissue. Analysis of individual cancer cells in the same tissue indicated marked heterogeneity in the expression of most class I enzymes indicating potential complications with the use of class- or isoform

The histone genes in HeLa cells are on individual transcriptional units

International Nuclear Information System (INIS)

Hackett, P.B.; Traub, P.; Gallwitz, D.

1978-01-01

The distances of the five major histone genes from their promotors have been investigated in order to determine whether in human cells these genes could be transcribed as a single polycistronic transcriptional unit. By measuring the decreases of both histone protein and histone mRNA synthesis as functions of the ultraviolet light dosage, it was possible to calculate the distances of the histone genes from their promotors. The inactivation kinetics for histone genes H1 and H3 are first-order, indicating a single type of transcriptional unit for each gene. The dose-response kinetics for genes H2A, H2B and H4 are first-order with two distinct rates; 10 to 15% of the genes for each of these histones appear to be much more sensitive to ultraviolet light inactivation than are the majority. It is concluded that the transcriptional units for 85 to 90% of the genes for H2A, H2B and H4 are similar. As determined by the inhibition of protein synthesis, the inactivation coefficients for the major component of each histone are: H1, 907 mm 2 /erg; H2A, 878 mm 2 /erg; H2B, 871 mm 2 /erg; H3, 965 mm 2 /erg; and H4, 792 mm 2 /erg. The sensitivities of histone mRNA synthesis to irradiation were measured by translation in vitro with similar results. The calculated target sizes for the genes (in base-pairs) are: H1, 1190; H2A, 1240; H2B, 1250; H3, 1130; and H4, 1380. This similarity in target sizes for all five of the histones genes indicates that they are primarily transcribed from individual transcriptional units. (author)

[PHI regulates histone methylation and acetylation in Burkitt lymphoma Daudi cell line].

Science.gov (United States)

Hong, Ling-Ling; Ma, Xu-Dong; Huang, Yi-Qun

2011-02-01

This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.

Targeting Histone Abnormality in Triple Negative Breast Cancer

Science.gov (United States)

2015-08-01

κB pathway in triple negative breast cancer . 8th International Nitric Oxide Conference & 6th International Nitrite/ Nitrate Conference, Cleveland, OH...1 AWARD NUMBER: W81XWH-14-1-0237 TITLE: Targeting Histone Abnormality in Triple-Negative Breast Cancer PRINCIPAL INVESTIGATOR: Yi...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Histone Abnormality in Triple-Negative Breast Cancer 5b. GRANT NUMBER W81XWH-14-1-0237 5c

FACT, the Bur kinase pathway, and the histone co-repressor HirC have overlapping nucleosome-related roles in yeast transcription elongation.

Directory of Open Access Journals (Sweden)

Jennifer R Stevens

Full Text Available Gene transcription is constrained by the nucleosomal nature of chromosomal DNA. This nucleosomal barrier is modulated by FACT, a conserved histone-binding heterodimer. FACT mediates transcription-linked nucleosome disassembly and also nucleosome reassembly in the wake of the RNA polymerase II transcription complex, and in this way maintains the repression of 'cryptic' promoters found within some genes. Here we focus on a novel mutant version of the yeast FACT subunit Spt16 that supplies essential Spt16 activities but impairs transcription-linked nucleosome reassembly in dominant fashion. This Spt16 mutant protein also has genetic effects that are recessive, which we used to show that certain Spt16 activities collaborate with histone acetylation and the activities of a Bur-kinase/Spt4-Spt5/Paf1C pathway that facilitate transcription elongation. These collaborating activities were opposed by the actions of Rpd3S, a histone deacetylase that restores a repressive chromatin environment in a transcription-linked manner. Spt16 activity paralleling that of HirC, a co-repressor of histone gene expression, was also found to be opposed by Rpd3S. Our findings suggest that Spt16, the Bur/Spt4-Spt5/Paf1C pathway, and normal histone abundance and/or stoichiometry, in mutually cooperative fashion, facilitate nucleosome disassembly during transcription elongation. The recessive nature of these effects of the mutant Spt16 protein on transcription-linked nucleosome disassembly, contrasted to its dominant negative effect on transcription-linked nucleosome reassembly, indicate that mutant FACT harbouring the mutant Spt16 protein competes poorly with normal FACT at the stage of transcription-linked nucleosome disassembly, but effectively with normal FACT for transcription-linked nucleosome reassembly. This functional difference is consistent with the idea that FACT association with the transcription elongation complex depends on nucleosome disassembly, and that the

Histone deacetylase inhibitors induced differentiation and accelerated mineralization of pulp-derived cells.

LENUS (Irish Health Repository)

Duncan, Henry F

2012-03-01

Histone deacetylase inhibitors (HDACis) alter the homeostatic balance between 2 groups of cellular enzymes, histone deacetylases (HDACs) and histone acetyltransferases (HATs), increasing transcription and influencing cell behavior. This study investigated the potential of 2 HDACis, valproic acid (VPA) and trichostatin A (TSA), to promote reparative processes in pulp cells as assayed by viability, cell cycle, and mineralization analyses.

