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Sample records for acetylated histone h3k9

  1. Genome-Wide Alteration of Histone H3K9 Acetylation Pattern in Mouse Offspring Prenatally Exposed to Arsenic

    PubMed Central

    Cronican, Andrea A.; Fitz, Nicholas F.; Carter, Alexis; Saleem, Muzamil; Shiva, Sruti; Barchowsky, Aaron; Koldamova, Radosveta; Schug, Jonathan; Lefterov, Iliya

    2013-01-01

    Chronic exposure to arsenic in drinking water, especially in utero or perinatal exposure, can initiate neurological and cognitive dysfunction, as well as memory impairment. Several epidemiological studies have demonstrated cognitive and learning deficits in children with early exposure to low to moderate levels of arsenic, but pathogenic mechanisms or etiology for these deficits are poorly understood. Since in vivo studies show a role for histone acetylation in cognitive performance and memory formation, we examined if prenatal exposure to arsenic causes changes in the epigenomic landscape. We exposed C57Bl6/J mice to 100 μg/L arsenic in the drinking water starting 1 week before conception till birth and applied chromatin immunoprecipitation followed by high-throughput massive parallel sequencing (ChIP-seq) to evaluate H3K9 acetylation pattern in the offspring of exposed and control mice. Arsenic exposure during embryonic life caused global hypo-acetylation at H3K9 and changes in functional annotation with highly significant representation of Krüppel associated box (KRAB) transcription factors in brain samples from exposed pups. We also found that arsenic exposure of adult mice impaired spatial and episodic memory, as well as fear conditioning performance. This is the first study to demonstrate: a) genome wide changes in H3K9 acetylation pattern in an offspring prenatally exposed to arsenic, and b) a connection between moderate arsenic exposure and cognitive impairment in adult mice. The results also emphasize the applicability of Next Generation Sequencing methodology in studies aiming to reveal the role of environmental factors, other than dietary restriction, in developmental reprogramming through histone modifications during embryonic development. PMID:23405071

  2. DNA demethylation and histone H3K9 acetylation determine the active transcription of the NKG2D gene in human CD8+ T and NK cells

    PubMed Central

    Fernández-Sánchez, Alba; Baragaño Raneros, Aroa; Carvajal Palao, Reyes; Sanz, Ana B.; Ortiz, Alberto; Ortega, Francisco; Suárez-Álvarez, Beatriz; López-Larrea, Carlos

    2013-01-01

    The human activating receptor NKG2D is mainly expressed by NK, NKT, γδ T and CD8+ T cells and, under certain conditions, by CD4+ T cells. This receptor recognizes a diverse family of ligands (MICA, MICB and ULBPs 1–6) leading to the activation of effector cells and triggering the lysis of target cells. The NKG2D receptor-ligand system plays an important role in the immune response to infections, tumors, transplanted graft and autoantigens. Elucidation of the regulatory mechanisms of NKG2D is therefore essential for therapeutic purposes. In this study, we speculate whether epigenetic mechanisms, such as DNA methylation and histone acetylation, participate in NKG2D gene regulation in T lymphocytes and NK cells. DNA methylation in the NKG2D gene was observed in CD4+ T lymphocytes and T cell lines (Jurkat and HUT78), while this gene was unmethylated in NKG2D-positive cells (CD8+ T lymphocytes, NK cells and NKL cell line) and associated with high levels of histone H3 lysine 9 acetylation (H3K9Ac). Treatment with the histone acetyltransferase (HAT) inhibitor curcumin reduces H3K9Ac levels in the NKG2D gene, downregulates NKG2D transcription and leads to a marked reduction in the lytic capacity of NKG2D-mediated NKL cells. These findings suggest that differential NKG2D expression in the different cell subsets is regulated by epigenetic mechanisms and that its modulation by epigenetic treatments might provide a new strategy for treating several pathologies. PMID:23235109

  3. Dose- and time- effect responses of DNA methylation and histone H3K9 acetylation changes induced by traffic-related air pollution

    PubMed Central

    Ding, Rui; Jin, Yongtang; Liu, Xinneng; Ye, Huaizhuang; Zhu, Ziyi; Zhang, Yuan; Wang, Ting; Xu, Yinchun

    2017-01-01

    As an important risk factor of respiratory disorders, traffic-related air pollution (TRAP) has caused extensive concerns. Epigenetic change has been considered a link between TRAP and respiratory diseases. However, the exact effects of TRAP on epigenetic changes are still unclear. Here we investigated the dose- and time- effect responses of TRAP on DNA methylations and H3K9 acetylation (H3K9ac) in both blood and lung tissues of rats. The findings showed that every 1 μg/m3 increase of TRAP components were associated with changes in %5 mC (95% CI) in LINE-1, iNOS, p16CDKN2A, and APC ranging from −0.088% (−0.150, −0.026) to 0.102 (0.049, 0.154), as well as 0.276 (0.053, 0.498) to 0.475 (0.103, 0.848) ng/mg increase of H3K9ac. In addition, every 1 more day exposure at high level of TRAP (in tunnel) also significantly changed the levels of DNA methylation (ranging from −0.842% to 0.248%) and H3K9ac (16.033 and 15.718 ng/mg pro in PBMC and lung tissue, respectively) changes. Season and/or sex could interact with air pollutants in affecting DNA methylation and H3K9ac. The findings showed that TRAP exposure is dose- and time- dependently associated with the changes of DNA methylation and H3K9ac. PMID:28256616

  4. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast.

    PubMed

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-04-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast.

  5. Acetylated Histone H3K9 is associated with meiotic recombination hotspots, and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

    PubMed Central

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

    Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast. PMID:23382177

  6. ESET/SETDB1 gene expression and histone H3 (K9) trimethylation in Huntington's disease.

    PubMed

    Ryu, Hoon; Lee, Junghee; Hagerty, Sean W; Soh, Byoung Yul; McAlpin, Sara E; Cormier, Kerry A; Smith, Karen M; Ferrante, Robert J

    2006-12-12

    Chromatin remodeling and transcription regulation are tightly controlled under physiological conditions. It has been suggested that altered chromatin modulation and transcription dysfunction may play a role in the pathogenesis of Huntington's disease (HD). Increased histone methylation, a well established mechanism of gene silencing, results in transcriptional repression. ERG-associated protein with SET domain (ESET), a histone H3 (K9) methyltransferase, mediates histone methylation. We show that ESET expression is markedly increased in HD patients and in transgenic R6/2 HD mice. Similarly, the protein level of trimethylated histone H3 (K9) was also elevated in HD patients and in R6/2 mice. We further demonstrate that both specificity protein 1 (Sp1) and specificity protein 3 (Sp3) act as transcriptional activators of the ESET promoter in neurons and that mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic, interferes with the DNA binding of these Sp family transcription factors, suppressing basal ESET promoter activity in a dose dependent manner. The combined pharmacological treatment with mithramycin and cystamine down-regulates ESET gene expression and reduces hypertrimethylation of histone H3 (K9). This polytherapy significantly ameliorated the behavioral and neuropathological phenotype in the R6/2 mice and extended survival over 40%, well beyond any existing reported treatment in HD mice. Our data suggest that modulation of gene silencing mechanisms, through regulation of the ESET gene is important to neuronal survival and, as such, may be a promising treatment in HD patients.

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

    PubMed Central

    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

    2016-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.17903.001 PMID:27648579

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

    SciTech Connect

    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.

  9. Histone H3-K9 methyltransferase ESET is essential for early development.

    PubMed

    Dodge, Jonathan E; Kang, Yong-Kook; Beppu, Hideyuki; Lei, Hong; Li, En

    2004-03-01

    Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites. ESET, G9a, SUV39-h1, SUV39-h2, and Eu-HMTase are histone methyltransferases that catalyze H3-K9 methylation in mammalian cells. Previous studies demonstrate that the SUV39-h proteins are preferentially targeted to the pericentric heterochromatin, and mice lacking both Suv39-h genes show cytogenetic abnormalities and an increased incidence of lymphoma. G9a methylates H3-K9 in euchromatin, and G9a null embryos die at 8.5 days postcoitum (dpc). G9a null embryo stem (ES) cells show altered DNA methylation in the Prader-Willi imprinted region and ectopic expression of the Mage genes. So far, an Eu-HMTase mouse knockout has not been reported. ESET catalyzes methylation of H3-K9 and localizes mainly in euchromatin. To investigate the in vivo function of Eset, we have generated an allele that lacks the entire pre- and post-SET domains and that expresses lacZ under the endogenous regulation of the Eset gene. We found that zygotic Eset expression begins at the blastocyst stage and is ubiquitous during postimplantation mouse development, while the maternal Eset transcripts are present in oocytes and persist throughout preimplantation development. The homozygous mutations of Eset resulted in peri-implantation lethality between 3.5 and 5.5 dpc. Blastocysts null for Eset were recovered but in less than Mendelian ratios. Upon culturing, 18 of 24 Eset(-/-) blastocysts showed defective growth of the inner cell mass and, in contrast to the approximately 65% recovery of wild-type and Eset(+/-) ES cells, no Eset(-/-) ES cell lines were obtained. Global H3-K9 trimethylation and DNA methylation at IAP repeats in Eset(-/-) blastocyst outgrowths were not dramatically altered. Together, these results suggest that Eset is required for peri-implantation development and the survival of ES cells.

  10. DNA methylation and histone H3-K9 modifications contribute to MUC17 expression.

    PubMed

    Kitamoto, Sho; Yamada, Norishige; Yokoyama, Seiya; Houjou, Izumi; Higashi, Michiyo; Goto, Masamichi; Batra, Surinder K; Yonezawa, Suguru

    2011-02-01

    MUC17 glycoprotein is a membrane-associated mucin that is mainly expressed in the digestive tract. It has been suggested that MUC17 expression is correlated with the malignancy potential of pancreatic ductal adenocarcinomas (PDACs). In the present study, we provided the first report of the MUC17 gene expression through epigenetic regulation such as promoter methylation, histone modification and microRNA (miRNA) expression. Near the transcriptional start site, the DNA methylation level of MUC17-negative cancer cell lines (e.g. PANC1) was high, whereas that of MUC17-positive cells (e.g. AsPC-1) was low. Histone H3-K9 (H3-K9) modification status was also closely related to MUC17 expression. Our results indicate that DNA methylation and histone H3-K9 modification in the 5' flanking region play a critical role in MUC17 expression. Furthermore, the hypomethylation status was observed in patients with PDAC. This indicates that the hypomethylation status in the MUC17 promoter could be a novel epigenetic marker for the diagnosis of PDAC. In addition, the result of miRNA microarray analysis showed that five potential miRNA candidates existed. It is also possible that the MUC17 might be post-transcriptionally regulated by miRNA targeting to the 3'-untranslated region of its mRNA. These understandings of the epigenetic changes of MUC17 may be of importance for the diagnosis of carcinogenic risk and the prediction of outcomes for cancer patients.

  11. Histone H3K9 modifications are a local chromatin event involved in ethanol-induced neuroadaptation of the NR2B gene.

    PubMed

    Qiang, Mei; Denny, Ashley; Lieu, Mai; Carreon, Stephanie; Li, Ji

    2011-09-01

    Expression of the NMDA receptor 2B (NR2B) gene is upregulated following chronic intermittent ethanol (CIE) treatment and withdrawal, which underlies behavioral alterations in addiction. The goal of this study was to characterize the changes of histone modifications induced by CIE treatment and its subsequent removal associated to the upregulation of NR2B gene transcription. To investigate the involvement of histone acetylation in the effect of ethanol on the NR2B gene, we examined the influence of CIE on histone acetylation in the 5' regulatory region of NR2B using a qChIP assay. CIE treatment and its subsequent removal produced a remarkable and selected increase in histone H3K9 acetylation. Interestingly, the majority of the increased H3K9 acetylation occurred after ethanol removal, which was coincident with a decrease in H3K9 methylation in the same time duration. Further examination of the mechanisms of ethanol-induced alterations on the histone modifications revealed that CIE-induced acetylation of H3K9 was not due to the changes in global enzyme activities or the expression of histone acetyltransferases (HATs) and deacetylase (HDACs). Instead, we found a significant downregulation in some histone methyltransferases (HMTs) at both the global level and the local chromatin of the NR2B gene following CIE treatment. Moreover, our experiments also indicated a decrease of G9a, Suv39 h1 and HDAC1-3 in the chromatin of the NR2B gene promoter, which may be responsible for the altered H3K9 modifications. Taken together, the findings suggest a mechanism where the changes in H3K9 modifications in the local chromatin of the NR2B gene underlie alcohol-induced neuroadaptation.

  12. The tumor suppressor, parafibromin, mediates histone H3 K9 methylation for cyclin D1 repression.

    PubMed

    Yang, Yong-Jin; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2010-01-01

    Parafibromin, a component of the RNA polymerase II-associated PAF1 complex, is a tumor suppressor linked to hyperparathyroidism-jaw tumor syndrome and sporadic parathyroid carcinoma. Parafibromin induces cell cycle arrest by repressing cyclin D1 via an unknown mechanism. Here, we show that parafibromin interacts with the histone methyltransferase, SUV39H1, and functions as a transcriptional repressor. The central region (128-227 amino acids) of parafibromin is important for both the interaction with SUV39H1 and transcriptional repression. Parafibromin associated with the promoter and coding regions of cyclin D1 and was required for the recruitment of SUV39H1 and the induction of H3 K9 methylation but not H3 K4 methylation. RNA interference analysis showed that SUV39H1 was critical for cyclin D1 repression. These data suggest that parafibromin plays an unexpected role as a repressor in addition to its widely known activity associated with transcriptional activation. Parafibromin as a part of the PAF1 complex might downregulate cyclin D1 expression by integrating repressive H3 K9 methylation during transcription.

  13. H3K9 acetylation change patterns in rats after exposure to traffic-related air pollution.

    PubMed

    Ding, Rui; Jin, Yongtang; Liu, Xinneng; Zhu, Ziyi; Zhang, Yuan; Wang, Ting; Xu, Yinchun

    2016-03-01

    Traffic-related air pollution (TRAP) has been acknowledged as a potential risk factor for numerous respiratory disorders including lung cancer; however, the exact mechanisms involved are still unclear. Here we investigated the effects of TRAP exposure on the H3K9 acetylation in rats. The exposure was performed in both spring and autumn with identical study procedures. In each season, 48 healthy Wistar rats were exposed to different levels of TRAP for 4 h, 7 d, 14 d, and 28 d, respectively. H3K9 acetylation levels in both the peripheral blood mononuclear cells (PBMCs) and lung tissues were quantified. Multiple linear regression was applied to assess the influence of air pollutants on H3K9 acetylation levels. The levels of PM2.5, PM10, and NO2 in the tunnel and crossroad groups were significantly higher than in the control group. The H3K9 acetylation levels were not significantly different between spring and autumn. When spring and autumn data were analyzed together, no significant association between the TRAP and H3K9 acetylation was found in 4h exposure window. However, in the 7 d exposure window, PM2.5 and PM10 exposures were associated with changes in H3K9 acetylation ranging from 0.276 (0.053, 0.498) to 0.475 (0.103, 0.848) per 1 μg/m(3) increase in the pollutant concentration. In addition, prolonged exposure of the rats in the tunnel showed that both PM2.5 and PM10 concentrations were positively associated with H3k9 acetylation in both PBMCs and lung tissues. The findings showed that 7-d and prolonged TRAP exposure could effectively increase the H3K9 acetylation level in both PBMCs and lung tissues of rats.

  14. A jumonji (Jarid2) protein complex represses cyclin D1 expression by methylation of histone H3-K9.

    PubMed

    Shirato, Haruki; Ogawa, Satoko; Nakajima, Kuniko; Inagawa, Masayo; Kojima, Mizuyo; Tachibana, Makoto; Shinkai, Yoichi; Takeuchi, Takashi

    2009-01-09

    Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

  15. Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscape alteration in bovine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology, we analyzed histone modification (acetylation) induced by butyrate and the large-scale mapping of the epigenomic landscape of normal histone H3 and acetylated histone H3K9 and H3K27. To d...

  16. Heterochromatic genome stability requires regulators of histone H3 K9 methylation.

    PubMed

    Peng, Jamy C; Karpen, Gary H

    2009-03-01

    Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

  17. GCN5 and E2F1 stimulate nucleotide excision repair by promoting H3K9 acetylation at sites of damage

    PubMed Central

    Guo, Ruifeng; Chen, Jie; Mitchell, David L.; Johnson, David G.

    2011-01-01

    Chromatin structure is known to be a barrier to DNA repair and a large number of studies have now identified various factors that modify histones and remodel nucleosomes to facilitate repair. In response to ultraviolet (UV) radiation several histones are acetylated and this enhances the repair of DNA photoproducts by the nucleotide excision repair (NER) pathway. However, the molecular mechanism by which UV radiation induces histone acetylation to allow for efficient NER is not completely understood. We recently discovered that the E2F1 transcription factor accumulates at sites of UV-induced DNA damage and directly stimulates NER through a non-transcriptional mechanism. Here we demonstrate that E2F1 associates with the GCN5 acetyltransferase in response to UV radiation and recruits GCN5 to sites of damage. UV radiation induces the acetylation of histone H3 lysine 9 (H3K9) and this requires both GCN5 and E2F1. Moreover, as previously observed for E2F1, knock down of GCN5 results in impaired recruitment of NER factors to sites of damage and inefficient DNA repair. These findings demonstrate a direct role for GCN5 and E2F1 in NER involving H3K9 acetylation and increased accessibility to the NER machinery. PMID:20972224

  18. The Methyltransferase Activity of Clr4Suv39h triggers RNAi Independently of Histone H3K9 Methylation

    PubMed Central

    Gerace, Erica L.; Halic, Mario; Moazed, Danesh

    2010-01-01

    Summary In fission yeast, pericentromeric dg and dh repeats are transcribed and give rise to small interfering RNAs (siRNAs) by a mechanism that depends on the Clr4suv39h histone H3 lysine 9 (H3K9) methyltransferase. Here we show that Clr4 activity promotes the assembly of a tripartite complex composed of the Clr4-containing CLRC complex and complexes involved in siRNA generation. However, unlike dh siRNAs, dg siRNAs accumulate to near wild-type levels in cells with H3K9 substitutions that cannot be methylated. Thus, Clr4 activity controls siRNA amplification from the different repeat regions by different mechanisms, H3K9 methylation-dependent versus -independent. Furthermore, artificial tethering of Rik1, a core subunit of the CLRC complex, to a euchromatic RNA mediates RNAi-dependent silencing that partially bypasses the requirement for other CLRC subunits. These findings establish Rik1 as a key link between CLRC and RNAi and reveal distinct centromeric siRNA amplification mechanisms that depend on the Clr4 methyltransferase activity. PMID:20705239

  19. Monoallele deletion of CBP leads to pericentromeric heterochromatin condensation through ESET expression and histone H3 (K9) methylation.

    PubMed

    Lee, Junghee; Hagerty, Sean; Cormier, Kerry A; Kim, Jinho; Kung, Andrew L; Ferrante, Robert J; Ryu, Hoon

    2008-06-15

    Chromatin remodeling is tightly controlled under physiological conditions. Alterations in chromatin structure are involved in the pathogenesis of neuronal systems. We found that the monoallelic deletion of CREB binding protein (CBP) results in the induction of ERG-associated protein with SET domain (ESET) and increases trimethylation of histone H3 (K9) and condensation of pericentromeric heterochromatin structure in neurons. Nested deletion and mutational analysis of the ESET promoter further demonstrated that the Ets-2 transcription factor regulates transcriptional activity of the ESET gene. In CBP+/- mice, Ets-2 occupancy in the ESET promoter DNA was markedly elevated. Our results suggest that CBP is a transcriptional repressor of ESET gene expression by limiting Ets-2 transcriptional activity, while CBP siRNA enhances basal and Ets-2-dependent ESET transcriptional activity. Altered expression of the ESET gene and hypertrimethylation of H3 (K9) correlate with striatal neuron atrophy and dysfunction in CBP+/- mice. These results establish an alternative pathway that loss of CBP leads to the pericentric heterochromatin condensation through ESET expression and trimethylation of H3 (K9).

  20. Involvement of histone methyltransferase GLP in HIV-1 latency through catalysis of H3K9 dimethylation

    SciTech Connect

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang

    2013-06-05

    Understanding the mechanism of HIV-1 latency is crucial to eradication of the viral reservoir in HIV-1-infected individuals. However, the role of histone methyltransferase (HMT) G9a-like protein (GLP) in HIV-1 latency is still unclear. In the present work, we established four clonal cell lines containing HIV-1 vector. We found that the integration sites of most clonal cell lines favored active gene regions. However, we also observed hypomethylation of CpG of HIV 5′LTR in all four clonal cell lines. Additionally, 5′-deoxy-5′-methylthioadenosine (MTA), a broad-spectrum histone methyltransferase inhibitor, was used to examine the role of histone methylation in HIV-1 latency. MTA was found to decrease the level of H3K9 dimethylation, causing reactivation of latent HIV-1 in C11 cells. GLP knockdown by small interfering RNA clearly induced HIV-1 LTR expression. Results suggest that GLP may play a significant role in the maintenance of HIV-1 latency by catalyzing dimethylation of H3K9. - Highlights: ► We have established an in vitro model of HIV-1 latency. ► The integration sites of most clonal cell lines favor in active gene regions. ► Hypomethylation occurs in CpG islands of HIV 5′LTR in all four clonal cell lines. ► MTA can reactivate latent HIV-1 by decreasing the level of H3K9 me2 in C11 cells. ► HMT GLP may play a significant role in the maintenance of HIV-1 latency.

  1. Ubiquitination of Lysine 867 of the Human SETDB1 Protein Upregulates Its Histone H3 Lysine 9 (H3K9) Methyltransferase Activity

    PubMed Central

    Ishimoto, Kenji; Kawamata, Natsuko; Uchihara, Yoshie; Okubo, Moeka; Fujimoto, Reiko; Gotoh, Eiko; Kakinouchi, Keisuke; Mizohata, Eiichi; Hino, Nobumasa; Okada, Yoshiaki; Mochizuki, Yasuhiro; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Inoue, Tsuyoshi; Tachibana, Keisuke; Doi, Takefumi

    2016-01-01

    Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin peptidase inhibitor, clade E, member 1 (SERPINE1) by methylating H3K9. These results suggest that the ubiquitination of SETDB1 at lysine 867 controls the expression of its target gene by activating its H3K9 methyltransferase activity. PMID:27798683

  2. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9.

    PubMed

    Tachibana, Makoto; Ueda, Jun; Fukuda, Mikiko; Takeda, Naoki; Ohta, Tsutomu; Iwanari, Hiroko; Sakihama, Toshiko; Kodama, Tatsuhiko; Hamakubo, Takao; Shinkai, Yoichi

    2005-04-01

    Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes.

  3. The histone H3K9 methylation and RNAi pathways regulate normalnucleolar and repeated DNA organization by inhibiting formation ofextrachromosomal DNAs

    SciTech Connect

    Peng, Jamy C.; Karpen, Gary H.

    2006-06-15

    In order to identify regulators of nuclear organization, Drosophila mutants in the Su(var)3-9 histone H3K9 methyltransferase, RNAi pathway components, and other regulators of heterochromatin-mediated gene silencing were examined for altered nucleoli and positioning of repeated DNAs. Animals lacking components of the H3K9 methylation and RNAi pathways contained disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared to wild type. We also observed a substantial increase in extrachromosomal repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 (Lig4), and ecc DNA formation is not induced by removal of cohesin. We conclude that H3K9 methylation of rDNA and satellites, maintained by Su(var)3-9, HP1, and the RNAi pathway, is necessary for the structural stability of repeated DNAs, which is mediated through suppression of non-homologous end joining (NHEJ). These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.

  4. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome

    PubMed Central

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency. PMID:27907109

  5. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

    PubMed

    Zhang, Haoyue; Sun, Linlin; Wang, Kun; Wu, Di; Trappio, Mason; Witting, Celeste; Cao, Kan

    2016-01-01

    Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

  6. Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the Nurses' Health Study.

    PubMed

    Healey, Megan A; Hu, Rong; Beck, Andrew H; Collins, Laura C; Schnitt, Stuart J; Tamimi, Rulla M; Hazra, Aditi

    2014-10-01

    Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

  7. Early postnatal feed restriction reduces liver connective tissue levels and affects H3K9 acetylation state of regulated genes associated with protein metabolism in low birth weight pigs.

    PubMed

    Nebendahl, Constance; Görs, Solvig; Albrecht, Elke; Krüger, Ricarda; Martens, Karen; Giller, Katrin; Hammon, Harald M; Rimbach, Gerald; Metges, Cornelia C

    2016-03-01

    Intrauterine growth retardation is associated with metabolic consequences in adulthood. Since our previous data indicate birth weight-dependent effects of feed restriction (R) on protein degradation processes in the liver, it should be investigated whether effects on connective tissue turnover are obvious and could be explained by global changes of histone H3K9me3 and H3K9ac states in regulated genes. For this purpose, female littermate pigs with low (U) or normal (N) birth weight were subjected to 3-week R (60% of ad libitum fed controls) with subsequent refeeding (REF) for further 5 weeks. The 3-week R-period induced a significant reduction of connective tissue area by 43% in the liver of U animals at 98 d of age, which was not found in age-matched N animals. Of note, after REF at 131 d of age, in previously feed-restricted U animals (UR), the percentage of mean connective tissue was only 53% of ad libitum fed controls (UK), indicating a persistent effect. In U animals, R induced H3K9 acetylation of regulated genes (e.g. XBP1, ERLEC1, GALNT2, PTRH2), which were inter alia associated with protein metabolism. In contrast, REF was mostly accompanied by deacetylation in U and N animals. Thus, our epigenetic data may give a first explanation for the observed birth weight-dependent differences in this connective tissue phenotype.

  8. Study of methyl transferase (G9aMT) and methylated histone (H3-K9) expressions in unexplained recurrent spontaneous abortion (URSA) and normal early pregnancy.

    PubMed

    Fatima, Nishat; Ahmed, S H; Salhan, Sudha; Rehman, S M F; Kaur, Jatinder; Owais, M; Chauhan, Shyam S

    2011-11-01

    We investigated the expression of methyl transferase G9a and methylated histone H3-K9 in fresh human decidual/endometrial tissue of 12 normal early pregnancies and 15 unexplained recurrent spontaneous abortions (URSA). The samples were obtained through dilatation and curettage and collected as per strict inclusion-exclusion criteria. The tissue was subjected to immunohistochemical analysis (IHC), western blotting (WB) and RT-PCR analysis. The results demonstrated methyl transferase G9a to have a lower expression in abortions when compared with that in normal pregnancy (P < 0.05). The sensitivity of RT-PCR, IHC and WB were respectively 66.67, 75 and 71.43%, while specificity of the same were 66.67, 60 and 78.92%, respectively. Methylated histone H3-K9 was significantly lower (P < 0.0001) in URSA tissues than in controls. This study suggests that methylation may cause URSA and indicates the need for further work to explore the role of methylation in URSA and its possible prevention through locally acting methylating/demethylating agents.

  9. Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing.

    PubMed

    Schotta, Gunnar; Ebert, Anja; Krauss, Veiko; Fischer, Andreas; Hoffmann, Jan; Rea, Stephen; Jenuwein, Thomas; Dorn, Rainer; Reuter, Gunter

    2002-03-01

    Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3-9 is the major heterochromatin-specific HMTase in Drosophila. SU (VAR)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3-7. Notably, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3-9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of HP1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3-9 HMTase in heterochromatin-induced gene silencing in Drosophila.

  10. Genome-wide mapping of histone H3K9me2 in acute myeloid leukemia reveals large chromosomal domains associated with massive gene silencing and sites of genome instability

    PubMed Central

    Popova, Evgenya Y.; Keasey, Nikki; Loughran, Thomas P.; Claxton, David F.

    2017-01-01

    A facultative heterochromatin mark, histone H3 lysine 9 dimethylation (H3K9me2), which is mediated by histone methyltransferases G9a/GLP (EHMT2/1), undergoes dramatic rearrangements during myeloid cell differentiation as observed by chromatin imaging. To determine whether these structural transitions also involve genomic repositioning of H3K9me2, we used ChIP-sequencing to map genome-wide topography of H3K9me2 in normal human granulocytes, normal CD34+ hematopoietic progenitors, primary myeloblasts from acute myeloid leukemia (AML) patients, and a model leukemia cell line K562. We observe that H3K9me2 naturally repositions from the previously designated “repressed” chromatin state in hematopoietic progenitors to predominant association with heterochromatin regions in granulocytes. In contrast, AML cells accumulate H3K9me2 on previously undefined large (> 100 Kb) genomic blocks that are enriched with AML-specific single nucleotide variants, sites of chromosomal translocations, and genes downregulated in AML. Specifically, the AML-specific H3K9me2 blocks are enriched with genes regulated by the proto-oncogene ERG that promotes stem cell characteristics. The AML-enriched H3K9me2 blocks (in contrast to the heterochromatin-associated H3K9me2 blocks enriched in granulocytes) are reduced by pharmacological inhibition of the histone methyltransferase G9a/GLP in K562 cells concomitantly with transcriptional activation of ERG and ETS1 oncogenes. Our data suggest that G9a/GLP mediate formation of transient H3K9me2 blocks that are preserved in AML myeloblasts and may lead to an increased rate of AML-specific mutagenesis and chromosomal translocations. PMID:28301528

  11. Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

    PubMed Central

    Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

    2015-01-01

    Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

  12. Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

    PubMed

    Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

    2015-01-01

    Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

  13. The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

    PubMed Central

    Congdon, Lauren M.; Sims, Jennifer K.; Tuzon, Creighton T.; Rice, Judd C.

    2014-01-01

    PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function. PMID:24423864

  14. Selective Interactions between Vertebrate Polycomb Homologs and the SUV39H1 Histone Lysine Methyltransferase Suggest that Histone H3-K9 Methylation Contributes to Chromosomal Targeting of Polycomb Group Proteins

    PubMed Central

    Sewalt, Richard G. A. B.; Lachner, Monika; Vargas, Mark; Hamer, Karien M.; den Blaauwen, Jan L.; Hendrix, Thijs; Melcher, Martin; Schweizer, Dieter; Jenuwein, Thomas; Otte, Arie P.

    2002-01-01

    Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures. PMID:12101246

  15. Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins.

    PubMed

    Sewalt, Richard G A B; Lachner, Monika; Vargas, Mark; Hamer, Karien M; den Blaauwen, Jan L; Hendrix, Thijs; Melcher, Martin; Schweizer, Dieter; Jenuwein, Thomas; Otte, Arie P

    2002-08-01

    Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.

  16. 5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

    PubMed

    Wozniak, R J; Klimecki, W T; Lau, S S; Feinstein, Y; Futscher, B W

    2007-01-04

    The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2'-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

  17. Histone acetylation in heterochromatin assembly

    PubMed Central

    Kim, Jeong-Hoon; Workman, Jerry L.

    2010-01-01

    Histone acetylation is generally considered a mark involved in activating gene expression by making chromatin structures less compact. In the April 1, 2010, issue of Genes & Development, Xhemalce and Kouzarides (pp. 647–652) demonstrate that the acetylation of histone H3 at Lys 4 (H3K4) plays a role in the formation of repressive heterochromatin in Schizosaccharomyces pombe. H3K4 acetylation mediates a switch of chromodomain proteins associated with methylated H3K9 during heterochromatin assembly. PMID:20395362

  18. Structural insight into coordinated recognition of trimethylated histone H3 lysine 9 (H3K9me3) by the plant homeodomain (PHD) and tandem tudor domain (TTD) of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) protein.

    PubMed

    Cheng, Jingdong; Yang, Yi; Fang, Jian; Xiao, Jianxiong; Zhu, Tingting; Chen, Fei; Wang, Ping; Li, Ze; Yang, Huirong; Xu, Yanhui

    2013-01-11

    UHRF1 is an important epigenetic regulator connecting DNA methylation and histone methylations. UHRF1 is required for maintenance of DNA methylation through recruiting DNMT1 to DNA replication forks. Recent studies have shown that the plant homeodomain (PHD) of UHRF1 recognizes the N terminus of unmodified histone H3, and the interaction is inhibited by methylation of H3R2, whereas the tandem tudor domain (TTD) of UHRF1 recognizes trimethylated histone H3 lysine 9 (H3K9me3). However, how the two domains of UHRF1 coordinately recognize histone methylations remains elusive. In this report, we identified that PHD largely enhances the interaction between TTD and H3K9me3. We present the crystal structure of UHRF1 containing both TTD and PHD (TTD-PHD) in complex with H3K9m3 peptide at 3.0 Å resolution. The structure shows that TTD-PHD binds to the H3K9me3 peptide with 1:1 stoichiometry with the two domains connected by the H3K9me3 peptide and a linker region. The TTD interacts with residues Arg-8 and trimethylated Lys-9, and the PHD interacts with residues Ala-1, Arg-2, and Lys-4 of the H3K9me3 peptide. The biochemical experiments indicate that PHD-mediated recognition of unmodified H3 is independent of the TTD, whereas TTD-mediated recognition of H3K9me3 PHD. Thus, both TTD and PHD are essential for specific recognition of H3K9me3 by UHRF1. Interestingly, the H3K9me3 peptide induces conformational changes of TTD-PHD, which do not affect the autoubiquitination activity or hemimethylated DNA binding affinity of UHRF1 in vitro. Taken together, our studies provide structural insight into the coordinated recognition of H3K9me3 by the TTD and PHD of UHRF1.

  19. GATA-1 Inhibits PU.1 Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia

    PubMed Central

    Burda, Pavel; Vargova, Jarmila; Curik, Nikola; Salek, Cyril; Papadopoulos, Giorgio Lucio; Strouboulis, John; Stopka, Tomas

    2016-01-01

    GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy. PMID:27010793

  20. Direct interrogation of the role of H3K9 in metazoan heterochromatin function

    PubMed Central

    Penke, Taylor J.R.; McKay, Daniel J.; Strahl, Brian D.; Matera, A. Gregory; Duronio, Robert J.

    2016-01-01

    A defining feature of heterochromatin is methylation of Lys9 of histone H3 (H3K9me), a binding site for heterochromatin protein 1 (HP1). Although H3K9 methyltransferases and HP1 are necessary for proper heterochromatin structure, the specific contribution of H3K9 to heterochromatin function and animal development is unknown. Using our recently developed platform to engineer histone genes in Drosophila, we generated H3K9R mutant flies, separating the functions of H3K9 and nonhistone substrates of H3K9 methyltransferases. Nucleosome occupancy and HP1a binding at pericentromeric heterochromatin are markedly decreased in H3K9R mutants. Despite these changes in chromosome architecture, a small percentage of H3K9R mutants complete development. Consistent with this result, expression of most protein-coding genes, including those within heterochromatin, is similar between H3K9R and controls. In contrast, H3K9R mutants exhibit increased open chromatin and transcription from piRNA clusters and transposons, resulting in transposon mobilization. Hence, transposon silencing is a major developmental function of H3K9. PMID:27566777

  1. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila

    PubMed Central

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S.; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2014-01-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing. PMID:24598257

  2. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila.

    PubMed

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2014-05-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing.

  3. A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

    PubMed

    Hong, Eun-Jin Erica; Villén, Judit; Gerace, Erica L; Gygi, Steven P; Moazed, Danesh

    2005-01-01

    The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

  4. KDM4C, a H3K9me3 Histone Demethylase, is Involved in the Maintenance of Human ESCC-Initiating Cells by Epigenetically Enhancing SOX2 Expression.

    PubMed

    Yuan, Xiang; Kong, Jinyu; Ma, Zhikun; Li, Na; Jia, Ruinuo; Liu, Yiwen; Zhou, Fuyou; Zhan, Qimin; Liu, Gang; Gao, Shegan

    2016-10-01

    Our studies investigating the existence of tumor-initiating cell (TIC) populations in human esophageal squamous cell carcinoma (ESCC) had identified a subpopulation of cells isolated from ESCC patient-derived tumor specimens marked by an ALDH(bri+) phenotype bear stem cell-like features. Importantly, KDM4C, a histone demethylase was enhanced in ALDH(bri+) subpopulation, suggesting that strategies interfering with KDM4C may be able to target these putative TICs. In the present study, by genetic and chemical means, we demonstrated that, KDM4C blockade selectively decreased the ESCC ALDH(bri+) TICs population in vitro and specifically targeted the TICs in ALDH(bri+)-derived xenograft, retarding engraftment. Subsequent studies of the KDM4C functional network identified a subset of pluripotency-associated genes (PAGs) and aldehyde dehydrogenase family members to be preferentially down-regulated in KDM4C inhibited ALDH(bri+) TICs. We further supported a model in which KDM4C maintains permissive histone modifications with a low level of H3K9 methylation at the promoters of several PAGs. Moreover, ectopic expression of SOX2 restored KDM4C inhibition-dependent ALDH(bri+) TIC properties. We further confirmed these findings by showing that the cytoplasmic and nuclear KDM4C staining increased with adverse pathologic phenotypes and poor patient survival. Such staining pattern of intracellular KDM4C appeared to overlap with the expression of SOX2 and ALDH1. Collectively, our findings provided the insights into the development of novel therapeutic strategies based on the inhibition of KDM4C pathway for the eliminating of ESCC TIC compartment.

  5. Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation, rapid target gene repression, and mouse viability

    PubMed Central

    Liu, Nan; Zhang, Zhuqiang; Jiang, Yonghua; Meng, Lingjun; Xiong, Jun; Zhao, Zuodong; Zhou, Xiaohua; Li, Jia; Li, Hong; Zheng, Yong; Chen, She; Cai, Tao; Gao, Shaorong

    2015-01-01

    GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability. PMID:25637356

  6. Structural Biology of Human H3K9 Methyltransferases

    SciTech Connect

    Wu, H.; Min, J; Lunin, V; Antoshenko, T; Dombrovsk, L; Zeng, H; Allali-Hassani, A; Campagna-Slater, V; Vedadi, M; et. al.

    2010-01-01

    SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. Post-translational modifications of histone proteins regulate chromatin compaction, mediate epigenetic regulation of transcription, and control cellular differentiation in health and disease. Methylation of histone tails is one of the fundamental events of epigenetic signaling. Tri-methylation of lysine 9 of histone 3 (H3K9) mediates chromatin recruitment of HP1, heterochromatin condensation and gene silencing. Similarly, methylation of H3K27 and H4K20 are associated with a repressed state of chromatin, whereas expressed genes are methylated at H3K4, H3K36 and H3K79. Histone methyltransferases are divided into protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs). HKMTs catalyze the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) to a substrate lysine and, with the exception of DOT1L, are all organized around a canonical SET domain. The structures of a number of HKMTs have been reported, including ternary complexes of human orthologs with co-factor and substrate peptides (SETD7-H3K4, SETD8-H4K20 and MLL1-H3K4), as well

  7. Distinct patterns of histone methylation and acetylation in human interphase nuclei.

    PubMed

    Skalníková, M; Bártová, E; Ulman, V; Matula, P; Svoboda, D; Harnicarová, A; Kozubek, M; Kozubek, S

    2007-01-01

    To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.

  8. Functional dynamics of H3K9 methylation during meiotic prophase progression.

    PubMed

    Tachibana, Makoto; Nozaki, Masami; Takeda, Naoki; Shinkai, Yoichi

    2007-07-25

    Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ-lineage displayed sterility due to a drastic loss of mature gametes. G9a-deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono- and di-methylation of H3K9 (H3K9me1 and 2) in G9a-deficient germ cells were significantly reduced and G9a-regulated genes were overexpressed during meiosis, suggesting that G9a-mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex-differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis.

  9. The level and distribution pattern of HP1β in the embryonic brain correspond to those of H3K9me1/me2 but not of H3K9me3.

    PubMed

    Bártová, Eva; Večeřa, Josef; Krejčí, Jana; Legartová, Soňa; Pacherník, Jiří; Kozubek, Stanislav

    2016-04-01

    We studied the histone signature of embryonic and adult brains to strengthen existing evidence of the importance of the histone code in mouse brain development. We analyzed the levels and distribution patterns of H3K9me1, H3K9me2, H3K9me3, and HP1β in both embryonic and adult brains. Western blotting showed that during mouse brain development, the levels of H3K9me1, H3K9me2, and HP1β exhibited almost identical trends, with the highest protein levels occurring at E15 stage. These trends differed from the relatively stable level of H3K9me3 at developmental stages E8, E13, E15, and E18. Compared with embryonic brains, adult brains were characterized by very low levels of H3K9me1/me2/me3 and HP1β. Manipulation of the embryonic epigenome through histone deacetylase inhibitor treatment did not affect the distribution patterns of the studied histone markers in embryonic ventricular ependyma. Similarly, Hdac3 depletion in adult animals had no effect on histone methylation in the adult hippocampus. Our results indicate that the distribution of HP1β in the embryonic mouse brain is related to that of H3K9me1/me2 but not to that of H3K9me3. The unique status of H3K9me3 in the brain was confirmed by its pronounced accumulation in the granular layer of the adult olfactory bulb. Moreover, among the studied proteins, H3K9me3 was the only posttranslational histone modification that was highly abundant at clusters of centromeric heterochromatin, called chromocenters. When we focused on the hippocampus, we found this region to be rich in H3K9me1 and H3K9me3, whereas H3K9me2 and HP1β were present at a very low level or even absent in the hippocampal blade. Taken together, these results revealed differences in the epigenome of the embryonic and adult mouse brain and showed that the adult hippocampus, the granular layer of the adult olfactory bulb, and the ventricular ependyma of the embryonic brain are colonized by specific epigenetic marks.

  10. Enrichment of H3K9me2 on Unsynapsed Chromatin in Caenorhabditis elegans Does Not Target de Novo Sites

    PubMed Central

    Guo, Yiqing; Yang, Bing; Li, Yini; Maine, Eleanor M.

    2015-01-01

    Many organisms alter the chromatin state of unsynapsed chromosomes during meiotic prophase, a phenomenon hypothesized to function in maintaining germline integrity. In Caenorhabditis elegans, histone H3 lysine 9 dimethylation (H3K9me2) is detected by immunolabeling as enriched on unsynapsed meiotic chromosomes. Loss of the SET domain protein, MET-2, greatly reduces H3K9me2 abundance and results in germline mortality. Here, we used him-8 mutations to disable X chromosome synapsis and performed a combination of molecular assays to map the sites of H3K9me2 accumulation, evaluate H3K9me2 abundance in germline vs. whole animals, and evaluate the impact of H3K9me2 loss on the germline transcriptome. Our data indicate that H3K9me2 is elevated broadly across the X chromosome and at defined X chromosomal sites in him-8 adults compared with controls. H3K9me2 levels are also elevated to a lesser degree at sites on synapsed chromosomes in him-8 adults compared with controls. These results suggest that MET-2 activity is elevated in him-8 mutants generally as well as targeted preferentially to the unsynapsed X. Abundance of H3K9me2 and other histone H3 modifications is low in germline chromatin compared with whole animals, which may facilitate genome reprogramming during gametogenesis. Loss of H3K9me2 has a subtle impact on the him-8 germline transcriptome, suggesting H3K9me2 may not be a major regulator of developmental gene expression in C. elegans. We hypothesize H3K9me2 may have a structural function critical for germline immortality, and a greater abundance of these marks may be required when a chromosome does not synapse. PMID:26156747

  11. Enrichment of H3K9me2 on Unsynapsed Chromatin in Caenorhabditis elegans Does Not Target de Novo Sites.

    PubMed

    Guo, Yiqing; Yang, Bing; Li, Yini; Xu, Xia; Maine, Eleanor M

    2015-07-08

    Many organisms alter the chromatin state of unsynapsed chromosomes during meiotic prophase, a phenomenon hypothesized to function in maintaining germline integrity. In Caenorhabditis elegans, histone H3 lysine 9 dimethylation (H3K9me2) is detected by immunolabeling as enriched on unsynapsed meiotic chromosomes. Loss of the SET domain protein, MET-2, greatly reduces H3K9me2 abundance and results in germline mortality. Here, we used him-8 mutations to disable X chromosome synapsis and performed a combination of molecular assays to map the sites of H3K9me2 accumulation, evaluate H3K9me2 abundance in germline vs. whole animals, and evaluate the impact of H3K9me2 loss on the germline transcriptome. Our data indicate that H3K9me2 is elevated broadly across the X chromosome and at defined X chromosomal sites in him-8 adults compared with controls. H3K9me2 levels are also elevated to a lesser degree at sites on synapsed chromosomes in him-8 adults compared with controls. These results suggest that MET-2 activity is elevated in him-8 mutants generally as well as targeted preferentially to the unsynapsed X. Abundance of H3K9me2 and other histone H3 modifications is low in germline chromatin compared with whole animals, which may facilitate genome reprogramming during gametogenesis. Loss of H3K9me2 has a subtle impact on the him-8 germline transcriptome, suggesting H3K9me2 may not be a major regulator of developmental gene expression in C. elegans. We hypothesize H3K9me2 may have a structural function critical for germline immortality, and a greater abundance of these marks may be required when a chromosome does not synapse.

