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Sample records for acetylates histone h4

  1. Acetylated histone H4 is reduced in human gastric adenomas and carcinomas.

    PubMed

    Ono, S; Oue, N; Kuniyasu, H; Suzuki, T; Ito, R; Matsusaki, K; Ishikawa, T; Tahara, E; Yasui, W

    2002-09-01

    Acetylation of core histones is closely linked to transcriptional activation of various genes. The acetylation levels of nucleosomal histones can be modified through a balance of histone acetyltransferases and deacetylases. To elucidate the role of histone acetylation in human gastric carcinogenesis, we studied the status of histone H4 acetylation in gastric carcinoma tissues and corresponding non-neoplastic mucosa. The status of histone acetylation was assessed by examining the expression of acetylated histone H4 through Western blotting and immunohistochemistry using an anti-acetylated histone H4 antibody. The levels of acetylated histone H4 expression were obviously reduced in 72% (13/18) of gastric carcinomas in comparison with non-neoplastic mucosa by Western blotting. In immunohistochemistry, acetylated histone H4 was clearly detected in the nuclei of both non-neoplastic epithelial and stromal cells, whereas the levels of acetylated histone H4 were heterogeneous or reduced in 66% (38/57) of gastric carcinomas and 46% (6/13) of gastric adenomas. Reduced expression of acetylated histone H4 was also observed in some areas of intestinal metaplasia adjacent to carcinomas. Reduction in the expression of acetylated histone H4 was significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. These results suggest that low levels of histone acetylation may be closely associated with the development and progression of gastric carcinomas, possibly through alteration of gene expression.

  2. Histone H4 lysine 20 acetylation is associated with gene repression in human cells

    PubMed Central

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  3. Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

    PubMed

    Kaimori, Jun-Ya; Maehara, Kazumitsu; Hayashi-Takanaka, Yoko; Harada, Akihito; Fukuda, Masafumi; Yamamoto, Satoko; Ichimaru, Naotsugu; Umehara, Takashi; Yokoyama, Shigeyuki; Matsuda, Ryo; Ikura, Tsuyoshi; Nagao, Koji; Obuse, Chikashi; Nozaki, Naohito; Takahara, Shiro; Takao, Toshifumi; Ohkawa, Yasuyuki; Kimura, Hiroshi; Isaka, Yoshitaka

    2016-01-01

    Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression. PMID:27064113

  4. Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

    PubMed Central

    Leung, Yiu Tak; Shi, Lihua; Maurer, Kelly; Song, Li; Zhang, Zhe; Petri, Michelle; Sullivan, Kathleen E

    2015-01-01

    Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1. PMID

  5. 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

  6. The Acetylation Landscape of the H4 Histone Tail: Disentangling the Interplay between the Specific and Cumulative Effects.

    PubMed

    Winogradoff, David; Echeverria, Ignacia; Potoyan, Davit A; Papoian, Garegin A

    2015-05-20

    Histone tails, the intrinsically disordered terminal regions of histone proteins, are key modulators of the structure and dynamics of chromatin and, consequently, are central to many DNA template-directed processes including replication, repair, and transcription. Acetylation of histone tails is a major post-translational modification (PTM) involved in regulating chromatin, yet it remains unclear how acetylation modifies the disordered state of histone tails and affects their function. We investigated the consequences of increasing acetylation on the isolated H4 histone tail by characterizing the conformational ensembles of unacetylated, mono-, di-, tri-, and tetra-acetylated H4 histone tails using Replica Exchange Molecular Dynamics (REMD) simulations. We found that progressive acetylation has a cumulative effect on the H4 tail, decreasing conformational heterogeneity, increasing helical propensity, and increasing hydrogen bond occupancies. The monoacetylation of lysine 16, however, has unique and specific effects: drastically decreasing the conformational heterogeneity of the H4 tail and leading to highly localized helical secondary structure and elongated conformations. We describe how the cumulative effects of acetylation arise from the charge reduction and increased hydrophobicity associated with adding acetyl groups, while the specific effects are a consequence of steric interactions that are sequence specific. Additionally, we found that increasing the level of acetylation results in the formation of spatially clustered lysines that could serve as recognition patches for binding of chromatin regulating proteins. Hence, we explore the mechanisms by which different acetylation patterns may result in specific recognition of the H4 histone tails by protein or DNA binding partners.

  7. Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation.

    PubMed

    Wakamori, Masatoshi; Fujii, Yoshifumi; Suka, Noriyuki; Shirouzu, Mikako; Sakamoto, Kensaku; Umehara, Takashi; Yokoyama, Shigeyuki

    2015-11-26

    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.

  8. 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

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

    PubMed

    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

    2016-04-21

    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

  10. Acetylated H4 histone and genomic DNA methylation patterns during bud set and bud burst in Castanea sativa.

    PubMed

    Santamaría, Ma Estrella; Hasbún, Rodrigo; Valera, Ma José; Meijón, Mónica; Valledor, Luis; Rodríguez, Jose L; Toorop, Peter E; Cañal, Ma Jesús; Rodríguez, Roberto

    2009-09-01

    The relationships between genomic DNA cytosine methylation, histone H4 acetylation and bud dormancy in Castanea sativa are described. Acetylated H4 histone and genomic DNA methylation patterns showed opposite abundance patterns during bud set and bud burst. Increased and decreased methylation levels in the apical buds coincided with bud set and bud burst, respectively. Intermediate axillary buds were characterized by constant levels of DNA methylation during burst of apical buds and reduced fluctuation in DNA methylation throughout the year, which coincided with the absence of macro-morphological changes. Furthermore, acetylated histone H4 (AcH4) levels from apical buds were higher during bud burst than during bud set, as was demonstrated by immunodetection. Results were validated with three additional C. sativa provenances. Thus, global DNA methylation and AcH4 levels showed opposite patterns and coincided with changes in bud dormancy in C. sativa.

  11. Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma

    PubMed Central

    Marquard, L; Gjerdrum, L M; Christensen, Ib J; Jensen, P B; Sehested, M; Ralfkiaer, E

    2008-01-01

    Prognostic significance of the therapeutic targets histone deacetylase 1, 2, 6 and acetylated histone H4 in cutaneous T-cell lymphoma Aims: Aberrant histone acetylation has been associated with malignancy and histone deacetylase (HDAC) inhibitors are currently being investigated in numerous clinical trials. So far, the malignancy most sensitive to HDAC inhibitors has been cutaneous T-cell lymphoma (CTCL). The reason for this sensitivity is unclear and studies on HDAC expression and histone acetylation in CTCL are lacking. The aim of this study was to address this issue. Methods and results: The immunohistochemical expression of HDAC1, HDAC2, HDAC6, and acetylated H4 was examined in 73 CTCLs and the results related to histological subtypes and overall survival. HDAC1 was most abundantly expressed (P < 0.0001), followed by HDAC2; HDAC6 and H4 acetylation were equally expressed. HDAC2 (P = 0.001) and H4 acetylation (P = 0.03) were significantly more common in aggressive than indolent CTCL subtypes. In contrast, no differences were observed for HDAC1 and HDAC6. In a Cox analysis, elevated HDAC6 was the only parameter showing significant influence on survival (P = 0.04). Conclusions: High expression of HDAC2 and acetylated H4 is more common in aggressive than indolent CTCL. HDAC6 expression is associated with a favorable outcome independent of the subtype. PMID:18671804

  12. The NuA4 Core Complex Acetylates Nucleosomal Histone H4 through a Double Recognition Mechanism.

    PubMed

    Xu, Peng; Li, Chengmin; Chen, Zhihong; Jiang, Shuanying; Fan, Shilong; Wang, Jiawei; Dai, Junbiao; Zhu, Ping; Chen, Zhucheng

    2016-09-15

    NuA4 catalyzes the acetylation of nucleosomes at histone H4, which is a well-established epigenetic event, controlling many genomic processes in Saccharomyces cerevisiae. Here we report the crystal structures of the NuA4 core complex and a cryoelectron microscopy structure with the nucleosome. The structures show that the histone-binding pocket of the enzyme is rearranged, suggesting its activation. The enzyme binds the histone tail mainly through the target lysine residue, with a preference for a small residue at the -1 position. The complex engages the nucleosome at the dish face and orients its catalytic pocket close to the H4 tail to achieve selective acetylation. The combined data reveal a space-sequence double recognition mechanism of the histone tails by a modifying enzyme in the context of the nucleosome. PMID:27594449

  13. Global Histone H4 Acetylation in the Olfactory Bulb of Lactating Rats with Different Patterns of Maternal Behavior.

    PubMed

    de Moura, Ana Carolina; da Silva, Ivy Reichert Vital; Reinaldo, Gustavo; Dani, Caroline; Elsner, Viviane Rostirola; Giovenardi, Márcia

    2016-10-01

    In rats, variations in the levels of neuromodulatory molecules and in the expression of their receptors are observed during pregnancy and postpartum. These changes may contribute to the development and management of maternal behavior. The frequency of licking the pups is used to evaluate maternal care, having mothers with low licking (LL) and high licking (HL) frequencies. Previously, we found that HL had increased levels of transcriptional expression of the receptors for serotonin (HTR1a, HTR1b), estrogen (Erα), dopamine (D1a), and prolactin (Prlr) than LL in the olfactory bulb (OB); however, the molecular mechanisms behind this phenomenon are unknown. Since evidences pointed out that epigenetic marks, which may alter gene expression, are modulated by environmental factors such as exercise, diet, maternal care, and xenobiotic exposure, our objective was to verify the acetylation levels of histone-H4 in the OB of LL and HL rats. Maternal behavior was studied for the first 7 postpartum days. LL (n = 4) and HL (n = 5) mothers were selected according to the behavior of licking their pups. Acetylation levels of histone-H4 were determined using the Global Histone-H4 Acetylation Assay Kit and expressed as ng/mg protein (mean ± SD). Analysis revealed that HL (278.36 ± 68.95) had increased H4 acetylation levels than LL (183.24 ± 73.05; p = 0.045). The enhanced expression of the previously studied receptors in the OB could be related, at least in part, to the hyperacetylation status of histone-H4 here observed. Afterward, the modulation of histone acetylation levels could exert a pivotal role through molecular mechanisms involved in the different patterns of maternal behavior.

  14. 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.

  15. Molecular dynamics simulations demonstrate the regulation of DNA-DNA attraction by H4 histone tail acetylations and mutations.

    PubMed

    Korolev, Nikolay; Yu, Hang; Lyubartsev, Alexander P; Nordenskiöld, Lars

    2014-10-01

    The positively charged N-terminal histone tails play a crucial role in chromatin compaction and are important modulators of DNA transcription, recombination, and repair. The detailed mechanism of the interaction of histone tails with DNA remains elusive. To model the unspecific interaction of histone tails with DNA, all-atom molecular dynamics (MD) simulations were carried out for systems of four DNA 22-mers in the presence of 20 or 16 short fragments of the H4 histone tail (variations of the 16-23 a. a. KRHRKVLR sequence, as well as the unmodified fragment a. a.13-20, GGAKRHRK). This setup with high DNA concentration, explicit presence of DNA-DNA contacts, presence of unstructured cationic peptides (histone tails) and K(+) mimics the conditions of eukaryotic chromatin. A detailed account of the DNA interactions with the histone tail fragments, K(+) and water is presented. Furthermore, DNA structure and dynamics and its interplay with the histone tail fragments binding are analysed. The charged side chains of the lysines and arginines play major roles in the tail-mediated DNA-DNA attraction by forming bridges and by coordinating to the phosphate groups and to the electronegative sites in the minor groove. Binding of all species to DNA is dynamic. The structure of the unmodified fully-charged H4 16-23 a.a. fragment KRHRKVLR is dominated by a stretched conformation. The H4 tail a. a. fragment GGAKRHRK as well as the H4 Lys16 acetylated fragment are highly flexible. The present work allows capturing typical features of the histone tail-counterion-DNA structure, interaction and dynamics.

  16. 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.

  17. A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation

    PubMed Central

    Wan, Guohui; Hu, Xiaoxiao; Liu, Yunhua; Han, Cecil; Sood, Anil K; Calin, George A; Zhang, Xinna; Lu, Xiongbin

    2013-01-01

    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. PMID:24097061

  18. 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.

  19. Nano-electrospray tandem mass spectrometric analysis of the acetylation state of histones H3 and H4 in stationary phase in Saccharomyces cerevisiae

    PubMed Central

    2011-01-01

    Background The involvement of histone acetylation in facilitating gene expression is well-established, particularly in the case of histones H3 and H4. It was previously shown in Saccharomyces cerevisiae that gene expression was significantly down-regulated and chromatin more condensed in stationary phase compared to exponential phase. We were therefore interested in establishing the acetylation state of histone H3 and H4 in stationary and in exponential phase, since the regulation of this modification could contribute to transcriptional shut-down and chromatin compaction during semi-quiescence. Results We made use of nano-spray tandem mass spectrometry to perform a precursor ion scan to detect an m/z 126 immonium ion, diagnostic of an Nε-acetylated lysine residue that allowed unambiguous identification of acetylated as opposed to tri-methylated lysine. The fragmentation spectra of peptides thus identified were searched with Mascot against the Swiss-Prot database, and the y-ion and b-ion fragmentation series subsequently analyzed for mass shifts compatible with acetylated lysine residues. We found that K9, K14 and K36 of histone H3 and K12 and K16 of histone H4 were acetylated in exponential phase (bulk histones), but could not detect these modifications in histones isolated from stationary phase cells at the sensitivity level of the mass spectrometer. The corresponding un-acetylated peptides were, however, observed. A significantly higher level of acetylation of these residues in exponential phase was confirmed by immuno-blotting. Conclusion H4K16 acetylation was previously shown to disrupt formation of condensed chromatin in vitro. We propose that de-acetylation of H4K16 allowed formation of condensed chromatin in stationary phase, and that acetylation of H3K9, H3K14, H3K36, and H4K12 reflected the active transcriptional state of the yeast genome in exponential phase. PMID:21726436

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. Active transgenes in zebrafish are enriched in acetylated histone H4 and dynamically associate with RNA Pol II and splicing complexes.

    PubMed

    Collas, P; Liang, M R; Vincent, M; Aleström, P

    1999-04-01

    We have investigated the functional organization of active and silent integrated luciferase transgenes in zebrafish, with the aim of accounting for the variegation of transgene expression in this species. We demonstrate the enrichment of transcriptionally active transgenes in acetylated histone H4 and the dynamic association of the transgenes with splicing factor SC35 and RNA Pol II. Analysis of interphase nuclei and extended chromatin fibers by immunofluorescence and in situ hybridization reveals a co-localization of transgenes with acetylated H4 in luciferase-expressing animals only. Enrichment of expressed transgenes in acetylated H4 is further demonstrated by their co-precipitation from chromatin using anti-acetylated H4 antibodies. Little correlation exists, however, between the level of histone acetylation and the degree of transgene expression. In transgene-expressing zebrafish, most transgenes co-localize with Pol II and SC35, whereas no such association occurs in non-expressing individuals. Inhibition of Pol II abolishes transgene expression and disrupts association of transgenes with SC35, although inactivated transgenes remains enriched in acetylated histones. Exposure of embryos to the histone deacetylation inhibitor TSA induces expression of most silent transgenes. Chromatin containing activated transgenes becomes enriched in acetylated histones and the transgenes recruit SC35 and Pol II. The results demonstrate a correlation between H4 acetylation and transgene activity, and argue that active transgenes dynamically recruit splicing factors and Pol II. The data also suggest that dissociation of splicing factors from transgenes upon Pol II inhibition is not a consequence of changes in H4 acetylation. PMID:10198286

  5. 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.

  6. Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Hwang, L R; Cha, S; Jong, J E; Jang, J H; Seo, T

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV. PMID:25283865

  7. Acetylation changes at lysine 5 of histone H4 associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Hwang, L R; Cha, S; Jong, J E; Jang, J H; Seo, T

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogenic agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease in humans. Similarly to other gammaherpesviruses such as Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS), KSHV displays two alternative life cycles, latent and lytic one. The transactivation from latency to the lytic phase is the result of transcriptional changes in the KSHV genome caused by the replication and transcriptional activator (RTA). During KSHV reactivation, epigenetic modifications of histone protein on the viral genome occur, which regulate the transcriptional activation of a number of lytic genes. The reactivation of EBV from latency to lytic cycle, induced by an immediate-early Zta protein, was shown to be accompanied by acetylation of specific lysines in histone H4. Accordingly, we hypothesized that the RTA-induced transactivation of KSHV could also be accompanied by histone acetylation. To validate this hypothesis, we assayed alterations of acetyl-histone H4-lysine 5 (acH4K5) during the RTA-mediated KSHV reactivation. While the modified histone protein in a total cell lysate was not distinguished between control and RTA-expressed cells, upregulated acH4K5 was detected on several lytic gene promoter regions during KSHV reactivation. Our results clearly indicate that this epigenetic change is related to transcription of genes expressed in the lytic cycle of KSHV.

  8. Hexavalent Chromium (Cr(VI)) Down-Regulates Acetylation of Histone H4 at Lysine 16 through Induction of Stressor Protein Nupr1.

    PubMed

    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

  9. 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

  10. Role of hMOF-dependent histone H4 lysine 16 acetylation in the maintenance of TMS1/ASC gene activity1

    PubMed Central

    Kapoor-Vazirani, Priya; Kagey, Jacob D.; Powell, Doris R.; Vertino, Paula M.

    2008-01-01

    Epigenetic silencing of tumor suppressor genes in human cancers is associated with aberrant methylation of promoter region CpG islands and local alterations in histone modifications. However, the mechanisms that drive these events remain unclear. Here, we establish an important role for histone H4 lysine 16 acetylation (H4K16Ac) and the histone acetyltransferase hMOF in the regulation of TMS1/ASC, a proapoptotic gene that undergoes epigenetic silencing in human cancers. In the unmethylated and active state, the TMS1 CpG island is spanned by positioned nucleosomes and marked by histone H3K4 methylation. H4K16Ac was uniquely localized to two sharp peaks that flanked the unmethylated CpG island and corresponded to strongly positioned nucleosomes. Aberrant methylation and silencing of TMS1 was accompanied by loss of the H4K16Ac peaks, loss of nucleosome positioning, hypomethylation of H3K4 and hypermethylation of H3K9. In addition, a single peak of histone H4 lysine 20 trimethylation was observed near the transcription start site. Downregulation of hMOF or another component of the MSL complex resulted in a gene-specific decrease in H4K16Ac, loss of nucleosome positioning and silencing of TMS1. Gene silencing induced by H4K16 deacetylation occurred independently of changes in histone methylation and DNA methylation and was reversed upon hMOF re-expression. These results indicate that the selective marking of nucleosomes flanking the CpG island by hMOF is required to maintain TMS1 gene activity, and suggest that the loss of H4K16Ac, mobilization of nucleosomes and transcriptional downregulation may be important events in the epigenetic silencing of certain tumor suppressor genes in cancer. PMID:18701507

  11. Structural insights into acetylated-histone H4 recognition by the bromodomain-PHD finger module of human transcriptional coactivator CBP.

    PubMed

    Plotnikov, Alexander N; Yang, Shuai; Zhou, Thomas Jiachi; Rusinova, Elena; Frasca, Antonio; Zhou, Ming-Ming

    2014-02-01

    Bromodomain functions as the acetyl-lysine binding domains to regulate gene transcription in chromatin. Bromodomains are rapidly emerging as new epigenetic drug targets for human diseases. However, owing to their transient nature and modest affinity, histone-binding selectivity of bromodomains has remained mostly elusive. Here, we report high-resolution crystal structures of the bromodomain-PHD tandem module of human transcriptional coactivator CBP bound to lysine-acetylated histone H4 peptides. The structures reveal that the PHD finger serves a structural role in the tandem module and that the bromodomain prefers lysine-acetylated motifs comprising a hydrophobic or aromatic residue at -2 and a lysine or arginine at -3 or -4 position from the acetylated lysine. Our study further provides structural insights into distinct modes of singly and diacetylated histone H4 recognition by the bromodomains of CBP and BRD4 that function differently as a transcriptional coactivator and chromatin organizer, respectively, explaining their distinct roles in control of gene expression in chromatin.

  12. Induction by fructose force-feeding of histone H3 and H4 acetylation at their lysine residues around the Slc2a5 gene and its expression in mice.

    PubMed

    Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2013-01-01

    It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.

  13. Acetylations of Ftz-F1 and histone H4K5 are required for the fine-tuning of ecdysone biosynthesis during Drosophila metamorphosis.

    PubMed

    Borsos, Barbara N; Pankotai, Tibor; Kovács, Dávid; Popescu, Christina; Páhi, Zoltán; Boros, Imre M

    2015-08-01

    The molting during Drosophila development is tightly regulated by the ecdysone hormone. Several steps of the ecdysone biosynthesis have been already identified but the regulation of the entire process has not been clarified yet. We have previously reported that dATAC histone acetyltransferase complex is necessary for the steroid hormone biosynthesis process. To reveal possible mechanisms controlled by dATAC we made assumptions that either dATAC may influence directly the transcription of Halloween genes involved in steroid hormone biosynthesis or it may exert an indirect effect on it by acetylating the Ftz-F1 transcription factor which regulates the transcription of steroid converting genes. Here we show that the lack of dATAC complex results in increased mRNA level and decreased protein level of Ftz-F1. In this context, decreased mRNA and increased protein levels of Ftz-F1 were detected upon treatment of Drosophila S2 cells with histone deacetylase inhibitor trichostatin A. We showed that Ftz-F1, the transcriptional activator of Halloween genes, is acetylated in S2 cells. In addition, we found that ecdysone biosynthetic Halloween genes are transcribed in S2 cells and their expression can be influenced by deacetylase inhibitors. Furthermore, we could detect H4K5 acetylation at the regulatory regions of disembodied and shade Halloween genes, while H3K9 acetylation is absent on these genes. Based on our findings we conclude that the dATAC HAT complex might play a dual regulatory role in Drosophila steroid hormone biosynthesis through the acetylation of Ftz-F1 protein and the regulation of the H4K5 acetylation at the promoters of Halloween genes. PMID:25959239

  14. Acetylations of Ftz-F1 and histone H4K5 are required for the fine-tuning of ecdysone biosynthesis during Drosophila metamorphosis.

    PubMed

    Borsos, Barbara N; Pankotai, Tibor; Kovács, Dávid; Popescu, Christina; Páhi, Zoltán; Boros, Imre M

    2015-08-01

    The molting during Drosophila development is tightly regulated by the ecdysone hormone. Several steps of the ecdysone biosynthesis have been already identified but the regulation of the entire process has not been clarified yet. We have previously reported that dATAC histone acetyltransferase complex is necessary for the steroid hormone biosynthesis process. To reveal possible mechanisms controlled by dATAC we made assumptions that either dATAC may influence directly the transcription of Halloween genes involved in steroid hormone biosynthesis or it may exert an indirect effect on it by acetylating the Ftz-F1 transcription factor which regulates the transcription of steroid converting genes. Here we show that the lack of dATAC complex results in increased mRNA level and decreased protein level of Ftz-F1. In this context, decreased mRNA and increased protein levels of Ftz-F1 were detected upon treatment of Drosophila S2 cells with histone deacetylase inhibitor trichostatin A. We showed that Ftz-F1, the transcriptional activator of Halloween genes, is acetylated in S2 cells. In addition, we found that ecdysone biosynthetic Halloween genes are transcribed in S2 cells and their expression can be influenced by deacetylase inhibitors. Furthermore, we could detect H4K5 acetylation at the regulatory regions of disembodied and shade Halloween genes, while H3K9 acetylation is absent on these genes. Based on our findings we conclude that the dATAC HAT complex might play a dual regulatory role in Drosophila steroid hormone biosynthesis through the acetylation of Ftz-F1 protein and the regulation of the H4K5 acetylation at the promoters of Halloween genes.

  15. The Molecular Basis for Histone H4- and H2A-Specific Amino-Terminal Acetylation by NatD

    PubMed Central

    Magin, Robert S.; Liszczak, Glen P.; Marmorstein, Ronen

    2014-01-01

    SUMMARY N-terminal acetylation is among the most common protein modifications in eukaryotes and is mediated by evolutionarily conserved N-terminal acetyltransferases (NATs). NatD is among the most selective NATs; its only known substrates are histones H4 and H2A, containing the N-terminal sequence SGRGK in humans. Here we characterize the molecular basis for substrate-specific acetylation by NatD by reporting its crystal structure bound to cognate substrates and performing related biochemical studies. A novel N-terminal segment wraps around the catalytic core domain to make stabilizing interactions, and the α1-α2 and β6-β7 loops adopt novel conformations to properly orient the histone N termini in the binding site. Ser1 and Arg3 of the histone make extensive contacts to highly conserved NatD residues in the substrate binding pocket, and flanking glycine residues also appear to contribute to substrate-specific binding by NatD, together defining a Ser-Gly-Arg-Gly recognition sequence. These studies have implications for understanding substrate-specific acetylation by NAT enzymes. PMID:25619998

  16. Neurogenin 3 Recruits CBP Co-activator to Facilitate Histone H3/H4 Acetylation in the Target Gene INSM1

    PubMed Central

    Breslin, Mary B.; Wang, Hong-Wei; Pierce, Amy; Aucoin, Rebecca; Lan, Michael S.

    2007-01-01

    INSM1 is a downstream target gene of ngn3. A promoter construct containing the −426/+40bp region transiently co-transfected into NIH-3T3 cells with a ngn3 expression plasmid resulted in a 12 fold increase in promoter activity. The ngn3/E47 heterodimer selectively binds and activates the E-box3 of the INSM1 promoter. The endogenous ngn3 and CBP co-activator occupy the INSM1 promoter, resulting in hyper-acetylation of histone H3/H4 chromatin in a human neuroblastoma cell line, IMR-32. Additionally, adenoviral ngn3 can induce endogenous INSM-1 expression in PANC-1 cells through the recruitment of CBP to the INSM1 promoter and increase the acetylation of the INSM1 promoter region. PMID:17300785

  17. Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb).

    PubMed

    Jeong, Jin Boo; Jeong, Hyung Jin; Park, Jae Ho; Lee, Sun Hee; Lee, Jeong Rak; Lee, Hee Kyeong; Chung, Gyu Young; Choi, Jeong Doo; de Lumen, Ben O

    2007-12-26

    Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention. PMID:18038993

  18. Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner.

    PubMed

    Byers, Jennifer; Eichinger, Daniel

    2008-01-01

    Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.

  19. Levels of histone acetylation in thyroid tumors.

    PubMed

    Puppin, Cinzia; Passon, Nadia; Lavarone, Elisa; Di Loreto, Carla; Frasca, Francesco; Vella, Veronica; Vigneri, Riccardo; Damante, Giuseppe

    2011-08-12

    Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells. PMID:21763277

  20. Histone acetylation: truth of consequences?

    PubMed

    Choi, Jennifer K; Howe, Leann J

    2009-02-01

    Eukaryotic DNA is packaged into a nucleoprotein structure known as chromatin, which is comprised of DNA, histones, and nonhistone proteins. Chromatin structure is highly dynamic, and can shift from a transcriptionally inactive state to an active form in response to intra- and extracellular signals. A major factor in chromatin architecture is the covalent modification of histones through the addition of chemical moieties, such as acetyl, methyl, ubiquitin, and phosphate groups. The acetylation of the amino-terminal tails of histones is a process that is highly conserved in eukaryotes, and was one of the earliest histone modifications characterized. Since its identification in 1964, a large body of evidence has accumulated demonstrating that histone acetylation plays an important role in transcription. Despite our ever-growing understanding of the nuclear processes involved in nucleosome acetylation, however, the exact biochemical mechanisms underlying the downstream effects of histone acetylation have yet to be fully elucidated. To date, histone acetylation has been proposed to function in 2 nonmutually exclusive manners: by directly altering chromatin structure, and by acting as a molecular tag for the recruitment of chromatin-modifying complexes. Here, we discuss recent research focusing on these 2 potential roles of histone acetylation and clarify what we actually know about the function of this modification.

  1. 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

  2. Histone acetylation and globin gene switching.

    PubMed Central

    Hebbes, T R; Thorne, A W; Clayton, A L; Crane-Robinson, C

    1992-01-01

    An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription. Images PMID:1549462

  3. 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.

  4. Genome-wide analysis of H4K5 acetylation associated with fear memory in mice

    PubMed Central

    2013-01-01

    Background Histone acetylation has been implicated in learning and memory in the brain, however, its function at the level of the genome and at individual genetic loci remains poorly investigated. This study examines a key acetylation mark, histone H4 lysine 5 acetylation (H4K5ac), genome-wide and its role in activity-dependent gene transcription in the adult mouse hippocampus following contextual fear conditioning. Results Using ChIP-Seq, we identified 23,235 genes in which H4K5ac correlates with absolute gene expression in the hippocampus. However, in the absence of transcription factor binding sites 150 bp upstream of the transcription start site, genes were associated with higher H4K5ac and expression levels. We further establish H4K5ac as a ubiquitous modification across the genome. Approximately one-third of all genes have above average H4K5ac, of which ~15% are specific to memory formation and ~65% are co-acetylated for H4K12. Although H4K5ac is prevalent across the genome, enrichment of H4K5ac at specific regions in the promoter and coding region are associated with different levels of gene expression. Additionally, unbiased peak calling for genes differentially acetylated for H4K5ac identified 114 unique genes specific to fear memory, over half of which have not previously been associated with memory processes. Conclusions Our data provide novel insights into potential mechanisms of gene priming and bookmarking by histone acetylation following hippocampal memory activation. Specifically, we propose that hyperacetylation of H4K5 may prime genes for rapid expression following activity. More broadly, this study strengthens the importance of histone posttranslational modifications for the differential regulation of transcriptional programs in cognitive processes. PMID:23927422

  5. The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation.

    PubMed

    Bowman, Andrew; Ward, Richard; Wiechens, Nicola; Singh, Vijender; El-Mkami, Hassane; Norman, David George; Owen-Hughes, Tom

    2011-02-18

    Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.

  6. Monitoring the effect of belinostat in solid tumors by H4 acetylation

    PubMed Central

    MARQUARD, LENA; PETERSEN, KAMILLE DUMONG; PERSSON, MORTEN; HOFF, KIRSTEN DAMGAARD; JENSEN, PETER BUHL; SEHESTED, MAXWELL

    2008-01-01

    Histone deacetylase (HDAC) inhibition is a novel entity in medical oncology, and several HDAC inhibitors are in clinical trials. One of them is the hydroxamic acid belinostat (PXD101) that has demonstrated therapeutic efficacy for several clinical indications. Acetylation of histones is a key event after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude mice carrying A2780 human ovarian cancer xenografts. Acetylated H4 was monitored in vivo by immunohistochemistry during treatment with belinostat, and compared with pharmacokinetics in plasma and tumor tissue. We found an increased level of acetylated H4 15 min after a single treatment (200 mg/kg i.v.) with maximum level reached after 1 h. H4 acetylation intensity reflected the belinostat concentration in plasma and tumor tissue. The threshold level for belinostat activity, indicated by acetylated H4, correlated with belinostat plasma concentrations above 1,000 ng/ml. In conclusion, examination of H4 acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors. PMID:18452428

  7. Dynamic changes in histone acetylation regulate origins of DNA replication

    PubMed Central

    Unnikrishnan, Ashwin; Gafken, Philip R.; Tsukiyama, Toshio

    2011-01-01

    While histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here, we describe a very efficient, single-step method to specifically purify histones located around an origin of replication from S. cerevisiae. Using high-resolution mass spectrometry, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that histone H3 and H4 acetylation is dynamically regulated around an origin of replication, at the level of multiply-acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S-phase. PMID:20228802

  8. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated.

  9. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated. PMID:27423860

  10. Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

    PubMed

    Natsume, Ryo; Eitoku, Masamitsu; Akai, Yusuke; Sano, Norihiko; Horikoshi, Masami; Senda, Toshiya

    2007-03-15

    CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA-histone-H3-H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain-an acetylated-histone-recognizing domain-of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure-function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 A resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3-H4 dimer's mutually exclusive interactions with another histone H3-H4 dimer and CIA-I. The carboxy-terminal beta-strand of histone H4 changes its partner from the beta-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3-H4 tetramer-disrupting activity. Mutants with weak histone H3-H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3-H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1-histone-H3-H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

  11. 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

  12. 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.

  13. Structural Basis of Histone H4 Recognition by p55

    SciTech Connect

    Song,J.; Garlick, J.; Kingston, R.

    2008-01-01

    p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. Here, we present a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a ?-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones.

  14. Kinetic analysis of histone acetylation turnover and Trichostatin A induced hyper- and hypoacetylation in alfalfa.

    PubMed

    Waterborg, Jakob H; Kapros, Tamás

    2002-01-01

    Dynamic histone acetylation is a characteristic of chromatin transcription. The first estimates for the rate of acetylation turnover of plants are reported, measured in alfalfa cells by pulse, pulse-chase, and steady-state acetylation labeling. Acetylation turnover half-lives of about 0.5 h were observed by all methods used for histones H3, H4, and H2B. This is consistent with the rate at which changes in gene expression occur in plants. Treatment with histone deacetylase inhibitor Trichostatin A (TSA) induced hyperacetylation at a similar rate. Replacement histone variant H3.2, preferentially localized in highly acetylated chromatin, displayed faster acetyl turnover. Histone H2A with a low level of acetylation was not subject to rapid turnover or hyperacetylation. Patterns of acetate labeling revealed fundamental differences between histone H3 versus histones H4 and H2B. In H3, acetylation of all molecules, limited by lysine methylation, had similar rates, independent of the level of lysine acetylation. Acetylation of histones H4 and H2B was seen in only a fraction of all molecules and involved multiacetylation. Acetylation turnover rates increased from mono- to penta- and hexaacetylated forms, respectively. TSA was an effective inhibitor of alfalfa histone deacetylases in vivo and caused a doubling in steady-state acetylation levels by 4-6 h after addition. However, hyperacetylation was transient due to loss of TSA inhibition. TSA-induced overexpression of cellular deacetylase activity produced hypoacetylation by 18 h treatment with enhanced acetate turnover labeling of alfalfa histones. Thus, application of TSA to change gene expression in vivo in plants may have unexpected consequences. PMID:12123281

  15. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

    PubMed Central

    Snyder, Nathaniel W.; Wei, Shuanzeng; Venneti, Sriram; Worth, Andrew J.; Yuan, Zuo-Fei; Lim, Hee-Woong; Liu, Shichong; Jackson, Ellen; Aiello, Nicole M.; Haas, Naomi B.; Rebbeck, Timothy R.; Judkins, Alexander; Won, Kyoung-Jae; Chodosh, Lewis A.; Garcia, Benjamin A.; Stanger, Ben Z.; Feldman, Michael D.; Blair, Ian A.; Wellen, Kathryn E.

    2014-01-01

    SUMMARY Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. PMID:24998913

  16. Sumoylated Human Histone H4 Prevents Chromatin Compaction by Inhibiting Long-range Internucleosomal Interactions*

    PubMed Central

    Dhall, Abhinav; Wei, Sijie; Fierz, Beat; Woodcock, Christopher L.; Lee, Tae-Hee; Chatterjee, Champak

    2014-01-01

    The structure of eukaryotic chromatin directly influences gene function, and is regulated by chemical modifications of the core histone proteins. Modification of the human histone H4 N-terminal tail region by the small ubiquitin-like modifier protein, SUMO-3, is associated with transcription repression. However, the direct effect of sumoylation on chromatin structure and function remains unknown. Therefore, we employed a disulfide-directed strategy to generate H4 homogenously and site-specifically sumoylated at Lys-12 (suH4ss). Chromatin compaction and oligomerization assays with nucleosomal arrays containing suH4ss established that SUMO-3 inhibits array folding and higher order oligomerization, which underlie chromatin fiber formation. Moreover, the effect of sumoylation differed from that of acetylation, and could be recapitulated with the structurally similar protein ubiquitin. Mechanistic studies at the level of single nucleosomes revealed that, unlike acetylation, the effect of SUMO-3 arises from the attenuation of long-range internucleosomal interactions more than from the destabilization of a compacted dinucleosome state. Altogether, our results present the first insight on the direct structural effects of histone H4 sumoylation and reveal a novel mechanism by which SUMO-3 inhibits chromatin compaction. PMID:25294883

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

    PubMed

    Druesne-Pecollo, Nathalie; Chaumontet, Catherine; Pagniez, Anthony; Vaugelade, Pierre; Bruneau, Aurélia; Thomas, Muriel; Cherbuy, Claire; Duée, Pierre-Henri; Martel, Paule

    2007-03-01

    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.

  18. 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.

  19. Histone deacetylase 3 indirectly modulates tubulin acetylation.

    PubMed

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-12-15

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

  20. 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

  1. 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

  2. Induction of histone acetylation on the sucrase-isomaltase gene in the postnatal rat jejunum.

    PubMed

    Yorita, Satoko; Mochizuki, Kazuki; Goda, Toshinao

    2009-04-23

    The rapid induction of the sucrase-isomaltase (SI) gene in rat jejunum from the onset to final period of weaning was associated with increases of the acetylation of histones H3 and H4 on the promoter/transcriptional region of the gene, suggesting that an abrupt jejunal induction of histone acetylation changes on the SI gene during this period may be concerned with the expression of the gene.

  3. Mass spectrometry identifies and quantifies 74 unique histone H4 isoforms in differentiating human embryonic stem cells.

    PubMed

    Phanstiel, Doug; Brumbaugh, Justin; Berggren, W Travis; Conard, Kevin; Feng, Xuezhu; Levenstein, Mark E; McAlister, Graeme C; Thomson, James A; Coon, Joshua J

    2008-03-18

    Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.

  4. Roles for Histone Acetylation in Regulation of Telomere Elongation and Two-cell State in Mouse ES Cells.

    PubMed

    Dan, Jiameng; Yang, Jiao; Liu, Yifei; Xiao, Andrew; Liu, Lin

    2015-10-01

    Mammalian telomeres and subtelomeres are marked by heterochromatic epigenetic modifications, including repressive DNA methylation and histone methylation (e.g., H3K9me3 and H4K20me3). Loss of these epigenetic marks results in increased rates of telomere recombination and elongation. Other than these repressive epigenetic marks, telomeric and subtelomeric H3 and H4 are underacetylated. Yet, whether histone acetylation also regulates telomere length has not been directly addressed. We thought to test the effects of histone acetylation levels on telomere length using histone deacetylase (HDAC) inhibitor (sodium butyrate, NaB) that mediates histone hyperacetylation and histone acetyltransferase (HAT) inhibitor (C646) that mediates histone hypoacetylation. We show that histone hyperacetylation dramatically elongates telomeres in wild-type ES cells, and only slightly elongates telomeres in Terc(-/-) ES cells, suggesting that Terc is involved in histone acetylation-induced telomere elongation. In contrast, histone hypoacetylation shortens telomeres in both wild-type and Terc(-/-) ES cells. Additionally, histone hyperacetylation activates 2-cell (2C) specific genes including Zscan4, which is involved in telomere recombination and elongation, whereas histone hypoacetylation represses Zscan4 and 2C genes. These data suggest that histone acetylation levels affect the heterochromatic state at telomeres and subtelomeres, and regulate gene expression at subtelomeres, linking histone acetylation to telomere length maintenance.

  5. Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development.