Mechanical Stability and Fibrinolytic Resistance of Clots Containing Fibrin, DNA, and Histones*

Science.gov (United States)

Longstaff, Colin; Varjú, Imre; Sótonyi, Péter; Szabó, László; Krumrey, Michael; Hoell, Armin; Bóta, Attila; Varga, Zoltán; Komorowicz, Erzsébet; Kolev, Krasimir

2013-01-01

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nm dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator. PMID:23293023

Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

Science.gov (United States)

Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

2010-03-01

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

Prepatterning of developmental gene expression by modified histones before zygotic genome activation

DEFF Research Database (Denmark)

Lindeman, Leif C.; Andersen, Ingrid S.; Reiner, Andrew H.

2011-01-01

A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive...... role of histone marks for ZGA. In zebrafish, pre-ZGA development for ten cell cycles provides an opportunity to examine whether genomic enrichment in modified histones is present before initiation of transcription. By profiling histone H3 trimethylation on all zebrafish promoters before and after ZGA......, we demonstrate here an epigenetic prepatterning of developmental gene expression. This involves pre-ZGA marking of transcriptionally inactive genes involved in homeostatic and developmental regulation by permissive H3K4me3 with or without repressive H3K9me3 or H3K27me3. Our data suggest that histone...

Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

Science.gov (United States)

Gould, Travis J; Lysov, Zakhar; Swystun, Laura L; Dwivedi, Dhruva J; Zarychanski, Ryan; Fox-Robichaud, Alison E; Liaw, Patricia C

2016-12-01

Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown. To address this, we investigated the ability of histones to modulate the procoagulant potential of THP-1 cells and peripheral blood monocytes, and examined the effects of plasmas obtained from septic patients to induce a procoagulant phenotype on monocytic cells. Tissue factor (TF) activity assays were performed on histone-treated THP-1 cells and blood monocytes. Exposure of monocytic cells to histones resulted in increases in TF activity, TF antigen, and phosphatidylserine exposure. Histones modulate the procoagulant activity via engagement of Toll-like receptors 2 and 4, and this effect was abrogated with inhibitory antibodies. Increased TF activity of histone-treated cells corresponded to enhanced thrombin generation in plasma determined by calibrated automated thrombography. Finally, TF activity was increased on monocytes exposed to plasma from septic patients, an effect that was attenuated in plasma from patients receiving unfractionated heparin (UFH). Our studies suggest that increased levels of extracellular histones found in sepsis contribute to dysregulated coagulation by increasing TF activity of monocytes. These procoagulant effects can be partially ameliorated in sepsis patients receiving UFH, thereby identifying extracellular histones as a potential therapeutic target for sepsis treatment.

Impact of plasma histones in human sepsis and their contribution to cellular injury and inflammation.

Science.gov (United States)

Ekaney, Michael Liembo; Otto, Gordon Philipp; Sossdorf, Maik; Sponholz, Christoph; Boehringer, Michael; Loesche, Wolfgang; Rittirsch, Daniel; Wilharm, Arne; Kurzai, Oliver; Bauer, Michael; Claus, Ralf Alexander

2014-09-24

Circulating histones have been identified as mediators of damage in animal models of sepsis and in patients with trauma-associated lung injury. Despite existing controversies on actual histone concentrations, clinical implications and mechanism of action in various disease conditions, histone levels in human sepsis, association with disease progression and mediated effects on endothelial and immune cells remain unreported. This study aimed to determine histone levels and its clinical implication in septic patients and to elucidate histone-mediated effects ex-vivo. Histone levels, endogenous activated protein C (APC) levels and clinical data from two independent cohorts of septic patients were obtained. Histone levels were compared with various control groups including healthy individuals, intensive care unit (ICU) patients without sepsis, ICU patients with multiple organ failure and patients with minor or multiple trauma, all without infection. Endothelial and monocytic cells were stimulated with histones. Cellular integrity and sepsis prototypical cytokines were evaluated. The mechanism of action of histones via Toll-like receptor 4 (TLR4) was evaluated using a function blocking antibody. Histone degradation in plasma was studied by immunoblotting. Histone H4 levels were significantly elevated in patients with sepsis (cohort I; n = 15 and cohort II; n = 19) versus ICU controls (n = 12), patients with multiple organ failure (n = 12) or minor trauma (n = 7), associated with need for renal replacement therapy and decrease in platelet count during disease progression, and remarkably were significantly associated with increased mortality rates in septic patients (ICU-, 28 day- and 90 day mortality rates). There was an inverse correlation between plasma histones and endogenous APC levels. Histone stimulation induced the release of sepsis prototypic cytokines and decreased cell integrity indicated by a significant increase of lactate dehydrogenase (LDH) and propidium

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

Directory of Open Access Journals (Sweden)

Laura Civiero

2017-12-01

Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots

Science.gov (United States)

Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R.; Bois, Philippe R. J.