  12. Inhibition of H3K9 methyltransferase G9a ameliorates methylglyoxal-induced peritoneal fibrosis

    PubMed Central

    Maeda, Kazuya; Doi, Shigehiro; Nakashima, Ayumu; Nagai, Takuo; Irifuku, Taisuke; Ueno, Toshinori; Masaki, Takao

    2017-01-01

    Activity of H3K9 histone methyltransferase G9a is reportedly induced by transforming growth factor-β1 (TGF-β1) and plays an important role in the progression of cancer and fibrosis. In this study, we investigated whether inhibition of G9a-mediated H3K9 methylation attenuates peritoneal fibrosis in mice and human peritoneal mesothelial cells (HPMCs). Nonadherent cells of peritoneal dialysis (PD) patients were isolated from PD effluent to examine expression of G9a. Peritoneal fibrosis was induced by peritoneal injection of methylglyoxal (MGO) in male C57/B6 mice for 3 weeks. BIX01294, a G9a inhibitor, was administered by subcutaneous injection. Effects of BIX01294 on MGO-induced pathological and functional changes in mice were evaluated by immunohistochemistry and a peritoneal equilibration test. HPMCs were isolated from human omentum, and the inhibitory effect of BIX01294 on TGF-β1-induced fibrotic changes was investigated in the HPMCs by western blotting. G9a was upregulated in nonadherent cells of human PD effluent, the peritoneum of MGO-injected mice, and TGF-β1-stimulated HPMCs. BIX01294 significantly reduced the submesothelial zone thickness and cell density in MGO-injected mice. Immunohistochemical staining revealed that BIX01294 treatment decreased not only mono-methylation of H3K9 (H3K9me1), but also the number of mesenchymal cells, accumulation of collagen, and infiltration of monocytes. In addition to the pathological changes, BIX01294 reduced the level of TGF-β1 in peritoneal fluid and improved peritoneal functions. Furthermore, BIX01294 inhibited TGF-β1-induced fibrotic changes along with suppression of H3K9me1 in HPMCs. Therefore, inhibition of H3K9 methyltransferase G9a suppresses peritoneal fibrosis through a reduction of H3K9me1. PMID:28278257

  13. Inhibition of H3K9 methyltransferase G9a ameliorates methylglyoxal-induced peritoneal fibrosis.

    PubMed

    Maeda, Kazuya; Doi, Shigehiro; Nakashima, Ayumu; Nagai, Takuo; Irifuku, Taisuke; Ueno, Toshinori; Masaki, Takao

    2017-01-01

    Activity of H3K9 histone methyltransferase G9a is reportedly induced by transforming growth factor-β1 (TGF-β1) and plays an important role in the progression of cancer and fibrosis. In this study, we investigated whether inhibition of G9a-mediated H3K9 methylation attenuates peritoneal fibrosis in mice and human peritoneal mesothelial cells (HPMCs). Nonadherent cells of peritoneal dialysis (PD) patients were isolated from PD effluent to examine expression of G9a. Peritoneal fibrosis was induced by peritoneal injection of methylglyoxal (MGO) in male C57/B6 mice for 3 weeks. BIX01294, a G9a inhibitor, was administered by subcutaneous injection. Effects of BIX01294 on MGO-induced pathological and functional changes in mice were evaluated by immunohistochemistry and a peritoneal equilibration test. HPMCs were isolated from human omentum, and the inhibitory effect of BIX01294 on TGF-β1-induced fibrotic changes was investigated in the HPMCs by western blotting. G9a was upregulated in nonadherent cells of human PD effluent, the peritoneum of MGO-injected mice, and TGF-β1-stimulated HPMCs. BIX01294 significantly reduced the submesothelial zone thickness and cell density in MGO-injected mice. Immunohistochemical staining revealed that BIX01294 treatment decreased not only mono-methylation of H3K9 (H3K9me1), but also the number of mesenchymal cells, accumulation of collagen, and infiltration of monocytes. In addition to the pathological changes, BIX01294 reduced the level of TGF-β1 in peritoneal fluid and improved peritoneal functions. Furthermore, BIX01294 inhibited TGF-β1-induced fibrotic changes along with suppression of H3K9me1 in HPMCs. Therefore, inhibition of H3K9 methyltransferase G9a suppresses peritoneal fibrosis through a reduction of H3K9me1.

  14. In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration.

    PubMed

    Suzuki, Miyuki; Takagi, Chiyo; Miura, Shinichirou; Sakane, Yuto; Suzuki, Makoto; Sakuma, Tetsushi; Sakamoto, Naoaki; Endo, Tetsuya; Kamei, Yasuhiro; Sato, Yuko; Kimura, Hiroshi; Yamamoto, Takashi; Ueno, Naoto; Suzuki, Ken-ichi T

    2016-04-01

    Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification-specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac-Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac-Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac-Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.

  15. H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

    PubMed

    Matsumura, Yoshihiro; Nakaki, Ryo; Inagaki, Takeshi; Yoshida, Ayano; Kano, Yuka; Kimura, Hiroshi; Tanaka, Toshiya; Tsutsumi, Shuichi; Nakao, Mitsuyoshi; Doi, Takefumi; Fukami, Kiyoko; Osborne, Timothy F; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Sakai, Juro

    2015-11-19

    Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

  16. Histone acetylation in neurodevelopment.

    PubMed

    Contestabile, Antonio; Sintoni, Silvia

    2013-01-01

    Post-translational modification of histones is a primary mechanism through which epigenetic regulation of DNA transcription does occur. Among these modifications, regulation of histone acetylation state is an important tool to influence gene expression. Epigenetic regulation of neurodevelopment contributes to the structural and functional shaping of the brain during neurogenesis and continues to impact on neural plasticity lifelong. Alterations of these mechanisms during neurodevelopment may result in later occurrence of neuropsychatric disorders. The present paper reviews and discusses available data on histone modifications, in particular histone acetylation, in neurogenesis considering results obtained in culture systems of neural progenitors as well as in in vivo studies. Possible teratogenic effects of altered histone acetylation state during development are also considered. The use during pregnancy of drugs such as valproic acid, which acts as a histone deacetylase inhibitor, may result during postnatal development in autistic-like symptoms. The effect of gestational administration of the drug has been, therefore, tested on adult hippocampal neurogenesis in animals showing behavioral impairment as a consequence of the drug administration at a specific stage of pregnancy. These experimental results show that adult neurogenesis in the hippocampal dentate gyrus is not quantitatively altered by gestational valproic acid administration. Future steps and goals of research on the role and mechanisms of histone acetylation in neurodevelopment are briefly discussed.

  17. UHRF2 decreases H3K9ac expression by interacting with it through the PHD and SRA/YDG domain in HepG2 hepatocellular carcinoma cells

    PubMed Central

    Zhang, Ting; Zhao, Linglin; Zeng, Shengyuan; Bai, Lu; Chen, Junxia; Zhang, Zheng; Wang, Yalan; Duan, Changzhu

    2017-01-01

    Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) is a multi-domain E3 ubiquitin ligase which is involved in epigenetic regulation and plays an essential role in tumorigenesis. However, the role of UHRF2 in histone H3 acetylation has not yet been fully elucidated and few studies have reported its role in hepatocellular carcinoma (HCC). In this study, we examined the correlation between UHRF2 and acetylated H3 in HCC. Immunohistochemistry and western blot analysis demonstrated that the levels of histone H3 lysine 9 acetylation (H3K9ac) and histone H3 lysine 14 acetylation (H3K14ac) were higher in the HCC tissues and HepG2 HCC cells compared with the adjacent non-tumor tissues and L02 normal cells. The level of UHRF2 was higher in the HCC tissues compared with the adjacent non-tumor tissues, but its expression did not exhibit a significant difference between the HepG2 HCC cells and the L02 normal cells. In addition, when comparing the HCC tissues, a higher expression of UHRF2 correlated with a lower expression of H3K9ac in the HCC tissues. The overexpression of UHRF2 increased the expression of H3K9ac in L02 normal cells (P<0.01), but decreased the expression of H3K9ac in HepG2 cancer cells (P<0.05). Moreover, immunofluorescence staining and co-immunoprecipitation assay indicated that UHRF2 co-localized and interacted with H3K9ac in L02 and HepG2 cells and the plant homeodomain (PHD) finger domain was the key domain for UHRF2 directly binding to H3K9ac. Taken together, these results suggest that UHRF2 decreases the expression of H3K9ac in HepG2 HCC cells and interacts with it through the PHD domain. PMID:28004105

  18. UHRF2 decreases H3K9ac expression by interacting with it through the PHD and SRA/YDG domain in HepG2 hepatocellular carcinoma cells.

    PubMed

    Zhang, Ting; Zhao, Linglin; Zeng, Shengyuan; Bai, Lu; Chen, Junxia; Zhang, Zheng; Wang, Yalan; Duan, Changzhu

    2017-01-01

    Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) is a multi-domain E3 ubiquitin ligase which is involved in epigenetic regulation and plays an essential role in tumorigenesis. However, the role of UHRF2 in histone H3 acetylation has not yet been fully elucidated and few studies have reported its role in hepatocellular carcinoma (HCC). In this study, we examined the correlation between UHRF2 and acetylated H3 in HCC. Immunohistochemistry and western blot analysis demonstrated that the levels of histone H3 lysine 9 acetylation (H3K9ac) and histone H3 lysine 14 acetylation (H3K14ac) were higher in the HCC tissues and HepG2 HCC cells compared with the adjacent non-tumor tissues and L02 normal cells. The level of UHRF2 was higher in the HCC tissues compared with the adjacent non-tumor tissues, but its expression did not exhibit a significant difference between the HepG2 HCC cells and the L02 normal cells. In addition, when comparing the HCC tissues, a higher expression of UHRF2 correlated with a lower expression of H3K9ac in the HCC tissues. The overexpression of UHRF2 increased the expression of H3K9ac in L02 normal cells (P<0.01), but decreased the expression of H3K9ac in HepG2 cancer cells (P<0.05). Moreover, immunofluorescence staining and co-immunoprecipitation assay indicated that UHRF2 co-localized and interacted with H3K9ac in L02 and HepG2 cells and the plant homeodomain (PHD) finger domain was the key domain for UHRF2 directly binding to H3K9ac. Taken together, these results suggest that UHRF2 decreases the expression of H3K9ac in HepG2 HCC cells and interacts with it through the PHD domain.

  19. Genistein mediated histone acetylation and demethylation activates tumor suppressor genes in prostate cancer cells.

    PubMed

    Kikuno, Nobuyuki; Shiina, Hiroaki; Urakami, Shinji; Kawamoto, Ken; Hirata, Hiroshi; Tanaka, Yuichiro; Majid, Shahana; Igawa, Mikio; Dahiya, Rajvir

    2008-08-01

    Genistein is a phytoestrogen that has been reported to suppress the AKT signaling pathway in several malignancies. However, the molecular mechanism of genistein action is not known. We tested the hypothesis that genistein activates expression of several aberrantly silenced tumor suppressor genes (TSGs) that have unmethylated promoters such as PTEN, CYLD, p53 and FOXO3a. We report here that genistein activates TSGs through remodeling of the heterochromatic domains at promoters in prostate cancer cells by modulating histone H3-Lysine 9 (H3-K9) methylation and deacetylation. Genistein activation involved demethylation and acetylation of H3-K9 at the PTEN and the CYLD promoter, while acetylation of H3-K9 at the p53 and the FOXO3a promoter occurred through reduction of endogenous SIRT1 activity. There was a decrease of SIRT1 expression and accumulation of SIRT1 in the cytoplasm from the nucleus. Increased expression of these TSGs was also reciprocally related to attenuation of phosphorylated-AKT and NF-kappaB binding activity in prostate cancer cells. This is the first report describing a novel epigenetic pathway that activates TSGs by modulating either histone H3-Lysine 9 (H3-K9) methylation or deacetylation at gene promoters leading to inhibition of the AKT signaling pathway. These findings strengthen the understanding of how genistein may be chemoprotective in prostate cancer.

  20. Histone acetylation in insect chromosomes.

    PubMed

    Allfrey, V G; Pogo, B G; Littau, V C; Gershey, E L; Mirsky, A E

    1968-01-19

    Acetylation of histones takes place along the salivary gland chromosomes of Chironomus thummi when RNA synthesis is active. It can be observed but not measured quantitatively by autoradiography of chromosome squashes. The "fixatives" commonly used in preparing squashes of insect chromosomes preferentially extract the highly acetylated "arginine-rich" histone fractions; the use of such fixatives may explain the reported absence of histone acetylation in Drosophila melanogaster.

  1. Genomic targets, and histone acetylation and gene expression profiling of neural HDAC inhibition.

    PubMed

    Lopez-Atalaya, Jose P; Ito, Satomi; Valor, Luis M; Benito, Eva; Barco, Angel

    2013-09-01

    Histone deacetylase inhibitors (HDACis) have been shown to potentiate hippocampal-dependent memory and synaptic plasticity and to ameliorate cognitive deficits and degeneration in animal models for different neuropsychiatric conditions. However, the impact of these drugs on hippocampal histone acetylation and gene expression profiles at the genomic level, and the molecular mechanisms that underlie their specificity and beneficial effects in neural tissue, remains obscure. Here, we mapped four relevant histone marks (H3K4me3, AcH3K9,14, AcH4K12 and pan-AcH2B) in hippocampal chromatin and investigated at the whole-genome level the impact of HDAC inhibition on acetylation profiles and basal and activity-driven gene expression. HDAC inhibition caused a dramatic histone hyperacetylation that was largely restricted to active loci pre-marked with H3K4me3 and AcH3K9,14. In addition, the comparison of Chromatin immunoprecipitation sequencing and gene expression profiles indicated that Trichostatin A-induced histone hyperacetylation, like histone hypoacetylation induced by histone acetyltransferase deficiency, had a modest impact on hippocampal gene expression and did not affect the transient transcriptional response to novelty exposure. However, HDAC inhibition caused the rapid induction of a homeostatic gene program related to chromatin deacetylation. These results illuminate both the relationship between hippocampal gene expression and histone acetylation and the mechanism of action of these important neuropsychiatric drugs.

  2. HP1 Binding to Chromatin Methylated at H3K9 Is Enhanced by Auxiliary Factors▿

    PubMed Central

    Eskeland, Ragnhild; Eberharter, Anton; Imhof, Axel

    2007-01-01

    A large portion of the eukaryotic genome is packaged into transcriptionally silent heterochromatin. Several factors that play important roles during the establishment and maintenance of this condensed form have been identified. Methylation of lysine 9 within histone H3 and the subsequent binding of the chromodomain protein heterochromatin protein 1 (HP1) are thought to initiate heterochromatin formation in vivo and to propagate a heterochromatic state lasting through several cell divisions. For the present study we analyzed the binding of HP1 to methylated chromatin in a fully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 binds only weakly to methylated chromatin. However, the addition of recombinant SU(VAR) protein, such as ACF1 or SU(VAR)3-9, facilitates HP1 binding to chromatin methylated at lysine 9 within the H3 N terminus (H3K9). We propose that HP1 has multiple target sites that contribute to its recognition of chromatin, only one of them being methylated at H3K9. These findings have implications for the mechanisms of recognition of specific chromatin modifications in vivo. PMID:17101786

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

    PubMed Central

    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

  4. A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin.

    PubMed

    Schotta, Gunnar; Lachner, Monika; Sarma, Kavitha; Ebert, Anja; Sengupta, Roopsha; Reuter, Gunter; Reinberg, Danny; Jenuwein, Thomas

    2004-06-01

    Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.

  5. Genomic Prevalence of Heterochromatic H3K9me2 and Transcription Do Not Discriminate Pluripotent from Terminally Differentiated Cells

    PubMed Central

    Tiwari, Vijay K.; Baubec, Tuncay; Roloff, Tim C.; Gaidatzis, Dimos; Stadler, Michael B.; Schübeler, Dirk

    2011-01-01

    Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change, many genes show altered expression upon differentiation. Quantitative sequencing of transcripts demonstrates however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global basal level transcription in stem cells. This suggests that cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity. PMID:21655081

  6. H3K9 Trimethylation Silences Fas Expression To Confer Colon Carcinoma Immune Escape and 5-Fluorouracil Chemoresistance.

    PubMed

    Paschall, Amy V; Yang, Dafeng; Lu, Chunwan; Choi, Jeong-Hyeon; Li, Xia; Liu, Feiyan; Figueroa, Mario; Oberlies, Nicholas H; Pearce, Cedric; Bollag, Wendy B; Nayak-Kapoor, Asha; Liu, Kebin

    2015-08-15

    The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL(+) and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

  7. BMP8B Is a Tumor Suppressor Gene Regulated by Histone Acetylation in Gastric Cancer.

    PubMed

    Wisnieski, Fernanda; Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Santos, Leonardo Caires; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Artigiani, Ricardo; Demachki, Sâmia; Assumpção, Paulo Pimentel; Lourenço, Laércio Gomes; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

    2017-04-01

    Different from genetic alterations, the reversible nature of epigenetic modifications provides an interesting opportunity for the development of clinically relevant therapeutics in different tumors. In this study, we aimed to screen and validate candidate genes regulated by the epigenetic marker associated with transcriptional activation, histone acetylation, in gastric cancer (GC). We first compared gene expression profile of trichostatin A-treated and control GC cell lines using microarray assay. Among the 55 differentially expressed genes identified in this analysis, we chose the up-regulated genes BMP8B and BAMBI for further analyses, that included mRNA and histone acetylation quantification in paired GC and nontumor tissue samples. BMP8B expression was reduced in GC compared to nontumor samples (P < 0.01). In addition, reduced BMP8B expression was associated with poorly differentiated GC (P = 0.02). No differences or histopathological associations were identified concerning BAMBI expression. Furthermore, acetylated H3K9 and H4K16 levels at BMP8B were increased in GC compared to nontumors (P < 0.05). However, reduced levels of acetylated H3K9 and H4K16 were associated with poorly differentiated GC (P < 0.05). Reduced levels of acetylated H3K9 was also associated with diffuse-type histological GC (P < 0.05). Notably, reduced BMP8B mRNA and acetylated H4K16 levels were positively correlated in poorly differentiated GC (P < 0.05). Our study demonstrated that BMP8B seems to be a tumor suppressor gene regulated by H4K16 acetylation in poorly differentiated GC. Therefore, BMP8B may be a potential target for TSA-based therapies in this GC sample subset. J. Cell. Biochem. 118: 869-877, 2017. © 2016 Wiley Periodicals, Inc.

  8. Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics.

    PubMed

    Abshiru, Nebiyu; Ippersiel, Kevin; Tang, Yong; Yuan, Hua; Marmorstein, Ronen; Verreault, Alain; Thibault, Pierre

    2013-04-09

    Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme-to-substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. This article is part of a Special Issue entitled: From protein structures to clinical applications.

  9. Chaperone-mediated acetylation of histones by Rtt109 identified by quantitative proteomics

    PubMed Central

    Abshiru, Nebiyu; Ippersiel, Kevin; Tang, Yong; Yuan, Hua; Marmorstein, Ronen; Verreault, Alain; Thibault, Pierre

    2014-01-01

    Rtt109 is a fungal-specific histone acetyltransferase (HAT) that associates with either Vps75 or Asf1 to acetylate histone H3. Recent biochemical and structural studies suggest that site-specific acetylation of H3 by Rtt109 is dictated by the binding chaperone where Rtt109-Asf1 acetylates K56, while Rtt109-Vps75 acetylates K9 and K27. To gain further insights into the roles of Vps75 and Asf1 in directing site-specific acetylation of H3, we used quantitative proteomics to profile the global and site-specific changes in H3 and H4 during in vitro acetylation assays with Rtt109 and its chaperones. Our analyses showed that Rtt109-Vps75 preferentially acetylates H3 K9 and K23, the former residue being the major acetylation site. At high enzyme to substrate ratio, Rtt109 also acetylated K14, K18, K27 and to a lower extent K56 of histone H3. Importantly, this study revealed that in contrast to Rtt109-Vps75, Rtt109-Asf1 displayed a far greater site-specificity, with K56 being the primary site of acetylation. For the first time, we also report the acetylation of histone H4 K12 by Rtt109-Vps75, whereas Rtt109-Asf1 showed no detectable activity toward H4. PMID:23036725

  10. Cdyl, a New Partner of the Inactive X Chromosome and Potential Reader of H3K27me3 and H3K9me2

    PubMed Central

    Escamilla-Del-Arenal, M.; da Rocha, S. T.; Spruijt, C. G.; Masui, O.; Renaud, O.; Smits, Arne H.; Margueron, R.

    2013-01-01

    X chromosome inactivation is a remarkable example of chromosome-wide gene silencing and facultative heterochromatin formation. Numerous histone posttranslational modifications, including H3K9me2 and H3K27me3, accompany this process, although our understanding of the enzymes that lay down these marks and the factors that bind to them is still incomplete. Here we identify Cdyl, a chromodomain-containing transcriptional corepressor, as a new chromatin-associated protein partner of the inactive X chromosome (Xi). Using mouse embryonic stem cell lines with mutated histone methyltransferase activities, we show that Cdyl relies on H3K9me2 for its general association with chromatin in vivo. For its association with Xi, Cdyl requires the process of differentiation and the presence of H3K9me2 and H3K27me3, which both become chromosomally enriched following Xist RNA coating. We further show that the removal of the PRC2 component Eed and subsequent loss of H3K27me3 lead to a reduction of both Cdyl and H3K9me2 enrichment on inactive Xi. Finally, we show that Cdyl associates with the H3K9 histone methyltransferase G9a and the MGA protein, both of which are also found on Xi. We propose that the combination of H3K9me2 and H3K27me3 recruits Cdyl to Xi, and this, in turn, may facilitate propagation of the H3K9me2 mark by anchoring G9a. PMID:24144980

  11. The Chp1 chromodomain binds the H3K9me tail and the nucleosome core to assemble heterochromatin

    PubMed Central

    Zocco, Manuel; Marasovic, Mirela; Pisacane, Paola; Bilokapic, Silvija; Halic, Mario

    2016-01-01

    To maintain genome stability, cells pack large portions of their genome into silent chromatin or heterochromatin. Histone H3 lysine 9 methylation, a hallmark of heterochromatin, is recognized by conserved readers called chromodomains. But how chromodomains interact with their actual binding partner, the H3K9 methylated nucleosome, remains elusive. We have determined the structure of a nucleosome trimethylated at lysine 9 of histone H3 (H3K9me3 Nucleosome) in a complex with the chromodomain of Chp1, a protein required for RNA interference-dependent heterochromatin formation in fission yeast. The cryo-electron microscopy structure reveals that the chromodomain of Chp1 binds the histone H3 lysine 9 methylated tail and the core of the nucleosome, primarily histones H3 and H2B. Mutations in chromodomain of Chp1 loops, which interact with the nucleosome core, abolished this interaction in vitro. Moreover, fission yeast cells with Chp1 loop mutations have a defect in Chp1 recruitment and heterochromatin formation. This study reveals the structural basis for heterochromatic silencing and suggests that chromodomains could read histone code in the H3 tail and the nucleosome core, which would provide an additional layer of regulation. PMID:27462451

  12. Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation.

    PubMed

    Yang, Feikun; Hao, Ru; Kessler, Barbara; Brem, Gottfried; Wolf, Eckhard; Zakhartchenko, Valeri

    2007-01-01

    The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned

  13. Detection of histone acetylation levels in the dorsal hippocampus reveals early tagging on specific residues of H2B and H4 histones in response to learning.

    PubMed

    Bousiges, Olivier; Neidl, Romain; Majchrzak, Monique; Muller, Marc-Antoine; Barbelivien, Alexandra; Pereira de Vasconcelos, Anne; Schneider, Anne; Loeffler, Jean-Philippe; Cassel, Jean-Christophe; Boutillier, Anne-Laurence

    2013-01-01

    The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain--notably in the hippocampus--and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.

  14. Utilizing Targeted Mass Spectrometry to Demonstrate Asf1-Dependent Increases in Residue Specificity for Rtt109-Vps75 Mediated Histone Acetylation

    PubMed Central

    Kuo, Yin-Ming; Henry, Ryan A.; Huang, Liangqun; Chen, Xu; Stargell, Laurie A.; Andrews, Andrew J.

    2015-01-01

    In Saccharomyces cerevisiae, Rtt109, a lysine acetyltransferase (KAT), associates with a histone chaperone, either Vps75 or Asf1. It has been proposed that these chaperones alter the selectivity of Rtt109 or which residues it preferentially acetylates. In the present study, we utilized a label-free quantitative mass spectrometry-based method to determine the steady-state kinetic parameters of acetylation catalyzed by Rtt109-Vps75 on H3 monomer, H3/H4 tetramer, and H3/H4-Asf1 complex. These results show that among these histone conformations, only H3K9 and H3K23 are significantly acetylated under steady-state conditions and that Asf1 promotes H3/H4 acetylation by Rtt109-Vps75. Asf1 equally increases the Rtt109-Vps75 specificity for both of these residues with a maximum stoichiometry of 1:1 (Asf1 to H3/H4), but does not alter the selectivity between these two residues. These data suggest that the H3/H4-Asf1 complex is a substrate for Rtt109-Vps75 without altering selectivity between residues. The deletion of either Rtt109 or Asf1 in vivo results in the same reduction of H3K9 acetylation, suggesting that Asf1 is required for efficient H3K9 acetylation both in vitro and in vivo. Furthermore, we found that the acetylation preference of Rtt109-Vps75 could be directed to H3K56 when those histones already possess modifications, such as those found on histones purified from chicken erythrocytes. Taken together, Vps75 and Asf1 both enhance Rtt109 acetylation for H3/H4, although via different mechanisms, but have little impact on the residue selectivity. Importantly, these results provide evidence that histone chaperones can work together via interactions with either the enzyme or the substrate to more efficiently acetylate histones. PMID:25781956

  15. Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

    PubMed Central

    Jackel, Jamie N.; Storer, Jessica M.; Coursey, Tami

    2016-01-01

    ABSTRACT In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin. IMPORTANCE In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but

  16. CDKN1A histone acetylation and gene expression relationship in gastric adenocarcinomas.

    PubMed

    Wisnieski, Fernanda; Calcagno, Danielle Queiroz; Leal, Mariana Ferreira; Santos, Leonardo Caires; Gigek, Carolina Oliveira; Chen, Elizabeth Suchi; Demachki, Sâmia; Artigiani, Ricardo; Assumpção, Paulo Pimentel; Lourenço, Laércio Gomes; Burbano, Rommel Rodríguez; Smith, Marília Cardoso

    2017-02-01

    CDKN1A is a tumor suppressor gene involved in gastric carcinogenesis and is a potential target for histone deacetylase inhibitor-based therapies. Upregulation of CDKN1A is generally observed in several cell lines after histone deacetylase inhibitor treatment; however, little is known about the histone acetylation status associated with this gene in clinical samples, including gastric tumor tissue samples. Therefore, our goal was to quantify the H3K9 and H4K16 acetylation levels associated with three CDKN1A regions in 21 matched pairs of gastric adenocarcinoma and corresponding adjacent non-tumor samples by chromatin immunoprecipitation and to correlate these data with the gene expression. Our results demonstrated that the -402, -20, and +182 CDKN1A regions showed a significantly increased acetylation level in at least one of the histones evaluated (p < 0.05, for all comparisons), and these levels were positively correlated in gastric tumors. However, an inverse correlation was detected between both H3K9 and H4K16 acetylation at the -402 CDKN1A region and mRNA levels in gastric tumors (r = -0.51, p = 0.02; r = -0.60, p < 0.01, respectively). Furthermore, increased H4K16 acetylation at the -20 CDKN1A region was associated with gastric tumors of patients without lymph node metastasis (p = 0.04). These results highlight the complexity of these processes in gastric adenocarcinoma and contribute to a better understanding of CDKN1A regulation in carcinogenesis.

  17. Structure of the Rtt109-AcCoA/Vps75 Complex and Implications for Chaperone-Mediated Histone Acetylation

    PubMed Central

    Tang, Yong; Holbert, Marc A.; Delgoshaie, Neda; Wurtele, Hugo; Guillemette, Benoît; Meeth, Katrina; Yuan, Hua; Drogaris, Paul; Lee, Eun-Hye; Durette, Chantal; Thibault, Pierre; Verreault, Alain; Cole, Philip A.; Marmorstein, Ronen

    2011-01-01

    Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27 and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ~12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3–H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones prior to deposition. PMID:21256037

  18. A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.

    PubMed

    Porter, Louise F; Galli, Giorgio G; Williamson, Sally; Selley, Julian; Knight, David; Elcioglu, Nursel; Aydin, Ali; Elcioglu, Mustafa; Venselaar, Hanka; Lund, Anders H; Bonshek, Richard; Black, Graeme C; Manson, Forbes D

    2015-12-01

    Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.

  19. A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome

    PubMed Central

    Porter, Louise F.; Galli, Giorgio G.; Williamson, Sally; Selley, Julian; Knight, David; Elcioglu, Nursel; Aydin, Ali; Elcioglu, Mustafa; Venselaar, Hanka; Lund, Anders H.; Bonshek, Richard; Black, Graeme C.; Manson, Forbes D.

    2015-01-01

    Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9–14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9–14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9–14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9–14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease. PMID:26395458

  20. The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

    PubMed Central

    Aronsen, Jan Magnus; Ferrini, Arianna; Brien, Patrick; Alkass, Kanar; Tomasso, Antonio; Agrawal, Asmita; Bergmann, Olaf; Reik, Wolf; Roderick, Hywel Llewelyn

    2016-01-01

    Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease. PMID:27893464

  1. Histone Acetylation Facilitates Rapid and Robust Memory CD8 T Cell Response through Differential Expression of Effector Molecules (Eomesodermin and Its Targets: Perforin and Granzyme B)1

    PubMed Central

    Araki, Yasuto; Fann, Monchou; Wersto, Robert; Weng, Nan-ping

    2008-01-01

    To understand the mechanism regulating the effector function of memory CD8 T cells, we examined expression and chromatin state of a key transcription factor (eomesodermin, EOMES) and two of its targets: perforin (PRF1) and granzyme B (GZMB). Accessible chromatin associated histone 3 lysine 9 acetylation (H3K9Ac) was found significantly higher at the proximal promoter and the first exon region of all three genes in memory CD8 T cells than in naive CD8 T cells. Correspondingly, EOMES and PRF1 were constitutively higher expressed in memory CD8 T cells than in naive CD8 T cells at resting and activated states. In contrast, higher expression of GZMB was induced in memory CD8 T cells than in naive CD8 T cells only after activation. Regardless of their constitutive or inducible expression, decreased H3K9Ac levels after treatment with a histone acetyl-transferase inhibitor (Curcumin) led to decreased expression of all three genes in activated memory CD8 T cells. These findings suggest that H3K9Ac associated accessible chromatin state serves as a corner stone for the differentially high expression of these effector genes in memory CD8 T cells. Thus, epigenetic changes mediated via histone acetylation may provide a chromatin “memory” for the rapid and robust transcriptional response of memory CD8 T cells. PMID:18523274

  2. FGF2 is a target and a trigger of epigenetic mechanisms associated with differences in emotionality: partnership with H3K9me3.

    PubMed

    Chaudhury, Sraboni; Aurbach, Elyse L; Sharma, Vikram; Blandino, Peter; Turner, Cortney A; Watson, Stanley J; Akil, Huda

    2014-08-12

    Posttranslational modifications of histone tails in chromatin template can result from environmental experiences such as stress and substance abuse. However, the role of epigenetic modifications as potential predisposing factors in affective behavior is less well established. To address this question, we used our selectively bred lines of high responder (bHR) and low responder (bLR) rats that show profound and stable differences in affective responses, with bLRs being prone to anxiety- and depression-like behavior and bHRs prone to addictive behavior. We first asked whether these phenotypes are associated with basal differences in epigenetic profiles. Our results reveal broad between-group differences in basal levels of trimethylated histone protein H3 at lysine 9 (H3K9me3) in hippocampus (HC), amygdala, and nucleus accumbens. Moreover, levels of association of H3K9me3 at Glucocorticoid Receptor (GR) and Fibroblast growth Factor 2 (FGF2) promoters differ reciprocally between bHRs and bLRs in these regions, consistent with these genes' opposing levels of expression and roles in modulating anxiety behavior. Importantly, this basal epigenetic pattern is modifiable by FGF2, a factor that modulates anxiety behavior. Thus, early-life FGF2, which decreases anxiety, altered the levels of H3K9me3 and its binding at FGF2 and GR promoters of bLRs rendering them more similar to bHRs. Conversely, knockdown of HC FGF2 altered both anxiety behavior and levels of H3K9me3 in bHRs, rendering them more bLR-like. These findings implicate FGF2 as a modifier of epigenetic mechanisms associated with emotional responsiveness, and point to H3K9me3 as a key player in the regulation of affective vulnerability.

  3. FGF2 is a target and a trigger of epigenetic mechanisms associated with differences in emotionality: Partnership with H3K9me3

    PubMed Central

    Chaudhury, Sraboni; Aurbach, Elyse L.; Sharma, Vikram; Blandino, Peter; Turner, Cortney A.; Watson, Stanley J.; Akil, Huda

    2014-01-01

    Posttranslational modifications of histone tails in chromatin template can result from environmental experiences such as stress and substance abuse. However, the role of epigenetic modifications as potential predisposing factors in affective behavior is less well established. To address this question, we used our selectively bred lines of high responder (bHR) and low responder (bLR) rats that show profound and stable differences in affective responses, with bLRs being prone to anxiety- and depression-like behavior and bHRs prone to addictive behavior. We first asked whether these phenotypes are associated with basal differences in epigenetic profiles. Our results reveal broad between-group differences in basal levels of trimethylated histone protein H3 at lysine 9 (H3K9me3) in hippocampus (HC), amygdala, and nucleus accumbens. Moreover, levels of association of H3K9me3 at Glucocorticoid Receptor (GR) and Fibroblast growth Factor 2 (FGF2) promoters differ reciprocally between bHRs and bLRs in these regions, consistent with these genes’ opposing levels of expression and roles in modulating anxiety behavior. Importantly, this basal epigenetic pattern is modifiable by FGF2, a factor that modulates anxiety behavior. Thus, early-life FGF2, which decreases anxiety, altered the levels of H3K9me3 and its binding at FGF2 and GR promoters of bLRs rendering them more similar to bHRs. Conversely, knockdown of HC FGF2 altered both anxiety behavior and levels of H3K9me3 in bHRs, rendering them more bLR-like. These findings implicate FGF2 as a modifier of epigenetic mechanisms associated with emotional responsiveness, and point to H3K9me3 as a key player in the regulation of affective vulnerability. PMID:25071177

  4. Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

    USGS Publications Warehouse

    Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

    2011-01-01

    In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

  5. Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer.

    PubMed

    Cong, Peiqing; Zhu, Kongju; Ji, Qianqian; Zhao, Haijing; Chen, Yaosheng

    2013-09-01

    Until now, the efficiency of animal cloning by somatic cell nuclear transfer (SCNT) has remained low. Efforts to improve cloning efficiency have demonstrated a positive role of trichostatin A (TSA), an inhibitor of deacetylases, on the development of nuclear transfer (NT) embryos in many species. Here, we report the effects of TSA on pre-implantation development of porcine NT embryos. Our results showed that treatment of reconstructed porcine embryos with 50 nmol/L TSA for 24 h after activation significantly improved the production of blastocysts (P < 0.05), while treating donor cells with the same solution resulted in increases in cleavage rates and blastomere numbers (P < 0.05). However, TSA treatment of both donor cells and SCNT embryos did not improve blastocyst production, nor did it increase blastomere numbers. Using indirect immunofluorescence, we found that TSA treatment of NT embryos could improve the reprogramming of histone acetylation at lysine 9 of histone 3 (H3K9) and affect nuclear swelling of transferred nuclei. However, no apparent effect of TSA treatment on H3K9 dimethylation (H3K9me2) was observed. These findings suggest a positive effect of TSA treatment (either treating NT embryos or donor cells) on the development of porcine NT embryos, which is achieved by improving epigenetic reprogramming.

  6. Suv39h-dependent H3K9me3 marks intact retrotransposons and silences LINE elements in mouse embryonic stem cells.

    PubMed

    Bulut-Karslioglu, Aydan; De La Rosa-Velázquez, Inti A; Ramirez, Fidel; Barenboim, Maxim; Onishi-Seebacher, Megumi; Arand, Julia; Galán, Carmen; Winter, Georg E; Engist, Bettina; Gerle, Borbala; O'Sullivan, Roderick J; Martens, Joost H A; Walter, Jörn; Manke, Thomas; Lachner, Monika; Jenuwein, Thomas

    2014-07-17

    Heterochromatin is required to restrict aberrant expression of retrotransposons, but it remains poorly defined due to the underlying repeat-rich sequences. We dissected Suv39h-dependent histone H3 lysine 9 trimethylation (H3K9me3) by genome-wide ChIP sequencing in mouse embryonic stem cells (ESCs). Refined bioinformatic analyses of repeat subfamilies indicated selective accumulation of Suv39h-dependent H3K9me3 at interspersed repetitive elements that cover ∼5% of the ESC epigenome. The majority of the ∼8,150 intact long interspersed nuclear elements (LINEs) and endogenous retroviruses (ERVs), but only a minor fraction of the >1.8 million degenerate and truncated LINEs/ERVs, are enriched for Suv39h-dependent H3K9me3. Transcriptional repression of intact LINEs and ERVs is differentially regulated by Suv39h and other chromatin modifiers in ESCs but governed by DNA methylation in committed cells. These data provide a function for Suv39h-dependent H3K9me3 chromatin to specifically repress intact LINE elements in the ESC epigenome.

  7. Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin

    PubMed Central

    Hiragami-Hamada, Kyoko; Soeroes, Szabolcs; Nikolov, Miroslav; Wilkins, Bryan; Kreuz, Sarah; Chen, Carol; De La Rosa-Velázquez, Inti A.; Zenn, Hans Michael; Kost, Nils; Pohl, Wiebke; Chernev, Aleksandar; Schwarzer, Dirk; Jenuwein, Thomas; Lorincz, Matthew; Zimmermann, Bastian; Walla, Peter Jomo; Neumann, Heinz; Baubec, Tuncay; Urlaub, Henning; Fischle, Wolfgang

    2016-01-01

    Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin. PMID:27090491

  8. Histone deacetylase inhibitor improves the development and acetylation levels of cat-cow interspecies cloned embryos.

    PubMed

    Wittayarat, Manita; Sato, Yoko; Do, Lanh Thi Kim; Morita, Yasuhiro; Chatdarong, Kaywalee; Techakumphu, Mongkol; Taniguchi, Masayasu; Otoi, Takeshige

    2013-08-01

    Abnormal epigenetic reprogramming, such as histone acetylation, might cause low efficiency of interspecies somatic cell nuclear transfer (iSCNT). This study was conducted to evaluate the effects of trichostatin A (TSA) on the developmental competence and histone acetylation of iSCNT embryos reconstructed from cat somatic cells and bovine cytoplasm. The iSCNT cat and parthenogenetic bovine embryos were treated with various concentrations of TSA (0, 25, 50, or 100 nM) for 24 h, respectively, following fusion and activation. Treatment with 50 nM TSA produced significantly higher rates of cleavage and blastocyst formation (84.3% and 4.6%, respectively) of iSCNT embryos than the rates of non-TSA-treated iSCNT embryos (63.8% and 0%, respectively). Similarly, the treatment of 50 nM TSA increased the blastocyst formation rate of parthenogenetic bovine embryos. The acetylation levels of histone H3 lysine 9 (H3K9) in the iSCNT embryos with the treatment of 50 nM TSA were similar to those of in vitro-fertilized embryos and significantly higher (p<0.05) than those of non-TSA-treated iSCNT embryos (control), irrespective of the embryonic development stage (two-cell, four-cell, and eight-cell stages). These results indicated that the treatment of 50 nM TSA postfusion was beneficial for development to the blastocyst stage of iSCNT cat embryos and correlated with the increasing levels of acetylation at H3K9.

  9. Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

    PubMed Central

    Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; Martin, Eric W.; Krietsch, Jana; Sanchez, Gabriela; Inman, Margaux; Tsang, Herman; Warr, Matthew; Passegué, Emmanuelle; Larabell, Carolyn A.; Forsberg, E. Camilla

    2015-01-01

    Summary Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increased cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation. PMID:26489895

  10. The HP1alpha-CAF1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin.

    PubMed

    Loyola, Alejandra; Tagami, Hideaki; Bonaldi, Tiziana; Roche, Danièle; Quivy, Jean Pierre; Imhof, Axel; Nakatani, Yoshihiro; Dent, Sharon Y R; Almouzni, Geneviève

    2009-07-01

    Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

  11. Association of Taf14 with acetylated histone H3 directs gene transcription and the DNA damage response

    PubMed Central

    Shanle, Erin K.; Andrews, Forest H.; Meriesh, Hashem; McDaniel, Stephen L.; Dronamraju, Raghuvar; DiFiore, Julia V.; Jha, Deepak; Wozniak, Glenn G.; Bridgers, Joseph B.; Kerschner, Jenny L.; Krajewski, Krzysztof; Martín, Glòria Mas; Morrison, Ashby J.; Kutateladze, Tatiana G.; Strahl, Brian D.

    2015-01-01

    The YEATS domain, found in a number of chromatin-associated proteins, has recently been shown to have the capacity to bind histone lysine acetylation. Here, we show that the YEATS domain of Taf14, a member of key transcriptional and chromatin-modifying complexes in yeast, is a selective reader of histone H3 Lys9 acetylation (H3K9ac). Structural analysis reveals that acetylated Lys9 is sandwiched in an aromatic cage formed by F62 and W81. Disruption of this binding in cells impairs gene transcription and the DNA damage response. Our findings establish a highly conserved acetyllysine reader function for the YEATS domain protein family and highlight the significance of this interaction for Taf14. PMID:26341557

  12. Promoter histone H3 lysine 9 di-methylation is associated with DNA methylation and aberrant expression of p16 in gastric cancer cells.

    PubMed

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2009-11-01

    In the course of gastric cancer development, gene silencing by DNA hypermethylation is an important mechanism. While DNA methylation often co-exists with histone modifications to regulate gene expression, the function of histone modifications in gene silencing in gastric cancer has not been evaluated in detail. p16, a well-known tumor suppressor gene, is frequently silenced in DNA hypermethylation manner in gastric cancer. Accordingly, we chose p16 to clarify whether there is a correlation among histone H3 lysine 9 (H3-K9) di-methylation, H3-K9 acetylation, DNA methylation and p16 expression in human gastric cancer. Three gastric cancer cells, MKN-45, SGC-7901 and BGC-823, were treated with 5-aza-2'-deoxycytidine (5-Aza-dC) and/or trichostatin A (TSA). We investigated p16 promoter DNA methylation status, p16 mRNA levels, regional and global levels of di-methyl-H3-K9 and acetyl-H3-K9 in four groups: i) 5-Aza-dC, ii) TSA, iii) the combination of 5-Aza-dC and TSA and iv) control group with no treatments. p16 silencing is characterized by DNA hypermethylation, H3-K9 hypoacetylation and H3-K9 hypermethylation at the promoter region. Treatment with TSA, increased H3-K9 acetylation at the hypermethylated promoter, but did not affect H3-K9 di-methylation or p16 expression. By contrast, treatment with 5-Aza-dC, reduced H3-K9 di-methylation, increased H3-K9 acetylation at the hypermethylated promoter and reactivated the expression of p16. Combined treatment restored the expression of p16 synergistically. In addition, 5-Aza-dC and the combined treatment did not result in global alteration of H3-K9 di-methylation. These results suggest that H3-K9 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence p16 in gastric cancer. The decreased H3-K9 di-methylation correlates with DNA demethylation and reactivation of p16. H3-K9 di-methylation as well as DNA methylation related to p16 silencing is limited to the promoter region. In addition to its effect

  13. Histone acetylation influences the transcriptional activation of POX in Beta vulgaris L. and Beta maritima L. under salt stress.

    PubMed

    Yolcu, Seher; Ozdemir, Filiz; Güler, Aybüke; Bor, Melike

    2016-03-01

    Acetylation of histone proteins is a type of chromatin modification which facilitates the activation of genes. Recent studies brought up the importance of this reversible and rapid process for the regulation of gene expression especially in plant defense against a variety of environmental stresses. Deciphering the exact mechanisms of chromatin modifications under abiotic stress conditions is important for improving crop plants' performance and yield. In a previous study we compared the salt stress responses of Beta vulgaris (sugar beet) and Beta maritima (wild beet). In accordance with those results we suggested that chromatin remodeling can be an active process in the regulation of genes related to salt stress tolerance of these plants. Therefore we performed ChIP assay in control and salt stressed (250 and 500 mM NaCl) plants and compared the enrichment of acetylation in the associated chromatin sites. We found that the transcriptional activation of one peroxidase (POX) encoding gene was associated with the elevated levels of acetylation in H3K9 and H3K27 sites. The acetylation patterns were remarkably different between two species in which the highest acetylation levels were found at H3K9 and H3K27 in wild beet and sugar beet respectively.

  14. H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

    PubMed Central

    Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

    2017-01-01

    DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

  15. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

    PubMed Central

    Seto, Edward; Yoshida, Minoru

    2014-01-01

    Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

  16. RelB/p52-mediated NF-κB signaling alters histone acetylation to increase the abundance of corticotropin-releasing hormone in human placenta.

    PubMed

    Di Stefano, Valeria; Wang, Bingbing; Parobchak, Nataliya; Roche, Natalie; Rosen, Todd

    2015-08-25

    Corticotropin-releasing hormone (CRH) produced in the placenta may be part of a clock that regulates the length of human gestation. Maternal plasma CRH abundance exponentially increases as pregnancy advances. Glucocorticoid stimulates CRH expression in full-term human placenta by promoting noncanonical (RelB/p52 heterodimer-mediated) nuclear factor κB (NF-κB) pathway activity. Using dexamethasone to mimic glucocorticoid exposure, we found that an epigenetic switch mediated the glucocorticoid-induced expression of CRH as gestation advances. The amount of acetylated histone H3 lysine 9 (H3K9) associated with the CRH promoter was greater in cytotrophoblasts from full-term placenta than in those from midterm placenta. Knocking down the lysine acetyltransferase CBP reduced H3K9 histone acetylation and prevented dexamethasone-induced CRH expression. Unexpectedly, knocking down the histone deacetylase HDAC1 or pharmacologically inhibiting type I and II HDACs also decreased the expression of CRH yet increased the acetylation of H3K9 and other histone regions. Both CBP and HDAC1 bound at the CRH promoter in a complex with the RelB/p52 heterodimer in a mutually dependent manner; knocking down any one factor in the complex prevented binding of the others as well as the dexamethasone-induced CRH expression. Our results suggest that glucocorticoids induce a transcription complex consisting of RelB/p52, CBP, and HDAC1 that triggers a dynamic acetylation-mediated epigenetic change to induce CRH expression in full-term human placenta.

  17. Role of histone modifications and DNA methylation in the regulation of O6-methylguanine-DNA methyltransferase gene expression in human stomach cancer cells.

    PubMed

    Meng, Chun-Feng; Zhu, Xin-Jiang; Peng, Guo; Dai, Dong-Qiu

    2010-05-01

    To determine a possible function of histone modifications in stomach carcinogenesis, we analyzed global and MGMT-promoter levels of di-methyl-H3-K9, di-methyl-H3-K4 and acetyl-H3-K9, as well as MGMT DNA methylation and mRNA expression following treatment with 5-aza-2' -deoxycytidine and/or Trichostatin A. We found that histone H3-K9 di-methylation, H3-K4 di-methylation, H3-K9 acetylation and DNA methylation work in combination to silence MGMT. The results indicate that histone modifications as well as DNA methylation may be involved in stomach carcinogenesis. In addition to its effect on DNA methylation, 5-aza-2' -deoxycytidine can act at histone modification level to reactivate MGMT expression in a region-specific and DNA methylation-dependent manner.

  18. ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia.

    PubMed

    Wan, Liling; Wen, Hong; Li, Yuanyuan; Lyu, Jie; Xi, Yuanxin; Hoshii, Takayuki; Joseph, Julia K; Wang, Xiaolu; Loh, Yong-Hwee E; Erb, Michael A; Souza, Amanda L; Bradner, James E; Shen, Li; Li, Wei; Li, Haitao; Allis, C David; Armstrong, Scott A; Shi, Xiaobing

    2017-03-09

    Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. Recognition of modified histones by 'reader' proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR-Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.