    PubMed

    Sachs, L M; Shi, Y B

    2000-11-21

    Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR). It has been postulated that TR-mediated gene regulation involves chromatin remodeling. In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation. Earlier studies have led us to propose a dual function model for TR during development. In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation. During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation. Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles. We further show that TH treatment leads to histone deacetylase release from TH response gene promoters. Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes. On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation. These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

  6. 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

  7. Antifungal properties of wheat histones (H1-H4) and purified wheat histone H1.

    PubMed

    De Lucca, Anthony J; Heden, Lars-Olof; Ingber, Bruce; Bhatnagar, Deepak

    2011-07-13

    Wheat ( Triticum spp.) histones H1, H2, H3, and H4 were extracted, and H1 was further purified. The effect of these histones on specific fungi that may or may not be pathogenic to wheat was determined. These fungi included Aspergillus flavus , Aspergillus fumigatus , Aspergillus niger , Fusarium oxysporum , Fusarium verticillioides , Fusarium solani , Fusarium graminearum , Penicillium digitatum , Penicillium italicum , and Greeneria uvicola . Non-germinated and germinating conidia of these fungi were bioassayed separately. The non-germinated and germinating conidia of all Fusarium species were highly susceptible to the mixture (H1-H4) as well as pure H1, with viability losses of 99-100% found to be significant (p < 0.001) at ≤10 μM or less for the histone mixture and pure H1. F. graminearum was the most sensitive to histone activity. The histones were inactive against all of the non-germinated Penicillium spp. conidia. However, they significantly reduced the viability of the germinating conidia of the Penicillium spp. conidia, with 95% loss at 2.5 μM. Non-germinated and germinating conidia viability of the Aspergillus spp. and G. uvicola were unaffected when exposed to histones up to 10 μM. Results indicate that Fusarium spp. pathogenic to wheat are susceptible to wheat histones, indicating that these proteins may be a resistance mechanism in wheat against fungal infection.

  8. Histone deacetylase 3 indirectly modulates tubulin acetylation

    PubMed Central

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-01-01

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation. PMID:26450925

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

    PubMed

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

    2016-06-01

    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 genes. These modifications include acetylation of histone H3 at lysine 27 (H3K27ac), which blocks Polycomb-mediated trimethylation of H3K27 (H3K27me3). H3K27ac is also widely used to identify active enhancers, and the assumption has been that profiling 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 histone H3 (lysine 64 (H3K64ac) and lysine 122 (H3K122ac)) marks active gene promoters and also a subset of active enhancers. Moreover, we find a new class of active functional enhancers that is marked by H3K122ac but lacks H3K27ac. This work suggests that, to identify enhancers, a more comprehensive analysis of histone acetylation is required than has previously been considered.

  10. Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation.

    PubMed

    Li, Zhenfei; Nie, Fen; Wang, Sheng; Li, Lin

    2011-02-22

    Histone methylation has an important role in transcriptional regulation. However, unlike H3K4 and H3K9 methylation, the role of H4K20 monomethylation (H4K20me-1) in transcriptional regulation remains unclear. Here, we show that Wnt3a specifically stimulates H4K20 monomethylation at the T cell factor (TCF)-binding element through the histone methylase SET8. Additionally, SET8 is crucial for activation of the Wnt reporter gene and target genes in both mammalian cells and zebrafish. Furthermore, SET8 interacts with lymphoid enhancing factor-1 (LEF1)/TCF4 directly, and this interaction is regulated by Wnt3a. Therefore, we conclude that SET8 is a Wnt signaling mediator and is recruited by LEF1/TCF4 to regulate the transcription of Wnt-activated genes, possibly through H4K20 monomethylation at the target gene promoters. Our findings also indicate that H4K20me-1 is a marker for gene transcription activation, at least in canonical Wnt signaling. PMID:21282610

  11. Acetylation of core histones in response to HDAC inhibitors is diminished in mitotic HeLa cells

    PubMed Central

    Patzlaff, Jason S.; Terrenoire, Edith; Turner, Bryan M.; Earnshaw, William C.; Paulson, James R.

    2010-01-01

    Histone acetylation is a key modification that regulates chromatin accessibility. Here we show that treatment with butyrate or other histone deacetylase (HDAC) inhibitors does not induce histone hyperacetylation in metaphase-arrested HeLa cells. When compared to similarly treated interphase cells, acetylation levels are significantly decreased in all four core histones and at all individual sites examined. However, the extent of the decrease varies, ranging from only slight reduction at H3K23 and H4K12 to no acetylation at H3K27 and barely detectable acetylation at H4K16. Our results show that the bulk effect is not due to increased or butyrate-insensitive HDAC activity, though these factors may play a role with some individual sites. We conclude that the lack of histone acetylation during mitosis is primarily due to changes in histone acetyltransferases (HATs) or changes in chromatin. The effects of protein phosphatase inhibitors on histone acetylation in cell lysates suggest that the reduced ability of histones to become acetylated in mitotic cells depends on protein phosphorylation. PMID:20452346

  12. 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

  13. Investigation of histone H4 hyperacetylation dynamics in the 5S rRNA genes family by chromatin immunoprecipitation assay.

    PubMed

    Burlibașa, Liliana; Suciu, Ilinca

    2015-12-01

    Oogenesis is a critical event in the formation of female gamete, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes dramatically concomitant with genome remodelling, while genomic information is stably maintained. The aim of the present study was to investigate the presence of H4 acetylation of the oocyte and somatic 5S rRNA genes in Triturus cristatus, using chromatin immunoprecipitation assay (ChIP). Our findings suggest that some epigenetic mechanisms such as histone acetylation could be involved in the transcriptional regulation of 5S rRNA gene families.

  14. Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment.

    PubMed

    Duan, Ruifeng; Liu, Xiaohua; Wang, Tianhui; Wu, Lei; Gao, Xiujie; Zhang, Zhiqing

    2016-09-01

    Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions. PMID:27161370

  15. 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

  16. CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

    PubMed

    Liu, Wallace H; Roemer, Sarah C; Port, Alex M; Churchill, Mair E A

    2012-12-01

    Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

  17. Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain.

    PubMed

    Tochio, Naoya; Umehara, Takashi; Munemasa, Yoshiko; Suzuki, Toru; Sato, Shin; Tsuda, Kengo; Koshiba, Seizo; Kigawa, Takanori; Nagai, Ryozo; Yokoyama, Shigeyuki

    2010-08-01

    Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel beta-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

  18. Induction of histone acetylation on the CRBPII gene in perinatal rat small intestine.

    PubMed

    Ogura, Yuko; Mochizuki, Kazuki; Goda, Toshinao

    2007-09-01

    The expression of genes associated with lipid and vitamin A metabolism is elevated when the small intestinal mucosa is maturing rapidly during the perinatal period. We have previously reported that cellular retinol-binding protein type II (CRBPII) mRNA levels rise abruptly in the rat small intestine during this period. In this study, we examined whether the acetylation of histones H3 and H4 is involved in the intestinal expression of CRBPII during the perinatal stage. The expression of cyclin D1 and cyclin B1 genes, which are markers of cell proliferation, decreased markedly during the perinatal period, whereas expression of CRBPII as well as villin, a marker of intestinal maturation, increased rapidly. Using a ChIP assay, we showed rapid induction of acetylation of the histones H3 and H4 which interacted with the promoter/enhancer region of the CRBPII gene at this time. The binding of CBP and p300, which have histone acetyltransferase activity, as well as binding of retinoid X receptor alpha (RXRalpha) increased on the CRBPII promoter/enhancer region during the perinatal period. These results suggest that CRBPII gene expression during the perinatal period is associated with abrupt acetylation of histones H3 and H4 followed by the binding of CBP/p300 and RXRalpha.

  19. Histone acetylation in the olfactory bulb of young rats facilitates aversive olfactory learning and synaptic plasticity.

    PubMed

    Wang, Y-J; Okutani, F; Murata, Y; Taniguchi, M; Namba, T; Kaba, H

    2013-03-01

    Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4 h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2 h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

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

    PubMed Central

    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

  1. H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida).

    PubMed

    Salvini, M; Bini, E; Santucci, A; Batistoni, R

    1997-04-01

    Two clones, obtained by polymerase chain reaction from macronuclear DNA of the unicellular ciliated protist Blepharisma japonicum, were isolated and sequenced. They correspond to fragments of two different putative H4 histone genes. The existence of multiple H4 histone genes was also suggested by Southern blot hybridisation experiments employing one of the obtained clones as a probe. Two B. japonicum H4 protein fragments, which were directly sequenced, show differences in the amino acid sequences too. The comparison of the obtained B. japonicum H4 partial amino acid sequences with each other, and with H4 from other ciliates and from representative microbial and multicellular organisms, highlights the larger histone heterogeneity of lower eukaryotes compared to that observed in higher organisms. PMID:9103980

  2. H4 histone in the macronucleus of Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida).

    PubMed

    Salvini, M; Bini, E; Santucci, A; Batistoni, R

    1997-04-01

    Two clones, obtained by polymerase chain reaction from macronuclear DNA of the unicellular ciliated protist Blepharisma japonicum, were isolated and sequenced. They correspond to fragments of two different putative H4 histone genes. The existence of multiple H4 histone genes was also suggested by Southern blot hybridisation experiments employing one of the obtained clones as a probe. Two B. japonicum H4 protein fragments, which were directly sequenced, show differences in the amino acid sequences too. The comparison of the obtained B. japonicum H4 partial amino acid sequences with each other, and with H4 from other ciliates and from representative microbial and multicellular organisms, highlights the larger histone heterogeneity of lower eukaryotes compared to that observed in higher organisms.

  3. Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance

    PubMed Central

    Earley, Keith; Lawrence, Richard J.; Pontes, Olga; Reuther, Rachel; Enciso, Angel J.; Silva, Manuela; Neves, Nuno; Gross, Michael; Viegas, Wanda; Pikaard, Craig S.

    2006-01-01

    Nucleolar dominance describes the silencing of one parental set of ribosomal RNA (rRNA) genes in a genetic hybrid, an epigenetic phenomenon that occurs on a scale second only to X-chromosome inactivation in mammals. An RNA interference (RNAi) knockdown screen revealed that the predicted Arabidopsis histone deacetylase, HDA6, is required for rRNA gene silencing in nucleolar dominance. In vivo, derepression of silenced rRNA genes upon knockdown of HDA6 is accompanied by nucleolus organizer region (NOR) decondensation, loss of promoter cytosine methylation, and replacement of histone H3 Lys 9 (H3K9) dimethylation with H3K4 trimethylation, H3K9 acetylation, H3K14 acetylation, and histone H4 tetra-acetylation. Consistent with these in vivo results, purified HDA6 deacetylates lysines modified by histone acetyltransferases whose substrates include H3K14, H4K5, and H4K12. HDA6 localizes, in part, to the nucleolus, supporting a model whereby HDA6 erases histone acetylation as a key step in an epigenetic switch mechanism that silences rRNA genes through concerted histone and DNA modifications. PMID:16648464

  4. 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

  5. Relationship of histone acetylation to DNA topology and transcription.

    PubMed

    Krajewski, W A; Luchnik, A N

    1991-12-01

    An autonomously replicating plasmid constructed from bovine papiloma virus (BPV) and pBR322 was stably maintained as a nuclear episome in a mouse cell culture. Addition to a cell culture of sodium butyrate (5 mM) induced an increase in plasmid DNA supercoiling of 3-5 turns, an increase in acetylation of cellular histones, and a decrease in plasmid transcription by 2- to 4-fold. After withdrawal of butyrate, DNA supercoiling began to fluctuate in a wave-like manner with an amplitude of up to 3 turns and a period of 3-4 h. These waves gradually faded by 24 h. The transcription of the plasmid and acetylation of cellular histones also oscillated with the same period. The wave-like alterations were not correlated with the cell cycle, for there was no resumption of DNA replication after butyrate withdrawal for at least 24 h. In vitro chemical acetylation of histones with acetyl adenylate also led to an increase in the superhelical density of plasmid DNA. The parallel changes in transcription, histone acetylation, and DNA supercoiling in vivo may indicate a functional innerconnection. Also, the observed in vivo variation in the level of DNA supercoiling directly indicates the possibility of its natural regulation in eukaryotic cells.

  6. 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

  7. Structural and biochemical analyses of monoubiquitinated human histones H2B and H4

    PubMed Central

    Machida, Shinichi; Sekine, Satoshi; Nishiyama, Yuuki; Horikoshi, Naoki; Kurumizaka, Hitoshi

    2016-01-01

    Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A–H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3–H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg2+-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin. PMID:27335322

  8. Point mutation of H3/H4 histones affects acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Liu, Xiangyong; Zhang, Xiaohua; Zhang, Zhaojie

    2014-10-10

    The molecular mechanism of acetic acid tolerance in yeast remains unclear despite of its importance for efficient cellulosic ethanol production. In this study, we examined the effects of histone H3/H4 point mutations on yeast acetic acid tolerance by comprehensively screening a histone H3/H4 mutant library. A total of 24 histone H3/H4 mutants (six acetic acid resistant and 18 sensitive) were identified. Compared to the wild-type strain, the histone acetic acid-resistant mutants exhibited improved ethanol fermentation performance under acetic acid stress. Genome-wide transcriptome analysis revealed that changes in the gene expression in the acetic acid-resistant mutants H3 K37A and H4 K16Q were mainly related to energy production, antioxidative stress. Our results provide novel insights into yeast acetic acid tolerance on the basis of histone, and suggest a novel approach to improve ethanol production by altering the histone H3/H4 sequences.

  9. PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies

    PubMed Central

    Rouhana, Labib; Weiss, Jennifer A.; King, Ryan S.; Newmark, Phillip A.

    2014-01-01

    The well-known regenerative abilities of planarian flatworms are attributed to a population of adult stem cells called neoblasts that proliferate and differentiate to produce all cell types. A characteristic feature of neoblasts is the presence of large cytoplasmic ribonucleoprotein granules named chromatoid bodies, the function of which has remained largely elusive. This study shows that histone mRNAs are a common component of chromatoid bodies. Our experiments also demonstrate that accumulation of histone mRNAs, which is typically restricted to the S phase of eukaryotic cells, is extended during the cell cycle of neoblasts. The planarian PIWI homologs SMEDWI-1 and SMEDWI-3 are required for proper localization of germinal histone H4 (gH4) mRNA to chromatoid bodies. The association between histone mRNA and chromatoid body components extends beyond gH4 mRNA, since transcripts of other core histone genes were also found in these structures. Additionally, piRNAs corresponding to loci of every core histone type have been identified. Altogether, this work provides evidence that links PIWI proteins and chromatoid bodies to histone mRNA regulation in planarian stem cells. The molecular similarities between neoblasts and undifferentiated cells of other organisms raise the possibility that PIWI proteins might also regulate histone mRNAs in stem cells and germ cells of other metazoans. PMID:24903754

  10. The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

    PubMed Central

    Liu, Wallace H; Roemer, Sarah C; Zhou, Yeyun; Shen, Zih-Jie; Dennehey, Briana K; Balsbaugh, Jeremy L; Liddle, Jennifer C; Nemkov, Travis; Ahn, Natalie G; Hansen, Kirk C; Tyler, Jessica K; Churchill, Mair EA

    2016-01-01

    The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication. DOI: http://dx.doi.org/10.7554/eLife.18023.001 PMID:27690308

  11. Alterations of global histone H4K20 methylation during prostate carcinogenesis

    PubMed Central

    2012-01-01

    Background Global histone modifications have been implicated in the progression of various tumour entities. Our study was designed to assess global methylation levels of histone 4 lysine 20 (H4K20me1-3) at different stages of prostate cancer (PCA) carcinogenesis. Methods Global H4K20 methylation levels were evaluated using a tissue microarray in patients with clinically localized PCA (n = 113), non-malignant prostate disease (n = 27), metastatic hormone-naive PCA (mPCA, n = 30) and castration-resistant PCA (CRPC, n = 34). Immunohistochemistry was performed to assess global levels of H4K20 methylation levels. Results Similar proportions of the normal, PCA, and mPCA prostate tissues showed strong H4K20me3 staining. CRPC tissue analysis showed the weakest immunostaining levels of H4K20me1 and H4K20me2, compared to other prostate tissues. H4K20me2 methylation levels indicated significant differences in examined tissues except for normal prostate versus PCA tissue. H4K20me1 differentiates CRPC from other prostate tissues. H4K20me1 was significantly correlated with lymph node metastases, and H4K20me2 showed a significant correlation with the Gleason score. However, H4K20 methylation levels failed to predict PSA recurrence after radical prostatectomy. Conclusions H4K20 methylation levels constitute valuable markers for the dynamic process of prostate cancer carcinogenesis. PMID:22413846

  12. Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

    PubMed

    Griffiths, G R; Huang, P C

    1979-08-25

    The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.

  13. Histone chaperone spt16 promotes redeposition of the original h3-h4 histones evicted by elongating RNA polymerase.

    PubMed

    Jamai, Adil; Puglisi, Andrea; Strubin, Michel

    2009-08-14

    Nucleosomes are surprisingly dynamic structures in vivo, showing transcription-independent exchange of histones H2A-H2B genome-wide and exchange of H3-H4 mainly within the promoters of transcribed genes. In addition, nucleosomes are disrupted in front of and reassembled behind the elongating RNA polymerase. Here we show that inactivation of histone chaperone Spt16 in yeast results in rapid loss of H2B and H3 from transcribed genes but also from inactive genes. In all cases, histone loss is blocked by a transcription inhibitor, indicating a transcription-dependent event. Thus, nucleosomes are efficiently evicted by the polymerase but do not reform in the absence of Spt16. Yet exchange of nucleosomal H2B with free histones occurs normally, and, unexpectedly, incorporation of new H3 increases at all loci tested. This points to Spt16 restoring normal nucleosome structure by redepositing the displaced H3-H4 histones, thereby preventing incorporation of new histones and perhaps changes in histone modification patterns associated with ongoing transcription.

  14. Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

    SciTech Connect

    Aoyama, Kazumasa; Fukumoto, Yasunori; Ishibashi, Kenichi; Kubota, Sho; Morinaga, Takao; Horiike, Yasuyoshi; Yuki, Ryuzaburo; Takahashi, Akinori; Nakayama, Yuji; Yamaguchi, Naoto

    2011-12-10

    c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

  15. Association of histone acetylation at the ACTA2 promoter region with epithelial mesenchymal transition of lens epithelial cells

    PubMed Central

    Ganatra, D A; Rajkumar, S; Patel, A R; Gajjar, D U; Johar, K; Arora, A I; Kayastha, F B; Vasavada, A R

    2015-01-01

    Purpose Epithelial mesenchymal transition (EMT) plays a central role in the development of fibrotic complications of the lens. The current study is designed to check whether EMT of lens epithelial cells (LECs) is regulated by epigenetic modifications and to evaluate the effect of Trichostatin-A (TSA) on the transforming growth factor-β (TGF-β)-induced EMT. Methods Fetal human LECs (FHL124) were treated with TGF-β2 in the presence or absence of TSA. Levels of mRNA, protein, as well as localization of α-smooth muscle actin (αSMA) were studied along with migration of LECs. Acetylation of histone H4 was analyzed and chromatin immunoprecipitation (ChIP) was carried out to study the level of acetylated histone H4 at the promoter of αSMA gene (ACTA2). Student's t-test was used for statistical analysis. Results TGF-β2 treatment resulted in myofibroblast-like changes and increased migratory capacity of FHL124. Protein and mRNA expression of αSMA increased, and immunofluorescence revealed presence of extensive stress fibers. TSA treatment preserved epithelial morphology, retarded cell migration, and abrogated an increase in αSMA levels. TSA led to the accumulation of acetylated histone H4 that was reduced on TGF-β2 treatment. However, increased level of histone H4 acetylation was found at the ACTA2 promoter region during TGF-β treatment. Conclusions The increased level of αSMA, a hallmark of EMT in LECs, is associated with increased level of histone H4 acetylation at its promoter region, and TSA helps in suppressing EMT by epigenetically reducing this level. TSA thus shows promising potential in management of fibrotic conditions of the lens. PMID:25853442

  16. Purifying selection and birth-and-death evolution in the histone H4 gene family.

    PubMed

    Piontkivska, Helen; Rooney, Alejandro P; Nei, Masatoshi

    2002-05-01

    Histones are small basic proteins encoded by a multigene family and are responsible for the nucleosomal organization of chromatin in eukaryotes. Because of the high degree of protein sequence conservation, it is generally believed that histone genes are subject to concerted evolution. However, purifying selection can also generate a high degree of sequence homogeneity. In this study, we examined the long-term evolution of histone H4 genes to determine whether concerted evolution or purifying selection was the major factor for maintaining sequence homogeneity. We analyzed the proportion (p(S)) of synonymous nucleotide differences between the H4 genes from 59 species of fungi, plants, animals, and protists and found that p(S) is generally very high and often close to the saturation level (p(S) ranging from 0.3 to 0.6) even though protein sequences are virtually identical for all H4 genes. A small proportion of genes showed a low level of p(S) values, but this appeared to be caused by recent gene duplication. Our findings suggest that the members of this gene family evolve according to the birth-and-death model of evolution under strong purifying selection. Using histone-like genes in archaebacteria as outgroups, we also showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes form separate clusters and that these classes of genes diverged nearly at the same time, before the eukaryotic kingdoms diverged.

  17. 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.

  18. Histone Acetyl Transferase (HAT) HBO1 and JADE1 in Epithelial Cell Regeneration

    PubMed Central

    Havasi, Andrea; Haegele, Joseph A.; Gall, Jonathan M.; Blackmon, Sherry; Ichimura, Takaharu; Bonegio, Ramon G.; Panchenko, Maria V.

    2014-01-01

    HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes in vitro and in vivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery. PMID:23159946

  19. Structural insights into recognition of acetylated histone ligands by the BRPF1 bromodomain

    PubMed Central

    Lubula, Mulu Y.; Eckenroth, Brian E.; Carlson, Samuel; Poplawski, Amanda; Chruszcz, Maksymilian; Glass, Karen C.

    2014-01-01

    BRPF1 is part of the MOZ HAT complex and contains a unique combination of domains typically found in chromatin-associated factors, which include PHD fingers, a bromodomain and a PWWP domain. Bromodomains are conserved structural motifs generally known to recognize acetylated histones, and the BRPF1 bromodomain preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We solved the X-ray crystal structures of the BRPF1 bromodomain in complex with the H2AK5ac and H4K12ac histone peptides. Site-directed mutagenesis on residues in the BRPF1 bromodomain-binding pocket was carried out to investigate the contribution of specific amino acids on ligand binding. Our results provide critical insights into the molecular mechanism of ligand binding by the BRPF1 bromodomain, and reveal that ordered water molecules are an essential component driving ligand recognition. PMID:25281266

  20. BBAP monoubiquitylates histone H4 at lysine 91 and selectively modulates the DNA damage response.

    PubMed

    Yan, Qingsheng; Dutt, Shilpee; Xu, Rong; Graves, Katherine; Juszczynski, Przemyslaw; Manis, John P; Shipp, Margaret A

    2009-10-01

    Although the BBAP E3 ligase and its binding partner BAL are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Because BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA-damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Because 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 and an associated DNA damage response. PMID:19818714

  1. 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.

  2. 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.

  3. 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. PMID:26748054

  4. A novel histone H4 mutant defective in nuclear division and mitotic chromosome transmission.

    PubMed Central

    Smith, M M; Yang, P; Santisteban, M S; Boone, P W; Goldstein, A T; Megee, P C

    1996-01-01

    The histone proteins are essential for the assembly and function of th e eukaryotic chromosome. Here we report the first isolation of a temperature-sensitive lethal histone H4 mutant defective in mitotic chromosome transmission Saccharomyces cerevisiae. The mutant requires two amino acid substitutions in histone H4: a lethal Thr-to-Ile change at position 82, which lies within one of the DNA-binding surfaces of the protein, and a substitution of Ala to Val at position 89 that is an intragenic suppressor. Genetic and biochemical evidence shows that the mutant histone H4 is temperature sensitive for function but not for synthesis, deposition, or stability. The chromatin structure of 2 micrometer circle minichromosomes is temperature sensitive in vivo, consistent with a defect in H4-DNA interactions. The mutant also has defects in transcription, displaying weak Spt- phenotypes. At the restrictive temperature, mutant cells arrest in the cell cycle at nuclear division, with a large bud, a single nucleus with 2C DNA content, and a short bipolar spindle. At semipermissive temperatures, the frequency of chromosome loss is elevated 60-fold in the mutant while DNA recombination frequencies are unaffected. High-copy CSE4, encoding an H3 variant related to the mammalian CENP-A kinetochore antigen, was found to suppress the temperature sensitivity of the mutant without suppressing the Spt- transcription defect. These genetic, biochemical, and phenotypic results indicate that this novel histone H4 mutant defines one or more chromatin-dependent steps in chromosome segregation. PMID:8622646

  5. Cigarette Smoke Component Acrolein Modulates Chromatin Assembly by Inhibiting Histone Acetylation*

    PubMed Central

    Chen, Danqi; Fang, Lei; Li, Hongjie; Tang, Moon-shong; Jin, Chunyuan

    2013-01-01

    Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an α,β-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function. PMID:23770671

  6. Cigarette smoke component acrolein modulates chromatin assembly by inhibiting histone acetylation.

    PubMed

    Chen, Danqi; Fang, Lei; Li, Hongjie; Tang, Moon-shong; Jin, Chunyuan

    2013-07-26

    Chromatin structure and gene expression are both regulated by nucleosome assembly. How environmental factors influence histone nuclear import and the nucleosome assembly pathway, leading to changes in chromatin organization and transcription, remains unknown. Acrolein (Acr) is an α,β-unsaturated aldehyde, which is abundant in the environment, especially in cigarette smoke. It has recently been implicated as a potential major carcinogen of smoking-related lung cancer. Here we show that Acr forms adducts with histone proteins in vitro and in vivo and preferentially reacts with free histones rather than with nucleosomal histones. Cellular fractionation analyses reveal that Acr exposure specifically inhibits acetylations of N-terminal tails of cytosolic histones H3 and H4, modifications that are important for nuclear import and chromatin assembly. Notably, Acr exposure compromises the delivery of histone H3 into chromatin and increases chromatin accessibility. Moreover, changes in nucleosome occupancy at several genomic loci are correlated with transcriptional responses to Acr exposure. Our data provide new insights into mechanisms whereby environmental factors interact with the genome and influence genome function.

  7. Histone chaperones Nap1 and Vps75 regulate histone acetylation during transcription elongation.

    PubMed

    Xue, Yu-Ming; Kowalska, Anna K; Grabowska, Kamila; Przybyt, Katarzyna; Cichewicz, Magda A; Del Rosario, Brian C; Pemberton, Lucy F

    2013-04-01

    Histone chaperones function in chromatin assembly and disassembly, suggesting they have important regulatory roles in transcription elongation. The Saccharomyces cerevisiae proteins Nap1 and Vps75 are structurally related, evolutionarily conserved histone chaperones. We showed that Nap1 genetically interacts with several transcription elongation factors and that both Nap1 and Vps75 interact with the RNA polymerase II kinase, CTK1. Loss of NAP1 or VPS75 suppressed cryptic transcription within the open reading frame (ORF) observed when strains are deleted for the kinase CTK1. Loss of the histone acetyltransferase Rtt109 also suppressed ctk1-dependent cryptic transcription. Vps75 regulates Rtt109 function, suggesting that they function together in this process. Histone H3 K9 was found to be the important lysine that is acetylated by Rtt109 during ctk1-dependent cryptic transcription. We showed that both Vps75 and Nap1 regulate the relative level of H3 K9 acetylation in the STE11 ORF. This supports a model in which Nap1, like Vps75, directly regulates Rtt109 activity or regulates the assembly of acetylated chromatin. Although Nap1 and Vps75 share many similarities, due to their distinct interactions with SET2, Nap1 and Vps75 may also play separate roles during transcription elongation. This work sheds further light on the importance of histone chaperones as general regulators of transcription elongation. PMID:23401858

  8. Acetylated H4K16 by MYST1 protects UROtsa cells from arsenic toxicity and is decreased following chronic arsenic exposure

    SciTech Connect

    Jo, William Jaime; Ren, Xuefeng; Chu, Feixia; Aleshin, Maria; Wintz, Henri; Burlingame, Alma; Smith, Martyn Thomas; Vulpe, Chris Dillon; Zhang Luoping

    2009-12-15

    Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2 gene is required for optimal growth of yeast in the presence of arsenite (As{sup III}). Yeast Sas2p is orthologous to human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is necessary for the resistance of yeast to As{sup III} through the modulation of chromatin state. We further explored the role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis in human bladder epithelial cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16 and induced sensitivity to As{sup III} and to its more toxic metabolite monomethylarsonous acid (MMA{sup III}) at doses relevant to high environmental human exposures. In addition, both As{sup III} and MMA{sup III} treatments decreased global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16 is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1 gene in human sensitivity to arsenic.

  9. Acetylated H4K16 by MYST1 protects UROtsa cells from arsenic toxicity and is decreased following chronic arsenic exposure.

    PubMed

    Jo, William Jaime; Ren, Xuefeng; Chu, Feixia; Aleshin, Maria; Wintz, Henri; Burlingame, Alma; Smith, Martyn Thomas; Vulpe, Chris Dillon; Zhang, Luoping

    2009-12-15

    Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2 gene is required for optimal growth of yeast in the presence of arsenite (As(III)). Yeast Sas2p is orthologous to human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is necessary for the resistance of yeast to As(III) through the modulation of chromatin state. We further explored the role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis in human bladder epithelial cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16 and induced sensitivity to As(III) and to its more toxic metabolite monomethylarsonous acid (MMA(III)) at doses relevant to high environmental human exposures. In addition, both As(III) and MMA(III) treatments decreased global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16 is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1 gene in human sensitivity to arsenic.

  10. Acetylated H4K16 by MYST1 Protects UROtsa Cells from the Carcinogen Arsenic and is Decreased Following Chronic Arsenic Exposure

    PubMed Central

    Jo, William Jaime; Ren, Xuefeng; Chu, Feixia; Aleshin, Maria; Wintz, Henri; Burlingame, Alma; Smith, Martyn Thomas; Vulpe, Chris Dillon; Zhang, Luoping

    2009-01-01

    Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2 gene is required for optimal growth of yeast in the presence of arsenite (AsIII). Yeast Sas2p is orthologous to human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is necessary for the resistance of yeast to AsIII through the modulation of chromatin. The role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis was further explored in human bladder epithelial cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16, and induced sensitivity to AsIII and to its more toxic metabolite monomethylarsonous acid (MMAIII) at doses relevant to high environmental human exposures. In addition, both AsIII and MMAIII treatments decreased global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16 is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1 gene in human sensitivity to arsenic. PMID:19732783

  11. DNA Mismatch Repair Interacts with CAF-1- and ASF1A-H3-H4-dependent Histone (H3-H4)2 Tetramer Deposition.

    PubMed

    Rodriges Blanko, Elena; Kadyrova, Lyudmila Y; Kadyrov, Farid A

    2016-04-22

    DNA mismatch repair (MMR) is required for the maintenance of genome stability and protection of humans from several types of cancer. Human MMR occurs in the chromatin environment, but little is known about the interactions between MMR and the chromatin environment. Previous research has suggested that MMR coincides with replication-coupled assembly of the newly synthesized DNA into nucleosomes. The first step in replication-coupled nucleosome assembly is CAF-1-dependent histone (H3-H4)2 tetramer deposition, a process that involves ASF1A-H3-H4 complex. In this work we used reconstituted human systems to investigate interactions between MMR and CAF-1- and ASF1A-H3-H4-dependent histone (H3-H4)2 tetramer deposition. We have found that MutSα inhibits CAF-1- and ASF1A-H3-H4-dependent packaging of a DNA mismatch into a tetrasome. This finding supports the idea that MMR occurs before the DNA mismatch is packaged into the tetrasome. Our experiments have also revealed that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers does not interfere with MMR reactions. In addition, we have established that unnecessary degradation of the discontinuous strand that takes place in both DNA polymerase δ (Pol δ)- and DNA polymerase ϵ (Pol ϵ)-dependent MMR reactions is suppressed by CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers. These data suggest that CAF-1- and ASF1A-H3-H4-dependent deposition of the histone (H3-H4)2 tetramers is compatible with MMR and protects the discontinuous daughter strand from unnecessary degradation by MMR machinery.

  12. The TIP60 complex regulates bivalent chromatin recognition by 53BP1 through direct H4K20me binding and H2AK15 acetylation

    PubMed Central

    Jacquet, Karine; Fradet-Turcotte, Amélie; Avvakumov, Nikita; Lambert, Jean-Philippe; Roques, Céline; Pandita, Raj K.; Paquet, Eric; Herst, Pauline; Gingras, Anne-Claude; Pandita, Tej K.; Legube, Gaëlle; Doyon, Yannick; Durocher, Daniel; Côté, Jacques

    2016-01-01

    SUMMARY The NuA4/TIP60 acetyltransferase complex is a key regulator of genome expression and stability. Here, we identified MBTD1 as a new stable subunit of the complex and gleaned intriguing insights into TIP60’s function. Harboring a histone reader domain for H4K20me1/2, MBTD1 allows TIP60 to associate with specific gene promoters and to promote the repair of DNA double strand breaks by homologous recombination. Interestingly, the non-homologous end joining factor 53BP1 engages chromatin through simultaneous binding of H4K20me2 and H2AK15ub, and it was postulated that Tip60-dependent acetylation of H4 regulates this binding. Our findings now indicate that the TIP60 complex is a potent regulator of DNA damage repair pathways in part by targeting the same histone mark as 53BP1. In addition, deposition of H2AK15ub by RNF168 inhibits chromatin acetylation by TIP60, while this residue can be acetylated by TIP60 in vivo, blocking its ubiquitylation. Altogether, these results uncover an intricate mechanism orchestrated by the TIP60 complex which regulates 53BP1-dependent repair pathway selection through incompatible bivalent binding and modification of chromatin. PMID:27153538

  13. Sodium butyrate-induced histone acetylation strengthens the expression of cocaine-associated contextual memory

    PubMed Central

    Itzhak, Yossef; Liddie, Shervin; Anderson, Karen L.

    2013-01-01

    The conditioned place preference (CPP) paradigm entails Pavlovian conditioning and allows evaluating the acquisition and extinction of drug-associated memory. Epigenetic regulation of chromatin structure by acetylation and deacetylation of histone proteins is critical for formation of long-term memory (LTM). We have recently shown that a single administration of the histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) facilitated extinction of fear-associated memory in mice. Using the CPP paradigm, the present study investigated the effect of NaB on cocaine-associated memory. C57B/6 mice were conditioned by either fixed daily doses of cocaine (5mg/kg × 4 and 15mg/kg × 4 days) or an escalating schedule (3,6,12 and 24mg/kg). Acute administration of NaB (1.2g/kg) prior to conditioning by fixed doses of cocaine increased the expression and impaired the extinction of place preference compared to control subjects. Subjects that were conditioned by 15mg/kg × 4 cocaine and received a single injection of NaB following the first or the second CPP test showed impaired extinction compared to control mice that received saline instead of NaB. Subjects that were conditioned by escalating schedule of cocaine and subsequently received repeated injections of NaB during daily reexposure to nonreinforced context showed either enhancement or no effect on place preference. Acute administration of NaB (1.2g/kg) to naïve mice resulted in marked increase in acetylation of histone H3 lysine 14 (H3K14) and histone H4 lysine 8 (H4K8) in hippocampus but not amygdala. Results suggest that regardless of the scheduling of either cocaine or NaB administration, NaB-induced histone hyperacetylation in the hippocampus may strengthen cocaine-associated contextual memory. PMID:23567105

  14. IDENTIFICATION OF HISTONE H3 LYSINE 36 ACETYLATION AS A HIGHLY CONSERVED HISTONE MODIFICATION*

    PubMed Central

    Morris, Stephanie A.; Rao, Bhargavi; Garcia, Benjamin A.; Hake, Sandra B.; Diaz, Robert L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Allis, C. David; Lieb, Jason D.; Strahl, Brian D.

    2010-01-01

    Histone lysine (K) acetylation is a major mechanism by which cells regulate the structure and function of chromatin, and new sites of acetylation continue to be discovered. Here we identify and characterize histone H3K36 acetylation (H3K36ac). By mass spectrometric analyses of H3 purified from Tetrahymena thermophila and Saccharomyces cerevisiae (yeast), we find that H3K36 can be acetylated or methylated. Using an antibody specific to H3K36ac, we show that this modification is conserved in mammals. In yeast, genome-wide ChIP-chip experiments show that H3K36ac is localized predominantly to the promoters of RNA polymerase II-transcribed genes, a pattern inversely related to that of H3K36 methylation. The pattern of H3K36ac localization is similar to that of other sites of H3 acetylation, including H3K9ac and H3K14ac. Using histone acetyltransferase complexes purified from yeast, we show that the Gcn5-containing SAGA complex that regulates transcription specifically acetylates H3K36 in vitro. Deletion of GCN5 completely abolishes H3K36ac in vivo. These data expand our knowledge of the genomic targets of Gcn5, show H3K36ac is highly conserved, and raise the intriguing possibility that the transition between H3K36ac and H3K36me acts as an “acetyl/methyl switch” governing chromatin function along transcription units. PMID:17189264

  15. Carbohydrate/fat ratio in the diet alters histone acetylation on the sucrase-isomaltase gene and its expression in mouse small intestine.

    PubMed

    Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2007-06-15

    A diet with a high carbohydrate/fat ratio enhances jejunal SI gene expression. Using ChIP assay, we revealed that the acetylation of histone H3 on transcriptional region and H4 on promoter region, respectively, of mouse SI gene are high. The acetylation of histone H3 and H4 as well as binding of HNF-1 and Cdx-2 on SI gene, was enhanced by increase in carbohydrate/fat ratio in the diet. These suggest that induction of SI gene by the diet rich in carbohydrate is associated with acetylation of histone H3 and H4 as well as binding of HNF-1 and Cdx-2 on SI gene.

  16. The cryptomonad histone H4-encoding gene: structure and chromosomal localization.

    PubMed

    Müller, S B; Rensing, S A; Maier, U G

    1994-12-15

    Cryptomonads are unicellular flagellates whose plastids are surrounded by four membranes. A periplastidal compartment, containing eukaryote-type ribosomes, starch grains and a so-called nucleomorph, is located between the inner and outer membrane pairs. The nucleomorph has been shown to be the vestigial nucleus of a eukaryotic endosymbiont. In order to obtain more information about the chromatin structure of the nucleomorph and the host nuclear chromosomes, we studied the distribution of the histone, H4. H4 was not detectable in the nucleomorph by immunolocalization, thus supporting earlier findings by Gibbs [In: Wiesner et al. (Eds.), Experimental Phycology 1, 1990, pp. 145-157]. Likewise, no H4 DNA was demonstrable in the nucleomorph by Southern hybridization. Sequence analysis, and Southern and Northern blot data of a cryptomonad gene, H4, indicate an intermediate position for these genes between animals and plants.

  17. 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.

  18. Histone Deacetylase (HDAC) Inhibitor Kinetic Rate Constants Correlate with Cellular Histone Acetylation but Not Transcription and Cell Viability

    PubMed Central

    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

    2013-01-01

    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. PMID:23897821

  19. PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression.

    PubMed

    Liu, Wen; Tanasa, Bogdan; Tyurina, Oksana V; Zhou, Tian Yuan; Gassmann, Reto; Liu, Wei Ting; Ohgi, Kenneth A; Benner, Chris; Garcia-Bassets, Ivan; Aggarwal, Aneel K; Desai, Arshad; Dorrestein, Pieter C; Glass, Christopher K; Rosenfeld, Michael G

    2010-07-22

    While reversible histone modifications are linked to an ever-expanding range of biological functions, the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1-S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression. PMID:20622854

  20. Enhanced histone acetylation in somatic cells induced by a histone deacetylase inhibitor improved inter-generic cloned leopard cat blastocysts.