2016-01-01

ABSTRACT Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined “hot spots.” In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots. PMID:27821479

Abundance of intrinsic structural disorder in the histone H1 subtypes.

Science.gov (United States)

Kowalski, Andrzej

2015-12-01

The intrinsically disordered proteins consist of partially structured regions linked to the unstructured stretches, which consequently form the transient and dynamic conformational ensembles. They undergo disorder to order transition upon binding their partners. Intrinsic disorder is attributed to histones H1, perceived as assemblers of chromatin structure and the regulators of DNA and proteins activity. In this work, the comparison of intrinsic disorder abundance in the histone H1 subtypes was performed both by the analysis of their amino acid composition and by the prediction of disordered stretches, as well as by identifying molecular recognition features (MoRFs) and ANCHOR protein binding regions (APBR) that are responsible for recognition and binding. Both human and model organisms-animals, plants, fungi and protists-have H1 histone subtypes with the properties typical of disordered state. They possess a significantly higher content of hydrophilic and charged amino acid residues, arranged in the long regions, covering over half of the whole amino acid residues in chain. Almost complete disorder corresponds to histone H1 terminal domains, including MoRFs and ANCHOR. Those motifs were also identified in a more ordered histone H1 globular domain. Compared to the control (globular and fibrous) proteins, H1 histones demonstrate the increased folding rate and a higher proportion of low-complexity segments. The results of this work indicate that intrinsic disorder is an inherent structural property of histone H1 subtypes and it is essential for establishing a protein conformation which defines functional outcomes affecting on DNA- and/or partner protein-dependent cell processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Investigation of Extracellular Histones in Pig-To-Baboon Organ Xenotransplantation.

Science.gov (United States)

Li, Tao; Lee, Whayoung; Hara, Hidetaka; Long, Cassandra; Ezzelarab, Mohamed; Ayares, David; Huang, Hai; Wang, Yi; Esmon, Charles T; Cooper, David K C; Iwase, Hayato

2017-10-01

Serum (extracellular) histone levels are increased in inflammatory states and in the presence of coagulation dysfunction, for example, trauma, chemical/ischemic injury, infection. There is increasing evidence of a systemic inflammatory response associated with the presence of a pig xenograft in a nonhuman primate. We evaluated extracellular histone levels in baboons with various pig xenografts. We measured serum histones in baboons with pig heterotopic heart (n = 8), life-supporting kidney (n = 5), orthotopic liver (n = 4), and artery patch (n = 9) grafts by enzyme-linked immunosorbent assay. C-reactive protein (CRP), free triiodothyronine (fT3), serum amyloid A (SAA), and platelet counts were also measured, all of which may provide an indication of an inflammatory state. We investigated the effect of histones on platelet aggregation and on cytotoxicity of pig cells in vitro. Serum histones increased when baboons developed consumptive coagulopathy (eg, thrombocytopenia) or infection. CRP levels tended to be higher and fT3 levels lower when consumptive coagulopathy developed. Measurement of SAA correlated fairly well with CRP and indicated the state of inflammation. Treatment of the recipient with tocilizumab reduced the level of serum histones, CRP, and SAA, and increased the level of fT3 and platelet counts. In vitro, histone-induced platelet aggregation and endothelial cell apoptosis were both significantly reduced by the NF-κB pathway inhibitor, parthenolide. These noninvasive assays may be useful for monitoring the health status of nonhuman primate recipients of pig organ grafts and may help in management after xenotransplantation. Tocilizumab and NF-κB inhibitors might prove valuable in reducing the inflammatory response to a pig xenograft.

A grammar inference approach for predicting kinase specific phosphorylation sites.

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner.

A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

Science.gov (United States)

Datta, Sutapa; Mukhopadhyay, Subhasis

2015-01-01

Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

ERK2-Mediated Phosphorylation of Transcriptional Coactivator Binding Protein PIMT/NCoA6IP at Ser298 Augments Hepatic Gluconeogenesis

Science.gov (United States)

Parsa, Kishore V. L.; Kain, Vasundhara; Behera, Soma; Suraj, Sashidhara Kaimal; Babu, Phanithi Prakash; Kar, Anand; Panda, Sunanda; Zhu, Yi-jun; Jia, Yuzhi; Thimmapaya, Bayar; Reddy, Janardan K.; Misra, Parimal

2013-01-01

PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser298 and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMTS298D) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMTS298D but not PIMTS298A augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser298 phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser298 is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia. PMID:24358311

Specific distribution of the Saccharomyces cerevisiae linker histone homolog HHO1p in the chromatin

OpenAIRE

Freidkin, Ilya; Katcoff, Don J.