  19. Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxiety-like and alcohol-drinking behaviours.

    PubMed

    Sakharkar, Amul J; Zhang, Huaibo; Tang, Lei; Baxstrom, Kathryn; Shi, Guangbin; Moonat, Sachin; Pandey, Subhash C

    2014-08-01

    Recent studies have demonstrated the involvement of epigenetic mechanisms in psychiatric disorders, including alcoholism. Here, we investigated the effects of histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) on amygdaloid HDAC-induced histone deacetylation and neuropeptide Y (NPY) expression and on anxiety-like and alcohol-drinking behaviours in alcohol-preferring (P) and -non-preferring (NP) rats. It was found that P rats displayed higher anxiety-like and alcohol-drinking behaviours, higher amygdaloid nuclear, but not cytosolic, HDAC activity, which was associated with increased HDAC2 protein levels and deficits in histone acetylation and NPY expression in the central (CeA) and medial nucleus of amygdala (MeA), as compared to NP rats. TSA treatment attenuated the anxiety-like and alcohol-drinking behaviours, with concomitant reductions in amygdaloid nuclear, but not cytosolic HDAC activity, and HDAC2, but not HDAC4, protein levels in the CeA and MeA of P rats, without effect in NP rats. TSA treatment also increased global histone acetylation (H3-K9 and H4-K8) and NPY expression in the CeA and MeA of P, but not in NP rats. Histone H3 acetylation within the NPY promoter was also innately lower in the amygdala of P rats compared with NP rats; which was normalized by TSA treatment. Voluntary ethanol intake in P, but not NP rats, produced anxiolytic effects and decreased the HDAC2 levels and increased histone acetylation in the CeA and MeA. These results suggest that higher HDAC2 expression-related deficits in histone acetylation may be involved in lower NPY expression in the amygdala of P rats, and operative in controlling anxiety-like and alcohol-drinking behaviours.

  20. Quisinostat treatment improves histone acetylation and developmental competence of porcine somatic cell nuclear transfer embryos.

    PubMed

    Jin, Long; Guo, Qing; Zhu, Hai-Ying; Xing, Xiao-Xu; Zhang, Guang-Lei; Xuan, Mei-Fu; Luo, Qi-Rong; Luo, Zhao-Bo; Wang, Jun-Xia; Yin, Xi-Jun; Kang, Jin-Dan

    2017-02-22

    Abnormal epigenetic modifications are considered a main contributing factor to low cloning efficiency. In the present study, we explored the effects of quisinostat, a novel histone deacetylase inhibitor, on blastocyst formation rate in porcine somatic-cell nuclear transfer (SCNT) embryos, on acetylation of histone H3 lysine 9 (AcH3K9), and on expression of POU5F1 protein and apoptosis-related genes BAX and BCL2. Our results showed that treatment with 10 nM quisinostat for 24 h significantly improved the development of reconstructed embryos compared to the untreated group (19.0 ± 1.6% vs. 10.2 ± 0.9%; P < 0.05). Quisinostat-treated SCNT embryos also possessed significantly increased AcH3K9 at the pseudo-pronuclear stage (P < 0.05), as well as improved immunostaining intensity for POU5F1 at the blastocyst stage (P < 0.05). While no statistical difference in BAX expression was observed, BCL2 transcript abundance was significantly different in the quisinostat-treated compared to the untreated control group. Of the 457 quisinostat-treated cloned embryos transferred into three surrogates, six fetuses developed from the one sow that became pregnant. These findings suggested that quisinostat can regulate gene expression and epigenetic modification, facilitating nuclear reprogramming and subsequently improving the developmental competence of pig SCNT embryos and blastocyst quality. This article is protected by copyright. All rights reserved.

  1. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  2. Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism.

    PubMed

    Chaturvedi, Pankaj; Kalani, Anuradha; Givvimani, Srikanth; Kamat, Pradip Kumar; Familtseva, Anastasia; Tyagi, Suresh C

    2014-04-01

    The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5'-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/-) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/- mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

  3. H3K9 trimethylation precedes DNA methylation during sheep oogenesis: HDAC1, SUV39H1, G9a, HP1, and Dnmts are involved in these epigenetic events.

    PubMed

    Russo, Valentina; Bernabò, Nicola; Di Giacinto, Oriana; Martelli, Alessandra; Mauro, Annunziata; Berardinelli, Paolo; Curini, Valentina; Nardinocchi, Delia; Mattioli, Mauro; Barboni, Barbara

    2013-01-01

    The oocyte, to become a fully mature gamete, has to acquire a correct pattern of DNA methylation on its genome; this epigenetic event represents the major point of the molecular mechanisms that occur during postnatal oogenesis. It is known that an intimate link exists between DNA methylation and histone posttranslational modifications, such as trimethylation of lysine 9 on histone 3 (H3K9me3), that is essential in the silencing of gene transcription. What remains unclear is the precise sequence of these two epigenetic events and the protein expression of the enzymes that catalyze this epigenetic maturation during oogenesis. To identify the key molecules involved in global DNA methylation and H3K9me3, a biological network-based computational model was realized. Then, the spatiotemporal distribution of the proteins, identified from the biological network, was assessed during postnatal oogenesis. The results obtained suggest the existence of a sequential cascade of events in which H3K9me3 is the primary step followed by DNA methylation. These two epigenetic marks are realized due to the recruitment of the HDAC1, SUV39H1, G9a, HP1, and Dnmt3a, which were always localized in the nuclei of the oocytes and were dependent on chromatin configuration. These results involving DNA methylation and H3K9me3 are crucial in defining the oocyte developmental competence.

  4. BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation

    PubMed Central

    Carey, Timothy S.; Cao, Zubing; Choi, Inchul; Ganguly, Avishek; Wilson, Catherine A.; Paul, Soumen

    2015-01-01

    During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling. PMID:26416882

  5. BRG1 Governs Nanog Transcription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation.

    PubMed

    Carey, Timothy S; Cao, Zubing; Choi, Inchul; Ganguly, Avishek; Wilson, Catherine A; Paul, Soumen; Knott, Jason G

    2015-12-01

    During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

  6. MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a

    SciTech Connect

    Chang, Yanqi; Sun, Lidong; Kokura, Kenji; Horton, John R.; Fukuda, Mikiko; Espejo, Alexsandra; Izumi, Victoria; Koomen, John M.; Bedford, Mark T.; Zhang, Xing; Shinkai, Yoichi; Fang, Jia; Cheng, Xiaodong

    2011-11-30

    DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a-MPP8-GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin.

  7. Neurospora chromosomes are organized by blocks of importin alpha-dependent heterochromatin that are largely independent of H3K9me3

    PubMed Central

    Galazka, Jonathan M.; Klocko, Andrew D.; Uesaka, Miki; Honda, Shinji; Selker, Eric U.; Freitag, Michael

    2016-01-01

    Eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that presumably result from interactions between the chromatin constituents—proteins, DNA, and RNA—within the physical constraints of the nucleus. We used chromosome conformation capture (3C) followed by high-throughput sequencing (Hi-C) with wild-type and mutant strains of Neurospora crassa to gain insight into the role of heterochromatin in the organization and function of the genome. We tested the role of three proteins thought to be important for establishment of heterochromatin, namely, the histone H3 lysine 9 methyltransferase DIM-5, Heterochromatin Protein 1 (HP1), which specifically binds to the product of DIM-5 (trimethylated H3 lysine 9 [H3K9me3]), and DIM-3 (importin alpha), which is involved in DIM-5 localization. The average genome configuration of the wild-type strain revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, subtelomeres, and interspersed heterochromatin. Surprisingly, loss of either H3K9me3 or HP1 had only mild effects on heterochromatin compaction, whereas dim-3 caused more drastic changes, specifically decreasing interactions between constitutive heterochromatic domains. Thus, associations between heterochromatic regions are a major component of the chromosome conformation in Neurospora, but two widely studied key heterochromatin proteins are not necessary, implying that undefined protein factors play key roles in maintaining overall chromosome organization. PMID:27260477

  8. Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases.

    PubMed

    Bártová, Eva; Pacherník, Jirí; Harnicarová, Andrea; Kovarík, Ales; Kovaríková, Martina; Hofmanová, Jirina; Skalníková, Magdalena; Kozubek, Michal; Kozubek, Stanislav

    2005-11-01

    The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

  9. `Up-regulation of histone acetylation induced by social defeat mediates the conditioned rewarding effects of cocaine.

    PubMed

    Montagud-Romero, S; Montesinos, J; Pascual, M; Aguilar, M A; Roger-Sanchez, C; Guerri, C; Miñarro, J; Rodríguez-Arias, M

    2016-10-03

    Social defeat (SD) induces a long-lasting increase in the rewarding effects of psychostimulants measured using the self-administration and conditioned place procedures (CPP). However, little is known about the epigenetic changes induced by social stress and about their role in the increased response to the rewarding effects of psychostimulants. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced behavioral changes, we addressed the hypothesis that SD induces transcriptional changes by histone modifications associated with the acquisition of place conditioning. After a fourth defeat, H3(K9) acetylation was decreased in the hippocampus, while there was an increase of HAT and a decrease of HDAC levels in the cortex. Three weeks after the last defeat, mice displayed an increase in histone H4(K12) acetylation and an upregulation of histone acetyl transferase (HAT) activity in the hippocampus. In addition, H3(K4)me3, which is closely associated with transcriptional initiation, was also augmented in the hippocampus three weeks after the last defeat. Inhibition of HAT by curcumin (100mg/kg) before each SD blocked the increase in the conditioned reinforcing effects of 1mg/kg of cocaine, while inhibition of HDAC by valproic acid (500mg/kg) before social stress potentiated cocaine-induced CPP. Preference was reinstated when animals received a priming dose of 0.5mg/kg of cocaine, an effect that was absent in untreated defeated mice. These results suggest that the experience of SD induces chromatin remodeling, alters histone acetylation and methylation, and modifies the effects of cocaine on place conditioning. They also point to epigenetic mechanisms as potential avenues leading to new treatments for the long-term effects of social stress on drug addiction.

  10. Swimming exercise ameliorates neurocognitive impairment induced by neonatal exposure to isoflurane and enhances hippocampal histone acetylation in mice.

    PubMed

    Zhong, T; Ren, F; Huang, C S; Zou, W Y; Yang, Y; Pan, Y D; Sun, B; Wang, E; Guo, Q L

    2016-03-01

    Isoflurane-induced neurocognitive impairment in the developing rodent brain is well documented, and regular physical exercise has been demonstrated to be a viable intervention for some types of neurocognitive impairment. This study was designed to investigate the potential protective effect of swimming exercise on both neurocognitive impairment caused by repeated neonatal exposure to isoflurane and the underlying molecular mechanism. Mice received 0.75% isoflurane exposures for 4h on postnatal days 7, 8, and 9. From the third month after anesthesia, the mice were subjected to regular swimming exercise for 4weeks, followed by a contextual fear condition (CFC) trial. We found that repeated neonatal exposure to isoflurane reduced freezing behavior during CFC testing and deregulated hippocampal histone H4K12 acetylation. Conversely, mice subjected to regular swimming exercise showed enhanced hippocampal H3K9, H4K5, and H4K12 acetylation levels, increased numbers of c-Fos-positive cells 1h after CFC training, and less isoflurane-induced memory impairment. We also observed increases in histone acetylation and of cAMP-response element-binding protein (CREB)-binding protein (CBP) during the swimming exercise program. The results suggest that neonatal isoflurane exposure-induced memory impairment was associated with dysregulation of H4K12 acetylation, which may lead to less hippocampal activation following learning tasks. Swimming exercise was associated with enhanced hippocampal histone acetylation and CBP expression. Exercise most likely ameliorated isoflurane-induced memory impairment by enhancing hippocampal histone acetylation and activating more neuron cells during memory formation.

  11. Study on the effects of blueberry treatment on histone acetylation modification of CCl4-induced liver disease in rats.

    PubMed

    Zhan, W; Liao, X; Tian, T; Yu, L; Liu, X; Li, B; Liu, J; Han, B; Xie, R J; Ji, Q H; Yang, Q

    2017-02-16

    The objective of this study was to investigate the effects of blueberry treatment on histone acetylation modification of carbon tetrachloride (CCl4)-induced liver disease in rats. Laboratory rats were randomly divided into control, hepatic fibrosis, blueberry treatment, blueberry intervention, and natural recovery groups. Rats in the model groups were treated with CCl4 administered subcutaneously at 4- and 8-week intervals, and then executed. Both the 4- and 8-week treatment groups were treated with blueberry juice for 8 weeks, and then executed after 12 and 16 weeks, respectively. Following the experiment, four liver function and hepatic fibrosis indices were measured. Liver index was calculated, hematoxylin-eosin staining was conducted, and H3K9, H3K14, and H3K18 expressions were evaluated among the nuclear proteins of the liver tissues. No differences in alanine transaminase were noted between the control and intervention groups, but significant differences were detected among the model, treatment, and natural recovery groups (P < 0.01). Significant differences were also observed in aspartate transaminase, hyaluronic acid, and collagen IV among the model, treatment, intervention, and natural recovery groups (P < 0.01, P < 0.01, P < 0.01). Liver index, and H3K9 and H3K14 expression were significantly different among the model groups (P < 0.05 and P < 0.01), whereas H3K18 expression was dramatically different among model, treatment, intervention, and natural recovery groups (P < 0.01). Following blueberry treatment, rat liver function and hepatic fibrosis improved, potentially indicating that blueberry components could regulate histone acetylation and improve liver pathologic changes in rats with CCl4-induced disease.

  12. DNA promoter and histone H3 methylation downregulate NGX6 in gastric cancer cells.

    PubMed

    Liu, Jian; Zhu, Xinjiang; Xu, Xiaoyang; Dai, Dongqiu

    2014-01-01

    Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate tumor metastasis suppressor gene. Our study was to determine whether DNA hypermethylation and histone modification at the NGX6 gene promoter play important roles in silencing NGX6 expression in gastric cancer. NGX6 expression was downregulated in all gastric cancer cells and 76.19 % tissues. In three GC cell lines, hypermethylated NGX6 loci were characterized by histone H3-K9 hypoacetylation and hypermethylation. Trichostatin A treatment could moderately increase H3-K9 acetylation at the silenced loci; however, it had no effect on DNA and H3-K9 methylation and minimal effects on NGX6 expression. In contrast, 5'aza-2'-deoxycytidine treatment could rapidly decrease DNA and H3-K9 methylation at the silenced loci, leading to the reexpression of NGX6. Combined treatment with 5'aza-2'-deoxycytidine and trichostatin A had synergistic effects on the reexpression of NGX6 at the hypermethylation loci. Our current study shows that NGX6 expression is downregulated in GC cancer cells and tissues due to NGX6 promoter methylation and H3-K9 methylation, but not H3-K9 acetylation. Our findings indicate that the downregulation of NGX6 expression contributes to the development and progression of gastric cancer. More studies are needed to determine the precise mechanism of NGX6 in the progression of gastric cancer.

  13. Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena

    PubMed Central

    1989-01-01

    In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly. PMID:2654136

  14. Involvement of histone methylation and phosphorylation in regulation of transcription by thyroid hormone receptor.

    PubMed

    Li, Jiwen; Lin, Qiushi; Yoon, Ho-Geun; Huang, Zhi-Qing; Strahl, Brian D; Allis, C David; Wong, Jiemin

    2002-08-01

    Previous studies have established an important role of histone acetylation in transcriptional control by nuclear hormone receptors. With chromatin immunoprecipitation assays, we have now investigated whether histone methylation and phosphorylation are also involved in transcriptional regulation by thyroid hormone receptor (TR). We found that repression by unliganded TR is associated with a substantial increase in methylation of H3 lysine 9 (H3-K9) and a decrease in methylation of H3 lysine 4 (H3-K4), methylation of H3 arginine 17 (H3-R17), and a dual modification of phosphorylation of H3 serine 10 and acetylation of lysine 14 (pS10/acK14). On the other hand, transcriptional activation by liganded TR is coupled with a substantial decrease in both H3-K4 and H3-K9 methylation and a robust increase in H3-R17 methylation and the dual modification of pS10/acK14. Trichostatin A treatment results in not only histone hyperacetylation but also an increase in methylation of H3-K4, increase in dual modification of pS10/acK14, and reduction in methylation of H3-K9, revealing an extensive interplay between histone acetylation, methylation, and phosphorylation. In an effort to understand the underlying mechanism for an increase in H3-K9 methylation during repression by unliganded TR, we demonstrated that TR interacts in vitro with an H3-K9-specific histone methyltransferase (HMT), SUV39H1. Functional analysis indicates that SUV39H1 can facilitate repression by unliganded TR and in so doing requires its HMT activity. Together, our data uncover a novel role of H3-K9 methylation in repression by unliganded TR and provide strong evidence for the involvement of multiple distinct histone covalent modifications (acetylation, methylation, and phosphorylation) in transcriptional control by nuclear hormone receptors.

  15. FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis

    PubMed Central

    Kato, Yasuko; Alavattam, Kris G.; Sin, Ho-Su; Meetei, Amom Ruhikanta; Pang, Qishen; Andreassen, Paul R.; Namekawa, Satoshi H.

    2015-01-01

    Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline. PMID:26123487

  16. Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires Saga/Ada-mediated histone acetylation.

    PubMed

    Nützmann, Hans-Wilhelm; Reyes-Dominguez, Yazmid; Scherlach, Kirstin; Schroeckh, Volker; Horn, Fabian; Gacek, Agnieszka; Schümann, Julia; Hertweck, Christian; Strauss, Joseph; Brakhage, Axel A

    2011-08-23

    Sequence analyses of fungal genomes have revealed that the potential of fungi to produce secondary metabolites is greatly underestimated. In fact, most gene clusters coding for the biosynthesis of antibiotics, toxins, or pigments are silent under standard laboratory conditions. Hence, it is one of the major challenges in microbiology to uncover the mechanisms required for pathway activation. Recently, we discovered that intimate physical interaction of the important model fungus Aspergillus nidulans with the soil-dwelling bacterium Streptomyces rapamycinicus specifically activated silent fungal secondary metabolism genes, resulting in the production of the archetypal polyketide orsellinic acid and its derivatives. Here, we report that the streptomycete triggers modification of fungal histones. Deletion analysis of 36 of 40 acetyltransferases, including histone acetyltransferases (HATs) of A. nidulans, demonstrated that the Saga/Ada complex containing the HAT GcnE and the AdaB protein is required for induction of the orsellinic acid gene cluster by the bacterium. We also showed that Saga/Ada plays a major role for specific induction of other biosynthesis gene clusters, such as sterigmatocystin, terrequinone, and penicillin. Chromatin immunoprecipitation showed that the Saga/Ada-dependent increase of histone 3 acetylation at lysine 9 and 14 occurs during interaction of fungus and bacterium. Furthermore, the production of secondary metabolites in A. nidulans is accompanied by a global increase in H3K14 acetylation. Increased H3K9 acetylation, however, was only found within gene clusters. This report provides previously undescribed evidence of Saga/Ada dependent histone acetylation triggered by prokaryotes.

  17. Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment.

    PubMed

    Stewart, M David; Li, Jiwen; Wong, Jiemin

    2005-04-01

    Histone H3 lysine 9 (H3-K9) methylation has been shown to correlate with transcriptional repression and serve as a specific binding site for heterochromatin protein 1 (HP1). In this study, we investigated the relationship between H3-K9 methylation, transcriptional repression, and HP1 recruitment by comparing the effects of tethering two H3-K9-specific histone methyltransferases, SUV39H1 and G9a, to chromatin on transcription and HP1 recruitment. Although both SUV39H1 and G9a induced H3-K9 methylation and repressed transcription, only SUV39H1 was able to recruit HP1 to chromatin. Targeting HP1 to chromatin required not only K9 methylation but also a direct protein-protein interaction between SUV39H1 and HP1. Targeting methyl-K9 or a HP1-interacting region of SUV39H1 alone to chromatin was not sufficient to recruit HP1. We also demonstrate that methyl-K9 can suppress transcription independently of HP1 through a mechanism involving histone deacetylation. In an effort to understand how H3-K9 methylation led to histone deacetylation in both H3 and H4, we found that H3-K9 methylation inhibited histone acetylation by p300 but not its association with chromatin. Collectively, these data indicate that H3-K9 methylation alone can suppress transcription but is insufficient for HP1 recruitment in the context of chromatin exemplifying the importance of chromatin-associated factors in reading the histone code.

  18. MeCP2 binds to nucleosome free (linker DNA) regions and to H3K9/H3K27 methylated nucleosomes in the brain.

    PubMed

    Thambirajah, Anita A; Ng, Marlee K; Frehlick, Lindsay J; Li, Andra; Serpa, Jason J; Petrotchenko, Evgeniy V; Silva-Moreno, Begonia; Missiaen, Kristal K; Borchers, Christoph H; Adam Hall, J; Mackie, Ryan; Lutz, Frank; Gowen, Brent E; Hendzel, Michael; Georgel, Philippe T; Ausió, Juan

    2012-04-01

    Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2'-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me(2) and H3K27me(3) in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications.

  19. High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

    PubMed

    Yu, Jingwen; Wu, Yanqing; Yang, Peixin

    2016-05-01

    Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses

  20. Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens.

    PubMed

    Martin, Tracey A; Jayanthi, Subramaniam; McCoy, Michael T; Brannock, Christie; Ladenheim, Bruce; Garrett, Tiffany; Lehrmann, Elin; Becker, Kevin G; Cadet, Jean Lud

    2012-01-01

    Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might

  1. Post-Translational Modifications of Histones in Human Sperm.

    PubMed

    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.

  2. A role for histone acetylation mechanisms in adolescent alcohol exposure-induced deficits in hippocampal brain-derived neurotrophic factor expression and neurogenesis markers in adulthood.

    PubMed

    Sakharkar, Amul J; Vetreno, Ryan P; Zhang, Huaibo; Kokare, Dadasaheb M; Crews, Fulton T; Pandey, Subhash C

    2016-12-01

    Binge drinking during adolescence is a risk factor for neuropsychiatric disorders that can develop later in life. Histone acetylation is an important epigenetic mechanism that contributes to neurodevelopment. We investigated the effects of adolescent intermittent ethanol (AIE) exposure, as opposed to normal saline (AIS) exposure, on histone acetylation-mediated regulation of brain-derived neurotrophic factor (BDNF) expression and developmental stages of neurogenesis (proliferating and immature neurons) in the hippocampus in adulthood. AIE exposure increased whole hippocampal histone deacetylase (HDAC) activity and decreased binding protein of cyclic adenosine monophosphate response element binding protein (CBP) and histone H3-K9 acetylation levels in the CA1, CA2, and CA3 regions of the hippocampus. BDNF protein and exon IV mRNA levels in the CA1 and CA3 regions of the hippocampus of AIE-exposed adult rats were decreased as compared to AIS-exposed adult rats. AIE-induced anxiety-like behaviors and deficits in histone H3 acetylation at BDNF exon IV promoter in the hippocampus during adulthood, which were reversed by treatment with the HDAC inhibitor, trichostatin A (TSA). Similarly, neurogenesis was inhibited by AIE in adulthood as demonstrated by the decrease in Ki-67 and doublecortin (DCX)-positive cells in the dentate gyrus, which was normalized by TSA treatment. These results indicate that AIE exposure increases HDACs and decreases CBP levels that may be associated with a decrease in histone H3 acetylation in the hippocampus. These epigenetic changes potentially decrease BDNF expression and inhibit neurogenesis in the hippocampus that may be involved in AIE-induced behavioral abnormalities, including anxiety, in adulthood.

  3. piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells.

    PubMed

    Pezic, Dubravka; Manakov, Sergei A; Sachidanandam, Ravi; Aravin, Alexei A

    2014-07-01

    Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.

  4. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  5. Histone H4 lysine 16 acetylation breaks the genome's silence

    PubMed Central

    Shia, Wei-Jong; Pattenden, Samantha G; Workman, Jerry L

    2006-01-01

    Acetylation at histone H4 lysine 16 is involved in many cellular processes in organisms as diverse as yeast and humans. A recent biochemical study pinpoints this particular acetylation mark as a switch for changing chromatin from a repressive to a transcriptionally active state. PMID:16689998

  6. Altering histone acetylation status in donor cells with suberoylanilide hydroxamic acid does not affect dog cloning efficiency.

    PubMed

    Kim, Min Jung; Oh, Hyun Ju; Kim, Geon A; Suh, Han Na; Jo, Young Kwang; Choi, Yoo Bin; Kim, Dong Hoon; Han, Ho Jae; Lee, Byeong Chun

    2015-10-15

    Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs, and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on dog cloning efficiency. Relative messenger RNA expressions of the bax1/bcl2 ratio and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 hours) were compared. Treatment with 1 μM for 20 hours showed significantly lower bax1/bcl2 and Dnmt1 transcript abundance. Acetylation of H3K9 was significantly increased after SAHA treatment, but H4K5, H4K8 and H4K16 were not changed. After SCNT using control or donor cells treated with SAHA, a total of 76 and 64 cloned embryos were transferred to seven and five recipients, respectively. Three fetuses were diagnosed in both control and SAHA-treated groups by ultrasonography 29 days after the embryo transfer, but there was no significant difference in the pregnancy rate (4.2% vs. 4.3%). In conclusion, although SAHA treatment as used in this study significantly decreased bax1/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 acetylation, it was not enough to increase in vivo developmental competence of cloned dog embryos.

  7. Histone Acetylation Inhibitors Promote Axon Growth in Adult DRG neurons

    PubMed Central

    Lin, Shen; Nazif, Kutaiba; Smith, Alexander; Baas, Peter W; Smith, George M

    2015-01-01

    Intrinsic mechanisms that guide damaged axons to regenerate following spinal cord injury remain poorly understood. Manipulation of posttranslational modifications of key proteins in mature neurons could re-invigorate growth machinery after injury. One such modification is acetylation, a reversible process controlled by two enzyme families acting in opposition, the Histone Deacetylases (HDACs) and the Histone Acetyl Transferases (HATs). While acetylated histones in the nucleus is associated with upregulation of growth promoting genes, de-acetylated tubulin in the axoplasm is associated with more labile microtubules, conducive to axon growth. In this study we investigated the effects of HAT inhibitors and HDAC inhibitors on cultured adult dorsal root ganglia (DRG) neurons. We found that inhibition of HATs, using Anacardic Acid or CPTH2, improved axon outgrowth, while inhibition of HDACs using TSA or Tubacin, inhibited axon growth. Furthermore, Anacardic Acid increased the number of axons able to cross an inhibitory chondroitin sulfate proteoglycan (CSPG) border. Histone acetylation, but not tubulin acetylation levels, was affected by HAT inhibitors, whereas tubulin acetylation levels were increased in the presence of HDAC inhibitor Tubacin. Although microtubule stabilizing drug taxol did not have an effect on the lengths of DRG axons, nocodazole decreased axon lengths. While the mechanistic basis will require future studies, our data show that inhibitors of HAT can augment axon growth in adult DRG neurons, with the potential of aiding axon growth over inhibitory substrates produced by the glial scar. PMID:25702820

  8. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

    SciTech Connect

    Ren, Aishu; Qiu, Yu; Cui, Hongjuan; Fu, Gang

    2015-03-27

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspase 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model.

  9. BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites.

    PubMed

    Emberly, Eldon; Blattes, Roxane; Schuettengruber, Bernd; Hennion, Magali; Jiang, Nan; Hart, Craig M; Käs, Emmanuel; Cuvier, Olivier

    2008-12-23

    Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest. Here, we combined chromatin immunoprecipitations and computational approaches to break down the binding signature of the Drosophila boundary element-associated factor (BEAF) subfamily. We identify a dual-core BEAF binding signature at 1,720 sites genome-wide, defined by five to six BEAF binding motifs bracketing 200 bp AT-rich nuclease-resistant spacers. Dual-cores are tightly linked to hundreds of genes highly enriched in cell-cycle and chromosome organization/segregation annotations. siRNA depletion of BEAF from cells leads to cell-cycle and chromosome segregation defects. Quantitative RT-PCR analyses in BEAF-depleted cells show that BEAF controls the expression of dual core-associated genes, including key cell-cycle and chromosome segregation regulators. beaf mutants that impair its insulating function by preventing proper interactions of BEAF complexes with the dual-cores produce similar effects in embryos. Chromatin immunoprecipitations show that BEAF regulates transcriptional activity by restricting the deposition of methylated histone H3K9 marks in dual-cores. Our results reveal a novel role for BEAF chromatin dual-cores in regulating a distinct set of genes involved in chromosome organization/segregation and the cell cycle.

  10. Comparative analysis of expression of histone H2a genes in mouse

    PubMed Central

    Nishida, Hiromi; Suzuki, Takahiro; Ookawa, Hiroki; Tomaru, Yasuhiro; Hayashizaki, Yoshihide

    2005-01-01

    Background At least 18 replication-dependent histone H2a genes are distributed in 3 Hist gene clusters on different chromosomes of the mouse genome. In this analysis we designed specific PCR primers for each histone H2a transcript and studied the expression levels and patterns using quantitative RT-PCR (qRT-PCR). In addition, we compared histone H3 K9 acetylation levels in the promoter regions of H2a genes by ChIP (chromatin immunoprecipitation) – quantitative PCR (qPCR) analysis. Results RT-PCR analysis indicated that all 20 histone H2a genes assessed in this study are expressed. The replication-dependent histone H2a genes have different expression levels but similar expression patterns. Among the 20 histone H2a genes, the expression-level of H2afz, a replication-independent gene, was highest, and that of Hist1h2aa, a replication-dependent gene, was lowest. Among 18 replication-dependent H2a genes, the expression level of Hist3h2a was highest. The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in promoter regions of both H2afz and Hist3h2a are clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa is similar to that in the γ-satellite region. Conclusion These results strongly suggest that histone H3 K9 acetylation plays a role in the expression of histone genes. PMID:16098230

  11. RAPID SEMISYNTHESIS OF ACETYLATED AND SUMOYLATED HISTONE ANALOGS

    PubMed Central

    Dhall, Abhinav; Weller, Caroline E.

    2016-01-01

    The density and diversity of post-translational modifications (PTMs) observed in histone proteins typically limits their purification to homogeneity from biological sources. Access to quantities of uniformly modified histones is, however, critical for investigating the downstream effects of histone PTMs on chromatin-templated processes. Therefore, a number of semisynthetic methodologies have been developed to generate histones bearing precisely defined PTMs or close analogs thereof. In this chapter, we present two optimized and rapid strategies for generating functional analogs of site-specifically acetylated and sumoylated histones. First, we describe a convergent strategy to site-specifically attach the small ubiquitin-like modifier-3 (SUMO-3) protein to the site of Lys12 in histone H4 by means of a disulfide linkage. We then describe the generation of thialysine analogs of histone H3 acetylated at Lys 14 or Lys 56, using thiol-ene coupling chemistry. Both strategies afford multi-milligram quantities of uniformly modified histones that are easily incorporated into mononucleosomes and nucleosome arrays for biophysical and biochemical investigations. These methods are readily extendable to any desired sites in the four core nucleosomal histones and their variant forms. PMID:27423861

  12. Epigenetic Regulation of Interleukin 6 by Histone Acetylation in Macrophages and Its Role in Paraquat-Induced Pulmonary Fibrosis

    PubMed Central

    Hu, Lingli; Yu, Yanfang; Huang, Huijie; Fan, Hanting; Hu, Li; Yin, Caiyong; Li, Kai; Fulton, David J. R.; Chen, Feng

    2017-01-01

    Overexpression of interleukin 6 (IL-6) has been proposed to contribute to pulmonary fibrosis and other fibrotic diseases. However, the regulatory mechanisms and the role of IL-6 in fibrosis remain poorly understood. Epigenetics refers to alterations of gene expression without changes in the DNA sequence. Alternation of chromatin accessibility by histone acetylation acts as a critical epigenetic mechanism to regulate various gene transcriptions. The goal of this study was to determine the impact of IL-6 in paraquat (PQ)-induced pulmonary fibrosis and to explore whether the epigenetic regulations may play a role in transcriptional regulation of IL-6. In PQ-treated lungs and macrophages, we found that the mRNA and protein expression of IL-6 was robustly increased in a time-dependent and a dose-dependent manner. Our data demonstrated that PQ-induced IL-6 expression in macrophages plays a central role in pulmonary fibrosis through enhanced epithelial-to-mesenchymal transition (EMT). IL-6 expression and its role to enhance PQ-induced pulmonary fibrosis were increased by histone deacetylase (HDAC) inhibition and prevented by histone acetyltransferase (HAT) inhibition. In addition, the ability of CRISPR-ON transcription activation system (CRISPR-ON) to promote transcription of IL-6 was enhanced by HDAC inhibitor and blocked by HAT inhibitor. Chromatin immunoprecipitation experiments revealed that HDAC inhibitor increased histones activation marks H3K4me3 and H3K9ac at IL-6 promoter regions. In conclusion, IL-6 functioning through EMT in PQ-induced pulmonary fibrosis was regulated dynamically by HDAC and HAT both in vitro and in vivo via epigenetically regulating chromatin accessibility. PMID:28194150

  13. ATRX binds to atypical chromatin domains at the 3′ exons of zinc finger genes to preserve H3K9me3 enrichment

    PubMed Central

    Chowdhury, Asif H.; Hasson, Dan; Dyer, Michael A.

    2016-01-01

    ABSTRACT ATRX is a SWI/SNF chromatin remodeler proposed to govern genomic stability through the regulation of repetitive sequences, such as rDNA, retrotransposons, and pericentromeric and telomeric repeats. However, few direct ATRX target genes have been identified and high-throughput genomic approaches are currently lacking for ATRX. Here we present a comprehensive ChIP-sequencing study of ATRX in multiple human cell lines, in which we identify the 3′ exons of zinc finger genes (ZNFs) as a new class of ATRX targets. These 3′ exonic regions encode the zinc finger motifs, which can range from 1–40 copies per ZNF gene and share large stretches of sequence similarity. These regions often contain an atypical chromatin signature: they are transcriptionally active, contain high levels of H3K36me3, and are paradoxically enriched in H3K9me3. We find that these ZNF 3′ exons are co-occupied by SETDB1, TRIM28, and ZNF274, which form a complex with ATRX. CRISPR/Cas9-mediated loss-of-function studies demonstrate (i) a reduction of H3K9me3 at the ZNF 3′ exons in the absence of ATRX and ZNF274 and, (ii) H3K9me3 levels at atypical chromatin regions are particularly sensitive to ATRX loss compared to other H3K9me3-occupied regions. As a consequence of ATRX or ZNF274 depletion, cells with reduced levels of H3K9me3 show increased levels of DNA damage, suggesting that ATRX binds to the 3′ exons of ZNFs to maintain their genomic stability through preservation of H3K9me3. PMID:27029610

  14. Effect of histone acetylation modification with MGCD0103, a histone deacetylase inhibitor, on nuclear reprogramming and the developmental competence of porcine somatic cell nuclear transfer embryos.

    PubMed

    Jin, Long; Zhu, Hai-Ying; Guo, Qing; Li, Xiao-Chen; Zhang, Yu-Chen; Cui, Cheng-Du; Li, Wen-Xue; Cui, Zheng-Yun; Yin, Xi-Jun; Kang, Jin-Dan

    2017-01-01

    Cloning remains as an important technique to enhance the reconstitution and distribution of animal population with high-genetic merit. One of the major detrimental factors of this technique is the abnormal epigenetic modifications. MGCD0103 is known as a histone deacetylase inhibitor. In this study, we investigated the effect of MGCD0103 on the in vitro blastocyst formation rate in porcine somatic cell nuclear transferred (SCNT) embryos and expression in acetylation of the histone H3 lysine 9 and histone H4 lysine 12. We compared the in vitro embryonic development of SCNT embryos treated with different concentrations of MGCD0103 for 24 hours. Our results reported that treating with 0.2-μM MGCD0103 for 24 hours effectively improved the development of SCNT embryos, in comparison to the control group (blastocyst formation rate, 25.5 vs. 10.7%, P < 0.05). Then we tested the in vitro development of SCNT embryos treated with 0.2-μM MGCD0103 for various intervals after activation. Treatment for 6 hours significantly improved the development of pig SCNT embryos, compared with the control group (blastocyst formation rate, 21.2 vs. 10.5%, P < 0.05). Furthermore, MGCD0103 supplementation significantly (P < 0.05) increases the average fluorescence intensity of AcH3K9 and AcH4K12 in embryos at the pseudo-pronuclear stage. To examine the in vivo development, MGCD0103-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and three fetuses developed. These results suggest that MGCD0103 can enhance the nuclear reprogramming and improve in vitro developmental potential of porcine SCNT embryos.

  15. Histone acetylation associated up-regulation of the cell wall related genes is involved in salt stress induced maize root swelling

    PubMed Central

    2014-01-01

    Background Salt stress usually causes crop growth inhibition and yield decrease. Epigenetic regulation is involved in plant responses to environmental stimuli. The epigenetic regulation of the cell wall related genes associated with the salt-induced cellular response is still little known. This study aimed to analyze cell morphological alterations in maize roots as a consequence of excess salinity in relation to the transcriptional and epigenetic regulation of the cell wall related protein genes. Results In this study, maize seedling roots got shorter and displayed swelling after exposure to 200 mM NaCl for 48 h and 96 h. Cytological observation showed that the growth inhibition of maize roots was due to the reduction in meristematic zone cell division activity and elongation zone cell production. The enlargement of the stele tissue and cortex cells contributed to root swelling in the elongation zone. The cell wall is thought to be the major control point for cell enlargement. Cell wall related proteins include xyloglucan endotransglucosylase (XET), expansins (EXP), and the plasma membrane proton pump (MHA). RT-PCR results displayed an up-regulation of cell wall related ZmEXPA1, ZmEXPA3, ZmEXPA5, ZmEXPB1, ZmEXPB2 and ZmXET1 genes and the down-regulation of cell wall related ZmEXPB4 and ZmMHA genes as the duration of exposure was increased. Histone acetylation is regulated by HATs, which are often correlated with gene activation. The expression of histone acetyltransferase genes ZmHATB and ZmGCN5 was increased after 200 mM NaCl treatment, accompanied by an increase in the global acetylation levels of histones H3K9 and H4K5. ChIP experiment showed that the up-regulation of the ZmEXPB2 and ZmXET1 genes was associated with the elevated H3K9 acetylation levels on the promoter regions and coding regions of these two genes. Conclusions These data suggested that the up-regulation of some cell wall related genes mediated cell enlargement to possibly mitigate the

  16. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies.

    PubMed

    Rothbart, Scott B; Lin, Shu; Britton, Laura-Mae; Krajewski, Krzysztof; Keogh, Michael-C; Garcia, Benjamin A; Strahl, Brian D

    2012-01-01

    Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

  17. Regulation of Histone Acetylation by Autophagy in Parkinson Disease*

    PubMed Central

    Park, Goonho; Tan, Jieqiong; Garcia, Guillermina; Kang, Yunyi; Salvesen, Guy; Zhang, Zhuohua

    2016-01-01

    Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP+)-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP+-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP+-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis. PMID:26699403

  18. Regulation of Histone Acetylation by Autophagy in Parkinson Disease.

    PubMed

    Park, Goonho; Tan, Jieqiong; Garcia, Guillermina; Kang, Yunyi; Salvesen, Guy; Zhang, Zhuohua

    2016-02-12

    Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP(+))-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP(+)-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP(+)-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis.

  19. The induction of H3K9 methylation by PIWIL4 at the p16{sup Ink4a} locus

    SciTech Connect

    Sugimoto, Keiki; Kage, Hidenori; Aki, Naomi; Sano, Atsushi; Kitagawa, Hiroshi; Nagase, Takahide; Yatomi, Yutaka; Ohishi, Nobuya; Takai, Daiya . E-mail: dtakai-ind@umin.ac.jp

    2007-08-03

    The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16{sup Ink4a} (CDKN2A) locus. The elevated level of histone methylation resulted in the downregulation of the p16{sup Ink4a} gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.

  20. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation.

    PubMed

    Bailey, Zachary S; Grinter, Michael B; VandeVord, Pamela J

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  1. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    PubMed Central

    Bailey, Zachary S.; Grinter, Michael B.; VandeVord, Pamela J.

    2016-01-01

    Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17

  2. Causal role of histone acetylations in enhancer function

    PubMed Central

    Pradeepa, Madapura M.

    2017-01-01

    ABSTRACT Enhancers control development and cellular function by spatiotemporal regulation of gene expression. Co-occurrence of acetylation of histone H3 at lysine 27 (H3K27ac) and mono methylation of histone H3 at lysine 4 (H3K4me1) has been widely used for identification of active enhancers. However, increasing evidence suggests that using this combination of marks alone for enhancer identification gives an incomplete picture of the active enhancer repertoire. We have shown that the H3 globular domain acetylations, H3K64ac and H3K122ac, and an H4 tail acetylation, H4K16ac, are enriched at active enhancers together with H3K27ac, and also at a large number of enhancers without detectable H3K27ac. We propose that acetylations at these lysine residues of histones H3 and H4 might function by directly affecting chromatin structure, nucleosome–nucleosome interactions, nucleosome stability, and transcription factor accessibility. PMID:27792455

  3. The loss of histone H3 lysine 9 acetylation due to dSAGA-specific dAda2b mutation influences the expression of only a small subset of genes

    PubMed Central

    Zsindely, Nóra; Pankotai, Tibor; Újfaludi, Zsuzsanna; Lakatos, Dániel; Komonyi, Orbán; Bodai, László; Tora, László; Boros, Imre M.

    2009-01-01

    In Drosophila, the dADA2b-containing dSAGA complex is involved in histone H3 lysine 9 and 14 acetylation. Curiously, although the lysine 9- and 14-acetylated histone H3 levels are drastically reduced in dAda2b mutants, these animals survive until a late developmental stage. To study the molecular consequences of the loss of histone H3 lysine 9 and 14 acetylation, we compared the total messenger ribonucleic acid (mRNA) profiles of wild type and dAda2b mutant animals at two developmental stages. Global gene expression profiling indicates that the loss of dSAGA-specific H3 lysine 9 and 14 acetylation results in the expression change (up- or down-regulation) of a rather small subset of genes and does not cause a general transcription de-regulation. Among the genes up-regulated in dAda2b mutants, particularly high numbers are those which play roles in antimicrobial defense mechanisms. Results of chromatin immunoprecipitation experiments indicate that in dAda2b mutants, the lysine 9-acetylated histone H3 levels are decreased both at dSAGA up- and down-regulated genes. In contrast to that, in the promoters of dSAGA-independent ribosomal protein genes a high level of histone H3K9ac is maintained in dAda2b mutants. Our data suggest that by acetylating H3 at lysine 9, dSAGA modifies Pol II accessibility to specific promoters differently. PMID:19740772

  4. Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

    PubMed

    Hsu, Chia-Chi; Wu, Pei-Shan; Chen, Tien-Chih; Yu, Chun-Wei; Tsai, Wen-Chieh; Wu, Keqiang; Wu, Wen-Luan; Chen, Wen-Huei; Chen, Hong-Hwa

    2014-01-01

    Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.

  5. Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

    PubMed

    Kadiyala, Chandra Sekhar Rao; Zheng, Ling; Du, Yunpeng; Yohannes, Elizabeth; Kao, Hung-Ying; Miyagi, Masaru; Kern, Timothy S

    2012-07-27

    Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.

  6. Global Levels of Histone Modifications in Peripheral Blood Mononuclear Cells of Subjects with Exposure to Nickel

    PubMed Central

    Arita, Adriana; Niu, Jingping; Qu, Qingshan; Zhao, Najuan; Ruan, Ye; Nadas, Arthur; Chervona, Yana; Wu, Fen; Sun, Hong; Hayes, Richard B.

    2011-01-01

    Background: Occupational exposure to nickel (Ni) is associated with an increased risk for lung and nasal cancers. Ni compounds exhibit weak mutagenic activity, cause gene amplification, and disrupt cellular epigenetic homeostasis. However, the Ni-induced changes in global histone modification levels have only been tested in vitro. Objective: This study was conducted in a Chinese population to determine whether occupational exposure to Ni is associated with alterations of global histone modification levels and to evaluate the inter- and intraindividual variance of global histone modification levels. Method: Forty-five subjects with occupational exposure to Ni and 75 referents were recruited. Urinary Ni and global H3K4 trimethylation, H3K9 acetylation, and H3K9 dimethylation levels were measured in peripheral blood mononuclear cells (PBMCs) of subjects. Results: H3K4me3 was elevated in Ni-exposed subjects (0.25% ± 0.11%) compared with referents (0.15% ± 0.04%; p = 0.0004), and H3K9me2 was decreased (Ni-exposed subjects, 0.11% ± 0.05%; referents, 0.15% ± 0.04%; p = 0.003). H3K4me3 was positively (r = 0.4, p = 0.0008) and H3K9ac was negatively (r = 0.1, p = 0.01) associated with urinary Ni. Interindividual variances of H3K4me3, H3K9ac, and H3K9me2 were larger compared with intraindividual variance in both exposure test groups, resulting in reliability coefficients (an estimate of consistency of a set of measurements) of 0.60, 0.67, and 0.79 for H3K4me3, H3K9ac, and H3K9me2, respectively, for Ni-exposed subjects and of 0.75, 0.74, and 0.97, respectively, for referent subjects. Conclusion: The results of this study indicate that occupational exposure to Ni is associated with alterations of global histone modification levels and that measurements of global levels of histone modifications are relatively stable over time in human PBMCs. PMID:22024396

  7. Cell differentiation along multiple pathways accompanied by changes in histone acetylation status.

    PubMed

    Legartová, Soňa; Kozubek, Stanislav; Franek, Michal; Zdráhal, Zbyněk; Lochmanová, Gabriela; Martinet, Nadine; Bártová, Eva

    2014-04-01

    Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4.

  8. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  9. Epigenetic engineering reveals a balance between histone modifications and transcription in kinetochore maintenance

    PubMed Central

    Molina, Oscar; Vargiu, Giulia; Abad, Maria Alba; Zhiteneva, Alisa; Jeyaprakash, A. Arockia; Masumoto, Hiroshi; Kouprina, Natalay; Larionov, Vladimir; Earnshaw, William C.