    PubMed

    Lee, Hyo-Sang; Yu, Xian-Feng; Bang, Jae-Il; Cho, Su-Jin; Deb, Gautam Kumar; Kim, Byeong-Woo; Kong, Il-Keun

    2010-11-01

    The objective was to determine whether alterations of histone acetylation status in donor cells affected inter-generic SCNT (igSCNT)-cloned embryo development. Leopard cat cells were treated with trichostatin A (TSA; a histone deacetylase inhibitor) for 48 h, and then donor cells were transferred into enucleated oocytes from domestic cats. Compared to non-treated cells, the acetylated histone 3 at lysine 9 (AcH3K9) and histone 4 at lysine 5 (AcH4K5) in the TSA group increased for up to 48 h (P < 0.05). The AcH3K9 signal ratios of igSCNT group was higher than control group 3 h after activation (P < 0.05). Treatment with TSA significantly increased total cell number of blastocysts (109.1 ± 6.9 vs. 71.8 ± 2.9, mean ± SEM), with no significant effects on rates of cleavage or blastocyst development (71.1 ± 2.8 vs. 67.6 ± 2.9 and 12.2 ± 2.6 vs. 11.0 ± 2.6, respectively). When igSCNT cloned embryos were transferred into a domestic cat oviduct and recovered after 8 d, blastocyst development rates and total cell numbers were greater in the TSA-igSCNT group (20.7 ± 3.0% and 2847.6 ± 37.2) than in the control igSCNT group (5.7 ± 2.2% and 652.1 ± 17.6, P < 0.05). Average total cell numbers of blastocysts were approximately 4.4-fold higher in the TSA-igSCNT group (2847.6 ± 37.2, n = 10) than in the control group (652.1 ± 17.6, n = 8; P < 0.05), but were ∼2.9-fold lower than in vivo cat blastocysts produced by intrauterine insemination (8203.8 ± 29.6, n = 5; P < 0.001). Enhanced histone acetylation levels of donor cells improved in vivo developmental competence and quality of inter-generic cloned embryos; however, fewer cells in blastocysts derived from igSCNT than blastocysts produced by insemination may reduce development potential following intergeneric cloning (none of the cloned embryos were maintained to term).

  1. Writers and Readers of Histone Acetylation: Structure, Mechanism, and Inhibition

    PubMed Central

    Marmorstein, Ronen; Zhou, Ming-Ming

    2014-01-01

    Histone acetylation marks are written by histone acetyltransferases (HATs) and read by bromodomains (BrDs), and less commonly by other protein modules. These proteins regulate many transcription-mediated biological processes, and their aberrant activities are correlated with several human diseases. Consequently, small molecule HAT and BrD inhibitors with therapeutic potential have been developed. Structural and biochemical studies of HATs and BrDs have revealed that HATs fall into distinct subfamilies containing a structurally related core for cofactor binding, but divergent flanking regions for substrate-specific binding, catalysis, and autoregulation. BrDs adopt a conserved left-handed four-helix bundle to recognize acetyllysine; divergent loop residues contribute to substrate-specific acetyllysine recognition. PMID:24984779

  2. Butyrate mediates decrease of histone acetylation centered on transcription start sites and down-regulation of associated genes

    PubMed Central

    Rada-Iglesias, Alvaro; Enroth, Stefan; Ameur, Adam; Koch, Christoph M.; Clelland, Gayle K.; Respuela-Alonso, Patricia; Wilcox, Sarah; Dovey, Oliver M.; Ellis, Peter D.; Langford, Cordelia F.; Dunham, Ian; Komorowski, Jan; Wadelius, Claes

    2007-01-01

    Butyrate is a histone deacetylase inhibitor (HDACi) with anti-neoplastic properties, which theoretically reactivates epigenetically silenced genes by increasing global histone acetylation. However, recent studies indicate that a similar number or even more genes are down-regulated than up-regulated by this drug. We treated hepatocarcinoma HepG2 cells with butyrate and characterized the levels of acetylation at DNA-bound histones H3 and H4 by ChIP-chip along the ENCODE regions. In contrast to the global increases of histone acetylation, many genomic regions close to transcription start sites were deacetylated after butyrate exposure. In order to validate these findings, we found that both butyrate and trichostatin A treatment resulted in histone deacetylation at selected regions, while nucleosome loss or changes in histone H3 lysine 4 trimethylation (H3K4me3) did not occur in such locations. Furthermore, similar histone deacetylation events were observed when colon adenocarcinoma HT-29 cells were treated with butyrate. In addition, genes with deacetylated promoters were down-regulated by butyrate, and this was mediated at the transcriptional level by affecting RNA polymerase II (POLR2A) initiation/elongation. Finally, the global increase in acetylated histones was preferentially localized to the nuclear periphery, indicating that it might not be associated to euchromatin. Our results are significant for the evaluation of HDACi as anti-tumourogenic drugs, suggesting that previous models of action might need to be revised, and provides an explanation for the frequently observed repression of many genes during HDACi treatment. PMID:17567991

  3. Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice

    PubMed Central

    Song, Xian Jun; Kuroha, Takeshi; Ayano, Madoka; Furuta, Tomoyuki; Nagai, Keisuke; Komeda, Norio; Segami, Shuhei; Miura, Kotaro; Ogawa, Daisuke; Kamura, Takumi; Suzuki, Takamasa; Higashiyama, Tetsuya; Yamasaki, Masanori; Mori, Hitoshi; Inukai, Yoshiaki; Wu, Jianzhong; Kitano, Hidemi; Sakakibara, Hitoshi; Jacobsen, Steven E.; Ashikari, Motoyuki

    2015-01-01

    Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1’s allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding. PMID:25535376

  4. Dietary resistant starch reduces histone acetylation on the glucose-dependent insulinotropic polypeptide gene in the jejunum.

    PubMed

    Shimada, Masaya; Mochizuki, Kazuki; Goda, Toshinao

    2009-12-01

    We have reported that dietary resistant starch (RS) reduces glucose-dependent insulinotropic polypeptide (GIP) mRNA levels along the jejunoileum in both normal and diabetic rats. In this study, we found that jejunal reduction of the GIP gene by feeding normal rats dietary RS was associated with decreases in histone H3 and H4 acetylation on the promoter/enhancer region of the gene.

  5. Histone Acetylation is Involved in Gibberellin-Regulated sodCp Gene Expression in Maize Aleurone Layers.

    PubMed

    Hou, Haoli; Wang, Pu; Zhang, Hao; Wen, Huan; Gao, Fei; Ma, Ningjie; Wang, Qing; Li, Lijia

    2015-11-01

    The cereal aleurone layer plays an important role in seed germination, and reactive oxygen species (ROS) in aleurone layers act as crucial signal molecules in this progression. Recent studies have revealed that epigenetic modification is involved in plant development and seed germination. However, little is known about a possible relationship between histone modification and the ROS signaling pathway in cereal aleurone layers during seed germination. Here, we found that the expression of both histone acetyltransferases (HATs) and histone deacetylases (HDACs) was increased gradually during seed germination, accompanied by an increase in global acetylation levels of histones H3 and H4 in maize aleurone layers. The acetylation was found to be promoted by GA(3) and suppressed by ABA. However, when the HDAC inhibitor trichostatin A (TSA) was used, the increased H3K9ac and H4K5ac level correlated with an inhibition of the germination. These results indicated that the overall histone acetylation in the aleurone layers is not required for germination. Similarly these two hormones, GA(3) and ABA, exerted opposed effects on the expression of the ROS-related gene sodCp. Furthermore, chromatin immunoprecipitation experiments showed that the promoter region of the sodCp gene was hyperacetylated during germination, and this acetylation was promoted by GA(3) and inhibited by both ABA and TSA. These results suggested that GA(3)-mediated expression of the sodCp gene in aleurone layers is associated with histone hyperacetylation on the promoter and coding region of this gene, consequently leading to an accumulation of H(2)O(2) which regulated production of α-amylase during seed germination.

  6. 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.

  7. 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.

  8. Histone H3 N-terminal acetylation sites especially K14 are important for rDNA silencing and aging.

    PubMed

    Xu, Heng-hao; Su, Trent; Xue, Yong

    2016-02-24

    Histone variants and histone modifications are essential components in the establishment and maintenance of the repressed status of heterochromatin. Among these histone variants and modifications, acetylation at histone H4K16 is uniquely important for the maintenance of silencing at telomere and mating type loci but not at the ribosomal DNA locus. Here we show that mutations at H3 N-terminal acetylation site K14 specifically disrupt rDNA silencing. However, the mutant ion at H3K14R doesn't affect the recruitment of Pol II repressor RENT (regulator of nucleolar silencing and telophase exit) complex at the rDNA region. Instead, the CAF-1(chromatin assembly factor I) subunit Cac2 level decreased in the H3K14R mutant. Further experiments revealed that the single mutation at H3K14 and multi-site mutations at H3 N-terminus including K14 also delayed replication-depend nucleosome assembly and advanced replicative life span. In conclusion, our data suggest that histone H3 N-terminal acetylation sites especially at K14 are important for rDNA silencing and aging.

  9. Histone H3 N-terminal acetylation sites especially K14 are important for rDNA silencing and aging

    PubMed Central

    Xu, Heng-hao; Su, Trent; Xue, Yong

    2016-01-01

    Histone variants and histone modifications are essential components in the establishment and maintenance of the repressed status of heterochromatin. Among these histone variants and modifications, acetylation at histone H4K16 is uniquely important for the maintenance of silencing at telomere and mating type loci but not at the ribosomal DNA locus. Here we show that mutations at H3 N-terminal acetylation site K14 specifically disrupt rDNA silencing. However, the mutant ion at H3K14R doesn’t affect the recruitment of Pol II repressor RENT (regulator of nucleolar silencing and telophase exit) complex at the rDNA region. Instead, the CAF-1(chromatin assembly factor I) subunit Cac2 level decreased in the H3K14R mutant. Further experiments revealed that the single mutation at H3K14 and multi-site mutations at H3 N-terminus including K14 also delayed replication-depend nucleosome assembly and advanced replicative life span. In conclusion, our data suggest that histone H3 N-terminal acetylation sites especially at K14 are important for rDNA silencing and aging. PMID:26906758

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

    PubMed

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

    2015-12-01

    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.

  11. 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.

  12. DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

    SciTech Connect

    Elsässer, Simon J; Huang, Hongda; Lewis, Peter W; Chin, Jason W; Allis, C David; Patel, Dinshaw J

    2013-01-24

    Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly90 in H3.3 and Glu225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

  13. Depletion of histone H4 and nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae.

    PubMed Central

    Han, M; Kim, U J; Kayne, P; Grunstein, M

    1988-01-01

    We have previously constructed a yeast strain (UKY403) whose sole histone H4 gene is under control of the GAL1 promoter. This yeast arrests in G2 upon glucose treatment as a result of histone H4 depletion. The yeast PHO5 gene contains phase nucleosomes covering promoter (UAS) sequences in the PHO5 repressed state and it has been suggested that nucleosomes prevent the binding of positively acting factors to these UAS sequences. Using UKY403 we examined the length of polynucleosomes and nucleosome phasing in the PHO5 upstream region by the use of micrococcal nuclease and indirect end-labeling. It was found that glucose arrest led to a severe disruption in PHO5 chromatin structure and that most nucleosomes had their position altered or were lost from the PHO5 promoter region. Cell undergoing nucleosome depletion synthesized large quantities of accurate PHO5 transcripts even under repressive, high inorganic phosphate conditions. Histone H4 depletion did not appear to affect the repression or activation of another inducible yeast gene, CUP1. Arrest with landmarks in early G1 (in the cell division cycle mutant cdc28) or in various stages of G2 (in cdc15, cdc17 and cdc20) does not activate PHO5; nor does arrest due to chromosome topology changes (in top2 or the top1top2 topoisomerase mutants). cdc14, which has its arrest landmark at a similar point in the cell cycle as cdc15, does derepress PHO5. However, since it also leads to derepression of CUP1 it is probably functioning through an independent mechanism. Therefore, our data suggest that nucleosomes regulate PHO5 transcription. Images PMID:3046934

  14. Histone Acetylation Modifiers in the Pathogenesis of Alzheimer’s Disease

    PubMed Central

    Lu, Xi; Wang, Li; Yu, Caijia; Yu, Daohai; Yu, Gang

    2015-01-01

    It is becoming more evident that histone acetylation, as one of the epigenetic modifications or markers, plays a key role in the etiology of Alzheimer’s disease (AD). Histone acetylases and histone deacetylases (HDACs) are the well-known covalent enzymes that modify the reversible acetylation of lysine residues in histone amino-terminal domains. In AD, however, the roles of these enzymes are controversial. Some recent studies indicate that HDAC inhibitors are neuroprotective by regulating memory and synaptic dysfunctions in cellular and animal models of AD; while on the other hand, increase of histone acetylation have been implicated in AD pathology. In this review, we focus on the recent advances on the roles of histone acetylation covalent enzymes in AD and discuss how targeting these enzymes can ultimately lead to therapeutic approaches for treating AD. PMID:26136662

  15. A unique binding mode enables MCM2 to chaperone histones H3–H4 at replication forks

    PubMed Central

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-01-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3–H4. Our first structure shows an H3–H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3–H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2–7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3–H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication. PMID:26167883

  16. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks.

    PubMed

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia; Hödl, Martina; Strandsby, Anne; González-Aguilera, Cristina; Chen, Shoudeng; Groth, Anja; Patel, Dinshaw J

    2015-08-01

    During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.

  17. `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

    2016-10-01

    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.

  18. `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

    2016-10-01

    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. PMID:27180319

  19. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  20. Chromosome mapping of ribosomal genes and histone H4 in the genus Radacridium (Romaleidae)

    PubMed Central

    Anjos, Allison; Loreto, Vilma; de Souza, Maria José

    2013-01-01

    In this study, two species of Romaleidae grasshoppers, Radacridium mariajoseae and R.nordestinum, were analyzed after CMA3/DA/DAPI sequential staining and fluorescence in situ hybridization (FISH) to determine the location of the 18S and 5S rDNA and histone H4 genes. Both species presented karyotypes composed of 2n = 23, X0 with exclusively acrocentric chromosomes. CMA3+ blocks were detected after CMA3/DA/DAPI staining in only one medium size autosome bivalent and in the X chromosome in R. mariajoseae. On the other hand, all chromosomes, except the L1 bivalent, of R. nordestinum presented CMA3+ blocks. FISH analysis showed that the 18S genes are restricted to the X chromosome in R. mariajoseae, whereas these genes were located in the L2, S9 and S10 autosomes in R. nordestinum. In R. mariajoseae, the 5S rDNA sites were localized in the in L1 and L2 bivalents and in the X chromosome. In R. nordestinum, the 5S genes were located in the L2, L3, M4 and M5 pairs. In both species the histone H4 genes were present in a medium size bivalent. Together, these data evidence a great variability of chromosome markers and show that the 18S and 5S ribosomal genes are dispersed in the Radacridium genome without a significant correlation. PMID:24130439

  1. Changed histone acetylation patterns in normal appearing white matter and early MS lesions

    PubMed Central

    Pedre, X; Mastronardi, F.; Bruck, W.; López-Rodas, G; Kuhlmann, T; Casaccia, P

    2011-01-01

    The epigenetic identity of oligodendrocytes is modulated by post-translational modifications of histones. Acetylation of histone H3 results from the balance between the activity of histone-acetyltransferases (HATs) and histone deacetylases (HDACs) and modulates transcriptional activation. We have previously shown that in rodents histone deacetylation favors oligodendrocyte differentiation, while acetylation is associated with increased levels of transcriptional inhibitors of oligodendrocyte differentiation. Here we report in humans brains, a shift towards histone acetylation in the white matter of the frontal lobes of aged subjects and in patients with chronic multiple sclerosis (MS). Increased immunoreactivity for acetylated histone H3 was observed in the nuclei of NogoA+ oligodendrocytes in a subset of MS samples. These changes were associated with high levels of transcriptional inhibitors of oligodendrocyte differentiation (i.e. TCF7L2, ID2 and SOX2) and higher HAT transcript levels (i.e. CBP, P300) in female MS patients compared to non-neurological controls and correlated with disease duration. Chromatin immunoprecipitation from samples of MS patients revealed enrichment of acetyl-histone H3 at the promoter of the increased target genes (i.e. TCF7L2). The data in chronic lesions contrasted with findings in early MS lesions, where a marked oligodendroglial histone deacetylation was observed. Together these data suggest that histone deacetylation is a process that occurs at the early stages of the disease and whose efficiency decreases with disease duration. PMID:21368055

  2. Thanatos-associated protein 7 associates with template activating factor-Ibeta and inhibits histone acetylation to repress transcription.

    PubMed

    Macfarlan, Todd; Parker, J Brandon; Nagata, Kyosuke; Chakravarti, Debabrata

    2006-02-01

    The posttranslational modifications of histones on chromatin or a lack thereof is critical in transcriptional regulation. Emerging studies indicate a role for histone-binding proteins in transcriptional activation and repression. We have previously identified template-activating factor-Ibeta (TAF-Ibeta, also called PHAPII, SET, and I(2)(pp2A)) as a component of a cellular complex called inhibitor of acetyltransferases (INHAT) that masks histone acetylation in vitro and blocks histone acetyltransferase (HAT)-dependent transcription in living cells. TAF-Ibeta has also been shown to associate with transcription factors, including nuclear receptors, to regulate their activities. To identify novel interactors of TAF-Ibeta, we employed a yeast two-hybrid screen and identified a previously uncharacterized human protein called thanatos-associated protein-7 (THAP7), a member of a large family of THAP domain-containing putative DNA-binding proteins. In this study we demonstrate that THAP7 associates with TAF-Ibeta in vitro and map their association domains to a C-terminal predicted coiled-coil motif on THAP7 and the central region of TAF-Ibeta. Similarly, stably transfected THAP7 associates with endogenous TAF-Ibeta in intact cells. Like TAF-Ibeta, THAP7 associates with histone H3 and histone H4 and inhibits histone acetylation. The histone-interacting domain of THAP7 is sufficient for this activity in vitro. Promoter-targeted THAP7 can also recruit TAF-Ibeta and silencing mediator of retinoid and thyroid receptors/nuclear hormone receptor corepressor (NCoR) proteins to promoters, and knockdown of TAF-Ibeta by small interfering RNA relieves THAP7-mediated repression, indicating that, like nuclear hormone receptors, THAP7 may represent a novel class of transcription factor that uses TAF-Ibeta as a corepressor to maintain histones in a hypoacetylated, repressed state. PMID:16195249

  3. Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot.

    PubMed

    Yamada, Takatomi; Mizuno, Ken-ichi; Hirota, Kouji; Kon, Ning; Wahls, Wayne P; Hartsuiker, Edgar; Murofushi, Hiromu; Shibata, Takehiko; Ohta, Kunihiro

    2004-04-21

    Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1.Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+ gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner. PMID:14988732

  4. Roles of histone acetylation and chromatin remodeling factor in a meiotic recombination hotspot

    PubMed Central

    Yamada, Takatomi; Mizuno, Ken-ichi; Hirota, Kouji; Kon, Ning; Wahls, Wayne P; Hartsuiker, Edgar; Murofushi, Hiromu; Shibata, Takehiko; Ohta, Kunihiro

    2004-01-01

    Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the recombination hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1·Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic recombination at M26 in a site-specific manner. PMID:14988732

  5. Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells.

    PubMed

    Rahim, Rayhana; Strobl, Jeannine S

    2009-09-01

    The antimalarial drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have potential applications in cancer treatment. The growth of MCF-7 and MDA-MB-231 human breast cancer cells in vitro was inhibited by CQ and HCQ and these cells were more sensitive than nontumorigenic MCF-10A breast epithelial cells. Furthermore, all-trans retinoic acid (ATRA) augmented the anticancer effects of CQ and HCQ as evidenced by significant reductions in Ki67-positive cancer cells and clonogenicity compared with cells treated with CQ or HCQ in the absence of ATRA. As an earlier study suggested that CQ, HCQ, and ATRA are breast cancer cell differentiation agents, these agents were screened in cell-free histone deacetylase (HDAC) and histone acetyltransferase (HAT) assays. ATRA, but not CQ or HCQ, inhibited HDAC activity in HeLa nuclear extracts. Growth inhibitory concentrations of HCQ and ATRA stimulated purified p300/CBP-associated factor, where CBP is the cAMP-response element binding protein, HAT activity. To investigate whether growth inhibitory concentrations of these agents influenced protein acetylation in cells, gel-purified histone H3 and histone H4 were analyzed using mass spectrometry. HCQ alone and HCQ+ATRA treatments altered the acetylation status in the N-terminal lysines of histones H3 and H4 compared with dimethyl sulfoxide (DMSO) controls. The results indicated that HCQ and ATRA regulate protein acetylation events in MCF-7 breast cancer cells, and identify a potential mechanism for their effects on breast cancer cell growth and differentiation. PMID:19584707

  6. DNA Methylation Is Linked to Deacetylation of Histone H3, but Not H4, on the Imprinted Genes Snrpn and U2af1-rs1

    PubMed Central

    Gregory, Richard I.; Randall, Tamzin E.; Johnson, Colin A.; Khosla, Sanjeev; Hatada, Izuho; O'Neill, Laura P.; Turner, Bryan M.; Feil, Robert

    2001-01-01

    The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpn is explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. The U2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5′ DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5′ untranslated region (5′-UTR) and 3′-UTR of U2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins. PMID:11463825

  7. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin.

    PubMed

    Svensson, J Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C; Allshire, Robin C; Ekwall, Karl

    2015-06-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.

  8. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

    PubMed Central

    Svensson, J. Peter; Shukla, Manu; Menendez-Benito, Victoria; Norman-Axelsson, Ulrika; Audergon, Pauline; Sinha, Indranil; Tanny, Jason C.; Allshire, Robin C.; Ekwall, Karl

    2015-01-01

    Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution, genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation–exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally, genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di, and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and that several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation. PMID:25778913

  9. The C terminus of the histone chaperone Asf1 cross-links to histone H3 in yeast and promotes interaction with histones H3 and H4.

    PubMed

    Dennehey, Briana K; Noone, Seth; Liu, Wallace H; Smith, Luke; Churchill, Mair E A; Tyler, Jessica K

    2013-02-01

    The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

  10. Histone Acetylation and CREB Binding Protein Are Required for Neuronal Resistance against Ischemic Injury

    PubMed Central

    Yildirim, Ferah; Ji, Shengbo; Kronenberg, Golo; Barco, Angel; Olivares, Roman; Benito, Eva; Dirnagl, Ulrich; Gertz, Karen; Endres, Matthias

    2014-01-01

    Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT) and deacetylase activities (HDAC). Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB)–binding protein (CBP) as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD) in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min) subthreshold occlusion of the middle cerebral artery (MCA), followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons. PMID:24748101

  11. Nuclear factor κB-dependent histone acetylation is specifically involved in persistent forms of memory.

    PubMed

    Federman, Noel; de la Fuente, Verónica; Zalcman, Gisela; Corbi, Nicoletta; Onori, Annalisa; Passananti, Claudio; Romano, Arturo

    2013-04-24

    Memory consolidation requires gene expression regulation by transcription factors, which eventually may induce chromatin modifications as histone acetylation. This mechanism is regulated by histone acetylases and deacetylases. It is not yet clear whether memory consolidation always recruits histone acetylation or it is only engaged in more persistent memories. To address this question, we used different strength of training for novel object recognition task in mice. Only strong training induced a long-lasting memory and an increase in hippocampal histone H3 acetylation. Histone acetylase inhibition in the hippocampus during consolidation impaired memory persistence, whereas histone deacetylase inhibition caused weak memory to persist. Nuclear factor κB (NF-κB) transcription factor inhibition impaired memory persistence and, concomitantly, reduced the general level of H3 acetylation. Accordingly, we found an important increase in H3 acetylation at a specific NF-κB-regulated promoter region of the Camk2d gene, which was reversed by NF-kB inhibition. These results show for the first time that histone acetylation is a specific molecular signature of enduring memories.

  12. 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. PMID:27402865

  13. 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.

  14. The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones.

    PubMed

    Topping, Traci B; Gloss, Lisa M

    2011-12-01

    The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:β-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α-synuclein-mediated neurotoxicity and fibrillation is considered. PMID:21953551

  15. The impact of solubility and electrostatics on fibril formation by the H3 and H4 histones

    PubMed Central

    Topping, Traci B; Gloss, Lisa M

    2011-01-01

    The goal of this study was to examine fibril formation by the heterodimeric eukaryotic histones (H2A-H2B and H3-H4) and homodimeric archaeal histones (hMfB and hPyA1). The histone fold dimerization motif is an obligatorily domain-swapped structure comprised of two fused helix:β-loop:helix motifs. Domain swapping has been proposed as a mechanism for the evolution of protein oligomers as well as a means to form precursors in the formation of amyloid-like fibrils. Despite sharing a common fold, the eukaryotic histones of the core nucleosome and archaeal histones fold by kinetic mechanisms of differing complexity with transient population of partially folded monomeric and/or dimeric species. No relationship was apparent between fibrillation propensity and equilibrium stability or population of kinetic intermediates. Only H3 and H4, as isolated monomers and as a heterodimer, readily formed fibrils at room temperature, and this propensity correlates with the significantly lower solubility of these polypeptides. The fibrils were characterized by ThT fluorescence, FTIR, and far-UV CD spectroscopies and electron microscopy. The helical histone fold comprises the protease-resistant core of the fibrils, with little or no protease protection of the poorly structured N-terminal tails. The highly charged tails inhibit fibrillation through electrostatic repulsion. Kinetic studies indicate that H3 and H4 form a co-fibril, with simultaneous incorporation of both histones. The potential impact of H3 and H4 fibrillation on the cytotoxicity of extracellular histones and α-synuclein-mediated neurotoxicity and fibrillation is considered. PMID:21953551

  16. Modulation of histone deacetylase 6 (HDAC6) nuclear import and tubulin deacetylase activity through acetylation.

    PubMed

    Liu, Yuanjing; Peng, Lirong; Seto, Edward; Huang, Suming; Qiu, Yi

    2012-08-17

    The reversible acetylation of histones and non-histone proteins by histone acetyltransferases and deacetylases (HDACs) plays a critical role in many cellular processes in eukaryotic cells. HDAC6 is a unique histone deacetylase with two deacetylase domains and a C-terminal zinc finger domain. HDAC6 resides mainly in the cytoplasm and regulates many important biological processes, including cell migration and degradation of misfold proteins. HDAC6 has also been shown to localize in the nucleus to regulate transcription. However, how HDAC6 shuttles between the nucleus and cytoplasm is largely unknown. In addition, it is not clear how HDAC6 enzymatic activity is modulated. Here, we show that HDAC6 can be acetylated by p300 on five clusters of lysine residues. One cluster (site B) of acetylated lysine is in the N-terminal nuclear localization signal region. These lysine residues in site B were converted to glutamine to mimic acetylated lysines. The mutations significantly reduced HDAC6 tubulin deacetylase activity and further impaired cell motility, but had no effect on histone deacetylase activity. More interestingly, these mutations retained HDAC6 in the cytoplasm by blocking the interaction with the nuclear import protein importin-α. The retention of HDAC6 in the cytoplasm by acetylation eventually affects histone deacetylation. Thus, we conclude that acetylation is an important post-translational modification that regulates HDAC6 tubulin deacetylase activity and nuclear import.

  17. 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…

  18. Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci

    PubMed Central

    Litt, Michael D.; Simpson, Melanie; Recillas-Targa, Félix; Prioleau, Marie-Noëlle; Felsenfeld, Gary

    2001-01-01

    We have studied developmentally regulated patterns of histone acetylation at high resolution across ∼54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken β-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5′ insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases. PMID:11331588

  19. Reconsolidation involves histone acetylation depending on the strength of the memory.

    PubMed

    Federman, N; Fustiñana, M S; Romano, A

    2012-09-01

    Gene expression is a necessary step for memory re-stabilization after retrieval, a process known as reconsolidation. Histone acetylation is a fundamental mechanism involved in epigenetic regulation of gene expression and has been implicated in memory consolidation. However, few studies are available in reconsolidation, all of them in vertebrate models. Additionally, the recruitment of histone acetylation as a function of different memory strengths has not been systematically analyzed before. Here we studied the role of histone acetylation in reconsolidation using a well-characterized memory model in invertebrate, the context-signal memory in the crab Chasmagnathus. Firstly, we found an increase in histone H3 acetylation 1h after memory reactivation returning to basal levels at 3 h. Strikingly, this increment was only detected during reconsolidation of a long-term memory induced by a strong training of 30 trials, but not for a short-term memory formed by a weak training of five trials or for a long-term memory induced by a standard training of 15 trials. Furthermore, we showed that a weak memory which was enhanced during consolidation by histone deacetylases inhibition, also recruited histone H3 acetylation in reconsolidation as the strong training does. Accordingly, we found the first evidence that the administration of a histone acetyl transferase inhibitor during memory reconsolidation impairs long-term memory re-stabilization. Finally, we found that strong training memory, at variance with the standard training memory, was resistant to extinction, indicating that such strong training induced in fact a stronger memory. In conclusion, the results presented here support that the participation of histone acetylation during reconsolidation is an evolutionary conserved feature and constitutes a specific molecular characteristic of strong memories.

  20. 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

  1. Chromatin movement visualized with photoactivable GFP-labeled histone H4.

    PubMed

    Wiesmeijer, Karien; Krouwels, Ilke M; Tanke, Hans J; Dirks, Roeland W

    2008-01-01

    The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

  2. Boric acid-dependent decrease in regulatory histone H3 acetylation is not mutagenic in yeast.

    PubMed

    Pointer, Benjamin R; Schmidt, Martin

    2016-07-01

    Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations. The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast. PMID:27190149

  3. Boric acid-dependent decrease in regulatory histone H3 acetylation is not mutagenic in yeast.

    PubMed

    Pointer, Benjamin R; Schmidt, Martin

    2016-07-01

    Candida albicans is a dimorphic yeast commonly found on human mucosal membranes that switches from yeast to hyphal morphology in response to environmental factors. The change to hyphal growth requires histone H3 modifications by the yeast-specific histone acetyltransferase Rtt109. In addition to its role in morphogenesis, Rtt109-dependent acetylation of histone H3 lysine residues 9 and 56 has regulatory functions during DNA replication and repair. Boric acid (BA) is a broad-spectrum agent that specifically inhibits C. albicans hyphal growth, locking the fungus in its harmless commensal yeast state. The present study characterizes the effect of BA on C. albicans histone acetylation in respect to specificity, time-course and significance. We demonstrate that sublethal concentrations of BA reduce H3K9/H3K56 acetylation, both on a basal level and in response to genotoxic stress. Acetylation at other selected histone sites were not affected by BA. qRT-PCR expression analysis of the DNA repair gene Rad51 indicated no elevated level of genotoxic stress during BA exposure. A forward-mutation analysis demonstrated the BA does not increase spontaneous or induced mutations. The findings suggest that DNA repair remains effective even when histone H3 acetylation decreases and dispels the notion that BA treatment impairs genome integrity in yeast.

  4. 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.

  5. 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.

  6. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    SciTech Connect

    Yao, Lushuai; Li, Yanyan; Du, Fengxia; Han, Xiao; Li, Xiaohua; Niu, Yuanjie; Ren, Shancheng; Sun, Yingli

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  7. Protein Arginine Methyltransferase Prmt5-Mep50 Methylates Histones H2A and H4 and the Histone Chaperone Nucleoplasmin in Xenopus laevis Eggs*

    PubMed Central

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T.; Hunt, Donald F.; Shechter, David

    2011-01-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame. PMID:22009756

  8. Protein arginine methyltransferase Prmt5-Mep50 methylates histones H2A and H4 and the histone chaperone nucleoplasmin in Xenopus laevis eggs.

    PubMed

    Wilczek, Carola; Chitta, Raghu; Woo, Eileen; Shabanowitz, Jeffrey; Chait, Brian T; Hunt, Donald F; Shechter, David

    2011-12-01

    Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

  9. Feeding rats dietary resistant starch reduces both the binding of ChREBP and the acetylation of histones on the Thrsp gene in the jejunum.

    PubMed

    Shimada, Masaya; Mochizuki, Kazuki; Goda, Toshinao

    2011-02-23

    We have previously reported that the thyroid hormone-responsive spot 14 protein (Thrsp) gene is expressed in rat jejunum. In this study, we found that jejunal mRNA and protein expressions of Thrsp were markedly reduced in rats fed a diet containing a high amount of resistant starch (RS), which is an indigestible starch, for 7 days, compared with those fed a regular starch diet. Furthermore, we found that the binding of carbohydrate response element binding protein (ChREBP), which is a key transcription factor for the Thrsp gene, and the acetylation of histones H3 and H4, which is one of the histone modifications for transactivation, on the Thrsp gene were reduced by feeding the RS diet. These results suggest that the reduction of jejunal Thrsp gene expression by feeding a diet rich in less-digestible starch is associated with decreases in the binding of ChREBP and the acetylation of histones on the gene.

  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. Structure–function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2

    PubMed Central

    Chen, Shoudeng; Rufiange, Anne; Huang, Hongda; Rajashankar, Kanagalaghatta R.; Nourani, Amine; Patel, Dinshaw J.

    2015-01-01

    Cells use specific mechanisms such as histone chaperones to abrogate the inherent barrier that the nucleosome poses to transcribing polymerases. The current model postulates that nucleosomes can be transiently disrupted to accommodate passage of RNA polymerases and that histones H3 and H4 possess their own chaperones dedicated to the recovery of nucleosomes. Here, we determined the crystal structure of the conserved C terminus of human Suppressors of Ty insertions 2 (hSpt2C) chaperone bound to an H3/H4 tetramer. The structural studies demonstrate that hSpt2C is bound to the periphery of the H3/H4 tetramer, mimicking the trajectory of nucleosomal-bound DNA. These structural studies have been complemented with in vitro binding and in vivo functional studies on mutants that disrupt key intermolecular contacts involving two acidic patches and hydrophobic residues on Spt2C. We show that contacts between both human and yeast Spt2C with the H3/H4 tetramer are required for the suppression of H3/H4 exchange as measured by H3K56ac and new H3 deposition. These interactions are also crucial for the inhibition of spurious transcription from within coding regions. Together, our data indicate that Spt2 interacts with the periphery of the H3/H4 tetramer and promotes its recycling in the wake of RNA polymerase. PMID:26109053

  12. Structure-function studies of histone H3/H4 tetramer maintenance during transcription by chaperone Spt2.

    PubMed

    Chen, Shoudeng; Rufiange, Anne; Huang, Hongda; Rajashankar, Kanagalaghatta R; Nourani, Amine; Patel, Dinshaw J

    2015-06-15

    Cells use specific mechanisms such as histone chaperones to abrogate the inherent barrier that the nucleosome poses to transcribing polymerases. The current model postulates that nucleosomes can be transiently disrupted to accommodate passage of RNA polymerases and that histones H3 and H4 possess their own chaperones dedicated to the recovery of nucleosomes. Here, we determined the crystal structure of the conserved C terminus of human Suppressors of Ty insertions 2 (hSpt2C) chaperone bound to an H3/H4 tetramer. The structural studies demonstrate that hSpt2C is bound to the periphery of the H3/H4 tetramer, mimicking the trajectory of nucleosomal-bound DNA. These structural studies have been complemented with in vitro binding and in vivo functional studies on mutants that disrupt key intermolecular contacts involving two acidic patches and hydrophobic residues on Spt2C. We show that contacts between both human and yeast Spt2C with the H3/H4 tetramer are required for the suppression of H3/H4 exchange as measured by H3K56ac and new H3 deposition. These interactions are also crucial for the inhibition of spurious transcription from within coding regions. Together, our data indicate that Spt2 interacts with the periphery of the H3/H4 tetramer and promotes its recycling in the wake of RNA polymerase.

  13. Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

    PubMed

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

    2016-03-22

    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 γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of 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.

  14. Quantitative Measurement of Histone Tail Acetylation Reveals Stage-Specific Regulation and Response to Environmental Changes during Drosophila Development.

    PubMed

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

    2016-03-22

    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 γ-irradiation altered histone acetylation patterns. Strikingly, γ-irradiation dramatically increased the level of acetylation at H3K18, a site linked to DNA repair via nonhomologous end joining. In mutant fly strains deficient in DNA repair proteins, however, this increase in the level of 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

  15. 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.

  16. 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.

  17. 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…

  18. Functional coupling of transcription factor HiNF-P and histone H4 gene expression during pre- and post-natal mouse development.

    PubMed

    Liu, Li-Jun; Xie, Ronglin; Hussain, Sadiq; Lian, Jane B; Rivera-Perez, Jaime; Jones, Stephen N; Stein, Janet L; Stein, Gary S; van Wijnen, Andre J

    2011-09-01

    Transcription factor Histone Nuclear Factor P (HiNF-P; gene symbol Hinfp) mediates cell cycle control of histone H4 gene expression to support the packaging of newly replicated DNA as chromatin. The HiNF-P/p220(NPAT) complex controls multiple H4 genes in established human cell lines and is critical for cell proliferation. The mouse Hinfp(LacZ) null allele causes early embryonic lethality due to a blastocyst defect. However, neither Hinfp function nor its temporal expression relative to histone H4 genes during fetal development has been explored. Here, we establish that expression of Hinfp is biologically coupled with expression of twelve functional mouse H4 genes during pre- and post-natal tissue-development. Both Hinfp and H4 genes are robustly expressed at multiple embryonic (E) days (from E5.5 to E15.5), coincident with ubiquitous LacZ staining driven by the Hinfp promoter. Five highly expressed mouse H4 genes (Hist1h4d, Histh4f, Hist1h4m and Hist2h4) account for >90% of total histone H4 mRNA throughout development. Post-natal expression of H4 genes in mice is most evident in lung, spleen, thymus and intestine, and with few exceptions (e.g., adult liver) correlates with Hinfp gene expression. Histone H4 gene expression decreases butHinfp levels remain constitutive upon cell growth inhibition in culture. The in vivo co-expression of Hinfp and histone H4 genes is consistent with the biological function of Hinfp as a principal transcriptional regulator of histone H4 gene expression during mouse development.

  19. Quantitating the specificity and selectivity of Gcn5-mediated acetylation of histone H3.

    PubMed

    Kuo, Yin-Ming; Andrews, Andrew J

    2013-01-01

    Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k(cat)/K(m)) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to -4 kcal/mol between K14 and K18, the highest and lowest measurable k(cat)/K(m). These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5. PMID:23437046

  20. Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

    PubMed

    Reynolds, J A; Bautista-Jimenez, Robin; Denlinger, D L

    2016-09-01

    The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation

  1. Changes in histone acetylation as potential mediators of pupal diapause in the flesh fly, Sarcophaga bullata.

    PubMed

    Reynolds, J A; Bautista-Jimenez, Robin; Denlinger, D L

    2016-09-01

    The growing appreciation that epigenetic processes are integral to the responses of many organisms to changes in the environment suggests a possible role for epigenetics in coordination of insect diapause. The results we present suggest that histone modification may be one type of epigenetic process that contributes to regulation of pupal diapause in the flesh fly, Sarcophaga bullata. Reduction in total histone H3 acetylation in diapausing pupae, shifts in mRNA expression profiles of genes encoding histone acetyltransferase (HAT) and histone deacetylase (HDAC) in pre-diapause, diapause and post-diapause flies compared to their nondiapause counterparts, and alterations in HDAC enzyme activity during and post-diapause lend support to the hypothesis that this specific type of histone modification is involved in regulating diapause programming, maintenance, and termination. Transcription of genes encoding HDAC1, HDAC3, HDAC6, and Sirtuin2 were all upregulated in photosensitive first instar larvae programmed to enter pupal diapause, suggesting that histone deacetylation may be linked to the early decision to enter diapause. A 50% reduction in transcription of hdac3 and a corresponding 30% reduction in HDAC activity during diapause suggest that removal of acetyl groups from histones primarily occurs prior to diapause entry and that further histone deacetylation is not necessary to maintain diapause. Transcription of the HDAC genes was quickly elevated when diapause was terminated, followed by an increase in enzyme activity after a short delay. A maternal effect operating in these flies prevents pupal diapause in progeny whose mothers experienced pupal diapause, even if the progeny are reared in strong diapause-inducing short-day conditions. Such nondiapausing pupae had HDAC transcription profiles nearly identical to the profiles seen in nondiapausing pupae generated under a long-day photoperiod. Together, these results provide consistent evidence for histone acetylation

  2. 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

  3. Long-term acarbose administration alleviating the impairment of spatial learning and memory in the SAMP8 mice was associated with alleviated reduction of insulin system and acetylated H4K8.