2001-01-01

In virtually all eukaryotic organisms, linker DNA between nucleosomes is associated with a histone termed linker histone or histone H1. In Saccharomyces cerevisiae, HHO1 encodes a putative linker histone with very significant homology to histone H1. The encoded protein is expressed in the nucleus, but has not been shown to affect global chromatin structure, nor has its deletion shown any detectable phenotype. In vitro chromatin assembly experiments with recombinant HHO1p have shown that it is...

Histone Deacetylase Inhibitors as Anticancer Drugs.

Science.gov (United States)

Eckschlager, Tomas; Plch, Johana; Stiborova, Marie; Hrabeta, Jan

2017-07-01

Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

Histone Deacetylase Inhibitors as Anticancer Drugs

Directory of Open Access Journals (Sweden)

Tomas Eckschlager

2017-07-01

Full Text Available Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC and histone acetyltransferases (HAT. HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

Energy Technology Data Exchange (ETDEWEB)

Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J [MSKCC; (Rockefeller); (MRC)

2013-01-24

Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

Directory of Open Access Journals (Sweden)

Yoda Satoshi

2008-11-01

Full Text Available Abstract Background Alterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis. Results We observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1 that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest. Conclusion Our results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

International Nuclear Information System (INIS)

Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

2015-01-01

Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

Missing value imputation for microarray gene expression data using histone acetylation information

Directory of Open Access Journals (Sweden)

Feng Jihua

2008-05-01

Full Text Available Abstract Background It is an important pre-processing step to accurately estimate missing values in microarray data, because complete datasets are required in numerous expression profile analysis in bioinformatics. Although several methods have been suggested, their performances are not satisfactory for datasets with high missing percentages. Results The paper explores the feasibility of doing missing value imputation with the help of gene regulatory mechanism. An imputation framework called histone acetylation information aided imputation method (HAIimpute method is presented. It incorporates the histone acetylation information into the conventional KNN(k-nearest neighbor and LLS(local least square imputation algorithms for final prediction of the missing values. The experimental results indicated that the use of acetylation information can provide significant improvements in microarray imputation accuracy. The HAIimpute methods consistently improve the widely used methods such as KNN and LLS in terms of normalized root mean squared error (NRMSE. Meanwhile, the genes imputed by HAIimpute methods are more correlated with the original complete genes in terms of Pearson correlation coefficients. Furthermore, the proposed methods also outperform GOimpute, which is one of the existing related methods that use the functional similarity as the external information. Conclusion We demonstrated that the using of histone acetylation information could greatly improve the performance of the imputation especially at high missing percentages. This idea can be generalized to various imputation methods to facilitate the performance. Moreover, with more knowledge accumulated on gene regulatory mechanism in addition to histone acetylation, the performance of our approach can be further improved and verified.

Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug

International Nuclear Information System (INIS)

Jafargholizadeh, Naser; Zargar, Seyed Jalal; Safarian, Shahrokh; Habibi-Rezaei, Mehran

2012-01-01

Highlights: ► For the first time, our results show mitoxantrone anticancer drug binds to histone H1, via hydrophobic, hydrogen, van der Waals and electrostatic interactions. ► Binding of mitoxantrone molecules to histone H1 is positive cooperative. ► Histone H1 may be considered as a new target for mitoxantrone at the chromatin level. - Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.

[Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

Science.gov (United States)

Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S

2014-01-01

The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

Neutralisation of the anti-coagulant effects of heparin by histones in blood plasma and purified systems.

Science.gov (United States)

Longstaff, Colin; Hogwood, John; Gray, Elaine; Komorowicz, Erzsebet; Varjú, Imre; Varga, Zoltán; Kolev, Krasimir

2016-03-01

Neutrophil extracellular traps (NETs) composed primarily of DNA and histones are a link between infection, inflammation and coagulation. NETs promote coagulation and approaches to destabilise NETs have been explored to reduce thrombosis and treat sepsis. Heparinoids bind histones and we report quantitative studies in plasma and purified systems to better understand physiological consequences. Unfractionated heparin (UFH) was investigated by activated partial thromboplastin time (APTT) and alongside low-molecular-weight heparins (LMWH) in purified systems with thrombin or factor Xa (FXa) and antithrombin (AT) to measure the sensitivity of UFH or LMWH to histones. A method was developed to assess the effectiveness of DNA and non-anticoagulant heparinoids as anti-histones. Histones effectively neutralised UFH, the IC50 value for neutralisation of 0.2 IU/ml UFH was 1.8 µg/ml histones in APTT and 4.6 µg/ml against 0.6 IU/ml UFH in a purified system. Histones also inhibited the activities of LMWHs with thrombin (IC50 6.1 and 11.0 µg/ml histones, for different LMWHs) or FXa (IC50 7.8 and 7.0 µg/ml histones). Direct interactions of UFH and LMWH with DNA and histones were explored by surface plasmon resonance, while rheology studies showed complex effects of histones, UFH and LMWH on clot resilience. A conclusion from these studies is that anticoagulation by UFH and LMWH will be compromised by high affinity binding to circulating histones even in the presence of DNA. A complete understanding of the effects of histones, DNA and heparins on the haemostatic system must include an appreciation of direct effects on fibrin and clot structure.