    2016-01-01

    Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic biology ‘in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoidtetO HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoidtetO array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the ‘epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres. PMID:27841270

  10. Gradual development of a genome-wide H3-K9 trimethylation pattern in paternally derived pig pronucleus.

    PubMed

    Jeong, Young Sun; Yeo, Seungeun; Park, Jung Sun; Lee, Kyung-Kwang; Kang, Yong-Kook

    2007-06-01

    The cytoplasm of a mature oocyte contains many protein complexes that are programmed to restructure incoming sperm chromatins on fertilization. Of the complicated biochemical events that these functional machineries control, the most impressive and important is epigenetic reprogramming. Despite its importance in epigenetic resetting, or "de-differentiation," of gamete genomes back to an incipient status, the mechanisms of epigenetic reprogramming do not seem to be conserved among mammals. Here, we report that, unlike in the mouse, the pig sperm-derived pronucleus is markedly trimethylated at lysine 9 of histone H3 (H3-m(3)K9), which might be associated with preservation of paternally derived cytosine methylation in pig zygotes. The male H3-m(3)K9 pattern is gradually established during pronucleus development, and this process occurs independently of DNA replication. Considering these unique epigenetic features, the pig zygote is, we believe, suited to serve as another model of epigenetic reprogramming that is antithetical to the well-characterized mouse model.

  11. Profiles of Epigenetic Histone Post-translational Modifications at Type 1 Diabetes Susceptible Genes*

    PubMed Central

    Miao, Feng; Chen, Zhuo; Zhang, Lingxiao; Liu, Zheng; Wu, Xiwei; Yuan, Yate-Ching; Natarajan, Rama

    2012-01-01

    Both genetic and environmental factors are implicated in type 1 diabetes (T1D). Because environmental factors can trigger epigenetic changes, we hypothesized that variations in histone post-translational modifications (PTMs) at the promoter/enhancer regions of T1D susceptible genes may be associated with T1D. We therefore evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy nondiabetic controls, and explored their connections to T1D. We used the chromatin immunoprecipitation-linked to microarray approach to profile key histone PTMs, including H3-lysine 4 trimethylation (H3K4me3), H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac), and H4K16Ac at genes within the T1D susceptible loci in lymphocytes, and H3K4me3, H3K9me2, H3K9Ac, and H4K16Ac at the insulin-dependent diabetes mellitus 1 region in monocytes of T1D patients and healthy controls separately. We screened for potential variations in histone PTMs using computational methods to compare datasets from T1D and controls. Interestingly, we observed marked variations in H3K9Ac levels at the upstream regions of HLA-DRB1 and HLA-DQB1 within the insulin-dependent diabetes mellitus 1 locus in T1D monocytes relative to controls. Additional experiments with THP-1 monocytes demonstrated increased expression of HLA-DRB1 and HLA-DQB1 in response to interferon-γ and TNF-α treatment that were accompanied by changes in H3K9Ac at the same promoter regions as that seen in the patient monocytes. These results suggest that the H3K9Ac status of HLA-DRB1 and HLA-DQB1, two genes highly associated with T1D, may be relevant to their regulation and transcriptional response toward external stimuli. Thus, the promoter/enhancer architecture and chromatin status of key susceptible loci could be important determinants in their functional association to T1D susceptibility. PMID:22431725

  12. Histone acetylation dependent energy landscapes in tri-nucleosome revealed by residue-resolved molecular simulations

    PubMed Central

    Chang, Le; Takada, Shoji

    2016-01-01

    Histone tail acetylation is a key epigenetic marker that tends to open chromatin folding and activate transcription. Despite intensive studies, precise roles of individual lysine acetylation in chromatin folding have only been poorly understood. Here, we revealed structural dynamics of tri-nucleosomes with several histone tail acetylation states and analyzed histone tail interactions with DNA by performing molecular simulations at an unprecedentedly high resolution. We found versatile acetylation-dependent landscapes of tri-nucleosome. The H4 and H2A tail acetylation reduced the contact between the first and third nucleosomes mediated by the histone tails. The H3 tail acetylation reduced its interaction with neighboring linker DNAs resulting in increase of the distance between consecutive nucleosomes. Notably, two copies of the same histone in a single nucleosome have markedly asymmetric interactions with DNAs, suggesting specific pattern of nucleosome docking albeit high inherent flexibility. Estimated transcription factor accessibility was significantly high for the H4 tail acetylated structures. PMID:27698366

  13. Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation[W][OPEN

    PubMed Central

    Gent, Jonathan I.; Madzima, Thelma F.; Bader, Rechien; Kent, Matthew R.; Zhang, Xiaoyu; Stam, Maike; McGinnis, Karen M.; Dawe, R. Kelly

    2014-01-01

    RNA-directed DNA methylation (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2. The data revealed that the majority of the genome exists in a heterochromatic state defined by inaccessible chromatin that is marked by H3K9me2 and H3K27me2 but that lacks RdDM. The minority of the genome marked by RdDM was predominantly near genes, and its overall chromatin structure appeared more similar to euchromatin than to heterochromatin. These and other data indicate that the densely staining chromatin defined as heterochromatin differs fundamentally from RdDM-targeted chromatin. We propose that small interfering RNAs perform a specialized role in repressing transposons in accessible chromatin environments and that the bulk of heterochromatin is incompatible with small RNA production. PMID:25465407

  14. L-Proline Induces a Mesenchymal-like Invasive Program in Embryonic Stem Cells by Remodeling H3K9 and H3K36 Methylation

    PubMed Central

    Comes, Stefania; Gagliardi, Miriam; Laprano, Nicola; Fico, Annalisa; Cimmino, Amelia; Palamidessi, Andrea; De Cesare, Dario; De Falco, Sandro; Angelini, Claudia; Scita, Giorgio; Patriarca, Eduardo J.; Matarazzo, Maria R.; Minchiotti, Gabriella

    2013-01-01

    Summary Metabolites are emerging as key mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. We found that the nonessential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells. This embryonic-stem-cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9 and H3K36 methylation status. Consistently, L-Pro-induced esMT is fully reversible either after L-Pro withdrawal or by addition of ascorbic acid (vitamin C), which in turn reduces H3K9 and H3K36 methylation, promoting a mesenchymal-like-to-embryonic-stem-cell transition (MesT). These findings suggest that L-Pro, which is produced by proteolytic remodeling of the extracellular matrix, may act as a microenvironmental cue to control stem cell behavior. PMID:24319666

  15. Histone H3 lysine 9 acetylation pattern suggests that X and B chromosomes are silenced during entire male meiosis in a grasshopper.

    PubMed

    Cabrero, J; Teruel, M; Carmona, F D; Jiménez, R; Camacho, J P M

    2007-01-01

    The facultative heterochromatic X chromosome in leptotene spermatocytes of the grasshopper Eyprepocnemis plorans showed marked hypoacetylation for lysine 9 in the H3 histone (H3-K9) with no sign of histone H2AX phosphorylation. Since H3-K9 hypoacetylation precedes the meiotic appearance of phosphorylated H2AX (gamma-H2AX), which marks the beginning of recombinational DNA double-strand breaks (DSBs), it seems that meiotic sex-chromosome inactivation (MSCI) in this grasshopper occurs prior to the beginning of recombination and hence synapsis (which in this species begins later than recombination). In addition, all constitutively heterochromatic chromosome regions harbouring a 180-bp tandem-repeat DNA and rDNA (B chromosomes and pericentromeric regions of A chromosomes) were H3-K9 hypoacetylated at early leptotene even though they will synapse at subsequent stages. This also suggests that meiotic silencing in this grasshopper might be independent of synapsis. The H3-K9 hypoacetylated state of facultative and constitutive heterochromatin persisted during subsequent meiotic stages and was even apparent in round spermatids. Finally, the fact that B chromosomes are differentially hypoacetylated in testis and embryo interphase cells suggests that they might be silenced early in development and remain this way for most (or all) life-cycle stages.

  16. Redistribution of H3K27me3 and acetylated histone H4 upon exposure to azacitidine and decitabine results in de-repression of the AML1/ETO target gene IL3.

    PubMed

    Buchi, Francesca; Masala, Erico; Rossi, Alessia; Valencia, Ana; Spinelli, Elena; Sanna, Alessandro; Gozzini, Antonella; Santini, Valeria

    2014-03-01

    Human acute myeloid leukemia is characterized by a block in maturation caused by genetic and epigenetic alterations. We studied the effects of low concentrations of the DNA methyltransferase (DNMT) inhibitors 5-azacitidine and decitabine on apoptosis and on chromatin remodeling in an AML1/ETO inducible model of human AML. While both DNMT inhibitors induced apoptosis, only azacitidine did so via caspase activation, possibly through its exclusive non-DNA depending effects. We evaluated histone marks for permissive chromatin, H3K4me3, and acetylated histone H4, and for non-permissive chromatin, H3K9me2, and H3K27me3, at the promoter of the IL3 gene, which is under the direct control of AML1/ETO and is critical for myeloid maturation. We observed that low concentrations of DNMT inhibitors induced a loss of H3K27me3 and gain of acetylated histone H4 at the IL3 promoter exclusively in AML1/ETO-positive cells, which was associated with transcriptional reactivation of the IL3 gene.

  17. A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.

    PubMed

    Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

    2014-12-01

    Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

  18. Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF

    PubMed Central

    Chatterjee, Nilanjana; North, Justin A.; Dechassa, Mekonnen Lemma; Manohar, Mridula; Prasad, Rashmi; Luger, Karolin; Ottesen, Jennifer J.; Poirier, Michael G.

    2015-01-01

    Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as “readers,” which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers. PMID:26416878

  19. The genomic landscape of histone modifications in human T cells

    PubMed Central

    Roh, Tae-Young; Cuddapah, Suresh; Cui, Kairong; Zhao, Keji

    2006-01-01

    To understand the molecular basis that supports the dynamic gene expression programs unique to T cells, we investigated the genomic landscape of activating histone modifications, including histone H3 K9/K14 diacetylation (H3K9acK14ac), H3 K4 trimethylation (H3K4me3), and the repressive histone modification H3 K27 trimethylation (H3K27me3) in primary human T cells. We show that H3K9acK14ac and H3K4me3 are associated with active genes required for T cell function and development, whereas H3K27me3 is associated with silent genes that are involved in development in other cell types. Unexpectedly, we find that 3,330 gene promoters are associated with all of these histone modifications. The gene expression levels are correlated with both the absolute and relative levels of the activating H3K4me3 and the repressive H3K27me3 modifications. Our data reveal that rapidly inducible genes are associated with the H3 acetylation and H3K4me3 modifications, suggesting they assume a chromatin structure poised for activation. In addition, we identified a subpopulation of chromatin regions that are associated with high levels of H3K4me3 and H3K27me3 but low levels of H3K9acK14ac. Therefore, these regions have a distinctive chromatin modification pattern and thus may represent a distinct class of chromatin domains. PMID:17043231

  20. Barrier-to-Autointegration Factor influences specific histone modifications

    PubMed Central

    Montes de Oca, Rocío; Andreassen, Paul R.

    2011-01-01

    Defects in the nuclear envelope or nuclear ‘lamina’ networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells. PMID:22127260

  1. Barrier-to-Autointegration Factor influences specific histone modifications.

    PubMed

    Montes de Oca, Rocío; Andreassen, Paul R; Wilson, Katherine L

    2011-01-01

    Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.

  2. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  3. In vivo treatment by diallyl disulfide increases histone acetylation in rat colonocytes

    SciTech Connect

    Druesne-Pecollo, Nathalie . E-mail: Nathalie.Pecollo@jouy.inra.fr; Chaumontet, Catherine; Pagniez, Anthony; Vaugelade, Pierre; Bruneau, Aurelia; Thomas, Muriel; Cherbuy, Claire; Duee, Pierre-Henri; Martel, Paule

    2007-03-02

    Diallyl disulfide (DADS) is an organosulfur compound from garlic which exhibits various anticarcinogenic properties including inhibition of tumor cell proliferation. DADS antiproliferative effects were previously associated with an increase in histone acetylation in two human tumor colon cell lines, suggesting that DADS-induced histone hyperacetylation could be one of the mechanisms involved in its protective properties on colon carcinogenesis. The effects of DADS on histone H4 and H3 acetylation levels were investigated in vivo in colonocytes isolated from non-tumoral rat. Administrated by intracaecal perfusion or gavage, DADS increases histone H4 and H3 acetylation in colonocytes. Moreover, data generated using cDNA expression arrays suggest that DADS could modulate the expression of a subset of genes. These results suggest the involvement of histone acetylation in modulation of gene expression by DADS in normal rat colonocytes, which might play a role in its biological effects as well as in its anticarcinogenic properties in vivo.

  4. Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.

    PubMed

    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

    2013-05-23

    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 α4 s/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.

  5. Aberrant histone modification in CD19+ B cells of patients with chronic lymphocytic leukemia

    PubMed Central

    Zhou, Keshu; Zhang, Qing; Liu, Yanyan; Xiong, Yuanyuan; Wu, Shengsheng; Yang, Jingke; Zhou, Hu; Liu, Xinjian; Wei, Xudong; Song, Yongping

    2017-01-01

    The aim of this study was to detect the alterations in histone methylation and acetylation in patients with chronic lymphocytic leukemia (CLL). Global histone H3/H4 acetylation and H3K4/H3K9 methylation were detected by the EpiQuik™ global histone H3/H4 acetylation and H3K4/H3K9 methylation assay kits. The mRNA expression of selected chromatin modifier genes was measured by real-time polymerase chain reaction (RT-PCR). Our results found that the global histone H3/H4 hypoacetylation in the CD19+ B cells of patients with CLL (P=0.028 and P=0.03, respectively) and the global histone H3K9 methylation in patients with CLL were significantly increased compared with controls (P=0.02), while there was no significant difference in the global histone H3K4 methylation between the two groups. The level of SIRT1 and EZH2 mRNA expression was upregulated in patients with CLL (P=0.03 and P=0.02, respectively), which increased significantly with progression from Binet stage A to stage C (P=0.015 and P=0.01, respectively) and Rai good to high risk stage (P=0.007 and P=0.008, respectively). The level of HDAC1 and HDAC7 mRNA expression was significantly increased (P=0.02 and P=0.008, respectively) and HDAC2 and P300 mRNA expression was reduced in patients with CLL (P=0.002 and P=0.001, respectively). In conclusion, it is observed that the aberrant histone modification plays an important role in the pathogenesis of CLL. PMID:28260932

  6. Histone Deacetylase Inhibitors Globally Enhance H3/H4 Tail Acetylation Without Affecting H3 Lysine 56 Acetylation

    PubMed Central

    Drogaris, Paul; Villeneuve, Valérie; Pomiès, Christelle; Lee, Eun-Hye; Bourdeau, Véronique; Bonneil, Éric; Ferbeyre, Gerardo; Verreault, Alain; Thibault, Pierre

    2012-01-01

    Histone deacetylase inhibitors (HDACi) represent a promising avenue for cancer therapy. We applied mass spectrometry (MS) to determine the impact of clinically relevant HDACi on global levels of histone acetylation. Intact histone profiling revealed that the HDACi SAHA and MS-275 globally increased histone H3 and H4 acetylation in both normal diploid fibroblasts and transformed human cells. Histone H3 lysine 56 acetylation (H3K56ac) recently elicited much interest and controversy due to its potential as a diagnostic and prognostic marker for a broad diversity of cancers. Using quantitative MS, we demonstrate that H3K56ac is much less abundant than previously reported in human cells. Unexpectedly, in contrast to H3/H4 N-terminal tail acetylation, H3K56ac did not increase in response to inhibitors of each class of HDACs. In addition, we demonstrate that antibodies raised against H3K56ac peptides cross-react against H3 N-terminal tail acetylation sites that carry sequence similarity to residues flanking H3K56. PMID:22355734

  7. Uncoupling histone turnover from transcription-associated histone H3 modifications.

    PubMed

    Ferrari, Paolo; Strubin, Michel

    2015-04-30

    Transcription in eukaryotes is associated with two major changes in chromatin organization. Firstly, nucleosomal histones are continuously replaced by new histones, an event that in yeast occurs predominantly at transcriptionally active promoters. Secondly, histones become modified post-translationally at specific lysine residues. Some modifications, including histone H3 trimethylation at lysine 4 (H3K4me3) and acetylation at lysines 9 (H3K9ac) and 14 (H3K14ac), are specifically enriched at active promoters where histones exchange, suggesting a possible causal relationship. Other modifications accumulate within transcribed regions and one of them, H3K36me3, is thought to prevent histone exchange. Here we explored the relationship between these four H3 modifications and histone turnover at a few selected genes. Using lysine-to-arginine mutants and a histone exchange assay, we found that none of these modifications plays a major role in either promoting or preventing histone turnover. Unexpectedly, mutation of H3K56, whose acetylation occurs prior to chromatin incorporation, had an effect only when introduced into the nucleosomal histone. Furthermore, we used various genetic approaches to show that histone turnover can be experimentally altered with no major consequence on the H3 modifications tested. Together, these results suggest that transcription-associated histone turnover and H3 modification are two correlating but largely independent events.

  8. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation

    PubMed Central

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid

    2016-01-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442–29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. PMID:27402865

  9. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

    PubMed

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla

    2016-10-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

  10. Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.

    PubMed

    Young, Isaac A; Mittal, Chitvan; Shogren-Knaak, Michael A

    2016-02-01

    Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.

  11. Increased acetyl and total histone levels in post-mortem Alzheimer's disease brain.

    PubMed

    Narayan, Pritika J; Lill, Claire; Faull, Richard; Curtis, Maurice A; Dragunow, Mike

    2015-02-01

    Histone acetylation is an epigenetic modification that plays a critical role in chromatin remodelling and transcriptional regulation. There is increasing evidence that epigenetic modifications may become compromised in aging and increase susceptibility to the development of neurodegenerative disorders such as Alzheimer's disease. Immunohistochemical labelling of free-floating sections from the inferior temporal gyrus (Alzheimer's disease, n=14; control, n=17) and paraffin-embedded tissue microarrays containing tissue from the middle temporal gyrus (Alzheimer's disease, n=29; control, n=28) demonstrated that acetyl histone H3 and acetyl histone H4 levels, as well as total histone H3 and total histone H4 protein levels, were significantly increased in post-mortem Alzheimer's disease brain tissue compared to age- and sex-matched neurologically normal control brain tissue. Changes in acetyl histone levels were proportional to changes in total histone levels. The increase in acetyl histone H3 and H4 was observed in Neuronal N immunopositive pyramidal neurons in Alzheimer's disease brain. Using immunolabelling, histone markers correlated significantly with the level of glial fibrillary acidic protein and HLA-DP, -DQ and -DR immunopositive cells and with the pathological hallmarks of Alzheimer's disease (hyperphosphorylated tau load and β-amyloid plaques). Given that histone acetylation changes were correlated with changes in total histone protein, it was important to evaluate if protein degradation pathways may be compromised in Alzheimer's disease. Consequently, significant positive correlations were also found between ubiquitin load and histone modifications. The relationship between histone acetylation and ubiquitin levels was further investigated in an in vitro model of SK-N-SH cells treated with the proteasome inhibitor Mg132 and the histone deacetylase inhibitor valproic acid. In this model, compromised protein degradation caused by Mg132 lead to elevated histone

  12. Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases.

    PubMed

    Pham, Tho X; Park, Young-Ki; Lee, Ji-Young

    2016-06-21

    We previously demonstrated that the organic extract of Spirulina platensis (SPE), an edible blue-green alga, possesses potent anti-inflammatory effects. In this study, we investigated if the regulation of histone deacetylases (HDACs) play a role in the anti-inflammatory effect of SPE in macrophages. Treatment of macrophages with SPE rapidly and dose-dependently reduced HDAC2, 3, and 4 proteins which preceded decreases in their mRNA levels. Degradation of HDAC4 protein was attenuated in the presence of inhibitors of calpain proteases, lysosomal acidification, and Ca(2+)/calmodulin-dependent protein kinase II, respectively, but not a proteasome inhibitor. Acetylated histone H3 was increased in SPE-treated macrophages to a similar level as macrophages treated with a pan-HDAC inhibitor, with concomitant inhibition of inflammatory gene expression upon LPS stimulation. Knockdown of HDAC3 increased basal and LPS-induced pro-inflammatory gene expression, while HDAC4 knockdown increased basal expression of interleukin-1β (IL-1β), but attenuated LPS-induced inflammatory gene expression. Chromatin immunoprecipitation showed that SPE decreased p65 binding and H3K9/K14 acetylation at the Il-1β and tumor necrosis factor α (Tnfα) promoters. Our results suggest that SPE increased global histone H3 acetylation by facilitating HDAC protein degradation, but decreases histone H3K9/K14 acetylation and p65 binding at the promoters of Il-1β and Tnfα to exert its anti-inflammatory effect.

  13. Characterization of post‐translational modifications on lysine 9 of histone H3 variants in mouse testis using matrix‐assisted laser desorption/ionization in‐source decay

    PubMed Central

    Kwak, Ho‐Geun

    2016-01-01

    Rationale Post‐translational modifications (PTMs) of histones result in changes to transcriptional activities and chromatin remodeling. Lysine 9 of histone H3 (H3K9) is subject to PTMs, such as methylation and acetylation, which influence histone activity during spermatogenesis. Characterization strategies for studying PTMs on H3K9 have been developed to provide epigenetic and proteomic information. Proteomic analysis has been used to limited success to study PTMs on H3K9; however, a comprehensive analytical approach is required to elucidate global patterns of PTMs of H3 variants during spermatogenesis. Methods Intact H3 variants in mouse testis were separated by high‐performance liquid chromatography on a reversed‐phase column with an ion‐pairing reagent. Modifications to H3K9 were identified via top‐down analysis using matrix‐assisted laser desorption/ionization in source decay (MALDI‐ISD). Results Mono‐, di‐, and tri‐methylations were identified at H3K9 in mouse testis and epididymis. These modifications were also observed in testis‐specific histone H3 (H3t). Specifically, tri‐methylation was more abundant on H3tK9 than on K9 of other H3 variants. Conclusions We introduce a method for rapid, simple, and comprehensive characterization of PTMs on the N‐termini of H3 variants using MALDI‐ISD. This approach provides novel and useful information, including K9 modifications on H3t, which would benefit epigenetic and proteomic research. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd. PMID:27643486

  14. Histone Acetylation is Recruited in Consolidation as a Molecular Feature of Stronger Memories

    ERIC Educational Resources Information Center

    Federman, Noel; Fustinana, Maria Sol; Romano, Arturo

    2009-01-01

    Gene expression is a key process for memory consolidation. Recently, the participation of epigenetic mechanisms like histone acetylation was evidenced in long-term memories. However, until now the training strength required and the persistence of the chromatin acetylation recruited are not well characterized. Here we studied whether histone…

  15. Synthesis, Acetylation, and Phosphorylation of Histone IV and Its Binding to DNA During Spermatogenesis in Trout*

    PubMed Central

    Louie, Andrew J.; Dixon, Gordon H.

    1972-01-01

    During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [3H]aminoacids and inorganic [32P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [3H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [3H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total 3H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an “obligatory” acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that ε-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH2-terminal region of histone IV to assume the correct conformation (in this case, an α-helix), and fit into the major groove of DNA. Deacetylation then “locks” histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known. Images PMID:4505675

  16. Expression of non-secreted IL-4 is associated with HDAC inhibitor-induced cell death, histone acetylation and c-Jun regulation in human gamma/delta T-cells

    PubMed Central

    Bhat, Jaydeep; Schütze, Stefan; Zeissig, Sebastian; Ammerpohl, Ole; Adam, Dieter; Kabelitz, Dieter

    2016-01-01

    Previously, the expression of a non-secreted IL-4 variant (IL-4δ13) has been described in association with apoptosis and age-dependent Th2 T-cell polarization. Signaling pathways involved in this process have so far not been studied. Here we report the induction of IL-4δ13 expression in human γδ T-cells upon treatment with a sublethal dose of histone deacetylase (HDACi) inhibitor valproic acid (VPA). Induction of IL-4δ13 was associated with increased cytoplasmic IL-4Rα and decreased IL-4 expression, while mRNA for mature IL-4 was concomitantly down-regulated. Importantly, only the simultaneous combination of apoptosis and necroptosis inhibitors prevented IL-4δ13 expression and completely abrogated VPA-induced global histone H3K9 acetylation mark. Further, our work reveals a novel involvement of transcription factor c-Jun in the signaling network of IL-4, HDAC1, caspase-3 and mixed lineage kinase domain-like protein (MLKL). This study provides novel insights into the effects of epigenetic modulator VPA on human γδ T-cell differentiation. PMID:27556516

  17. The PHD finger of p300 influences its ability to acetylate histone and non-histone targets.

    PubMed

    Rack, Johannes G M; Lutter, Timo; Kjæreng Bjerga, Gro Elin; Guder, Corina; Ehrhardt, Christine; Värv, Signe; Ziegler, Mathias; Aasland, Rein

    2014-12-12

    In enzymes that regulate chromatin structure, the combinatorial occurrence of modules that alter and recognise histone modifications is a recurrent feature. In this study, we explored the functional relationship between the acetyltransferase domain and the adjacent bromodomain/PHD finger (bromo/PHD) region of the transcriptional coactivator p300. We found that the bromo/PHD region of p300 can bind to the acetylated catalytic domain in vitro and augment the catalytic activity of the enzyme. Deletion of the PHD finger, but not the bromodomain, impaired the ability of the enzyme to acetylate histones in vivo, whilst it enhanced p300 self-acetylation. A point mutation in the p300 PHD finger that is related to the Rubinstein-Taybi syndrome resulted in increased self-acetylation but retained the ability to acetylate histones. Hence, the PHD finger appears to negatively regulate self-acetylation. Furthermore, our data suggest that the PHD finger has a role in the recruitment of p300 to chromatin.

  18. Behavioral Neuroadaptation to Alcohol: From Glucocorticoids to Histone Acetylation

    PubMed Central

    Mons, Nicole; Beracochea, Daniel

    2016-01-01

    neuroadaptive changes during withdrawal from chronic alcohol intake. It then highlights the role of cAMP–PKA–CREB signaling cascade and histone acetylation within the PFC and limbic structures in alcohol-induced anxiety and behavioral impairments, and how an understanding of functional alterations of these pathways might lead to better treatments for neuropsychiatric disorders. PMID:27766083

  19. Epigenetic Disruptions of Histone Signatures for the Trophectoderm and Inner Cell Mass in Mouse Parthenogenetic Embryos

    PubMed Central

    Chen, Yi-Hui

    2015-01-01

    Epigenetic asymmetry has been shown to be associated with the first lineage allocation event in preimplantation development, that is, the formation of the trophectoderm (TE) and inner cell mass (ICM) lineages in the blastocyst. Since parthenogenesis causes aberrant segregation between the TE and ICM lineages, we examined several development-associated histone modifications in parthenotes, including those involved in (i) transcriptional activation [acetylated histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac), trimethylated histone H3 lysine 4 (H3K4Me3), and dimethylated histone H3 arginine 26 (H3R26Me2)] and (ii) transcriptional repression [trimethylated histone H3 lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3), and mono-ubiquitinated histone H2A lysine 119 (H2AK119u1)]. Here, we report that in parthenotes, H3R26Me2 expression decreased from the morula stage, while expression patterns and levels of H3K9Ac, H3K27Me3, and H2AK119u1 were unchanged until the blastocyst stage; whereas H3K14Ac, H3K4Me3, and H3K9Me3 showed normal patterns and levels of expressions. Relative to the decrease of H3K9Ac in the ICM and increase in the TE of parthenotes, we detected reduced expression of TAT-interactive protein 60 acetyltransferase and histone deacetylase 1 deacetylase in the ICM and TE of parthenotes, respectively. Relative to the decrease of H3R26Me2, we also observed decreased expression of coactivator-associated arginine methyltransferase 1 methyltransferase and increased expression of the Wnt effector transcription factor 7L2 and miR-181c microRNA in parthenotes. Furthermore, relative to the decrease in H3K27Me3 and H2AK119u1, we found increased phosphorylation of Akt1 and enhancer of zeste homolog 2 in parthenogenetic TE. Therefore, our findings that histone signatures are impaired in parthenotes provide a mechanistic explanation for aberrant lineage segregation and TE defects. PMID:25315067

  20. H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

    PubMed Central

    Zhang, Xian; Li, Binkui; Rezaeian, Abdol Hossein; Xu, Xiaohong; Chou, Ping-Chieh; Jin, Guoxiang; Han, Fei; Pan, Bo-Syong; Wang, Chi-Yun; Long, Jie; Zhang, Anmei; Huang, Chih-Yang; Tsai, Fuu-Jen; Tsai, Chang-Hai; Logothetis, Christopher; Lin, Hui-Kuan

    2017-01-01

    Dynamic changes in histone modifications under various physiological cues play important roles in gene transcription and cancer. Identification of new histone marks critical for cancer development is of particular importance. Here we show that, in a glucose-dependent manner, E3 ubiquitin ligase NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues, which specifically recruits histone acetyltransferase GCN5 for subsequent H3 acetylation. Genome-wide analysis of chromatin immunoprecipitation followed by sequencing reveals that NEDD4 regulates glucose-induced H3 K9 acetylation at transcription starting site and enhancer regions. Integrative analysis of ChIP-seq and microarray data sets also reveals a consistent role of NEDD4 in transcription activation and H3 K9 acetylation in response to glucose. Functionally, we show that NEDD4-mediated H3 ubiquitination, by transcriptionally activating IL1α, IL1β and GCLM, is important for tumour sphere formation. Together, our study reveals the mechanism for glucose-induced transcriptome reprograming and epigenetic regulation in cancer by inducing NEDD4-dependent H3 ubiquitination. PMID:28300060

  1. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    SciTech Connect

    Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang; and others

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

  2. Establishment and application of a flow cytometry-based method for detecting histone acetylation levels.

    PubMed

    Jianhui, Li; Chunfu, Wang; Fan, Bai; Yan, Zhuang; Zhuojun, Mao; Yongtao, Sun

    2016-06-20

    Histone deacetylase inhibitors, which have also received attention in AIDS and other diseases, are a new class of anticancer drugs developed in recent years. However, there is still a lack of a unified and reliable method for detecting histone acetylation levels in basic and clinical research. In this study, we developed a flow cytometry-based method to detect histone acetylation levels by comparing different sample processing temperature (on ice vs. room temperature), permeabilization method (intracellular vs. nuclear), antibody dose (antibody titration) and antibody incubation time (time gradient) using whole blood and peripheral blood mononuclear cells. In addition, we applied this optimized method in in vitro experiment and clinical trial of Chidamide (the only China FDA approved HDACi), the result of which confirmed that the flow cytometry-based method for detecting histone acetylation levels is a reliable, fast and convenient method which can be used in basic and clinical research.

  3. Evaluating the Role of Epigenetic Histone Modifications in the Metabolic Memory of Type 1 Diabetes

    PubMed Central

    Miao, Feng; Chen, Zhuo; Genuth, Saul; Paterson, Andrew; Zhang, Lingxiao; Wu, Xiwei; Li, Sierra Min; Cleary, Patricia; Riggs, Arthur; Harlan, David M.; Lorenzi, Gayle; Kolterman, Orville; Sun, Wanjie; Lachin, John M.; Natarajan, Rama

    2014-01-01

    We assessed whether epigenetic histone posttranslational modifications are associated with the prolonged beneficial effects (metabolic memory) of intensive versus conventional therapy during the Diabetes Control and Complications Trial (DCCT) on the progression of microvascular outcomes in the long-term Epidemiology of Diabetes Interventions and Complications (EDIC) study. We performed chromatin immunoprecipitation linked to promoter tiling arrays to profile H3 lysine-9 acetylation (H3K9Ac), H3 lysine-4 trimethylation (H3K4Me3), and H3K9Me2 in blood monocytes and lymphocytes obtained from 30 DCCT conventional treatment group subjects (case subjects: mean DCCT HbA1c level >9.1% [76 mmol/mol] and progression of retinopathy or nephropathy by EDIC year 10 of follow-up) versus 30 DCCT intensive treatment subjects (control subjects: mean DCCT HbA1c level <7.3% [56 mmol/mol] and without progression of retinopathy or nephropathy). Monocytes from case subjects had statistically greater numbers of promoter regions with enrichment in H3K9Ac (active chromatin mark) compared with control subjects (P = 0.0096). Among the patients in the two groups combined, monocyte H3K9Ac was significantly associated with the mean HbA1c level during the DCCT and EDIC (each P < 2.2E-16). Of note, the top 38 case hyperacetylated promoters (P < 0.05) included >15 genes related to the nuclear factor-κB inflammatory pathway and were enriched in genes related to diabetes complications. These results suggest an association between HbA1c level and H3K9Ac, and a possible epigenetic explanation for metabolic memory in humans. PMID:24458354

  4. Romidepsin reduces histone deacetylase activity, induces acetylation of histones, inhibits proliferation, and activates apoptosis in immortalized epithelial endometriotic cells.

    PubMed

    Imesch, Patrick; Fink, Daniel; Fedier, André

    2010-12-01

    Romidepsin inhibited HDAC activity, produced acetylation of the histone proteins, up-regulated p21, and down-regulated cyclins B1 and D1, resulting in proliferation inhibition and apoptosis activation in 11z immortalized epithelial endometriotic cells. Our findings provide evidence that endometriotic cells are sensitive to the epigenetic effects of romidepsin and suggest that endometriosis may be therapeutically targeted by romidepsin.

  5. The tor pathway regulates gene expression by linking nutrient sensing to histone acetylation.

    PubMed

    Rohde, John R; Cardenas, Maria E

    2003-01-01

    The Tor pathway mediates cell growth in response to nutrient availability, in part by inducing ribosomal protein (RP) gene expression via an unknown mechanism. Expression of RP genes coincides with recruitment of the Esa1 histone acetylase to RP gene promoters. We show that inhibition of Tor with rapamycin releases Esa1 from RP gene promoters and leads to histone H4 deacetylation without affecting promoter occupancy by Rap1 and Abf1. Genetic and biochemical evidence identifies Rpd3 as the major histone deacetylase responsible for reversing histone H4 acetylation at RP gene promoters in response to Tor inhibition by rapamycin or nutrient limitation. Our results illustrate that the Tor pathway links nutrient sensing with histone acetylation to control RP gene expression and cell growth.

  6. The histone 3 lysine 9 methyltransferase inhibitor chaetocin improves prognosis in a rat model of high salt diet-induced heart failure

    PubMed Central

    Ono, Tomohiko; Kamimura, Naomi; Matsuhashi, Tomohiro; Nagai, Toshihiro; Nishiyama, Takahiko; Endo, Jin; Hishiki, Takako; Nakanishi, Tsuyoshi; Shimizu, Noriaki; Tanaka, Hirotoshi; Ohta, Shigeo; Suematsu, Makoto; Ieda, Masaki; Sano, Motoaki; Fukuda, Keiichi; Kaneda, Ruri

    2017-01-01

    Histone acetylation has been linked to cardiac hypertrophy and heart failure. However, the pathological implications of changes in histone methylation and the effects of interventions with histone methyltransferase inhibitors for heart failure have not been fully clarified. Here, we focused on H3K9me3 status in the heart and investigated the effects of the histone H3K9 methyltransferase inhibitor chaetocin on prognoses in Dahl salt-sensitive rats, an animal model of chronic heart failure. Chaetocin prolonged survival and restored mitochondrial dysfunction. ChIP-seq analysis demonstrated that chronic stress to the heart induced H3K9me3 elevation in thousands of repetitive elements, including intronic regions of mitochondria-related genes, such as the gene encoding peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. Furthermore, chaetocin reversed this effect on these repetitive loci. These data suggested that excessive heterochromatinization of repetitive elements of mitochondrial genes in the failing heart may lead to the silencing of genes and impair heart function. Thus, chaetocin may be a potential therapeutic agent for chronic heart failure. PMID:28051130

  7. Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

    SciTech Connect

    Hong, Gia-Ming

    2012-05-01

    Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2

  8. The Role of Histone Acetylation in Memory Formation and Cognitive Impairments

    PubMed Central

    Peixoto, Lucia; Abel, Ted

    2013-01-01

    Long-term memory formation requires transcription and protein synthesis. Over the past few decades, a great amount of knowledge has been gained regarding the molecular players that regulate the transcriptional program linked to memory consolidation. Epigenetic mechanisms have been shown to be essential for the regulation of neuronal gene expression, and histone acetylation has been one of the most studied and best characterized. In this review, we summarize the lines of evidence that have shown the relevance of histone acetylation in memory in both physiological and pathological conditions. Great advances have been made in identifying the writers and erasers of histone acetylation marks during learning. However, the identities of the upstream regulators and downstream targets that mediate the effect of changes in histone acetylation during memory consolidation remain restricted to a handful of molecules. We outline a general model by which corepressors and coactivators regulate histone acetylation during memory storage and discuss how the recent advances in high-throughput sequencing have the potential to radically change our understanding of how epigenetic control operates in the brain. PMID:22669172

  9. Effects of histone acetylation on superoxide dismutase 1 gene expression in the pathogenesis of senile cataract

    PubMed Central

    Rong, Xianfang; Qiu, Xiaodi; Jiang, Yongxiang; Li, Dan; Xu, Jie; Zhang, Yinglei; Lu, Yi

    2016-01-01

    Histone acetylation plays key roles in gene expression, but its effects on superoxide dismutase 1 (SOD1) expression in senile cataract remains unknown. To address this problem, the study was to investigate the influence of histone acetylation on SOD1 expression and its effects in the pathogenesis of senile cataract. Senile cataract was classified into three types—nuclear cataract (NC), cortical cataract (CC), and posterior subcapsular cataract (SC)—using the Lens Opacities Classification System III. In senile cataracts, SOD1 expression decreased significantly. Both H3 and H4 were deacetylated at −600 bp of the SOD1 promoter of cataract lenses, and hypoacetylated at −1500, −1200, and −900 bp. In hypoacetylated histones, the hypoacetylation pattern differed among the cataracts. In vitro, anacardic acid (AA) significantly reduced H3 and H4 acetylation at the SOD1 promoter, decreased protein expression, and induced cataract formation in rabbits. AA also inhibited HLEC viability and increased cell apoptosis. In contrast, trichostatin A (TSA) was able to efficaciously stop AA’s effects on both rabbit lenses and HLECs. Decreased histone acetylation at the SOD1 promoter is associated with declined SOD1 expression in senile cataracts. Histone acetylation plays an essential role in the regulation of SOD1 expression and in the pathogenesis of senile cataracts. PMID:27703255

  10. Histone acetylation is recruited in consolidation as a molecular feature of stronger memories.

    PubMed

    Federman, Noel; Fustiñana, Maria Sol; Romano, Arturo

    2009-10-01

    Gene expression is a key process for memory consolidation. Recently, the participation of epigenetic mechanisms like histone acetylation was evidenced in long-term memories. However, until now the training strength required and the persistence of the chromatin acetylation recruited are not well characterized. Here we studied whether histone acetylation is involved in consolidation in invertebrates, whether it depends on the training strength, and whether it is a permanent or transient mechanism. We used a well-characterized memory model in invertebrates, the context-signal memory in crabs. Our results show no changes in histone 3 (H3) acetylation during consolidation of a standard training protocol. However, strong training induced a significant increase in H3 acetylation 1-h post-training, returning to basal levels afterward. Accordingly, the administration of histone deacetylase inhibitors sodium butyrate (NaB) and trichostatin A allowed a weak training to induce long-term memory. NaB enhanced memory in two phases during consolidation. These findings support that H3 acetylation (1) is involved in consolidation, (2) occurs only after strong training, (3) is a transient process, and (4) memory is enhanced in two phases. The coincidence of these phases with other mechanisms of gene expression is discussed.

  11. Histone H3 globular domain acetylation identifies a new class of enhancers

    PubMed Central

    Pradeepa, Madapura M; Grimes, Graeme R; Kumar, Yatendra; Olley, Gabrielle; Taylor, Gillian C A; Schneider, Robert; Bickmore, Wendy A

    2016-01-01

    Histone acetylation is generally associated with active chromatin, but most studies have focused on the acetylation of histone tails. Various histone H3 and H4 tail acetylations mark the promoters of active genes1. This includes acetylation of H3 on lysine 27 (H3K27ac), which blocks the deposition of polycomb mediated H3K27me32. H3K27ac is also widely used to identify active enhancers3,4, and the assumption has been that profiling of H3K27ac is a comprehensive way of cataloguing the set of active enhancers in mammalian cell types. Here we show that acetylation of lysine residues in the globular domain of H3 (H3K64ac and H3K122ac) marks active gene promoters and also a subset of active enhancers. Moreover, we find a novel class of active functional enhancers that are marked by H3K122ac but lack H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than was previously considered. PMID:27089178

  12. Immunocytochemical and immunogold analyses of histone H4 acetylation during Chara vulgaris spermiogenesis.

    PubMed

    Wojtczak, Agnieszka

    2016-03-01

    Histone acetylation is one of the epigenetic modifications which play a significant role in chromatin remodeling during spermiogenesis. Acetylation of the histone H4 makes the exchange of nucleoproteins easy. Research on mouse spermatogenesis showed that H4 histone acetylated at Lys 12 (H4K12ac) was specific only to spermatids. Immunocytochemical studies of Chara vulgaris spermatids with the use of antibodies against the histone H4K12ac revealed positive reactions in spermatid nuclei at stages I-VII. This reaction, connected with nuclear condensation, was much stronger at the early stages of spermiogenesis than later on. Moreover, it showed that at the stages V-VII in spermatid nuclei the presence of the histone H4K12ac corresponded with DNA double-strand breaks. Electron microscopy studies with the use of immunogold technique revealed an almost twofold difference between the mean total numbers of gold grains in the examined chromatin in both stages. This study showed nearly equal distribution of gold grains on condensed and non-condensed chromatin of spermatids at the stage III/IV (48.11% and 51.89%, respectively). In the later stage-VI, when chromatin condensation proceeded, labeling of condensed chromatin reached 57.27%, while in the case of non-condensed chromatin it dropped to 42.73%. The percentage analysis also revealed an increase (above 9%) in condensed chromatin labeling in relation to the stage III/IV. Intensive acetylation of histone H4 at the early stages is correlated with DNA DSBs and transcriptional activity. It facilitates chromatin loosening, which enables the correct course of chromatin remodeling at a later stage. Histone γH2AX also influences chromatin structure in many biological processes in different cell types. Current studies reveal other similarities regarding histone H4 acetylation, not only between Chara and mammals but between invertebrates (molluscs) and vertebrates (bony fishes) as well.

  13. Chromatin immunoprecipitation microarrays for identification of genes silenced by histone H3 lysine 9 methylation.

    PubMed

    Kondo, Yutaka; Shen, Lanlan; Yan, Pearlly S; Huang, Tim Hui-Ming; Issa, Jean-Pierre J

    2004-05-11

    Switching from acetylation to methylation at histone H3 lysine 9 (K9) has recently been shown to contribute to euchromatin gene silencing. To identify genes silenced by K9 modifications, we probed a human CpG island microarray with DNA obtained by chromatin immunoprecipitation (ChIP) in a cancer cell line using an anti-H3-K9 methylated antibody or an anti-H3-K9 acetylated antibody. Of the 27 clones with the highest signal ratio of K9 methylation over acetylation (Me/Ac), 13 contained repetitive sequences. Among 14 nonrepetitive clones, we identified 11 genes (seven known and four previously undescribed), one EST, and two unknown fragments. Using ChIP-PCR, all 18 examined clones showed higher ratios of H3-K9 Me/Ac than the active gene control, P21, thus confirming the microarray data. In addition, we found a strong correlation between the K9 Me/Ac ratio and CpG island DNA methylation (R = 0.92, P < 0.01), and five of seven genes examined (megalin, thrombospondin-4, KR18, latrophilin-3, and phosphatidylinositol-3-OH kinase P101 subunit) showed lack of expression by RT-PCR and reactivation by DNA methylation and/or histone deacetylase inhibition, suggesting that these genes are true targets of silencing through histone modifications. All five genes also showed significant DNA methylation in a cell line panel and in primary colon cancers. Our data suggest that CpG island microarray coupled with ChIP can identify novel targets of gene silencing in cancer. This unbiased approach confirms the tight coupling between DNA methylation and histone modifications in cancer and could be used to probe gene silencing in nonneoplastic conditions as well.

  14. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids.

    PubMed Central

    Oliva, R; Mezquita, C

    1982-01-01

    In order to study the relationship between acetylation of histones, chromatin structure and gene activity, the distribution and turnover of acetyl groups among nucleosomal core histones and the extent of histone H4 acetylation were examined in rooster testis cell nuclei at different stages of spermatogenesis. Histone H4 was the predominant acetylated histone in mature testes. Hyperacetylation of H4 and rapid turnover of its acetyl groups are not univocally correlated with transcriptional activity since they were detected in both genetically active testicular cells and genetically inactive elongated spermatids. During the transition from nucleohistone to nucleoprotamine in elongated spermatids the chromatin undergoes dramatic structural changes with exposition of binding sites on DNA (1). Hyperacetylation of H4 and rapid turnover of its acetyl groups could be correlated with the particular conformation of chromatin in elongated spermatids and might represent a necessary condition for binding of chromosomal proteins to DNA. Images PMID:7162988

  15. Quantitative measurement of histone tail acetylation reveals stage-specific regulation and response to environmental changes during Drosophila development

    PubMed Central

    Henry, Ryan A.; Singh, Tanu; Kuo, Yin-Ming; Biester, Alison; O’Keefe, Abigail; Lee, Sandy; Andrews, Andrew J.; O’Reilly, Alana M.

    2016-01-01

    Histone modification plays a major role in regulating gene transcription and ensuring the healthy development of an organism. Numerous studies have suggested that histones are dynamically modified during developmental events to control gene expression levels in a temporal and spatial manner. However, the study of histone acetylation dynamics using currently available techniques is hindered by the difficulty of simultaneously measuring acetylation of the numerous potential sites of modification present in histones. Here, we present a methodology that allows us to combine mass spectrometry-based histone analysis with Drosophila developmental genetics. Using this system, we characterized histone acetylation patterns during multiple developmental stages of the fly. Additionally, we utilized this analysis to characterize how treatments with pharmacological agents or environmental changes such as gamma-irradiation altered histone acetylation patterns. Strikingly, gamma-irradiation dramatically increased acetylation at H3K18, a site linked to DNA repair via non-homologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in H3K18 acetylation was lost. These results demonstrate the efficacy of our combined mass spectrometry system with a Drosophila model system, and provide interesting insight into the changes in histone acetylation during development, as well as the effects of both pharmacological and environmental agents on global histone acetylation. PMID:26836402

  16. Hippocampal histone acetylation regulates object recognition and the estradiol-induced enhancement of object recognition.