    PubMed

    Yan, Wen-Wen; Chen, Gui-Hai; Wang, Fang; Tong, Jing-Jing; Tao, Fei

    2015-04-01

    Age-associated memory impairment (AAMI) not only reduces the quality of life for the elderly but also increases the costs of healthcare for society. Methods that can regulate glucose metabolism, insulin/insulin-like growth factor 1 (IGF-1) system and acetylated histone H4 lysine 8 (H4K8ac), one of the most well-researched facets of histone acetylation modification associating with cognition, tend to ameliorate the AAMI. Here, we used SAMP8 mice, the excellent animal model of aging and AAMI, to study the effect of long-term treatment with acarbose, an inhibitor of a-glucosidase, on AAMI and explore whether blood glucose, insulin/IGF-1 system and H4K8ac are associated with potential effects. The treatment group received acarbose (20mg/kg/d, dissolved in drinking water) at the age of 3-month until 9-month old before the behavioral test, and the controls only received water. Compared to the young controls (3-month-old, n=11), the old group (9-month-old, n=8) had declined abilities of spatial learning and memory and levels of serum insulin, hippocampal insulin receptors (InsRs) and H4K8ac. Interestingly, the acarbose group (9-month-old, n=9) showed better abilities of spatial learning and memory and higher levels of insulin, InsRs and H4K8ac relative to the old controls. Good performance of spatial learning and memory was positively correlated with the elevated insulin, InsRs and H4K8ac. All these results suggested that long-term administration of acarbose could alleviate the age-related impairment of spatial learning and memory in the SAMP8 mice, and the alleviated reduction of an insulin system and H4K8ac might be associated with the alleviation.

  4. Focus on acetylation: the role of histone deacetylase inhibitors in cancer therapy and beyond.

    PubMed

    Konstantinopoulos, Panagiotis A; Karamouzis, Michalis V; Papavassiliou, Athanasios G

    2007-05-01

    Reversal of tumorigenic epigenetic alterations is an exciting strategy for anticancer drug development. Pharmacologic inhibition of histone deacetylases (HDACs) induces differentiation, proliferation arrest and apoptosis of cancer cells. In addition to their effects on histones, HDAC inhibitors increase the acetylation level of several non-histone proteins, such as transcription factors, cytoskeletal proteins and molecular chaperones, which are crucial in tumorigenesis. Most importantly, the therapeutic potential of HDAC inhibitors goes well beyond carcinogenesis and may include neurodegenerative and inflammatory disorders. This editorial discusses the implication of HDACs in carcinogenesis, the molecular basis of the selectivity of HDAC inhibitors and their possible therapeutic role in non-malignant pathologic conditions.

  5. Discovery and Mechanism of Natural Products as Modulators of Histone Acetylation

    PubMed Central

    Salvador, Lilibeth A.; Luesch, Hendrik

    2013-01-01

    Small molecules that modulate histone acetylation by targeting key enzymes mediating this posttranslational modification – histone acetyltransferases and histone deacetylases – are validated chemotherapeutic agents for the treatment of cancer. This area of research has seen a rapid increase in interest in the past decade, with the structurally diverse natural products-derived compounds at its forefront. These secondary metabolites from various biological sources target this epigenetic modification through distinct mechanisms of enzyme regulation by utilizing a diverse array of pharmacophores. We review the discovery of these compounds and discuss their modes of inhibition together with their downstream biological effects. PMID:22594471

  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. Crystal structure of DPF3b in complex with an acetylated histone peptide.

    PubMed

    Li, Weiguo; Zhao, Anthony; Tempel, Wolfram; Loppnau, Peter; Liu, Yanli

    2016-09-01

    Histone acetylation plays an important role in chromatin dynamics and is associated with active gene transcription. This modification is written by acetyltransferases, erased by histone deacetylases and read out by bromodomain containing proteins, and others such as tandem PHD fingers of DPF3b. Here we report the high resolution crystal structure of the tandem PHD fingers of DPF3b in complex with an H3K14ac peptide. In the complex structure, the histone peptide adopts an α-helical conformation, unlike previously observed by NMR, but similar to a previously reported MOZ-H3K14ac complex structure. Our crystal structure adds to existing evidence that points to the α-helix as a natural conformation of histone tails as they interact with histone-associated proteins. PMID:27402533

  8. Pharmacological doses of gamma-hydroxybutyrate (GHB) potentiate histone acetylation in the rat brain by histone deacetylase inhibition.

    PubMed

    Klein, Christian; Kemmel, Véronique; Taleb, Omar; Aunis, Dominique; Maitre, Michel

    2009-08-01

    Several small chain fatty acids, including butyrate, valproate, phenylbutyrate and its derivatives, inhibit several HDAC activities in the brain at a several hundred micromolar concentration. Gamma-hydroxy-butyrate (GHB), a natural compound found in the brain originating from the metabolism of GABA, is structurally related to these fatty acids. The average physiological tissue concentration of GHB in the brain is below 50 microM, but when GHB is administered or absorbed for therapeutic or recreative purposes, its concentration reaches several hundred micromolars. In the present scenario, we demonstrate that pharmacological concentrations of GHB significantly induce brain histone H3 acetylation with a heterogeneous distribution in the brain and reduce in vitro HDAC activity. The degree of HDAC inhibition was also different according to the region of the brain considered. Taking into account the multiple physiological and functional roles attributed to the modification of histone acetylation and its consequences at the level of gene expression, we propose that part of the therapeutic or toxic effects of high concentrations of GHB in the brain after therapeutic administration of the drug could be partly due to GHB-induced epigenetic factors. In addition, we hypothesize that GHB, being naturally synthesized in the cytosolic compartment of certain neurons, could penetrate into the nuclei and may reach sufficient levels that could significantly modulate histone acetylation and may participate in the epigenetic modification of gene expression.

  9. Dynamics of histone H2A, H4 and HS1ph during spermatogenesis with a focus on chromatin condensation and maturity of spermatozoa

    PubMed Central

    Zhang, Zhao-Hui; Mu, Shu-Mei; Guo, Ming-Shen; Wu, Jiang-li; Li, Yan-qin; Zhang, Han; Wang, Ying; Kang, Xian-Jiang

    2016-01-01

    Histones and histone phosphorylation play vital roles during animal spermatogenesis and spermatozoa maturation. The dynamic distribution of histones H2A and H4 and phosphorylated H2A and H4 at serine 1 (HS1ph) was explored in mammalian and Decapoda germ cells, with a special focus on the distribution of H2A, H4 and HS1ph between mouse condensed spermatozoa chromatin and crab non-condensed spermatozoa chromatin. The distribution of histone marks was also analysed in mature spermatozoa with different chromatin structures. Histone H2A and H4 marks were closely associated with the relatively loose chromatin structure in crab spermatozoa. The significant decrease in the HS1ph signal during spermatogenesis suggests that eliminating most of these epigenetic marks in the nucleusis closely associated with spermatozoa maturity. PMID:27121047

  10. The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protist Trichomonas vaginalis.

    PubMed

    Marinets, A; Müller, M; Johnson, P J; Kulda, J; Scheiner, O; Wiedermann, G; Duchêne, M

    1996-12-01

    Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones from Trichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which contained T. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of the T. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists like Entamoeba histolytica, Trypanosoma cruzi, and Leishmania infantum.

  11. Epigenetic effects of dietary butyrate on hepatic histone acetylation and enzymes of biotransformation in chicken.

    PubMed

    Mátis, Gábor; Neogrády, Zsuzsanna; Csikó, György; Gálfi, Péter; Fébel, Hedvig; Jemnitz, Katalin; Veres, Zsuzsanna; Kulcsár, Anna; Kenéz, Akos; Huber, Korinna

    2013-12-01

    The aim of the study was to investigate the in vivo epigenetic influences of dietary butyrate supplementation on the acetylation state of core histones and the activity of drug-metabolising microsomal cytochrome P450 (CYP) enzymes in the liver of broiler chickens in the starter period. One-day-old Ross 308 broilers were fed a starter diet without or with sodium butyrate (1.5 g/kg feed) for 21 days. After slaughtering, nucleus and microsome fractions were isolated from the exsanguinated liver by multi-step differential centrifugation. Histone acetylation level was detected from hepatocyte nuclei by Western blotting, while microsomal CYP activity was examined by specific enzyme assays. Hyperacetylation of hepatic histone H2A at lysine 5 was observed after butyrate supplementation, providing modifications in the epigenetic regulation of cell function. No significant changes could be found in the acetylation state of the other core histones at the acetylation sites examined. Furthermore, butyrate did not cause any changes in the drugmetabolising activity of hepatic microsomal CYP2H and CYP3A37 enzymes, which are mainly involved in the biotransformation of most xenobiotics in chicken. These data indicate that supplementation of the diet with butyrate probably does not have any pharmacokinetic interactions with simultaneously applied xenobiotics.

  12. 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…

  13. Histone Acetylation and Its Modifiers in the Pathogenesis of Diabetic Nephropathy

    PubMed Central

    Li, Xiaoxia; Li, Chaoyuan

    2016-01-01

    Diabetic nephropathy (DN) remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors contributing to DN is required to develop more effective therapeutic options. It is becoming more evident that histone acetylation (HAc), as one of the epigenetic mechanisms, is thought to be associated with the etiology of diabetic vascular complications such as diabetic retinopathy (DR), diabetic cardiomyopathy (DCM), and DN. Histone acetylases (HATs) and histone deacetylases (HDACs) are the well-known regulators of reversible acetylation in the amino-terminal domains of histone and nonhistone proteins. In DN, however, the roles of histone acetylation (HAc) and these enzymes are still controversial. Some new evidence has revealed that HATs and HDACs inhibitors are renoprotective in cellular and animal models of DN, while, on the other hand, upregulation of HAc has been implicated in the pathogenesis of DN. In this review, we focus on the recent advances on the roles of HAc and their covalent enzymes in the development and progression of DN in certain cellular processes including fibrosis, inflammation, hypertrophy, and oxidative stress and discuss how targeting these enzymes and their inhibitors can ultimately lead to the therapeutic approaches for treating DN. PMID:27379253

  14. Histone Acetylation and Its Modifiers in the Pathogenesis of Diabetic Nephropathy.

    PubMed

    Li, Xiaoxia; Li, Chaoyuan; Sun, Guangdong

    2016-01-01

    Diabetic nephropathy (DN) remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors contributing to DN is required to develop more effective therapeutic options. It is becoming more evident that histone acetylation (HAc), as one of the epigenetic mechanisms, is thought to be associated with the etiology of diabetic vascular complications such as diabetic retinopathy (DR), diabetic cardiomyopathy (DCM), and DN. Histone acetylases (HATs) and histone deacetylases (HDACs) are the well-known regulators of reversible acetylation in the amino-terminal domains of histone and nonhistone proteins. In DN, however, the roles of histone acetylation (HAc) and these enzymes are still controversial. Some new evidence has revealed that HATs and HDACs inhibitors are renoprotective in cellular and animal models of DN, while, on the other hand, upregulation of HAc has been implicated in the pathogenesis of DN. In this review, we focus on the recent advances on the roles of HAc and their covalent enzymes in the development and progression of DN in certain cellular processes including fibrosis, inflammation, hypertrophy, and oxidative stress and discuss how targeting these enzymes and their inhibitors can ultimately lead to the therapeutic approaches for treating DN. PMID:27379253

  15. Effects of histone acetylation on chromatin topology in vivo.

    PubMed

    Lutter, L C; Judis, L; Paretti, R F

    1992-11-01

    Recently a model for eukaryotic transcriptional activation has been proposed in which histone hyperacetylation causes release of nucleosomal supercoils, and this unconstrained tension in turn stimulates transcription (V. G. Norton, B. S. Imai, P. Yau, and E. M. Bradbury, Cell 57:449-457, 1989; V. G. Norton, K. W. Marvin, P. Yau, and E. M. Bradbury, J. Biol. Chem. 265:19848-19852, 1990). These studies analyzed the effect of histone hyperacetylation on the change in topological linking number which occurs during nucleosome assembly in vitro. We have tested this model by determining the effect of histone hyperacetylation on the linking number change which occurs during assembly in vivo. We find that butyrate treatment of cells infected with simian virus 40 results in hyperacetylation of the histones of the extracted viral minichromosome as expected. However, the change in constrained supercoils of the minichromosome DNA is minimal, a result which is inconsistent with the proposed model. These results indicate that the proposed mechanism of transcriptional activation is unlikely to take place in the cell.

  16. Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab, Eriocheir sinensis

    PubMed Central

    Wu, Jiang-Li; Kang, Xian-Jiang; Guo, Ming-Shen; Mu, Shu-Mei; Zhang, Zhao-Hui

    2015-01-01

    During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin. PMID:25993499

  17. The histone H4 Lys 20 methyltransferase PR-Set7 regulates replication origins in mammalian cells.

    PubMed

    Tardat, Mathieu; Brustel, Julien; Kirsh, Olivier; Lefevbre, Christine; Callanan, Mary; Sardet, Claude; Julien, Eric

    2010-11-01

    The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4-Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insensitive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins.

  18. HISTONE DEACETYLASE6-Defective Mutants Show Increased Expression and Acetylation of ENHANCER OF TRIPTYCHON AND CAPRICE1 and GLABRA2 with Small But Significant Effects on Root Epidermis Cellular Pattern1

    PubMed Central

    Li, Dong-Xu; Chen, Wen-Qian; Xu, Zhi-Hong; Bai, Shu-Nong

    2015-01-01

    Cellular patterning in the Arabidopsis (Arabidopsis thaliana) root epidermis is dependent on positional information, the transmission of which involves histone acetylation. Here, we report that HISTONE DEACETYLASE6 (HDA6) has significant effects on this cellular patterning. Mutation of HDA6 led to ectopic hair cells in the nonhair positions of root epidermis in Arabidopsis, based on an analysis of paraffin sections stained with Toluidine Blue. While HDA6 was present throughout the root tip, epidermis-specific complementation with HDA6 could rescue the hda6 phenotype. Both transcript levels and expression patterns of ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) and GLABRA2 (GL2) in the root tip were affected in hda6. Consistent with these changes in expression, HDA6 directly bound to the promoter regions of ETC1 and GL2, and acetylation of histone H3 on these promoter regions and acetylation of histone H4 on the ETC1 promoter region was increased in the hda6 mutant. Taken together, these results indicate that HDA6 affects the cellular patterning of Arabidopsis root epidermis through altering the histone acetylation status of ETC1 and GL2 promoters and thereby affects the expression of these two components of the core transcription factor network determining epidermal cell fates. Our findings thus provide new insights into the role of histone acetylation in root epidermis cell patterning. PMID:26143251

  19. Transcriptional activation of the enterocyte differentiation marker intestinal alkaline phosphatase is associated with changes in the acetylation state of histone H3 at a specific site within its promoter region in vitro.

    PubMed

    Hinnebusch, Brian F; Henderson, J Welles; Siddique, Aleem; Malo, Madhu S; Zhang, Wenying; Abedrapo, Mario A; Hodin, Richard A

    2003-02-01

    expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between -1378 and -1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program.

  20. 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.

  1. 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

  2. Histone acetylation is involved in TCDD‑induced cleft palate formation in fetal mice.

    PubMed

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

    2016-08-01

    The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8‑tetrachlorodibenzo‑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

  3. 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.

  4. 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

  5. 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.

  6. Curcumin-induced histone acetylation inhibition improves stress-induced gastric ulcer disease in rats.

    PubMed

    He, Ping; Zhou, Renmin; Hu, Guorui; Liu, Zhifeng; Jin, Yu; Yang, Guang; Li, Mei; Lin, Qian

    2015-03-01

    Curcumin is known to possess anti‑inflammatory properties. Despite the fact that curcumin is known to be a strong inhibitor of H+, K+‑ATPase activity, the mechanism underlying the curcumin‑induced inhibition of the transcription of the H+, K+‑ATPase α subunit in gastric mucosal parietal cells remains unclear. The present study investigated the possible mechanism by which curcumin inhibits stomach H+, K+‑ATPase activity during the acute phase of gastric ulcer disease. A rat model of stress‑induced gastric ulcers was produced, in which the anti‑ulcer effects of curcumin were examined. Curcumin‑induced inhibition of the H+, K+‑ATPase promoter via histone acetylation, was verified using a chromatin immunoprecipitation assay. The results showed that curcumin improved stress‑induced gastric ulcer disease in rats, as demonstrated by increased pH values and reduced gastric mucosal hemorrhage and ulcer index. These effects were accompanied by a significant reduction in the level of histone H3 acetylation at the site of the H+, K+‑ATPase promoter and in the expression of the gastric H+,K+‑ATPase α subunit gene and protein. In conclusion, curcumin downregulated the acetylation of histone H3 at the site of the H+, K+‑ATPase promoter gene, thereby inhibiting the transcription and expression of the H+, K+‑ATPase gene. Curcumin was shown to have a preventive and therapeutic effect in gastric ulcer disease.

  7. A nucleosome-like structure containing DNA and the arginine-rich histones H3 and H4.

    PubMed

    Moss, T; Stephens, R M; Crane-Robinson, C; Bradbury, E M

    1977-07-01

    The low-angle X-ray diffraction pattern from fibres of reconstituted H3/H4/DNA complexes is very similar to that of chromatin and has well defined maxima at 10.6, 5.4, 3.4 and 2.6 nm. Staphyloccal nuclease digestion of reconstituted H3/H4/DNA yields DNA fragments of length 49, 69, 100, 128, 193 and 255 b.p. as principal components. Comparison of the relative amounts of DNA fragments shows that the larger components (100 and 128 b.p.) increase with respect to the smaller (49 and 69 b.p.) as the histone to DNA ratio increases. A structural unit containing intergral of 65 b.p. of DNA and tetrameric (H3/H4)2 is proposed such that longer DNA fragments result from multiples of this unit. The principal nucleo-protein particle resulting from nuclease digestion contains 128/139 b.p. of DNA and has electrophoretic mobility very close to that of 'core' nucleosome. It probably represents a dimer of the basic structural unit.

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

    PubMed

    Barber, Cynthia M; Turner, Fiona B; Wang, Yanming; Hagstrom, Kirsten; Taverna, Sean D; Mollah, Sahana; Ueberheide, Beatrix; Meyer, Barbara J; Hunt, Donald F; Cheung, Peter; Allis, C David

    2004-05-01

    Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase. PMID:15133681

  9. The histone H3 lysine 56 acetylation pathway is regulated by target of rapamycin (TOR) signaling and functions directly in ribosomal RNA biogenesis.

    PubMed

    Chen, Hongfeng; Fan, Meiyun; Pfeffer, Lawrence M; Laribee, R Nicholas

    2012-08-01

    Epigenetic changes in chromatin through histone post-translational modifications are essential for altering gene transcription in response to environmental cues. How histone modifications are regulated by environmental stimuli remains poorly understood yet this process is critical for delineating how epigenetic pathways are influenced by the cellular environment. We have used the target of rapamycin (TOR) pathway, which transmits environmental nutrient signals to control cell growth, as a model to delineate mechanisms underlying this phenomenon. A chemical genomics screen using the TOR inhibitor rapamycin against a histone H3/H4 mutant library identified histone H3 lysine 56 acetylation (H3K56ac) as a chromatin modification regulated by TOR signaling. We demonstrate this acetylation pathway functions in TOR-dependent cell growth in part by contributing directly to ribosomal RNA (rRNA) biogenesis. Specifically, H3K56ac creates a chromatin environment permissive to RNA polymerase I transcription and nascent rRNA processing by regulating binding of the high mobility group protein Hmo1 and the small ribosomal subunit (SSU) processome complex. Overall, these studies identify a novel chromatin regulatory role for TOR signaling and support a specific function for H3K56ac in ribosomal DNA (rDNA) gene transcription and nascent rRNA processing essential for cell growth.

  10. 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

  11. 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

  12. 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. PMID:23790014

  13. 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.

  14. Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts.

    PubMed

    Wada, Takuma Tsuzuki; Araki, Yasuto; Sato, Kojiro; Aizaki, Yoshimi; Yokota, Kazuhiro; Kim, Yoon Taek; Oda, Hiromi; Kurokawa, Riki; Mimura, Toshihide

    2014-02-21

    Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA. PMID:24513290

  15. Jejunal induction of SI and SGLT1 genes in rats by high-starch/low-fat diet is associated with histone acetylation and binding of GCN5 on the genes.

    PubMed

    Inoue, Seiya; Mochizuki, Kazuki; Goda, Toshinao

    2011-01-01

    The intestinal expression of genes involved in carbohydrate digestion and absorption, such as sucrase-isomaltase (SI) and sodium-dependent glucose cotransporter (SGLT1), is higher in rodents fed a high-starch/low-fat (HS) diet than in those fed a low-starch/high-fat (LS) diet. In the present study, we investigated whether the HS diet-induced induction of SI and SGLT1 in the rat jejunum is coordinately regulated by nuclear transcription factors, histone acetylation, or histone acetyltransferases. HS diet intake induced jejunal expression of a histone acetyltransferase, general control of amino acid synthesis (GCN5), concurrently with the SI and SGLT1 genes; however, gene expression of nuclear transcription factors such as hepatocyte nuclear factor-1, caudal type homeobox-2, and GATA-binding protein-4 was unaffected by the HS diet. Acetylation of histones H3/H4 and binding of acetyltransferase GCN5 on the promoter/enhancer and transcribed regions of SI and SGLT1 genes were significantly higher in HS diet-fed rats than in LS diet-fed rats, but transcription factor binding was not affected by the HS diet. Our results suggest that the concomitant induction of SI and SGLT1 genes in the jejunum by the HS diet is closely associated with the binding of GCN5 and acetylation of histones H3/H4 on these genes.

  16. Histone H3 Acetylation and H3 K4 Methylation Define Distinct Chromatin Regions Permissive for Transgene Expression

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

    Histone modifications are associated with distinct transcription states and serve as heritable epigenetic markers for chromatin structure and function. While H3 K9 methylation defines condensed heterochromatin that is able to silence a nearby gene, how gene silencing within euchromatin regions is achieved remains elusive. We report here that histone H3 K4 methylation or K9/K14 acetylation defines distinct chromatin regions permissive or nonpermissive for transgene expression. A permissive chromatin region is enriched in H3 K4 methylation and H3 acetylation, while a nonpermissive region is poor in or depleted of these two histone modifications. The histone modification states of the permissive chromatin can spread to transgenic promoters. However, de novo histone H3 acetylation and H3 K4 methylation at a transgenic promoter in a nonpermissive chromatin region are stochastic, leading to variegated transgene expression. Moreover, nonpermissive chromatin progressively silences a transgene, an event that is accompanied by the reduction of H3 K4 methylation and H3 acetylation levels at the transgenic promoter. These repressive effects of nonpermissive chromatin cannot be completely countered by strong transcription activators, indicating the dominance of the chromatin effects. We therefore propose a model in which histone H3 acetylation and H3 K4 methylation localized to discrete sites in the mammalian genome mark distinct chromatin functions that dictate transgene expression or silencing. PMID:16914722

  17. 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

  18. Piccolo NuA4-catalyzed acetylation of nucleosomal histones: critical roles of an Esa1 Tudor/chromo barrel loop and an Epl1 enhancer of polycomb A (EPcA) basic region.

    PubMed

    Huang, Jiehuan; Tan, Song

    2013-01-01

    Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously determined that the Tudor domain of Esa1 and the EPcA homology domain of Epl1 play critical roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA.

  19. Piccolo NuA4-Catalyzed Acetylation of Nucleosomal Histones: Critical Roles of an Esa1 Tudor/Chromo Barrel Loop and an Epl1 Enhancer of Polycomb A (EPcA) Basic Region

    PubMed Central

    Huang, Jiehuan

    2013-01-01

    Piccolo NuA4 is an essential yeast histone acetyltransferase (HAT) complex that targets histones H4 and H2A in nucleosome substrates. While Piccolo NuA4's catalytic subunit Esa1 alone is unable to acetylate nucleosomal histones, its accessory subunits, Yng2 and Epl1, enable Esa1 to bind to and to act on nucleosomes. We previously determined that the Tudor domain of Esa1 and the EPcA homology domain of Epl1 play critical roles in Piccolo NuA4's ability to act on the nucleosome. In this work, we pinpoint a loop within the Esa1 Tudor domain and a short basic region at the N terminus of the Epl1 EPcA domain as necessary for this nucleosomal HAT activity. We also show that this Esa1 Tudor domain loop region is positioned close to nucleosomal DNA and that the Epl1 EPcA basic region is in proximity to the N-terminal histone H2A tail, the globular region of histone H4, and also to nucleosomal DNA when Piccolo NuA4 interacts with the nucleosome. Since neither region identified is required for Piccolo NuA4 to bind to nucleosomes and yet both are needed to acetylate nucleosomes, these regions may function after the enzyme binds nucleosomes to disengage substrate histone tails from nucleosomal DNA. PMID:23109429

  20. 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

  1. Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila

    PubMed Central

    Li, Yulong; Armstrong, Robin L.; Duronio, Robert J.; MacAlpine, David M.

    2016-01-01

    The methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7. PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which has been partially attributed to defects in origin selection and activation. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. We find that deregulation of H4K20 methylation had no impact on origin activation throughout the genome. Instead, depletion of PR-Set7 and loss of H4K20me1 results in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. Coincident with the ATR-dependent cell cycle arrest, we find increased DNA damage that is specifically limited to late replicating regions of the Drosophila genome, suggesting that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains. PMID:27131378

  2. ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation.

    PubMed

    Clarke, Christopher J; Shamseddine, Achraf A; Jacob, Joseph J; Khalife, Gabrielle; Burns, Tara A; Hannun, Yusuf A

    2016-05-01

    Neutral sphingomyelinase-2 (nSMase2) is a key ceramide-producing enzyme in cellular stress responses. While many posttranslational regulators of nSMase2 are known, emerging evidence suggests a more protracted regulation of nSMase2 at the transcriptional level. Previously, we reported that nSMase2 is induced by all-trans retinoic acid (ATRA) in MCF7 cells and implicated nSMase2 in ATRA-induced growth arrest. Here, we further investigated how ATRA regulates nSMase2. We find that ATRA regulates nSMase2 transcriptionally through the retinoic acid receptor-α, but this is independent of previously identified transcriptional regulators of nSMase2 (Sp1, Sp3, Runx2) and is not through increased promoter activity. Epigenetically, the nSMase2 gene is not repressively methylated in MCF7 cells. However, inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) induced nSMase2 comparably to ATRA; furthermore, combined ATRA and TSA treatment was not additive, suggesting ATRA regulates nSMase2 through direct modulation of histone acetylation. Confirming this, the histone acetyltransferases CREB-binding protein and p300 were required for ATRA induction of nSMase2. Finally, use of class-specific HDAC inhibitors suggested that HDAC4 and/or HDAC5 are negative regulators of nSMase2 expression. Collectively, these results identify a novel pathway of nSMase2 regulation and suggest that physiological or pharmacological modulation of histone acetylation can directly affect nSMase2 levels. PMID:27013100

  3. 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

  4. 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

  5. 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. PMID:26914410

  6. Barcelona conference on epigenetics and cancer: 50 years of histone acetylation

    PubMed Central

    Perez-Salvia, Montserrat; Simó-Riudalbas, Laia; Ausió, Juan; Esteller, Manel

    2015-01-01

    The Barcelona Conference on Epigenetics and Cancer (BCEC) was held in Barcelona, Spain, on October 1st and 2nd, 2014. The meeting was co-organized by the Cancer Epigenetics and Biology Program (PEBC-IDIBELL) and B·Debate, an initiative of Biocat, with the support of "la Caixa" Foundation. The scientific committee was comprised of leading scientists in the field of epigenetics: Dr. Manel Esteller, director of PEBC-IDIBELL, Dr. Alejandro Vaquero and Dr. Esteban Ballestar, from PEBC-IDIBELL, Juan Ausió from the University of Victoria (Canada), and Marcus Buschbeck, from the Institute of Predictive and Personalized Medicine of Cancer (IMPPC), as BCEC series coordinator. This meeting was the second edition of the BCEC series, which was launched by 5 leading Barcelonan institutes to bring together leading investigators in the fields of epigenetics and chromatin research. The topics discussed during the meeting included the current challenges, opportunities, and perspectives surrounding the study of histone modifications (focusing in acetylation), chromatin structure and gene expression, and the involvement of histone acetylation in physiology and diseases, such as cancer or neurological diseases. PMID:25942103

  7. Rewiring AMPK and mitochondrial retrograde signaling for metabolic control of aging and histone acetylation in respiratory-defective cells.

    PubMed

    Friis, R Magnus N; Glaves, John Paul; Huan, Tao; Li, Liang; Sykes, Brian D; Schultz, Michael C

    2014-04-24

    Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG) response and the AMPK (Snf1) pathway prevents abnormal histone deacetylation in ρ(0) cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ(0) cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ(0) cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.

  8. The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

    PubMed Central

    Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

    2013-01-01

    HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

  9. 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.

  10. A mutational mimic analysis of histone H3 post-translational modifications: specific sites influence the conformational state of H3/H4, causing either positive or negative supercoiling of DNA.

    PubMed

    White, Rachel H; Keberlein, Melissa; Jackson, Vaughn

    2012-10-16

    Histone H3 has specific sites of post-translational modifications that serve as epigenetic signals to cellular machinery to direct various processes. Mutational mimics of these modifications (glutamine for acetylation, methionine and leucine for methylation, and glutamic acid for phosphorylation) were constructed at the relevant sites of the major histone variant, H3.2, and their effects on the conformational equilibrium of the H3/H4 tetramer at physiological ionic strength were determined when bound to or free of DNA. The deposition vehicle used for this analysis was NAP1, nucleosome assembly protein 1. Acetylation mimics in the N-terminus preferentially stabilized the left-handed conformer (DNA negatively supercoiled), and mutations within the globular region preferred the right-handed conformer (DNA positively supercoiled). The methylation mimics in the N-terminus tended to maintain characteristics similar to those of wild-type H3/H4; i.e., the conformational equilibrium maintains similar levels of both left- and right-handed conformers. Phosphorylation mimics facilitated a mixed effect, i.e., when at serines, the left-handed conformer, and at threonines, a mixture of both conformers. When double mutations were present, the conformational equilibrium was shifted dramatically, either leftward or rightward depending on the specific sites. In contrast, these mutations tended not to affect the direction and extent of supercoiling for variants H3.1 and H3.3. Variant H3.3 promoted only the left-handed conformer, and H3.1 tended to maintain both conformers. Additional experiments indicate the importance of a propagation mechanism for ensuring the formation of a particular superhelical state over an extended region of the DNA. The potential relevance of these results to the maintenance of epigenetic information on a gene is discussed.

  11. A mutational mimic analysis of histone H3 post-translational modifications: specific sites influence the conformational state of H3/H4, causing either positive or negative supercoiling of DNA.

    PubMed

    White, Rachel H; Keberlein, Melissa; Jackson, Vaughn

    2012-10-16

    Histone H3 has specific sites of post-translational modifications that serve as epigenetic signals to cellular machinery to direct various processes. Mutational mimics of these modifications (glutamine for acetylation, methionine and leucine for methylation, and glutamic acid for phosphorylation) were constructed at the relevant sites of the major histone variant, H3.2, and their effects on the conformational equilibrium of the H3/H4 tetramer at physiological ionic strength were determined when bound to or free of DNA. The deposition vehicle used for this analysis was NAP1, nucleosome assembly protein 1. Acetylation mimics in the N-terminus preferentially stabilized the left-handed conformer (DNA negatively supercoiled), and mutations within the globular region preferred the right-handed conformer (DNA positively supercoiled). The methylation mimics in the N-terminus tended to maintain characteristics similar to those of wild-type H3/H4; i.e., the conformational equilibrium maintains similar levels of both left- and right-handed conformers. Phosphorylation mimics facilitated a mixed effect, i.e., when at serines, the left-handed conformer, and at threonines, a mixture of both conformers. When double mutations were present, the conformational equilibrium was shifted dramatically, either leftward or rightward depending on the specific sites. In contrast, these mutations tended not to affect the direction and extent of supercoiling for variants H3.1 and H3.3. Variant H3.3 promoted only the left-handed conformer, and H3.1 tended to maintain both conformers. Additional experiments indicate the importance of a propagation mechanism for ensuring the formation of a particular superhelical state over an extended region of the DNA. The potential relevance of these results to the maintenance of epigenetic information on a gene is discussed. PMID:23003102

  12. 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. PMID:26943799

  13. 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.

  14. 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.

  15. Inhibition of different histone acetyltransferases (HATs) uncovers transcription-dependent and -independent acetylation-mediated mechanisms in memory formation.

    PubMed

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

    2016-02-01

    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 garcinol, an inhibitor of the HATs of the p300 (EP300 binding protein)/CBP (CREB-binding protein) family, and the HATs of the PCAF (p300/CBP-associated factor) family. As comparative agent we applied C646, a specific inhibitor that selectively blocks HATS of the p300/CBP family. Immunochemical analysis reveals differences in histone H3 acetylation in the honeybee brain, in response to the injection of either C646 or garcinol. Behavioral assessment reveals that the two drugs cause memory impairment of different nature when injected after associative conditioning: processes disturbed by garcinol are annihilated by the established transcription blocker actinomycin D and thus seem to require transcription processes. Actions of C646 are unaltered by actinomycin D, and thus seem to be independent of transcription. The outcome of our different approaches as summarized suggests that distinct HATs contribute to different acetylation-mediated processes in memory formation. We further deduce that the acetylation-mediated processes in memory formation comprise transcription-dependent and transcription-independent mechanisms.

  16. Increasing histone acetylation of cloned embryos, but not donor cells, by sodium butyrate improves their in vitro development in pigs.

    PubMed

    Das, Ziban Chandra; Gupta, Mukesh Kumar; Uhm, Sang Jun; Lee, Hoon Taek

    2010-02-01

    Previous studies have demonstrated that increased histone acetylation in donor cells or cloned embryos, by applying a histone deacetylase inhibitor (HDACi) such as trichostatin A (TSA), significantly enhances their developmental competence. However, its effect may vary with the type of HDACi and the target species, with some research showing nonsignificant or detrimental effects of TSA on in vitro and in vivo development of embryos. In this study, we show that sodium salt of butyric acid, a short-chain fatty acid produced naturally in the body by bacterial degradation of dietary fibers in the colon and rectum, increases histone acetylation in pig fibroblast and embryos at a concentration of 1.0 and 5.0 mM, respectively. However, treatment of donor cells with NaBu did not affect the rate of blastocyst formation or embryo quality in terms of histone acetylation and total nuclei per blastocyst (p > 0.05). On the contrary, treatment of cloned pig embryos with NaBu for 4 h significantly enhanced (p < 0.01) the rate of blastocyst formation (18.3 +/- 2.1 vs. 11.2 +/- 3.0%), although the total nuclei number per blastocyst did not differ. More importantly, blastocysts generated from NaBu-treated cloned embryos had increased levels of histone acetylation that was comparable to those of in vitro fertilized (IVF) embryos (36.7 +/- 3.6 vs. 45.9 +/- 2.5). In conclusion, our data suggest that histone hyperacetylation by NaBu treatment of cloned embryos, but not donor cell, enhances their in vitro development up to blastocyst stage.

  17. Histone acetyltransferase mediated regulation of FOXP3 acetylation and Treg function

    PubMed Central

    Xiao, Yan; Li, Bin; Zhou, Zhaocai; Hancock, Wayne W.; Zhang, Hongtao; Greene, Mark I.

    2010-01-01

    Regulatory T cells (Tregs) are required for the maintenance of immune homeostasis as first clearly described by Herman Waldmann’s laboratory. Dysfunction of Treg cells also leads to fatal autoimmunity in humans and mice. Conversely, the activation of different classes of Tregs operative systemically and within the cancer microenvironment can suppress host anti-tumor immune responses and promote tumor progression. Therefore, the development of new therapeutic approaches to regulate the activity of Treg cells may have considerable clinical potential. FOXP3 is the key transcriptional regulator of Treg development and function. The activity of FOXP3 is regulated by acetylation, a process catalyzed by distinct types of histone/protein acetyltransferases (HATs) that regulate the functions of many transcription factors, independently of FOXP3, as well as non-histone proteins, in addition to their effects on chromatin accessibility. Interactions between FOXP3 and these enzymes determine the suppressive function of FOXP3. Clearly, small molecules targeting these enzymes are candidates for the regulation of Treg function in vaccines and tumor therapies. PMID:20869864

  18. Histone genes in three sea star species: cluster arrangement, transcriptional polarity, and analyses of the flanking regions of H3 and H4 genes.

    PubMed

    Cool, D; Banfield, D; Honda, B M; Smith, M J

    1988-01-01

    The arrangement of core histone genes and their transcriptional polarity has been determined for three species of sea stars (Pisaster ochraceus, P. brevispinus, and Dermasterias imbricata) representing two orders which diverged over 500 million years ago. Each species has approximately 500 core histones cluster repeats per haploid genome. The close phylogenetic relationship between the Pisaster species is evident from the correspondence of restriction sites in the repeat element, identical arrangement of core histones, and high degree of sequence homology in both the coding and spacer regions of the H3 gene. The Dermasterias repeat has the same gene order and transcriptional polarity of core histones, but its restriction map is significantly different. Moreover, the Dermasterias H3 gene has the same amino acid sequence, but in comparison to Pisaster nucleotide sequences, shows a high level of silent substitutions. Analyses of the nucleotide sequence of the 5' and 3' regions surrounding the H3 gene from each species demonstrate the presence of appropriately spaced consensus and processing signal segments. The 3' spacer segment of the Dermasterias H4 gene contains an unusual, threefold tandemly repeated, 21-nucleotide, AT-rich sequence. No similar sequence is seen in the P. brevispinus H4 3' region, but these two species show a striking regularity of distribution of five different homologous oligomers in the 3' spacer.

  19. Potential role of PCTAIRE-2, PCTAIRE-3 and P-Histone H4 in amyloid precursor protein-dependent Alzheimer pathology

    PubMed Central

    Chaput, Dale; Kirouac, Lisa; Stevens, Stanley M.; Padmanabhan, Jaya

    2016-01-01

    Amyloid Precursor Protein (APP) is regulated in a mitosis-specific manner and plays a role in proliferative signaling in cells. Though APP-derived Aβ generation has a well-established role in neurodegeneration, the mechanistic role of APP in this process is not fully understood. Here, we performed an unbiased, comprehensive analysis of the phosphoproteome signature in APP-null neuroblastoma cells (B103) compared to those expressing APP-695 isoform (B103-695) to determine if APP expression affects protein phosphorylation. Stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry-based phosphoproteomic analysis with PolyMAC identified a total of 2,478 phosphopeptides in the B103 and B103-695 cell culture model system. We observed that phosphorylation of PCTAIRE-2 (CDK17), PCTAIRE-3 (CDK18), and Histone H4 are significantly elevated in B103-695 cells; western blot analysis confirmed overexpression of PCTAIREs and increased phosphorylation of Histone H4. More importantly, analysis of primary neurons treated with Aβ, as well as brain samples from MCI (mild cognitive impaired) and AD patients recapitulated these results, showing increased levels of PCTAIREs and P-Histone H4. These novel findings identify a hitherto uncharacterized mechanism by which APP and/or Aβ may promote AD neurodegeneration, and raises the possibility that their inhibition may protect against pathology development in AD. PMID:26885753

  20. 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

  1. ChIp-seq of bovine cells (MDBK) to study butyrate-induced histone modification with 10 datasets

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Next-generation sequencing was combined with chromatin immunoprecipitation (ChIP) technology to analyze histone modification (acetylation) induced by butyrate and to map the epigenomic landscape of normal histone H3, H4 in rumen cells of the cow. Ten variants of histone H3 and H4 modification were m...