A computational model for histone mark propagation reproduces the distribution of heterochromatin in different human cell types.

Science.gov (United States)

Schwämmle, Veit; Jensen, Ole Nørregaard

2013-01-01

Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.

Enhancing dopaminergic signaling and histone acetylation promotes long-term rescue of deficient fear extinction

Science.gov (United States)

Whittle, N; Maurer, V; Murphy, C; Rainer, J; Bindreither, D; Hauschild, M; Scharinger, A; Oberhauser, M; Keil, T; Brehm, C; Valovka, T; Striessnig, J; Singewald, N

2016-01-01

Extinction-based exposure therapy is used to treat anxiety- and trauma-related disorders; however, there is the need to improve its limited efficacy in individuals with impaired fear extinction learning and to promote greater protection against return-of-fear phenomena. Here, using 129S1/SvImJ mice, which display impaired fear extinction acquisition and extinction consolidation, we revealed that persistent and context-independent rescue of deficient fear extinction in these mice was associated with enhanced expression of dopamine-related genes, such as dopamine D1 (Drd1a) and -D2 (Drd2) receptor genes in the medial prefrontal cortex (mPFC) and amygdala, but not hippocampus. Moreover, enhanced histone acetylation was observed in the promoter of the extinction-regulated Drd2 gene in the mPFC, revealing a potential gene-regulatory mechanism. Although enhancing histone acetylation, via administering the histone deacetylase (HDAC) inhibitor MS-275, does not induce fear reduction during extinction training, it promoted enduring and context-independent rescue of deficient fear extinction consolidation/retrieval once extinction learning was initiated as shown following a mild conditioning protocol. This was associated with enhanced histone acetylation in neurons of the mPFC and amygdala. Finally, as a proof-of-principle, mimicking enhanced dopaminergic signaling by L-dopa treatment rescued deficient fear extinction and co-administration of MS-275 rendered this effect enduring and context-independent. In summary, current data reveal that combining dopaminergic and epigenetic mechanisms is a promising strategy to improve exposure-based behavior therapy in extinction-impaired individuals by initiating the formation of an enduring and context-independent fear-inhibitory memory. PMID:27922638

Specific mixing facilitates the comparative quantification of phosphorylation sites with significant dysregulations

Energy Technology Data Exchange (ETDEWEB)

Liu, Jing [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Bo [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Liu, Zheyi; Dong, Mingming; Mao, Jiawei; Zhou, Ye; Chen, Jin [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Wang, Fangjun, E-mail: wangfj@dicp.ac.cn [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China); Zou, Hanfa [Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R& A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Dalian 116023 (China)

2017-01-15

Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations. - Highlights: • All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9). • The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved. • The newly expressed or disappeared proteins and protein

Differential patterns of histone acetylation in inflammatory bowel diseases

Directory of Open Access Journals (Sweden)

Adcock Ian M

2011-01-01

Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

SIMAC - A phosphoproteomic strategy for the rapid separation of mono-phosphorylated from multiply phosphorylated peptides

DEFF Research Database (Denmark)

Thingholm, Tine E; Jensen, Ole N; Robinson, Phillip J

2008-01-01

spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy - SIMAC - for sequential separation of mono-phosphorylated peptides and multiply phosphorylated peptides from...... and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a three-fold increase in recovery of multiply phosphorylated peptides....

Circulating Histones Are Major Mediators of Cardiac Injury in Patients With Sepsis.

Science.gov (United States)

Alhamdi, Yasir; Abrams, Simon T; Cheng, Zhenxing; Jing, Shengjie; Su, Dunhao; Liu, Zhiyong; Lane, Steven; Welters, Ingeborg; Wang, Guozheng; Toh, Cheng-Hock