    PubMed

    Zhao, Zaorui; Fan, Lu; Fortress, Ashley M; Boulware, Marissa I; Frick, Karyn M

    2012-02-15

    Histone acetylation has recently been implicated in learning and memory processes, yet necessity of histone acetylation for such processes has not been demonstrated using pharmacological inhibitors of histone acetyltransferases (HATs). As such, the present study tested whether garcinol, a potent HAT inhibitor in vitro, could impair hippocampal memory consolidation and block the memory-enhancing effects of the modulatory hormone 17β-estradiol E2. We first showed that bilateral infusion of garcinol (0.1, 1, or 10 μg/side) into the dorsal hippocampus (DH) immediately after training impaired object recognition memory consolidation in ovariectomized female mice. A behaviorally effective dose of garcinol (10 μg/side) also significantly decreased DH HAT activity. We next examined whether DH infusion of a behaviorally subeffective dose of garcinol (1 ng/side) could block the effects of DH E2 infusion on object recognition and epigenetic processes. Immediately after training, ovariectomized female mice received bilateral DH infusions of vehicle, E2 (5 μg/side), garcinol (1 ng/side), or E2 plus garcinol. Forty-eight hours later, garcinol blocked the memory-enhancing effects of E2. Garcinol also reversed the E2-induced increase in DH histone H3 acetylation, HAT activity, and levels of the de novo methyltransferase DNMT3B, as well as the E2-induced decrease in levels of the memory repressor protein histone deacetylase 2. Collectively, these findings suggest that histone acetylation is critical for object recognition memory consolidation and the beneficial effects of E2 on object recognition. Importantly, this work demonstrates that the role of histone acetylation in memory processes can be studied using a HAT inhibitor.

  17. Regulation of RNA polymerase II activation by histone acetylation in single living cells.

    PubMed

    Stasevich, Timothy J; Hayashi-Takanaka, Yoko; Sato, Yuko; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Nagase, Takahiro; Nozaki, Naohito; McNally, James G; Kimura, Hiroshi

    2014-12-11

    In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

  18. Inhibition of monomethylarsonous acid (MMA(III))-induced cell malignant transformation through restoring dysregulated histone acetylation.

    PubMed

    Ge, Yichen; Gong, Zhihong; Olson, James R; Xu, Peilin; Buck, Michael J; Ren, Xuefeng

    2013-10-04

    Inorganic arsenic (iAs) and its high toxic metabolite, monomethylarsonous acid (MMA(III)), are able to induce malignant transformation of human cells. Chronic exposure to these chemicals is associated with an increased risk of developing multiple cancers in human. However, the mechanisms contributing to iAs/MMA(III)-induced cell malignant transformation and carcinogenesis are not fully elucidated. We recently showed that iAs/MMA(III) exposure to human cells led to a decreased level of histone acetylation globally, which was associated with an increased sensitivity to arsenic cytotoxicity. In the current study, it demonstrated that prolonged exposure to low-level MMA(III) in human urothelial cells significantly increased the expression and activity of histone deacetylases (HDACs) with an associated reduction of histone acetylation levels both globally and lysine specifically. Administration of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), at 4 weeks after the initial MMA(III) treatment inhibited the MMA(III)-mediated up-regulation of the expression and activities of HDACs, leading to increase histone acetylation and prevention of MMA(III)-induced malignant transformation. These new findings suggest that histone acetylation dysregulation may be a key mechanism in MMA(III)-induced malignant transformation and carcinogenesis, and that HDAC inhibitors could be targeted to prevent or treat iAs-related cancers.

  19. Binding Mode of Acetylated Histones to Bromodomains: Variations on a Common Motif.

    PubMed

    Marchand, Jean-Rémy; Caflisch, Amedeo

    2015-08-01

    Bromodomains, epigenetic readers that recognize acetylated lysine residues in histone tails, are potential drug targets in cancer and inflammation. Herein we review the crystal structures of human bromodomains in complex with histone tails and analyze the main interaction motifs. The histone backbone is extended and occupies, in one of the two possible orientations, the bromodomain surface groove lined by the ZA and BC loops. The acetyl-lysine side chain is buried in the cavity between the four helices of the bromodomain, and its oxygen atom accepts hydrogen bonds from a structural water molecule and a conserved asparagine residue in the BC loop. In stark contrast to this common binding motif, a large variety of ancillary interactions emerge from our analysis. In 10 of 26 structures, a basic side chain (up to five residues up- or downstream in sequence with respect to the acetyl-lysine) interacts with the carbonyl groups of the C-terminal turn of helix αB. Furthermore, the complexes reveal many heterogeneous backbone hydrogen bonds (direct or water-bridged). These interactions contribute unselectively to the binding of acetylated histone tails to bromodomains, which provides further evidence that specific recognition is modulated by combinations of multiple histone modifications and multiple modules of the proteins involved in transcription.

  20. Functional Roles of Acetylated Histone Marks at Mouse Meiotic Recombination Hot Spots.

    PubMed

    Getun, Irina V; Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R; Cleveland, John L; Bois, Philippe R J

    2017-02-01

    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.

  1. Inhibition of Different Histone Acetyltransferases (HATs) Uncovers Transcription-Dependent and -Independent Acetylation-Mediated Mechanisms in Memory Formation

    ERIC Educational Resources Information Center

    Merschbaecher, Katja; Hatko, Lucyna; Folz, Jennifer; Mueller, Uli

    2016-01-01

    Acetylation of histones changes the efficiency of the transcription processes and thus contributes to the formation of long-term memory (LTM). In our comparative study, we used two inhibitors to characterize the contribution of different histone acetyl transferases (HATs) to appetitive associative learning in the honeybee. For one we applied…

  2. Histone hypoacetylation-activated genes are repressed by acetyl-CoA- and chromatin-mediated mechanism

    PubMed Central

    Mehrotra, Swati; Galdieri, Luciano; Zhang, Tiantian; Zhang, Man; Pemberton, Lucy F.; Vancura, Ales

    2014-01-01

    Transcriptional activation is typically associated with increased acetylation of promoter histones. However, this paradigm does not apply to transcriptional activation of all genes. In this study we have characterized a group of genes that are repressed by histone acetylation. These histone hypoacetylation-activated genes (HHAAG) are normally repressed during exponential growth, when the cellular level of acetyl-CoA is high and global histone acetylation is also high. The HHAAG are induced during diauxic shift, when the levels of acetyl-CoA and global histone acetylation decrease. The histone hypoacetylation-induced activation of HHAAG is independent of Msn2/Msn4. The repression of HSP12, one of the HHAAG, is associated with well-defined nucleosomal structure in the promoter region, while histone hypoacetylation-induced activation correlates with delocalization of positioned nucleosomes or with reduced nucleosome occupancy. Correspondingly, unlike the majority of yeast genes, HHAAG are transcriptionally upregulated when expression of histone genes is reduced. Taken together, these results suggest a model in which histone acetylation is required for proper positioning of promoter nucleosomes and repression of HHAAG. PMID:24907648

  3. Histone acetylation: from code to web and router via intrinsically disordered regions.

    PubMed

    Horikoshi, Masami

    2013-01-01

    Structural changes of chromatin, which consists of nucleosomes and nucleosome-associated factors, lead to functional changes that are important determinants of eukaryotic gene regulation. These structural changes are regulated by modifications of histones and DNA, both of which are components of nucleosomes, as well as by replacement of histone variants and the actions of noncoding RNAs. In studies of chromatin modifications, a great deal of attention has been paid to histone acetylation. Progress in understanding this subject has been extensive, including i) elucidation of the relationship of histone acetylation and gene activity; ii) the first isolation of a histonemodifying enzyme; iii) the first identification of a factor that recognizes a modified site; iv) elucidation of the mechanism by which histone modification leads to structural changes in nucleosomes; and v) elucidation of the mechanism of border formation between euchromatin and heterochromatin. Histone acetylation is considered to be fundamental in several fields, including studies of a) the role of chromatin and epigenetics in higher-order biochemical systems such as transcription, DNA replication, and repair; b) biological phenomena such as cell proliferation and differentiation; and c) cancer and aging, potentially leading to clinical applications. In this review, I will discuss the histone code hypothesis, at one time believed to represent a unified theory regarding the functions of histone modification. In addition, I will describe the "modification web theory, " by which the problems in the histone code hypothesis can be overcome, as well as the "signal router theory, " which explains the mechanisms of formation, development, and evolution of the modification web from a structural viewpoint. Lastly, I will illustrate how these novel theories partially explain the robustness of biological systems against various perturbations, and elucidate the strategy that a cell employs to avoid fatal

  4. Histone Acetylation Induced Transformation of B-DNA to Z-DNA in Cells Probed through FT-IR Spectroscopy.

    PubMed

    Zhang, Fengqiu; Huang, Qing; Yan, Jingwen; Chen, Zhu

    2016-04-19

    A nucleosome is made up of DNA and histones, and acetylation of histones perturbs the interaction of DNA and histones and thus affects the chromatin conformation and function. However, whether or how acetylation induces DNA conformation changes is still elusive. In this work, we applied FT-IR spectroscopy to monitor the DNA signals in cells as the histone acetylation was regulated by trichostatin A (TSA), a reversible inhibitor to histone deacetylases (HDACs). Our results unambiguously demonstrate the significant transformation of B-DNA to Z-DNA upon histone acetylation in the TSA treated HeLa cells. This is the first report providing the explicit experimental evidence for such a B-Z transformation of DNA in the epigenetic states of cells.

  5. Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus.

    PubMed

    Pieterse, E; Hofstra, J; Berden, J; Herrmann, M; Dieker, J; van der Vlag, J

    2015-01-01

    In addition to disturbed apoptosis and insufficient clearance of apoptotic cells, there is recent evidence for a role of neutrophils in the aetiopathogenesis of systemic lupus erythematosus (SLE). In response to various stimuli, neutrophils can rapidly release DNA fibres decorated with citrullinated histones and anti-microbial peptides. These structures are referred to as neutrophil extracellular traps (NETs). In addition to apoptotic cell-derived microparticles, these NETs may comprise a further source of autoantigens, able to drive the autoimmune response in SLE. Our group recently identified specific histone modifications occurring during apoptosis that play an important role in the autoimmune response in SLE. In the current study, we evaluated the presence and immunostimulatory potential of these previously identified histone modifications in NETs. Compared to NETs from healthy donors, the histones present in NETs formed by SLE-derived neutrophils contain increased amounts of acetylated and methylated residues, which we previously observed to be associated with apoptosis and SLE. Treatment of neutrophils with histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), prior to induction of NETosis, induced NETs containing hyperacetylated histones, endowed with an increased capacity to activate macrophages. This implies that specific histone modifications, in particular acetylation, might enhance the immunostimulatory potential of NETs in SLE.

  6. Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

    PubMed Central

    Mäntylä, Elina; Salokas, Kari; Oittinen, Mikko; Aho, Vesa; Mäntysaari, Pekka; Palmujoki, Lassi; Kalliolinna, Olli; Ihalainen, Teemu O.; Niskanen, Einari A.; Timonen, Jussi

    2016-01-01

    ABSTRACT The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCE Viral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. PMID:26842481

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

    SciTech Connect

    Higashi, Tsunehito; Matsunaga, Sachihiro; Isobe, Keisuke; Morimoto, Akihiro; Shimada, Tomoko; Kataoka, Shogo; Watanabe, Wataru; Uchiyama, Susumu; Itoh, Kazuyoshi; Fukui, Kiichi . E-mail: kfukui@bio.eng.osaka-u.ac.jp

    2007-06-08

    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.

  8. DNA methylation and histone acetylation work in concert to regulate memory formation and synaptic plasticity.

    PubMed

    Miller, Courtney A; Campbell, Susan L; Sweatt, J David

    2008-05-01

    A clear understanding is developing concerning the importance of epigenetic-related molecular mechanisms in transcription-dependent long-term memory formation. Chromatin modification, in particular histone acetylation, is associated with transcriptional activation, and acetylation of histone 3 (H3) occurs in Area CA1 of the hippocampus following contextual fear conditioning training. Conversely, DNA methylation is associated with transcriptional repression, but is also dynamically regulated in Area CA1 following training. We recently reported that inhibition of the enzyme responsible for DNA methylation, DNA methyltransferase (DNMT), in the adult rat hippocampus blocks behavioral memory formation. Here, we report that DNMT inhibition also blocks the concomitant memory-associated H3 acetylation, without affecting phosphorylation of its upstream regulator, extracellular signal-regulated kinase (ERK). Interestingly, the DNMT inhibitor-induced deficit in memory consolidation, along with deficits in long-term potentiation, can be rescued by pharmacologically increasing levels of histone acetylation prior to DNMT inhibition. These observations suggest that DNMT activity is not only necessary for memory and plasticity, but that DNA methylation may work in concert with histone modifications to regulate plasticity and memory formation in the adult rat hippocampus.

  9. RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

    PubMed Central

    Khan, Dilshad H.; Gonzalez, Carolina; Cooper, Charlton; Sun, Jian-Min; Chen, Hou Yu; Healy, Shannon; Xu, Wayne; Smith, Karen T.; Workman, Jerry L.; Leygue, Etienne; Davie, James R.

    2014-01-01

    Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA. PMID:24234443

  10. 17ß-Estradiol Regulates Histone Alterations Associated with Memory Consolidation and Increases "Bdnf" Promoter Acetylation in Middle-Aged Female Mice

    ERIC Educational Resources Information Center

    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…

  11. Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly

    PubMed Central

    Li, Qing; Zhou, Hui; Wurtele, Hugo; Davies, Brian; Horazdovsky, Bruce; Verreault, Alain; Zhang, Zhiguo

    2008-01-01

    SUMMARY Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106. PMID:18662540

  12. JNK1 regulates histone acetylation in trigeminal neurons following chemical stimulation.

    PubMed

    Wu, Jing; Zhang, Xuan; Nauta, Haring J; Lin, Qing; Li, Junfa; Fang, Li

    2008-11-28

    Trigeminal nerve fibers in nasal and oral cavities are sensitive to various environmental hazardous stimuli, which trigger many neurotoxic problems such as chronic migraine headache and trigeminal irritated disorders. However, the role of JNK kinase cascade and its epigenetic modulation of histone remodeling in trigeminal ganglion (TG) neurons activated by environmental neurotoxins remains unknown. Here we investigated the role of JNK/c-Jun cascade in the regulation of acetylation of H3 histone in TG neurons following in vitro stimulation by a neuro-inflammatory agent, mustard oil (MO). We found that MO stimulation elicited JNK/c-Jun pathway significantly by enhancing phospho-JNK1, phospho-c-Jun expression, and c-Jun activity, which were correlated with an elevated acetylated H3 histone in TG neurons. However, increases in phospho-c-Jun and c-Jun activity were significantly blocked by a JNK inhibitor, SP600125. We also found that altered H3 histone remodeling, assessed by H3 acetylation in triggered TG neurons, was reduced by SP600125. The study suggests that the activated JNK signaling in regulation of histone remodeling may contribute to neuro-epigentic changes in peripheral sensory neurons following environmental neurotoxic exposure.

  13. Platelet activating factor-induced expression of p21 is correlated with histone acetylation.

    PubMed

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M; Liu, Jingwei; Neelapu, Sattva S; Ullrich, Stephen E

    2017-02-03

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome.

  14. Platelet activating factor-induced expression of p21 is correlated with histone acetylation

    PubMed Central

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M.; Liu, Jingwei; Neelapu, Sattva S.; Ullrich, Stephen E.

    2017-01-01

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome. PMID:28157211

  15. Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection

    PubMed Central

    2014-01-01

    Background There is renewed interest towards understanding the host-pathogen interaction in the light of epigenetic modifications. Although epithelial tissue is the major site for host-pathogen interactions, there is handful of studies to show how epithelial cells respond to pathogens. Bacterial infection in the mammary gland parenchyma induces local and subsequently systemic inflammation that results in a complex disease called mastitis. Globally Staphylococcus aureus is the single largest mastitis pathogen and the infection can ultimately result in either subclinical or chronic and sometimes lifelong infection. Results In the present report we have addressed the differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus, isolated from field samples. Immunohistochemical and immunoblotting analysis showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Global gene expression analysis in S. aureus infected mice mammary tissue revealed a selective set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Furthermore, we have identified several differentially expressed known miRNAs and 3 novel miRNAs in S. aureus infected mice mammary tissue by small RNA sequencing. By employing these gene expression data, an attempt has been made to delineate the gene regulatory networks in the strain specific inflammatory response. Apparently, one of the isolates of S. aureus activated the NF-κB signaling leading to drastic inflammatory response and induction of immune surveillance, which could possibly lead to rapid clearance of the pathogen. The other strain repressed most of the inflammatory response, which might help in its sustenance in the host tissue. Conclusion Taken together, our studies shed substantial lights to understand the mechanisms of strain specific differential inflammatory

  16. Cell shape regulates global histone acetylation in human mammaryepithelial cells

    SciTech Connect

    Le Beyec, Johanne; Xu, Ren; Lee, Sun-Young; Nelson, Celeste M.; Rizki, Aylin; Alcaraz, Jordi; Bissell, Mina J.

    2007-02-28

    Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

  17. Inhibitors of Histone Deacetylases Attenuate Noise-Induced Hearing Loss.

    PubMed

    Chen, Jun; Hill, Kayla; Sha, Su-Hua

    2016-08-01

    Loss of auditory sensory hair cells is the major pathological feature of noise-induced hearing loss (NIHL). Currently, no established clinical therapies for prevention or amelioration of NIHL are available. The absence of treatments is due to our lack of a comprehensive understanding of the molecular mechanisms underlying noise-induced damage. Our previous study indicates that epigenetic modification of histones alters hair cell survival. In this study, we investigated the effect of noise exposure on histone H3 lysine 9 acetylation (H3K9ac) in the inner ear of adult CBA/J mice and determined if inhibition of histone deacetylases by systemic administration of suberoylanilide hydroxamic acid (SAHA) could attenuate NIHL. Our results showed that H3K9ac was decreased in the nuclei of outer hair cells (OHCs) and marginal cells of the stria vascularis in the basal region after exposure to a traumatic noise paradigm known to induce permanent threshold shifts (PTS). Consistent with these results, levels of histone deacetylases 1, 2, and 3 (HDAC1, HDAC2 and HDAC3) were increased predominately in the nuclei of cochlear cells. Silencing of HDAC1, HDAC2, or HDAC3 with siRNA reduced the expression of the target HDAC in OHCs, but did not attenuate noise-induced PTS, whereas treatment with the pan-HDAC inhibitor SAHA, also named vorinostat, reduced OHC loss, and attenuated PTS. These findings suggest that histone acetylation is involved in the pathogenesis of noise-induced OHC death and hearing loss. Pharmacological targeting of histone deacetylases may afford a strategy for protection against NIHL.

  18. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription.

    PubMed

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-03-25

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001.

  19. Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription

    PubMed Central

    Di Cerbo, Vincenzo; Mohn, Fabio; Ryan, Daniel P; Montellier, Emilie; Kacem, Salim; Tropberger, Philipp; Kallis, Eleni; Holzner, Monika; Hoerner, Leslie; Feldmann, Angelika; Richter, Florian Martin; Bannister, Andrew J; Mittler, Gerhard; Michaelis, Jens; Khochbin, Saadi; Feil, Robert; Schuebeler, Dirk; Owen-Hughes, Tom; Daujat, Sylvain; Schneider, Robert

    2014-01-01

    Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states. DOI: http://dx.doi.org/10.7554/eLife.01632.001 PMID:24668167

  20. Life span extension by targeting a link between metabolism and histone acetylation in Drosophila.

    PubMed

    Peleg, Shahaf; Feller, Christian; Forne, Ignasi; Schiller, Evelyn; Sévin, Daniel C; Schauer, Tamas; Regnard, Catherine; Straub, Tobias; Prestel, Matthias; Klima, Caroline; Schmitt Nogueira, Melanie; Becker, Lore; Klopstock, Thomas; Sauer, Uwe; Becker, Peter B; Imhof, Axel; Ladurner, Andreas G

    2016-03-01

    Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. Here, we assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, we find that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, we decreased the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau. We find that these targeted interventions both alleviate the observed aging-associated changes and promote longevity. Our findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span.

  1. Insights into the epigenetic mechanisms involving histone lysine methylation and demethylation in ischemia induced damage and repair has therapeutic implication.

    PubMed

    Chakravarty, Sumana; Jhelum, Priya; Bhat, Unis Ahmad; Rajan, Wenson D; Maitra, Swati; Pathak, Salil S; Patel, Anant B; Kumar, Arvind

    2017-01-01

    Cerebral ischemic stroke is one of the leading causes of death and disability worldwide. Therapeutic interventions to minimize ischemia-induced neural damage are limited due to poor understanding of molecular mechanisms mediating complex pathophysiology in stroke. Recently, epigenetic mechanisms mostly histone lysine (K) acetylation and deacetylation have been implicated in ischemic brain damage and have expanded the dimensions of potential therapeutic intervention to the systemic/local administration of histone deacetylase inhibitors. However, the role of other epigenetic mechanisms such as histone lysine methylation and demethylation in stroke-induced damage and subsequent recovery process is elusive. Here, we established an Internal Carotid Artery Occlusion (ICAO) model in CD1 mouse that resulted in mild to moderate level of ischemic damage to the striatum, as suggested by magnetic resonance imaging (MRI), TUNEL and histopathological staining along with an evaluation of neurological deficit score (NDS), grip strength and rotarod performance. The molecular investigations show dysregulation of a number of histone lysine methylases (KMTs) and few of histone lysine demethylases (KDMs) post-ICAO with significant global attenuation in the transcriptionally repressive epigenetic mark H3K9me2 in the striatum. Administration of Dimethyloxalylglycine (DMOG), an inhibitor of KDM4 or JMJD2 class of histone lysine demethylases, significantly ameliorated stroke-induced NDS by restoring perturbed H3K9me2 levels in the ischemia-affected striatum. Overall, these results highlight the novel role of epigenetic regulatory mechanisms controlling the epigenetic mark H3K9me2 in mediating the stroke-induced striatal damage and subsequent repair following mild to moderate cerebral ischemia.

  2. Genome-wide prediction of conserved and nonconserved enhancers by histone acetylation patterns

    PubMed Central

    Roh, Tae-young; Wei, Gang; Farrell, Catherine M.; Zhao, Keji

    2007-01-01

    Comparative genomic studies have been useful in identifying transcriptional regulatory elements in higher eukaryotic genomes, but many important regulatory elements cannot be detected by such analyses due to evolutionary variations and alignment tool limitations. Therefore, in this study we exploit the highly conserved nature of epigenetic modifications to identify potential transcriptional enhancers. By using a high-resolution genome-wide mapping technique, which combines the chromatin immunoprecipitation and serial analysis of gene expression assays, we have recently determined the distribution of lysine 9/14-diacetylated histone H3 in human T cells. We showed the existence of 46,813 regions with clusters of histone acetylation, termed histone acetylation islands, some of which correspond to known transcriptional regulatory elements. In the present study, we find that 4679 sequences conserved between human and pufferfish coincide with histone acetylation islands, and random sampling shows that 33% (13/39) of these can function as transcriptional enhancers in human Jurkat T cells. In addition, by comparing the human histone acetylation island sequences with mouse genome sequences, we find that despite the conservation of many of these regions between these species, 21,855 of these sequences are not conserved. Furthermore, we demonstrate that about 50% (26/51) of these nonconserved sequences have enhancer activity in Jurkat cells, and that many of the orthologous mouse sequences also have enhancer activity in addition to conserved epigenetic modification patterns in mouse T-cell chromatin. Therefore, by combining epigenetic modification and sequence data, we have established a novel genome-wide method for identifying regulatory elements not discernable by comparative genomics alone. PMID:17135569

  3. Histone acetylation is involved in TCDD-induced cleft palate formation in fetal mice

    PubMed Central

    Yuan, Xingang; Qiu, Lin; Pu, Yalan; Liu, Cuiping; Zhang, Xuan; Wang, Chen; Pu, Wei; Fu, Yuexian

    2016-01-01

    The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8-tetrachlo-rodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. Pregnant mice (n=10) were randomly divided into two groups: i) TCDD group, mice were treated with 28 µg/kg TCDD on gestation day (GD) 10 by oral gavage; ii) control group, mice were treated with an equal volume of corn oil. On GD 16.5, the fetal mice were evaluated for the presence of a cleft palate. An additional 36 pregnant mice were divided into the control and TCDD groups, and palate samples were collected on GD 13.5, GD 14.5 and GD 15.5, respectively. Transforming growth factor-β3 (TGF-β3) mRNA expression, TGF-β3 promoter methylation, histone acetyltransferase (HAT) activity and histone H3 (H3) acetylation in the palates were evaluated in the two groups. The incidence of a cleft palate in the TCDD group was 93.55%, and no cases of cleft palate were identified in the control group. On GD 13.5 and GD 14.5, TGF-β3 mRNA expression, HAT activity and acetylated H3 levels were significantly increased in the TCDD group compared with the control. Methylated bands were not observed in the TCDD or control groups. In conclusion, at the critical period of palate fusion (GD 13.5–14.5), TCDD significantly increased TGF-β3 gene expression, HAT activity and H3 acetylation. Therefore, histone acetylation may be involved in TCDD-induced cleft palate formation in fetal mice. PMID:27279340

  4. Effect of histone acetylation on the formation and removal of B(a)P chromatin adducts.

    PubMed Central

    Kootstra, A

    1982-01-01

    The modification of core histone proteins in mouse 10T1/2 cells and human lung epitheloid (A549) cells by B(a)PDE in vivo and in vitro was found to be similar. Only histones H2A and H3 were extensively modified. Also other proteins, possibly A24 protein and the minor histone H1 species seem to be binding relatively high levels of this ultimate carcinogen. Butyrate treatment which causes hyperacetylation of the core histones, did not change the specificity of B(a)PDE binding to core histones, nor did it affect the initial level of DNA modification. The acetylated species of histone H3 were all accessible to B(a)PDE, suggesting that these epsilon-amino-groups of the lysine residues are not the targets of the B(a)PDE. The rate of removal of B(a)P-DNA adducts was not affected by butyrate treatment in either normal human or XP fibroblasts. Furthermore the B(a)P-core histones were not preferentially removed from normal human fibroblast chromatin during a 24 h post-treatment incubation. Images PMID:6285308

  5. The RSC Complex Exploits Histone Acetylation to Abrogate the Nucleosomal Barrier to RNA Polymerase II Elongation

    PubMed Central

    Carey, Michael; Li, Bing; Workman, Jerry L.

    2007-01-01

    Summary The coordinated action of histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling enzymes in promoter-dependent transcription initiation represents a paradigm for how epigenetic information regulates gene expression. However, little is known about how such enzymes function during transcription elongation. Here we investigated the role of RSC, a bromodomain-containing ATPase, in nucleosome transcription in vitro. Purified S. cerevisiae RNA polymerase II (pol II) arrests at two primary locations on a positioned mononucleosome. RSC stimulates passage of pol II through these sites. The function of RSC in elongation requires the energy of ATP hydrolysis. Moreover, the SAGA and NuA4 HATs strongly stimulated RSC’s effect on elongation. The stimulation correlates closely with Acetyl-CoA-dependent recruitment of RSC to nucleosomes. Thus, RSC can recognize acetylated nucleosomes and facilitate passage of pol II through them. These data support the view that histone modifications regulate accessibility of the coding region to pol II. PMID:17081996

  6. Trichostatin A affects histone acetylation and gene expression in porcine somatic cell nucleus transfer embryos.

    PubMed

    Cervera, R P; Martí-Gutiérrez, N; Escorihuela, E; Moreno, R; Stojkovic, M

    2009-11-01

    Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming after somatic cell nucleus transfer (SCNT) and may underlie the observed reduced viability of cloned embryos. In the current study, we tested the effects of the histone deacetylase-inhibitor trichostatin A (TSA) on preimplantation development and on histone acetylation and the gene expression of nucleus transfer (NT) porcine (Sus scrofa) embryos. Our results showed that 5 nM TSA for 26 h after reconstitution resulted in embryos (NTTSA) that reached the blastocyst stage at a higher level (48.1% vs. 20.2%) and increased number of cells (105.0 vs. 75.3) than that of the control (NTC) embryos. In addition, and unlike the NTC embryos, the treated embryos displayed a global acetylated histone H4 at lysine 8 profile similar to the in vitro-fertilized (IVF) and cultured embryos during the preimplantation development. Finally, we determined that several transcription factors exert a dramatic amount of genetic control over pluripotency, including Oct4, Nanog, Cdx2, and Rex01, the imprinting genes Igf2 and Igf2r, and the histone deacetyltransferase gene Hdac2. The NT blastocysts showed similar levels of Oct4, Cdx2, and Hdac2 but lower levels of Nanog than those of the IVF blastocyst. However, the NTTSA blastocysts showed similar levels of Rex01, Igf2, and Igf2r as those of IVF blastocysts, whereas the NTC blastocysts showed significantly lower levels for those genes. Our results suggest that TSA improves porcine SCNT preimplantation development and affects the acetylated status of the H4K8, rendering acetylation levels similar to those of the IVF counterparts.

  7. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics.

    PubMed

    Bernier, Morgan; Luo, Yi; Nwokelo, Kingsley C; Goodwin, Michelle; Dreher, Sarah J; Zhang, Pei; Parthun, Mark R; Fondufe-Mittendorf, Yvonne; Ottesen, Jennifer J; Poirier, Michael G

    2015-12-09

    H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1.

  8. Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea

    PubMed Central

    2010-01-01

    Background The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression. Results The results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals. Conclusion The epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation. PMID:20067625

  9. MOF Acetylates the Histone Demethylase LSD1 to Suppress Epithelial-to-Mesenchymal Transition.

    PubMed

    Luo, Huacheng; Shenoy, Anitha K; Li, Xuehui; Jin, Yue; Jin, Lihua; Cai, Qingsong; Tang, Ming; Liu, Yang; Chen, Hao; Reisman, David; Wu, Lizi; Seto, Edward; Qiu, Yi; Dou, Yali; Casero, Robert A; Lu, Jianrong

    2016-06-21

    The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression.

  10. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics

    PubMed Central

    Bernier, Morgan; Luo, Yi; Nwokelo, Kingsley C.; Goodwin, Michelle; Dreher, Sarah J.; Zhang, Pei; Parthun, Mark R.; Fondufe-Mittendorf, Yvonne; Ottesen, Jennifer J.; Poirier, Michael G.

    2015-01-01

    H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1. PMID:26648124

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

    PubMed

    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

  12. Association of global levels of histone modifications with recurrence-free survival in stage IIB and III esophageal squamous cell carcinomas.

    PubMed

    I, Hoseok; Ko, Eunkyung; Kim, Yujin; Cho, Eun Yoon; Han, Joungho; Park, Joobae; Kim, Kwhanmien; Kim, Duk-Hwan; Shim, Young Mog

    2010-02-01

    This study was aimed at understanding the effects of histone modifications on recurrence-free survival (RFS) after esophagectomy in esophageal squamous cell carcinoma (ESCC). The acetylation of histone H3 lysine (H3K9Ac), histone H3 lysine 18 (H3K18Ac), and histone H4 lysine 12 (H4K12Ac), and the dimethylation of histone H3 lysine 9 (H3K9diMe) and histone H4 arginine 3 (H4R3diMe) were analyzed by immunohistochemistry in 237 ESCCs. The K-means clustering algorithm was used to identify unique patterns of histone modifications. At a median follow-up of 5.1 years, 109 (46%) of 237 patients had developed recurrence of disease. Mean global levels of H3K9Ac, H3K18Ac, H3K9diMe, H4K12Ac, and H4R3diMe were 81.5%, 65.1%, 80.3%, 45.9%, and 27.4%, respectively. In the analysis of individual histones, a 1% increase in the global level of H3K18Ac in pathologic stage III worsened RFS at 1.009 times [95% confidence interval (CI), 1.001-1.016; P = 0.03], after adjusting for age, sex, and operative method. Cluster analysis also showed significant effects of histone modifications on RFS. For stage IIB cancers, Cox proportional hazards analysis showed that RFS of cluster 1, with high global levels of H3K18Ac and H4R3diMe, was 2.79 times poorer (95% CI, 1.14-6.27; P = 0.008) than that of cluster 2, with low levels. RFS for stage III cancers was also poorer in cluster 1 than cluster 2 (adjusted hazard ratio, 2.42; 95% CI, 1.10-5.34; P = 0.02). In conclusion, the present study suggests that global levels of histone modifications in ESCC may be an independent prognostic factor of RFS.

  13. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability.

    PubMed

    Lauffer, Benjamin E L; Mintzer, Robert; Fong, Rina; Mukund, Susmith; Tam, Christine; Zilberleyb, Inna; Flicke, Birgit; Ritscher, Allegra; Fedorowicz, Grazyna; Vallero, Roxanne; Ortwine, Daniel F; Gunzner, Janet; Modrusan, Zora; Neumann, Lars; Koth, Christopher M; Lupardus, Patrick J; Kaminker, Joshua S; Heise, Christopher E; Steiner, Pascal

    2013-09-13

    Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.

  14. Histone H4 acetylation required for chromatin decompaction during DNA replication.

    PubMed

    Ruan, Kun; Yamamoto, Takaharu G; Asakawa, Haruhiko; Chikashige, Yuji; Kimura, Hiroshi; Masukata, Hisao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-07-30

    Faithful DNA replication is a prerequisite for cell proliferation. Several cytological studies have shown that chromosome structures alter in the S-phase of the cell cycle. However, the molecular mechanisms behind the alteration of chromosome structures associated with DNA replication have not been elucidated. Here, we investigated chromatin structures and acetylation of specific histone residues during DNA replication using the meiotic nucleus of the fission yeast Schizosaccharomyces pombe. The S. pombe meiotic nucleus provides a unique opportunity for measuring the levels of compaction of chromatin along the chromosome in a defined orientation. By direct measurement of chromatin compaction in living cells, we demonstrated that decompaction of chromatin occurs during meiotic DNA replication. This chromatin decompaction was suppressed by depletion of histone acetyltransferase Mst1 or by arginine substitution of specific lysine residues (K8 and K12) of histone H4. These results suggest that acetylation of histone H4 residues K8 and K12 plays a critical role in loosening chromatin structures during DNA replication.

  15. Acetylation of EGF Receptor Contributes to Tumor Cell Resistance to Histone Deacetylase Inhibitors

    PubMed Central

    Song, Hui; Li, Chia-Wei; Labaff, Adam M.; Lim, Seung-Oe; Li, Long-Yuan; Kan, Shu-Fen; Chen, Yue; Zhang, Kai; Lang, Jingyu; Xie, Xiaoming; Wang, Yan; Huo, Long-Fei; Hsu, Sheng-Chieh; Chen, Xiaomin; Zhao, Yingming; Hung, Mien-Chie

    2011-01-01

    Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication. PMID:21094134

  16. Alterations of histone modifications by cobalt compounds

    PubMed Central

    Li, Qin; Ke, Qingdong; Costa, Max

    2009-01-01

    In the present study, we examined the effects of CoCl2 on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl2 (≥200 μM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 μM of CoCl2 for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen. PMID:19376846

  17. Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements

    PubMed Central

    Myers, Fiona A.; Lefevre, Pascal; Mantouvalou, Evangelia; Bruce, Kimberley; Lacroix, Claire; Bonifer, Constanze; Thorne, Alan W.; Crane-Robinson, Colyn

    2006-01-01

    Native chromatin IP assays were used to define changes in core histone acetylation at the lysozyme locus during developmental maturation of chicken macrophages and stimulation to high-level expression by lipo-polysaccharide. In pluripotent precursors the lysozyme gene (Lys) is inactive and there is no acetylation of core histones at the gene, its promoter or at the upstream cis-control elements. In myeloblasts, where there is a very low level of Lys expression, H4 acetylation appears at the cis-control elements but not at the Lys gene or its promoter: neither H3 nor H2B become significantly acetylated in myeloblasts. In mature macrophages, Lys expression increases 5-fold: H4, H2B and H2A.Z are all acetylated at the cis-control elements but H3 remains unacetylated except at the −2.4 S silencer. Stimulation with LPS increases Lys expression a further 10-fold: this is accompanied by a rise in H3 acetylation throughout the cis-control elements; H4 and H2B acetylation remain substantial but acetylation at the Lys gene and its promoter remains low. Acetylation is thus concentrated at the cis-control elements, not at the Lys gene or its immediate promoter. H4 acetylation precedes H3 acetylation during development and H3 acetylation is most directly linked to high-level Lys expression. PMID:16914441

  18. Histone H3 Acetyl K9 and Histone H3 Tri Methyl K4 as Prognostic Markers for Patients with Cervical Cancer

    PubMed Central

    Beyer, Susanne; Zhu, Junyan; Mayr, Doris; Kuhn, Christina; Schulze, Sandra; Hofmann, Simone; Dannecker, Christian; Jeschke, Udo; Kost, Bernd P.

    2017-01-01

    Chromatin remodeling alters gene expression in carcinoma tissue. Although cervical cancer is the fourth most common cancer in women worldwide, a systematic study about the prognostic value of specific changes in the chromatin structure, such as histone acetylation or histone methylation, is missing. In this study, the expression of histone H3 acetyl K9, which is known to denote active regions at enhancers and promoters, and histone H3 tri methyl K4, which preferentially identifies active gene promoters, were examined as both show high metastatic potential. A panel of patients with cervical cancer was selected and the importance of the histone modifications concerning survival-time (overall survival and relapse-free survival) was analyzed in 250 cases. Histone H3 acetyl K9 staining was correlated with low grading, low FIGO (TNM classification and the International Federation of Gynecology and Obstetrics) status, negative N-status and low T-status in cervical cancer, showing a higher expression in adenocarcinoma than in squamous cell carcinoma. Cytoplasmic expression of histone H3 tri methyl K4 in a cervical cancer specimen was correlated with advanced T-status and poor prognosis. While cytoplasmic H3K4me3 expression seemed to be a marker of relapse-free survival, nuclear expression showed a correlation to poor prognosis in overall survival. Within this study, we analyzed the chemical modification of two histone proteins that are connected to active gene expression. Histone H3 acetyl K9 was found to be an independent marker of overall survival. Histone H3 tri methyl K4 was correlated with poor prognosis and it was found to be an independent marker of relapse-free survival. Therefore, we could show that chromatin remodeling plays an important role in cervical cancer biology. PMID:28241481

  19. Histone H3 Acetyl K9 and Histone H3 Tri Methyl K4 as Prognostic Markers for Patients with Cervical Cancer.

    PubMed

    Beyer, Susanne; Zhu, Junyan; Mayr, Doris; Kuhn, Christina; Schulze, Sandra; Hofmann, Simone; Dannecker, Christian; Jeschke, Udo; Kost, Bernd P

    2017-02-23

    Chromatin remodeling alters gene expression in carcinoma tissue. Although cervical cancer is the fourth most common cancer in women worldwide, a systematic study about the prognostic value of specific changes in the chromatin structure, such as histone acetylation or histone methylation, is missing. In this study, the expression of histone H3 acetyl K9, which is known to denote active regions at enhancers and promoters, and histone H3 tri methyl K4, which preferentially identifies active gene promoters, were examined as both show high metastatic potential. A panel of patients with cervical cancer was selected and the importance of the histone modifications concerning survival-time (overall survival and relapse-free survival) was analyzed in 250 cases. Histone H3 acetyl K9 staining was correlated with low grading, low FIGO (TNM classification and the International Federation of Gynecology and Obstetrics) status, negative N-status and low T-status in cervical cancer, showing a higher expression in adenocarcinoma than in squamous cell carcinoma. Cytoplasmic expression of histone H3 tri methyl K4 in a cervical cancer specimen was correlated with advanced T-status and poor prognosis. While cytoplasmic H3K4me3 expression seemed to be a marker of relapse-free survival, nuclear expression showed a correlation to poor prognosis in overall survival. Within this study, we analyzed the chemical modification of two histone proteins that are connected to active gene expression. Histone H3 acetyl K9 was found to be an independent marker of overall survival. Histone H3 tri methyl K4 was correlated with poor prognosis and it was found to be an independent marker of relapse-free survival. Therefore, we could show that chromatin remodeling plays an important role in cervical cancer biology.

  20. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

    PubMed Central

    Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

    2016-01-01

    Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

  1. Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development

    PubMed Central

    Karmodiya, Krishanpal; Anamika, Krishanpal; Muley, Vijaykumar; Pradhan, Saurabh J.; Bhide, Yoshita; Galande, Sanjeev

    2014-01-01

    In this study, we have investigated genome-wide occurrence of Histone Acetyltransferases (HATs) in genomes of Mus musculus and Danio rerio on the basis of presence of HAT domain. Our study identified a group of proteins that lacks characteristic features of known HAT families, relatively smaller in size and has no other associated domains. Most of the proteins in this unclassified group are Camello proteins, which are not yet known and classified as functional HATs. Our in vitro and in vivo analysis revealed that Camello family proteins are active HATs and exhibit specificity towards histone H4. Interestingly, Camello proteins are among the first identified HATs showing perinuclear localization. Moreover, Camello proteins are evolutionarily conserved in all chordates and are observed for the first time in cnidarians in phylogeny. Furthermore, knockdown of Camello protein (CMLO3) in zebrafish embryos exhibited defects in axis elongation and head formation. Thus, our study identified a novel family of active HATs that is specific for histone H4 acetylation, exhibits perinuclear localization and is essential for zebrafish development. PMID:25123547

  2. Crx activates opsin transcription by recruiting HAT-containing co-activators and promoting histone acetylation

    PubMed Central

    Peng, Guang-Hua; Chen, Shiming

    2008-01-01

    The homeodomain transcription factor Crx is required for expression of many photoreceptor genes in the mammalian retina. The mechanism by which Crx activates transcription remains to be determined. Using protein–protein interaction assays, Crx was found to interact with three co-activator proteins (complexes): STAGA, Cbp and p300, all of which possess histone acetyl-transferase (HAT) activity. To determine the role of Crx–HAT interactions in target gene chromatin modification and transcriptional activation, quantitative RT–PCR and chromatin immunoprecipitation were performed on Crx target genes, rod and cone opsins, in developing mouse retina. Although cone opsins are transcribed earlier than rhodopsin during development, the transcription of each gene is preceded by the same sequence of events in their promoter and enhancer regions: (i) binding of Crx, followed by (ii) binding of HATs, (iii) the acetylation of histone H3, then (iv) binding of other photoreceptor transcription factors (Nrl and Nr2e3) and RNA polymerase II. In Crx knockout mice (Crx−/−), the association of HATs and AcH3 with target promoter/enhancer regions was significantly decreased, which correlates with aberrant opsin transcription and photoreceptor dysfunction in these mice. Similar changes to the opsin chromatin were seen in Y79 retinoblastoma cells, where opsin genes are barely transcribed. These defects in Y79 cells can be reversed by expressing a recombinant Crx or applying histone deacetylase inhibitors. Altogether, these results suggest that one mechanism for Crx-mediated transcriptional activation is to recruit HATs to photoreceptor gene chromatin for histone acetylation, thereby inducing and maintaining appropriate chromatin configurations for transcription. PMID:17656371

  3. Enhancing dopaminergic signaling and histone acetylation promotes long-term rescue of deficient fear extinction

    PubMed Central

    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

  4. Anti-Inflammatory Effects of Spirulina platensis Extract via the Modulation of Histone Deacetylases

    PubMed Central

    Pham, Tho X.; Park, Young-Ki; Lee, Ji-Young

    2016-01-01

    We previously demonstrated that the organic extract of Spirulina platensis (SPE), an edible blue-green alga, possesses potent anti-inflammatory effects. In this study, we investigated if the regulation of histone deacetylases (HDACs) play a role in the anti-inflammatory effect of SPE in macrophages. Treatment of macrophages with SPE rapidly and dose-dependently reduced HDAC2, 3, and 4 proteins which preceded decreases in their mRNA levels. Degradation of HDAC4 protein was attenuated in the presence of inhibitors of calpain proteases, lysosomal acidification, and Ca2+/calmodulin-dependent protein kinase II, respectively, but not a proteasome inhibitor. Acetylated histone H3 was increased in SPE-treated macrophages to a similar level as macrophages treated with a pan-HDAC inhibitor, with concomitant inhibition of inflammatory gene expression upon LPS stimulation. Knockdown of HDAC3 increased basal and LPS-induced pro-inflammatory gene expression, while HDAC4 knockdown increased basal expression of interleukin-1β (IL-1β), but attenuated LPS-induced inflammatory gene expression. Chromatin immunoprecipitation showed that SPE decreased p65 binding and H3K9/K14 acetylation at the Il-1β and tumor necrosis factor α (Tnfα) promoters. Our results suggest that SPE increased global histone H3 acetylation by facilitating HDAC protein degradation, but decreases histone H3K9/K14 acetylation and p65 binding at the promoters of Il-1β and Tnfα to exert its anti-inflammatory effect. PMID:27338466

  5. Dynamic Competing Histone H4 K5K8 Acetylation and Butyrylation Are Hallmarks of Highly Active Gene Promoters

    PubMed Central

    Goudarzi, Afsaneh; Zhang, Di; Huang, He; Barral, Sophie; Kwon, Oh Kwang; Qi, Shankang; Tang, Zhanyun; Buchou, Thierry; Vitte, Anne-Laure; He, Tieming; Cheng, Zhongyi; Montellier, Emilie; Gaucher, Jonathan; Curtet, Sandrine; Debernardi, Alexandra; Charbonnier, Guillaume; Puthier, Denis; Petosa, Carlo; Panne, Daniel; Rousseaux, Sophie; Roeder, Robert G.; Zhao, Yingming; Khochbin, Saadi

    2016-01-01

    Summary Recently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation. Here, we focus on histone butyrylation in the context of sperm cell differentiation. Specifically, we investigate the butyrylation of histone H4 lysine 5 and 8 at gene promoters where acetylation guides the binding of Brdt, a bromodomain-containing protein, thereby mediating stage-specific gene expression programs and post-meiotic chromatin reorganization. Genome-wide mapping data show that highly active Brdt-bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at H4 K5 and H4 K8. Despite acting as a direct stimulator of transcription, histone butyrylation competes with acetylation, especially at H4 K5, to prevent Brdt binding. Additionally, H4 K5K8 butyrylation also marks retarded histone removal during late spermatogenesis. Hence, alternating H4 acetylation and butyrylation, while sustaining direct gene activation and dynamic bromodomain binding, could impact the final male epigenome features. PMID:27105113

  6. Acetylation of LYS-16 of H4 Histone Tail May Sequester the Tail and Inhibit its Interactions with Neighboring Nucleosomes

    NASA Astrophysics Data System (ADS)

    Potoyan, Davit; Papoian, Garegin

    2012-02-01

    Histone tails are highly flexible N terminal protrusions of histone proteins, which help to fold DNA into dense superstructures known as chromatin. On a molecular scale histone tails are poly-electrolites with high degree of conformational disorder, allowing them to function as bio-molecular ``switches,'' regulating various genetic regulatory processes via diverse types of covalent modifications. Because of being intrinsically disordered, the structural and dynamical aspects of histone tails are still poorly understood. Using multiple explicit solvent and coarse-grained MD simulations we have investigated the impact of the acetylation of LYS-16 residue on the conformational and DNA-binding propensities of H4 histone tail. The potential of mean force computed as a function of distance between a model DNA and histone tail center of mass showed a dramatic enhancement of binding affinity upon mono-acetylation of the H4 tail. The estimated binding free energy gain for the wild type is 2kT, while for the acetylated it reaches 4-5 kT. Additionally our structural analysis shows that acetylation is driving the chain into collapsed states, which get enriched in secondary structural elements upon binding to the DNA. We suggest a non-electrostatic mechanism that explains the enhanced binding affinity of the acetylated H4 tail. At last our findings lead us to propose a hypothesis that can potentially account for the celebrated chromatin ``fiber loosening effects'' observed in many experiments.