  2. Histone acetylation may suppress human glioma cell proliferation when p21 WAF/Cip1 and gelsolin are induced.

    PubMed Central

    Kamitani, Hideki; Taniura, Seijiro; Watanabe, Kenji; Sakamoto, Makoto; Watanabe, Takashi; Eling, Thomas

    2002-01-01

    Histone deacetylase inhibitors that increase histone acetylation on transformed cells are being investigated as unique anticancer drugs. The aim of this investigation was to evaluate an antiproliferative activity of the histone deacetylase inhibitors sodium butyrate (NaBT) and trichostatin A on 5 glioma cell lines, T98G, A172, U-87 MG, U-118 MG, and U-373 MG, with the examination of the altered expressions in p21 and gelsolin genes. Treatment with 5-mM NaBT and 40 ng/ml trichostatin A for 48 h caused more than a 50% growth inhibition in 5 cell lines as measured by cell proliferation assays. An increase in histone acetylation was confirmed in each cell line. After treatment with 5 mM NaBT, T98G, A172, and U118 cells undergo apoptosis as indicated by DNA ladder formation. Treatment with NaBT and trichostatin A also decreased DNA synthesis as examined by the fluorescence-activated cell sorting analysis in T98G and U87 cells. In addition to the suppression of cell growth, the up regulation of p21 and gelsolin expression was observed after treatment with NaBT, especially in T98G cells. Maximum expression of p21 and gelsolin was observed within 24 h after treatment. Results from our in vitro studies indicate that the treatment of human glioma cells with one of the histone deacetylase inhibitors suppresses cell growth with decreasing DNA synthesis and stimulates apoptosis, and that associated molecular mechanisms responsible for these effects include increased histone acetylation as well as enhanced expression of p21 and gelsolin. PMID:11916500

  3. Nucleosome free region dominates histone acetylation in targeting SWR1 to yeast promoters for H2A.Z replacement

    PubMed Central

    Ranjan, Anand; Mizuguchi, Gaku; FitzGerald, Peter C.; Wei, Debbie; Wang, Feng; Huang, Yingzi; Luk, Ed; Woodcock, Christopher L; Wu, Carl

    2013-01-01

    Summary The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. While the multi-subunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S. cerevisiae promoters has been unclear. A sensitive assay for competitive binding of di-nucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the ATPase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and post-translational histone modifications. PMID:24034247

  4. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. PMID:27382434

  5. 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. PMID:21825172

  6. 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

  7. CXCL8 histone H3 acetylation is dysfunctional in airway smooth muscle in asthma: regulation by BET.

    PubMed

    Clifford, Rachel L; Patel, Jamie K; John, Alison E; Tatler, Amanda L; Mazengarb, Lisa; Brightling, Christopher E; Knox, Alan J

    2015-05-01

    Asthma is characterized by airway inflammation and remodeling and CXCL8 is a CXC chemokine that drives steroid-resistant neutrophilic airway inflammation. We have shown that airway smooth muscle (ASM) cells isolated from asthmatic individuals secrete more CXCL8 than cells from nonasthmatic individuals. Here we investigated chromatin modifications at the CXCL8 promoter in ASM cells from nonasthmatic and asthmatic donors to further understand how CXCL8 is dysregulated in asthma. ASM cells from asthmatic donors had increased histone H3 acetylation, specifically histone H3K18 acetylation, and increased binding of histone acetyltransferase p300 compared with nonasthmatic donors but no differences in CXCL8 DNA methylation. The acetylation reader proteins Brd3 and Brd4 were bound to the CXCL8 promoter and Brd inhibitors inhibited CXCL8 secretion from ASM cells by disrupting Brd4 and RNA polymerase II binding to the CXCL8 promoter. Our results show a novel dysregulation of CXCL8 transcriptional regulation in asthma characterized by a promoter complex that is abnormal in ASM cells isolated from asthmatic donors and can be modulated by Brd inhibitors. Brd inhibitors may provide a new therapeutic strategy for steroid-resistant inflammation.

  8. 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.

  9. Neutron scattering studies of the H2a-H2b and (H3-H4)/sub 2/ histone complexes

    SciTech Connect

    Carlson, R.D.

    1982-01-01

    Neutron scattering experiments have shown that both the (H3-H4)/sub 2/ and H2a-H2b histone complexes are quite asymmetric in solution. The (H3-H4)/sub 2/ tetramer is an oblate or flattened structure, with a radius of gyration almost as large as that of the core octamer. If the tetramer is primarily globular, it must have an axial ratio of about 1:5. It is more likely, however, that this asymmetry results in part from N-terminal arms that extend outward approximately within the major plane of the particle. If this is the case, less asymmetric models for the globular part of the tetramer, including a dislocated disk, can be made consistent with the scattering data. The H2a-H2b dimer, on the other hand, is an elongated structure. 48 references, 12 figures, 1 table.

  10. Immunohistochemical evaluation of global DNA methylation and histone acetylation in papillary urothelial neoplasm of low malignant potential.

    PubMed

    Barbisan, F; Mazzucchelli, R; Santinelli, A; Stramazzotti, D; Scarpelli, M; Lopez-Beltran, A; Cheng, L; Montironi, R

    2008-01-01

    A preceding study has shown that karyometry detected subvisual differences in chromatin organization status between non-recurrent and recurrent papillary urothelial neoplasm of low malignant potential (PUNLMP). The status of chromatin organization depends on epigenetic events, such as DNA methylation and histone acetylation. The aim of this study is to explore global DNA methylation and global histone acetylation in non-recurrent and recurrent PUNLMP. 5-methylcytosine (5MeC) and acetylated histone H3 lysine 9 (AcH3K9) were investigated by immunohistochemistry (IHC) in 20 PUNLMP cases (10 non-recurrent and 10 recurrent), in 5 cases of normal urothelium (NU) and in 5 cases of muscle invasive pT2 urothelial carcinoma (UC). For global DNA methylation, the mean percentage of positive nuclei in the cells adjacent to the stroma increased from NU (79%) through non-recurrent and recurrent PUNLMP (86% and 93%, respectively) to UC (97%). The percentages of positive nuclei in the intermediate cell layers and in the superficial cells in the four groups were similar to those adjacent to the stroma. The proportion of nuclei with weak-to-moderate intensity was far greater than that of those strongly stained and increased steadily from NU to UC. For global histone acetylation, the mean percentage of positive nuclei was highest in non-recurrent PUNLMP (i.e. 90%) and lowest in recurrent PUNLMP (i.e. 81%). In NU and UC the mean percentages of positive nuclei were 84% and 86%, respectively. The percentage of positive nuclei decreased from the cell layer adjacent to the stroma to the superficial cell layer. The proportion of nuclei with weak-to-moderate intensity was slightly greater than that of those strongly stained. In comparison with global DNA methylation, the proportion of strongly stained nuclei was much higher. In conclusion, there are differences in global DNA methylation and histone acetylation patterns between non-recurrent and recurrent PUNLMP. Further studies are needed to

  11. Postnatal Isoflurane Exposure Induces Cognitive Impairment and Abnormal Histone Acetylation of Glutamatergic Systems in the Hippocampus of Adolescent Rats.

    PubMed

    Liang, Bing; Fang, Jie

    2016-09-01

    Isoflurane can elicit cognitive impairment. However, the pathogenesis in the brain remains inconclusive. The present study investigated the mechanism of glutamate neurotoxicity in adolescent male rats that underwent postnatal isoflurane exposure and the role of sodium butyrate (NaB) in cognitive impairment induced by isoflurane exposure. Seven-day-old rats were exposed to 1.7 % isoflurane for 35 min every day for four consecutive days, and then glutamate neurotoxicity was examined in the hippocampus. Morris water maze analysis showed cognitive impairments in isoflurane-exposed rats. High-performance liquid chromatography found higher hippocampal glutamate concentrations following in vitro and in vivo isoflurane exposure. The percentage of early apoptotic hippocampal neurons was markedly increased after isoflurane exposure. Decreased acetylation and increased HDAC2 activity were observed in the hippocampus of isoflurane-exposed rats and hippocampal neurons. Furthermore, postnatal isoflurane exposure decreased histone acetylation of hippocampal neurons in the promoter regions of GLT-1 and mGLuR1/5, but not mGLuR2/3. Treatment with NaB not only restored the histone acetylation of the GLT-1 and mGLuR1/5 promoter regions and glutamate excitatory neurotoxicity in hippocampal neurons, but also improved cognitive impairment in vivo. Moreover, NaB may be a potential therapeutic drug for cognitive impairment caused by isoflurane exposure. These results suggest that postnatal isoflurane exposure contributes to cognitive impairment via decreasing histone acetylation of glutamatergic systems in the hippocampus of adolescent rats. PMID:27307148

  12. 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. PMID:26773543

  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. The Histone Demethylase UTX Promotes Brown Adipocyte Thermogenic Program Via Coordinated Regulation of H3K27 Demethylation and Acetylation.

    PubMed

    Zha, Lin; Li, Fenfen; Wu, Rui; Artinian, Liana; Rehder, Vincent; Yu, Liqing; Liang, Houjie; Xue, Bingzhong; Shi, Hang

    2015-10-01

    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.

  15. Relationships of new sibling species of Paramecium jenningsi based on sequences of the histone H4 gene fragment.

    PubMed

    Maciejewska, Agnieszka

    2007-06-01

    Paramecium jenningsi Diller & Earl, 1958 belongs to the "aurelia" subgroup of the genus, together with Paramecium caudatum, Paramecium multimicronucleatum, Paramecium schewiakoffi and species of the Paramecium aurelia complex. The original assumption that the morphospecies P. jenningsi was a single genetic species was questioned because a comparison of genome analyses suggested the possibility that this morphospecies contained two sibling species. To refine understanding of relationships between the strains of P. jenningsi, a molecular phylogenetic analysis was conducted using H4 gene sequences. Some polymorphic sites were found among the compared sequences, and specific patterns of single nucleotide polymorphism (SNP) markers characterize two groups of strains of P. jenningsi. Phylogenetic trees constructed by different methods identified two clearly different groups (from Japan and mainland Asia) whatever the method used. The sequences of the H4 gene analyzed in the present study are closely related, and provide a good subject for phylogenetic analysis. The presence of two isolated groups of strains in the P. jenningsi group can reveal the evolutionary relationship between them; it confirms the presence of two sibling species among the known strains of P. jenningsi, and the close relationships between them and species of the P. aurelia complex.

  16. Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.

    PubMed

    Corbet, Cyril; Pinto, Adán; Martherus, Ruben; Santiago de Jesus, João Pedro; Polet, Florence; Feron, Olivier

    2016-08-01

    Bioenergetic preferences of cancer cells foster tumor acidosis that in turn leads to dramatic reduction in glycolysis and glucose-derived acetyl-coenzyme A (acetyl-CoA). Here, we show that the main source of this critical two-carbon intermediate becomes fatty acid (FA) oxidation in acidic pH-adapted cancer cells. FA-derived acetyl-CoA not only fuels the tricarboxylic acid (TCA) cycle and supports tumor cell respiration under acidosis, but also contributes to non-enzymatic mitochondrial protein hyperacetylation, thereby restraining complex I activity and ROS production. Also, while oxidative metabolism of glutamine supports the canonical TCA cycle in acidic conditions, reductive carboxylation of glutamine-derived α-ketoglutarate sustains FA synthesis. Concomitance of FA oxidation and synthesis is enabled upon sirtuin-mediated histone deacetylation and consecutive downregulation of acetyl-CoA carboxylase ACC2 making mitochondrial fatty acyl-CoA degradation compatible with cytosolic lipogenesis. Perturbations of these regulatory processes lead to tumor growth inhibitory effects further identifying FA metabolism as a critical determinant of tumor cell proliferation under acidosis. PMID:27508876

  17. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification.

    PubMed

    Dreveny, Ingrid; Deeves, Sian E; Fulton, Joel; Yue, Baigong; Messmer, Marie; Bhattacharya, Amit; Collins, Hilary M; Heery, David M

    2014-01-01

    Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators.

  18. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification

    PubMed Central

    Dreveny, Ingrid; Deeves, Sian E.; Fulton, Joel; Yue, Baigong; Messmer, Marie; Bhattacharya, Amit; Collins, Hilary M.; Heery, David M.

    2014-01-01

    Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators. PMID:24150941

  19. Genome-wide alteration of histone H3K9 acetylation pattern in mouse offspring prenatally exposed to arsenic.

    PubMed

    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.

  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. Melatonin inhibits TPA-induced oral cancer cell migration by suppressing matrix metalloproteinase-9 activation through the histone acetylation.

    PubMed

    Yeh, Chia-Ming; Lin, Chiao-Wen; Yang, Jia-Sin; Yang, Wei-En; Su, Shih-Chi; Yang, Shun-Fa

    2016-04-19

    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

  3. Mixtures of SCFA, composed according to physiologically available concentrations in the gut lumen, modulate histone acetylation in human HT29 colon cancer cells.

    PubMed

    Kiefer, Jeannette; Beyer-Sehlmeyer, Gabriele; Pool-Zobel, Beatrice L

    2006-11-01

    Intake of fibre has beneficial properties on gut health. Butyrate, a product of bacterial gut fermentation, is thought to contribute to positive effects by retarding growth and enhancing apoptosis of tumour cells. One mechanism is seen in its capacity to modulate histone acetylation and thereby transcriptional activity of genes. Next to butyrate, propionate and acetate are also major products of gut fermentation and together they may exert different potencies of cellular effects than butyrate alone. Since virtually nothing is known on combination effects by SCFA mixtures, here we had the aim to assess how physiological relevant concentrations and mixtures of SCFA modulate histone acetylation in human colon cells. HT29 colon cancer cells were incubated with mixtures of butyrate, acetate and propionate and with the individual compounds as controls. Histone acetylation was determined with acid-urea gel electrophoresis and immunoblotting. Acetylated histones slowly increased over 24 h and persisted up to 72 h in butyrate-treated HT29 cells. Butyrate (5-40 mM) and propionate (20-40 mM) enhanced histone acetylation significantly after 24 h incubation, whereas acetate (2.5-80 mM) was ineffective. Mixtures of these SCFA also modulated histone acetylation, mainly due to additive effects of butyrate and propionate, but not due to acetate. In conclusion, physiological concentrations of propionate together with butyrate could have more profound biological activities than generally assumed. Together, these SCFA could possibly mediate important processes related to an altered transcriptional gene activation and thus contribute to biological effects possibly related to cancer progression or prevention.

  4. Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

    PubMed

    Burgos, Emmanuel S; Wilczek, Carola; Onikubo, Takashi; Bonanno, Jeffrey B; Jansong, Janina; Reimer, Ulf; Shechter, David

    2015-04-10

    The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.

  5. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    PubMed Central

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-01

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways. PMID:26784169

  6. Histone H3K9 acetylation level modulates gene expression and may affect parasite growth in human malaria parasite Plasmodium falciparum.

    PubMed

    Srivastava, Sandeep; Bhowmick, Krishanu; Chatterjee, Snehajyoti; Basha, Jeelan; Kundu, Tapas K; Dhar, Suman K

    2014-12-01

    Three-dimensional positioning of the nuclear genome plays an important role in the epigenetic regulation of genes. Although nucleographic domain compartmentalization in the regulation of epigenetic state and gene expression is well established in higher organisms, it remains poorly understood in the pathogenic parasite Plasmodium falciparum. In the present study, we report that two histone tail modifications, H3K9Ac and H3K14Ac, are differentially distributed in the parasite nucleus. We find colocalization of active gene promoters such as Tu1 (tubulin-1 expressed in the asexual stages) with H3K9Ac marks at the nuclear periphery. By contrast, asexual stage inactive gene promoters such as Pfg27 (gametocyte marker) and Pfs28 (ookinete marker) occupy H3K9Ac devoid zones at the nuclear periphery. The histone H3K9 is predominantly acetylated by the PCAF/GCN5 class of lysine acetyltransferases, which is well characterized in the parasite. Interestingly, embelin, a specific inhibitor of PCAF/GCN5 family histone acetyltransferase, selectively decreases total H3K9Ac acetylation levels (but not H3K14Ac levels) around the var gene promoters, leading to the downregulation of var gene expression, suggesting interplay among histone acetylation status, as well as subnuclear compartmentalization of different genes and their activation in the parasites. Finally, we found that embelin inhibited parasitic growth at the low micromolar range, raising the possibility of using histone acetyltransferases as a target for antimalarial therapy.

  7. 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.

  8. Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.

    PubMed

    Mikula, Michal; Majewska, Aneta; Ledwon, Joanna Karolina; Dzwonek, Artur; Ostrowski, Jerzy

    2014-12-01

    Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.

  9. GITR subverts Foxp3+ Tregs to boost Th9 immunity through regulation of histone acetylation

    PubMed Central

    Xiao, Xiang; Shi, Xiaomin; Fan, Yihui; Zhang, Xiaolong; Wu, Minhao; Lan, Peixiang; Minze, Laurie; Fu, Yang-Xin; Ghobrial, Rafik M.; Liu, Wentao; Li, Xian Chang

    2015-01-01

    Glucocorticoid-induced TNFR-related protein (GITR) is a costimulatory molecule with diverse effects on effector T cells and regulatory T cells (Tregs), but the underlying mechanism remains poorly defined. Here we demonstrate that GITR ligation subverts the induction of Foxp3+ Tregs and directs the activated CD4+ T cells to Th9 cells. Such GITR-mediated iTreg to Th9 induction enhances anti-tumour immunity in vivo. Mechanistically, GITR upregulates the NF-κB family member p50, which recruits histone deacetylases to the Foxp3 locus to produce a ‘closed' chromatin structure. Furthermore, GITR ligation also activates STAT6, and STAT6 renders Il9 locus accessible via recruitment of histone acetyltransferase p300, and together with inhibition of Foxp3, GITR induces strong Th9 responses. Thus, Th9 cells and iTregs are developmentally linked and GITR can subvert tolerogenic conditions to boost Th9 immunity. PMID:26365427

  10. Treadmill exercise alters histone acetylation differently in rats exposed or not exposed to aversive learning context.

    PubMed

    de Meireles, Louisiana Carolina Ferreira; Bertoldi, Karine; Elsner, Viviane Rostirola; Moysés, Felipe dos Santos; Siqueira, Ionara Rodrigues

    2014-12-01

    Epigenetic modifications have been linked to memory formation after learning context exposure and to exercise effects on memory performance. The aim of this study was to investigate the effect of treadmill exercise (20 min/day during 2 weeks) on H3K14 acetylation and H3S10 phosphorylation levels in the hippocampi of 3-month-old Wistar rats exposed and not exposed to aversive learning context. Male Wistar rats aged 2-3 months were assigned to non-exercised (sedentary) and exercised (running daily for 20 min for 2 weeks) groups. Single-trial step-down inhibitory avoidance (IA) conditioning was employed as an aversive memory model. Epigenetic parameters were determined 30 min after the IA test. A decrease in the H3K14 acetylation in the hippocampus 24 h after IA training (30 min after test session) was observed. Exercise reversed the IA effect, and no effect was observed in the non-IA exposed group. Our data support the hypothesis that modulation of H3K14 acetylation levels in the hippocampus might be related, at least in part, to exercise effects on aversive memory.

  11. Resetting the epigenetic histone code in the MRL-lpr/lpr mouse model of lupus by histone deacetylase inhibition.

    PubMed

    Garcia, Benjamin A; Busby, Scott A; Shabanowitz, Jeffrey; Hunt, Donald F; Mishra, Nilamadhab

    2005-01-01

    The baseline level of gene expression varies between healthy controls and systemic lupus erythematosus (SLE) patients, and among SLE patients themselves. These variations may explain the different clinical manifestations and severity of disease observed in SLE. Epigenetic mechanisms, which involve DNA and histone modifications, are predictably associated with distinct transcriptional states. To understand the interplay between various histone modifications, including acetylation and methylation, and lupus disease, we performed differential expression histone modification analysis in splenocytes from the MRL-lpr/lpr mouse model of lupus. Using stable isotope labeling in combination with mass spectrometry, we found global site-specific hypermethylation (except H3 K4 methylation) and hypoacetylation in histone H3 and H4 MRL-lpr/lpr mice compared to control MRL/MPJ mice. Moreover, we have identified novel histone modifications such as H3 K18 methylation, H4 K31 methylation, and H4 K31 acetylation that are differentially expressed in MRL-lpr/lpr mice compared to controls. Finally, in vivo administration of the histone deacetylase inhibitor trichostatin A (TSA) corrected the site-specific hypoacetylation states on H3 and H4 in MRL-lpr/lpr mice with improvement of disease phenotype. Thus, this study is the first to establish the association between aberrant histone codes and pathogenesis of autoimmune disease SLE. These aberrant post-translational histone modifications can therefore be reset with histone deacetylase inhibition in vivo.

  12. Treating Colon Cancer Cells with FK228 Reveals a Link between Histone Lysine Acetylation and Extensive Changes in the Cellular Proteome.

    PubMed

    Wang, Tian-yun; Jia, Yan-long; Zhang, Xi; Sun, Qiu-li; Li, Yi-chun; Zhang, Jun-he; Zhao, Chun-peng; Wang, Xiao-yin; Wang, Li

    2015-12-17

    The therapeutic value of FK228 as a cancer treatment option is well known, and various types of cancer have been shown to respond to this drug. However, the complete mechanism of FK228 and the affect it has on histone lysine acetylation and the colon cancer cell proteome are largely unknown. In the present study, we used stable isotope labeling by amino acids in cell culture (SILAC) and affinity enrichment followed by high-resolution liquid chromatograph-mass spectrometer (LC-MS)/MS analysis to quantitate the changes in the lysine acetylome in HCT-8 cells after FK228 treatment. A total of 1,194 lysine acetylation sites in 751 proteins were quantified, with 115 of the sites in 85 proteins being significantly upregulated and 38 of the sites in 32 proteins being significantly downregulated in response to FK228 treatment. Interestingly, 47 histone lysine acetylation sites were identified in the core histone proteins. We also found a novel lysine acetylation site on H2BK121. These significantly altered proteins are involved in multiple biological functions as well as a myriad of metabolic and enzyme-regulated pathways. Taken together, the link between FK228 function and the downstream changes in the HCT-8 cell proteome observed in response to FK228 treatment is established.

  13. Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin

    PubMed Central

    Robert, Carine; Nagaria, Pratik K.; Pawar, Nisha; Adewuyi, Adeoluwa; Gojo, Ivana; Meyers, David J.; Cole, Philip A.; Rassool, Feyruz V.

    2016-01-01

    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. PMID:27064363

  14. 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.

  15. 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. PMID:27064363

  16. Novel curcumin analog C66 prevents diabetic nephropathy via JNK pathway with the involvement of p300/CBP-mediated histone acetylation.

    PubMed

    Wang, Yangwei; Wang, Yonggang; Luo, Manyu; Wu, Hao; Kong, Lili; Xin, Ying; Cui, Wenpeng; Zhao, Yunjie; Wang, Jingying; Liang, Guang; Miao, Lining; Cai, Lu

    2015-01-01

    Glomerulosclerosis and interstitial fibrosis represent the key events in development of diabetic nephropathy (DN), with connective tissue growth factor (CTGF), plasminogen activator inhibitor-1 (PAI-1) and fibronectin 1 (FN-1) playing important roles in these pathogenic processes. To investigate whether the plant metabolite curcumin, which exerts epigenetic modulatory properties when applied as a pharmacological agent, may prevent DN via inhibition of the JNK pathway and epigenetic histone acetylation, diabetic and age-matched non-diabetic control mice were administered a 3-month course of curcumin analogue (C66), c-Jun N-terminal kinase inhibitor (JNKi, sp600125), or vehicle alone. At treatment end, half of the mice were sacrificed for analysis and the other half were maintained without treatment for an additional 3 months. Renal JNK phosphorylation was found to be significantly increased in the vehicle-treated diabetic mice, but not the C66- and JNKi-treated diabetic mice, at both the 3-month and 6-month time points. C66 and JNKi treatment also significantly prevented diabetes-induced renal fibrosis and dysfunction. Diabetes-related increases in histone acetylation, histone acetyl transferases' (HATs) activity, and the p300/CBP HAT expression were also significantly attenuated by C66 or JNKi treatment. Chromatin immunoprecipitation assays showed that C66 and JNKi treatments decreased H3-lysine9/14-acetylation (H3K9/14Ac) level and p300/CBP occupancy at the CTGF, PAI-1 and FN-1 gene promoters. Thus, C66 may significantly and persistently prevent renal injury and dysfunction in diabetic mice via down-regulation of diabetes-related JNK activation and consequent suppression of the diabetes-related increases in HAT activity, p300/CBP expression, and histone acetylation.

  17. 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.

  18. Spt6 prevents transcription-coupled loss of posttranslationally modified histone H3

    PubMed Central

    Kato, Hiroaki; Okazaki, Kosuke; Iida, Tetsushi; Nakayama, Jun-ichi; Murakami, Yota; Urano, Takeshi

    2013-01-01

    The tail of histone H3 is an ideal medium for storing epigenetic information because displacement of histone H3 is heavily restricted during transcription. To maintain the locus-specific modifications of histone H3, histone molecules should be retained locally at the original position through multiple rounds of transcription. Here, we found that fission yeast Spt6, a highly conserved RNA polymerase II-interacting histone H3–H4 chaperone, is essential for the maintenance of Lys-4 and Lys-9 methylation of histone H3 in euchromatin and heterochromatin, respectively. In euchromatin, loss of Lys-4 methylated histone H3 and deposition of newly synthesized Lys-56 acetylated histone H3 induced by Spt6 inactivation were coupled with transcription. While in heterochromatin, Spt6 prevents histone turnover and cryptic transcription in parallel with Clr3 histone deacetylase. We propose that Spt6 retains posttranslationally modified histone H3 during transcription to maintain epigenome integrity. PMID:23851719

  19. Photosynthetic Genes and Genes Associated with the C4 Trait in Maize Are Characterized by a Unique Class of Highly Regulated Histone Acetylation Peaks on Upstream Promoters.

    PubMed

    Perduns, Renke; Horst-Niessen, Ina; Peterhansel, Christoph

    2015-08-01

    Histone modifications contribute to gene regulation in eukaryotes. We analyzed genome-wide histone H3 Lysine (Lys) 4 trimethylation and histone H3 Lys 9 acetylation (two modifications typically associated with active genes) in meristematic cells at the base and expanded cells in the blade of the maize (Zea mays) leaf. These data were compared with transcript levels of associated genes. For individual genes, regulations (fold changes) of histone modifications and transcript levels were much better correlated than absolute intensities. When focusing on regulated histone modification sites, we identified highly regulated secondary H3 Lys 9 acetylation peaks on upstream promoters (regulated secondary upstream peaks [R-SUPs]) on 10% of all genes. R-SUPs were more often found on genes that were up-regulated toward the blade than on down-regulated genes and specifically, photosynthetic genes. Among those genes, we identified six genes encoding enzymes of the C4 cycle and a significant enrichment of genes associated with the C4 trait derived from transcriptomic studies. On the DNA level, R-SUPs are frequently associated with ethylene-responsive elements. Based on these data, we suggest coevolution of epigenetic promoter elements during the establishment of C4 photosynthesis.

  20. Sodium butyrate promotes the differentiation of rat bone marrow mesenchymal stem cells to smooth muscle cells through histone acetylation.

    PubMed

    Liu, Jingxia; Wang, Yanzhou; Wu, Yuzhang; Ni, Bing; Liang, Zhiqing

    2014-01-01

    Establishing an effective method to improve stem cell differentiation is crucial in stem cell transplantation. Here we aimed to explore whether and how sodium butyrate (NaB) induces rat bone marrow mesenchymal stem cells (MSCs) to differentiate into bladder smooth muscle cells (SMCs). We found that NaB significantly suppressed MSC proliferation and promoted MSCs differentiation into SMCs, as evidenced by the enhanced expression of SMC specific genes in the MSCs. Co-culturing the MSCs with SMCs in a transwell system promoted the differentiation of MSCs into SMCs. NaB again promoted MSC differentiation in this system. Furthermore, NaB enhanced the acetylation of SMC gene-associated H3K9 and H4, and decreased the expression of HDAC2 and down-regulated the recruitment of HDAC2 to the promoter regions of SMC specific genes. Finally, we found that NaB significantly promoted MSC depolarization and increased the intracellular calcium level of MSCs upon carbachol stimulation. These results demonstrated that NaB effectively promotes MSC differentiation into SMCs, possibly by the marked inhibition of HDAC2 expression and disassociation of HDAC2 recruitment to SMC specific genes in MSCs, which further induces high levels of H3K9ace and H4ace and the enhanced expression of target genes, and this strategy could potentially be applied in clinical tissue engineering and cell transplantation. PMID:25548915

  1. Superantigen-induced CD4+ T cell tolerance is associated with DNA methylation and histone hypo-acetylation at cytokine gene loci.

    PubMed

    Thomas, R M; Saouaf, S J; Wells, A D

    2007-10-01

    Anergy is an important mechanism of peripheral tolerance in which T cells lose the capacity to produce proinflammatory cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFNgamma). To determine whether the induction of T-cell anergy in vivo is associated with epigenetic changes that oppose cytokine gene expression, we measured DNA methylation and histone acetylation at the IL2 and IFNgamma loci in CD4+ T cells from mice tolerant to a viral superantigen. Tolerant T cells exhibited more DNA methylation and less histone acetylation at the regulatory regions of the IL2 and IFNgamma genes than effector T cells, which are able to produce IL-2 and IFNgamma. These data show that T-cell anergy in this model is associated with epigenetic modifications that oppose gene expression, and suggest that these mechanisms may be important in the maintenance of tolerance. PMID:17671507

  2. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer.

    PubMed

    Tang, Hui; Fang, Huasheng; Yin, Eric; Brasier, Allan R; Sowers, Lawrence C; Zhang, Kangling

    2014-06-01

    Histone acetylation and methylation play an important role in the regulation of gene expression. Irregular patterns of histone global acetylation and methylation have frequently been seen in various diseases. Quantitative analysis of these patterns is of high value for the evaluation of disease development and of outcomes from therapeutic treatment. Targeting histone acetylation and methylation by selected reaction monitoring (SRM) is one of the current quantitative methods. Here, we reported the use of the multiplexed parallel reaction monitoring (PRM) method on the QExactive mass spectrometer to target previously known lysine acetylation and methylation sites of histone H3 and H4 for the purpose of establishing precursor-product pairs for SRM. 55 modified peptides among which 29 were H3 K27/K36 modified peptides were detected from 24 targeted precursor ions included in the inclusion list. The identification was carried out directly from the trypsin digests of core histones that were separated without derivatization on a homemade capillary column packed with Waters YMC ODS-AQ reversed phase materials. Besides documenting the higher-energy c-trap dissociation (HCD) MS(2) spectra of previously known histone H3/H4 acetylated and methylated tryptic peptides, we identified novel H3 K18 methylation, H3 K27 monomethyl/acetyl duel modifications, H2B K23 acetylation, and H4 K20 acetylation in mammalian histones. The information gained from these experiments sets the foundation for quantification of histone modifications by targeted mass spectrometry methods directly from core histone samples. PMID:24823915

  3. Re-feeding rats a high-sucrose diet after 3 days of starvation enhances histone H3 acetylation in transcribed region and expression of jejunal GLUT5 gene.

    PubMed

    Honma, Kazue; Masuda, Yuriko; Mochizuki, Kazuki; Goda, Toshinao

    2014-01-01

    Fasting for 3 days leads to reduction in the expression of GLUT5 and SGLT1 genes in jejunum. Re-feeding a high-sucrose diet in fasted rats enhanced mRNA levels and histone H3 acetylation on transcribed region of GLUT5 gene within 24 h, but not in SGLT1. Responsiveness of jejunal GLUT5 gene is associated with changes in histone H3 acetylation on transcribed region.

  4. The Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex requires the Enhancer of Polycomb A domain and chromodomain to acetylate nucleosomes.

    PubMed

    Selleck, William; Fortin, Israël; Sermwittayawong, Decha; Côté, Jacques; Tan, Song

    2005-07-01

    Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding.

  5. The Saccharomyces cerevisiae Piccolo NuA4 Histone Acetyltransferase Complex Requires the Enhancer of Polycomb A Domain and Chromodomain To Acetylate Nucleosomes

    PubMed Central

    Selleck, William; Fortin, Israël; Sermwittayawong, Decha; Côté, Jacques; Tan, Song

    2005-01-01

    Chromatin modification complexes are key gene regulatory factors which posttranslationally modify the histone component of chromatin with epigenetic marks. To address what features of chromatin modification complexes are responsible for the specific recognition of nucleosomes compared to naked histones, we have performed a functional dissection of the Esa1-containing Saccharomyces cerevisiae Piccolo NuA4 histone acetyltransferase complex. Our studies define the Piccolo determinants sufficient to assemble its three subunits into a complex as well as Piccolo determinants sufficient to specifically acetylate a chromatin template. We find that the conserved Enhancer of Polycomb A (EPcA) homology region of the Epl1 component and the N-terminal 165 amino acids of the Yng2 component of Piccolo are sufficient with Esa1 to specifically act on nucleosomes. We also find that the Esa1 chromodomain plays a critical role in Piccolo's ability to distinguish between histones and nucleosomes. In particular, specific point mutations in the chromodomain putative hydrophobic cage which strongly hinder growth in yeast greatly reduce histone acetyltransferase activity on nucleosome substrates, independent of histone methylation or other modifications. However, the chromodomain is not required for Piccolo to bind to nucleosomes, suggesting a role for the chromodomain in a catalysis step after nucleosome binding. PMID:15964809

  6. Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis

    PubMed Central

    Che, Jun; Smith, Stephanie; Kim, Yoo Jung; Shim, Eun Yong; Myung, Kyungjae; Lee, Sang Eun

    2015-01-01

    Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR. PMID:25705897

  7. 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

  8. 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

  9. Emerging role of linker histone variant H1x as a biomarker with prognostic value in astrocytic gliomas. A multivariate analysis including trimethylation of H3K9 and H4K20.

    PubMed

    Sepsa, Athanasia; Levidou, Georgia; Gargalionis, Antonis; Adamopoulos, Christos; Spyropoulou, Anastasia; Dalagiorgou, Georgia; Thymara, Irene; Boviatsis, Efstathios; Themistocleous, Marios S; Petraki, Kalliopi; Vrettakos, George; Samaras, Vassilis; Zisakis, Athanassios; Patsouris, Efstratios; Piperi, Christina; Korkolopoulou, Penelope

    2015-01-01

    Although epigenetic alterations play an essential role in gliomagenesis, the relevance of aberrant histone modifications and the respective enzymes has not been clarified. Experimental data implicates histone H3 lysine (K) methyltransferases SETDB1 and SUV39H1 into glioma pathobiology, whereas linker histone variant H1.0 and H4K20me3 reportedly affect prognosis. We investigated the expression of H3K9me3 and its methyltransferases along with H4K20me3 and H1x in 101 astrocytic tumors with regard to clinicopathological characteristics and survival. The effect of SUV39H1 inhibition by chaetocin on the proliferation, colony formation and migration of T98G cells was also examined. SETDB1 and cytoplasmic SUV39H1 levels increased from normal brain through low-grade to high-grade tumors, nuclear SUV39H1 correlating inversely with grade. H3K9me3 immunoreactivity was higher in normal brain showing no association with grade, whereas H1x and H4K20me3 expression was higher in grade 2 than in normal brain or high grades. These expression patterns of H1x, H4K20me3 and H3K9me3 were verified by Western immunoblotting. Chaetocin treatment significantly reduced proliferation, clonogenic potential and migratory ability of T98G cells. H1x was an independent favorable prognosticator in glioblastomas, this effect being validated in an independent set of 66 patients. Diminished nuclear SUV39H1 expression adversely affected survival in univariate analysis. In conclusion, H4K20me3 and H3K9 methyltransferases are differentially implicated in astroglial tumor progression. Deregulation of H1x emerges as a prognostic biomarker. PMID:25602259

  10. Induction of the BCMO1 gene during the suckling-weaning transition in rats is associated with histone H3 K4 methylation and subsequent coactivator binding and histone H3 acetylation to the gene.

    PubMed

    Mochizuki, Hiroko; Mochizuki, Kazuki; Suruga, Kazuhito; Igarashi, Miki; Takase, Sachiko; Goda, Toshinao

    2012-01-01

    The cells involved in nutrient absorption in the small intestine of rats undergo rapid maturation during the suckling-weaning transition period, i.e., 2-4 wk after birth. During this period, the serum thyroid hormone level is increased. However, the molecular mechanisms involved in the regulation of β-carotene 15,15'-monooxygenase 1 (BCMO1) gene expression in the small intestine remain unknown. In this study, we found that jejunal β-carotene 15,15' dioxygenase activity and the gene expression of BCMO1 were significantly increased during this transition period between days 13 and 27 after birth. A chromatin immunoprecipitation assay revealed that di- and tri-methylation of histone H3 at lysine 4 (K4) and the binding of thyroid hormone receptor (TR) α-1 binding on the promoter/enhancer and/or transcribed regions of the BCMO1 gene were enhanced from the earlier stage of weaning (i.e., 20 d after birth), prior to an enhancement of the acetylation of histone H3 and the binding of coactivator (SRC-1 and CBP) to the promoter/enhancer and/or transcribed regions of the BCMO1 gene, which was apparent at 27 d after birth. These results suggest that histone H3 K4 methylation and TRα-1 binding on the BCMO1 gene during the suckling-weaning transient period in rats predisposes to subsequent coactivator recruitment and histone H3 acetylation on the gene.

  11. 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.

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

    PubMed

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

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation. PMID:26918332

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

    PubMed Central

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

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation. PMID:26918332

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

    PubMed

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

    2016-01-01

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

  15. The Autotaxin–Lysophosphatidic Acid Axis Modulates Histone Acetylation and Gene Expression during Oligodendrocyte Differentiation

    PubMed Central

    Wheeler, Natalie A.; Lister, James A.

    2015-01-01

    During development, oligodendrocytes (OLGs), the myelinating cells of the CNS, undergo a stepwise progression during which OLG progenitors, specified from neural stem/progenitor cells, differentiate into fully mature myelinating OLGs. This progression along the OLG lineage is characterized by well synchronized changes in morphology and gene expression patterns. The latter have been found to be particularly critical during the early stages of the lineage, and they have been well described to be regulated by epigenetic mechanisms, especially by the activity of the histone deacetylases HDAC1 and HDAC2. The data presented here identify the extracellular factor autotaxin (ATX) as a novel upstream signal modulating HDAC1/2 activity and gene expression in cells of the OLG lineage. Using the zebrafish as an in vivo model system as well as rodent primary OLG cultures, this functional property of ATX was found to be mediated by its lysophospholipase D (lysoPLD) activity, which has been well characterized to generate the lipid signaling molecule lysophosphatidic acid (LPA). More specifically, the lysoPLD activity of ATX was found to modulate HDAC1/2 regulated gene expression during a time window coinciding with the transition from OLG progenitor to early differentiating OLG. In contrast, HDAC1/2 regulated gene expression during the transition from neural stem/progenitor to OLG progenitor appeared unaffected by ATX and its lysoPLD activity. Thus, together, our data suggest that an ATX–LPA–HDAC1/2 axis regulates OLG differentiation specifically during the transition from OLG progenitor to early differentiating OLG and via a molecular mechanism that is evolutionarily conserved from at least zebrafish to rodent. SIGNIFICANCE STATEMENT The formation of the axon insulating and supporting myelin sheath by differentiating oligodendrocytes (OLGs) in the CNS is considered an essential step during vertebrate development. In addition, loss and/or dysfunction of the myelin sheath has

  16. Absolute quantification of acetylation and phosphorylation of the histone variant H2AX upon ionizing radiation reveals distinct cellular responses in two cancer cell lines.