2015-10-01

To investigate the impact of circulating histones on cardiac injury and dysfunction in a murine model and patients with sepsis. Prospective, observational clinical study with in vivo and ex vivo translational laboratory investigations. General ICU and university research laboratory. Sixty-five septic patients and 27 healthy volunteers. Twelve-week-old male C57BL/6N mice. Serial blood samples from 65 patients with sepsis were analyzed, and left ventricular function was assessed by echocardiography. Patients' sera were incubated with cultured cardiomyocytes in the presence or absence of antihistone antibody, and cellular viability was assessed. Murine sepsis was initiated by intraperitoneal Escherichia coli injection (10(8) colony-forming unit/mouse) in 12-week-old male C57BL/6N mice, and the effect of antihistone antibody (10 mg/kg) was studied. Murine blood samples were collected serially, and left ventricular function was assessed by intraventricular catheters and electrocardiography. Circulating histones and cardiac troponins in human and murine plasma were quantified. In 65 patients with sepsis, circulating histones were significantly elevated compared with healthy controls (n = 27) and linearly correlated with cardiac troponin T levels (rs = 0.650; p histone levels were significantly associated with new-onset left ventricular dysfunction (p = 0.001) and arrhythmias (p = 0.01). Left ventricular dysfunction only predicted adverse outcomes when combined with elevated histones or cardiac troponin levels. Furthermore, patients' sera directly induced histone-specific cardiomyocyte death ex vivo, which was abrogated by antihistone antibodies. In vivo studies on septic mice confirmed the cause-effect relationship between circulating histones and the development of cardiac injury, arrhythmias, and left ventricular dysfunction. Circulating histones are novel and important mediators of septic cardiomyopathy, which can potentially be utilized for prognostic and therapeutic

Phosphorylation of stress protein pp80 is related to promotion of transformation

International Nuclear Information System (INIS)

Smith, B.M.; Gindhart, T.D.; Hirano, K.; Colburn, N.H.

1986-01-01

The JB6 mouse epidermal cell system is an in vitro model of late stage promotion, and includes cell lines sensitive (P+) or resistant (P-) to phorbol ester-induced anchorage independent transformation, and transformed (T/sub x/) lines. Certain promoter-induced changes in phosphoproteins, identified by gel electrophoresis, are unique to cells of one phenotype, and occur only with specific promoters. An 80Kd protein is inversely correlated with phenotype: P- cells have a constitutively higher level (p 35 S-methionine. pp80 shares properties with the 80Kd heat stress protein: molecular weight relative abundance, and isoelectric point (4.5). Pharmacological analogs of calcium, the lanthanides, promote transformation of JB6 cells, but have no effect on phosphorylation of the 80Kd protein. If pp80 is on the promotion pathway, it is limited to a specific subset of transformation promoters

About a significance of the avian linker histone (H1) polymorphic ...

Indian Academy of Sciences (India)

60

structural disorder may specify histone H1 interaction with both DNA and partnering proteins through ... from the studies conducted on mammalian model, including the human H1 variants. However ..... Thus, the disparate layout of histone H1.

Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation, linker histone H1-induced compaction, and 30-nm fiber formation of the nucleosome array

International Nuclear Information System (INIS)

Hizume, Kohji; Nakai, Tonau; Araki, Sumiko; Prieto, Eloise; Yoshikawa, Kenichi; Takeyasu, Kunio

2009-01-01

In order to reveal the roles of histone tails in the formation of higher-order chromatin structures, we employed atomic force microscopy (AFM), and an in vitro reconstitution system to examine the properties of reconstituted chromatin composed of tail-less histones and a long DNA (106-kb plasmid) template. The tail-less nucleosomes did not aggregate at high salt concentrations or with an excess amount of core histones, in contrast with the behavior of nucleosomal arrays composed of nucleosomes containing normal, N-terminal tails. Analysis of our nucleosome distributions reveals that the attractive interaction between tail-less nucleosomes is weakened. Addition of linker histone H1 into the tail-less nucleosomal array failed to promote the formation of 30 nm chromatin fibers that are usually formed in the normal nucleosomal array. These results demonstrate that the attractive interaction between nucleosomes via histone tails plays a critical role in the formation of the uniform 30-nm chromatin fiber.

Characterization of Chromatin Structure-associated Histone Modifications in Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Chang Pyo Hong

2012-09-01

Full Text Available Chromatin structure and dynamics that are influenced by epigenetic marks, such as histone modification and DNA methylation, play a crucial role in modulating gene transcription. To understand the relationship between histone modifications and regulatory elements in breast cancer cells, we compared our chromatin immunoprecipitation sequencing (ChIP-Seq histone modification patterns for histone H3K4me1, H3K4me3, H3K9/16ac, and H3K27me3 in MCF-7 cells with publicly available formaldehyde-assisted isolation of regulatory elements (FAIRE-chip signals in human chromosomes 8, 11, and 12, identified by a method called FAIRE. Active regulatory elements defined by FAIRE were highly associated with active histone modifications, like H3K4me3 and H3K9/16ac, especially near transcription start sites. The H3K9/16ac-enriched genes that overlapped with FAIRE signals (FAIRE-H3K9/14ac were moderately correlated with gene expression levels. We also identified functional sequence motifs at H3K4me1-enriched FAIRE sites upstream of putative promoters, suggesting that regulatory elements could be associated with H3K4me1 to be regarded as distal regulatory elements. Our results might provide an insight into epigenetic regulatory mechanisms explaining the association of histone modifications with open chromatin structure in breast cancer cells.

Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

Science.gov (United States)

Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

2013-10-01

In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Subcellular distribution of histone-degrading enzyme activities from rat liver

International Nuclear Information System (INIS)

Heinrich, P.C.; Raydt, G.; Puschendorf, B.; Jusic, M.

1976-01-01

Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. (orig.) [de

Selective Biological Responses of Phagocytes and Lungs to Purified Histones.

Science.gov (United States)

Fattahi, Fatemeh; Grailer, Jamison J; Lu, Hope; Dick, Rachel S; Parlett, Michella; Zetoune, Firas S; Nuñez, Gabriel; Ward, Peter A

2017-01-01

Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1β was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4. © 2017 S. Karger AG, Basel.

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

DEFF Research Database (Denmark)

Larsen, L; Tonnesen, M; Ronn, S G

2007-01-01

B (NFkappaB) is a critical signalling molecule in inflammation and is required for expression of the gene encoding inducible NO synthase (iNOS) and of pro-apoptotic genes. NFkappaB has recently been shown to associate with chromatin-modifying enzymes histone acetyltransferases and histone...... by immunoblotting and by immunoblotting combined with electrophoretic mobility shift assay, respectively. Viability was analysed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and apoptosis by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and histone...

Histone deacetylase inhibitors in multiple myeloma

Directory of Open Access Journals (Sweden)

Sarah Deleu

2009-06-01

Full Text Available Novel drugs such as bortezomib and high dose chemotherapy combined with stem cell transplantation improved the outcome of multiple myeloma patients in the past decade. However, multiple myeloma often remains incurable due to the development of drug resistance governed by the bone marrow micro-environment. Therefore targeting new pathways to overcome this resistance is needed. Histone deacetylase (HDAC inhibitors represent a new class of anti-myeloma agents. Inhibiting HDACs results in histone hyperacetylation and alterations in chromatine structure, which, in turn, cause growth arrest differentiation and/or apoptosis in several tumor cells. Here we summarize the molecular actions of HDACi as a single agent or in combination with other drugs in different in vitro and in vivo myeloma models and in (preclinical trials.

Phosphorylation of SU(VAR3-9 by the chromosomal kinase JIL-1.

Directory of Open Access Journals (Sweden)

Joern Boeke

2010-04-01

Full Text Available The histone methyltransferase SU(VAR3-9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR3-9's activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR3-9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR3-9 N-terminus. Among several other proteins known to bind Su(VAR3-9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR3-9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR3-9 and JIL-1.

Analysis of the histone protein tail and DNA in nucleosome using molecular dynamics simulation

Science.gov (United States)

Fujimori, R.; Komatsu, Y.; Fukuda, M.; Miyakawa, T.; Morikawa, R.; Takasu, M.

2013-02-01

We study the effect of the tails of H3 and H4 histones in the nucleosomes, where DNA and histones are packed in the form of chromatin. We perform molecular dynamics simulations of the complex of DNA and histones and calculate the mean square displacement and the gyration radius of the complex of DNA and histones for the cases with tails intact and the cases with tails missing. Our results show that the H3 tails are important for the motion of the histones. We also find that the motion of one tail is affected by other tails, although the tails are distanced apart, suggesting the correlated motion in biological systems.

Real-Time Tracking of Parental Histones Reveals Their Contribution to Chromatin Integrity Following DNA Damage.

Science.gov (United States)

Adam, Salomé; Dabin, Juliette; Chevallier, Odile; Leroy, Olivier; Baldeyron, Céline; Corpet, Armelle; Lomonte, Patrick; Renaud, Olivier; Almouzni, Geneviève; Polo, Sophie E

2016-10-06

Chromatin integrity is critical for cell function and identity but is challenged by DNA damage. To understand how chromatin architecture and the information that it conveys are preserved or altered following genotoxic stress, we established a system for real-time tracking of parental histones, which characterize the pre-damage chromatin state. Focusing on histone H3 dynamics after local UVC irradiation in human cells, we demonstrate that parental histones rapidly redistribute around damaged regions by a dual mechanism combining chromatin opening and histone mobilization on chromatin. Importantly, parental histones almost entirely recover and mix with new histones in repairing chromatin. Our data further define a close coordination of parental histone dynamics with DNA repair progression through the damage sensor DDB2 (DNA damage-binding protein 2). We speculate that this mechanism may contribute to maintaining a memory of the original chromatin landscape and may help preserve epigenome stability in response to DNA damage. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

International Nuclear Information System (INIS)

Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

2009-01-01

Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

[Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

Science.gov (United States)

Fedina, A B; Gazarian, G G

1976-01-01

Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

Energy Technology Data Exchange (ETDEWEB)

Shrestha, Chandan [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Ito, Takashi [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Kawahara, Ko-ichi [Department of Biomedical Engineering, Osaka Institute of Technology, Osaka (Japan); Shrestha, Binita; Yamakuchi, Munekazu; Hashiguchi, Teruto [Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Maruyama, Ikuro, E-mail: rinken@m3.kufm.kagoshima-u.ac.jp [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan)

2013-08-09

Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.

Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

International Nuclear Information System (INIS)

Shrestha, Chandan; Ito, Takashi; Kawahara, Ko-ichi; Shrestha, Binita; Yamakuchi, Munekazu; Hashiguchi, Teruto; Maruyama, Ikuro

2013-01-01

Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis

Inhibition of histone deacetylases stimulates HBV replication independent of protein X

NARCIS (Netherlands)

van de Klundert, Maarten A. A.; Swart, Marjolein; Zaaijer, Hans L.; Kootstra, Neeltje A.

2015-01-01

Aim: HBV expresses an accessory protein called X (HBx), which supports HBV replication by increasing transcription from episomal templates. Here, we investigate whether HBx augments HBV replication by interfering with the deacetylation of HBV DNA associated histones by histone deacetylases (HDACs).

HISTONE DEACETYLASE 9 represses seedling traits in Arabidopsis thaliana dry seeds

NARCIS (Netherlands)

van Zanten, Martijn; Zöll, C.; Wang, Z.; Philipp, C.; Carles, A.; Li, Y.; Kornet, N.G.; Liu, Y.; Soppe, W.J.J.

2014-01-01

Plant life is characterized by major phase changes. We studied the role of histone deacetylase (HDAC) activity in the transition from seed to seedling in Arabidopsis. Pharmacological inhibition of HDAC stimulated germination of freshly harvested seeds. Subsequent analysis revealed that histone

Structural Basis of Histone Demethylase KDM6B Histone 3 Lysine 27 Specificity

DEFF Research Database (Denmark)

Jones, Sarah E; Olsen, Lars; Gajhede, Michael

2018-01-01

KDM subfamily 6 enzymes KDM6A and KDM6B specifically catalyze demethylation of di- and trimethylated lysine on histone 3 lysine 27 (H3K27me3/2) and play an important role in repression of developmental genes. Despite identical amino acid sequence in the immediate surroundings of H3K9me3/2 (ARKS...

Comparative proteomic analysis of histone post-translational modifications upon ischemia/reperfusion-induced retinal injury

DEFF Research Database (Denmark)

Zhao, Xiaolu; Sidoli, Simone; Wang, Leilei

2014-01-01

We present a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in rat retinas affected by ischemia and reperfusion (I/R) injury. Retinal I/R injury contributes to serious ocular diseases, which can lead to vision loss and blindness. We applied linear...... ion trap-orbitrap hybrid tandem mass spectrometry (MS/MS) to quantify 131 single histone marks and 143 combinations of multiple histone marks in noninjured and injured retinas. We observed 34 histone PTMs that exhibited significantly (p

Biochemical systems approaches for the analysis of histone modification readout.

Science.gov (United States)

Soldi, Monica; Bremang, Michael; Bonaldi, Tiziana

2014-08-01

Chromatin is the macromolecular nucleoprotein complex that governs the organization of genetic material in the nucleus of eukaryotic cells. In chromatin, DNA is packed with histone proteins into nucleosomes. Core histones are prototypes of hyper-modified proteins, being decorated by a large number of site-specific reversible and irreversible post-translational modifications (PTMs), which contribute to the maintenance and modulation of chromatin plasticity, gene activation, and a variety of other biological processes and disease states. The observations of the variety, frequency and co-occurrence of histone modifications in distinct patterns at specific genomic loci have led to the idea that hPTMs can create a molecular barcode, read by effector proteins that translate it into a specific transcriptional state, or process, on the underlying DNA. However, despite the fact that this histone-code hypothesis was proposed more than 10 years ago, the molecular details of its working mechanisms are only partially characterized. In particular, two questions deserve specific investigation: how the different modifications associate and synergize into patterns and how these PTM configurations are read and translated by multi-protein complexes into a specific functional outcome on the genome. Mass spectrometry (MS) has emerged as a versatile tool to investigate chromatin biology, useful for both identifying and validating hPTMs, and to dissect the molecular determinants of histone modification readout systems. We review here the MS techniques and the proteomics methods that have been developed to address these fundamental questions in epigenetics research, emphasizing approaches based on the proteomic dissection of distinct native chromatin regions, with a critical evaluation of their present challenges and future potential. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. Copyright © 2014 Elsevier B.V. All rights reserved.

The emerging role of histone lysine demethylases in prostate cancer

Directory of Open Access Journals (Sweden)

Crea Francesco

2012-08-01

Full Text Available Abstract Early prostate cancer (PCa is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC. Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3. Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a

Mechanisms of transcriptional repression by histone lysine methylation

DEFF Research Database (Denmark)

Hublitz, Philip; Albert, Mareike; Peters, Antoine H F M

2009-01-01

. In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences......, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly...

Characterization of Complete Histone Tail Proteoforms Using Differential Ion Mobility Spectrometry