  7. Histone H3 lysine 56 acetylation and the response to DNA replication fork damage.

    PubMed

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien; St-Hilaire, Edlie; Lee, Eun-Hye; Tsao, Sarah; Dorn, Jonas; Maddox, Paul; Lisby, Michael; Pasero, Philippe; Verreault, Alain

    2012-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.

  8. Histone H3 Lysine 56 Acetylation and the Response to DNA Replication Fork Damage

    PubMed Central

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien; St-Hilaire, Edlie; Lee, Eun-Hye; Tsao, Sarah; Dorn, Jonas; Maddox, Paul; Lisby, Michael; Pasero, Philippe

    2012-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication. PMID:22025679

  9. Association of Reading Disabilities With Regions Marked by Acetylated H3 Histones in KIAA0319

    PubMed Central

    Couto, Jillian M.; Livne-Bar, Izzy; Huang, Katherine; Xu, Zhaodong; Cate-Carter, Tasha; Feng, Yu; Wigg, Karen; Humphries, Tom; Tannock, Rosemary; Kerr, Elizabeth N.; Lovett, Maureen W.; Bremner, Rod; Barr, Cathy L.

    2017-01-01

    Reading disabilities (RDs) have been associated with chromosome 6p with recent studies pointing to two genes, DCDC2 and KIAA0319. In this study, markers across the 6p region were tested for association with RD. Our strongest findings were for association with markers in KIAA0319, although with the opposite alleles compared with a previous study. We also found association with markers in VMP, but not with DCDC2. Current evidence indicates that differential regulation of KIAA0319 and DCDC2 contributes to RD, thus we used chromatin immunoprecipitation coupled with genomic tiling arrays (ChIP-chip) to map acetylated histones, a molecular marker for regulatory elements, across a 500 kb genomic region covering the RD locus on 6p. This approach identified several regions marked by acetylated histones that mapped near associated markers, including intron 7 of DCDC2 and the 5′ region of KIAA0319. The latter is located within the 70 kb region previously associated with differential expression of KIAA0319. Interestingly, five markers associated with RD in independent studies were also located within the 2.7 kb acetylated region, and six additional associated markers, including the most significant one in this study, were located within a 22 kb haplotype block that encompassed this region. Our data indicates that this putative regulatory region is a likely site of genetic variation contributing to RD in our sample, further narrowing the candidate region. PMID:19588467

  10. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    PubMed Central

    Chen, Zhuo; Zuo, Xuezhi; He, Dongliang; Ding, Shibin; Xu, Fangyi; Yang, Huiqin; Jin, Xin; Fan, Ying; Ying, Li; Tian, Chong; Ying, Chenjiang

    2017-01-01

    Bisphenol A (BPA), a typical environmental endocrine-disrupting chemical, induces epigenetic inheritance. Whether histone acetylation plays a role in these effects of BPA is largely unknown. Here, we investigated histone acetylation in male rats after long-term exposure to a ‘safe’ dose of BPA. Twenty adult male rats received either BPA (50 μg/kg·bw/day) or a vehicle diet for 35 weeks. Decreased protein lysine-acetylation levels at approximately ~17 kDa and ~25 kDa, as well as decreased histone acetylation of H3K9, H3K27 and H4K12, were detected by Western blot analysis of testes from the treated rats compared with controls. Additionally, increased protein expression of deacetylase Sirt1 and reduced binding of Sirt1, together with increased binding of estrogen receptor β (ERβ) to caveolin-1 (Cav-1), a structural protein component of caveolar membranes, were detected in treated rats compared with controls. Moreover, decreased acetylation of Cav-1 was observed in the treated rats for the first time. Our study showed that long-term exposure to a ‘safe’ dose of BPA reduces histone acetylation in the male reproductive system, which may be related to the phenotypic paternal-to-offspring transmission observed in our previous study. The evidence also suggested that these epigenetic effects may be meditated by Sirt1 via competition with ERβ for binding to Cav-1. PMID:28067316

  11. Long-term exposure to a ‘safe’ dose of bisphenol A reduced protein acetylation in adult rat testes

    NASA Astrophysics Data System (ADS)

    Chen, Zhuo; Zuo, Xuezhi; He, Dongliang; Ding, Shibin; Xu, Fangyi; Yang, Huiqin; Jin, Xin; Fan, Ying; Ying, Li; Tian, Chong; Ying, Chenjiang

    2017-01-01

    Bisphenol A (BPA), a typical environmental endocrine-disrupting chemical, induces epigenetic inheritance. Whether histone acetylation plays a role in these effects of BPA is largely unknown. Here, we investigated histone acetylation in male rats after long-term exposure to a ‘safe’ dose of BPA. Twenty adult male rats received either BPA (50 μg/kg·bw/day) or a vehicle diet for 35 weeks. Decreased protein lysine-acetylation levels at approximately ~17 kDa and ~25 kDa, as well as decreased histone acetylation of H3K9, H3K27 and H4K12, were detected by Western blot analysis of testes from the treated rats compared with controls. Additionally, increased protein expression of deacetylase Sirt1 and reduced binding of Sirt1, together with increased binding of estrogen receptor β (ERβ) to caveolin-1 (Cav-1), a structural protein component of caveolar membranes, were detected in treated rats compared with controls. Moreover, decreased acetylation of Cav-1 was observed in the treated rats for the first time. Our study showed that long-term exposure to a ‘safe’ dose of BPA reduces histone acetylation in the male reproductive system, which may be related to the phenotypic paternal-to-offspring transmission observed in our previous study. The evidence also suggested that these epigenetic effects may be meditated by Sirt1 via competition with ERβ for binding to Cav-1.

  12. Characterization of histone modifications associated with DNA damage repair genes upon exposure to gamma rays in Arabidopsis seedlings

    PubMed Central

    Mondal, Suvendu; Go, Young Sam; Lee, Seung Sik; Chung, Byung Yeoup; Kim, Jin-Hong

    2016-01-01

    Dynamic histone modifications play an important role in controlling gene expression in response to various environmental cues. This mechanism of regulation of gene expression is important for sessile organisms, like land plants. We have previously reported consistent upregulation of various marker genes in response to gamma rays at various post-irradiation times. In the present study, we performed various chromatin modification analyses at selected loci using the standard chromatin immunoprecipitation procedure, and demonstrate that upregulation of these genes is associated with histone H3 lysine 4 tri-methylation (H3K4me3) at the gene body or transcription start sites of these loci. Further, at specific AtAgo2 loci, both H3K4me3 and histone H3 lysine 9 acetylation (H3K9ac) are important in controlling gene expression in response to gamma irradiation. There was no change in DNA methylation in these selected loci. We conclude that specific histone modification such as H3K4me3 and H3K9ac may be more important in activating gene expression in these selected loci in response to gamma irradiation than a change in DNA methylation. PMID:27534791

  13. Distribution of acetylated histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes

    PubMed Central

    Vignali, Marissa; Steger, David J.; Neely, Kristen E.; Workman, Jerry L.

    2000-01-01

    We analyzed the targeting of histone acetyltransferase (HAT) complexes by DNA-binding activators during transcriptional activation and the resulting distribution of acetylated histones. An in vitro competition assay was developed to acetylate and transcribe a nucleosomal array template in the presence of excess non-specific chromatin, which mimics in vivo conditions. Stimulation of transcription from the nucleosomal array template under competitive conditions by the SAGA and NuA4 HAT complexes depended on the presence of the Gal4-VP16 activator, which recognizes sites in the promoter and directly interacts with these HATs. Importantly, the stimulation of transcription by SAGA and NuA4 depended on the presence of Gal4-VP16 during histone acetylation, and Gal4-VP16-bound nucleosomal templates were acetylated preferentially by SAGA and NuA4 relative to the competitor chromatin. While targeting of the SAGA complex led to H3 acetylation of promoter-proximal nucleosomes, targeting of the NuA4 complex led to a broader domain of H4 acetylation of >3 kbp. Thus, either promoter-proximal H3 acetylation by SAGA or broadly distributed acetylation of H4 by NuA4 activated transcription from chromatin templates. PMID:10835360

  14. EIN2-dependent regulation of acetylation of histone H3K14 and non-canonical histone H3K23 in ethylene signalling

    PubMed Central

    Zhang, Fan; Qi, Bin; Wang, Likai; Zhao, Bo; Rode, Siddharth; Riggan, Nathaniel D.; Ecker, Joseph R.; Qiao, Hong

    2016-01-01

    Ethylene gas is essential for many developmental processes and stress responses in plants. EIN2 plays a key role in ethylene signalling but its function remains enigmatic. Here, we show that ethylene specifically elevates acetylation of histone H3K14 and the non-canonical acetylation of H3K23 in etiolated seedlings. The up-regulation of these two histone marks positively correlates with ethylene-regulated transcription activation, and the elevation requires EIN2. Both EIN2 and EIN3 interact with a SANT domain protein named EIN2 nuclear associated protein 1 (ENAP1), overexpression of which results in elevation of histone acetylation and enhanced ethylene-inducible gene expression in an EIN2-dependent manner. On the basis of these findings we propose a model where, in the presence of ethylene, the EIN2 C terminus contributes to downstream signalling via the elevation of acetylation at H3K14 and H3K23. ENAP1 may potentially mediate ethylene-induced histone acetylation via its interactions with EIN2 C terminus. PMID:27694846

  15. Histone H4 acetylation dynamics determined by stable isotope labeling with amino acids in cell culture and mass spectrometry.

    PubMed

    Su, Xiaodan; Zhang, Liwen; Lucas, David M; Davis, Melanie E; Knapp, Amy R; Green-Church, Kari B; Marcucci, Guido; Parthun, Mark R; Byrd, John C; Freitas, Michael A

    2007-04-01

    This paper describes an integrated approach that couples stable isotope labeling with amino acids in cell culture to acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular medium and in medium in which lysine was substituted with deuterium-labeled lysine. Histone deacetylase (HDAC) activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture medium for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and from labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF MS. Detailed information was obtained for both the change of histone H4 acetylation specific to the N terminus and the global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. This study provides a quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli.

  16. MRG15 activates the cdc2 promoter via histone acetylation in human cells

    SciTech Connect

    Pena, AndreAna N.; Tominaga, Kaoru; Pereira-Smith, Olivia M.

    2011-07-01

    Chromatin remodeling is required for transcriptional activation and repression. MRG15 (MORF4L1), a chromatin modulator, is a highly conserved protein and is present in complexes containing histone acetyltransferases (HATs) as well as histone deacetylases (HDACs). Loss of expression of MRG15 in mice and Drosophila results in embryonic lethality and fibroblast and neural stem/progenitor cells cultured from Mrg15 null mouse embryos exhibit marked proliferative defects when compared with wild type cells. To determine the role of MRG15 in cell cycle progression we performed chromatin immunoprecipitation with an antibody to MRG15 on normal human fibroblasts as they entered the cell cycle from a quiescent state, and analyzed various cell cycle gene promoters. The results demonstrated a 3-fold increase in MRG15 occupancy at the cdc2 promoter during S phase of the cell cycle and a concomitant increase in acetylated histone H4. H4 lysine 12 was acetylated at 24 h post-serum stimulation while there was no change in acetylation of lysine 16. HDAC1 and 2 were decreased at this promoter during cell cycle progression. Over-expression of MRG15 in HeLa cells activated a cdc2 promoter-reporter construct in a dose-dependent manner, whereas knockdown of MRG15 resulted in decreased promoter activity. In order to implicate HAT activity, we treated cells with the HAT inhibitor anacardic acid and determined that HAT inhibition results in loss of expression of cdc2 mRNA. Further, chromatin immunoprecipitation with Tip60 localizes the protein to the same 110 bp stretch of the cdc2 promoter pulled down by MRG15. Additionally, we determined that cotransfection of MRG15 with the known associated HAT Tip60 had a cooperative effect in activating the cdc2 promoter. These results suggest that MRG15 is acting in a HAT complex involving Tip60 to modify chromatin via acetylation of histone H4 at the cdc2 promoter to activate transcription.

  17. Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes

    PubMed Central

    Messier, Terri L.; Gordon, Jonathan A. R.; Boyd, Joseph R.; Tye, Coralee E.; Browne, Gillian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.

    2016-01-01

    The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways. PMID:26783963

  18. Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization.

    PubMed

    Wu, Fang; Caron, Cécile; De Robertis, Christine; Khochbin, Saadi; Rousseaux, Sophie

    2008-12-01

    Before fertilization, the genome packaging of male and female gametes is very different. Indeed, whereas the female haploid genome is associated with histones in a somatic-like chromatin structure, most of the male genome is tightly bound to protamines. However, it has recently been demonstrated that the pericentric heterochromatin regions of the male genome are associated with specific H2A-like histone variants, named H2AL1 and H2AL2, suggesting a heterogeneous organization. The fate and role of the sex-specific genome packaging transmitted by germinal cells to the embryo are not well understood. The aim of the present study was to follow reprogramming of the parental genomes in early embryos after in vivo fertilization. We show here that two typical epigenetic markers, trimethylated lysine 9 of histone H3 (TriMethylH3K9) and acetylated H4, are asymmetrically distributed between the parental genomes in one-cell mouse embryos, confirming data from embryos obtained after intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Indeed, whereas the maternal genome is highly enriched with trimethylH3K9, this mark is not detected in the paternal genome. On the contrary, histone H4 incorporated in the paternal genome is highly acetylated at an early stage, while in the maternal pronucleus, the level of acetylated H4 remains low in early one-cell embryos and becomes enriched at a later stage. Moreover, our results suggest a very quick disappearance of histone H2A variants H2AL1 and H2Al2 from the paternal pericentric heterochromatin regions after sperm-egg fusion.

  19. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.

    PubMed

    Tachibana, Makoto; Sugimoto, Kenji; Nozaki, Masami; Ueda, Jun; Ohta, Tsutomu; Ohki, Misao; Fukuda, Mikiko; Takeda, Naoki; Niida, Hiroyuki; Kato, Hiroyuki; Shinkai, Yoichi

    2002-07-15

    Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

  20. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing

    PubMed Central

    Tie, Feng; Banerjee, Rakhee; Stratton, Carl A.; Prasad-Sinha, Jayashree; Stepanik, Vincent; Zlobin, Andrei; Diaz, Manuel O.; Scacheri, Peter C.; Harte, Peter J.

    2009-01-01

    Summary Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing. PMID:19700617

  1. Brd4 engagement from chromatin targeting to transcriptional regulation: selective contact with acetylated histone H3 and H4

    PubMed Central

    2009-01-01

    Bromodomain-containing protein 4 (Brd4) contains two tandem bromodomains (BD1 and BD2) that bind preferentially to acetylated lysine residues found in histones and nonhistone proteins. This molecular recognition allows Brd4 to associate with acetylated chromatin throughout the cell cycle and regulates transcription at targeted loci. Recruitment of positive transcription elongation factor b, and possibly the general initiation cofactor Mediator as well, plays an important role in Brd4-regulated transcription. Selective contacts with acetyl-lysines in nucleosomal histones and chromatin-binding factors likely provide a molecular switch modulating the steps from chromatin targeting to transcriptional regulation, thus further expanding the ‘acetylation code’ for combinatorial regulation in eukaryotes. PMID:20495683

  2. POWERDRESS and HDA9 interact and promote histone H3 deacetylation at specific genomic sites in Arabidopsis

    PubMed Central

    Kim, Yun Ju; Wang, Ruozhong; Gao, Lei; Li, Dongming; Xu, Chi; Mang, Hyunggon; Jeon, Jien; Chen, Xiangsong; Kwak, June M.; Mo, Beixin; Xiao, Langtao

    2016-01-01

    Histone acetylation is a major epigenetic control mechanism that is tightly linked to the promotion of gene expression. Histone acetylation levels are balanced through the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Arabidopsis HDAC genes (AtHDACs) compose a large gene family, and distinct phenotypes among AtHDAC mutants reflect the functional specificity of individual AtHDACs. However, the mechanisms underlying this functional diversity are largely unknown. Here, we show that POWERDRESS (PWR), a SANT (SWI3/DAD2/N-CoR/TFIII-B) domain protein, interacts with HDA9 and promotes histone H3 deacetylation, possibly by facilitating HDA9 function at target regions. The developmental phenotypes of pwr and hda9 mutants were highly similar. Three lysine residues (K9, K14, and K27) of H3 retained hyperacetylation status in both pwr and hda9 mutants. Genome-wide H3K9 and H3K14 acetylation profiling revealed elevated acetylation at largely overlapping sets of target genes in the two mutants. Highly similar gene-expression profiles in the two mutants correlated with the histone H3 acetylation status in the pwr and hda9 mutants. In addition, PWR and HDA9 modulated flowering time by repressing AGAMOUS-LIKE 19 expression through histone H3 deacetylation in the same genetic pathway. Finally, PWR was shown to physically interact with HDA9, and its SANT2 domain, which is homologous to that of subunits in animal HDAC complexes, showed specific binding affinity to acetylated histone H3. We therefore propose that PWR acts as a subunit in a complex with HDA9 to result in lysine deacetylation of histone H3 at specific genomic targets. PMID:27930340

  3. Arabidopsis NF-YCs Mediate the Light-Controlled Hypocotyl Elongation via Modulating Histone Acetylation.

    PubMed

    Tang, Yang; Liu, Xuncheng; Liu, Xu; Li, Yuge; Wu, Keqiang; Hou, Xingliang

    2017-02-13

    Light is a crucial environmental signal that promotes photomorphogenesis, the developmental process with a series of light-dependent alterations for plants to adapt various external challenges. Chromatin modification has been proposed to be involved in such light-mediated growth, but the underlying mechanism is still elusive. In this study, we identified four Arabidopsis thaliana Nuclear Factor-YC homologs, NF-YC1, NF-YC3, NF-YC4, and NF-YC9 (NF-YCs), which function redundantly as repressors of light-controlled hypocotyl elongation via histone deacetylation. Obvious etiolation phenotypes are observed in NF-YCs loss-of-function mutant seedlings grown under light conditions, including significant elongated hypocotyls and fewer opened cotyledons. We found that NF-YCs interact with histone deacetylase HDA15 in the light, co-target the promoters of a set of hypocotyl elongation-related genes, and modulate the levels of histone H4 acetylation on the associated chromatins, thus repressing gene expression. In contrast, NF-YC-HDA15 complex is dismissed from the target genes in the dark, resulting in increased level of H4 acetylation and consequent etiolated growth. Further analyses revealed that transcriptional repression activity of NF-YCs on the light-controlled hypocotyl elongation partially depends on the deacetylation activity of HDA15, and loss of HDA15 function could rescue the short-hypocotyl phenotype of NF-YCs overexpression plants. Taken together, our results indicate that NF-YC1, NF-YC3, NF-YC4, and NF-YC9 function as transcriptional co-repressors by interacting with HDA15 to inhibit hypocotyl elongation in photomorphogenesis during the early seedling stage. Our findings highlight that NF-YCs can modulate plant development in response to environmental cues via epigenetic regulation.

  4. Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae

    PubMed Central

    Cavero, Santiago; Herruzo, Esther; Ontoso, David; San-Segundo, Pedro A.

    2016-01-01

    In meiotic cells, the pachytene checkpoint or meiotic recombination checkpoint is a surveillance mechanism that monitors critical processes, such as recombination and chromosome synapsis, which are essential for proper distribution of chromosomes to the meiotic progeny. Failures in these processes lead to the formation of aneuploid gametes. Meiotic recombination occurs in the context of chromatin; in fact, the histone methyltransferase Dot1 and the histone deacetylase Sir2 are known regulators of the pachytene checkpoint in Saccharomyces cerevisiae. We report here that Sas2-mediated acetylation of histone H4 at lysine 16 (H4K16ac), one of the Sir2 targets, modulates meiotic checkpoint activity in response to synaptonemal complex defects. We show that, like sir2, the H4-K16Q mutation, mimicking constitutive acetylation of H4K16, eliminates the delay in meiotic cell cycle progression imposed by the checkpoint in the synapsis-defective zip1 mutant. We also demonstrate that, like in dot1, zip1-induced phosphorylation of the Hop1 checkpoint adaptor at threonine 318 and the ensuing Mek1 activation are impaired in H4-K16 mutants. However, in contrast to sir2 and dot1, the H4-K16R and H4-K16Q mutations have only a minor effect in checkpoint activation and localization of the nucleolar Pch2 checkpoint factor in ndt80-prophase-arrested cells. We also provide evidence for a cross-talk between Dot1-dependent H3K79 methylation and H4K16ac and show that Sir2 excludes H4K16ac from the rDNA region on meiotic chromosomes. Our results reveal that proper levels of H4K16ac orchestrate this meiotic quality control mechanism and that Sir2 impinges on additional targets to fully activate the checkpoint. PMID:28357333

  5. [Possible involvement of histone acetylation in the development of emotional resistance to stress stimuli].

    PubMed

    Miyagawa, Kazuya; Tsuji, Minoru; Takeda, Hiroshi

    2012-11-01

    Recent research has demonstrated that complex 'epigenetic' mechanisms, which regulate gene transcription without altering the DNA code, could play a critical role in the pathophysiology of psychiatric disorders. We previously reported that pretreatment of mice with 5-HT(1A) receptor agonists 24 hr before testing suppressed the decrease in emotional behaviors induced by exposure to acute restraint stress. In addition, DNA microarray analysis showed that such a pretreatment with 5-HT(1A) receptor agonist produces changes in several gene transcriptions in the hippocampus including the reduction of histone deacetylase 10. Based on these findings, we recently carried out studies focused on the relationship between the development of emotional resistance to stress and histone acetylation induced by a 5-HT(1A) receptor agonist as well as a histone deacetylase inhibitor. The findings suggest that 5-HT(1A) receptor agonists may be useful for preventing mental illnesses that arise due to a decreased resistance and adaptability to stress. Moreover, the notion that chromatin remodeling is an important mechanism in mediating emotionality under stressful situations is further supported.

  6. Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscapes alteration in bovine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Volatile short-chain fatty acids (VFAs, acetate, propionate, and butyrate) are nutrients especially critical to ruminants. Beyond their nutritional impact, clear evidence is beginning to link modifications in chromatin structure induced by butyrate to cell cycle progression, DNA replication and over...

  7. NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3.

    PubMed

    Ginsburg, Daniel S; Anlembom, Timi Elvuchio; Wang, Jianing; Patel, Sanket R; Li, Bing; Hinnebusch, Alan G

    2014-11-21

    Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nucleosomes and its recruitment to coding sequences and attendant acetylation of histone H3 in vivo. Thus, H3 methylation exerts opposing effects of enhancing nucleosome acetylation by both NuA4 and SAGA as well as stimulating nucleosome deacetylation by multiple HDACs to maintain the proper level of histone acetylation in transcribed coding sequences.

  8. Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

    PubMed

    Bheda, Poonam; Swatkoski, Stephen; Fiedler, Katherine L; Boeke, Jef D; Cotter, Robert J; Wolberger, Cynthia

    2012-04-17

    Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

  9. Histone acetyl transferase 1 is essential for mammalian development, genome stability, and the processing of newly synthesized histones H3 and H4.

    PubMed

    Nagarajan, Prabakaran; Ge, Zhongqi; Sirbu, Bianca; Doughty, Cheryl; Agudelo Garcia, Paula A; Schlederer, Michaela; Annunziato, Anthony T; Cortez, David; Kenner, Lukas; Parthun, Mark R

    2013-06-01

    Histone acetyltransferase 1 is an evolutionarily conserved type B histone acetyltransferase that is thought to be responsible for the diacetylation of newly synthesized histone H4 on lysines 5 and 12 during chromatin assembly. To understand the function of this enzyme in a complex organism, we have constructed a conditional mouse knockout model of Hat1. Murine Hat1 is essential for viability, as homozygous deletion of Hat1 results in neonatal lethality. The lungs of embryos and pups genetically deficient in Hat1 were much less mature upon histological evaluation. The neonatal lethality is due to severe defects in lung development that result in less aeration and respiratory distress. Many of the Hat1(-/-) neonates also display significant craniofacial defects with abnormalities in the bones of the skull and jaw. Hat1(-/-) mouse embryonic fibroblasts (MEFs) are defective in cell proliferation and are sensitive to DNA damaging agents. In addition, the Hat1(-/-) MEFs display a marked increase in genome instability. Analysis of histone dynamics at sites of replication-coupled chromatin assembly demonstrates that Hat1 is not only responsible for the acetylation of newly synthesized histone H4 but is also required to maintain the acetylation of histone H3 on lysines 9, 18, and 27 during replication-coupled chromatin assembly.

  10. Recognition of Histone H3K4 Trimethylation by the Plant Homeodomain of PHF2 Modulates Histone Demethylation

    SciTech Connect

    Wen, Hong; Li, Jingzhi; Song, Tanjing; Lu, Ming; Kan, Pu-Yeh; Lee, Min Gyu; Sha, Bingdong; Shi, Xiaobing

    2010-10-28

    Distinct lysine methylation marks on histones create dynamic signatures deciphered by the 'effector' modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the 'cross-talk' of histone modifications.

  11. Radiosensitization by SAHA in Experimental Colorectal Carcinoma Models-In Vivo Effects and Relevance of Histone Acetylation Status

    SciTech Connect

    Folkvord, Sigurd; Ree, Anne Hansen; Furre, Torbjorn; Halvorsen, Thomas; Flatmark, Kjersti

    2009-06-01

    Purpose: Histone deacetylase inhibitors are being evaluated as antitumor agents in ongoing clinical trials, and promising preclinical results, combined with favorable toxicity profiles, have rendered the drugs as interesting candidates for combination with other treatment modalities, such as radiotherapy. The aim of the present study was to evaluate the radiosensitizing properties of suberoylanilide hydroxamic acid (SAHA) and the possible requirement of histone hyperacetylation at radiation exposure. Methods and materials: Radiosensitization by SAHA was assessed in a colorectal carcinoma cell line and in two colorectal xenograft models by analysis of clonogenic survival and tumor growth delay, respectively. Histone acetylation status at radiation exposure was evaluated by Western blot. Results: In vitro, radiosensitization was demonstrated when cells were preincubated with SAHA, and, in the xenografts, tumor growth was delayed when the mice were treated with fractionated radiation combined with daily SAHA injections compared with radiation alone. Surprisingly, the SAHA-dependent growth delay was still present when radiation was delivered at restored baseline acetylation levels compared with maximal histone hyperacetylation. Conclusion: SAHA was an effective radiosensitizer in experimental colorectal carcinoma models, suggesting that histone deacetylase inhibition might constitute a valuable supplement to current multimodal treatment strategies in rectal cancer. The presence of histone hyperacetylation at radiation was not required to obtain an increased radiation response, questioning the validity of using histone hyperacetylation as a molecular marker for radiosensitivity.

  12. H3 Histone Tail Conformation within the Nucleosome and the Impact of K14 Acetylation Studied Using Enhanced Sampling Simulation

    PubMed Central

    Ikebe, Jinzen; Sakuraba, Shun; Kono, Hidetoshi

    2016-01-01

    Acetylation of lysine residues in histone tails is associated with gene transcription. Because histone tails are structurally flexible and intrinsically disordered, it is difficult to experimentally determine the tail conformations and the impact of acetylation. In this work, we performed simulations to sample H3 tail conformations with and without acetylation. The results show that irrespective of the presence or absence of the acetylation, the H3 tail remains in contact with the DNA and assumes an α-helix structure in some regions. Acetylation slightly weakened the interaction between the tail and DNA and enhanced α-helix formation, resulting in a more compact tail conformation. We inferred that this compaction induces unwrapping and exposure of the linker DNA, enabling DNA-binding proteins (e.g., transcription factors) to bind to their target sequences. In addition, our simulation also showed that acetylated lysine was more often exposed to the solvent, which is consistent with the fact that acetylation functions as a post-translational modification recognition site marker. PMID:26967163

  13. N-acetyl-heparin attenuates acute lung injury caused by acid aspiration mainly by antagonizing histones in mice.

    PubMed

    Zhang, Yanlin; Zhao, Zanmei; Guan, Li; Mao, Lijun; Li, Shuqiang; Guan, Xiaoxu; Chen, Ming; Guo, Lixia; Ding, Lihua; Cong, Cuicui; Wen, Tao; Zhao, Jinyuan

    2014-01-01

    Acute lung injury (ALI) is the leading cause of death in intensive care units. Extracellular histones have recently been recognized to be pivotal inflammatory mediators. Heparin and its derivatives can bind histones through electrostatic interaction. The purpose of this study was to investigate 1) the role of extracellular histones in the pathogenesis of ALI caused by acid aspiration and 2) whether N-acetyl-heparin (NAH) provides more protection than heparin against histones at the high dose. ALI was induced in mice via intratracheal instillation of hydrochloric acid (HCl). Lethality rate, blood gas, myeloperoxidase (MPO) activity, lung edema and pathological changes were used to evaluate the degree of ALI. Heparin/NAH was administered intraperitoneally, twice a day, for 3 days or until death. Acid aspiration caused an obvious increase in extracellular histones. A significant correlation existed between the concentration of HCl aspirated and the circulating histones. Heparin/NAH (10 mg/kg) improved the lethality rate, blood gas, MPO activity, lung edema and pathological score. At a dose of 20 mg/kg, NAH still provided protection, however heparin tended to aggravate the injury due to hemorrhagic complications. The specific interaction between heparin and histones was verified by the binding assay. In summary, high levels of extracellular histones can be pathogenic in ALI caused by acid aspiration. By neutralizing extracellular histones, heparin/NAH can offer similar protection at the moderate doses. At the high dose, NAH provides better protection than heparin.

  14. The Essential Gene wda Encodes a WD40 Repeat Subunit of Drosophila SAGA Required for Histone H3 Acetylation

    PubMed Central

    Guelman, Sebastián; Suganuma, Tamaki; Florens, Laurence; Weake, Vikki; Swanson, Selene K.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2006-01-01

    Histone acetylation provides a switch between transcriptionally repressive and permissive chromatin. By regulating the chromatin structure at specific promoters, histone acetyltransferases (HATs) carry out important functions during differentiation and development of higher eukaryotes. HAT complexes are present in organisms as diverse as Saccharomyces cerevisiae, humans, and flies. For example, the well-studied yeast SAGA is related to three mammalian complexes. We previously identified Drosophila melanogaster orthologues of yeast SAGA components Ada2, Ada3, Spt3, and Tra1 and demonstrated that they associate with dGcn5 in a high-molecular-weight complex. To better understand the function of Drosophila SAGA (dSAGA), we sought to affinity purify and characterize this complex in more detail. A proteomic approach led to the identification of an orthologue of the yeast protein Ada1 and the novel protein encoded by CG4448, referred to as WDA (will decrease acetylation). Embryos lacking both alleles of the wda gene exhibited reduced levels of histone H3 acetylation and could not develop into adult flies. Our results point to a critical function of dSAGA and histone acetylation during Drosophila development. PMID:16980620

  15. DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

    PubMed

    Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

    2016-08-01

    Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

  16. Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter

    SciTech Connect

    Astrand, Carolina; Belikov, Sergey; Wrange, Orjan

    2009-09-10

    Transcription from the mouse mammary tumor virus (MMTV) promoter is induced by the glucocorticoid receptor (GR). This switch was reconstituted in Xenopus oocytes. Previously, we showed that Nuclear Factor 1 (NF1) and Octamer Transcription Factor 1 (Oct1) bind constitutively to the MMTV promoter and thereby induce translational nucleosome positioning representing an intermediary, i.e. preset, state of nucleosome organization. Here we further characterize this NF1 and Oct1 induced preset chromatin in relation to the inactive and the hormone-activated state. The preset chromatin exhibits increased histone acetylation but does not cause dissociation of histone H1 as oppose to the hormone-activated state. Furthermore, upon hormone induction the preset MMTV chromatin displays an enhanced and prolonged GR binding capacity and transcription during an intrinsic and time-dependent silencing of the injected template. The silencing process correlates with a reduced histone acetylation. However, a histone deacetylase inhibitor, trichostatin A (TSA), does not counteract silencing in spite of its distinct stimulation of GR-DNA binding. The latter indicates the importance of histone acetylation to maintain DNA access for inducible factor binding. We discuss how constitutively bound factors such as NF1 and Oct1 may participate in the maintenance of tissue specificity of hormone responsive genes.

  17. The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association.

    PubMed

    Allahverdi, Abdollah; Yang, Renliang; Korolev, Nikolay; Fan, Yanping; Davey, Curt A; Liu, Chuan-Fa; Nordenskiöld, Lars

    2011-03-01

    Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na(+), K(+), Mg(2+), Ca(2+), cobalt-hexammine(3+), spermidine(3+) and spermine(4+). Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K(+), (as well as Rb(+) and Cs(+)) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K(+) (and Rb(+)/Cs(+)) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome-nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

  18. Regulation of proliferation and histone acetylation in embryonic neural precursors by CREB/CREM signaling

    PubMed Central

    Parlato, Rosanna; Mandl, Claudia; Hölzl-Wenig, Gabriele; Liss, Birgit; Tucker, Kerry L; Ciccolini, Francesca

    2014-01-01

    The transcription factor CREB (cAMP-response element binding protein) regulates differentiation, migration, survival and activity-dependent gene expression in the developing and mature nervous system. However, its specific role in the proliferation of embryonic neural progenitors is still not completely understood. Here we investigated how CREB regulates proliferation of mouse embryonic neural progenitors by a conditional mutant lacking Creb gene in neural progenitors. In parallel, we explored possible compensatory effects by the genetic ablation of another member of the same gene family, the cAMP-responsive element modulator (Crem). We show that CREB loss differentially impaired the proliferation, clonogenic potential and self-renewal of precursors derived from the ganglionic eminence (GE), in comparison to those derived from the cortex. This phenotype was associated with a specific reduction of histone acetylation in the GE of CREB mutant mice, and this reduction was rescued in vivo by inhibition of histone deacetylation. These observations indicate that the impaired proliferation could be caused by a reduced acetyltransferase activity in Creb conditional knock-out mice. These findings support a crucial role of CREB in controlling embryonic neurogenesis and propose a novel mechanism by which CREB regulates embryonic neural development. PMID:27504469

  19. Repetitive transcranial magnetic stimulation induces long-lasting changes in protein expression and histone acetylation

    PubMed Central

    Etiévant, Adeline; Manta, Stella; Latapy, Camille; Magno, Luiz Alexandre V.; Fecteau, Shirley; Beaulieu, Jean-Martin

    2015-01-01

    The use of non-invasive brain stimulation like repetitive transcranial magnetic stimulation (rTMS) is an increasingly popular set of methods with promising results for the treatment of neurological and psychiatric disorders. Despite great enthusiasm, the impact of non-invasive brain stimulation on its neuronal substrates remains largely unknown. Here we show that rTMS applied over the frontal cortex of awaken mice induces dopamine D2 receptor dependent persistent changes of CDK5 and PSD-95 protein levels specifically within the stimulated brain area. Importantly, these modifications were associated with changes of histone acetylation at the promoter of these genes and prevented by administration of the histone deacetylase inhibitor MS-275. These findings show that, like several other psychoactive treatments, repeated rTMS sessions can exert long-lasting effects on neuronal substrates. This underscores the need of understanding these effects in the development of future clinical applications as well as in the establishment of improved guidelines to use rTMS in non-medical settings. PMID:26585834

  20. Modulation of histone H3 lysine 56 acetylation as an antifungal therapeutic strategy

    PubMed Central

    Wurtele, Hugo; Tsao, Sarah; Lépine, Guylaine; Mullick, Alaka; Tremblay, Jessy; Drogaris, Paul; Lee, Eun-Hye; Thibault, Pierre; Verreault, Alain; Raymond, Martine

    2014-01-01

    Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections. PMID:20601951

  1. Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

    SciTech Connect

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

    2008-04-15

    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.

  2. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown.

    PubMed

    Plazas-Mayorca, Mariana D; Bloom, Joshua S; Zeissler, Ulrike; Leroy, Gary; Young, Nicolas L; DiMaggio, Peter A; Krugylak, Leonid; Schneider, Robert; Garcia, Benjamin A

    2010-09-01

    Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.

  3. Chromatin Landscape Defined by Repressive Histone Methylation during Oligodendrocyte Differentiation

    PubMed Central

    Liu, Jia; Magri, Laura; Zhang, Fan; Marsh, Nidaa O.; Albrecht, Stefanie; Huynh, Jimmy L.; Kaur, Jasbir; Kuhlmann, Tanja; Zhang, Weijia; Slesinger, Paul A.

    2015-01-01

    In many cell types, differentiation requires an interplay between extrinsic signals and transcriptional changes mediated by repressive and activating histone modifications. Oligodendrocyte progenitors (OPCs) are electrically responsive cells receiving synaptic input. The differentiation of these cells into myelinating oligodendrocytes is characterized by temporal waves of gene repression followed by activation of myelin genes and progressive decline of electrical responsiveness. In this study, we used chromatin isolated from rat OPCs and immature oligodendrocytes, to characterize the genome-wide distribution of the repressive histone marks, H3K9me3 and H3K27me3, during differentiation. Although both marks were present at the OPC stage, only H3K9me3 marks (but not H3K27me3) were found to be increased during differentiation, at genes related to neuronal lineage and regulation of membrane excitability. Consistent with these findings, the levels and activity of H3K9 methyltransferases (H3K9 HMT), but not H3K27 HMT, increased more prominently upon exposure to oligodendrocyte differentiating stimuli and were detected in stage-specific repressive protein complexes containing the transcription factors SOX10 or YY1. Silencing H3K9 HMT, but not H3K27 HMT, impaired oligodendrocyte differentiation and functionally altered the response of oligodendrocytes to electrical stimulation. Together, these results identify repressive H3K9 methylation as critical for gene repression during oligodendrocyte differentiation. PMID:25568127

  4. Characterization of Drosophila melanogaster JmjC+N histone demethylases.

    PubMed

    Lloret-Llinares, Marta; Carré, Clément; Vaquero, Alejandro; de Olano, Natalia; Azorín, Fernando

    2008-05-01

    In this article, we characterize histone demethylase activity of the entire family of JmjC+N proteins of Drosophila melanogaster. Our results show that Lid (little imaginal discs), which is structurally homologous to JARID1, demethylates H3K4me3. However, contrary to what would be inferred from its demethylase activity, lid contributes to the establishment of transcriptionally competent chromatin states as: (i) is required for histone H3 acetylation; (ii) contributes to expression of the homoeotic gene Ultrabithorax (Ubx); and (iii) antagonizes heterochromatin-mediated gene silencing (PEV). These results, which are consistent with the identification of lid as a trithorax group (trxG) gene, are discussed in the context of current models for the contribution of H3K4me3 to the regulation of gene expression. Here, we also show that the two Drosophila JMJD2 homologues, dJMJD2(1)/CG15835 and dJMJD2(2)/CG33182, are capable of demethylating both H3K9me3 and H3K36me3. dJMJD2(1)/CG15835 regulates heterochromatin organization, as its over-expression induces spreading of HP1, out of heterochromatin, into euchromatin, without affecting the actual pattern of histone modifications of heterochromatin. dJMJD2(1)/CG15835 is excluded from heterochromatin and localizes to multiple euchromatic sites, where it regulates H3K36 methylation. These results indicate that dJMJD2(1)/CG15835 contributes to delimit hetero- and euchromatic territories through the regulation of H3K36 methylation in euchromatin. On the other hand, dJARID2/CG3654 shows no demethylase activity on H3K4me3, H3K9me3, H3K27me3, H3K36me3 and H4K20me3.

  5. Royal Jelly Constituents Increase the Expression of Extracellular Superoxide Dismutase through Histone Acetylation in Monocytic THP-1 Cells.

    PubMed

    Makino, Junya; Ogasawara, Rie; Kamiya, Tetsuro; Hara, Hirokazu; Mitsugi, Yukari; Yamaguchi, Eiji; Itoh, Akichika; Adachi, Tetsuo

    2016-04-22

    Extracellular superoxide dismutase (EC-SOD) is one of the main SOD isozymes and plays an important role in the prevention of cardiovascular diseases by accelerating the dismutation reaction of superoxide. Royal jelly includes 10-hydroxy-2-decenoic acid (10H2DA, 2), which regulates the expression of various types of genes in epigenetics through the effects of histone deacetylase (HDAC) antagonism. The expression of EC-SOD was previously reported to be regulated epigenetically through histone acetylation in THP-1 cells. Therefore, we herein evaluated the effects of the royal jelly constituents 10-hydroxydecanoic acid (10HDA, 1), sebacic acid (SA, 3), and 4-hydroperoxy-2-decenoic acid ethyl ester (4-HPO-DAEE, 4), which is a derivative of 2, on the expression of EC-SOD in THP-1 cells. The treatment with 1 mM 1, 2, or 3 or 100 μM 4 increased EC-SOD expression and histone H3 and H4 acetylation levels. Moreover, the enrichment of acetylated histone H4 was observed in the proximal promoter region of EC-SOD and was caused by the partial promotion of ERK phosphorylation (only 4) and inhibition of HDAC activities, but not by the expression of HDACs. Overall, 4 exerted stronger effects than 1, 2, or 3 and has potential as a candidate or lead compound against atherosclerosis.

  6. Histone H3 trimethylation at lysine 9 marks the inactive metaphase X chromosome in the marsupial Monodelphis domestica.

    PubMed

    Zakharova, Irina S; Shevchenko, Alexander I; Shilov, Alexander G; Nesterova, Tatyana B; Vandeberg, John L; Zakian, Suren M

    2011-04-01

    In somatic cells of female marsupial and eutherian mammals, X chromosome inactivation (XCI) occurs. XCI results in the transcriptional silencing of one of the two X chromosomes and is accompanied by specific covalent histone modifications attributable to the inactive chromatin state. Because data about repressed chromatin of the inactive X chromosome (Xi) in marsupials are sparse, we examined in more detail the distribution of active and inactive chromatin markers on metaphase X chromosomes of an American marsupial, Monodelphis domestica. Consistent with data reported previously both for eutherian and marsupial mammals, we found that the Xi of M. domestica lacks active histone markers-H3K4 dimethylation and H3K9 acetylation. We did not observe on metaphase spreads enrichment of the Xi with H3K27 trimethylation which is involved in XCI in eutherians and was detected on the Xi in the interphase nuclei of mature female M. domestica in an earlier study. Moreover, we found that the Xi of M. domestica was specifically marked with H3K9 trimethylation, which is known to be a component of the Xi chromatin in eutherians and is involved in both marsupials and eutherians in meiotic sex chromosome inactivation which has been proposed as an ancestral mechanism of XCI.

  7. Differential Promoter Methylation and Histone Modification Contribute to the Brain Specific Expression of the Mouse Mbu-1 Gene

    PubMed Central

    Kim, Byungtak; Kang, Seongeun; Kim, Sun Jung

    2012-01-01

    Mbu-1 (Csrnp-3) is a mouse gene that was identified in our previous study as showing highly restricted expression to the central nervous system. In this study, to elucidate the regulatory mechanism for tissue specificity of the gene, epigenetic approaches that identify the profiles of CpG methylation, as well as histone modifications at the promoter region were conducted. Methylation-specific PCR revealed that the CpG sites in brain tissues from embryo to adult stages showed virtually no methylation (0.052–0.67%). Lung (9.0%) and pancreas (3.0%) also showed lower levels. Other tissues such as liver, kidney, and heart showed much higher methylation levels ranging from approximately 39–93%. Treatment of 5-aza-2′-deoxycytidine (5-Aza-dC) significantly decreased promoter methylation, reactivating Mbu-1 expression in NG108-15 and Neuro-2a neuronal cells. Chromatin immunoprecipitation assay revealed that 5-Aza-dC decreased levels of acetylated H3K9 and methylated H3K4, and increased methylated H3K9. This result indicates that CpG methylation converses with histone modifications in an opposing sense of regulating Mbu-1 expression. PMID:23076708

  8. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

    PubMed Central

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A.; Klein, Brianna J.; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G.; Li, Wei; Bedford, Mark T.; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

  9. Curcumin modulates the effect of histone modification on the expression of chemokines by type II alveolar epithelial cells in a rat COPD model

    PubMed Central

    Gan, Lixing; Li, Chengye; Wang, Jian; Guo, Xuejun

    2016-01-01

    Background Studies have suggested that histone modification has a positive impact on various aspects associated with the progression of COPD. Histone deacetylase 2 (HDAC2) suppresses proinflammatory gene expression through deacetylation of core histones. Objective To investigate the effect of histone modification on the expression of chemokines in type II alveolar epithelial cells (AEC II) in a rat COPD model and regulation of HDAC2 expression by curcumin in comparison with corticosteroid. Materials and methods The rat COPD model was established by cigarette smoke exposure and confirmed by histology and pathophysioloy. AEC II were isolated and cultured in vitro from the COPD models and control animals. The cells were treated with curcumin, corticosteroid, or trichostatin A, and messenger RNA (mRNA) expression of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2α (MIP-2α) was assessed by quantitative real-time polymerase chain reaction (RT-PCR). The expression of HDAC2 was measured by Western blot. Chromatin immunoprecipitation was used to detect H3/H4 acetylation and H3K9 methylation in the promoter region of three kinds of chemokine genes (IL-8, MCP-1, and MIP-2α). Results Compared to the control group, the mRNAs of MCP-1, IL-8, and MIP-2α were upregulated 4.48-fold, 3.14-fold, and 2.83-fold, respectively, in the AEC II from COPD model. The protein expression of HDAC2 in the AEC II from COPD model was significantly lower than from the control group (P<0.05). The decreased expression of HDAC2 was negatively correlated with the increased expression of IL-8, MCP-1, and MIP-2α mRNAs (all P<0.05). The level of H3/H4 acetylation was higher but H3K9 methylation in the promoter region of chemokine genes was lower in the cells from COPD model than from the control group (all P<0.05). Curcumin downregulated the expression of MCP-1, IL-8, and MIP-2α, and the expression was further enhanced in the presence of

  10. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells

    PubMed Central

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  11. 17β-Estradiol regulates histone alterations associated with memory consolidation and increases Bdnf promoter acetylation in middle-aged female mice.