    PubMed

    Matsuda, Shun; Furuya, Kanji; Ikura, Masae; Matsuda, Tomonari; Ikura, Tsuyoshi

    2015-11-01

    Histone modifications change upon the cellular response to ionizing radiation, and their cellular amounts could reflect the DNA damage response activity. We previously reported a sensitive and reliable method for the absolute quantification of γH2AX within cells, using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The technique has broad adaptability to a variety of biological systems and can quantitate different modifications of histones. In this study, we applied it to quantitate the levels of γH2AX and K5-acetylated H2AX, and to compare the radiation responses between two cancer cell lines: HeLa and U-2 OS. The two cell lines have distinct properties in terms of their H2AX modifications. HeLa cells have relatively high γH2AX (3.1 %) against the total H2AX even in un-irradiated cells, while U-2 OS cells have an essentially undetectable level (nearly 0 %) of γH2AX. In contrast, the amounts of acetylated histones are lower in HeLa cells (9.3 %) and higher in U-2 OS cells (24.2 %) under un-irradiated conditions. Furthermore, after ionizing radiation exposure, the time-dependent increases and decreases in the amounts of histone modifications differed between the two cell lines, especially at the early time points. These results suggest that each biological system has distinct kinase/phosphatase and/or acetylase/deacetylase activities. In conclusion, for the first time, we have succeeded in simultaneously monitoring the absolute amounts of phosphorylated and acetylated cellular H2AX after ionizing radiation exposure. This multi-criteria assessment enables precise comparisons of the effects of radiation between any biological systems.

  17. 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

  18. 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

  19. 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

  20. Crystallization and preliminary X-ray diffraction analysis of the BRPF1 bromodomain in complex with its H2AK5ac and H4K12ac histone-peptide ligands

    PubMed Central

    Lubula, Mulu Y.; Poplawaski, Amanda; Glass, Karen C.

    2014-01-01

    The bromodomain-PHD finger protein 1 (BRPF1) is an essential subunit of the monocytic leukemia zinc (MOZ) histone acetyltransferase (HAT) complex and is required for complex formation and enzymatic activation. BRPF1 contains a structurally conserved bromodomain, which recognizes specific acetyllysine residues on histone proteins. The MOZ HAT plays a direct role in hematopoiesis, and deregulation of its activity is linked to the development of acute myeloid leukemia. However, the molecular mechanism of histone-ligand recognition by the BRPF1 bromodomain is currently unknown. The 117-amino-acid BRPF1 bromodomain was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments of the BRPF1 bromodomain in complex with its H4K12ac and H2AK5ac histone ligands yielded crystals that were suitable for high-resolution X-ray diffraction analysis. The BRPF1 bromodomain–H4K12ac crystals belonged to the tetragonal space group P43212, with unit-cell parameters a = 75.1, b = 75.1, c = 86.3 Å, and diffracted to a resolution of 1.94 Å. The BRPF1 bromodomain–H2AK5ac crystals grew in the monoclinic space group P21, with unit-cell parameters a = 60.9, b = 55.6, c = 82.1 Å, β = 93.6°, and diffracted to a resolution of 1.80 Å. Complete data sets were collected from both crystal forms using synchrotron radiation on beamline X29 at Brookhaven National Laboratory (BNL). PMID:25286946

  1. Anti-inflammatory effect of 2-methoxy-4-vinylphenol via the suppression of NF-κB and MAPK activation, and acetylation of histone H3.

    PubMed

    Jeong, Jin Boo; Hong, Se Chul; Jeong, Hyung Jin; Koo, Jin Suk

    2011-12-01

    Although inflammation acts as host defense mechanism against infection or injury and is primarily a self limiting process, inadequate resolution of inflammatory responses leads to various chronic disorders. This work aimed to elucidate the anti-inflammatory effects of 2-methoxy-4-vinylphenol (2M4VP) isolated from pine needles in LPS-stimulated RAW264.7 cells. Some key pro-inflammatory mediators including nitric oxide (NO), prostaglandins (PGE(2)), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied by sandwich ELISA and western blot. In addition, suppression of NF-κB and MAPK activation, and histone acetylation was studied by western blot analysis and immunostaining. 2M4VP dosedependently inhibited NO and PGE(2) production and also blocked LPS-induced iNOS and COX-2 expression. In addition, 2M4VP potently inhibited the translocation of NF-κB p65 into the nucleus by IκB degradation following IκB-α phosphorylation and the phosphorylation of MAPKs such as p38, ERK1/2, and JNK. Also, 2M4VP inhibited hyper-acetylation of histone H3 (Lys9/Lys14) induced by LPS. Taken together, our results suggest that 2M4VP, a naturally occurring phenolic compound, exert potent anti-inflammatory effects by inhibiting LPS-induced NO, PGE(2), iNOS, and COX-2 in RAW264.7 cells. These effects are mediated by suppression of NF-κB and MAPK activation and histone acetylation.

  2. Ginsenoside Rg3 Inhibits Melanoma Cell Proliferation through Down-Regulation of Histone Deacetylase 3 (HDAC3) and Increase of p53 Acetylation

    PubMed Central

    Shan, Xiu; Fu, Yuan-Shan; Aziz, Faisal; Wang, Xiao-Qi; Yan, Qiu; Liu, Ji-Wei

    2014-01-01

    Malignant melanoma is an aggressive and deadly form of skin cancer, and despite recent advances in available therapies, is still lacking in completely effective treatments. Rg3, a monomer extracted from ginseng roots, has been attempted for the treatment of many cancers. It is reported that the expressions of histone deacetylase 3 (HDAC3) and p53 acetylation correlate with tumor cell growth. However, the antitumor effect of Rg3 on melanoma and the mechanism by which it regulates HDAC3 expression and p53 acetylation remain unknown. We found high expression of HDAC3 in human melanoma tissues to be significantly correlated to lymph node metastasis and clinical stage of disease (p<0.05). In melanoma cells, Rg3 inhibited cell proliferation and induced G0/G1 cell cycle arrest. Rg3 also decreased the expression of HDAC3 and increased the acetylation of p53 on lysine (k373/k382). Moreover, suppression of HDAC3 by either siRNA or a potent HDAC3 inhibitor (MS-275) inhibited cell proliferation, increased p53 acetylation and transcription activity. In A375 melanoma xenograft studies, we demonstrated that Rg3 and HDAC3 short hairpin RNA (shHDAC3) inhibited the growth of xenograft tumors with down-regulation of HDAC3 expression and up-regulation of p53 acetylation. In conclusion, Rg3 has antiproliferative activity against melanoma by decreasing HDAC3 and increasing acetylation of p53 both in vitro and in vivo. Thus, Rg3 serves as a potential therapeutic agent for the treatment of melanoma. PMID:25521755

  3. The SANT domain of Ada2 is required for normal acetylation of histones by the yeast SAGA complex.

    PubMed

    Sterner, David E; Wang, Xun; Bloom, Melissa H; Simon, Gabriel M; Berger, Shelley L

    2002-03-01

    Transcription is regulated through chromatin remodeling and histone modification, mediated by large protein complexes. Histone and nucleosome interaction has been shown to be mediated by specific chromatin domains called bromodomains and chromodomains. Here we provide evidence for a similar function of two additional domains within the yeast SAGA complex, containing the histone acetyltransferase Gcn5. We have analyzed deletion and substitution mutations within Gcn5 and Ada2, an interacting protein within SAGA, and have identified substrate recognition functions within the SANT domain of Ada2 and regions of the histone acetyltransferase domain of Gcn5 that are distinct from catalytic function itself. These results suggest that histone and nucleosomal substrate recognition by SAGA involves multiple conserved domains and proteins, beyond those that have been previously identified. PMID:11777910

  4. Nucleosomal histone proteins of L. donovani: a combination of recombinant H2A, H2B, H3 and H4 proteins were highly immunogenic and offered optimum prophylactic efficacy against Leishmania challenge in hamsters.

    PubMed

    Baharia, Rajendra K; Tandon, Rati; Sahasrabuddhe, Amogh A; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    The present study includes cloning and expression of recombinant Leishmania donovani histone proteins (rLdH2B, rLdH3, rLdH2A and rLdH4), assessment of their immunogenicity in Leishmania infected cured patients/endemic contacts as well as in cured hamsters and finally evaluation of their prophylactic efficacy in hamsters against L. donovani challenge. All recombinant proteins were expressed and purified from the heterologous bacterial host system. Leishmania infected cured patients/endemic contacts as well as cured hamsters exhibited significantly higher proliferative responses to individual recombinant histones and their pooled combination (rLdH2B+rLdH3+rLdH2A+rLdH4) than those of L.donovani infected hosts. The L.donovani soluble antigens (SLD) stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLdH2B, rLdH3, rLdH2A, rLdH4 and pooled combination (rLdH2-4) stimulated the production of Th1 cytokines IFN-γ, IL-12 and TNF-α but not Th2 cytokines IL-4 or IL-10. The immunogenicity of these histone proteins along with their combination was also checked in cured hamsters where they stimulated higher lymphoproliferation and Nitric oxide production in lymphocytes of cured hamsters than that of infected controls. Moreover, significantly increased IgG2 response, an indicative of cell mediated immunity, was observed in cured hamsters against these individual proteins and their combination as compared to infected hamsters. Further, it was demonstrated that rLdH2B, rLdH3, rLdH2A and rLdH4 and pooled combination were able to provide considerable protection for hamsters against L. donovani challenge. The efficacy was supported by the increased inducible Nitric Oxide Synthase (iNOS) mRNA transcripts and Th1-type cytokines--IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β. Hence, it is inferred that pooled rLdH2-4 elicits Thl-type of immune responses exclusively and confer considerable protection

  5. Feeding rats dietary resistant starch shifts the peak of SGLT1 gene expression and histone H3 acetylation on the gene from the upper jejunum toward the ileum.

    PubMed

    Shimada, Masaya; Mochizuki, Kazuki; Goda, Toshinao

    2009-09-01

    Sodium glucose cotransporter 1 (SGLT1) participates in the incorporation of glucose from the lumen to enterocytes in the small intestine. We examined whether dietary resistant starch (RS), an autoclaved high amylose starch that is digested more slowly than regular cornstarch in the small intestine, alters SGLT1 mRNA levels along the jejunum-ileum of rats. The SGLT1 mRNA level was lower in the upper jejunum in rats fed an RS diet than in those fed a regular cornstarch diet, whereas it was higher in the lower jejunum/upper ileum. Furthermore, using chromatin immunoprecipitation (ChIP) assay, we demonstrated that histone H3 acetylation on the promoter/enhancer and transcriptional regions was reduced in the upper jejunum and elevated in the lower jejunum/upper ileum by feeding rats an RS diet. On the other hand, HNF-1 binding on the region around transcription start site of the SGLT1 gene was not altered in each jejunoileal segment by feeding rats an RS diet. Our results suggest that a shift of the expressional peak of the SGLT1 gene from the upper jejunum toward the ileum by dietary RS is associated with a change of histone H3 acetylation rather than that of HNF-1 binding on the gene.

  6. Sodium butyrate up-regulates cathelicidin gene expression via activator protein-1 and histone acetylation at the promoter region in a human lung epithelial cell line, EBC-1.

    PubMed

    Kida, Yutaka; Shimizu, Takashi; Kuwano, Koichi

    2006-05-01

    The antimicrobial protein cathelicidin is considered to play an important role in the defense mechanisms against bacterial infection. Recent studies show that sodium butyrate induces cathelicidin gene expression in human colonic, gastric and hepatic cells. However, little is known about the precise regulatory mechanisms underlying sodium butyrate-induced cathelicidin gene expression. In this study, we examined the regulatory mechanisms involved in sodium butyrate-induced cathelicidin gene expression using a human lung epithelial cell line, EBC-1. Our results indicate that sodium butyrate induces both cathelicidin mRNA and protein expression. Moreover, deletion or mutation of a putative activator protein-1 (AP-1) binding site in the cathelicidin gene promoter abrogated the response to sodium butyrate stimulation. Three different mitogen-activated protein (MAP) kinase inhibitors suppressed sodium butyrate-induced transactivation of the cathelicidin promoter. Electrophoretic mobility shift assays (EMSA) showed that nuclear extracts prepared from sodium butyrate-stimulated EBC-1 cells generated specific binding to probe including a putative AP-1 binding site in the cathelicidin gene promoter. Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that sodium butyrate augmented histone acetylation of the cathelicidin promoter in EBC-1 cells. Therefore, these results indicate that AP-1 and histone acetylation of the cathelicidin promoter play a critical role in the regulation of inducible cathelicidin gene expression in EBC-1 cells stimulated with sodium butyrate.

  7. Histone deacetylase inhibitors valproic acid and depsipeptide sensitize retinoblastoma cells to radiotherapy by increasing H2AX phosphorylation and p53 acetylation-phosphorylation.

    PubMed

    Kawano, Takeshi; Akiyama, Masaharu; Agawa-Ohta, Miyuki; Mikami-Terao, Yoko; Iwase, Satsuki; Yanagisawa, Takaaki; Ida, Hiroyuki; Agata, Naoki; Yamada, Hisashi

    2010-10-01

    Although p53 is intact in most cases of retinoblastoma, it is largely inactivated by the ubiqutin-proteasome system through interaction with murine double minute 2 (MDM2) and murine double minute X (MDMX). The present study showed that the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and depsipeptide (FK228) synergistically enhanced ionizing radiation (IR)-induced apoptosis, associated with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Y79 and WER1-Rb1 human retinoblastoma cells. Both VPA and FK228 enhanced IR-induced phosphorylation of histone H2AX on Ser139 preceding apoptosis. Exposure of cells to IR in the presence of VPA or FK228 induced the accumulation of p53 acetylated at Lys382 and phosphorylated at Ser46 through the reduction of binding affinity with MDM2 and MDMX. These results suggest that acetylation of p53 by HDAC inhibitors is a promising new therapeutic target in refractory retinoblastoma. PMID:20811699

  8. Standardised extract of Bacopa monniera (CDRI-08) improves contextual fear memory by differentially regulating the activity of histone acetylation and protein phosphatases (PP1α, PP2A) in hippocampus.

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Venkataraman, Jois Shreyas; Rajan, Koilmani Emmanuvel

    2014-05-01

    Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15-29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus.

  9. Standardised extract of Bacopa monniera (CDRI-08) improves contextual fear memory by differentially regulating the activity of histone acetylation and protein phosphatases (PP1α, PP2A) in hippocampus.

    PubMed

    Preethi, Jayakumar; Singh, Hemant K; Venkataraman, Jois Shreyas; Rajan, Koilmani Emmanuvel

    2014-05-01

    Contextual fear conditioning is a paradigm for investigating cellular mechanisms involved in hippocampus-dependent memory. Earlier, we showed that standardised extract of Bacopa monniera (CDRI-08) improves hippocampus-dependent learning in postnatal rats by elevating the level of serotonin (5-hydroxytryptamine, 5-HT), activate 5-HT3A receptors, and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. In this study, we have further examined the molecular mechanism of CDRI-08 in hippocampus-dependent memory and compared to the histone deacetylase (HDACs) inhibitor sodium butyrate (NaB). To assess the hippocampus-dependent memory, wistar rat pups were subjected to contextual fear conditioning (CFC) following daily (postnatal days 15-29) administration of vehicle solution (0.5 % gum acacia + 0.9 % saline)/CDRI-08 (80 mg/kg, p.o.)/NaB (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior compared to control group when re-exposed to the same context. Administration of CDRI-08/NaB resulted in activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and up-regulation of p300, Ac-H3 and Ac-H4 levels, and down-regulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A) in hippocampus following CFC. This would subsequently result in an increased brain-derived neurotrophic factor (Bdnf) (exon IV) mRNA in hippocampus. Altogether, our results indicate that CDRI-08 enhances hippocampus-dependent contextual memory by differentially regulating histone acetylation and protein phosphatases in hippocampus. PMID:24610280

  10. Inhalable Metal-Rich Air Particles and Histone H3K4 Dimethylation and H3K9 Acetylation in a Cross-sectional Study of Steel Workers

    PubMed Central

    Cantone, Laura; Nordio, Francesco; Hou, Lifang; Apostoli, Pietro; Bonzini, Matteo; Tarantini, Letizia; Angelici, Laura; Bollati, Valentina; Zanobetti, Antonella; Schwartz, Joel; Bertazzi, Pier A.

    2011-01-01

    Background: Epidemiology investigations have linked exposure to ambient and occupational air particulate matter (PM) with increased risk of lung cancer. PM contains carcinogenic and toxic metals, including arsenic and nickel, which have been shown in in vitro studies to induce histone modifications that activate gene expression by inducing open-chromatin states. Whether inhalation of metal components of PM induces histone modifications in human subjects is undetermined. Objectives: We investigated whether the metal components of PM determined activating histone modifications in 63 steel workers with well-characterized exposure to metal-rich PM. Methods: We determined histone 3 lysine 4 dimethylation (H3K4me2) and histone 3 lysine 9 acetylation (H3K9ac) on histones from blood leukocytes. Exposure to inhalable metal components (aluminum, manganese, nickel, zinc, arsenic, lead, iron) and to total PM was estimated for each study subject. Results: Both H3K4me2 and H3K9ac increased in association with years of employment in the plant (p-trend = 0.04 and 0.006, respectively). H3K4me2 increased in association with air levels of nickel [β = 0.16; 95% confidence interval (CI), 0.03–0.3], arsenic (β = 0.16; 95% CI, 0.02–0.3), and iron (β = 0.14; 95% CI, 0.01–0.26). H3K9ac showed nonsignificant positive associations with air levels of nickel (β = 0.24; 95% CI, –0.02 to 0.51), arsenic (β = 0.21; 95% CI, –0.06 to 0.48), and iron (β = 0.22; 95% CI, –0.03 to 0.47). Cumulative exposures to nickel and arsenic, defined as the product of years of employment by metal air levels, were positively correlated with both H3K4me2 (nickel: β = 0.16; 95% CI, 0.01–0.3; arsenic: β = 0.16; 95% CI, 0.03–0.29) and H3K9ac (nickel: β = 0.27; 95% CI, 0.01–0.54; arsenic: β = 0.28; 95% CI, 0.04–0.51). Conclusions: Our results indicate histone modifications as a novel epigenetic mechanism induced in human subjects by long-term exposure to inhalable nickel and arsenic. PMID

  11. Treatment of nuclear-donor cells or cloned zygotes with chromatin-modifying agents increases histone acetylation but does not improve full-term development of cloned cattle.

    PubMed

    Sangalli, Juliano Rodrigues; De Bem, Tiago Henrique Camara; Perecin, Felipe; Chiaratti, Marcos Roberto; Oliveira, Lilian de Jesus; de Araújo, Reno Roldi; Valim Pimentel, José Rodrigo; Smith, Lawrence Charles; Meirelles, Flávio Vieira

    2012-06-01

    Although somatic cell nuclear transfer (SCNT) is a promising tool, its potential use is hampered by the high mortality rates during the development to term of cloned offspring. Abnormal epigenetic reprogramming of donor nuclei after SCNT is thought to be the main cause of this low efficiency. We hypothesized that chromatin-modifying agents (CMAs) targeting chromatin acetylation and DNA methylation could alter the chromatin configuration and turn them more amenable to reprogramming. Thus, bovine fibroblasts were treated with 5-aza-2'-deoxycytidine (AZA) plus trichostatin (TSA) or hydralazine (HH) plus valproic acid (VPA) whereas, in another trial, cloned bovine zygotes were treated with TSA. The treatment of fibroblasts with either AZA+TSA or HH+VPA increased histone acetylation, but did not affect the level of DNA methylation. However, treatment with HH+VPA decreased cellular viability and proliferation. The use of these cells as nuclear donors showed no positive effect on pre- and postimplantation development. Regarding the treatment of cloned zygotes with TSA, treated one-cell embryos showed an increase in the acetylation patterns, but not in the level of DNA methylation. Moreover, this treatment revealed no positive effect on pre- and postimplantation development. This work provides evidence the treatment of either nuclear donor cells or cloned zygotes with CMAs has no positive effect on pre- and postimplantation development of cloned cattle.

  12. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding.

    PubMed

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M; Nandicoori, Vinay K; Khosla, Sanjeev

    2015-04-30

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.

  13. The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

    PubMed Central

    Sharma, Garima; Upadhyay, Sandeep; Srilalitha, M.; Nandicoori, Vinay K.; Khosla, Sanjeev

    2015-01-01

    To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation. PMID:25824946

  14. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGES

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  15. 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

  16. Potential role for PADI-mediated histone citrullination in preimplantation development

    PubMed Central

    2012-01-01

    Background The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. Results In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. Conclusions Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation

  17. Interaction of the histone (H3-H4)2 tetramer of the nucleosome with positively supercoiled DNA minicircles: Potential flipping of the protein from a left- to a right-handed superhelical form.

    PubMed

    Hamiche, A; Carot, V; Alilat, M; De Lucia, F; O'Donohue, M F; Revet, B; Prunell, A

    1996-07-23

    We have studied the ability of the histone (H3-H4)2 tetramer, the central part of the nucleosome of eukaryotic chromatin, to form particles on DNA minicircles of negative and positive superhelicities, and the effect of relaxing these particles with topoisomerase I. The results show that even modest positive torsional stress from the DNA, and in particular that generated by DNA thermal fluctuations, can trigger a major, reversible change in the conformation of the particle. Neither a large excess of naked DNA, nor a crosslink between the two H3s prevented the transition from one form to the other. This suggested that during the transition, the histones neither dissociated from the DNA nor were even significantly reshuffled. Moreover, the particles reconstituted on negatively and positively supercoiled minicircles look similar under electron microscopy. These data agree best with a transition involving a switch of the wrapped DNA from a left- to a right-handed superhelix. It is further proposed, based on the left-handed overall superhelical conformation of the tetramer within the octamer [Arents, G., Burlingame, R. W., Wang, B. C., Love, W. E. & Moudrianakis, E. N. (1991) Proc. Natl.Acad. Sci. USA 88, 10148-10152] that this change in DNA topology is mediated by a similar change in the topology of the tetramer itself, which may occur through a rotation (or a localized deformation) of the two H3-H4 dimers about their H3-H3 interface. Potential implications of this model for nucleosome dynamics in vivo are discussed. PMID:8755519

  18. Histone H3 lysine 14 (H3K14) acetylation facilitates DNA repair in a positioned nucleosome by stabilizing the binding of the chromatin Remodeler RSC (Remodels Structure of Chromatin).

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

    Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

  19. Hepatocyte nuclear factor-1alpha is required for expression but dispensable for histone acetylation of the lactase-phlorizin hydrolase gene in vivo.

    PubMed

    Bosse, Tjalling; van Wering, Herbert M; Gielen, Marieke; Dowling, Lauren N; Fialkovich, John J; Piaseckyj, Christina M; Gonzalez, Frank J; Akiyama, Taro E; Montgomery, Robert K; Grand, Richard J; Krasinski, Stephen D

    2006-05-01

    Hepatocyte nuclear factor-1alpha (HNF-1alpha) is a modified homeodomain-containing transcription factor that has been implicated in the regulation of intestinal genes. To define the importance and underlying mechanism of HNF-1alpha for the regulation of intestinal gene expression in vivo, we analyzed the expression of the intestinal differentiation markers and putative HNF-1alpha targets lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) in hnf1alpha null mice. We found that in adult jejunum, LPH mRNA in hnf1alpha(-/-) mice was reduced 95% compared with wild-type controls (P < 0.01, n = 4), whereas SI mRNA was virtually identical to that in wild-type mice. Furthermore, SI mRNA abundance was unchanged in the absence of HNF-1alpha along the length of the adult mouse small intestine as well as in newborn jejunum. We found that HNF-1alpha occupies the promoters of both the LPH and SI genes in vivo. However, in contrast to liver and pancreas, where HNF-1alpha regulates target genes by recruitment of histone acetyl transferase activity to the promoter, the histone acetylation state of the LPH and SI promoters was not affected by the presence or absence of HNF-1alpha. Finally, we showed that a subset of hypothesized intestinal target genes is regulated by HNF-1alpha in vivo and that this regulation occurs in a defined tissue-specific and developmental context. These data indicate that HNF-1alpha is an activator of a subset of intestinal genes and induces these genes through an alternative mechanism in which it is dispensable for chromatin remodeling.

  20. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27.

    PubMed

    Aronson, B E; Rabello Aronson, S; Berkhout, R P; Chavoushi, S F; He, A; Pu, W T; Verzi, M P; Krasinski, S D

    2014-11-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal ('jejunal') identity while repressing a distal intestinal ('ileal') identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal versus ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- versus up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wild-type control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27.

  1. 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.

  2. Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

    PubMed

    Foy, Rebecca L; Song, Ihn Young; Chitalia, Vipul C; Cohen, Herbert T; Saksouk, Nehme; Cayrou, Christelle; Vaziri, Cyrus; Côté, Jacques; Panchenko, Maria V

    2008-10-24

    Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

  3. Genomewide Histone H3 Lysine 9 Acetylation Profiling in CD4+ T Cells Revealed Endoplasmic Reticulum Stress Deficiency in Patients with Acute-on-chronic Liver Failure.

    PubMed

    Jin, L; Wang, K; Liu, H; Chen, T; Yang, Y; Ma, X; Wang, J; Li, Y; Du, D; Zhao, Y; He, Y

    2015-11-01

    Acute-on-chronic liver failure (ACLF) displayed 'sepsis-like' immune paralysis. Little is known about the role of CD4+ T lymphocytes, the primary regulator of innate and adopted immune system, played in ACLF. Acetylation of histone H3 lysine 9 (H3K9ac), a key epigenetic modification, tightly controls gene transcription. Whether and how does H3K9ac modification regulate CD4+ T cells in ACLF remains unclear. PBMCs were isolated from patients with ACLF, immune tolerance of chronic hepatitis B (CHB-T) and immune active of chronic hepatitis B (CHB-A). Then, CD4+ T lymphocytes were purified by magnetic microbeads, and the purity was confirmed by flow cytometry. H3K9ac variations were analysed in CD4+ T cells using chromatin immunoprecipitation microarray and then confirmed by quantitative PCR. Whole-genome H3K9 acetylation analyses were conducted by bioinformatics. A total of 70 genes were differently modified in H3K9ac between CHB-A and ACLF groups, while 44 genes were differently modified in H3K9ac between CHB-T and ACLF groups. Clustering algorithm analysis showed patients with ACLF displayed 'sepsis-like' immune paralysis. Functional analysis showed endoplasmic reticulum (ER) stress, or downstream pathway-related genes, such as BIP, ATF4, PER1, CSNK1D, IRF3, BNIP1, AKT1 and UBC, were differentially modified in ACLF. We profiled H3K9 acetyl modification in CD4+ T lymphocytes from HBV-infected patients with three different immune states, that is ACLF, immune tolerance and immune active phases. ACLF displayed 'sepsis-like' immune paralysis. ER stress in CD4+ T lymphocytes attributed to ACLF. This study provides some useful clues for revealing the mechanisms underlying ACLF. PMID:26173605

  4. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    SciTech Connect

    Yamagata, Kazutsune; Kitabayashi, Issay

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  5. 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.

  6. Comment on "A histone acetylation switch regulates H2A.Z deposition by the SWR-C remodeling enzyme".

    PubMed

    Wang, Feng; Ranjan, Anand; Wei, Debbie; Wu, Carl

    2016-07-22

    Watanabe et al (Reports, 12 April 2013, p. 195) study the yeast SWR1/SWR-C complex responsible for depositing the histone variant H2A.Z by replacing nucleosomal H2A with H2A.Z. They report that reversal of H2A.Z replacement is mediated by SWR1 and related INO80 on an H2A.Z nucleosome carrying H3K56Q. Using multiple assays and reaction conditions, we find no evidence of such reversal of H2A.Z exchange. PMID:27463665

  7. Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.

    PubMed

    Ng, Danny W-K; Chandrasekharan, Mahesh B; Hall, Timothy C

    2006-01-01

    The phaseolin (phas) promoter drives copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues, in which a nucleosome is positioned over its three-phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis thaliana leaves was accomplished by ectopic expression of the transcription factor Phaseolus vulgaris ABI3-like factor (ALF) and application of abscisic acid (ABA). Placement of hemagglutinin-tagged ALF expression under the control of an estradiol-inducible promoter permitted chromatin immunoprecipitation analysis of chronological changes in histone modifications, notably increased acetylation of H3-K9 and H4-K12, as phas chromatin was remodeled (potentiated). A different array of changes, including acetylation of H3-K14 and methylation of H3-K4, was found to be associated with ABA-mediated activation. Thus, temporal separation of phas potentiation from activation revealed that histone H3 and H4 Lys residues are not globally hyperacetylated during phas expression. Whereas decreases in histone H3 and H4 levels were detected during ALF-mediated remodeling, slight increases occurred after ABA-mediated activation, suggesting the restoration of histone-phas interactions or the replacement of histones in the phas chromatin. The observed histone modifications provide insight into factors involved in the euchromatinization and activation of a plant gene and expand the evidence for histone code conservation among eukaryotes. PMID:16326929

  8. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    SciTech Connect

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of

  9. Ethanol induced acetylation of histone at G9a exon1 and G9a-mediated histone H3 dimethylation leads to neurodegeneration in neonatal mice.

    PubMed

    Subbanna, S; Nagre, N N; Shivakumar, M; Umapathy, N S; Psychoyos, D; Basavarajappa, B S

    2014-01-31

    The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), comparable to a time point within the third trimester of human pregnancy, induces neurodegeneration. However, the molecular mechanisms underlying the deleterious effects of ethanol on the developing brain are poorly understood. In our previous study, we showed that a high dose administration of ethanol at P7 enhances G9a and leads to caspase-3-mediated degradation of dimethylated H3 on lysine 9 (H3K9me2). In this study, we investigated the potential role of epigenetic changes at G9a exon1, G9a-mediated H3 dimethylation on neurodegeneration and G9a-associated proteins in the P7 brain following exposure to a low dose of ethanol. We found that a low dose of ethanol induces mild neurodegeneration in P7 mice, enhances specific acetylation of H3 on lysine 14 (H3K14ace) at G9a exon1, G9a protein levels, augments the dimethylation of H3K9 and H3 lysine 27 (H3K27me2). However, neither dimethylated H3K9 nor K27 underwent degradation. Pharmacological inhibition of G9a activity prior to ethanol treatment prevented H3 dimethylation and neurodegeneration. Further, our immunoprecipitation data suggest that G9a directly associates with DNA methyltransferase (DNMT3A) and methyl-CpG-binding protein 2 (MeCP2). In addition, DNMT3A and MeCP2 protein levels were enhanced by a low dose of ethanol that was shown to induce mild neurodegeneration. Collectively, these epigenetic alterations lead to association of G9a, DNMT3A and MeCP2 to form a larger repressive complex and have a significant role in low-dose ethanol-induced neurodegeneration in the developing brain.

  10. GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27

    PubMed Central

    Aronson, B. E.; Aronson, S. Rabello; Berkhout, R. P.; Chavoushi, S. F.; He, A.; Pu, W. T.; Verzi, M. P.; Krasinski, S. D.

    2015-01-01

    GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal (‘jejunal’) identity while repressing a distal intestinal (‘ileal’) identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal vs. ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- vs. up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wildtype control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27. PMID:24878542

  11. Chromatin immunoprecipitation analysis of the tobacco PR-1a- and the truncated CaMV 35S promoter reveals differences in salicylic acid-dependent TGA factor binding and histone acetylation.

    PubMed

    Butterbrodt, Thomas; Thurow, Corinna; Gatz, Christiane

    2006-07-01

    Salicylic acid (SA) is a plant signalling molecule needed for the induction of defence responses upon attack by a variety of pathogens. Truncation of the Cauliflower Mosaic Virus (CaMV) 35S promoter down to 90 bp has identified activation sequence-1 (as-1) as an autonomous SA-responsive cis element. The as-1-like elements are found in a number of SA-inducible promoters like e.g. the tobacco PR-1a promoter. They are recognized by basic/leucine zipper (bZIP) transcription factors of the TGA family. In tobacco leaves, TGA2.2 is the most abundant TGA factor. TGA2.2 is required for the expression of as-1-containing promoters. Here we unravel clear differences between the "truncated" CaMV 35S and the PR-1a promoter with respect to in vivo TGA binding and histone acetylation. Chromatin immunoprecipitation (ChIP) analysis revealed SA-inducible recruitment of tobacco TGA2.2 as well as SA-inducible histone acetylation at the PR-1a promoter. In contrast, no influence of SA on TGA2.2 binding and histone acetylation was detectable at the "truncated" CaMV 35S promoter. The finding of SA-independent TGA factor binding in the absence of additional flanking regulatory sequences suggests that transcriptional activation is not necessarily mediated by inducible DNA binding of TGA factors. Plants with severely reduced TGA2.2 protein levels also showed SA-induced histone acetylation at the PR-1a promoter indicating that regulatory events independent from TGA2.2 function are initiated at the PR-1a promoter.

  12. Mycobacterium tuberculosis EIS gene inhibits macrophage autophagy through up-regulation of IL-10 by increasing the acetylation of histone H3.

    PubMed

    Duan, Liang; Yi, Min; Chen, Juan; Li, Shengjin; Chen, Weixian

    2016-05-13

    Autophagy plays a crucial role in the progress of Mycobacterium tuberculosis (MTB) infection. Recently, MTB enhanced intracellular survival (EIS) protein was reported to be secreted from MTB cells and linked to the inhibition of autophagy and the intracellular persistence of the pathogen. Here, we investigated the mechanism of EIS-mediated inhibition of autophagy in a human phorbol myristate acetate (PMA)-treated THP-1 cell line as well as in murine macrophages. We confirmed that the presence of EIS led to the inhibition of rapamycin (Rapa)-induced autophagy, while IL-10 gene expression was increased and Akt/mTOR/p70S6K pathway was activated during the process. IL-10 gene silencing led to a significant recovery of EIS-mediated autophagy suppression and decreased activity of the Akt/mTOR/p70S6K pathway. IL-10 promoter activity was unaffected by EIS. Remarkably, EIS increased the acetylation level of histone H3 (Ac-H3), which binds to the SP1 and STAT3 region of the human IL-10 gene promoter sequence. Thus, EIS protein possibly increased IL-10 expression through the regulation of Ac-H3 of its promoter. Our data demonstrated that one possible mechanism of the MTB evasion of autophagy is that the EIS protein up-regulates IL-10 via Ac-H3 and thus activates Akt/mTOR/p70S6K pathway. PMID:27079235

  13. Differential Modulation by Akkermansia muciniphila and Faecalibacterium prausnitzii of Host Peripheral Lipid Metabolism and Histone Acetylation in Mouse Gut Organoids

    PubMed Central

    Lukovac, Sabina; Belzer, Clara; Pellis, Linette; Keijser, Bart J.; de Vos, Willem M.; Montijn, Roy C.

    2014-01-01

    ABSTRACT The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. PMID:25118238

  14. Histone H3 K27 acetylation marks a potent enhancer element on the adipogenic master regulator gene Pparg2

    PubMed Central

    Ramlee, Muhammad Khairul; Zhang, Qiongyi; Idris, Muhammad; Peng, Xu; Sim, Choon Kiat; Han, Weiping; Xu, Feng

    2014-01-01

    PPARγ2 is expressed almost exclusively in adipose tissue and plays a central role in adipogenesis. Despite intensive studies over the last 2 decades, the mechanism regulating the expression of the Pparg2 gene, especially the role of cis-regulatory elements, is still not completely understood. Here, we report a comprehensive investigation of the enhancer elements within the murine Pparg2 gene. Utilizing the combined techniques of sequence conservation analysis and chromatin marker examination, we identified a potent enhancer element that augmented the expression of a reporter gene under the control of the Pparg2 promoter by 20-fold. This enhancer element was first identified as highly conserved non-coding sequence 10 (CNS10) and was later shown to be enriched with the enhancer marker H3 K27 acetylation. Further studies identified a binding site for p300 as the essential enhancer element in CNS10. Moreover, p300 physically binds to CNS10 and is required for the enhancer activity of CNS10. The depletion of p300 by siRNA resulted in significantly impaired activation of Pparg2 at the early stages of 3T3-L1 adipogenesis. In summary, our study identified a novel enhancer element on the murine Pparg2 gene and suggested a novel mechanism for the regulation of Pparg2 expression by p300 in 3T3-L1 adipogenesis. PMID:25485585

  15. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase.

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

    Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

  16. Quantification of histone modifications by parallel-reaction monitoring: a method validation.

    PubMed

    Sowers, James L; Mirfattah, Barsam; Xu, Pei; Tang, Hui; Park, In Young; Walker, Cheryl; Wu, Ping; Laezza, Fernanda; Sowers, Lawrence C; Zhang, Kangling

    2015-10-01

    Abnormal epigenetic reprogramming is one of the major causes leading to irregular gene expression and regulatory pathway perturbations, in the cells, resulting in unhealthy cell development or diseases. Accurate measurements of these changes of epigenetic modifications, especially the complex histone modifications, are very important, and the methods for these measurements are not trivial. By following our previous introduction of PRM to targeting histone modifications (Tang, H.; Fang, H.; Yin, E.; Brasier, A. R.; Sowers, L. C.; Zhang, K. Multiplexed parallel reaction monitoring targeting histone modifications on the QExactive mass spectrometer. Anal. Chem. 2014, 86 (11), 5526-34), herein we validated this method by varying the protein/trypsin ratios via serial dilutions. Our data demonstrated that PRM with SILAC histones as the internal standards allowed reproducible measurements of histone H3/H4 acetylation and methylation in the samples whose histone contents differ at least one-order of magnitude. The method was further validated by histones isolated from histone H3 K36 trimethyltransferase SETD2 knockout mouse embryonic fibroblasts (MEF) cells. Furthermore, histone acetylation and methylation in human neural stem cells (hNSC) treated with ascorbic acid phosphate (AAP) were measured by this method, revealing that H3 K36 trimethylation was significantly down-regulated by 6 days of treatment with vitamin C.

  17. 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

  18. Histone Deacetylases

    PubMed Central

    Parbin, Sabnam; Kar, Swayamsiddha; Shilpi, Arunima; Sengupta, Dipta; Deb, Moonmoon; Rath, Sandip Kumar

    2014-01-01

    In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer. PMID:24051359

  19. 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. PMID:25501842

  20. Quickly evolving histones, nucleosome stability and chromatin folding: all about histone H2A.Bbd.

    PubMed

    González-Romero, Rodrigo; Méndez, Josefina; Ausió, Juan; Eirín-López, José M

    2008-04-30

    Histone H2A.Bbd (Barr body-deficient) is a novel histone variant which is largely excluded from the inactive X chromosome of mammals. Discovered only 6 years ago, H2A.Bbd displays very unusual structural and functional properties, for instance, it is relatively shorter and only 48% identical compared to H2A, lacking both the typical C-terminal tail of the H2A family and the very last sequence of the docking domain, making it the most specialized among all histone variants known to date. Indeed, molecular evolutionary analyses have shown that H2A.Bbd is a highly hypervariable and quickly evolving protein exclusive to mammalian lineages, in striking contrast to all other histones. Different studies have described a deposition pattern of H2A.Bbd in the chromatin that overlaps with regions of histone H4 acetylation suggesting its association with transcriptionally active euchromatic regions of the genome. In this regard, it is believed that this histone variant plays an important role in determining such regions by destabilizing the nucleosome and locally unfolding the chromatin fiber. This review provides a concise, comprehensive and timely summary of the work published on H2A.Bbd structure and function. Special emphasis is placed on its chromatin deposition patterns in relation to gene expression profiles and its evolutionary history, as well as on the dynamics of H2A.Bbd-containing nucleosomes.

  1. Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apc-minus mice.