    PubMed

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

    2014-09-01

    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 17β-estradiol (E2) to enhance object recognition memory consolidation requires histone H3 acetylation in the dorsal hippocampus. However, the extent to which histone acetylation is regulated by E2 in middle-aged females is unknown. The mnemonic benefits of E2 in aging females appear to be greatest in middle age, and so pinpointing the molecular mechanisms through which E2 enhances memory at this age could lead to the development of safer and more effective treatments for maintaining memory function without the side effects of current therapies. Here, we show that dorsal hippocampal infusion of E2 rapidly enhanced object recognition and spatial memory, and increased histone H3 acetylation in the dorsal hippocampus, while also significantly reducing levels of histone deacetylase (HDAC2 and HDAC3) proteins. E2 specifically increased histone H3 acetylation at Bdnf promoters pII and pIV in the dorsal hippocampus of both young and middle-aged mice, despite age-related decreases in pI and pIV acetylation. Furthermore, levels of mature BDNF and pro-BDNF proteins in the dorsal hippocampus were increased by E2 in middle-aged females. Together, these data suggest that the middle-aged female dorsal hippocampus remains epigenetically responsive to E2, and that E2 may enhance memory in middle-aged females via epigenetic regulation of Bdnf.

  12. The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122.

    PubMed

    Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin

    2017-03-29

    Deposition of (H3-H4)2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3-H4)2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

  13. Alcohol-Induced Histone Acetylation Reveals a Gene Network Involved in Alcohol Tolerance

    PubMed Central

    Ghezzi, Alfredo; Krishnan, Harish R.; Lew, Linda; Prado, Francisco J.; Ong, Darryl S.; Atkinson, Nigel S.

    2013-01-01

    Sustained or repeated exposure to sedating drugs, such as alcohol, triggers homeostatic adaptations in the brain that lead to the development of drug tolerance and dependence. These adaptations involve long-term changes in the transcription of drug-responsive genes as well as an epigenetic restructuring of chromosomal regions that is thought to signal and maintain the altered transcriptional state. Alcohol-induced epigenetic changes have been shown to be important in the long-term adaptation that leads to alcohol tolerance and dependence endophenotypes. A major constraint impeding progress is that alcohol produces a surfeit of changes in gene expression, most of which may not make any meaningful contribution to the ethanol response under study. Here we used a novel genomic epigenetic approach to find genes relevant for functional alcohol tolerance by exploiting the commonalities of two chemically distinct alcohols. In Drosophila melanogaster, ethanol and benzyl alcohol induce mutual cross-tolerance, indicating that they share a common mechanism for producing tolerance. We surveyed the genome-wide changes in histone acetylation that occur in response to these drugs. Each drug induces modifications in a large number of genes. The genes that respond similarly to either treatment, however, represent a subgroup enriched for genes important for the common tolerance response. Genes were functionally tested for behavioral tolerance to the sedative effects of ethanol and benzyl alcohol using mutant and inducible RNAi stocks. We identified a network of genes that are essential for the development of tolerance to sedation by alcohol. PMID:24348266

  14. A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation.

    PubMed

    Wan, Guohui; Hu, Xiaoxiao; Liu, Yunhua; Han, Cecil; Sood, Anil K; Calin, George A; Zhang, Xinna; Lu, Xiongbin

    2013-10-30

    A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti-cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA-JADE, that is induced after DNA damage in an ataxia-telangiectasia mutated (ATM)-dependent manner. LncRNA-JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA-JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA-JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA-JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA-JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA-JADE may contribute to breast tumorigenesis.

  15. Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

    SciTech Connect

    Bian, Chuanbing; Xu, Chao; Ruan, Jianbin; Lee, Kenneth K.; Burke, Tara L.; Tempel, Wolfram; Barsyte, Dalia; Li, Jing; Wu, Minhao; Zhou, Bo O.; Fleharty, Brian E.; Paulson, Ariel; Allali-Hassani, Abdellah; Zhou, Jin-Qiu; Mer, Georges; Grant, Patrick A.; Workman, Jerry L.; Zang, Jianye; Min, Jinrong

    2011-09-28

    The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

  16. Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

    PubMed Central

    Bian, Chuanbing; Xu, Chao; Ruan, Jianbin; Lee, Kenneth K; Burke, Tara L; Tempel, Wolfram; Barsyte, Dalia; Li, Jing; Wu, Minhao; Zhou, Bo O; Fleharty, Brian E; Paulson, Ariel; Allali-Hassani, Abdellah; Zhou, Jin-Qiu; Mer, Georges; Grant, Patrick A; Workman, Jerry L; Zang, Jianye; Min, Jinrong

    2011-01-01

    The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation. PMID:21685874

  17. Replisome Function During Replicative Stress Is Modulated by Histone H3 Lysine 56 Acetylation Through Ctf4

    PubMed Central

    Luciano, Pierre; Dehé, Pierre-Marie; Audebert, Stéphane; Géli, Vincent; Corda, Yves

    2015-01-01

    Histone H3 lysine 56 acetylation in Saccharomyces cerevisiae is required for the maintenance of genome stability under normal conditions and upon DNA replication stress. Here we show that in the absence of H3 lysine 56 acetylation replisome components become deleterious when replication forks collapse at natural replication block sites. This lethality is not a direct consequence of chromatin assembly defects during replication fork progression. Rather, our genetic analyses suggest that in the presence of replicative stress H3 lysine 56 acetylation uncouples the Cdc45–Mcm2-7–GINS DNA helicase complex and DNA polymerases through the replisome component Ctf4. In addition, we discovered that the N-terminal domain of Ctf4, necessary for the interaction of Ctf4 with Mms22, an adaptor protein of the Rtt101-Mms1 E3 ubiquitin ligase, is required for the function of the H3 lysine 56 acetylation pathway, suggesting that replicative stress promotes the interaction between Ctf4 and Mms22. Taken together, our results indicate that Ctf4 is an essential member of the H3 lysine 56 acetylation pathway and provide novel mechanistic insights into understanding the role of H3 lysine 56 acetylation in maintaining genome stability upon replication stress. PMID:25697176

  18. Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.

    PubMed Central

    Bartsch, J; Truss, M; Bode, J; Beato, M

    1996-01-01

    The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855250

  19. Trichostatin A induces apoptosis of p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion.

    PubMed

    Yee, Su-Bog; Kim, Myung Soo; Baek, Soo Jin; Kim, Gyoo Cheon; Yoo, Ki Soo; Yoo, Young Hyun; Park, Bong Soo

    2004-11-01

    Although inhibition of histone deacetylase has been demonstrated to induce apoptosis of various cancer cells, there is no report on its effect on mast cell demise to date. Here we studied whether a histone deacetylase inhibitor Trichostatin A (TSA) produces apoptosis in p815 mastocytoma cells. TSA prominently increased the amount of acetylated histones, H3, H4, H2A and H2B, in p815 mastocytoma cells. TSA reduced the viability of p815 mastocytoma cells, and many apoptotic manifestations such as generation of DNA fragmentation, activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and increase of DNA hypoploidy proved that the reduction of viability resulted from apoptosis. Whereas TSA treatment increased the expression level of Bad, it decreased the level of Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. The reduction of mitochondrial membrane potential, the release of cytochrome c and Smac/DIABLO to cytosol, and mitochondrial localization of Bad were also shown. Taken together, TSA induces apoptosis on p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion. Our data therefore provide the possibility that TSA could be considered as a novel therapeutic strategy for mastocytoma from its apoptosis-inducing activity.

  20. Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance.

    PubMed

    Samoilov, Mikhail; Churilova, Anna; Gluschenko, Tatjana; Vetrovoy, Oleg; Dyuzhikova, Natalia; Rybnikova, Elena

    2016-03-01

    Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180mm Hg, 3h) and neuroprotective preconditioning mode (three episodes of 360mm Hg for 2h spaced at 24h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II-III, V) and hippocampus (CA1, CA3) by 3-24h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24h, whereas acetylation of H3K24 was intense but transient, peaked at 3h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance.

  1. Neonatal isolation decreases cued fear conditioning and frontal cortical histone 3 lysine 9 methylation in adult female rats.

    PubMed

    Kao, Gour-Shenq; Cheng, Ling-Yi; Chen, Li-Hsien; Tzeng, Wen-Yu; Cherng, Chienfang G; Su, Chien-Chou; Wang, Ching-Yi; Yu, Lung

    2012-12-15

    Early life stress is thought to enhance adult susceptibility to stress and stress-related mood disorders. In this study, fear-potentiated startle was used to model the acquisition of a traumatic event-related memory in female rats experiencing early life stress. Daily 1-hr maternal and sibling separation throughout day 2-9 postpartum (D2-9 PP) caused a decrease in the fear-potentiated startle, but not acoustic startle baseline, in adult female rats. The separation procedure did not affect corticosterone secretion but produced an increase in serum estradiol concentration. Moreover, the separation procedure did not affect histone 3 lysine 9 (H3K9) acetylation but decreased H3K9 mono- and tri-methylation in frontal cortices. Treatment with 5-aza-2'-deoxycytidine (AZA) (5mg/kg at alternative days from D2PP to D9PP or 10mg/kg at D5PP and D9PP), a DNA methylation inhibitor, did not affect the separation-decreased fear-potentiated startle. Treatment with valproic acid (VPA), a histone deacetylase inhibitor, at 3 dosing regimens (300mg/kg at D2-9PP; 100mg/kg at D2-4PP, 200mg/kg at D5-7PP, 300mg/kg at D8-9PP; 100mg/kg at D2-5PP, 200mg/kg at D6-9PP) prior to daily separation reversed such a decrease in fear-potentiated startle. The lowest effective VPA dosing regimen used (100mg/kg at D2-5PP, 200mg/kg at D6-9PP) reversed the separation-decreased H3K9 mono- and tri-methylation in frontal cortices. Eight-day VPA (300mg/kg/day) and AZA (5mg/kg/day) administrations starting at D28PP were ineffective in altering the separation-decreased fear-potentiated startle. We, hereby, suggest that decreased frontal cortical H3K9 mono- and tri-methylation may be involved in early life separation-decreased fear memory of adult rats.

  2. Ketogenic diet improves the spatial memory impairment caused by exposure to hypobaric hypoxia through increased acetylation of histones in rats

    PubMed Central

    Zhao, Ming; Huang, Xin; Cheng, Xiang; Lin, Xiao; Zhao, Tong; Wu, Liying; Yu, Xiaodan; Wu, Kuiwu; Fan, Ming

    2017-01-01

    Exposure to hypobaric hypoxia causes neuron cell damage, resulting in impaired cognitive function. Effective interventions to antagonize hypobaric hypoxia-induced memory impairment are in urgent need. Ketogenic diet (KD) has been successfully used to treat drug-resistant epilepsy and improves cognitive behaviors in epilepsy patients and other pathophysiological animal models. In the present study, we aimed to explore the potential beneficial effects of a KD on memory impairment caused by hypobaric hypoxia and the underlying possible mechanisms. We showed that the KD recipe used was ketogenic and increased plasma levels of ketone bodies, especially β-hydroxybutyrate. The results of the behavior tests showed that the KD did not affect general locomotor activity but obviously promoted spatial learning. Moreover, the KD significantly improved the spatial memory impairment caused by hypobaric hypoxia (simulated altitude of 6000 m, 24 h). In addition, the improving-effect of KD was mimicked by intraperitoneal injection of BHB. The western blot and immunohistochemistry results showed that KD treatment not only increased the acetylated levels of histone H3 and histone H4 compared to that of the control group but also antagonized the decrease in the acetylated histone H3 and H4 when exposed to hypobaric hypoxia. Furthermore, KD-hypoxia treatment also promoted PKA/CREB activation and BDNF protein expression compared to the effects of hypoxia alone. These results demonstrated that KD is a promising strategy to improve spatial memory impairment caused by hypobaric hypoxia, in which increased modification of histone acetylation plays an important role. PMID:28355243

  3. Chromatin condensation in terminally differentiating mouse erythroblasts does not involve special architectural proteins but depends on histone deacetylation

    SciTech Connect

    Popova, Evgenya Y.; Krauss, Sharon Wald; Short, Sarah A.; Lee, Gloria; Villalobos, Jonathan; Etzell, Joan; Koury, Mark J.; Ney, Paul A.; Chasis, Joel Anne; Grigoryev, Sergei A.

    2008-08-21

    Terminal erythroid differentiation in vertebrates is characterized by progressive heterochromatin formation, chromatin condensation and, in mammals, culminates in nuclear extrusion. To date, although mechanisms regulating avian erythroid chromatin condensation have been identified, little is known regarding this process during mammalian erythropoiesis. To elucidate the molecular basis for mammalian erythroblast chromatin condensation, we used Friend virus-infected murine spleen erythroblasts that undergo terminal differentiation in vitro. Chromatin isolated from early and late stage erythroblasts had similar levels of linker and core histones, only a slight difference in nucleosome repeats, and no significant accumulation of known developmentally-regulated architectural chromatin proteins. However, histone H3(K9) dimethylation markedly increased while histone H4(K12) acetylation dramatically decreased and became segregated from the histone methylation as chromatin condensed. One histone deacetylase, HDAC5, was significantly upregulated during the terminal stages of Friend virus-infected erythroblast differentiation. Treatment with histone deacetylase inhibitor, trichostatin A, blocked both chromatin condensation and nuclear extrusion. Based on our data, we propose a model for a unique mechanism in which extensive histone deacetylation at pericentromeric heterochromatin mediates heterochromatin condensation in vertebrate erythroblasts that would otherwise be mediated by developmentally-regulated architectural proteins in nucleated blood cells.

  4. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

    SciTech Connect

    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

  5. Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3*

    PubMed Central

    Pradeepa, Madapura M.; Nikhil, Gupta; Hari Kishore, Annavarapu; Bharath, Giriyapura N.; Kundu, Tapas K.; Rao, Manchanahalli R. Satyanarayana

    2009-01-01

    The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids. Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis. PMID:19710011

  6. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum

    PubMed Central

    Levine, Amir A.; Guan, Zhonghui; Barco, Angel; Xu, Shiqin; Kandel, Eric R.; Schwartz, James H.

    2005-01-01

    Remodeling chromatin is essential for cAMP-regulated gene expression, necessary not only for development but also for memory storage and other enduring mental states. Histone acetylation and deacetylation mediate long-lasting forms of synaptic plasticity in Aplysia as well as cognition in mice. Here, we show that histone acetylation by the cAMP-response element binding protein (CREB)-binding protein (CBP) mediates sensitivity to cocaine by regulating expression of the fosB gene and its splice variant, ΔfosB, a transcription factor previously implicated in addiction. Using the chromatin immunoprecipitation assay with antibodies against histone H4 or CBP, we find that CBP is recruited to the fosB promoter to acetylate histone H4 in response to acute exposure to cocaine. We show that mutant mice that lack one allele of the CBP gene and have normal levels of fosB expression are less sensitive to chronic (10-day) administration of cocaine than are wild-type mice. This decreased sensitivity is correlated with decreased histone acetylation and results in decreased fosB expression and diminished accumulation of ΔfosB. Thus, CBP, which forms part of the promoter complex with CREB, mediates sensitivity to cocaine by acetylating histones. PMID:16380431

  7. The Histone Demethylase UTX Promotes Brown Adipocyte Thermogenic Program Via Coordinated Regulation of H3K27 Demethylation and Acetylation*

    PubMed Central

    Zha, Lin; Li, Fenfen; Wu, Rui; Artinian, Liana; Rehder, Vincent; Yu, Liqing; Liang, Houjie; Xue, Bingzhong; Shi, Hang

    2015-01-01

    Brown adipocytes function to dissipate energy as heat through adaptive thermogenesis. Understanding the molecular mechanisms underlying the brown fat thermogenic program may provide insights for the development of therapeutic approaches in the treatment of obesity. Most studies investigating the mechanisms underlying brown fat development focus on genetic mechanisms; little is known about the epigenetic mechanisms in this process. We have discovered that ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase for di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), plays a potential role in regulating brown adipocyte thermogenic program. We found that UTX is up-regulated during brown adipocyte differentiation and by cold exposure in both brown adipose tissue (BAT) and white adipose tissue (WAT) of mice, suggesting a potential role in thermogenesis. Inactivation of UTX down-regulates brown fat specific gene expression, while overexpression of UTX does the opposite. Notably, activation of β adrenergic signaling recruits UTX to the UCP1 and PGC1α promoters, leading to decreased H3K27me3, a histone transcriptional repressive mark. UTX demethylates H3K27me3 and subsequently interacts with the histone acetyltransferase (HAT) protein CBP, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulate brown adipocyte thermogenic program through coordinated control of demethylating H3K27me3 and acetylating H3K27, switching the transcriptional repressive state to the transcriptional active state at the promoters of UCP1 and PGC1α. We conclude that UTX may play a potential role in regulation of brown adipocyte gene expression and may mediate β adrenergic activation of brown fat function. PMID:26306033

  8. Oxidative stress alters global histone modification and DNA methylation.

    PubMed

    Niu, Yingmei; DesMarais, Thomas L; Tong, Zhaohui; Yao, Yixin; Costa, Max

    2015-05-01

    The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H2O2) for 3h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2',7'-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H2O2 inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.

  9. Exercise-induced modulation of histone H4 acetylation status and cytokines levels in patients with schizophrenia.

    PubMed

    Lavratti, Caroline; Dorneles, Gilson; Pochmann, Daniela; Peres, Alessandra; Bard, Andréia; de Lima Schipper, Lucas; Dal Lago, Pedro; Wagner, Luciane Carniel; Elsner, Viviane Rostirola

    2017-01-01

    The present study aimed to investigate the short and long-term effects of a concurrent exercise protocol on global histone H4 acetylation levels and inflammatory markers (interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma (IFN-γ) and cortisol) in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMC) and in peripheral blood of patients with schizophrenia (SZ), as well the intervention impact on anthropometric characteristics. Seventeen individuals were submitted to the intervention three times a week and blood samples were collected pre, 30, 60 and 90days after the intervention started. A remarkable reduction on body mass index and body mass were observed following intervention. The protocol also induced a histone H4 hypoacetylation status in PBMC all times evaluated when compared to the pre intervention period. Although the IL-4 and cortisol levels were not altered in response to the intervention, a reduction in IL-6 production during the 60 and 90days compared to the pre intervention period was observed. Finally, diminished IFN-γ production was found in the 90days period compared to the pre intervention and 30days after periods. In addition, systemic IL-6 levels were lower at 60 and 90days compared to the pre intervention. The concurrent exercise protocol was able to improve anthropometric characteristics in patients with SZ, engaging the modulation of cytokine and histone H4 acetylation levels.

  10. Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres

    PubMed Central

    Shang, Wei-Hao; Hori, Tetsuya; Westhorpe, Frederick G.; Godek, Kristina M.; Toyoda, Atsushi; Misu, Sadahiko; Monma, Norikazu; Ikeo, Kazuho; Carroll, Christopher W.; Takami, Yasunari; Fujiyama, Asao; Kimura, Hiroshi; Straight, Aaron F.; Fukagawa, Tatsuo

    2016-01-01

    Centromeres are specified epigenetically through the deposition of the centromere-specific histone H3 variant CENP-A. However, how additional epigenetic features are involved in centromere specification is unknown. Here, we find that histone H4 Lys5 and Lys12 acetylation (H4K5ac and H4K12ac) primarily occur within the pre-nucleosomal CENP-A–H4–HJURP (CENP-A chaperone) complex, before centromere deposition. We show that H4K5ac and H4K12ac are mediated by the RbAp46/48–Hat1 complex and that RbAp48-deficient DT40 cells fail to recruit HJURP to centromeres and do not incorporate new CENP-A at centromeres. However, C-terminally-truncated HJURP, that does not bind CENP-A, does localize to centromeres in RbAp48-deficient cells. Acetylation-dead H4 mutations cause mis-localization of the CENP-A–H4 complex to non-centromeric chromatin. Crucially, CENP-A with acetylation-mimetic H4 was assembled specifically into centromeres even in RbAp48-deficient DT40 cells. We conclude that H4K5ac and H4K12ac, mediated by RbAp46/48, facilitates efficient CENP-A deposition into centromeres. PMID:27811920

  11. A histone methylation network regulates transgenerational epigenetic memory in C. elegans.

    PubMed

    Greer, Eric L; Beese-Sims, Sara E; Brookes, Emily; Spadafora, Ruggero; Zhu, Yun; Rothbart, Scott B; Aristizábal-Corrales, David; Chen, Shuzhen; Badeaux, Aimee I; Jin, Qiuye; Wang, Wei; Strahl, Brian D; Colaiácovo, Monica P; Shi, Yang

    2014-04-10

    How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here, we identified multiple chromatin-modifying factors, including H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase, and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. Our findings uncover a network of chromatin regulators that control the transgenerational flow of epigenetic information and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates transgenerational effects on fertility.

  12. A histone methylation network regulates transgenerational epigenetic memory in C. elegans

    PubMed Central

    Greer, Eric L.; Beese-Sims, Sara E.; Brookes, Emily; Spadafora, Ruggero; Zhu, Yun; Rothbart, Scott B.; Aristizábal-Corrales, David; Chen, Shuzhen; Badeaux, Aimee I.; Jin, Qiuye; Wang, Wei; Strahl, Brian D.; Colaiácovo, Monica P.; Shi, Yang

    2014-01-01

    Summary How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans Histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here we identified multiple chromatin-modifying factors, including novel H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. Our findings uncover a network of chromatin regulators that control the trans-generational flow of epigenetic information, and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates trans-generational effects on fertility. PMID:24685137

  13. Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain

    PubMed Central

    Tyler, Christina R.; Hafez, Alexander K.; Solomon, Elizabeth R.; Allan, Andrea M.

    2015-01-01

    Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity alters the epigenome, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50 ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and expression of associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3 K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region- specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3 K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development induces alterations in the adult brain via histone modifications and chromatin modifiers a sex- and

  14. Thymoquinone Promotes Pancreatic Cancer Cell Death and Reduction of Tumor Size through Combined Inhibition of Histone Deacetylation and Induction of Histone Acetylation

    PubMed Central

    Relles, Daniel; Gong, Qiaoke

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is virtually therapy-resistant. As noninvasive lesions progress to malignancy, the precursor period provides a window for cancer therapies that can interfere with neoplastic progression. Thymoquinone (Tq), a major bioactive component of essential oil from Nigella sativa's seeds, has demonstrated antineoplastic activities in multiple cancers. In this study, we investigated antineoplastic potential of Tq in human PDAC cell lines, AsPC-1 and MiaPaCa-2. Tq (10–50 μM) inhibited cell viability and proliferation and caused partial G2 cycle arrest in dose-dependent manner in both cell lines. Cells accumulated in subG0/G1 phase, indicating apoptosis. This was associated with upregulation of p53 and downregulation of Bcl-2. Independently of p53, Tq increased p21 mRNA expression 12-fold. Tq also induced H4 acetylation (lysine 12) and downregulated HDACs activity, reducing expression of HDACs 1, 2, and 3 by 40–60%. In vivo, Tq significantly reduced tumor size in 67% of established tumor xenografts (P < 0.05), along with increased H4 acetylation and reduced HDACs expression. Our results showed that Tq mediated posttranslational modification of histone acetylation, inhibited HDACs expression, and induced proapoptotic signaling pathways. These molecular targets demonstrate rationale for using Tq as a promising antineoplastic agent to prevent postoperative cancer recurrence and to prolong survival of PDAC patients after surgical resection. PMID:28105374

  15. Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice.

    PubMed

    Itzhak, Yossef; Anderson, Karen L; Kelley, Jonathan B; Petkov, Martin

    2012-05-01

    Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice.

  16. Histone H3K56 Acetylation, CAF1, and Rtt106 Coordinate Nucleosome Assembly and Stability of Advancing Replication Forks

    PubMed Central

    Clemente-Ruiz, Marta; González-Prieto, Román; Prado, Félix

    2011-01-01

    Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage. PMID:22102830

  17. Acetylation mimics within individual core histone tail domains indicate distinct roles in regulating the stability of higher-order chromatin structure.

    PubMed

    Wang, Xiaodong; Hayes, Jeffrey J

    2008-01-01

    Nucleosome arrays undergo salt-dependent self-association into large oligomers in a process thought to recapitulate essential aspects of higher-order tertiary chromatin structure formation. Lysine acetylation within the core histone tail domains inhibits self-association, an effect likely related to its role in facilitating transcription. As acetylation of specific tail domains may encode distinct functions, we investigated biochemical and self-association properties of model nucleosome arrays containing combinations of native and mutant core histones with lysine-to-glutamine substitutions to mimic acetylation. Acetylation mimics within the tail domains of H2B and H4 caused the largest inhibition of array self-association, while modification of the H3 tail uniquely affected the stability of DNA wrapping within individual nucleosomes. In addition, the effect of acetylation mimics on array self-association is inconsistent with a simple charge neutralization mechanism. For example, acetylation mimics within the H2A tail can have either a positive or negative effect on self-association, dependent upon the acetylation state of the other tails and nucleosomal repeat length. Finally, we demonstrate that glutamine substitutions and lysine acetylation within the H4 tail domain have identical effects on nucleosome array self-association. Our results indicate that acetylation of specific tail domains plays distinct roles in the regulation of chromatin structure.

  18. Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

    PubMed

    Liu, Honglin; Kim, Jin-Moon; Aoki, Fugaku

    2004-05-01

    Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic

  19. 14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

    PubMed Central

    Karam, Caline S.; Kellner, Wendy A.; Takenaka, Naomi; Clemmons, Alexa W.; Corces, Victor G.

    2010-01-01

    Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation. PMID:20532201

  20. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation

    PubMed Central

    Yeh, Chia-Ming; Lin, Chiao-Wen; Yang, Jia-Sin; Yang, Wei-En; Su, Shih-Chi; Yang, Shun-Fa

    2016-01-01

    Melatonin exerts antimetastatic effects on liver and breast cancer and also inhibits matrix metalloproteinase (MMP) activity. However, the detailed impacts and underlying mechanisms of melatonin on oral cancer cell metastasis are still unclear. This study showed that melatonin attenuated the 12-O-tetradecanoylphorbol-13-acetate-induced migration of oral cancer cell lines, HSC-3 and OECM-1. Zymography, quantitative real-time PCR, and Western blotting analyses revealed that melatonin lessened MMP-9 enzyme activity as well as the expression of MMP-9 mRNA and protein. Furthermore, melatonin suppressed the phosphorylation of the ERK1/2 signalling pathway, which dampened MMP-9 gene transcription by affecting the expression of transcriptional coactivators, such as CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300), and decreasing histone acetylation in HSC-3 and OECM-1 cells. Examinations on clinical samples exhibited that MMP-9, CREBBP, and EP300 were significantly increased in oral cancer tissues. Moreover, the relative level of CREBBP was positively correlated with the expression of MMP-9 and EP300. In conclusion, we demonstrated that melatonin inhibits the motility of HSC-3 and OECM-1 cells in vitro through a molecular mechanism that involves attenuation of MMP-9 expression and activity mediated by decreased histone acetylation. PMID:26980735

  1. The Arabidopsis acetylated histone-binding protein BRAT1 forms a complex with BRP1 and prevents transcriptional silencing

    PubMed Central

    Zhang, Cui-Jun; Hou, Xiao-Mei; Tan, Lian-Mei; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-01-01

    Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes. PMID:27273316

  2. SAS-mediated acetylation of histone H4 Lys 16 is required for H2A.Z incorporation at subtelomeric regions in Saccharomyces cerevisiae

    PubMed Central

    Shia, Wei-Jong; Li, Bing; Workman, Jerry L.

    2006-01-01

    The yeast SAS (Something About Silencing) complex and the histone variant H2A.Z have both previously been linked to an antisilencing function at the subtelomeric regions. SAS is an H4 Lys 16-specific histone acetyltransferase complex. Here we demonstrate that the H4 Lys 16 acetylation by SAS is required for efficient H2A.Z incorporation near telomeres. The presence of H4 Lys 16 acetylation and H2A.Z synergistically prevent the ectopic propagation of heterochromatin. Overall, our data suggest a novel antisilencing mechanism near telomeres. PMID:16980580

  3. Glucocorticoid Receptor Signaling Represses the Antioxidant Response by Inhibiting Histone Acetylation Mediated by the Transcriptional Activator NRF2.

    PubMed

    Alam, Md Morshedul; Okazaki, Keito; Nguyen, Linh Thi Thao; Ota, Nao; Kitamura, Hiroshi; Murakami, Shohei; Shima, Hiroki; Igarashi, Kazuhiko; Sekine, Hiroki; Motohashi, Hozumi

    2017-03-17

    NRF2 (nuclear factor erythroid 2-related factor 2) is a key transcriptional activator that mediates the inducible expression of antioxidant genes. NRF2 is normally ubiquitinated by KEAP1 (Kelch-like ECH-associated protein 1) and subsequently degraded by proteasomes. Inactivation of KEAP1 by oxidative stress or electrophilic chemicals allows NRF2 to activate transcription through binding to antioxidant response elements (AREs) and recruiting histone acetyltransferase CBP (CREB-binding protein). While KEAP1-dependent regulation is a major determinant of NRF2 activity, NRF2-mediated transcriptional activation varies from context to context, suggesting other intracellular signaling cascades may impact NRF2 function. To identify a signaling pathway that modifies NRF2 activity, we immunoprecipitated endogenous NRF2 and its interacting proteins from mouse liver and identified glucocorticoid receptor (GR) as a novel NRF2-binding partner. We found that glucocorticoids (GC), dexamethasone (Dex) and betamethasone (Bet), antagonize diethyl maleate (DEM)-induced activation of NRF2 target genes in a GR-dependent manner. Dex treatment enhanced GR recruitment to AREs without affecting chromatin binding of NRF2, resulting in the inhibition of CBP recruitment and histone acetylation at AREs. This repressive effect was canceled by the addition of HDAC inhibitors. Thus, GR signaling decreases NRF2 transcriptional activation through reducing the NRF2-dependent histone acetylation. Consistent with these observations, GR signaling blocked NRF2-mediated cytoprotection from oxidative stress. This study suggests that an impaired antioxidant response by NRF2 and a resulting decrease in cellular antioxidant capacity account for the side effects of GCs, providing a novel viewpoint for the pathogenesis of hypercorticosteroidism.

  4. Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation

    PubMed Central

    Wakamori, Masatoshi; Fujii, Yoshifumi; Suka, Noriyuki; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki

    2015-01-01

    Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs. PMID:26607036

  5. Combinatorial patterns of histone acetylations and methylations in the human genome

    PubMed Central

    Wang, Zhibin; Zang, Chongzhi; Rosenfeld, Jeffrey A; Schones, Dustin E; Barski, Artem; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Peng, Weiqun; Zhang, Michael Q; Zhao, Keji

    2008-01-01

    Histones are characterized by numerous posttranslational modifications that influence gene transcription1,2. However, because of the lack of global distribution data in higher eukaryotic systems3, the extent to which gene-specific combinatorial patterns of histone modifications exist remains to be determined. Here, we report the patterns derived from the analysis of 39 histone modifications in human CD4+ T cells. Our data indicate that a large number of patterns are associated with promoters and enhancers. In particular, we identify a common modification module consisting of 17 modifications detected at 3,286 promoters. These modifications tend to colocalize in the genome and correlate with each other at an individual nucleosome level. Genes associated with this module tend to have higher expression, and addition of more modifications to this module is associated with further increased expression. Our data suggest that these histone modifications may act cooperatively to prepare chromatin for transcriptional activation. PMID:18552846

  6. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression*

    PubMed Central

    Zheng, Yupeng; Fornelli, Luca; Compton, Philip D.; Sharma, Seema; Canterbury, Jesse; Mullen, Christopher; Zabrouskov, Vlad; Fellers, Ryan T.; Thomas, Paul M.; Licht, Jonathan D.; Senko, Michael W.; Kelleher, Neil L.

    2016-01-01

    Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such “trivalent hypermethylation” (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry

  7. Egr-1 and RNA POL II facilitate glioma cell GDNF transcription induced by histone hyperacetylation in promoter II.

    PubMed

    Zhang, Bao-Le; Guo, Ting-Wen; Gao, Le-Le; Ji, Guang-Quan; Gu, Xiao-He; Shao, Yu-Qi; Yao, Rui-Qin; Gao, Dian-Shuai

    2017-02-06

    The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

  8. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

    NASA Astrophysics Data System (ADS)

    Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

    2016-04-01

    UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

  9. Methylation of histone H3 lysine 9 occurs during translation.

    PubMed

    Rivera, Carlos; Saavedra, Francisco; Alvarez, Francisca; Díaz-Celis, César; Ugalde, Valentina; Li, Jianhua; Forné, Ignasi; Gurard-Levin, Zachary A; Almouzni, Geneviève; Imhof, Axel; Loyola, Alejandra

    2015-10-30

    Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed.

  10. Methylation of histone H3 lysine 9 occurs during translation

    PubMed Central

    Rivera, Carlos; Saavedra, Francisco; Alvarez, Francisca; Díaz-Celis, César; Ugalde, Valentina; Li, Jianhua; Forné, Ignasi; Gurard-Levin, Zachary A.; Almouzni, Geneviève; Imhof, Axel; Loyola, Alejandra

    2015-01-01

    Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed. PMID:26405197

  11. Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

    SciTech Connect

    Li, Jing; Li, Zhihong; Ruan, Jianbin; Xu, Chao; Tong, Yufeng; Pan, Patricia W.; Tempel, Wolfram; Crombet, Lissete; Min, Jinrong; Zang, Jianye

    2012-02-27

    M-phase phosphoprotein 8 (MPP8) was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15). The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two {beta} strands from the two protomer subunits. Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

  12. Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

    PubMed

    Okubo, Takumi; Hayashi, Daiki; Yaguchi, Takayuki; Fujita, Yudai; Sakaue, Motoharu; Suzuki, Takehito; Tsukamoto, Atsushi; Murayama, Ohoshi; Lynch, Jonathan; Miyazaki, Yoko; Tanaka, Kazuaki; Takizawa, Tatsuya

    2016-01-01

    Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

  13. Specific histone modifications play critical roles in the control of encystation and antigenic variation in the early-branching eukaryote Giardia lamblia.

    PubMed

    Carranza, Pedro G; Gargantini, Pablo R; Prucca, César G; Torri, Alessandro; Saura, Alicia; Svärd, Staffan; Lujan, Hugo D

    2016-12-01

    During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post-transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post-translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD(+)-dependent and NAD(+)-independent histone deacetylases regulate encystation; however, a unique NAD(+)-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite.

  14. Matrix Attachment Region-Dependent Function of the Immunoglobulin μ Enhancer Involves Histone Acetylation at a Distance without Changes in Enhancer Occupancy

    PubMed Central

    Fernández, Luis A.; Winkler, Michael; Grosschedl, Rudolf

    2001-01-01

    Nuclear matrix attachment regions (MARs), which flank the immunoglobulin μ heavy-chain enhancer on either side, are required for the activation of the distal variable-region (VH) promoter in transgenic mice. Previously, we have shown that the MARs extend a local domain of chromatin accessibility at the μ enhancer to more distal sites. In this report, we examine the influence of MARs on the formation of a nucleoprotein complex at the enhancer and on the acetylation of histones, which have both been implicated in contributing to chromatin accessibility. By in vivo footprint analysis of transgenic μ gene constructs, we show that the occupancy of factor-binding sites at the μ enhancer is similar in transcriptionally active wild-type and transcriptionally inactive ΔMAR genes. Chromatin immunoprecipitation experiments indicate, however, that the acetylation of histones at enhancer-distal nucleosomes is enhanced 10-fold in the presence of MARs, whereas the levels of histone acetylation at enhancer-proximal nucleosomes are similar for wild-type and ΔMAR genes. Taken together, these data indicate that the function of MARs in mediating long-range chromatin accessibility and transcriptional activation of the VH promoter involves the generation of an extended domain of histone acetylation, independent of changes in the occupancy of the μ enhancer. PMID:11113195

  15. TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation.

    PubMed

    Yamamura, Yoshiko; Motegi, Katsumi; Kani, Kouichi; Takano, Hideyuki; Momota, Yukihiro; Aota, Keiko; Yamanoi, Tomoko; Azuma, Masayuki

    2012-08-01

    Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

  16. Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter

    PubMed Central

    Bouchard, Caroline; Dittrich, Oliver; Kiermaier, Astrid; Dohmann, Karen; Menkel, Annette; Eilers, Martin; Lüscher, Bernhard

    2001-01-01

    Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. We now show that Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc. PMID:11511535

  17. The interplay of histone modifications – writers that read

    PubMed Central

    Zhang, Tianyi; Cooper, Sarah; Brockdorff, Neil

    2015-01-01

    Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. PMID:26474904

  18. Inhibition of N-Terminal Lysines Acetylation and Transcription Factor Assembly by Epirubicin Induced Deranged Cell Homeostasis

    PubMed Central

    Khan, Shahper N.; Danishuddin, Mohd; Varshney, Bhavna; Lal, Sunil K.; Khan, Asad U.

    2012-01-01

    Epirubicin (EPI), an anthracycline antitumour antibiotic, is a known intercalating and DNA damaging agent. Here, we study the molecular interaction of EPI with histones and other cellular targets. EPI binding with histone core protein was predicted with spectroscopic and computational techniques. The molecular distance r, between donor (histone H3) and acceptor (EPI) was estimated using Förster’s theory of non-radiation energy transfer and the detailed binding phenomenon is expounded. Interestingly, the concentration dependent reduction in the acetylated states of histone H3 K9/K14 was observed suggesting more repressed chromatin state on EPI treatment. Its binding site near N-terminal lysines is further characterized by thermodynamic determinants and molecular docking studies. Specific DNA binding and inhibition of transcription factor (Tf)-DNA complex formation implicates EPI induced transcriptional inhibition. EPI also showed significant cell cycle arrest in drug treated cells. Chromatin fragmentation and loss of membrane integrity in EPI treated cells is suggestive of their commitment to cell death. This study provides an analysis of nucleosome dynamics during EPI treatment and provides a novel insight into its action. PMID:23251640

  19. GENERAL CONTROL NONREPRESSED PROTEIN5-Mediated Histone Acetylation of FERRIC REDUCTASE DEFECTIVE3 Contributes to Iron Homeostasis in Arabidopsis1

    PubMed Central

    Wang, Tianya; Liu, Zhenshan; Xu, Jianqin; Yao, Yingyin; Peng, Huiru; Xin, Mingming; Ni, Zhongfu

    2015-01-01

    Iron homeostasis is essential for plant growth and development. Here, we report that a mutation in GENERAL CONTROL NONREPRESSED PROTEIN5 (GCN5) impaired iron translocation from the root to the shoot in Arabidopsis (Arabidopsis thaliana). Illumina high-throughput sequencing revealed 879 GCN5-regulated candidate genes potentially involved in iron homeostasis. Chromatin immunoprecipitation assays indicated that five genes (At3G08040, At2G01530, At2G39380, At2G47160, and At4G05200) are direct targets of GCN5 in iron homeostasis regulation. Notably, GCN5-mediated acetylation of histone 3 lysine 9 and histone 3 lysine 14 of FERRIC REDUCTASE DEFECTIVE3 (FRD3) determined the dynamic expression of FRD3. Consistent with the function of FRD3 as a citrate efflux protein, the iron retention defect in gcn5 was rescued and fertility was partly restored by overexpressing FRD3. Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate. Collectively, these data suggest that GCN5 plays a critical role in FRD3-mediated iron homeostasis. Our results provide novel insight into the chromatin-based regulation of iron homeostasis in Arabidopsis. PMID:26002909

  20. Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation1,2,3

    PubMed Central

    Wee, Caroline L.; Teo, Shaun; Oey, Nicodemus E.; Wright, Graham D.; VanDongen, Hendrika M.A.

    2014-01-01

    Abstract Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the β-spectrin isoform βSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and βSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation. PMID:26464963

  1. Expression of PCAF, p300 and Gcn5 and more highly acetylated histone H4 in pediatric tumors.

    PubMed

    Armas-Pineda, C; Arenas-Huertero, F; Pérezpeñia-Diazconti, M; Chico-Ponce de León, F; Sosa-Sáinz, G; Lezama, P; Recillas-Targa, F

    2007-06-01

    Any deregulation of histone acetyltransferases (HATs) could affect several processes in tumors. In this paper, the expression of the PCAF, p300 and Gcn5 HATs by RT-PCR in 34 tumor samples was evaluated. Samples of both central nervous system tumors (CNST, 13 cases) and Wilm's tumors (WT, 11 cases) over-expressed PCAF up to 1.6-, and Gcn5 up to 1.3-fold, respectively. In 9 out of 10 samples of benign tumors (BT), PCAF was not expressed. The p300 gene was the least expressed in all tumors. The medians of expression of PCAF (124.0 DU) and Gcn5 (127.0 DU) genes were higher in CNST than in both WT (102.0 and 101.0 DU, respectively) and BT (70.0 and 82.4 DU, respectively). There was a trend to decrease the expression of PCAF and Gcn5 genes in CNST, according to: chemotherapy (110.0 and 96.0 DU, respectively), chemo plus radiotherapy (124.0 and 115.0 DU, respectively) or no treatment (134.0 and 142.0 DU, respectively) in the tumors. A similar trend was observed in WT. Finally, we revealed more highly acetylated forms of histone H4 in CNST and WT. The over-expression of PCAF could represent a new molecular tumor marker in malignant tumors, especially in CNST in pediatric patients.

  2. Histone H1-mediated epigenetic regulation controls germline stem cell self-renewal by modulating H4K16 acetylation

    PubMed Central

    Sun, Jin; Wei, Hui-Min; Xu, Jiang; Chang, Jian-Feng; Yang, Zhihao; Ren, Xingjie; Lv, Wen-Wen; Liu, Lu-Ping; Pan, Li-Xia; Wang, Xia; Qiao, Huan-Huan; Zhu, Bing; Ji, Jun-Yuan; Yan, Dong; Xie, Ting; Sun, Fang-Lin; Ni, Jian-Quan

    2015-01-01

    Epigenetics plays critical roles in controlling stem cell self-renewal and differentiation. Histone H1 is one of the most critical chromatin regulators, but its role in adult stem cell regulation remains unclear. Here we report that H1 is intrinsically required in the regulation of germline stem cells (GSCs) in the Drosophila ovary. The loss of H1 from GSCs causes their premature differentiation through activation of the key GSC differentiation factor bam. Interestingly, the acetylated H4 lysine 16 (H4K16ac) is selectively augmented in the H1-depleted GSCs. Furthermore, overexpression of mof reduces H1 association on chromatin. In contrast, the knocking down of mof significantly rescues the GSC loss phenotype. Taken together, these results suggest that H1 functions intrinsically to promote GSC self-renewal by antagonizing MOF function. Since H1 and H4K16 acetylation are highly conserved from fly to human, the findings from this study might be applicable to stem cells in other systems. PMID:26581759

  3. H4K5 histone acetylation of BRG1 is associated with heroin administration rather than addiction.

    PubMed

    Xu, Limin; Hong, Qingxiao; Chen, Xiaoying; Xu, Xuting; Liu, Huifen; Zhou, Wenhua; Duan, Shiwei

    2016-09-01

    Diacetylmorphine hydrochloride (heroin) addiction is a chronic relapsing brain disorder that is a heavy public health burden worldwide. Brm/SWI2-related gene-1 (BRG1) is a tumor suppressor gene that can influence embryogenesis and the development of the cerebellum. The current study aimed to investigate the effect of histone H4 lysine 5 (H4K5) modifications on the BRG1 gene in brain tissue of the ventral tegmental area (VTA) of heroin-addicted rats. A total of 21 male Sprague Dawley rats were raised in a standard manner and underwent heroin self-administration training. Rats were randomly divided into three equal groups: Group A, self-administered delivery of heroin; group B, yoked delivery of heroin; and group C, yoked delivery of saline. The VTA was harvested and subjected to chromatin immunoprecipitation (ChIP) analysis. Gene expression was evaluated by quantitative polymerase chain reaction. We calculated the recovery rate, which indicated the percentage of the total input BRG1 recovered by ChIP. Our results showed that BRG1 was less associated with H4K5 histone modification in the group of rats that underwent heroin self-administration than in the other two groups (A vs. B, P=0.031; A vs. C, P=0.067). The recovery fold changes of the self-administration group and the passive-administration group were significantly different from those of the group with yoked saline (A vs. C, P=0.013; B vs. C, P=0.009; A vs. B, P=0.731). The results of the current study demonstrated that H4K5 histone acetylation of BRG1 in the VTA may be associated with heroin administration, but not addiction.

  4. H4K5 histone acetylation of BRG1 is associated with heroin administration rather than addiction

    PubMed Central

    Xu, Limin; Hong, Qingxiao; Chen, Xiaoying; Xu, Xuting; Liu, Huifen; Zhou, Wenhua; Duan, Shiwei

    2016-01-01

    Diacetylmorphine hydrochloride (heroin) addiction is a chronic relapsing brain disorder that is a heavy public health burden worldwide. Brm/SWI2-related gene-1 (BRG1) is a tumor suppressor gene that can influence embryogenesis and the development of the cerebellum. The current study aimed to investigate the effect of histone H4 lysine 5 (H4K5) modifications on the BRG1 gene in brain tissue of the ventral tegmental area (VTA) of heroin-addicted rats. A total of 21 male Sprague Dawley rats were raised in a standard manner and underwent heroin self-administration training. Rats were randomly divided into three equal groups: Group A, self-administered delivery of heroin; group B, yoked delivery of heroin; and group C, yoked delivery of saline. The VTA was harvested and subjected to chromatin immunoprecipitation (ChIP) analysis. Gene expression was evaluated by quantitative polymerase chain reaction. We calculated the recovery rate, which indicated the percentage of the total input BRG1 recovered by ChIP. Our results showed that BRG1 was less associated with H4K5 histone modification in the group of rats that underwent heroin self-administration than in the other two groups (A vs. B, P=0.031; A vs. C, P=0.067). The recovery fold changes of the self-administration group and the passive-administration group were significantly different from those of the group with yoked saline (A vs. C, P=0.013; B vs. C, P=0.009; A vs. B, P=0.731). The results of the current study demonstrated that H4K5 histone acetylation of BRG1 in the VTA may be associated with heroin administration, but not addiction. PMID:27588112

  5. Effects of Carbocysteine and Beclomethasone on Histone Acetylation/Deacetylation Processes in Cigarette Smoke Exposed Bronchial Epithelial Cells.