    PubMed

    Myzak, Melinda C; Dashwood, W Mohaiza; Orner, Gayle A; Ho, Emily; Dashwood, Roderick H

    2006-03-01

    Sulforaphane (SFN) is an isothiocyanate from broccoli that induces phase 2 detoxification enzymes. We recently reported that SFN acts as a histone deacetylase (HDAC) inhibitor in human colon cancer cells in vitro, and the present study sought to extend these findings in vivo. In mice treated with a single oral dose of 10 mumol SFN, there was significant inhibition of HDAC activity in the colonic mucosa after 6 h, and immunoblots revealed a concomitant increase in acetylated histones H3 and H4, which returned to control levels by 48 h. Longer-term treatment with SFN in the diet resulted in levels of acetylated histones and p21(WAF1) in the ileum, colon, prostate, and peripheral blood mononuclear cells that were elevated compared with controls. Consistent with these findings, SFN suppressed tumor development in Apc(min) mice, and there was an increase in acetylated histones in the polyps, including acetylated histones specifically associated with the promoter region of the P21 and bax genes. These results provide the first evidence for HDAC inhibition by SFN in vivo and imply that such a mechanism might contribute to the cancer chemoprotective and therapeutic effects of SFN, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

  2. Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation.

    PubMed

    Feng, Xuejiao; Wang, Xiaoli; Cao, Xinyuan; Xia, Yankai; Zhou, Rong; Chen, Ling

    2015-12-01

    Perfluorooctane sulfonate (PFOS) at a high dose of 10 mg/kg has been reported to affect the neuroendocrine system and exert toxic effects in rodents. The present study examined the influence of chronic exposure to a low-dose of PFOS (0.1 mg/kg/day) on female reproductive endocrine and function. Herein, we show that adult female mice exposed to PFOS by gavage for 4 months (PFOS-mice) exhibited a prolongation of diestrus without signs of toxic effects. The numbers of mature follicles and corpora luteum were significantly reduced in PFOS-mice with increase of atresic follicles. The levels of serum estrogen (E2) and progesterone at proestrus and diestrus were reduced in PFOS-mice. In comparison with controls, PFOS-mice showed a significant decrease in the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), and gonadotrophin-releasing hormone, the number of kisspeptin neurons and the level of kiss1 mRNA in anteroventral periventricular nucleus at proestrus but not at diestrus, which could be corrected with the normalization to E2. PFOS-mice did not generate an LH-surge at proestrus, which could be rescued by the application of E2 or kisspeptin-10. Notably, the level of ovarian steroidogenic acute regulatory (StAR) mRNA was decreased in PFOS-mice with the reduction of histone H3K14 acetylation in StAR promoter relative to control mice, whereas the P450scc expression and histone H3K14 acetylation showed no difference between the groups. The present study provides evidence that the chronic exposure to the low-dose of PFOS through selectively reducing histone acetylation of StAR suppresses the biosynthesis of E2 to impair the follicular development and ovulation.

  3. Chronic Exposure of Female Mice to an Environmental Level of Perfluorooctane Sulfonate Suppresses Estrogen Synthesis Through Reduced Histone H3K14 Acetylation of the StAR Promoter Leading to Deficits in Follicular Development and Ovulation.

    PubMed

    Feng, Xuejiao; Wang, Xiaoli; Cao, Xinyuan; Xia, Yankai; Zhou, Rong; Chen, Ling

    2015-12-01

    Perfluorooctane sulfonate (PFOS) at a high dose of 10 mg/kg has been reported to affect the neuroendocrine system and exert toxic effects in rodents. The present study examined the influence of chronic exposure to a low-dose of PFOS (0.1 mg/kg/day) on female reproductive endocrine and function. Herein, we show that adult female mice exposed to PFOS by gavage for 4 months (PFOS-mice) exhibited a prolongation of diestrus without signs of toxic effects. The numbers of mature follicles and corpora luteum were significantly reduced in PFOS-mice with increase of atresic follicles. The levels of serum estrogen (E2) and progesterone at proestrus and diestrus were reduced in PFOS-mice. In comparison with controls, PFOS-mice showed a significant decrease in the levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), and gonadotrophin-releasing hormone, the number of kisspeptin neurons and the level of kiss1 mRNA in anteroventral periventricular nucleus at proestrus but not at diestrus, which could be corrected with the normalization to E2. PFOS-mice did not generate an LH-surge at proestrus, which could be rescued by the application of E2 or kisspeptin-10. Notably, the level of ovarian steroidogenic acute regulatory (StAR) mRNA was decreased in PFOS-mice with the reduction of histone H3K14 acetylation in StAR promoter relative to control mice, whereas the P450scc expression and histone H3K14 acetylation showed no difference between the groups. The present study provides evidence that the chronic exposure to the low-dose of PFOS through selectively reducing histone acetylation of StAR suppresses the biosynthesis of E2 to impair the follicular development and ovulation. PMID:26358002

  4. Molecular Modeling of Differentially Phosphorylated Serine 10 and Acetylated lysine 9/14 of Histone H3 Regulates their Interactions with 14-3-3ζ, MSK1, and MKP1

    PubMed Central

    Sharma, Ajit K.; Mansukh, Abhilasha; Varma, Ashok; Gadewal, Nikhil; Gupta, Sanjay

    2013-01-01

    Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure. PMID:24027420

  5. A histone code in meiosis: the histone kinase, NHK-1, is required for proper chromosomal architecture in Drosophila oocytes

    PubMed Central

    Ivanovska, Irena; Khandan, Tulasi; Ito, Takashi; Orr-Weaver, Terry L.

    2005-01-01

    To promote faithful propagation of the genetic material during sexual reproduction, meiotic chromosomes undergo specialized morphological changes that ensure accurate segregation of homologous chromosomes. The molecular mechanisms that establish the meiotic chromosomal structures are largely unknown. We describe a mutation in a recently identified Histone H2A kinase, nhk-1, in Drosophila that leads to female sterility due to defects in the formation of the meiotic chromosomal structures. The metaphase I arrest and the karyosome, a critical prophase I chromosomal structure, require nucleosomal histone kinase-1 (NHK-1) function. The defects are a result of failure to disassemble the synaptonemal complex and to load condensin onto the mutant chromosomes. Embryos laid by nhk-1-/- mutant females arrest with aberrant polar bodies and mitotic spindles, revealing that mitosis is affected as well. We analyzed the role of Histone H2A phosphorylation with respect to the histone code hypothesis and found that it is required for acetylation of Histone H3 and Histone H4 in meiosis. These studies reveal a critical role for histone modifications in chromosome dynamics in meiosis and mitosis. PMID:16230526

  6. Histone chaperones link histone nuclear import and chromatin assembly.

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

    Histone chaperones are proteins that shield histones from nonspecific interactions until they are assembled into chromatin. After their synthesis in the cytoplasm, histones are bound by different histone chaperones, subjected to a series of posttranslational modifications and imported into the nucleus. These evolutionarily conserved modifications, including acetylation and methylation, can occur in the cytoplasm, but their role in regulating import is not well understood. As part of histone import complexes, histone chaperones may serve to protect the histones during transport, or they may be using histones to promote their own nuclear localization. In addition, there is evidence that histone chaperones can play an active role in the import of histones. Histone chaperones have also been shown to regulate the localization of important chromatin modifying enzymes. This review is focused on the role histone chaperones play in the early biogenesis of histones, the distinct cytoplasmic subcomplexes in which histone chaperones have been found in both yeast and mammalian cells and the importins/karyopherins and nuclear localization signals that mediate the nuclear import of histones. We also address the role that histone chaperone localization plays in human disease. This article is part of a Special Issue entitled: Histone chaperones and chromatin assembly.

  7. 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

  8. The Histone Database: an integrated resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Levine, Kevin M; Morales, Mario; Zhang, Suiyuan; Moreland, R Travis; Baxevanis, Andreas D; Landsman, David

    2011-01-01

    Eukaryotic chromatin is composed of DNA and protein components-core histones-that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.

  9. The forced swimming-induced behavioural immobility response involves histone H3 phospho-acetylation and c-Fos induction in dentate gyrus granule neurons via activation of the N-methyl-D-aspartate/extracellular signal-regulated kinase/mitogen- and stress-activated kinase signalling pathway.

    PubMed

    Chandramohan, Yalini; Droste, Susanne K; Arthur, J Simon C; Reul, Johannes M H M

    2008-05-01

    The hippocampus is involved in learning and memory. Previously, we have shown that the acquisition of the behavioural immobility response after a forced swim experience is associated with chromatin modifications and transcriptional induction in dentate gyrus granule neurons. Given that both N-methyl-D-aspartate (NMDA) receptors and the extracellular signal-regulated kinases (ERK) 1/2 signalling pathway are involved in neuroplasticity processes underlying learning and memory, we investigated in rats and mice whether these signalling pathways regulate chromatin modifications and transcriptional events participating in the acquisition of the immobility response. We found that: (i) forced swimming evoked a transient increase in the number of phospho-acetylated histone H3-positive [P(Ser10)-Ac(Lys14)-H3(+)] neurons specifically in the middle and superficial aspects of the dentate gyrus granule cell layer; (ii) antagonism of NMDA receptors and inhibition of ERK1/2 signalling blocked forced swimming-induced histone H3 phospho-acetylation and the acquisition of the behavioural immobility response; (iii) double knockout (DKO) of the histone H3 kinase mitogen- and stress-activated kinases (MSK) 1/2 in mice completely abolished the forced swimming-induced increases in histone H3 phospho-acetylation and c-Fos induction in dentate granule neurons and the behavioural immobility response; (iv) blocking mineralocorticoid receptors, known not to be involved in behavioural immobility in the forced swim test, did not affect forced swimming-evoked histone H3 phospho-acetylation in dentate neurons; and (v) the pharmacological manipulations and gene deletions did not affect behaviour in the initial forced swim test. We conclude that the forced swimming-induced behavioural immobility response requires histone H3 phospho-acetylation and c-Fos induction in distinct dentate granule neurons through recruitment of the NMDA/ERK/MSK 1/2 pathway.

  10. Enzyme kinetics and inhibition of histone acetyltransferase KAT8

    PubMed Central

    Wapenaar, Hannah; van der Wouden, Petra E.; Groves, Matthew R.; Rotili, Dante; Mai, Antonello; Dekker, Frank J.

    2016-01-01

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of AA was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  11. Histone acetyltransferases regulate HIV-1 enhancer activity in vitro

    PubMed Central

    Sheridan, Philip L.; Mayall, Timothy P.; Verdin, Eric; Jones, Katherine A.

    1997-01-01

    Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity. PMID:9407026

  12. Methylation of histone H3 at lysine 4 and expression of the maltase-glucoamylase gene are reduced by dietary resistant starch.

    PubMed

    Shimada, Masaya; Mochizuki, Kazuki; Goda, Toshinao

    2013-03-01

    Methylated histone H3 at lysine 4 (K4) is associated with euchromatin and is involved in the transactivation of genes. However, it is unknown whether histone methylation is involved with changes in gene expression induced by nutrients. In this study, we examined whether methylations of histone H3 at K4 on maltase-glucoamylase (Mgam), which is responsible for the digestion of starch in the small intestine, as well as Mgam expression were altered by feeding rats an indigestible starch (resistant starch, RS). The mRNA and protein levels and the activities of MGAM were reduced in rats fed an RS diet compared with those fed a regular starch diet. Furthermore, we found that decreases in di- and tri-methylation of histone H3 at K4, as well as reduced acetylation of histones H3 and H4 on the Mgam gene were associated with a reduction of Mgam gene expression. These results suggest that the reductions of jejunal MGAM levels and activities caused by the RS diet are regulated at the mRNA level through a decrease in methylation of histone H3 at K4 and reduced acetylation of histones H3 and H4 on the Mgam gene.

  13. Simvastatin enhances NMDA receptor GluN2B expression and phosphorylation of GluN2B and GluN2A through increased histone acetylation and Src signaling in hippocampal CA1 neurons.

    PubMed

    Chen, Tingting; Zhang, Baofeng; Li, Guoxi; Chen, Lei; Chen, Ling

    2016-08-01

    Simvastatin (SV) can improve cognitive deficits in Alzheimer's disease patients and mice. Herein, we report that the administration of SV (20 mg/kg) for 5 days in mice (SV-mice) or the treatment of slices with SV (10 μM) for 4 h (SV-slices) could increase the density of NMDA-evoked inward currents (INMDA) in hippocampal CA1 pyramidal cells, which were blocked by farnesol (FOH) that converts farnesyl pyrophosphate (FPP), but not geranylgeraniol (GGOH) that increases geranylgeranylpyrophosphate (GGPP). Sensitivity of INMDA to ifenprodil in SV-mice or SV-slices was significantly increased. The levels of hippocampal GluN2B and GluN2A or Src phosphorylation in SV-mice or SV-slices were higher than controls, which were sensitive to FOH. The Src inhibitor PP2 could inhibit the SV-enhanced phosphorylation of GluN2B and GluN2A and SV-augmented INMDA, but PI3K inhibitor LY294002 did not. The levels of GluN2B mRNA and protein were elevated in SV-mice, which was abolished by FOH, but not by GGOH or PP2. Furthermore, the histone H3K9 and H3K27 acetylation of GluN2B promoter was increased in SV-mice, which was suppressed by FOH rather than GGOH or PP2. In control mice and slices, the reduction of FPP by farnesyl transferase inhibitor could increase the levels of GluN2B expression, the histone H3K9 and H3K27 acetylation and enhance the phosphorylation of GluN2B, GluN2A and Src. The findings indicate that the administration of SV can enhance GluN2B expression and GluN2B and GluN2A phosphorylation leading to augmentation of NMDAR activity through reducing FPP to increase histone acetylation of GluN2B and Src signaling.

  14. Interplay Between Histone H3 Lysine 56 Deacetylation and Chromatin Modifiers in Response to DNA Damage

    PubMed Central

    Simoneau, Antoine; Delgoshaie, Neda; Celic, Ivana; Dai, Junbiao; Abshiru, Nebiyu; Costantino, Santiago; Thibault, Pierre; Boeke, Jef D.; Verreault, Alain; Wurtele, Hugo

    2015-01-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage–induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage–induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage. PMID:25786853

  15. Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    PubMed Central

    2013-01-01

    Background MYST1 (also known as hMOF), a member of the MYST family of histone acetyltransferases (HATs) as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC). Simultaneously, we investigated the correlation between the expression of hMOF and clear cell RCC (ccRCC) biomarker carbohydrase IX (CA9) in RCC. Materials and methods The frozen RCC tissues and RCC cell lines as materials, the reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunohistochemical staining approaches were used. Results RT-PCR results indicate that hMOF gene expression levels frequently downregulated in 90.5% of patients (19/21) with RCC. The reduction of hMOF protein in both RCC tissues and RCC cell lines is tightly correlated with acetylation of histone H4K16. In addition, overexpression of CA9 was detected in 100% of ccRCC patients (21/21). However, transient transfection of hMOF in ccRCC 786–0 cells did not affect both the gene and protein expression of CA9. Conclusion hMOF as an acetyltransferase of H4K16 might be involved in the pathogenesis of kidney cancer, and this epigenetic changes might be a new CA9-independent RCC diagnostic maker. PMID:23394073

  16. Exposure of preimplantation embryos to low-dose bisphenol A impairs testes development and suppresses histone acetylation of StAR promoter to reduce production of testosterone in mice.

    PubMed

    Hong, Juan; Chen, Fang; Wang, Xiaoli; Bai, Yinyang; Zhou, Rong; Li, Yingchun; Chen, Ling

    2016-05-15

    Previous studies have shown that bisphenol A (BPA) is a potential endocrine disruptor and testicular toxicant. The present study focused on exploring the impact of exposure to low dose of BPA on male reproductive development during the early embryo stage and the underlying mechanisms. BPA (20 μg/kg/day) was orally administered to female mice on days 1-5 of gestation. The male offspring were euthanized at PND10, 20, 24, 35 or PND50. We found that the mice exposed to BPA before implantation (BPA-mice) displayed retardation of testicular development with reduction of testosterone level. The diameter and epithelium height of seminiferous tubules were reduced in BPA-mice at PND35. The numbers of spermatogenic cells at different stages were significantly reduced in BPA-mice at PND50. BPA-mice showed a persistent reduction in serum and testicular testosterone levels starting from PND24, whereas GnRH mRNA was significantly increased at PND35 and PND50. The expressions of testicular StAR and P450scc in BPA-mice also decreased relative to those of the controls at PND35 and PND50. Further analysis found that the levels of histone H3 and H3K14 acetylation (Ac-H3 and H3K14ac) in the promoter of StAR were decreased relative to those of control mice, whereas the level of Ac-H3 in the promoter of P450scc was not significantly different between the groups. These results provide evidence that exposure to BPA in preimplantation embryo retards the development of testes by reducing histone acetylation of the StAR promoter to disrupt the testicular testosterone synthesis.

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  18. Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα

    PubMed Central

    Chen, Yi; Li, Jiaqi; Qian, Shanhu; Shi, Yifen; Sun, Lan; Han, Yixiang; Zhang, Shenghui; Yu, Kang

    2016-01-01

    Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity. PMID:27058040

  19. Histone Deacetylase Inhibitors Trichostatin A and MCP30 Relieve Benzene-Induced Hematotoxicity via Restoring Topoisomerase IIα.

    PubMed

    Chen, Jingjing; Zheng, Zhouyi; Chen, Yi; Li, Jiaqi; Qian, Shanhu; Shi, Yifen; Sun, Lan; Han, Yixiang; Zhang, Shenghui; Yu, Kang

    2016-01-01

    Dysfunction of histone acetylation inhibits topoisomerase IIα (Topo IIα), which is implicated in benzene-induced hematotoxicity in patients with chronic benzene exposure. Whether histone deacetylase (HDAC) inhibitors can relieve benzene-induced hematotoxicity remains unclear. Here we showed that hydroquinone, a main metabolite of benzene, increased the HDAC activity, decreased the Topo IIα expression and induced apoptosis in human bone marrow mononuclear cells in vitro, and treatment with two HDAC inhibitors, namely trichostatin A (TSA) or a mixture of ribosome-inactivating proteins MCP30, almost completely reversed these effects. We further established a benzene poisoning murine model by inhaling benzene vapor in a container and found that benzene poisoning decreased the expression and activity of Topo IIα, and impaired acetylation of histone H4 and H3. The analysis of regulatory factors of Topo IIα promoter found that benzene poisoning decreased the mRNA levels of SP1 and C-MYB, and increased the mRNA level of SP3. Both TSA and MCP30 significantly enhanced the acetylation of histone H3 and H4 in Topo IIα promoter and increased the expression and activity of Topo IIα in benzene poisoning mice, which contributed to relieve the symptoms of hematotoxicity. Thus, treatment with HDAC inhibitors represents an attractive approach to reduce benzene-induced hematotoxicity.

  20. Yeast Enhancer of Polycomb defines global Esa1-dependent acetylation of chromatin

    PubMed Central

    Boudreault, Alexandre A.; Cronier, Dominique; Selleck, William; Lacoste, Nicolas; Utley, Rhea T.; Allard, Stéphane; Savard, Julie; Lane, William S.; Tan, Song; Côté, Jacques

    2003-01-01

    Drosophila Enhancer of Polycomb, E(Pc), is a suppressor of position-effect variegation and an enhancer of both Polycomb and trithorax mutations. A homologous yeast protein, Epl1, is a subunit of the NuA4 histone acetyltransferase complex. Epl1 depletion causes cells to accumulate in G2/M and global loss of acetylated histones H4 and H2A. In relation to the Drosophila protein, mutation of Epl1 suppresses gene silencing by telomere position effect. Epl1 protein is found in the NuA4 complex and a novel highly active smaller complex named Piccolo NuA4 (picNuA4). The picNuA4 complex contains Esa1, Epl1, and Yng2 as subunits and strongly prefers chromatin over free histones as substrate. Epl1 conserved N-terminal domain bridges Esa1 and Yng2 together, stimulating Esa1 catalytic activity and enabling acetylation of chromatin substrates. A recombinant picNuA4 complex shows characteristics similar to the native complex, including strong chromatin preference. Cells expressing only the N-terminal half of Epl1 lack NuA4 HAT activity, but possess picNuA4 complex and activity. These results indicate that the essential aspect of Esa1 and Epl1 resides in picNuA4 function. We propose that picNuA4 represents a nontargeted histone H4/H2A acetyltransferase activity responsible for global acetylation, whereas the NuA4 complex is recruited to specific genomic loci to perturb locally the dynamic acetylation/deacetylation equilibrium. PMID:12782659

  1. Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy

    PubMed Central

    Peng, Chang; Zhu, Jing; Sun, Hui-Chao; Huang, Xu-Pei; Zhao, Wei-An; Zheng, Min; Liu, Ling-Juan; Tian, Jie

    2014-01-01

    Background Cardiovascular malformations can be caused by abnormalities in Gata4 expression during fetal development. In a previous study, we demonstrated that ethanol exposure could lead to histone hyperacetylation and Gata4 over-expression in fetal mouse hearts. However, the potential mechanisms of histone hyperacetylation and Gata4 over-expression induced by ethanol remain unclear. Methods and Results Pregnant mice were gavaged with ethanol or saline. Fetal mouse hearts were collected for analysis. The results of ethanol fed groups showed that global HAT activity was unusually high in the hearts of fetal mice while global HDAC activity remained unchanged. Binding of P300, CBP, PCAF, SRC1, but not GCN5, were increased on the Gata4 promoter relative to the saline treated group. Increased acetylation of H3K9 and increased mRNA expression of Gata4, α-MHC, cTnT were observed in these hearts. Treatment with the pan-histone acetylase inhibitor, anacardic acid, reduced the binding of P300, PCAF to the Gata4 promoter and reversed H3K9 hyperacetylation in the presence of ethanol. Interestingly, anacardic acid attenuated over-expression of Gata4, α-MHC and cTnT in fetal mouse hearts exposed to ethanol. Conclusions Our results suggest that P300 and PCAF may be critical regulatory factors that mediate Gata4 over-expression induced by ethanol exposure. Alternatively, P300, PCAF and Gata4 may coordinate over-expression of cardiac downstream genes in mouse hearts exposed to ethanol. Anacardic acid may thus protect against ethanol-induced Gata4, α-MHC, cTnT over-expression by inhibiting the binding of P300 and PCAF to the promoter region of these genes. PMID:25101666

  2. Beyond Histone and Deacetylase: An Overview of Cytoplasmic Histone Deacetylases and Their Nonhistone Substrates

    PubMed Central

    Yao, Ya-Li; Yang, Wen-Ming

    2011-01-01

    Acetylation of lysines is a prominent form of modification in mammalian proteins. Deacetylation of proteins is catalyzed by histone deacetylases, traditionally named after their role in histone deacetylation, transcriptional modulation, and epigenetic regulation. Despite the link between histone deacetylases and chromatin structure, some of the histone deacetylases reside in various compartments in the cytoplasm. Here, we review how these cytoplasmic histone deacetylases are regulated, the identification of nonhistone substrates, and the functional implications of their nondeacetylase enzymatic activities. PMID:21234400

  3. Molecular Basis of Histone Tail Recognition by Human TIP5 PHD Finger and Bromodomain of the Chromatin Remodeling Complex NoRC

    PubMed Central

    Tallant, Cynthia; Valentini, Erica; Fedorov, Oleg; Overvoorde, Lois; Ferguson, Fleur M.; Filippakopoulos, Panagis; Svergun, Dmitri I.; Knapp, Stefan; Ciulli, Alessio

    2015-01-01

    Summary Binding of the chromatin remodeling complex NoRC to RNA complementary to the rDNA promoter mediates transcriptional repression. TIP5, the largest subunit of NoRC, is involved in recruitment to rDNA by interactions with promoter-bound TTF-I, pRNA, and acetylation of H4K16. TIP5 domains that recognize posttranslational modifications on histones are essential for recruitment of NoRC to chromatin, but how these reader modules recognize site-specific histone tails has remained elusive. Here, we report crystal structures of PHD zinc finger and bromodomains from human TIP5 and BAZ2B in free form and bound to H3 and/or H4 histones. PHD finger functions as an independent structural module in recognizing unmodified H3 histone tails, and the bromodomain prefers H3 and H4 acetylation marks followed by a key basic residue, KacXXR. Further low-resolution analyses of PHD-bromodomain modules provide molecular insights into their trans histone tail recognition, required for nucleosome recruitment and transcriptional repression of the NoRC complex. PMID:25533489

  4. Trichostatin A, a histone deacetylase inhibitor, suppresses JAK2/STAT3 signaling via inducing the promoter-associated histone acetylation of SOCS1 and SOCS3 in human colorectal cancer cells.

    PubMed

    Xiong, Hua; Du, Wan; Zhang, Yan-Jie; Hong, Jie; Su, Wen-Yu; Tang, Jie-Ting; Wang, Ying-Chao; Lu, Rong; Fang, Jing-Yuan

    2012-02-01

    Aberrant janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is involved in the oncogenesis of several cancers. Suppressors of cytokine signaling (SOCS) genes and SH2-containing protein tyrosine phosphatase 1 (SHP1) proteins, which are negative regulators of JAK/STAT signaling, have been reported to have tumor suppressor functions. However, in colorectal cancer (CRC) cells, the mechanisms that regulate SOCS and SHP1 genes, and the cause of abnormalities in the JAK/STAT signaling pathway, remain largely unknown. The present study shows that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, leads to the hyperacetylation of histones associated with the SOCS1 and SOCS3 promoters, but not the SHP1 promoter in CRC cells. This indicates that histone modifications are involved in the regulation of SOCS1 and SOCS3. Moreover, upregulation of SOCS1 and SOCS3 expression was achieved using TSA, which also significantly downregulated JAK2/STAT3 signaling in CRC cells. We also demonstrate that TSA suppresses the growth of CRC cells, and induces G1 cell cycle arrest and apoptosis through the regulation of downstream targets of JAK2/STAT3 signaling, including Bcl-2, survivin and p16(ink4a) . Therefore, our data demonstrate that TSA may induce SOCS1 and SOCS3 expression by inducing histone modifications and consequently inhibits JAK2/STAT3 signaling in CRC cells. These results also establish a mechanistic link between the inhibition of JAK2/STAT3 signaling and the anticancer action of TSA in CRC cells.

  5. Autoantibodies against Modified Histone Peptides in SLE Patients Are Associated with Disease Activity and Lupus Nephritis

    PubMed Central

    Dieker, Jürgen; Berden, Jo H.; Bakker, Marinka; Briand, Jean-Paul; Muller, Sylviane; Voll, Reinhard; Sjöwall, Christopher; Herrmann, Martin; Hilbrands, Luuk B.; van der Vlag, Johan

    2016-01-01

    Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis. PMID:27780265

  6. The Influence of Ionic Environment and Histone Tails on Columnar Order of Nucleosome Core Particles.

    PubMed

    Berezhnoy, Nikolay V; Liu, Ying; Allahverdi, Abdollah; Yang, Renliang; Su, Chun-Jen; Liu, Chuan-Fa; Korolev, Nikolay; Nordenskiöld, Lars

    2016-04-26

    The nucleosome core particle (NCP) is the basic building block of chromatin. Nucleosome-nucleosome interactions are instrumental in chromatin compaction, and understanding NCP self-assembly is important for understanding chromatin structure and dynamics. Recombinant NCPs aggregated by multivalent cations form various ordered phases that can be studied by x-ray diffraction (small-angle x-ray scattering). In this work, the effects on the supramolecular structure of aggregated NCPs due to lysine histone H4 tail acetylations, histone H2A mutations (neutralizing the acidic patch of the histone octamer), and the removal of histone tails were investigated. The formation of ordered mainly hexagonal columnar NCP phases is in agreement with earlier studies; however, the highly homogeneous recombinant NCP systems used in this work display a more compact packing. The long-range order of the NCP columnar phase was found to be abolished or reduced by acetylation of the H4 tails, acidic patch neutralization, and removal of the H3 and H2B tails. Loss of nucleosome stacking upon removal of the H3 tails in combination with other tails was observed. In the absence of the H2A tails, the formation of an unknown highly ordered phase was observed. PMID:27119633

  7. Arabidopsis MRG domain proteins bridge two histone modifications to elevate expression of flowering genes.

    PubMed

    Xu, Yifeng; Gan, Eng-Seng; Zhou, Jie; Wee, Wan-Yi; Zhang, Xiaoyu; Ito, Toshiro

    2014-01-01

    Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3'-end enrichment in animals. The MRG15 family proteins function as 'reader' proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5' regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function.

  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. PMID:25656994

  9. Transcriptional regulation of cell cycle genes in response to abiotic stresses correlates with dynamic changes in histone modifications in maize.

    PubMed

    Zhao, Lin; Wang, Pu; Hou, Haoli; Zhang, Hao; Wang, Yapei; Yan, Shihan; Huang, Yan; Li, Hui; Tan, Junjun; Hu, Ao; Gao, Fei; Zhang, Qi; Li, Yingnan; Zhou, Hong; Zhang, Wei; Li, Lijia

    2014-01-01

    The histone modification level has been shown to be related with gene activation and repression in stress-responsive process, but there is little information on the relationship between histone modification and cell cycle gene expression responsive to environmental cues. In this study, the function of histone modifications in mediating the transcriptional regulation of cell cycle genes under various types of stress was investigated in maize (Zea mays L.). Abiotic stresses all inhibit the growth of maize seedlings, and induce total acetylation level increase compared with the control group in maize roots. The positive and negative regulation of the expression of some cell cycle genes leads to perturbation of cell cycle progression in response to abiotic stresses. Chromatin immunoprecipitation analysis reveals that dynamic histone acetylation change in the promoter region of cell cycle genes is involved in the control of gene expression in response to external stress and different cell cycle genes have their own characteristic patterns for histone acetylation. The data also showed that the combinations of hyperacetylation and hypoacetylation states of specific lysine sites on the H3 and H4 tails on the promoter regions of cell cycle genes regulate specific cell cycle gene expression under abiotic stress conditions, thus resulting in prolonged cell cycle duration and an inhibitory effect on growth and development in maize seedlings. PMID:25171199

  10. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, attenuates postoperative cognitive dysfunction in aging mice

    PubMed Central

    Jia, Min; Liu, Wen-Xue; Sun, He-Liang; Chang, Yan-Qing; Yang, Jiao-Jiao; Ji, Mu-Huo; Yang, Jian-Jun; Feng, Chen-Zhuo

    2015-01-01

    Postoperative cognitive dysfunction (POCD) is a recognized clinical entity characterized with cognitive deficits after anesthesia and surgery, especially in aged patients. Previous studies have shown that histone acetylation plays a key role in hippocampal synaptic plasticity and memory formation. However, its role in POCD remains to be determined. Here, we show that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, attenuates POCD in aging Mice. After exposed to the laparotomy, a surgical procedure involving an incision into abdominal walls to examine the abdominal organs, 16- but not 3-month old male C57BL/6 mice developed obvious cognitive impairments in the test of long-term contextual fear conditioning. Intracerebroventricular (i.c.v.) injection of SAHA at the dose of (20 μg/2 μl) 3 h before and daily after the laparotomy restored the laparotomy-induced reduction of hippocampal acetyl-H3 and acetyl-H4 levels and significantly attenuated the hippocampus-dependent long-term memory (LTM) impairments in 16-month old mice. SAHA also reduced the expression of cleaved caspase-3, inducible nitric oxide synthase (iNOS) and N-methyl-D-aspartate (NMDA) receptor-calcium/calmodulin dependent kinase II (CaMKII) pathway, and increased the expression of brain-derived neurotrophic factor (BDNF), synapsin 1, and postsynaptic density 95 (PSD95). Taken together, our data suggest that the decrease of histone acetylation contributes to POCD and may serve as a target to improve the neurological outcome of POCD. PMID:26441515

  11. Trimethylation of histone H3K4 is associated with the induction of fructose-inducible genes in rat jejunum.

    PubMed

    Yoshinaga, Yumiko; Mochizuki, Kazuki; Goda, Toshinao

    2012-03-23

    We previously reported that fructose force-feeding rapidly induces jejunal Slc2a5 gene expression in rats. In this study, we conducted microarray analyses using total RNA to identify genes upregulated in rat jejunum by fructose force-feeding. Rats were force-fed fructose, glucose or distilled water for 6h. Genes such as Slc2a5, Cdkn1c, Cabp2, Ranbp3, Vwce and Gcgr were induced by force-feeding with fructose compared with glucose or distilled water. Chromatin immunoprecipitation assays revealed that trimethylation of histone H3K4, and acetylation of histones H3 and H4, on the transcribed region of these fructose-inducible genes were enhanced by force-feeding of fructose, but not glucose or distilled water. These results suggest that the induction of genes in the rat jejunum by fructose force-feeding is coordinately regulated by histone modifications, particularly trimethylation of histone H3K4.

  12. Analysis of histones and histone variants in plants.

    PubMed

    Trivedi, Ila; Rai, Krishan Mohan; Singh, Sunil Kumar; Kumar, Verandra; Singh, Mala; Ranjan, Amol; Lodhi, Niraj; Sawant, Samir V

    2012-01-01

    Histone proteins are the major protein components of chromatin - the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. For many years, histones were considered passive structural components of eukaryotic chromatin. In recent years, it has been demonstrated that dynamic association of histones and their variants to the genome plays a very important role in gene regulation. Histones are extensively modified during posttranslation viz. acetylation, methylation, phosphorylation, ubiquitylation, etc., and the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Different biochemical techniques have been developed to purify and separate histone proteins; here, we describe techniques for analysis of histones from plant tissues.

  13. Distinct and Redundant Roles of the Two MYST Histone Acetyltransferases Esa1 and Sas2 in Cell Growth and Morphogenesis of Candida albicans

    PubMed Central

    Wang, Xiongjun; Chang, Peng; Ding, Jianping

    2013-01-01

    Candida albicans is associated with humans, as both a harmless commensal organism and a pathogen. Adaption to human body temperature is extremely important for its growth and morphogenesis. Saccharomyces cerevisiae Esa1, a member of the MYST family HATs (histone acetyltransferases) and the catalytic subunit of the NuA4 complex, and its homologues in other eukaryotes have been shown to be essential for cell growth. To investigate the functional roles of two MYST family HATs, Esa1 and Sas2 in C. albicans, we deleted ESA1 and SAS2 in the C. albicans genome and performed cell growth analyses. Our results demonstrated that C. albicans Esa1 is not essential for general growth but is essential for filamentous growth. The esa1/esa1 mutant cells exhibited sensitivity to thermal, genotoxic, and oxidative stresses but tolerance to cold, osmotic, and cell wall stresses. In contrast, the sas2/sas2 mutant adapted to growth at higher temperatures and promoted filament formation at lower temperatures, resembling the phenotype of a C. albicans strain overexpressing ESA1. Cells with deletions of both ESA1 and SAS2 were inviable, reflecting the functional redundancy in cell growth. C. albicans Esa1 and Sas2 have distinct and synergistic effects on histone acetylation at H4K5, H4K12, and H4K16. Esa1 contributes mainly to acetylation of H4K5 and H4K12, whereas Sas2 contributes to acetylation of H4K16. Our findings suggest that C. albicans Esa1 and Sas2 play opposite roles in cell growth and morphogenesis and contribute coordinately to histone acetylation and gene regulation. PMID:23355007

  14. The Histone Database: a comprehensive resource for histones and histone fold-containing proteins.

    PubMed

    Mariño-Ramírez, Leonardo; Hsu, Benjamin; Baxevanis, Andreas D; Landsman, David

    2006-03-01

    The Histone Database is a curated and searchable collection of full-length sequences and structures of histones and nonhistone proteins containing histone-like folds, compiled from major public databases. Several new histone fold-containing proteins have been identified, including the huntingtin-interacting protein HYPM. Additionally, based on the recent crystal structure of the Son of Sevenless protein, an interpretation of the sequence analysis of the histone fold domain is presented. The database contains an updated collection of multiple sequence alignments for the four core histones (H2A, H2B, H3, and H4) and the linker histones (H1/H5) from a total of 975 organisms. The database also contains information on the human histone gene complement and provides links to three-dimensional structures of histone and histone fold-containing proteins. The Histone Database is a comprehensive bioinformatics resource for the study of structure and function of histones and histone fold-containing proteins. The database is available at http://research.nhgri.nih.gov/histones/.

  15. 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

  16. The epigenetic effects of aspirin: the modification of histone H3 lysine 27 acetylation in the prevention of colon carcinogenesis in azoxymethane- and dextran sulfate sodium-treated CF-1 mice.

    PubMed

    Guo, Yue; Liu, Yue; Zhang, Chengyue; Su, Zheng-Yuan; Li, Wenji; Huang, Mou-Tuan; Kong, Ah-Ng

    2016-06-01

    Colorectal cancer (CRC) is the third most common cancer worldwide. Chronic inflammation appears to enhance the risk of CRC. Emerging evidence has suggested that epigenetic mechanisms play an important role in CRC. Aspirin [acetylsalicylic acid (ASA)] has been shown to prevent CRC; however, the epigenetic mechanisms of its action remain unknown. This study investigated the protective role of ASA in azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted colitis-associated colon cancer (CAC) and examined the epigenetic effects, particularly on histone 3 lysine 27 acetylation (H3K27ac), underlying the preventive effect of ASA. CF-1 mice were fed with AIN-93M diet with or without 0.02% ASA from 1 week prior to AOM initiation until the mice were killed 20 weeks after AOM injection. Our results showed that AOM/DSS + ASA significantly suppressed inflammatory colitis symptoms and tumor multiplicity. AOM/DSS + ASA reduced AOM/DSS-induced protein expression and the activity of histone deacetylases (HDACs) and globally restored H3K27ac. Furthermore, AOM/DSS + ASA inhibited AOM/DSS-induced enrichment of H3K27ac in the promoters of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) that corresponded to the dramatic suppression of the messenger RNA (mRNA) and protein levels. Surprisingly, no significant changes in the H3K27ac abundance in the prostaglandin-endoperoxide synthase 2 (Cox-2) promoters or in the Cox-2 mRNA and protein expression were observed. Collectively, our results suggest that a potential novel epigenetic mechanism underlies the chemopreventive effects of ASA, and this mechanism attenuates CAC in AOM/DSS-induced CF-1 mice via the inhibition of HDACs and the modification of H3K27ac marks that suppress iNOS, TNF-α and IL-6. PMID:27207670

  17. FASIL-MS: An Integrated Proteomic and Bioinformatic Workflow To Universally Quantitate In Vivo-Acetylated Positional Isomers.

    PubMed

    Vitko, Dijana; Májek, Peter; Schirghuber, Erika; Kubicek, Stefan; Bennett, Keiryn L

    2016-08-01

    Dynamic changes in histone post-translational modifications (PTMs) regulate gene transcription leading to fine-tuning of biological processes such as DNA replication and cell cycle progression. Moreover, specific histone modifications constitute docking sites for recruitment of DNA damage repair proteins and mediation of subsequent cell survival. Therefore, understanding and monitoring changes in histone PTMs that can alter cell proliferation and thus lead to disease progression are of considerable medical interest. In this study, stable isotope labeling with N-acetoxy-D3-succinimide (D3-NAS) was utilized to efficiently derivatize unmodified lysine residues at the protein level. The sample preparation method was streamlined to facilitate buffer exchange between the multiple steps of the protocol by coupling chemical derivatization to filter-aided sample preparation (FASP). Additionally, the mass spectrometry method was adapted to simultaneously coisolate and subsequently cofragment all differentially H3/D3-acetylated histone peptide clusters. Combination of these multiplexed MS(2) spectra with the implementation of a data analysis algorithm enabled the quantitation of each and every in vivo-acetylated DMSO- and SAHA-treated H4(4-17) and H3(18-26) peptide. We have termed our new approach FASIL-MS for filter-aided stable isotopic labeling coupled to mass spectrometry. FASIL-MS enables the universal and site-specific quantitation of peptides with multiple in vivo-acetylated lysine residues. Data are available via ProteomeXchange (PXD003611). PMID:27302567

  18. Neonatal exposure to benzo[a]pyrene decreases the levels of serum testosterone and histone H3K14 acetylation of the StAR promoter in the testes of SD rats.