    PubMed

    Pace, E; Di Vincenzo, S; Ferraro, M; Siena, L; Chiappara, G; Dino, P; Vitulo, P; Bertani, A; Saibene, F; Lanata, L; Gjomarkaj, M

    2016-12-07

    Histone deacetylase expression/activity may control inflammation, cell senescence, and responses to corticosteroids. Cigarette smoke exposure, increasing oxidative stress, may negatively affect deacetylase expression/activity. The effects of cigarette smoke extracts (CSE), carbocysteine, and beclomethasone dipropionate on chromatin remodelling processes in human bronchial epithelial cells are largely unknown. The present study was aimed to assess the effects of cigarette smoke, carbocysteine, and beclomethasone dipropionate on histone deacetylase 3 (HDAC3) expression/activity, N-CoR (nuclear receptor corepressor) expression, histone acetyltransferases (HAT) (p300/CBP) expression, p-CREB and IL-1 m-RNA expression, neutrophil chemotaxis. Increased p-CREB expression was observed in the bronchial epithelium of smokers. CSE increased p-CREB expression and decreased HDAC3 expression and activity and N-CoR m-RNA and protein expression. At the same time, CSE increased the expression of the HAT, p300/CBP. All these events increased acetylation processes within the cells and were associated to increased IL-1 m-RNA expression and neutrophil chemotaxis. The incubation of CSE exposed cells with carbocysteine and beclomethasone counteracted the effects of cigarette smoke on HDAC3 and N-CoR but not on p300/CBP. The increased deacetylation processes due to carbocysteine and beclomethasone dipropionate incubation is associated to reduced p-CREB, IL-1 m-RNA expression, neutrophil chemotaxis. These findings suggest a new role of combination therapy with carbocysteine and beclomethasone dipropionate in restoring deacetylation processes compromised by cigarette smoke exposure. This article is protected by copyright. All rights reserved.

  6. Effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and the interactions between two nucleosomes.

    PubMed

    Lee, Ju Yeon; Wei, Sijie; Lee, Tae-Hee

    2011-04-01

    We characterized the effect of histone acetylation on the structure of a nucleosome and the interactions between two nucleosomes. In this study, nucleosomes reconstituted with the Selex "Widom 601" sequence were acetylated with the Piccolo NuA4 complex, which acetylates mainly H4 N-terminal tail lysine residues and some H2A/H3 N-terminal tail lysine residues. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies topology change of the DNA. Interactions between two nucleosomes in solution were also monitored to discover multiple transient dinucleosomal states that can be categorized to short-lived and long-lived (∼1 s) states. The formation of dinucleosomes is strongly Mg(2+)-dependent, and unacetylated nucleosomes favor the formation of long-lived dinucleosomes 4-fold as much as the acetylated ones. These results suggest that the acetylation of histones by Piccolo NuA4 disturbs not only the structure of a nucleosome but also the interactions between two nucleosomes. Lastly, we suggest a structural model for a stable dinucleosomal state where the two nucleosomes are separated by ∼2 nm face-to-face and rotated by 34° with respect to each other.

  7. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

    PubMed

    Hayashi-Takanaka, Yoko; Yamagata, Kazuo; Wakayama, Teruhiko; Stasevich, Timothy J; Kainuma, Takashi; Tsurimoto, Toshiki; Tachibana, Makoto; Shinkai, Yoichi; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2011-08-01

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

  8. Similar DNA methylation and histone H3 lysine 9 dimethylation patterns in tripronuclear and corrected bipronuclear human zygotes.

    PubMed

    Chen, Xinjie; Fan, Yong; Long, Xiaolin; Sun, Xiaofang

    2010-06-01

    After fertilization, male and female gametes undergo extensive reprogramming to restore totipotency. Both DNA methylation and histone modification are important epigenetic reprogramming events. Previous studies have reported that the paternal pronucleus of the human zygote is actively demethylated to some extent, while the maternal pronucleus remains methylated. However, to our knowledge, the relationship between DNA methylation and H3K9 dimethylation patterns in human embryos has not been reported. In this study, we examined the dynamic DNA methylation and H3K9 dimethylation patterns in triploid and bipronucleated zygotes and early developing embryos. We sought to gain further insight into the relationship between DNA methylation and H3K9 dimethylation and to investigate whether removing a pronucleus from triploid zygotes affects DNA methylation and H3K9 dimethylation patterns. We found that active DNA demethylation of the two male pronuclei occurred in tripronuclear human zygotes while the female pronucleus remained methylated at 20 h post-insemination. In tripronuclear human zygotes, H3K9 was hypomethylated in the two paternal pronuclei relative to the maternal pronucleus. Our data show that there are no differences in the DNA methylation and H3K9 dimethylation patterns between tripronuclear and corrected bipronuclear human zygotes. However, correction of 3PN human zygotes dispermic in origin could not improve subsequent embryo development. In conclusion, DNA methylation and H3K9 dimethylation patterns are well correlated in tripronuclear zygotes and embryos; early embryo development is not affected by removal of a male pronucleus. Our results imply that limited developmental potential of either 3PN or corrected 2PN embryos may not be caused by the abnormalities in DNA methylation or H3K9 dimethylation modification.

  9. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

    PubMed

    Robert, Carine; Nagaria, Pratik K; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J; Cole, Philip A; Rassool, Feyruz V

    2016-06-01

    Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

  10. Histone H3 Lysine 9 Methyltransferase DIM5 Is Required for the Development and Virulence of Botrytis cinerea

    PubMed Central

    Zhang, Xiaoli; Liu, Xinqiang; Zhao, Yanli; Cheng, Jiasen; Xie, Jiatao; Fu, Yanping; Jiang, Daohong; Chen, Tao

    2016-01-01

    Histone methylation is widely present in animals, plants and fungi, and the methylation modification of histone H3 has important biological functions. Methylation of Lys9 of histone H3 (H3K9) has been proven to regulate chromatin structure, gene silencing, transcriptional activation, plant metabolism, and other processes. In this work, we investigated the functions of a H3K9 methyltransferase gene BcDIM5 in Botrytis cinerea, which contains a PreSET domain, a SET domain and a PostSET domain. Characterization of BcDIM5 knockout transformants showed that the hyphal growth rate and production of conidiophores and sclerotia were significantly reduced, while complementary transformation of BcDIM5 could restore the phenotypes to the levels of wild type. Pathogenicity assays revealed that BcDIM5 was essential for full virulence of B. cinerea. BcDIM5 knockout transformants exhibited decreased virulence, down-regulated expression of some pathogenic genes and drastically decreased H3K9 trimethylation level. However, knockout transformants of other two genes heterochromatin protein 1 (HP1) BcHP1 and DNA methyltransferase (DIM2) BcDIM2 did not exhibit significant change in the growth phenotype and virulence compared with the wild type. Our results indicate that H3K9 methyltransferase BcDIM5 is required for H3K9 trimethylation to regulate the development and virulence of B. cinerea. PMID:27597848

  11. Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

    PubMed Central

    Rothstein, Rodney; Aguilera, Andrés

    2013-01-01

    DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation. PMID:23357952

  12. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity*

    PubMed Central

    Samant, Sadhana A.; Pillai, Vinodkumar B.; Sundaresan, Nagalingam R.; Shroff, Sanjeev G.; Gupta, Mahesh P.

    2015-01-01

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms. PMID:25911107

  13. Histone Deacetylase 3 (HDAC3)-dependent Reversible Lysine Acetylation of Cardiac Myosin Heavy Chain Isoforms Modulates Their Enzymatic and Motor Activity.

    PubMed

    Samant, Sadhana A; Pillai, Vinodkumar B; Sundaresan, Nagalingam R; Shroff, Sanjeev G; Gupta, Mahesh P

    2015-06-19

    Reversible lysine acetylation is a widespread post-translational modification controlling the activity of proteins in different subcellular compartments. We previously demonstrated that a class II histone deacetylase (HDAC), HDAC4, and a histone acetyltransferase, p300/CREB-binding protein-associated factor, associate with cardiac sarcomeres and that a class I and II HDAC inhibitor, trichostatin A, enhances contractile activity of myofilaments. In this study we show that a class I HDAC, HDAC3, is also present at cardiac sarcomeres. By immunohistochemical and electron microscopic analyses, we found that HDAC3 was localized to A-band of sarcomeres and capable of deacetylating myosin heavy chain (MHC) isoforms. The motor domains of both cardiac α- and β-MHC isoforms were found to be reversibly acetylated. Biomechanical studies revealed that lysine acetylation significantly decreased the Km for the actin-activated ATPase activity of MHC isoforms. By in vitro motility assay, we found that lysine acetylation increased the actin-sliding velocity of α-myosin by 20% and β-myosin by 36% compared with their respective non-acetylated isoforms. Moreover, myosin acetylation was found to be sensitive to cardiac stress. During induction of hypertrophy, myosin isoform acetylation increased progressively with duration of stress stimuli independently of isoform shift, suggesting that lysine acetylation of myosin could be an early response of myofilaments to increase contractile performance of the heart. These studies provide the first evidence for localization of HDAC3 at myofilaments and uncover a novel mechanism modulating the motor activity of cardiac MHC isoforms.

  14. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    SciTech Connect

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-06-15

    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.

  15. Enhanced Efficacy of 5-Fluorouracil in Combination with a Dual Histone Deacetylase and Phosphatidylinositide 3-Kinase Inhibitor (CUDC-907) in Colorectal Cancer Cells

    PubMed Central

    Hamam, Rimi; Ali, Dalia; Vishnubalaji, Radhakrishnan; Alsaaran, Zaid F.; Chalisserry, Elna Paul; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M.

    2017-01-01

    Background/Aims: 5-Fluorouracil (5-FU) is widely used in the treatment of patients with colorectal cancer (CRC). However, the efficacy of 5-FU as a single agent is limited, with multiple undesired side effects. Therefore, the aim of the current study was to assess the efficacy of CUDC-907 (a dual inhibitor of histone deacetylase and phosphatidylinositide 3-kinase) in combination with 5-FU against CRC cells. Materials and Methods: Cell viability was determined using AlamarBlue and colony formation assays. Acridine orange/ethidium bromide staining and flow cytometry were used to measure apoptotic and necrotic events, as well as cell cycle progression. Immunoblotting was used to assess acetylation of histone H3 and phosphorylation of AKT. Results: Our data revealed enhanced toxicity of CUDC-907 against HCT116, RKO, COLO-205, and HT-29 CRC cells when combined with 5-FU. Similarly, the colony formation capability of HCT116 cells was suppressed by the combination treatment. Cells treated with CUDC-907 and 5-FU underwent apoptosis and necrosis, and exhibited increased polyploidy. Furthermore, CRC cells treated with CUDC-907 exhibited a higher degree of histone H3 lysine 9 acetylation (H3K9ac) and reduced AKT phosphorylation (Ser473). Conclusion: Our data revealed, for the first time, the enhanced inhibitory effect of CUDC-907 against CRC cells when combined with 5-FU, supporting the application of this combination as a potential therapeutic strategy in CRC treatment. PMID:28139498

  16. Changes in cardiac Nav1.5 expression, function, and acetylation by pan-histone deacetylase inhibitors.

    PubMed

    Xu, Qin; Patel, Dakshesh; Zhang, Xian; Veenstra, Richard D

    2016-11-01

    Histone deacetylase (HDAC) inhibitors are small molecule anticancer therapeutics that exhibit limiting cardiotoxicities including QT interval prolongation and life-threatening cardiac arrhythmias. Because the molecular mechanisms for HDAC inhibitor-induced cardiotoxicity are poorly understood, we performed whole cell patch voltage-clamp experiments to measure cardiac sodium currents (INa) from wild-type neonatal mouse ventricular or human-induced pluripotent stem cell-derived cardiomyocytes treated with trichostatin A (TSA), vorinostat (VOR), or romidepsin (FK228). All three pan-HDAC inhibitors dose dependently decreased peak INa density and shifted the voltage activation curve 3- to 8-mV positive. Increases in late INa were not observed despite a moderate slowing of the inactivation rate at low activating potentials (<-40 mV). Scn5a mRNA levels were not significantly altered but NaV1.5 protein levels were significantly reduced. Immunoprecipitation with anti-NaV1.5 and Western blotting with anti-acetyl-lysine antibodies indicated that NaV1.5 acetylation is increased in vivo after HDAC inhibition. FK228 inhibited total cardiac HDAC activity with two apparent IC50s of 5 nM and 1.75 μM, consistent with previous findings with TSA and VOR. FK228 also decreased ventricular gap junction conductance (gj), again consistent with previous findings. We conclude that pan-HDAC inhibition reduces cardiac INa density and NaV1.5 protein levels without affecting late INa amplitude and, thus, probably does not contribute to the reported QT interval prolongation and arrhythmias associated with pan-HDAC inhibitor therapies. Conversely, reductions in gj may enhance the occurrence of triggered activity by limiting electrotonic inhibition and, combined with reduced INa, slow myocardial conduction and increase vulnerability to reentrant arrhythmias.

  17. Potential role of adolescent alcohol exposure-induced amygdaloid histone modifications in anxiety and alcohol intake during adulthood.

    PubMed

    Pandey, Subhash C; Sakharkar, Amul J; Tang, Lei; Zhang, Huaibo

    2015-10-01

    Binge drinking is common during adolescence and can lead to the development of psychiatric disorders, including alcoholism in adulthood. Here, the role and persistent effects of histone modifications during adolescent intermittent ethanol (AIE) exposure in the development of anxiety and alcoholism in adulthood were investigated. Rats received intermittent ethanol exposure during post-natal days 28-41, and anxiety-like behaviors were measured after 1 and 24 h of the last AIE. The effects of AIE on anxiety-like and alcohol-drinking behaviors in adulthood were measured with or without treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Amygdaloid brain regions were collected to measure HDAC activity, global and gene-specific histone H3 acetylation, expression of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated (Arc) protein and dendritic spine density (DSD). Adolescent rats displayed anxiety-like behaviors after 24 h, but not 1 h, of last AIE with a concomitant increase in nuclear and cytosolic amygdaloid HDAC activity and HDAC2 and HDAC4 levels leading to deficits in histone (H3-K9) acetylation in the central (CeA) and medial (MeA), but not in basolateral nucleus of amygdala (BLA). Interestingly, some of AIE-induced epigenetic changes such as, increased nuclear HDAC activity, HDAC2 expression, and decreased global histone acetylation persisted in adulthood. In addition, AIE decreased BDNF exons I and IV and Arc promoter specific histone H3 acetylation that was associated with decreased BDNF, Arc expression and DSD in the CeA and MeA during adulthood. AIE also induced anxiety-like behaviors and enhanced ethanol intake in adulthood, which was attenuated by TSA treatment via normalization of deficits in histone H3 acetylation of BDNF and Arc genes. These novel results indicate that AIE induces long-lasting effects on histone modifications and deficits in synaptic events in the amygdala, which are

  18. Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1

    PubMed Central

    Chen, Danqi; Kluz, Thomas; Fang, Lei; Zhang, Xiaoru; Sun, Hong; Jin, Chunyuan; Costa, Max

    2016-01-01

    The environmental and occupational carcinogen Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled. In spite of a considerable research effort, the mechanisms of Cr(VI)-induced carcinogenesis remain largely unknown. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons and is induced by a variety of stressors. Studies in animal models have suggested that Nupr1 is a key factor in the development of lung and pancreatic cancers, with little known about the underlying molecular mechanisms. Here we report that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI) through epigenetic mechanisms. Interestingly, Cr(VI) exposure also results in the loss of acetylation at histone H4K16, which is considered a ‘hallmark’ of human cancer. Cr(VI)-induced reduction of H4K16 acetylation appears to be caused by the induction of Nupr1, since (a) overexpression of Nupr1 decreased the levels of both H4K16 acetylation and the histone acetyltransferase MOF (male absent on the first; also known as Kat8, Myst 1), which specifically acetylates H4K16; (b) the loss of acetylation of H4K16 upon Cr(VI) exposure is greatly compromised by knockdown of Nupr1. Moreover, Nupr1-induced reduction of H4K16 acetylation correlates with the transcriptional down-regulation at several genomic loci. Notably, overexpression of Nupr1 induces anchorage-independent cell growth and knockdown of Nupr1 expression prevents Cr(VI)-induced cell transformation. We propose that Cr(VI) induces Nupr1 and rapidly perturbs gene expression by downregulating H4K16 acetylation, thereby contributing to Cr(VI)-induced carcinogenesis. PMID:27285315

  19. Arsenic alters global histone modifications in lymphocytes in vitro and in vivo.

    PubMed

    Pournara, Angeliki; Kippler, Maria; Holmlund, Teresa; Ceder, Rebecca; Grafström, Roland; Vahter, Marie; Broberg, Karin; Wallberg, Annika E

    2016-08-01

    Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1-proteins that are part of a gene expression silencing complex.

  20. Saccharomyces cerevisiae TORC1 Controls Histone Acetylation by Signaling Through the Sit4/PP6 Phosphatase to Regulate Sirtuin Deacetylase Nuclear Accumulation

    PubMed Central

    Workman, Jason J.; Chen, Hongfeng; Laribee, R. Nicholas

    2016-01-01

    The epigenome responds to changes in the extracellular environment, yet how this information is transmitted to the epigenetic regulatory machinery is unclear. Using a Saccharomyces cerevisiae yeast model, we demonstrate that target of rapamycin complex 1 (TORC1) signaling, which is activated by nitrogen metabolism and amino acid availability, promotes site-specific acetylation of histone H3 and H4 N-terminal tails by opposing the activity of the sirtuin deacetylases Hst3 and Hst4. TORC1 does so through suppression of the Tap42-regulated Sit4 (PP6) phosphatase complex, as sit4Δ rescues histone acetylation under TORC1-repressive conditions. We further demonstrate that TORC1 inhibition, and subsequent PP6 activation, causes a selective, rapid, nuclear accumulation of Hst4, which correlates with decreased histone acetylation. This increased Hst4 nuclear localization precedes an elevation in Hst4 protein expression, which is attributed to reduced protein turnover, suggesting that nutrient signaling through TORC1 may limit Hst4 nuclear accumulation to facilitate Hst4 degradation and maintain histone acetylation. This pathway is functionally relevant to TORC1 signaling since the stress sensitivity of a nonessential TORC1 mutant (tco89Δ) to hydroxyurea and arsenic can be reversed by combining tco89Δ with either hst3Δ, hst4Δ, or sit4Δ. Surprisingly, while hst3Δ or hst4Δ rescues the sensitivity tco89Δ has to low concentrations of the TORC1 inhibitor rapamycin, sit4Δ fails to do so. These results suggest Sit4 provides an additional function necessary for TORC1-dependent cell growth and proliferation. Collectively, this study defines a novel mechanism by which TORC1 suppresses a PP6-regulated sirtuin deacetylase pathway to couple nutrient signaling to epigenetic regulation. PMID:27343235

  1. Quantitative analysis of histone H3 and H4 post-translational modifications in doxorubicin-resistant leukemia cells.

    PubMed

    Liu, Tao; Guo, Qingcheng; Guo, Huaizu; Hou, Sheng; Li, Jing; Wang, Hao

    2016-04-01

    The epigenetic remodeling of chromatin through histone modifications has been widely implicated in drug resistance of cancer cells. However, whether epigenetic mechanisms contribute specifically to doxorubicin resistance in leukemia has not been carefully examined. Using a stable and sensitive workflow based on LC-MS, we quantitatively compared the extents of methylation and acetylation of histone H3 and H4 in acute leukemia cell line HL60 and its doxorubicin-resistant derivative, HL60/ADR, as well as the chronic leukemia cell line K562 and its doxorubicin-resistant derivative, K562/ADR. We found that increased levels of H3K9 methylation, H3K14, H3K18 and H3K23 acetylation, and potentially H4K20 methylation, are associated with drug resistance in both cells. Our results demonstrated that the doxorubicin-resistant acute and chronic leukemia cell lines may share a common epigenetic mechanism that involves a combination of transcriptional activation and silencing.

  2. The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

    PubMed Central

    Lian, Hui-Yong; Robertson, E. Douglas; Hiraga, Shin-ichiro; Alvino, Gina M.; Collingwood, David; McCune, Heather J.; Sridhar, Akila; Brewer, Bonita J.; Raghuraman, M. K.; Donaldson, Anne D.

    2011-01-01

    DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length–mediated control of replication timing requires the TG1–3 repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG1–3 tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG1–3 repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed. PMID:21441303

  3. Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer.

    PubMed

    Ma, Li; Yuan, Lili; An, Jing; Barton, Michelle C; Zhang, Qingyuan; Liu, Zhaoliang

    2016-11-01

    Acetylated H3 lysine 23 (H3K23ac) is a specific histone post-translational modification recognized by oncoprotein TRIM24. However, it is not clear whether H3K23ac levels are correlated with TRIM24 expression and what role H3K23ac may have in cancer. In this study, we collected breast carcinoma samples from 121 patients and conducted immunohistochemistry to determine the levels of TRIM24 and H3K23ac in breast cancer. Our results demonstrated that TRIM24 expression is positively correlated with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict shorter overall survival of breast cancer patients. We also showed that both TRIM24 and H3K23ac are higher in HER2-positive patients, and their levels were positively correlated with HER2 levels in breast cancer. Moreover, TRIM24 expression is associated with estrogen receptor (ER) and progesterone receptor (PR) statuses in both our cohort and The Cancer Genome Atlas (TCGA) breast carcinoma. In summary, our results revealed an important role of TRIM24 and H3K23ac in breast cancer and provided further evidence that TRIM24 small-molecule inhibitors may benefit ER- and PR-negative or HER2-positive breast cancer patients.

  4. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  5. Identification of Components of the Murine Histone Deacetylase 6 Complex: Link between Acetylation and Ubiquitination Signaling Pathways

    PubMed Central

    Seigneurin-Berny, Daphné; Verdel, André; Curtet, Sandrine; Lemercier, Claudie; Garin, Jérôme; Rousseaux, Sophie; Khochbin, Saadi

    2001-01-01

    The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination. PMID:11689694

  6. The histone acetylranseferase hMOF acetylates Nrf2 and regulates anti-drug responses in human non-small cell lung cancer

    PubMed Central

    Chen, Zhiwei; Ye, Xiangyun; Tang, Naiwang; Shen, Shengping; Li, Ziming; Niu, Xiaomin; Lu, Shun; Xu, Ling

    2014-01-01

    BACKGROUND AND PURPOSE The histone acetyltransferase MOF is a member of the MYST family. In mammals, MOF plays critical roles by acetylating histone H4 at K16 and non-histone substrates such as p53. Here we have investigated the role of MOF in human lung cancer and possible new substrates of hMOF. EXPERIMENTAL APPROACH Samples of human non-small cell lung cancer (NSCLC) were used to correlate MOF with clinicopathological parameters and NF–E2-related factor 2 (Nrf2) downstream genes. 293T-cells were used to study interactions between MOF and Nrf2, and acetylation of Nrf2 by MOF. Mouse embryonic fibroblast and A549 cells were utilized to assess involvement of MOF in antioxidative and anti-drug responses. A549 cells were used to analysis the role of MOF in anti-drug response in vitro and in vivo. KEY RESULTS hMOF was overexpressed in human NSCLC tissues and was associated with large tumour size, advanced disease stage and metastasis, and with poor prognosis. hMOF levels were positively correlated with Nrf2-downstream genes. MOF/hMOF physically interacted with and acetylated Nrf2 at Lys588. MOF-mediated acetylation increased nuclear retention of Nrf2 and transcription of its downstream genes. Importantly, MOF/hMOF was essential for anti-oxidative and anti-drug responses in vitro and regulated tumour growth and drug resistance in vivo in an Nrf2-dependent manner. CONCLUSION AND IMPLICATIONS hMOF was overexpressed in human NSCLC and was a predictor of poor survival. hMOF-mediated Nrf2 acetylation and nuclear retention are essential for anti-oxidative and anti-drug responses. hMOF may provide a therapeutic target for the treatment of NSCLC. PMID:24571482

  7. Alteration of histone acetylation pattern during long-term serum-free culture conditions of human fetal placental mesenchymal stem cells.

    PubMed

    Zhu, Yongzhao; Song, Xumei; Han, Fei; Li, Yukui; Wei, Jun; Liu, Xiaoming

    2015-01-01

    Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.

  8. Switch from Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells

    PubMed Central

    Xu, Dawei; Popov, Nikita; Hou, Mi; Wang, Qian; Björkholm, Magnus; Gruber, Astrid; Menkel, Annette R.; Henriksson, Marie

    2001-01-01

    Recent evidence suggests that the Myc and Mad1 proteins are implicated in the regulation of the gene encoding the human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase. We have analyzed the in vivo interaction between endogenous c-Myc and Mad1 proteins and the hTERT promoter in HL60 cells with the use of the chromatin immunoprecipitation assay. The E-boxes at the hTERT proximal promoter were occupied in vivo by c-Myc in exponentially proliferating HL60 cells but not in cells induced to differentiate by DMSO. In contrast, Mad1 protein was induced and bound to the hTERT promoter in differentiated HL60 cells. Concomitantly, the acetylation of the histones at the promoter was significantly reduced. These data suggest that the reciprocal E-box occupancy by c-Myc and Mad1 is responsible for activation and repression of the hTERT gene in proliferating and differentiated HL60 cells, respectively. Furthermore, the histone deacetylase inhibitor trichostatin A inhibited deacetylation of histones at the hTERT promoter and attenuated the repression of hTERT transcription during HL60 cell differentiation. In addition, trichostatin A treatment activated hTERT transcription in resting human lymphocytes and fibroblasts. Taken together, these results indicate that acetylation/deacetylation of histones is operative in the regulation of hTERT expression. PMID:11274400

  9. Histone Acetylation and Chromatin Remodeling Are Required for UV-B–Dependent Transcriptional Activation of Regulated Genes in Maize[W

    PubMed Central

    Casati, Paula; Campi, Mabel; Chu, Feixia; Suzuki, Nagi; Maltby, David; Guan, Shenheng; Burlingame, Alma L.; Walbot, Virginia

    2008-01-01

    The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B–tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B–sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B–tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B–upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B–tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B. PMID:18398050

  10. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    SciTech Connect

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul; Lee, Mee-Hee; Lee, Yoo-Hyun; Lee, Jeongmin; Jun, Woojin; Kim, Sunoh; Yoon, Ho-Geun

    2011-07-08

    Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  11. The preRC protein ORCA organizes heterochromatin by assembling histone H3 lysine 9 methyltransferases on chromatin

    PubMed Central

    Giri, Sumanprava; Aggarwal, Vasudha; Pontis, Julien; Shen, Zhen; Chakraborty, Arindam; Khan, Abid; Mizzen, Craig; Prasanth, Kannanganattu V; Ait-Si-Ali, Slimane; Ha, Taekjip; Prasanth, Supriya G

    2015-01-01

    Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure. DOI: http://dx.doi.org/10.7554/eLife.06496.001 PMID:25922909

  12. Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3 predominantly mark the pericentromeric chromatin during mitosis in monokinetic plants.

    PubMed

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2016-12-01

    Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and three monocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomal distribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinatorial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromeric chromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immunolabelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages of mitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the whole chromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion's CENH3 antibody on mitotic chromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3 detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromeric chromatin might have attributed through 'phospho-acetyl' cross talk that ultimately facilitate the sister chromatid cohesion at pericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14ac histone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. The plausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogenetics with special reference to chromatin dynamics have been discussed.

  13. The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3–H4 tetramers and Asf1–H3–H4 complexes

    PubMed Central

    Hammond, Colin M.; Sundaramoorthy, Ramasubramanian; Larance, Mark; Lamond, Angus; Stevens, Michael A.; El-Mkami, Hassane; Norman, David G.; Owen-Hughes, Tom

    2016-01-01

    Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers. In the presence of equimolar H3–H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3–H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3–H4 tetramer predominates. We show the Vps75–H3–H4 interaction is compatible with the histone chaperone Asf1 and deduce a structural model of the Vps75–Asf1-H3–H4 (VAH) co-chaperone complex using the Pulsed Electron-electron Double Resonance (PELDOR) technique and cross-linking MS/MS distance restraints. The model provides a molecular basis for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3–H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75 to interact with dimeric or tetrameric H3–H4 using the same interaction surface. PMID:27036862

  14. Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events.

    PubMed

    Li, Yong; Hassan, Yousef I; Moriyama, Hideaki; Zempleni, Janos

    2013-08-01

    Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to histones H3 and H4, thereby creating rare histone biotinylation marks in the epigenome. These marks co-localize with K9-methylated histone H3 (H3K9me), an abundant gene repression mark. The abundance of H3K9me marks in transcriptionally competent loci decreases when HCS is knocked down and when cells are depleted of biotin. Here we tested the hypothesis that the creation of H3K9me marks is at least partially explained by physical interactions between HCS and histone-lysine N-methyltransferases. Using a novel in silico protocol, we predicted that HCS-interacting proteins contain a GGGG(K/R)G(I/M)R motif. This motif, with minor variations, is present in the histone-lysine N-methyltransferase EHMT1. Physical interactions between HCS and the N-terminal, ankyrin and SET domains in EHMT1 were confirmed using yeast-two-hybrid assays, limited proteolysis assays and co-immunoprecipitation. The interactions were stronger between HCS and the N-terminus in EHMT1 compared with the ankyrin and SET domains, consistent with the localization of the HCS-binding motif in the EHMT1 N-terminus. HCS has the catalytic activity to biotinylate K161 within the binding motif in EHMT1. Mutation of K161 weakened the physical interaction between EHMT1 and HCS, but it is unknown whether this effect was caused by loss of biotinylation or loss of the motif. Importantly, HCS knockdown decreased the abundance of H3K9me marks in repeats, suggesting that HCS plays a role in creating histone methylation marks in these loci. We conclude that physical interactions between HCS and EHMT1 mediate epigenomic synergies between biotinylation and methylation events.

  15. mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression.

    PubMed

    Wang, Hengbin; An, Woojin; Cao, Ru; Xia, Li; Erdjument-Bromage, Hediye; Chatton, Bruno; Tempst, Paul; Roeder, Robert G; Zhang, Yi

    2003-08-01

    Methylation of histone tails plays an important role in chromatin structure and function. Previously, we reported that ESET/SETDB1 is a histone methyltransferase (HMTase). Here, we show that SETDB1 tightly associates with the human homolog of mAM, a murine ATFa-associated factor. Although recombinant ESET can methylate lysine 9 of histone H3 (H3-K9), its activity is severely compromised when compared to that of the ESET/mAM complex. mAM stimulates ESET enzymatic activity by increasing the Vmax and decreasing the Km. Importantly, mAM facilitates the ESET-dependent conversion of dimethyl H3-K9 to the trimethyl state both in vitro and in vivo. Chromatin-based transcription and ChIP analyses demonstrate that mAM enhances ESET-mediated transcriptional repression in a SAM-dependent manner, and this repression correlates with H3-K9 trimethylation at the promoter. Thus, our studies establish that promoter H3-K9 trimethylation is the cause of transcriptional repression and that mAM/hAM facilitates conversion of H3-K9 dimethyl to trimethyl by ESET/SETDB1.

  16. Histone hyperacetylation modulates spinal type II metabotropic glutamate receptor alleviating stress-induced visceral hypersensitivity in female rats

    PubMed Central

    Cao, Dong-Yuan; Bai, Guang; Ji, Yaping; Karpowicz, Jane

    2016-01-01

    Stress is often a trigger to exacerbate chronic pain including visceral hypersensitivity associated with irritable bowel syndrome, a female predominant functional bowel disorder. Epigenetic mechanisms that mediate stress responses are a potential target to interfere with visceral pain. The purpose of this study was to examine the effect of a histone deacetylase inhibitor, suberoylanilide hydroxamic acid, on visceral hypersensitivity induced by a subchronic stressor in female rats and to investigate the involvement of spinal glutamate receptors. Three daily sessions of forced swim induced visceral hypersensitivity. Intrathecal suberoylanilide hydroxamic acid prevented or reversed the stress-induced visceral hypersensitivity, increased spinal histone 3 acetylation and increased mGluR2 and mGluR3 expression. Chromatin immunoprecipitation (ChIP) analysis revealed enrichment of H3K9Ac and H3K18Ac at several promoter Grm2 and Grm3 regions. The mGluR2/3 antagonist LY341495 reversed the inhibitory effect of suberoylanilide hydroxamic acid on the stress-induced visceral hypersensitivity. In surprising contrast, stress and/or suberoylanilide hydroxamic acid had no effect on spinal NMDA receptor expression or function. These data reveal histone modification modulates mGluR2/3 expression in the spinal cord to attenuate stress-induced visceral hypersensitivity. HDAC inhibitors may provide a potential approach to relieve visceral hypersensitivity associated with irritable bowel syndrome. PMID:27385724

  17. Histone deacetylase inhibitors modulate the transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-a gene: interactive roles of modified histones, histone acetyltransferase, p300, AND Sp1.

    PubMed

    Kumar, Prerna; Tripathi, Satyabha; Pandey, Kailash N

    2014-03-07

    Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) and produces the intracellular second messenger, cGMP, which regulates cardiovascular homeostasis. We sought to determine the function of histone deacetylases (HDACs) in regulating Npr1 (coding for GC-A/NPRA) gene transcription, using primary mouse mesangial cells treated with class-specific HDAC inhibitors (HDACi). Trichostatin A, a pan inhibitor, and mocetinostat (MGCD0103), a class I HDAC inhibitor, significantly enhanced Npr1 promoter activity (by 8- and 10-fold, respectively), mRNA levels (4- and 5.3-fold, respectively), and NPRA protein (2.7- and 3.5-fold, respectively). However, MC1568 (class II HDAC inhibitor) had no discernible effect. Overexpression of HDAC1 and HDAC2 significantly attenuated Npr1 promoter activity, whereas HDAC3 and HDAC8 had no effect. HDACi-treated cultured cells in vitro and intact animals in vivo showed significantly reduced binding of HDAC1 and -2 and increased accumulation of acetylated H3-K9/14 and H4-K12 at the Npr1 promoter. Deletional analyses of the Npr1 promoter along with ectopic overexpression and inhibition of Sp1 confirmed that HDACi-induced Npr1 gene transcription is accomplished by Sp1 activation. Furthermore, HDACi attenuated the interaction of Sp1 with HDAC1/2 and promoted Sp1 association with p300 and p300/cAMP-binding protein-associated factor; it also promoted the recruitment of p300 and p300/cAMP-binding protein-associated factor to the Npr1 promoter. Our results demonstrate that trichostatin A and MGCD0103 enhanced Npr1 gene expression through inhibition of HDAC1/2 and increased both acetylation of histones (H3-K9/14, H4-K12) and Sp1 by p300, and their recruitment to Npr1 promoter. Our findings define a novel epigenetic regulatory mechanism that governs Npr1 gene transcription.

  18. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation.

    PubMed

    Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A; Shen, Li; Inoue, Azusa; Zhang, Yi

    2014-11-06

    Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by in vitro fertilization (IVF) but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells and its removal by ectopically expressed H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency.

  19. Two Independent Regions of Simian Virus 40 T Antigen Increase CBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, and Immortalize Primary Cells

    PubMed Central

    Sáenz Robles, Maria Teresa; Shivalila, Chikdu; Wano, Jeremy; Sorrells, Shelly; Roos, Alison

    2013-01-01

    Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation. PMID:24089570

  20. Discovery of a new class of histone deacetylase inhibitors with a novel zinc binding group

    PubMed Central

    Li, Youxuan; Woster, Patrick M.

    2015-01-01

    Small molecules featuring a hydroxamic acid or a benzamide zinc binding group (ZBG) are the most thoroughly studied histone deacetylase (HDAC) inhibitors. However, concerns about the pharmacokinetic liabilities of the hydroxamic acid moiety and potential metabolic toxicity of the aniline portion of benzamide HDAC inhibitors have stimulated research efforts aimed at discovering alternative ZBGs. Here we report the 2-(oxazol-2-yl)phenol moiety as a novel ZBG that can be used to produce compounds that are potent HDAC inhibitors. A series of analogues with this novel ZBG have been synthesized, and these analogues exhibit selective inhibition against HDAC1 as well as the class IIb HDACs (HDAC6 and HDAC10). Compound 10 possesses an IC50 value of 7.5 μM in the MV-4-11 leukemia cell line, and induces a comparable amount of acetylated histone 3 lysine 9 (H3K9) and p21Waf1/CIP1 as 0.5 μM of SAHA. Modeling of compound 10 in the active site of HDAC2 demonstrates that the 2-(oxazol-2-yl)phenol moiety has a zinc-binding pattern similar to benzamide HDAC inhibitors. PMID:26005563

  1. The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome.

    PubMed

    Kumari, Daman; Usdin, Karen

    2010-12-01

    Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5' UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin.

  2. Neurorestoration induced by the HDAC inhibitor sodium valproate in the lactacystin model of Parkinson’s is associated with histone acetylation and up-regulation of neurotrophic factors

    PubMed Central

    Harrison, Ian F; Crum, William R; Vernon, Anthony C; Dexter, David T

    2015-01-01

    Background and Purpose Histone hypoacetylation is associated with Parkinson's disease (PD), due possibly to an imbalance in the activities of enzymes responsible for histone (de)acetylation; correction of which may be neuroprotective/neurorestorative. This hypothesis was tested using the anti-epileptic drug sodium valproate, a known histone deacetylase inhibitor (HDACI), utilizing a delayed-start study design in the lactacystin rat model of PD. Experimental Approach The irreversible proteasome inhibitor lactacystin was unilaterally injected into the substantia nigra of Sprague–Dawley rats that subsequently received valproate for 28 days starting 7 days after lactacystin lesioning. Longitudinal motor behavioural testing, structural MRI and post-mortem assessment of nigrostriatal integrity were used to track changes in this model of PD and quantify neuroprotection/restoration. Subsequent cellular and molecular analyses were performed to elucidate the mechanisms underlying valproate's effects. Key Results Despite producing a distinct pattern of structural re-modelling in the healthy and lactacystin-lesioned brain, delayed-start valproate administration induced dose-dependent neuroprotection/restoration against lactacystin neurotoxicity, characterized by motor deficit alleviation, attenuation of morphological brain changes and restoration of dopaminergic neurons in the substantia nigra. Molecular analyses revealed that valproate alleviated lactacystin-induced histone hypoacetylation and induced up-regulation of brain neurotrophic/neuroprotective factors. Conclusions and Implications The histone acetylation and up-regulation of neurotrophic/neuroprotective factors associated with valproate treatment culminate in a neuroprotective and neurorestorative phenotype in this animal model of PD. As valproate induced structural re-modelling of the brain, further research is required to determine whether valproate represents a viable candidate for disease treatment; however

  3. Ablation of CBP in forebrain principal neurons causes modest memory and transcriptional defects and a dramatic reduction of histone acetylation but does not affect cell viability.

    PubMed

    Valor, Luis M; Pulopulos, Matias M; Jimenez-Minchan, Maria; Olivares, Roman; Lutz, Beat; Barco, Angel

    2011-02-02

    Rubinstein-Taybi syndrome (RSTS) is an inheritable disease associated with mutations in the gene encoding the CREB (cAMP response element-binding protein)-binding protein (CBP) and characterized by growth impairment, learning disabilities, and distinctive facial and skeletal features. Studies in mouse models for RSTS first suggested a direct role for CBP and histone acetylation in cognition and memory. Here, we took advantage of the genetic tools for generating mice in which the CBP gene is specifically deleted in postmitotic principal neurons of the forebrain to investigate the consequences of the loss of CBP in the adult brain. In contrast to the conventional CBP knock-out mice, which exhibit very early embryonic lethality, postnatal forebrain-restricted CBP mutants were viable and displayed no overt abnormalities. We identified the dimer of histones H2A and H2B as the preferred substrate of the histone acetyltransferase domain of CBP. Surprisingly, the loss of CBP and subsequent histone hypoacetylation had a very modest impact in the expression of a number of immediate early genes and did not affect neuronal viability. In addition, the behavioral characterization of these mice dissociated embryonic and postnatal deficits caused by impaired CBP function, narrowed down the anatomical substrate of specific behavioral defects, and confirmed the special sensitivity of object recognition memory to CBP deficiency. Overall, our study provides novel insights into RSTS etiology and clarifies some of the standing questions concerning the role of CBP and histone acetylation in activity-driven gene expression, memory formation, and neurodegeneration.

  4. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases

    SciTech Connect

    Horton, John R.; Upadhyay, Anup K.; Qi, Hank H.; Zhang, Xing; Shi, Yang; Cheng, Xiaodong

    2010-04-12

    Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially 'read' by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2. The presence of H3K4me3 on the same peptide as H3K9me2 makes the doubly methylated peptide a markedly better substrate of PHF8, whereas the presence of H3K4me3 has the opposite effect, diminishing the H3K9me2 demethylase activity of KIAA1718 without adversely affecting its H3K27me2 activity. The difference in substrate specificity between the two is explained by PHF8 adopting a bent conformation, allowing each of its domains to engage its respective target, whereas KIAA1718 adopts an extended conformation, which prevents its access to H3K9me2 by its jumonji domain when its PHD engages H3K4me3.

  5. Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome

    PubMed Central

    Liang, Gangning; Lin, Joy C. Y.; Wei, Vivian; Yoo, Christine; Cheng, Jonathan C.; Nguyen, Carvell T.; Weisenberger, Daniel J.; Egger, Gerda; Takai, Daiya; Gonzales, Felicidad A.; Jones, Peter A.

    2004-01-01

    Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (≈25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5′ regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes. PMID:15123803

  6. Berberine reverses abnormal expression of L-type pyruvate kinase by DNA demethylation and histone acetylation in the livers of the non-alcoholic fatty disease rat

    PubMed Central

    Zhang, Yuhao; Chang, Xinxia; Song, Xiao; Chen, Chen; Chen, Hongyan; Lu, Zhiqiang; Gao, Xin; Lu, Daru

    2015-01-01

    Berberine (BBR) can potentially be used as a drug against non-alcoholic fatty liver disease (NAFLD) and diabetes. Our previous study found that BBR could change the pattern of DNA methylation. But the mechanisms underlying berberine are still far from completely understood. In this study, the function of L-PK in cell metabolism was explored, and high-fat-diet induced SD rats NAFLD models were created. The NAFLD rats were randomly grouped to be oral administration with BBR at a dosage of 200 mg/kg daily. Then DNA methylation and histone acetylation around the L-type Pyruvate Kinase (L-PK) gene were examined. In the results, we found that L-PK had a positive effect on cell proliferation, glucose utilization and triglyceride metabolism. However, the expression of L-PK was reduced in the livers of NAFLD rats, in accord with the decrease of DNA hypermethylation and histone deacetylation in the regulatory regions of L-PK. Notably, BBR treatment can restore the expression of L-PK by the demethylation of L-PK promoter and the increase in acetylation levels of histone H3 and H4 around L-PK, which indicated that BBR may be a potential drug for epigenetic-included diseases. PMID:26221297

  7. A Ribbon-like Structure in the Ejective Organelle of the Green Microalga Pyramimonas parkeae (Prasinophyceae) Consists of Core Histones and Polymers Containing N-acetyl-glucosamine.

    PubMed

    Yamagishi, Takahiro; Kurihara, Akira; Kawai, Hiroshi

    2015-11-01

    The green microalga, Pyramimonas parkeae (Prasinophyceae) has an ejective organelle containing a coiled ribbon structure resembling the ejectisome in Cryptophyta. This structure is discharged from the cell by a stimulus and extends to form a tube-like structure, but the molecular components of the structure have not been identified. Tricine-SDS-PAGE analysis indicated that the ribbon-like structure of P. parkeae contains some proteins and low molecular acidic polymers. Edman degradation, LC/MS/MS analyses and immunological studies demonstrated that their proteins are core histones (H3, H2A, H2B and H4). In addition, monosaccharide composition analysis of the ribbon-like structures and degradation by lysozyme strongly indicated that the ribbon-like structure consist of β (1-4) linked polymers containing N-acetyl-glucosamine. Purified polymers and recombinant histones formed glob-like or filamentous structures. Therefore we conclude that the ribbon-like structure of P. parkeae mainly consists of a complex of core histones (H3, H2A, H2B and H4) and polymers containing N-acetyl-glucosamine, and suggest to name the ejective organelle in P. parkeae the "histrosome" to distinguish it from the ejectisome in Cryptophyta.

  8. Lunasin sensitivity in non-small cell lung cancer cells is linked to suppression of integrin signaling and changes in histone acetylation.

    PubMed

    Inaba, Junichi; McConnell, Elizabeth J; Davis, Keith R

    2014-12-18

    Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin's mechanism of action.

  9. Lunasin Sensitivity in Non-Small Cell Lung Cancer Cells Is Linked to Suppression of Integrin Signaling and Changes in Histone Acetylation

    PubMed Central

    Inaba, Junichi; McConnell, Elizabeth J.; Davis, Keith R.

    2014-01-01

    Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, β1 and β3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of β1 and β3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin’s mechanism of action. PMID:25530619

  10. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells.

    PubMed

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-07-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTOR(high), aH3/aH4(low)) and tumour-suppressing signals (Akt/mTOR(low) , aH3/aH4(high)) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTOR(high)) or mTOR inhibitor (aH3/aH4(low)) alone.

  11. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells

    PubMed Central

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTORhigh, aH3/aH4low) and tumour-suppressing signals (Akt/mTORlow, aH3/aH4high) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone. PMID:24779401

  12. ATRX ADD Domain Links an Atypical Histone Methylation Recognition Mechanism to Human Mental-Retardation Syndrome

    SciTech Connect

    S Iwase; B Xiang; S Ghosh; T Ren; P Lewis; J Cochrane; C Allis; D Picketts; D Patel; et al.

    2011-12-31

    ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD{sub ATRX}), whose function has remained elusive. Here we identify ADD{sub ATRX} as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD{sub ATRX} bound to H3{sub 1-15}K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.

  13. ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome

    SciTech Connect

    Iwase, Shigeki; Xiang, Bin; Ghosh, Sharmistha; Ren, Ting; Lewis, Peter W.; Cochrane, Jesse C.; Allis, C. David; Picketts, David J.; Patel, Dinshaw J.; Li, Haitao; Shi, Yang

    2011-07-19

    ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD{sub ATRX}), whose function has remained elusive. Here we identify ADD{sub ATRX} as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD{sub ATRX} bound to H3{sub 1-15}K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.

  14. Tungsten exposure causes a selective loss of histone demethylase protein.

    PubMed

    Laulicht-Glick, Freda; Wu, Feng; Zhang, Xiaoru; Jordan, Ashley; Brocato, Jason; Kluz, Thomas; Sun, Hong; Costa, Max