    PubMed

    Liang, Jiren; Zhu, Hongyan; Li, Cuizhen; Ding, Yicheng; Zhou, Zhijun; Wu, Qing

    2012-12-16

    Although benzo[a]pyrene (BaP) is an environmental endocrine disrupter, it has been unclear whether neonatal exposure to BaP affects the testosterone level and, if so, whether this influence persists into adulthood. In this present study, we gave neonatal rats (through oral gavages) doses of 0, 5, 10, or 25mg/kg day of BaP in corn oil from postnatal day 1 (PND 1) to PND 7. The rats were sacrificed at PND 8, PND 35, and PND 90. BaP exposure was confirmed through the induction of liver and testis CYP1A1 mRNA expression at PND 8 (i.e., immediately after exposure). The testicular daily sperm production and the sperm counts of the epididymis cauda at PND 90 were significantly lower than those of the control. The serum testosterone levels decreased markedly at PND 8, PND 35, and PND 90 after neonatal BaP exposure relative to those of the control. The mRNA expressions of StAR also decreased relative to those of the control at PND 8, PND 35, and PND 90, although the mRNA expressions of P450c17 and 17β-HSD were suppressed significantly only at PND 8. To further elucidate the mechanism of the persistent decrease in the mRNA expression of StAR, we determined the histone acetylation level in the StAR promoter. The extent of acetylation of H3K14 in the determined region decreased after neonatal exposure to BaP; this phenomenon persisted to the adult stage. Our results indicate that neonatal exposure to BaP damages testosterone production and sperm counts in the long term, possibly as a result of epigenetic regulation in the StAR promoter region.

  19. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex*

    PubMed Central

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.

    2010-01-01

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  20. Regulation of the p19Arf/p53 pathway by histone acetylation underlies neural stem cell behavior in senescence-prone SAMP8 mice

    PubMed Central

    Soriano-Cantón, Raúl; Perez-Villalba, Ana; Morante-Redolat, José Manuel; Marqués-Torrejón, María Ángeles; Pallás, Mercé; Pérez-Sánchez, Francisco; Fariñas, Isabel

    2015-01-01

    Brain aging is associated with increased neurodegeneration and reduced neurogenesis. B1/neural stem cells (B1-NSCs) of the mouse subependymal zone (SEZ) support the ongoing production of olfactory bulb interneurons, but their neurogenic potential is progressively reduced as mice age. Although age-related changes in B1-NSCs may result from increased expression of tumor suppressor proteins, accumulation of DNA damage, metabolic alterations, and microenvironmental or systemic changes, the ultimate causes remain unclear. Senescence-accelerated-prone mice (SAMP8) relative to senescence-accelerated-resistant mice (SAMR1) exhibit signs of hastened senescence and can be used as a model for the study of aging. We have found that the B1-NSC compartment is transiently expanded in young SAMP8 relative to SAMR1 mice, resulting in disturbed cytoarchitecture of the SEZ, B1-NSC hyperproliferation, and higher yields of primary neurospheres. These unusual features are, however, accompanied by premature loss of B1-NSCs. Moreover, SAMP8 neurospheres lack self-renewal and enter p53-dependent senescence after only two passages. Interestingly, in vitro senescence of SAMP8 cells could be prevented by inhibition of histone acetyltransferases and mimicked in SAMR1 cells by inhibition of histone deacetylases (HDAC). Our data indicate that expression of the tumor suppressor p19, but not of p16, is increased in SAMP8 neurospheres, as well as in SAMR1 neurospheres upon HDAC inhibition, and suggest that the SAMP8 phenotype may, at least in part, be due to changes in chromatin status. Interestingly, acute HDAC inhibition in vivo resulted in changes in the SEZ of SAMR1 mice that resembled those found in young SAMP8 mice. PMID:25728253

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

    PubMed Central

    Zhang, Hao; Wang, Pu; Hou, Haoli; Wen, Huan; Zhou, Hong; Gao, Fei; Wu, Jinping; Qiu, Zhengming; Li, Lijia

    2016-01-01

    Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph) signals was altered under OA treatment. Reactive oxygen species (ROS) was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL) assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac) level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation. PMID:27196101

  2. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis.

  3. The enhancing effect of genistein on apoptosis induced by trichostatin A in lung cancer cells with wild type p53 genes is associated with upregulation of histone acetyltransferase.

    PubMed

    Wu, Tzu-Chin; Lin, Yi-Chin; Chen, Hsiao-Ling; Huang, Pei-Ru; Liu, Shang-Yu; Yeh, Shu-Lan

    2016-02-01

    Genistein has been shown to enhance the antitumor activity of trichostatin A (TSA) in human lung carcinoma A549 cells. However, whether the combined treatment exerts the same effect in other lung cancer cells is unclear. In the present study we first compared the enhancing effect of genistein on the antitumor effect of TSA in ABC-1, NCI-H460 (H460) and A549 cells. Second, we investigated whether the effects of genistein are associated with increased histone/non-histone protein acetylation. We found that the enhancing effect of genistein on cell-growth-arrest in ABC-1 cells (p53 mutant) was less than in A549 and H460 cells. Genistein enhanced TSA induced apoptosis in A549 and H460 cells rather than in ABC-1 cells. After silencing p53 expression in A549 and H460 cells, the enhancing effect of genistein was diminished. In addition, genistein increased TSA-induced histone H3/H4 acetylation in A549 and H460 cells. Genistein also increased p53 acetylation in H460 cells. The inhibitor of acetyltransferase, anacardic acid, diminished the enhancing effect of genistein on all TSA-induced histone/p53 acetylation and apoptosis. Genistein in combination with TSA increased the expression of p300 protein, an acetyltransferase, in A549 and NCI-H460 cells. Furthermore, we demonstrated that genistein also enhanced the antitumor effect of genistein in A549-tumor-bearing mice. Taken together, these results suggest that the enhancing effects of genistein on TSA-induced apoptosis in lung cancer cells were p53-dependent and were associated with histone/non-histone protein acetylation.

  4. Histone H3 methylation at lysine 4 on the SLC2A5 gene in intestinal Caco-2 cells is involved in SLC2A5 expression.

    PubMed

    Inamochi, Yuko; Mochizuki, Kazuki; Osaki, Ayumi; Ishii, Takeshi; Nakayama, Tsutomu; Goda, Toshinao

    2010-01-29

    Histone H3 methylation at lysine 4 (K4) is associated with euchromatic regions and is thought to be important for the transcriptional activation of genes during differentiation. In this study, we found that di- and tri-methylation of histone H3 at K4 and acetylation of histones H3 and H4 from the promoter/enhancer to the transcribed region close to the transcription initiation site of the solute carrier family 2, member 5 (SLC2A5) gene, and its expression, were induced by differentiation of intestine-like Caco-2 cells. These effects were accompanied by contact inhibition of cell growth of these cells. Furthermore, these modifications were induced by co-treatment with a synthetic glucocorticoid hormone dexamethasone and a p44/42 mitogen-activated protein kinase inhibitor PD89059. Our results suggest that methylation of histone H3 at K4 and acetylation of histones H3 and H4 are involved in SLC2A5 gene induction associated with intestinal differentiation of Caco-2 cells.

  5. Proteinase-activated receptors induce interleukin-8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation.

    PubMed

    Wang, Hongying; Moreau, France; Hirota, Christina L; MacNaughton, Wallace K

    2010-06-01

    Proteinase-activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)-8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL-8 expression is not known. In HT-29 colonic epithelial cells, PAR(1) or PAR(2) agonists stimulated the expression of IL-8 through a NF-kappaB-dependent pathway without inducing IkappaB degradation and disassociation of IkappaB from NF-kappaB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser-276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR-induced IL-8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK-induced nuclear p65 phosphorylation. In conclusion, activation of either PAR(1) or PAR(2) stimulated the transcriptional up-regulation of IL-8 in HT-29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF-kappaB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.

  6. Depletion of Cellular Iron by Curcumin Leads to Alteration in Histone Acetylation and Degradation of Sml1p in Saccharomyces cerevisiae

    PubMed Central

    Azad, Gajendra Kumar; Singh, Vikash; Golla, Upendarrao; Tomar, Raghuvir S.

    2013-01-01

    Curcumin, a naturally occurring polyphenolic compound, is known to possess diverse pharmacological properties. There is a scarcity of literature documenting the exact mechanism by which curcumin modulates its biological effects. In the present study, we have used yeast as a model organism to dissect the mechanism underlying the action of curcumin. We found that the yeast mutants of histone proteins and chromatin modifying enzymes were sensitive to curcumin and further supplementation of iron resulted in reversal of the changes induced by curcumin. Additionally, treatment of curcumin caused the iron starvation induced expression of FET3, FRE1 genes. We also demonstrated that curcumin induces degradation of Sml1p, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. The degradation of Sml1p was mediated through proteasome and vacuole dependent protein degradation pathways. Furthermore, curcumin exerts biological effect by altering global proteome profile without affecting chromatin architecture. These findings suggest that the medicinal properties of curcumin are largely contributed by its cumulative effect of iron starvation and epigenetic modifications. PMID:23520547

  7. Histone-histone interactions in a lower eukaryote, Tetrahymena thermophila.

    PubMed

    Glover, C V; Gorovsky, M A

    1978-12-26

    The six pairwise interactions of Tetrahymena macronuclear histones H2A (formerly HX), H2B, H3, and H4 have been studied using the techniques of circular dichroism and continuous variation. Parallel experiments have been performed with calf thymus histones as controls, and the 12 possible interspecies pairs have also been examined. The behavior of three of the ciliate histones, H2A, H2B, and H3 is virtually identical with that of their vertebrate counterparts. Tetrahymena H4 exhibits a pattern of interactions identical with that of calf thymus H4, but displays significant quantitative differences in the strength of those interactions. Although we have not entirely eliminated the possibility that these differences may be artifactual, we suggest that they reflect the unique primary structure of Tetrahymena H4. These results extend the pattern of histone-histone interactions defined for plants, animals, and fungi to include the protists as well and imply the strong evolutionary conservatism of this pattern in spite of the electrophoretic and compositional differences which exist between homologous histones of different organisms. In addition, they demonstrate conclusively that histone HX should be redefined as Tetrahymena H2A.

  8. Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

    PubMed

    Zunder, Rachel M; Rine, Jasper

    2012-11-01

    In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

  9. Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    PubMed Central

    Passacquale, Gabriella; Phinikaridou, Alkystis; Warboys, Christina; Cooper, Margaret; Lavin, Begona; Alfieri, Alessio; Andia, Marcelo E; Botnar, Rene M; Ferro, Albert

    2015-01-01

    Background and Purpose There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression. Experimental Approach Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE−/− mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel. Key Results TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE−/− mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE−/− mice. Conclusions and Implications Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE−/− mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes. PMID:25824964

  10. Rational design and validation of a Tip60 histone acetyltransferase inhibitor

    NASA Astrophysics Data System (ADS)

    Gao, Chunxia; Bourke, Emer; Scobie, Martin; Famme, Melina Arcos; Koolmeister, Tobias; Helleday, Thomas; Eriksson, Leif A.; Lowndes, Noel F.; Brown, James A. L.

    2014-06-01

    Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

  11. Acetylation Mimics Within a Single Nucleosome Alter Local DNA Accessibility In Compacted Nucleosome Arrays

    PubMed Central

    Mishra, Laxmi N.; Pepenella, Sharon; Rogge, Ryan; Hansen, Jeffrey C.; Hayes, Jeffrey J.

    2016-01-01

    The activation of a silent gene locus is thought to involve pioneering transcription factors that initiate changes in the local chromatin structure to increase promoter accessibility and binding of downstream effectors. To better understand the molecular requirements for the first steps of locus activation, we investigated whether acetylation of a single nucleosome is sufficient to alter DNA accessibility within a condensed 25-nucleosome array. We found that acetylation mimics within the histone H4 tail domain increased accessibility of the surrounding linker DNA, with the increased accessibility localized to the immediate vicinity of the modified nucleosome. In contrast, acetylation mimics within the H3 tail had little effect, but were able to synergize with H4 tail acetylation mimics to further increase accessibility. Moreover, replacement of the central nucleosome with a nucleosome free region also resulted in increased local, but not global DNA accessibility. Our results indicate that modification or disruption of only a single target nucleosome results in significant changes in local chromatin architecture and suggest that very localized chromatin modifications imparted by pioneer transcription factors are sufficient to initiate a cascade of events leading to promoter activation. PMID:27708426

  12. Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics.

    PubMed

    Hadnagy, Annamaria; Beaulieu, Raymond; Balicki, Danuta

    2008-04-01

    Over the past few years, the histone deacetylase (HDAC) inhibitors have occupied an important place in the effort to develop novel, but less toxic, anticancer therapy. HDAC inhibitors block HDACs, which are the enzymes responsible for histone deacetylation, and therefore they modulate gene expression. The cellular effects of HDAC inhibitors include growth arrest and the induction of differentiation. Early successes in cancer therapeutics obtained using these drugs alone or in combination with other anticancer drugs emphasize the important place of posttranslational modifications of histones in cancer therapy. Histone tail modifications along with DNA methylation are the most studied epigenetic events related to cancer progression. Moreover, extranuclear functions of histones have also been described. Because HDAC inhibitors block HDACs and thereby increase histone acetylation, we propose a model wherein exogenous acetylated histones or other related acetylated proteins that are introduced into the nucleus become HDAC substrates and thereby compete with endogenous histones for HDACs. This competition may lead to the increased acetylation of the endogenous histones, as in the case of HDAC inhibitor therapy. Moreover, other mechanisms of action, such as binding to chromatin and modulating gene expression, are also possible for exogenously introduced histones.

  13. 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

  14. 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.

  15. CHROMATIN CONDENSATION IN TERMINALLY DIFFERENTIATING MOUSE ERYTHROBLASTS DOES NOT INVOLVE SPECIAL ARCHITECTURAL PROTEINS BUT DEPENDS ON HISTONE DEACETYLATION

    PubMed Central

    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.

    2009-01-01

    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. PMID:19172406

  16. Molecular insights into the recognition of N-terminal histone modifications by the BRPF1 bromodomain

    PubMed Central

    Poplawski, Amanda; Hu, Kaifeng; Lee, Woonghee; Natesan, Senthil; Peng, Danni; Carlson, Samuel; Shi, Xiaobing; Balaz, Stefan; Markley, John L.; Glass, Karen C.

    2014-01-01

    The monocytic leukemic zinc-finger (MOZ) histone acetyltransferase (HAT) acetylates free histones H3, H4, H2A, and H2B in vitro and is associated with up-regulation of gene transcription. The MOZ HAT functions as a quaternary complex with the bromodomain-PHD finger protein 1 (BRPF1), inhibitor of growth 5 (ING5), and hEaf6 subunits. BRPF1 links the MOZ catalytic subunit to the ING5 and hEaf6 subunits, thereby promoting MOZ HAT activity. Human BRPF1 contains multiple effector domains with known roles in gene transcription, and chromatin binding and remodeling. However, the biological function of the BRPF1 bromodomain remains unknown. Our findings reveal novel interactions of the BRPF1 bromodomain with multiple acetyllysine residues on the N-terminus of histones, and show it preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We used chemical shift perturbation data from NMR titration experiments to map the BRPF1 bromodomain ligand binding pocket and identified key residues responsible for coordination of the post-translationally modified histones. Extensive molecular dynamics simulations were used to generate structural models of bromodomain-histone ligand complexes, to analyze H-bonding and other interactions, and to calculate the binding free energies. Our results outline the molecular mechanism driving binding specificity of the BRPF1 bromodomain for discrete acetyllysine residues on the N-terminal histone tails. Together these data provide insights on how histone recognition by the bromodomain directs the biological function of BRPF1, ultimately targeting the MOZ HAT complex to chromatin substrates. PMID:24333487

  17. Inhibition of maize histone deacetylases by HC toxin, the host-selective toxin of Cochliobolus carbonum.

    PubMed Central

    Brosch, G; Ransom, R; Lechner, T; Walton, J D; Loidl, P

    1995-01-01

    HC toxin, the host-selective toxin of the maize pathogen Cochliobolus carbonum, inhibited maize histone deacetylase (HD) at 2 microM. Chlamydocin, a related cyclic tetrapeptide, also inhibited HD activity. The toxins did not affect histone acetyltransferases. After partial purification of histone deacetylases HD1-A, HD1-B, and HD2 from germinating maize embryos, we demonstrated that the different enzymes were similarly inhibited by the toxins. Inhibitory activities were reversibly eliminated by treating toxins with 2-mercaptoethanol, presumably by modifying the carbonyl group of the epoxide-containing amino acid Aeo (2-amino-9,10-epoxy-8-oxodecanoic acid). Kinetic studies revealed that inhibition of HD was of the uncompetitive type and reversible. HC toxin, in which the epoxide group had been hydrolyzed, completely lost its inhibitory activity; when the carbonyl group of Aeo had been reduced to the corresponding alcohol, the modified toxin was less active than native toxin. In vivo treatment of embryos with HC toxin caused the accumulation of highly acetylated histone H4 subspecies and elevated acetate incorporation into H4 in susceptible-genotype embryos but not in the resistant genotype. HDs from chicken and the myxomycete Physarum polycephalum were also inhibited, indicating that the host selectivity of HC toxin is not determined by its inhibitory effect on HD. Consistent with these results, we propose a model in which HC toxin promotes the establishment of pathogenic compatibility between C. carbonum and maize by interfering with reversible histone acetylation, which is implicated in the control of fundamental cellular processes, such as chromatin structure, cell cycle progression, and gene expression. PMID:8535144

  18. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote

    PubMed Central

    Schübeler, Dirk; MacAlpine, David M.; Scalzo, David; Wirbelauer, Christiane; Kooperberg, Charles; van Leeuwen, Fred; Gottschling, Daniel E.; O'Neill, Laura P.; Turner, Bryan M.; Delrow, Jeffrey; Bell, Stephen P.; Groudine, Mark

    2004-01-01

    The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity. PMID:15175259

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

    PubMed Central

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

    2011-01-01

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

  20. Histone deacetylase inhibitors sodium butyrate and valproic acid delay spontaneous cell death in purified rat retinal ganglion cells

    PubMed Central

    Boyle, Jennifer; Pielen, Amelie; Lagrèze, Wolf Alexander

    2011-01-01

    Purpose Histone deacetylase inhibitors (HDACi) have neuroprotective effects under various neurodegenerative conditions, e.g., after optic nerve crush (ONC). HDACi-mediated protection of central neurons by increased histone acetylation has not previously been demonstrated in rat retinal ganglion cells (RGCs), although epigenetic changes were shown to be associated with cell death after ONC. We investigated whether HDACi can delay spontaneous cell death in purified rat RGCs and analyzed concomitant histone acetylation levels. Methods RGCs were purified from newborn (postnatal day [P] 0–P2) rat retinas by immunopanning with antibodies against Thy-1.1 and culturing in serum-free medium for 2 days. RGCs were treated with HDACi, each at several different concentrations: 0.1–10 mM sodium butyrate (SB), 0.1–2 mM valproic acid (VPA), or 0.5–10 nM trichostatin A (TSA). Negative controls were incubated in media alone, while positive controls were incubated in 0.05–0.4 IU/µl erythropoietin. Survival was quantified by counting viable cells using phase-contrast microscopy. The expression of acetylated histone proteins (AcH) 3 and 4 was analyzed in RGCs by immunohistochemistry. Results SB and VPA enhanced RGC survival in culture, with both showing a maximum effect at 0.1 mM (increase in survival to 188% and 163%, respectively). Their neuroprotective effect was comparable to that of erythropoietin at 0.05 IU/µl. TSA 0.5–1.0 nM showed no effect on RGC survival, and concentrations ≥5 nM increased RGC death. AcH3 and AcH4 levels were only significantly increased in RGCs treated with 0.1 mM SB. VPA 0.1 mM produced only a slight effect on histone acetylation. Conclusions Millimolar concentrations of SB and VPA delayed spontaneous cell death in purified RGCs; however, significantly increased histone acetylation levels were only detectable in RGCs after SB treatment. As the potent HDACi TSA was not neuroprotective, mechanisms other than histone acetylation may be the

  1. SPOTing Acetyl-Lysine Dependent Interactions.

    PubMed

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-08-17

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  2. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation. PMID:27600229

  3. SPOTing Acetyl-Lysine Dependent Interactions

    PubMed Central

    Picaud, Sarah; Filippakopoulos, Panagis

    2015-01-01

    Post translational modifications have been recognized as chemical signals that create docking sites for evolutionary conserved effector modules, allowing for signal integration within large networks of interactions. Lysine acetylation in particular has attracted attention as a regulatory modification, affecting chromatin structure and linking to transcriptional activation. Advances in peptide array technologies have facilitated the study of acetyl-lysine-containing linear motifs interacting with the evolutionary conserved bromodomain module, which specifically recognizes and binds to acetylated sequences in histones and other proteins. Here we summarize recent work employing SPOT peptide technology to identify acetyl-lysine dependent interactions and document the protocols adapted in our lab, as well as our efforts to characterize such bromodomain-histone interactions. Our results highlight the versatility of SPOT methods and establish an affordable tool for rapid access to potential protein/modified-peptide interactions involving lysine acetylation.

  4. Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    PubMed Central

    Ghule, Prachi N.; Xie, Rong-Lin; Medina, Ricardo; Colby, Jennifer L.; Jones, Stephen N.; Lian, Jane B.; Stein, Janet L.; van Wijnen, Andre J.

    2014-01-01

    Fidelity of chromatin organization is crucial for normal cell cycle progression, and perturbations in packaging of DNA may predispose to transformation. Histone H4 protein is the most highly conserved chromatin protein, required for nucleosome assembly, with multiple histone H4 gene copies encoding identical protein. There is a long-standing recognition of the linkage of histone gene expression and DNA replication. A fundamental and unresolved question is the mechanism that couples histone biosynthesis with DNA replication and fidelity of cell cycle control. Here, we conditionally ablated the obligatory histone H4 transcription factor HINFP to cause depletion of histone H4 in mammalian cells. Deregulation of histone H4 results in catastrophic cellular and molecular defects that lead to genomic instability. Histone H4 depletion increases nucleosome spacing, impedes DNA synthesis, alters chromosome complement, and creates replicative stress. Our study provides functional evidence that the tight coupling between DNA replication and histone synthesis is reciprocal. PMID:24797072

  5. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity

    PubMed Central

    Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C.; Joncas, France-Hélène; Saksouk, Nehmé; Holliday, Michael; Paquet, Eric; Yan, Kezhi; Tong, Qiong; Klein, Brianna J.; Tan, Song; Yang, Xiang-Jiao; Kutateladze, Tatiana G.; Côté, Jacques

    2013-01-01

    Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)–Zn knuckle–PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1–BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit. PMID:24065767

  6. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

    PubMed

    Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C; Joncas, France-Hélène; Saksouk, Nehmé; Holliday, Michael; Paquet, Eric; Yan, Kezhi; Tong, Qiong; Klein, Brianna J; Tan, Song; Yang, Xiang-Jiao; Kutateladze, Tatiana G; Côté, Jacques

    2013-09-15

    Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit.

  7. Chatting histone modifications in mammals

    PubMed Central

    Izzo, Annalisa

    2010-01-01

    Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks. PMID:21266346

  8. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer

    PubMed Central

    Miller, Kyle M.

    2016-01-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer. PMID:27631103

  9. Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer.

    PubMed

    Gong, Fade; Chiu, Li-Ya; Miller, Kyle M

    2016-09-01

    Chromatin-based DNA damage response (DDR) pathways are fundamental for preventing genome and epigenome instability, which are prevalent in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the addition and removal of acetyl groups on lysine residues, a post-translational modification important for the DDR. Acetylation can alter chromatin structure as well as function by providing binding signals for reader proteins containing acetyl-lysine recognition domains, including the bromodomain (BRD). Acetylation dynamics occur upon DNA damage in part to regulate chromatin and BRD protein interactions that mediate key DDR activities. In cancer, DDR and acetylation pathways are often mutated or abnormally expressed. DNA damaging agents and drugs targeting epigenetic regulators, including HATs, HDACs, and BRD proteins, are used or are being developed to treat cancer. Here, we discuss how histone acetylation pathways, with a focus on acetylation reader proteins, promote genome stability and the DDR. We analyze how acetylation signaling impacts the DDR in the context of cancer and its treatments. Understanding the relationship between epigenetic regulators, the DDR, and chromatin is integral for obtaining a mechanistic understanding of genome and epigenome maintenance pathways, information that can be leveraged for targeting acetylation signaling, and/or the DDR to treat diseases, including cancer.

  10. Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

    PubMed Central

    Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

    2014-01-01

    To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the

  11. mChIP-KAT-MS, a method to map protein interactions and acetylation sites for lysine acetyltransferases

    PubMed Central

    Mitchell, Leslie; Huard, Sylvain; Cotrut, Michael; Pourhanifeh-Lemeri, Roghayeh; Steunou, Anne-Lise; Hamza, Akil; Lambert, Jean-Philippe; Zhou, Hu; Ning, Zhibin; Basu, Amrita; Côté, Jacques; Figeys, Daniel A.; Baetz, Kristin

    2013-01-01

    Recent global proteomic and genomic studies have determined that lysine acetylation is a highly abundant posttranslational modification. The next challenge is connecting lysine acetyltransferases (KATs) to their cellular targets. We hypothesize that proteins that physically interact with KATs may not only predict the cellular function of the KATs but may be acetylation targets. We have developed a mass spectrometry-based method that generates a KAT protein interaction network from which we simultaneously identify both in vivo acetylation sites and in vitro acetylation sites. This modified chromatin-immunopurification coupled to an in vitro KAT assay with mass spectrometry (mChIP-KAT-MS) was applied to the Saccharomyces cerevisiae KAT nucleosome acetyltransferase of histone H4 (NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome and in vitro-enriched acetylome, identifying over 70 previously undescribed physical interaction partners for the complex and over 150 acetyl lysine residues, of which 108 are NuA4-specific in vitro sites. Through this method we determine NuA4 acetylation of its own subunit Epl1 is a means of self-regulation and identify a unique link between NuA4 and the spindle pole body. Our work demonstrates that this methodology may serve as a valuable tool in connecting KATs with their cellular targets. PMID:23572591

  12. Detection of histone H3 phosphorylation in cultured cells and tissue sections by immunostaining.

    PubMed

    Padmanabhan, Jaya

    2009-01-01

    Growth factor stimulation results in phosphorylation of histone H3 at ser 10 and this correlated with expression of immediate early genes suggesting that this phosphorylation is associated with transcriptional activation. Although Western immunoblot analysis allows the detection of protein modifications in histones, in order to determine the localization of histones during different phases of cell cycle or during treatment of cells with different drugs we have to use immunohistochemistry. The protocol described here allows the detection of phosphorylated histones in tissue-cultured cells and tissue sections by fluorescent or bright-field immunostaining analysis. Here we used a serine 10 specific P-histone H3 antibody to determine the localization of this phosphoprotein in an asynchronously growing H4 glioma cell line and brain sections. It has been shown that long-term potentiation (LTP) is associated with gene transcription, and histone acetylation plays a major role in LTP formation (Wood et al., Learn Mem 13:241-244, 2006; Wood et al., Hippocampus 15:610-621, 2005; Alarcon et al., Neuron 42:947-959, 2004; Korzus et al., Neuron 42:961-972, 2004). Stimulus-induced phosphorylation of histone H3 at serine 10 has also been implicated in hippocampal neurons and striatal neurons (Li et al., J Neurochem 90:1117-1131, 2004; Crosio et al., J Cell Sci 116:4905-4914, 2003). Co-staining with a cell-specific antibody will allow us to determine the type of cells that show activation of histone phosphorylation in the brain.

  13. 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.

  14. Histone Deacetylases and Mechanisms of Regulation of Gene Expression (Histone deacetylases in cancer)

    PubMed Central

    Chen, Hong Ping; Zhao, Yu Tina; Zhao, Ting C

    2016-01-01

    In recent years, it has become widely recognized that histone modification plays a pivotal role in controlling gene expression, and is involved in a wide spectrum of disease regulation. Histone acetylation is a major modification that affects gene transcription and is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDAC). HATs acetylate lysines of histone proteins, resulting in relaxation of chromatin structure, and they also facilitate gene activation. Conversely, HDACs remove acetyl groups from hyperacetylated histones and suppress general gene transcription. In addition to histones, numerous non-histone proteins can be acetylated and deacetylated, and they are also involved in a wide range of disease regulation. To date, there are 18 HDACs in mammals classified into four classes based on homology to yeast HDACs. Accumulating evidence has revealed that HDACs play crucial roles in a variety of biological processes including inflammation, cell proliferation, apoptosis, and carcinogenesis. In this review, we summarize the current state of knowledge of HDACs in carcinogenesis and describe the involvement of HDACs in cancer-associated molecular processes. It is hoped than our understanding of the role of HDACs in cancer will lead to the design of more potent and specific drugs targeting selective HDAC proteins for the treatment of the disease. PMID:25746103

  15. Insights into neuroepigenetics through human histone deacetylase PET imaging.

    PubMed

    Wey, Hsiao-Ying; Gilbert, Tonya M; Zürcher, Nicole R; She, Angela; Bhanot, Anisha; Taillon, Brendan D; Schroeder, Fredrick A; Wang, Changing; Haggarty, Stephen J; Hooker, Jacob M

    2016-08-10

    Epigenetic dysfunction is implicated in many neurological and psychiatric diseases, including Alzheimer's disease and schizophrenia. Consequently, histone deacetylases (HDACs) are being aggressively pursued as therapeutic targets. However, a fundamental knowledge gap exists regarding the expression and distribution of HDACs in healthy individuals for comparison to disease states. Here, we report the first-in-human evaluation of neuroepigenetic regulation in vivo. Using positron emission tomography with [(11)C]Martinostat, an imaging probe selective for class I HDACs (isoforms 1, 2, and 3), we found that HDAC expression is higher in cortical gray matter than in white matter, with conserved regional distribution patterns within and between healthy individuals. Among gray matter regions, HDAC expression was lowest in the hippocampus and amygdala. Through biochemical profiling of postmortem human brain tissue, we confirmed that [(11)C]Martinostat selectively binds HDAC isoforms 1, 2, and 3, the HDAC subtypes most implicated in regulating neuroplasticity and cognitive function. In human stem cell-derived neural progenitor cells, pharmacologic-level doses of Martinostat induced changes in genes closely associated with synaptic plasticity, including BDNF (brain-derived neurotrophic factor) and SYP (synaptophysin), as well as genes implicated in neurodegeneration, including GRN (progranulin), at the transcript level, in concert with increased acetylation at both histone H3 lysine 9 and histone H4 lysine 12. This study quantifies HDAC expression in the living human brain and provides the foundation for gaining unprecedented in vivo epigenetic information in health and disease. PMID:27510902

  16. Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair.

    PubMed

    Gursoy-Yuzugullu, Ozge; Ayrapetov, Marina K; Price, Brendan D

    2015-06-16

    The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4-Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.

  17. Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes.

    PubMed

    Chittuluru, Johnathan R; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L; Fried, Michael G; Tan, Song; Côté, Jacques; Asturias, Francisco J

    2011-10-09

    We have used EM and biochemistry to characterize the structure of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes, and we have determined the interaction of NuA4 with the nucleosome core particle (NCP). The ATM-related Tra1 subunit, which is shared with the SAGA coactivator complex, forms a large domain joined to a second region that accommodates the catalytic subcomplex Piccolo and other NuA4 subunits. EM analysis of a NuA4-NCP complex shows the NCP bound at the periphery of NuA4. EM characterization of Piccolo and Piccolo-NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N terminus of Piccolo subunit enhancer of Polycomb-like 1 (Epl1) is essential for NCP interaction, whereas the subunit yeast homolog of mammalian Ing1 2 (Yng2) apparently positions Piccolo for efficient acetylation of histone H4 or histone H2A tails. Taken together, these results provide an understanding of the NuA4 subunit organization and the NuA4-NCP interactions.

  18. Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen-Induced Arthritis.

    PubMed

    Liu, Ya-Fei; Wen, Cai-Yu-Zhu; Chen, Zhe; Wang, Yu; Huang, Ying; Hu, Yong-Hong; Tu, Sheng-Hao

    2016-01-01

    Background. Wutou decoction (WTD) has been wildly applied in the treatment of rheumatoid arthritis and experimental arthritis in rats for many years. Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis; however, the effects of WTD on epigenetic changes are unclear. This study is set to explore the effects of WTD on DNA methylation and histone modifications in rats with collagen-induced arthritis (CIA). Methods. The CIA model was established by the stimulation of collagen and adjuvant. The knee synovium was stained with hematoxylin and eosin. The DNA methyltransferase 1 (DNMT1) and methylated CpG binding domain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR. The global DNA histone H3-K4/H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected. Results. Our data demonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (P < 0.05). The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (P < 0.05). Moreover, H3 acetylation of PBMCs was overexpressed in WTD compared with CIA (P < 0.05). Conclusions. WTD may modulate DNA methylation and histone modifications, functioning as anti-inflammatory potential. PMID:27042192

  19. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms.

    PubMed

    Wang, Huanyu; Ge, Zhongqi; Walsh, Scott T R; Parthun, Mark R

    2012-01-01

    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

  20. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

  1. Biotinylation of histones in human cells. Effects of cell proliferation.

    PubMed

    Stanley, J S; Griffin, J B; Zempleni, J

    2001-10-01

    An enzymatic mechanism has been proposed by which biotinidase may catalyze biotinylation of histones. Here, human cells were found to covalently bind biotin to histones H1, H2A, H2B, H3, and H4. Cells respond to proliferation with increased biotinylation of histones; biotinylation increases early in the cell cycle and remains increased during the cycle. Notwithstanding the catalytic role of biotinidase in biotinylation of histones, mRNA encoding biotinidase and biotinidase activity did not parallel the increased biotinylation of histones in proliferating cells. Biotinylation of histones might be regulated by enzymes other than biotinidase or by the rate of histone debiotinylation.

  2. Chromatin assembly factor 1 interacts with histone H3 methylated at lysine 79 in the processes of epigenetic silencing and DNA repair.

    PubMed

    Zhou, Hui; Madden, Benjamin J; Muddiman, David C; Zhang, Zhiguo

    2006-03-01

    In eukaryotic cells, chromatin is classified into euchromatin, which is active in transcription, and heterochromatin that silences transcription. Histones in these two domains contain distinct modifications. Chromatin assembly factor 1 (CAF-1) is a highly conserved protein that functions in DNA replication, DNA repair, and heterochromatin silencing. CAF-1 binds histones H3 and H4 and deposits histones onto DNA to form nucleosomes. However, modifications on H3 and H4 associated with CAF-1 are not known. Here, we have purified a complex containing CAF-1 and H3 and H4 from yeast cells and determined the modifications present on these histones using linear ion trap FT-ICR mass spectrometry. H4 that copurified with CAF-1 was a mixture of isoforms acetylated at lysines 5, 8, 12, and 16, whereas an H3 peptide methylated at lysine 79 and an H3 peptide acetylated at lysine 56 were detected. In yeast cell extracts, these two H3 modifications peaked in the late S phase with different kinetics. Moreover, the association of CAF-1 with H3 methylated at lysine 79 appeared to occur in the late S phase. Finally, cells lacking both Dot1p, the methyltransferase that methylates H3 lysine 79, and Cac1p, the large subunit of CAF-1, exhibited a dramatic loss of telomeric silencing and increased sensitivity to DNA damaging agents. Together, these data indicate that CAF-1 interacts with H3 methylated at lysine 79 during the processes of epigenetic silencing and DNA repair. PMID:16503640

  3. Histone variants: key players of chromatin.

    PubMed

    Biterge, Burcu; Schneider, Robert

    2014-06-01

    Histones are fundamental structural components of chromatin. Eukaryotic DNA is wound around an octamer of the core histones H2A, H2B, H3, and H4. Binding of linker histone H1 promotes higher order chromatin organization. In addition to their structural role, histones impact chromatin function and dynamics by, e.g., post-translational histone modifications or the presence of specific histone variants. Histone variants exhibit differential expression timings (DNA replication-independent) and mRNA characteristics compared to canonical histones. Replacement of canonical histones with histone variants can affect nucleosome stability and help to create functionally distinct chromatin domains. In line with this, several histone variants have been implicated in the regulation of cellular processes such as DNA repair and transcriptional activity. In this review, we focus on recent progress in the study of core histone variants H2A.X, H2A.Z, macroH2A, H3.3, and CENP-A, as well as linker histone H1 variants, their functions and their links to development and disease.

  4. Extent of histone modifications and H1/sup 0/ content during cell cycle progression in the presence of butyrate

    SciTech Connect

    D'Anna, J.A.; Gurley, L.R.; Tobey, R.A.

    1983-01-01

    The effects of butyrate upon the extents of phosphorylation of histones H1 and H1/sup 0/ during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain >98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1/sup 0/, H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1/sup 0/ phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1/sup 0/ in chromatin during the S or G2 phases of the cell cycle. 38 references, 4 figures, 1 table.

  5. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    PubMed

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

  6. Trypanosomatid histones.

    PubMed

    Alsford, Sam; Horn, David

    2004-07-01

    The histones are responsible for packaging and regulating access to eukaryotic genomes. Trypanosomatids are flagellated protists that diverged early from the eukaryotic lineage and include parasites that cause disease in humans and other mammals. Here, we review the properties of histones in parasitic trypanosomatids, from gene organization and sequence to expression, post-translational modification and function within chromatin. Phylogenetic and experimental analysis indicates that certain specifically conserved histone sequence motifs, particularly within the N-terminal 'tail' domains, possibly represent functionally important modification substrates conserved throughout the eukaryotic lineage. For example, histone H3 contains a highly conserved methylation substrate. Trypanosomatids also possess at least three variant histones. Among these is an orthologue of H2A.Z, a histone involved in protecting 'active' chromatin from silencing in yeast. Histones provide docking platforms for a variety of regulatory factors. The presence of histone modification and variant histones in trypanosomatids therefore represents evidence for a network that provides the discrimination required to regulate transcription, recombination, repair and chromosome replication and segregation.

  7. Modification of Histones during Spermiogenesis in Trout: A Molecular Mechanism for Altering Histone Binding to DNA

    PubMed Central

    Sung, Michael T.; Dixon, Gordon H.

    1970-01-01

    At a late stage of spermatogenesis in rainbow-trout testis, the entire complement of histones is replaced by newly synthesized protamine and histones are extensively phosphorylated and acetylated. Tryptic digestion of purified histones labeled by incubation of testicular cells with [32P]phosphate shows that phosphorylation occurs at a small number of seryl residues. Histone I (lysine-rich) is phosphorylated in the sequence Lys-Ser(PO4)-Pro-Lys, which is located in the lysine-rich C-terminal region of the molecule. Histones IIb1 (slightly lysine-rich) and IV (glycine, arginine-rich) give rise to the same phosphopeptide, Ac-Ser(PO4)-Gly-Arg, which comprises the amino terminus of each histone. Thermolysin digests of phosphohistones IIb1 and IV also released a phosphopeptide with composition corresponding to the first six residues of histone IV: Ac-Ser(PO4)-Gly-Arg-Gly-Lys-Gly. An α-helical model of the N-terminal region of histone IV shows that this region is a possible DNA-binding site. Phosphorylation at serine 1 together with ε-amino acetylation at lysines 5, 8, 12, and 16 (observed in histone IV from trout testis) could profoundly modify ionic interactions and lead to an „unzipping” of histone IV from DNA Images PMID:5274484

  8. Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

    PubMed Central

    Guan, Zhiyun

    2015-01-01

    Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects