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Sample records for dynamic histone h3

  1. Dynamic deposition of histone variant H3.3 accompanies developmental remodeling of the Arabidopsis transcriptome.

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    Heike Wollmann

    Full Text Available In animals, replication-coupled histone H3.1 can be distinguished from replication-independent histone H3.3. H3.3 variants are enriched at active genes and their promoters. Furthermore, H3.3 is specifically incorporated upon gene activation. Histone H3 variants evolved independently in plants and animals, and it is unclear whether different replication-independent H3.3 variants developed similar properties in both phyla. We studied Arabidopsis H3 variants in order to find core properties of this class of histones. Here we present genome-wide maps of H3.3 and H3.1 enrichment and the dynamic changes of their profiles upon cell division arrest. We find H3.3 enrichment to positively correlate with gene expression and to be biased towards the transcription termination site. In contrast with H3.1, heterochromatic regions are mostly depleted of H3.3. We report that, in planta, dynamic changes in H3.3 profiles are associated with the extensive remodeling of the transcriptome that occurs during cell differentiation. We propose that H3.3 dynamics are linked to transcription and are involved in resetting covalent histone marks at a genomic scale during plant development. Our study suggests that H3 variants properties likely result from functionally convergent evolution.

  2. The differential mobilization of histones H3.1 and H3.3 by herpes simplex virus 1 relates histone dynamics to the assembly of viral chromatin.

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    Conn, Kristen L; Hendzel, Michael J; Schang, Luis M

    2013-01-01

    During lytic infections, HSV-1 genomes are assembled into unstable nucleosomes. The histones required for HSV-1 chromatin assembly, however, are in the cellular chromatin. We have shown that linker (H1) and core (H2B and H4) histones are mobilized during HSV-1 infection, and proposed that the mobilized histones are available for assembly into viral chromatin. However, the actual relevance of histone mobilization remained unknown. We now show that canonical H3.1 and variant H3.3 are also mobilized during HSV-1 infection. Mobilization required no HSV-1 protein expression, although immediate early or early proteins enhanced it. We used the previously known differential association of H3.3 and H3.1 with HSV-1 DNA to test the relevance of histone mobilization. H3.3 binds to HSV-1 genomes first, whereas H3.1 only binds after HSV-1 DNA replication initiates. Consistently, H3.3 and H3.1 were differentially mobilized. H3.1 mobilization decreased with HSV-1 DNA replication, whereas H3.3 mobilization was largely unaffected by it. These results support a model in which previously mobilized H3.1 is immobilized by assembly into viral chromatin during HSV-1 DNA replication, whereas H3.3 is mobilized and assembled into HSV-1 chromatin throughout infection. The differential mobilizations of H3.3 and H3.1 are consistent with their differential assembly into viral chromatin. These data therefore relate nuclear histone dynamics to the composition of viral chromatin and provide the first evidence that histone mobilization relates to viral chromatin assembly.

  3. Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation

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    Jiang, Yan-Ke; Zou, Jian-Wei; Wu, Yu-Qian; Zhang, Na; Yu, Qing-Sen; Jiang, Yong-Jun

    Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3-HP1 trajectory. It is clear from the 10-ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications-a stable methylation and a dynamic phosphorylation mark-establish a regulatory mechanism of protein-protein interactions.

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

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    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.

  5. Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum

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    Salcedo-Amaya, Adriana M; van Driel, Marc A; Alako, Blaise T

    2009-01-01

    Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. To explore the epigenome of Plasmodium falciparum asexual stages, we performed MS analysis of histone modifications and found a general preponderance of H3/H4 acetylation and H3K4...

  6. Differential dynamics of histone H3 methylation at positions K4 and K9 in the mouse zygote

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    Walter Jörn

    2004-09-01

    Full Text Available Abstract Background In the mouse zygote the paternal genome undergoes dramatic structural and epigenetic changes. Chromosomes are decondensed, protamines replaced by histones and DNA is rapidly and actively demethylated. The epigenetic asymmetry between parental genomes remains at least until the 2-cell stage suggesting functional differences between paternal and maternal genomes during early cleavage stages. Results Here we analyzed the timing of histone deposition on the paternal pronucleus and the dynamics of histone H3 methylation (H3/K4mono-, H3/K4tri- and H3/K9di-methylation immediately after fertilization. Whereas maternal chromatin maintains all types of histone H3 methylation throughout the zygotic development, paternal chromosomes acquire new and unmodified histones shortly after fertilization. In the following hours we observe a gradual increase in H3/K4mono-methylation whereas H3/K4tri-methylation is not present before latest pronuclear stages. Histone H3/K9di-methylation is completely absent from the paternal pronucleus, including metaphase chromosomes of the first mitotic stage. Conclusion Parallel to the epigenetic asymmetry in DNA methylation, chromatin modifications are also different between both parental genomes in the very first hours post fertilization. Whereas methylation at H3/K4 gradually becomes similar between both genomes, H3/K9 methylation remains asymmetric.

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

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    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

  8. Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

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    Deng Wen LI; Qin YANG; Jia Tong CHEN; Hao ZHOU; Ru Ming LIU; Xi Tai HUANG

    2005-01-01

    The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determine its function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzed by indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylation begins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phosphoH3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms "sandwich-like structure" when the chromosomes condensed. With the cleavage progressing, the "ladders" of the histone contract into a bigger bright dot. Then the histone aggregates and some of compacted microtubules in the midbody region are composed into a "bar-like"complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the "bar",inside which locates microtubules and modified histones, to finish the cytokinesis and keep the "bar complex" out of the cells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may take part in the formation of midbody and play a crucial role in cytokinesis.

  9. Dynamic changes of histone H3 marks during Caenorhabditis elegans lifecycle revealed by middle-down proteomics

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    Sidoli, Simone; Vandamme, Julien; Elisabetta Salcini, Anna;

    2016-01-01

    We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval......-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during...... that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms. This article is protected by copyright. All...

  10. Clipping of flexible tails of histones H3 and H4 affects the structure and dynamics of the nucleosome.

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    Nurse, Nathan P; Jimenez-Useche, Isabel; Smith, Ian Tad; Yuan, Chongli

    2013-03-05

    Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail's lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

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    Catherine A Hazzalin

    2005-12-01

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

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

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    2005-12-01

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

  13. Acetylated histone H3 increases nucleosome dissociation

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

    2009-03-01

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

  14. The HIRA complex that deposits the histone H3.3 is conserved in Arabidopsis and facilitates transcriptional dynamics

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    Xin Nie

    2014-08-01

    Full Text Available In animals, replication-independent incorporation of nucleosomes containing the histone variant H3.3 enables global reprogramming of histone modifications and transcriptional profiles. H3.3 enrichment over gene bodies correlates with gene transcription in animals and plants. In animals, H3.3 is deposited into chromatin by specific protein complexes, including the HIRA complex. H3.3 variants evolved independently and acquired similar properties in animals and plants, questioning how the H3.3 deposition machinery evolved in plants and what are its biological functions. We performed phylogenetic analyses in the plant kingdom and identified in Arabidopsis all orthologs of human genes encoding members of the HIRA complex. Genetic analyses, biochemical data and protein localisation suggest that these proteins form a complex able to interact with H3.3 in Arabidopsis in a manner similar to that described in mammals. In contrast to animals, where HIRA is required for fertilization and early development, loss of function of HIRA in Arabidopsis causes mild phenotypes in the adult plant and does not perturb sexual reproduction and embryogenesis. Rather, HIRA function is required for transcriptional reprogramming during dedifferentiation of plant cells that precedes vegetative propagation and for the appropriate transcription of genes responsive to biotic and abiotic factors. We conclude that the molecular function of the HIRA complex is conserved between plants and animals. Yet plants diversified HIRA functions to enable asexual reproduction and responsiveness to the environment in response to the plant sessile lifestyle.

  15. Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer.

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    Zhou, Naru; Cao, Zubing; Wu, Ronghua; Liu, Xing; Tao, Jia; Chen, Zhen; Song, Dandan; Han, Fei; Li, Yunsheng; Fang, Fugui; Zhang, Xiaorong; Zhang, Yunhai

    2014-08-01

    Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development.

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

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

    2010-01-01

    SUZ12 is a core component of the polycomb repressive complex 2 (PRC2) and is required for the differentiation of mouse embryonic stem cells (ESCs). PRC2 is associated with transcriptional repression via methylation of H3 Lys-27. We applied quantitative mass spectrometry to investigate the effects....... The combined use of ETD and CID MS/MS increased the total number of identified modified peptides. Comparative quantitative analysis of histones from wild type and Suz12-deficient ESCs using stable isotope labeling with amino acids in cell culture and LC-MS/MS revealed a dramatic reduction of H3K27me2 and H3K27...... analysis of the dynamics of coexisting post-translational modifications in proteins....

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

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    Chauhan, Sakshi

    2016-09-02

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

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

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    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.

  19. Methylation of histone H3 lysine 9 occurs during translation

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    Rivera, Carlos; Saavedra, Francisco; Alvarez, Francisca; Díaz-Celis, César; Ugalde, Valentina; Li, Jianhua; Forné, Ignasi; Gurard-Levin, Zachary A.; Almouzni, Geneviève; Imhof, Axel; Loyola, Alejandra

    2015-01-01

    Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed. PMID:26405197

  20. Histone chaperones: assisting histone traffic and nucleosome dynamics.

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    Gurard-Levin, Zachary A; Quivy, Jean-Pierre; Almouzni, Geneviève

    2014-01-01

    The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

  1. Structure and function of human histone H3.Y nucleosome.

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    Kujirai, Tomoya; Horikoshi, Naoki; Sato, Koichi; Maehara, Kazumitsu; Machida, Shinichi; Osakabe, Akihisa; Kimura, Hiroshi; Ohkawa, Yasuyuki; Kurumizaka, Hitoshi

    2016-07-27

    Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes.

  2. Structural basis for recognition of H3K56-acetylated histone H3-H4 by the chaperone Rtt106

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    Su, Dan; Hu, Qi; Li, Qing; Thompson, James R; Cui, Gaofeng; Fazly, Ahmed; Davies, Brian A; Botuyan, Maria Victoria; Zhang, Zhiguo; Mer, Georges [Mayo

    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.

  3. Crystal structure of the nucleosome containing histone H3 with crotonylated lysine 122.

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    Suzuki, Yuya; Horikoshi, Naoki; Kato, Daiki; Kurumizaka, Hitoshi

    2016-01-15

    The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

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    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3...

  5. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

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    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

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

    Science.gov (United States)

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

    2013-04-01

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

  7. JMJD3 is a histone H3 Lysine 27 (H3K27) demethylase

    Institute of Scientific and Technical Information of China (English)

    Yang Xiang; Ziqi Zhu; Gang Han; Hanqing Lin; Longyong Xu; Charlie Degui Chen

    2007-01-01

    Histone methylation iS an important epigenetic phenomenon that participates in a diverse array of cellular processesand has been found to be associated with cancer.Recent identification of several histone demethylases has proved thathistone methylation is a reversible process.Through a candidate approach,we have biochemically identified JMJD3 asan H3K27 demethylase.Transfection of JMJD3 into HeLa cells caused a specific reduction of trimethyl H3K27,but hadno effect on di-and monomethyl H3K27,or histone lysine methylations on H3K4 and H3K9.The enzymatic activityrequires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding.In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation.In addition,we foundthat JMJD3 is upregulated in prostate cancer,and its expression iS higher in metastatic prostate cancer.Thus,we identi-fied JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated inprostate cancer.

  8. Histone H3R2 Symmetric Dimethylation and Histone H3K4 Trimethylation Are Tightly Correlated in Eukaryotic Genomes

    OpenAIRE

    Chih-Chi Yuan; Adam G.W. Matthews; Yi Jin; Chang Feng Chen; Brad A. Chapman; Toshiro K. Ohsumi; Karen C. Glass; Tatiana G. Kutateladze; Mark L. Borowsky; Kevin Struhl; Marjorie A. Oettinger

    2012-01-01

    The preferential in vitro interaction of the PHD finger of RAG2, a subunit of the V(D)J recombinase, with histone H3 tails simultaneously trimethylated at lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2sK4me3) predicted the existence of the previously unknown histone modification H3R2me2s. Here, we report the in vivo identification of H3R2me2s . Consistent with the binding specificity of the RAG2 PHD finger, high levels of H3R2me2sK4me3 are found at antigen receptor gene segmen...

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

    Science.gov (United States)

    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.

  10. Precision mapping of coexisting modifications in histone H3 tails from embryonic stem cells by ETD-MS/MS

    DEFF Research Database (Denmark)

    Jung, Hye Ryung; Sidoli, Simone; Haldbo, Simon

    2013-01-01

    Post-translational modifications (PTMs) of histones play a major role in regulating chromatin dynamics and influence processes such as transcription and DNA replication. Here, we report 114 distinct combinations of coexisting PTMs of histone H3 obtained from mouse embryonic stem (ES) cells. Histone......, and K36, suggesting interdependence between histone methylation marks. We hypothesize that the most abundant coexisting PTMs may provide a signature for the permissive state of mouse ES cells....

  11. Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis

    Directory of Open Access Journals (Sweden)

    Jun Ueda

    2017-01-01

    Full Text Available Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  13. Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape

    Science.gov (United States)

    Lu, Chao; Jain, Siddhant U.; Hoelper, Dominik; Bechet, Denise; Molden, Rosalynn C.; Ran, Leili; Murphy, Devan; Venneti, Sriram; Hameed, Meera; Pawel, Bruce R.; Wunder, Jay S.; Dickson, Brendan C.; Lundgren, Stefan M.; Jani, Krupa S.; De Jay, Nicolas; Papillon-Cavanagh, Simon; Andrulis, Irene L.; Sawyer, Sarah L.; Grynspan, David; Turcotte, Robert E.; Nadaf, Javad; Fahiminiyah, Somayyeh; Muir, Tom W.; Majewski, Jacek; Thompson, Craig B.; Chi, Ping; Garcia, Benjamin A.; Allis, C. David; Jabado, Nada; Lewis, Peter W.

    2016-01-01

    Several types of pediatric cancers reportedly contain high frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here, we report that the H3 lysine 36 to methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. Following the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of Polycomb Repressive Complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas where novel K36M/I mutations in H3.1 are identified. PMID:27174990

  14. R loops are linked to histone H3 S10 phosphorylation and chromatin condensation.

    Science.gov (United States)

    Castellano-Pozo, Maikel; Santos-Pereira, José M; Rondón, Ana G; Barroso, Sonia; Andújar, Eloisa; Pérez-Alegre, Mónica; García-Muse, Tatiana; Aguilera, Andrés

    2013-11-21

    R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.

  15. Evolutionary dynamics of 5S rDNA location in acridid grasshoppers and its relationship with H3 histone gene and 45S rDNA location.

    Science.gov (United States)

    Cabral-de-Mello, Diogo C; Cabrero, Josefa; López-León, María Dolores; Camacho, Juan Pedro M

    2011-07-01

    We analyze the chromosomal location of 5S rDNA clusters in 29 species of grasshoppers belonging to the family Acrididae. There was extensive variation among species for the number and location of 5S rDNA sites. Out of 148 sites detected, 75% were proximally located, 21.6% were interstitial, and only 3.4% were distal. The number of 5S rDNA sites per species varied from a single chromosome pair (in six species) to all chromosome pairs (in five species), with a range of intermediate situations. Thirteen chromosomes from eight species carried two 5S rDNA clusters. At intraspecific level, differences among populations were detected in Eyprepocnemis plorans, and some heteromorphisms have also been observed in some species. Double FISH for 5S rDNA and H3 histone gene DNA, performed on 17 of these 29 species, revealed that both markers are sometimes placed in a same chromosome but at different location, whereas they appeared to co-localize in five species (Calliptamus barbarus, Heteracris adpersa, Aiolopus strepens, Oedipoda charpentieri and O. coerulescens). Double fiber-FISH in A. strepens and O. coerulescens showed that the two DNAs are closely interspersed with variable relative amounts of both classes of DNA. Finally, no correlation was observed between the number of 5S and 45S rDNA clusters in 23 species where this information was available. These results are discussed in the light of possible mechanisms of spread that led to the extensive variation in the number of clusters observed for both rDNA types in acridid grasshoppers.

  16. Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity

    Directory of Open Access Journals (Sweden)

    Parthun Mark R

    2004-07-01

    Full Text Available Abstract Background The acetylation of the core histone NH2-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. Results To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. Conclusions Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p.

  17. The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.

    Science.gov (United States)

    Bowman, Andrew; Lercher, Lukas; Singh, Hari R; Zinne, Daria; Timinszky, Gyula; Carlomagno, Teresa; Ladurner, Andreas G

    2016-04-20

    Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.

  18. Dot1-dependent histone H3K79 methylation promotes the formation of meiotic double-strand breaks in the absence of histone H3K4 methylation in budding yeast.

    Directory of Open Access Journals (Sweden)

    Mohammad Bani Ismail

    Full Text Available Epigenetic marks such as histone modifications play roles in various chromosome dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and K79 is involved in the initiation of recombination and the recombination checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes as well as methylation-defective mutants of histone H3. We confirmed the role of Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs in meiosis for the initiation of meiotic recombination, and we showed the involvement of Dot1 (H3K79 methylation in DSB formation in the absence of Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4 methylation-defective mutants are defective in SC elongation, although they seem to have moderate reduction of DSBs. This suggests that high levels of DSBs mediated by histone H3K4 methylation promote SC elongation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    Gat-Viks, Irit; Vingron, Martin

    2009-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Susanne Beyer

    2017-02-01

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

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

    Science.gov (United States)

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

    2017-02-23

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

  4. The histone chaperone Vps75 forms multiple oligomeric assemblies capable of mediating exchange between histone H3-H4 tetramers and Asf1-H3-H4 complexes

    DEFF Research Database (Denmark)

    Hammond, Colin M; Sundaramoorthy, Ramasubramanian; Larance, Mark

    2016-01-01

    Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers...... catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3-H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75....... In the presence of equimolar H3-H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3-H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3-H4 tetramer predominates. We show the Vps75-H3-H4 interaction is compatible with the histone...

  5. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Huang, Hongda; Strømme, Caroline B; Saredi, Giulia

    2015-01-01

    , 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......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...... 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...

  8. Effect of phenylhexyl isothiocyanate on aberrant histone H3 methylation in primary human acute leukemia

    Directory of Open Access Journals (Sweden)

    Zou Yong

    2012-07-01

    Full Text Available Abstract Background We have previously studied the histone acetylation in primary human leukemia cells. However, histone H3 methylation in these cells has not been characterized. Methods This study examined the methylation status at histone H3 lysine 4 (H3K4 and histone H3 lysine 9 (H3K9 in primary acute leukemia cells obtained from patients and compared with those in the non-leukemia and healthy cells. We further characterized the effect of phenylhexyl isothiocyanate (PHI, Trichostatin A (TSA, and 5-aza-2’-deoxycytidine (5-Aza on the cells. Results We found that methylation of histone H3K4 was virtually undetectable, while methylation at H3K9 was significantly higher in primary human leukemia cells. The histone H3K9 hypermethylation and histone H3K4 hypomethylation were observed in both myeloid and lymphoid leukemia cells. PHI was found to be able to normalize the methylation level in the primary leukemia cells. We further showed that PHI was able to enhance the methyltransferase activity of H3K4 and decrease the activity of H3K9 methyltransferase. 5-Aza had similar effect on H3K4, but minimal effect on H3K9, whereas TSA had no effect on H3K4 and H3K9 methyltransferases. Conclusions This study revealed opposite methylation level of H3K4 and H3K9 in primary human leukemia cells and demonstrated for the first time that PHI has different effects on the methyltransferases for H3K4 and H3K9.

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

    Science.gov (United States)

    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 H3 globular domain acetylation identifies a new class of enhancers.

    Science.gov (United States)

    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.

  11. Autoantibodies against citrullinated histone H3 in rheumatoid arthritis and periodontitis patients

    NARCIS (Netherlands)

    Janssen, Koen M. J.; de Smit, Menke J.; Withaar, Coenraad; Brouwer, Elisabeth; van Winkelhoff, Arie J.; Vissink, Arjan; Westra, Johanna

    Aim: To determine the presence of citrullinated histones in inflamed periodontal tissue and to determine the presence of anti-citrullinated histone autoantibodies in sera from patients with rheumatoid arthritis (RA) and periodontitis (PD) patients. Methods: The presence of citrullinated histone H3,

  12. Histone H3 Lysine Methylation in Cognition and Intellectual Disability Disorders

    Science.gov (United States)

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

    2013-01-01

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

  13. Autoantibodies against citrullinated histone H3 in rheumatoid arthritis and periodontitis patients

    NARCIS (Netherlands)

    Janssen, Koen M J; de Smit, Menke J; Withaar, Coenraad; Brouwer, Elisabeth; van Winkelhoff, Arie Jan; Vissink, Arjan; Westra, Johanna

    2017-01-01

    AIM: To determine presence of citrullinated histones in inflamed periodontal tissue, and to determine presence of anti-citrullinated histone autoantibodies in sera from rheumatoid arthritis (RA) and periodontitis (PD)-patients . METHODS: Presence of citrullinated histone H3, PAD4 and CD68 was determ

  14. Apoptotic phosphorylation of histone H3 on Ser-10 by protein kinase Cδ.

    Directory of Open Access Journals (Sweden)

    Choon-Ho Park

    Full Text Available Phosphorylation of histone H3 on Ser-10 is regarded as an epigenetic mitotic marker and is tightly correlated with chromosome condensation during both mitosis and meiosis. However, it was also reported that histone H3 Ser-10 phosphorylation occurs when cells are exposed to various death stimuli, suggesting a potential role in the regulation of apoptosis. Here we report that histone H3 Ser-10 phosphorylation is mediated by the pro-apoptotic kinase protein kinase C (PKC δ during apoptosis. We observed that PKCδ robustly phosphorylates histone H3 on Ser-10 both in vitro and in vivo. Ectopic expression of catalytically active PKCδ efficiently induces condensed chromatin structure in the nucleus. We also discovered that activation of PKCδ is required for histone H3 Ser-10 phosphorylation after treatment with DNA damaging agents during apoptosis. Collectively, these findings suggest that PKCδ is the kinase responsible for histone H3 Ser-10 phosphoryation during apoptosis and thus contributes to chromatin condensation together with other apoptosis-related histone modifications. As a result, histone H3 Ser-10 phosphorylation can be designated a new 'apoptotic histone code' mediated by PKCδ.

  15. The Testis-Enriched Histone Demethylase, KDM4D, Regulates Methylation of Histone H3 Lysine 9 During Spermatogenesis in the Mouse but Is Dispensable for Fertility1

    Science.gov (United States)

    Iwamori, Naoki; Zhao, Ming; Meistrich, Marvin L.; Matzuk, Martin M.

    2011-01-01

    Epigenetic modifications, and methylation of histones in particular, dynamically change during spermatogenesis. Among various methylations of histone H3, methylation of histone H3 lysine 9 (H3K9) and its regulation are essential for spermatogenesis. Trimethytransferases as well as dimethyltransferase are required for meiotic progression. In addition, didemethylase of H3K9 is also critical for spermatogenesis through transcriptional regulation of spermatid-specific genes. However, the requirement for demethylation of trimethylated H3K9 (H3K9me3) during spermatogenesis remains to be elucidated. Here, we report the targeted disruption of KDM4D, a testis-enriched tridemethylase of H3K9. Kdm4d-null mice are viable and fertile and do not show any obvious phenotype. However, H3K9me3 accumulates significantly in Kdm4d-null round spermatids, and the distribution of methylated H3K9 in germ cells is dramatically changed. Nevertheless, the progression of spermatogenesis and the number of spermatozoa are normal, likely secondary to the earlier nuclear localization of another H3K9 tridemethylase, KDM4B, in Kdm4d-null elongating spermatids. These results suggest that demethylation of H3K9me3 in round spermatids is dispensable for spermatogenesis but that possible defects in Kdm4d-null elongating spermatids could be rescued by functional redundancy of the KDM4B demethylase. PMID:21293030

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

    Science.gov (United States)

    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.

  17. Both H4K20 mono-methylation and H3K56 acetylation mark transcription-dependent histone turnover in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hanna [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kwon, Chang Seob [Department of Chemistry and Biology, Korea Science Academy of KAIST, Busan, 614-822 (Korea, Republic of); Choi, Yoonjung, E-mail: jjungii@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Daeyoup, E-mail: daeyoup@kaist.ac.kr [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2016-08-05

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5′ promoter and 3′ termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast.

  18. Histone methyltransferase G9a contributes to H3K27 methylation in vivo

    Institute of Scientific and Technical Information of China (English)

    Hui Wu; Bing Zhu; Xiuzhen Chen; Jun Xiong; Yingfeng Li; Hong Li; Xiaojun Ding; Sheng Liu; She Chen; Shaorong Gao

    2011-01-01

    @@ Dear Editor, Histone modifications play a vital role in the conformation and function of their associated chromatin templates[1].Histone H3K27 methylation mediated by the PRC2 complex is critical for transcriptional regulation,Polycomb silencing,Drosophila segmentation,mammalian X inactivation and cancer[1].Interestingly,H3K27me1(H3 mono-methylated at residue K27)levels in vivo remain unaffected after PRC2 disruption[2,3],which is an indication for the existence of other contributing histone methyltransferase(s)to H3K27me1.

  19. Histone H3 phosphorylation in the rat pineal gland: adrenergic regulation and diurnal variation.

    Science.gov (United States)

    Chik, C L; Arnason, T G; Dukewich, W G; Price, D M; Ranger, A; Ho, A K

    2007-04-01

    In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

  20. Sgf29 binds histone H3K4me2/3 and is required for SAGA complex recruitment and histone H3 acetylation

    Energy Technology Data Exchange (ETDEWEB)

    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 (Toronto); (Stowers); (UST - China); (UV); (Chinese Aca. Sci.); (MCCM)

    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.

  1. The Program for Processing Newly-synthesized Histones H3.1 and H4

    Science.gov (United States)

    Campos, Eric I.; Fillingham, Jeffrey; Li, Guohong; Zheng, Haiyan; Voigt, Philipp; Kuo, Wei-Hung W.; Seepany, Harshika; Gao, Zhonghua; Day, Loren A.; Greenblatt, Jack F.

    2010-01-01

    The mechanism by which newly synthesized histones are imported into the nucleus and deposited onto replicating chromatin alongside segregating nucleosomal counterparts is poorly understood, yet this program is expected to bear on the putative epigenetic nature of histone posttranslational modifications. In order to define the events by which naïve pre-deposition histones are imported into the nucleus, we biochemically purified and characterized the gamut of histone H3.1-containing complexes from human cytoplasmic fractions and identified their associated histone PTMs. Through reconstitution assays, biophysical analyses, and live cell manipulations, we describe in detail this series of events, namely the assembly of H3-H4 dimers, the acetylation of histones by the HAT1 holoenzyme, and the transfer of histones between chaperones that culminates with their karyopherin-mediated nuclear import. We further demonstrate the high degree of conservation for this pathway between higher and lower eukaryotes. PMID:20953179

  2. PHD Finger Recognition of Unmodified Histone H3R2 Links UHRF1 to Regulation of Euchromatic Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Rajakumara, Eerappa; Wang, Zhentian; Ma, Honghui; Hu, Lulu; Chen, Hao; Lin, Yan; Guo, Rui; Wu, Feizhen; Li, Haitao; Lan, Fei; Shi, Yujiang Geno; Xu, Yanhui; Patel, Dinshaw J.; Shi, Yang (MSKCC); (Constellation); (Fudan); (Tsinghua)

    2011-08-29

    Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD{sub UHRF1}), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD{sub UHRF1} bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD{sub UHRF1} binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

  3. CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin

    NARCIS (Netherlands)

    O. Weth (Oliver); C. Paprotka (Christine); K. Günther (Katharina); A. Schulte (Astrid); M. Baierl (Manuel); J. Leers (Joerg); N.J. Galjart (Niels); R. Renkawitz (Rainer)

    2014-01-01

    textabstractInsulators functionally separate active chromatin domains frominactive ones. The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function. CTCF binding sites are depleted for the histone modification H3K27me3 and are enriched for the histone variant

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-09-20

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

  5. Cytogenetic mapping of 5S and 18S rRNAs and H3 histone genes in 4 ancient Proscopiidae grasshopper species: contribution to understanding the evolutionary dynamics of multigene families.

    Science.gov (United States)

    Cabral-de-Mello, D C; Martins, C; Souza, M J; Moura, R C

    2011-01-01

    This paper reports on the chromosomal location of 18S rRNA, 5S rRNA and H3 histone multigene families in 4 species of a relatively ancient and diversified group of grasshoppers belonging to the family Proscopiidae. The 5S rRNA and H3 histone genes were highly conserved in the number of sites and chromosomal position in the 4th chromosome pair in all species analyzed, whereas the 18S rRNA genes showed slightly more variation because they were present on one or 2 chromosome pairs, depending on the species. The 5S and 18S rRNA gene families occurred in different chromosomes; in contrast, H3 histone and 5S rRNA genes co-localized in the same chromosomal position, with an apparently interspersed organization. Considering that the Proscopiidae family is a relatively ancient group compared with the Acrididae family, the association of the H3 histone and 5S rRNA multigene families can represent a basal condition for grasshoppers, although more research is needed on other representatives of this insect group to confirm this statement. The presence of such an association of 5S rDNA and H3 histone in mussels and arthropods (beetles, grasshoppers and crustaceans) suggests that this linked configuration could represent an ancestral pattern for invertebrates. These results provide new insights into the understanding of the genome organization and the evolution of multigene families in grasshoppers and in insects as a whole. Copyright © 2010 S. Karger AG, Basel.

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

    Science.gov (United States)

    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.

  7. Genome-Wide Identification, Evolutionary, and Expression Analyses of Histone H3 Variants in Plants

    Directory of Open Access Journals (Sweden)

    Jinteng Cui

    2015-01-01

    Full Text Available Histone variants alter the nucleosome structure and play important roles in chromosome segregation, transcription, DNA repair, and sperm compaction. Histone H3 is encoded by many genes in most eukaryotic species and is the histone that contains the largest variety of posttranslational modifications. Compared with the metazoan H3 variants, little is known about the complex evolutionary history of H3 variants proteins in plants. Here, we study the identification, evolutionary, and expression analyses of histone H3 variants from genomes in major branches in the plant tree of life. Firstly we identified all the histone three related (HTR genes from the examined genomes, then we classified the four groups variants: centromeric H3, H3.1, H3.3 and H3-like, by phylogenetic analysis, intron information, and alignment. We further demonstrated that the H3 variants have evolved under strong purifying selection, indicating the conservation of HTR proteins. Expression analysis revealed that the HTR has a wide expression profile in maize and rice development and plays important roles in development.

  8. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells.

    Science.gov (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene

    2016-04-01

    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  9. Stage and gene specific signatures defined by histones H3K4me2 and H3K27me3 accompany mammalian retina maturation in vivo.

    Science.gov (United States)

    Popova, Evgenya Y; Xu, Xuming; DeWan, Andrew T; Salzberg, Anna C; Berg, Arthur; Hoh, Josephine; Zhang, Samuel S; Barnstable, Colin J

    2012-01-01

    The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo.

  10. Stage and gene specific signatures defined by histones H3K4me2 and H3K27me3 accompany mammalian retina maturation in vivo.

    Directory of Open Access Journals (Sweden)

    Evgenya Y Popova

    Full Text Available The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo.

  11. The histone H3 N-terminal tail: a computational analysis of the free energy landscape and kinetics.

    Science.gov (United States)

    Zheng, Yuqing; Cui, Qiang

    2015-05-28

    Histone tails are the short peptide protrusions outside of the nucleosome core particle and they play a critical role in regulating chromatin dynamics and gene activity. A histone H3 N-terminal tail, like other histone tails, can be covalently modified on different residues to activate or repress gene expression. Previous studies have indicated that, despite its intrinsically disordered nature, the histone H3 N-terminal tail has regions of notable secondary structural propensities. To further understand the structure-dynamics-function relationship in this system, we have carried out 75.6 μs long implicit solvent simulations and 29.3 μs long explicit solvent simulations. The extensive samplings allow us to better characterize not only the underlying free energy landscape but also kinetic properties through Markov state models (MSM). Dihedral principal component analysis (dPCA) and locally scaled diffusion map (LSDMap) analysis yield consistent results that indicate an overall flat free energy surface with several shallow basins that correspond to conformations with a high α-helical propensity in two regions of the peptide. Kinetic information extracted from Markov state models reveals rapid transitions between different metastable states with mean first passage times spanning from several hundreds of nanoseconds to hundreds of microseconds. These findings shed light on how the dynamical nature of the histone H3 N-terminal tail is related to its function. The complementary nature of dPCA, LSDMap and MSM for the analysis of biomolecules is also discussed.

  12. Dynamic Regulation of Histone Modifications in Xenopus Oocytes through Histone Exchange

    Science.gov (United States)

    Stewart, M. David; Sommerville, John; Wong, Jiemin

    2006-01-01

    Histone H3 lysine 9 (H3K9) methylation has broad roles in transcriptional repression, gene silencing, maintenance of heterochromatin, and epigenetic inheritance of heterochromatin. Using Xenopus laevis oocytes, we have previously shown that targeting G9a, an H3K9 histone methyltransferase, to chromatin increases H3K9 methylation and consequently represses transcription. Here we report that treatment with trichostatin A induces histone acetylation and is sufficient to activate transcription repressed by G9a, and this activation is accompanied by a reduction in dimethyl H3K9 (H3K9me2). We tested the possibility that the reduction in H3K9me2 was due to the replacement of methylated H3 with unmethylated H3.3. Surprisingly, we found that both free H3 and H3.3 are continually exchanged with chromatin-associated histones. This dynamic exchange of chromatin-associated H3 with free H3/H3.3 was not affected by alterations in transcriptional activity, elongation, acetylation, H3K9 methylation, or DNA replication. In support of this continual histone exchange model, we show that maintenance of H3K9 methylation at a specific site requires the continual presence of an H3K9 histone methyltransferase. Upon dissociation of the methyltransferase, H3K9 methylation decreases. Taken together, our data suggest that chromatin-associated and non-chromatin-associated histones are continually exchanged in the Xenopus oocyte, creating a highly dynamic chromatin environment. PMID:16943430

  13. Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis

    OpenAIRE

    Wang, Lifeng; Xu, Shiliyang; Lee, Ji-Eun; Baldridge, Anne; Grullon, Sean; Peng, Weiqun; Ge, Kai

    2012-01-01

    PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels dec...

  14. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  15. Human tNASP promotes in vitro nucleosome assembly with histone H3.3.

    Science.gov (United States)

    Kato, Daiki; Osakabe, Akihisa; Tachiwana, Hiroaki; Tanaka, Hiroki; Kurumizaka, Hitoshi

    2015-02-10

    Nuclear autoantigenic sperm proteins (NASPs) are members of the acidic histone chaperones, which promote nucleosome assembly. In humans, two splicing variants proposed for the somatic and testicular isoforms, sNASP and tNASP, respectively, have been found, and the shorter form, sNASP, reportedly promotes nucleosome assembly with the histone H3 isoforms, H3.1, H3.2, and H3.3. However, the biochemical properties of the longer form, tNASP, have not been reported. tNASP is considered to exist specifically in the testis. Our present results revealed that the tNASP protein is ubiquitously produced in various human tissues, in addition to testis. Unexpectedly, we found that the nucleosome assembly activity of purified tNASP was extremely low with the canonical histone H3.1 or H3.2, but was substantially detected with the replacement histone H3.3 variant. A mutational analysis revealed that the H3.3 Ile89 residue, corresponding to the H3.1 Val89 residue, is responsible for the tNASP-mediated nucleosome assembly with H3.3. A histone deposition assay showed that the H3.3-H4 complex is more efficiently deposited onto DNA by tNASP than the H3.1-H4 complex. These results provide evidence that tNASP is ubiquitously produced in various types of human tissues and promotes in vitro nucleosome assembly with H3 variant specificity.

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

    Science.gov (United States)

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

    2016-01-21

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

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

    DEFF Research Database (Denmark)

    Wurtele, Hugo; Kaiser, Gitte Schalck; Bacal, Julien;

    2012-01-01

    but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes......In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA...... damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin...

  18. Altered nucleosomes of active nucleolar chromatin contain accessible histone H3 in its hyperacetylated forms

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, E.M.; Sterner, R.; Allfrey, V.G.

    1987-05-25

    Chromatin of the organism Physarum polycephalum contains a class of conformationally altered nucleosomes previously localized to the transcribing regions of ribosomal genes in nucleoli. When nuclei are treated with 2-iodo(2-tritium)acetate, the histone H3 sulfhydryl group of the altered nucleosomes is derivatized while that of folded nucleosomes is not, and the labeled histones can then be identified by autoradiography of gels that separate H3 isoforms. The H3 derivatized is predominantly of tri- and tetraacetylated forms. In contrast, total free histone reacted with iodoacetate shows no preferential labeling of isoforms. Selective reaction of acetylated H3 is prevalent in both nucleolar and non-nucleolar chromatin. The results link specific patterns of H3 acetylation to changes in nucleosome conformation that occur during transcription.

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

    Science.gov (United States)

    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

  20. Histone H3 gene in the Pacific oyster, Crassostrea gigas Thunberg, 1793: molecular and cytogenetic characterisations

    Directory of Open Access Journals (Sweden)

    Karine Bouilly

    2010-12-01

    Full Text Available The Pacific oyster, Crassostrea gigas Thunberg, 1793 (2n = 20 is an economically important mollusc species cultured throughout the world. The most frequently used technique for molecular cytogenetic studies is fluorescence in situ hybridisation which offers new opportunities for the identification of oyster chromosomes. In oysters, it has been used to locate telomeric sequences, satellite DNA, simple sequence repeats, ribosomal RNA genes, and bacteriophage P1 clones. However, regarding chromosome identification, no study has been done with histone H3 gene. Histone H3 is among the most conserved eukaryotic proteins. Most histone H3 genes are repeatedly organised into clusters, which make them an ideal chromosomal marker. In bivalves, some data exist concerning sequence information but little knowledge is available concerning the physical mapping of histone genes. The histone H3 gene was sequenced in C. gigas and phylogenetic analysis revealed that C. gigas was more closely related to Ostrea edulis Linnaeus, 1758 and species of the genus Mytilus Linnaeus, 1758. In C. gigas, the histone H3 gene was mapped on two different pairs of chromosomes, one at an interstitial site on the long arm of chromosome pair 4, and the other on the telomeres of the smaller chromosome pair (pair 10. Polymorphism was detected on the telomeres of pair 10, once it was possible to observe single or double signals. Comparative chromosomal mapping should improve our understanding of bivalve genome organisation.

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

    Science.gov (United States)

    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.

  2. Double chromodomains cooperate to recognize the methylated histone H3 tail

    Energy Technology Data Exchange (ETDEWEB)

    Flanagan, John F.; Mi, Li-Zhi; Chruszcz, Maksymilian; Cymborowski, Marcin; Clines, Katrina L.; Kim, Youngchang; Minor, Wladek; Rastinejad, Fraydoon; Khorasanizadeh, Sepideh (ANL/SBC); (UV)

    2010-07-19

    Chromodomains are modules implicated in the recognition of lysine-methylated histone tails and nucleic acids. CHD (for chromo-ATPase/helicase-DNA-binding) proteins regulate ATP-dependent nucleosome assembly and mobilization through their conserved double chromodomains and SWI2/SNF2 helicase/ATPase domain. The Drosophila CHD1 localizes to the interbands and puffs of the polytene chromosomes, which are classic sites of transcriptional activity. Other CHD isoforms (CHD3/4 or Mi-2) are important for nucleosome remodelling in histone deacetylase complexes. Deletion of chromodomains impairs nucleosome binding and remodelling by CHD proteins. Here we describe the structure of the tandem arrangement of the human CHD1 chromodomains, and its interactions with histone tails. Unlike HP1 and Polycomb proteins that use single chromodomains to bind to their respective methylated histone H3 tails, the two chromodomains of CHD1 cooperate to interact with one methylated H3 tail. We show that the human CHD1 double chromodomains target the lysine 4-methylated histone H3 tail (H3K4me), a hallmark of active chromatin. Methylammonium recognition involves two aromatic residues, not the three-residue aromatic cage used by chromodomains of HP1 and Polycomb proteins. Furthermore, unique inserts within chromodomain 1 of CHD1 block the expected site of H3 tail binding seen in HP1 and Polycomb, instead directing H3 binding to a groove at the inter-chromodomain junction.

  3. Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila

    Directory of Open Access Journals (Sweden)

    Philip Yuk Kwong Yung

    2015-06-01

    Full Text Available Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28 whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

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

    Science.gov (United States)

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

    2016-08-17

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

  5. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes.

    Science.gov (United States)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-12-01

    Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  6. Chromosomal organization of the 18S and 5S rRNAs and histone H3 genes in Scarabaeinae coleopterans: insights into the evolutionary dynamics of multigene families and heterochromatin

    Directory of Open Access Journals (Sweden)

    Martins Cesar

    2011-10-01

    Full Text Available Abstract Background Scarabaeinae beetles show a high level of macro-chromosomal variability, although the karyotypic organization of heterochromatin and multigene families (rDNAs and histone genes is poorly understood in this group. To better understand the chromosomal organization and evolution in this group, we analyzed the karyotypes, heterochromatin distribution and chromosomal locations of the rRNAs and histone H3 genes in beetles belonging to eight tribes from the Scarabaeinae subfamily (Coleoptera, Scarabaeidae. Results The number of 18S rRNA gene (a member of the 45S rDNA unit sites varied from one to 16 and were located on the autosomes, sex chromosomes or both, although two clusters were most common. Comparison of the 45S rDNA cluster number and the diploid numbers revealed a low correlation value. However, a comparison between the number of 45S rDNA sites per genome and the quantity of heterochromatin revealed (i species presenting heterochromatin restricted to the centromeric/pericentromeric region that contained few rDNA sites and (ii species with a high quantity of heterochromatin and a higher number of rDNA sites. In contrast to the high variability for heterochromatin and 45S rDNA cluster, the presence of two clusters (one bivalent cluster co-located on autosomal chromosomes with the 5S rRNA and histone H3 genes was highly conserved. Conclusions Our results indicate that the variability of the 45S rDNA chromosomal clusters is not associated with macro-chromosomal rearrangements but are instead related to the spread of heterochromatin. The data obtained also indicate that both heterochromatin and the 45S rDNA loci could be constrained by similar evolutionary forces regulating spreading in the distinct Scarabaeinae subfamily lineages. For the 5S rRNA and the histone H3 genes, a similar chromosomal organization could be attributed to their association/co-localization in the Scarabaeinae karyotypes. These data provide evidence that

  7. CFP1 Regulates Histone H3K4 Trimethylation and Developmental Potential in Mouse Oocytes

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    Chao Yu

    2017-08-01

    Full Text Available Trimethylation of histone H3 at lysine-4 (H3K4me3 is associated with eukaryotic gene promoters and poises their transcriptional activation during development. To examine the in vivo function of H3K4me3 in the absence of DNA replication, we deleted CXXC finger protein 1 (CFP1, the DNA-binding subunit of the SETD1 histone H3K4 methyltransferase, in developing oocytes. We find that CFP1 is required for H3K4me3 accumulation and the deposition of histone variants onto chromatin during oocyte maturation. Decreased H3K4me3 in oocytes caused global downregulation of transcription activity. Oocytes lacking CFP1 failed to complete maturation and were unable to gain developmental competence after fertilization, due to defects in cytoplasmic lattice formation, meiotic division, and maternal-zygotic transition. Our study highlights the importance of H3K4me3 in continuous histone replacement for transcriptional regulation, chromatin remodeling, and normal developmental progression in a non-replicative system.

  8. Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27) Deacetylation and Methylation.

    Science.gov (United States)

    Li, Fenfen; Wu, Rui; Cui, Xin; Zha, Lin; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-02-26

    Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity, and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of the brown adipocyte thermogenic program. The Hdac1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or β3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing Hdac1 profoundly blocks, whereas deleting Hdac1 significantly enhances, β-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. β-Adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from (and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to) Ucp1 and Pgc1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates the brown adipocyte thermogenic program, and inhibiting Hdac1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching the transcriptional repressive state to the active state at the promoters of Ucp1 and Pgc1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis.

  9. The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin.

    Science.gov (United States)

    Bao, Xiaomin; Cai, Weili; Deng, Huai; Zhang, Weiguo; Krencik, Robert; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-11-21

    The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.

  10. The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms

    Directory of Open Access Journals (Sweden)

    Postberg Jan

    2010-08-01

    Full Text Available Abstract Background The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins. Results In the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes. Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts (animals, choanozoa, fungi, nuclearids, Amoebozoa. H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as Trichoplax. H3.1 and H3.1t are probably restricted to mammals. We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are

  11. Structural Insights into Selective Histone H3 Recognition by the Human Polybromo bromodomain 2

    Energy Technology Data Exchange (ETDEWEB)

    Charlop-Powers, Z.; Zeng, L; Zhang, Q; Zhou, M

    2010-01-01

    The Polybromo (PB) protein functions as a key component of the human PBAF chromatin remodeling complex in regulation of gene transcription. PB is made up of modular domains including six bromodomains that are known as acetyl-lysine binding domains. However, histone-binding specificity of the bromodomains of PB has remained elusive. In this study, we report biochemical characterization of all six PB bromodomains' binding to a suite of lysine-acetylated peptides derived from known acetylation sites on human core histones. We demonstrate that bromodomain 2 of PB preferentially recognizes acetylated lysine 14 of histone H3 (H3K14ac), a post-translational mark known for gene transcriptional activation. We further describe the molecular basis of the selective H3K14ac recognition of bromodomain 2 by solving the protein structures in both the free and bound forms using X-ray crystallography and NMR, respectively.

  12. Qualitative and quantitative analysis of lysine acetylation and methylation in yeast histone H3

    Science.gov (United States)

    Zhang, Kangling

    2008-01-01

    Histone post-translational modifications play important roles in cell functions and the modification patterns vary significantly among different organisms. It is important that histone modification patterns be identified. Flowing our previous work-identification of acetylation and methylation sites of histone H3 in a typical transcription most inactive chromatin isolated from chicken erythrocytes, here, we report using mass spectrometry to qualitatively and quantitatively analyze histone modification pattern of H3 in a typical transcription most active chromatin isolated from Saccharomyces cerevisiae. We compared the modification patterns of histone H3 between these two functionally opposite chromatins and observed that acetylation level at K9, K14, K27, K56 and methylation level at K4 and K79 are significantly higher in S. cerevisiae than in chicken erythrocytes, methylation at K9 is higher in chicken erythrocytes than in S. cerevisiae and methylation level at K36 is unchanged in these two chromatins. Contrary to other sites, acetylation levels at K18 and K23 are higher in chicken erythrocytes than in S. cerevisiae. Our data revealed the difference of acetylation and methylation pattern of individual H3 lysine between two distinct chromatins, one with more inactive form versus the other with more active form.

  13. Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

    Directory of Open Access Journals (Sweden)

    Ysabel Montoya

    2000-08-01

    Full Text Available Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V. peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V. braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2006-12-12

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

  16. Ornithine decarboxylase antizyme induces hypomethylation of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2 in human oral cancer cell line.

    Directory of Open Access Journals (Sweden)

    Daisuke Yamamoto

    Full Text Available BACKGROUND: Methylation of CpG islands of genome DNA and lysine residues of histone H3 and H4 tails regulates gene transcription. Inhibition of polyamine synthesis by ornithine decarboxylase antizyme-1 (OAZ in human oral cancer cell line resulted in accumulation of decarboxylated S-adenosylmethionine (dcSAM, which acts as a competitive inhibitor of methylation reactions. We anticipated that accumulation of dcSAM impaired methylation reactions and resulted in hypomethylation of genome DNA and histone tails. METHODOLOGY/PRINCIPAL FINDINGS: Global methylation state of genome DNA and lysine residues of histone H3 and H4 tails were assayed by Methylation by Isoschizomers (MIAMI method and western blotting, respectively, in the presence or absence of OAZ expression. Ectopic expression of OAZ mediated hypomethylation of CpG islands of genome DNA and histone H3 lysine 9 dimethylation (H3K9me2. Protein level of DNA methyltransferase 3B (DNMT3B and histone H3K9me specific methyltransferase G9a were down-regulated in OAZ transfectant. CONCLUSIONS/SIGNIFICANCE: OAZ induced hypomethylation of CpG islands of global genome DNA and H3K9me2 by down-regulating DNMT3B and G9a protein level. Hypomethylation of CpG islands of genome DNA and histone H3K9me2 is a potent mechanism of induction of the genes related to tumor suppression and DNA double strand break repair.

  17. Lysyl oxidase-like 2 (LOXL2) oxidizes trimethylated lysine 4 in histone H3.

    Science.gov (United States)

    Herranz, Nicolás; Dave, Natàlia; Millanes-Romero, Alba; Pascual-Reguant, Laura; Morey, Lluis; Díaz, Víctor M; Lórenz-Fonfría, Víctor; Gutierrez-Gallego, Ricardo; Jerónimo, Celia; Iturbide, Ane; Di Croce, Luciano; García de Herreros, Antonio; Peiró, Sandra

    2016-12-01

    Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation.

  18. Differential contributions of histone H3 and H4 residues to heterochromatin structure.

    Science.gov (United States)

    Yu, Qun; Olsen, Lars; Zhang, Xinmin; Boeke, Jef D; Bi, Xin

    2011-06-01

    Transcriptional silencing in Saccharomyces cerevisiae is mediated by heterochromatin. There is a plethora of information regarding the roles of histone residues in transcriptional silencing, but exactly how histone residues contribute to heterochromatin structure is not resolved. We address this question by testing the effects of a series of histone H3 and H4 mutations involving residues in their aminoterminal tails, on the solvent-accessible and lateral surfaces of the nucleosome, and at the interface of the H3/H4 tetramer and H2A/H2B dimer on heterochromatin structure and transcriptional silencing. The general state, stability, and conformational heterogeneity of chromatin are examined with a DNA topology-based assay, and the primary chromatin structure is probed by micrococcal nuclease. We demonstrate that the histone mutations differentially affect heterochromatin. Mutations of lysine 16 of histone H4 (H4-K16) and residues in the LRS (loss of rDNA silencing) domain of nucleosome surface markedly alter heterochromatin structure, supporting the notion that H4-K16 and LRS play key roles in heterochromatin formation. Deletion of the aminoterminal tail of H3 moderately alters heterochromatin structure. Interestingly, a group of mutations in the globular domains of H3 and H4 that abrogate or greatly reduce transcriptional silencing increase the conformational heterogeneity and/or reduce the stability of heterochromatin without affecting its overall structure. Surprisingly, yet another series of mutations abolish or reduce silencing without significantly affecting the structure, stability, or conformational heterogeneity of heterochromatin. Therefore, histone residues may contribute to the structure, stability, conformational heterogeneity, or other yet-to-be-characterized features of heterochromatin important for transcriptional silencing.

  19. Regional control of histone H3 lysine 27 methylation in Neurospora.

    Science.gov (United States)

    Jamieson, Kirsty; Rountree, Michael R; Lewis, Zachary A; Stajich, Jason E; Selker, Eric U

    2013-04-09

    Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis, worms, and mammals, but is absent from yeasts that have been examined. We identified and analyzed H3K27me3 in the filamentous fungus Neurospora crassa and in other Neurospora species. H3K27me3 covers 6.8% of the N. crassa genome, encompassing 223 domains, including 774 genes, all of which are transcriptionally silent. N. crassa H3K27me3-marked genes are less conserved than unmarked genes and only ∼35% of genes marked by H3K27me3 in N. crassa are also H3K27me3-marked in Neurospora discreta and Neurospora tetrasperma. We found that three components of the Neurospora Polycomb repressive complex 2 (PRC2)--[Su-(var)3-9; E(z); Trithorax] (SET)-7, embryonic ectoderm development (EED), and SU(Z)12 (suppressor of zeste12)--are required for H3K27me3, whereas the fourth component, Neurospora protein 55 (an N. crassa homolog of p55/RbAp48), is critical for H3K27me3 only at subtelomeric domains. Loss of H3K27me3, caused by deletion of the gene encoding the catalytic PRC2 subunit, set-7, resulted in up-regulation of 130 genes, including genes in both H3K27me3-marked and unmarked regions.

  20. Relationship between lunasin's sequence and its inhibitory activity of histones H3 and H4 acetylation.

    Science.gov (United States)

    Hernández-Ledesma, Blanca; Hsieh, Chia-Chien; de Lumen, Ben O

    2011-07-01

    Dysfunction of histone acetyltransferases (HATs) or histone deacetylases (HDACs) involved in histones acetylation has been associated with cancer. Inhibitors of these enzymes are becoming potential targets for new therapies. This study reports by Western-Blot analysis, that peptide lunasin is mainly an in vitro inhibitor of histone H4 acetylation by P300/cAMP-response element-binding protein (CBP)-associated factor (PCAF), with IC₅₀ values dependent on the lysine position sensitive to be acetylated (0.83 μM (H4-Lys 8), 1.27 μM (H4-Lys 12) and 0.40 μM (H4-Lys 5, 8, 12, 16)). Lunasin is also capable of inhibiting H3 acetylation (IC₅₀ of 5.91 μM (H3-Lys 9) and 7.81 μM (H3-Lys 9, 14)). Studies on structure-activity relationship establish that lunasin's sequence are essential for inhibiting H4 acetylation whereas poly-D sequence is the main active sequence responsible for H3 acetylation inhibition. Lunasin also inhibits H3 and H4 acetylation and cell proliferation (IC₅₀ of 181 μM) in breast cancer MDA-MB-231 cells. Moreover, this peptide decreases expression of cyclins and cyclin dependent kinases-4 and -6, implicated in cell cycle pathways. Results from this study demonstrates lunasin's role as modulator of histone acetylation and protein expression that might contribute on its chemopreventive properties against breast cancer. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Methylation of histone H3K23 blocks DNA damage in pericentric heterochromatin during meiosis

    Science.gov (United States)

    Papazyan, Romeo; Voronina, Ekaterina; Chapman, Jessica R; Luperchio, Teresa R; Gilbert, Tonya M; Meier, Elizabeth; Mackintosh, Samuel G; Shabanowitz, Jeffrey; Tackett, Alan J; Reddy, Karen L; Coyne, Robert S; Hunt, Donald F; Liu, Yifan; Taverna, Sean D

    2014-01-01

    Despite the well-established role of heterochromatin in protecting chromosomal integrity during meiosis and mitosis, the contribution and extent of heterochromatic histone posttranslational modifications (PTMs) remain poorly defined. Here, we gained novel functional insight about heterochromatic PTMs by analyzing histone H3 purified from the heterochromatic germline micronucleus of the model organism Tetrahymena thermophila. Mass spectrometric sequencing of micronuclear H3 identified H3K23 trimethylation (H3K23me3), a previously uncharacterized PTM. H3K23me3 became particularly enriched during meiotic leptotene and zygotene in germline chromatin of Tetrahymena and C. elegans. Loss of H3K23me3 in Tetrahymena through deletion of the methyltransferase Ezl3p caused mislocalization of meiosis-induced DNA double-strand breaks (DSBs) to heterochromatin, and a decrease in progeny viability. These results show that an evolutionarily conserved developmental pathway regulates H3K23me3 during meiosis, and our studies in Tetrahymena suggest this pathway may function to protect heterochromatin from DSBs. DOI: http://dx.doi.org/10.7554/eLife.02996.001 PMID:25161194

  2. 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).

    Science.gov (United States)

    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.

  3. Modification of histones by sugar β-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated.

    Science.gov (United States)

    Zhang, Suisheng; Roche, Kevin; Nasheuer, Heinz-Peter; Lowndes, Noel Francis

    2011-10-28

    The monosaccharide, β-N-acetylglucosamine (GlcNAc), can be added to the hydroxyl group of either serines or threonines to generate an O-linked β-N-acetylglucosamine (O-GlcNAc) residue (Love, D. C., and Hanover, J. A. (2005) Sci. STKE 2005 312, 1-14; Hart, G. W., Housley, M. P., and Slawson, C. (2007) Nature 446, 1017-1022). This post-translational protein modification, termed O-GlcNAcylation, is reversible, analogous to phosphorylation, and has been implicated in many cellular processes. Here, we present evidence that in human cells all four core histones of the nucleosome are substrates for this glycosylation in the relative abundance H3, H4/H2B, and H2A. Increasing the intracellular level of UDP-GlcNAc, the nucleotide sugar donor substrate for O-GlcNAcylation enhanced histone O-GlcNAcylation and partially suppressed phosphorylation of histone H3 at serine 10 (H3S10ph). Expression of recombinant H3.3 harboring an S10A mutation abrogated histone H3 O-GlcNAcylation relative to its wild-type version, consistent with H3S10 being a site of histone O-GlcNAcylation (H3S10glc). Moreover, O-GlcNAcylated histones were lost from H3S10ph immunoprecipitates, whereas immunoprecipitation of either H3K4me3 or H3K9me3 (active or inactive histone marks, respectively) resulted in co-immunoprecipitation of O-GlcNAcylated histones. We also examined histone O-GlcNAcylation during cell cycle progression. Histone O-GlcNAcylation is high in G(1) cells, declines throughout the S phase, increases again during late S/early G(2), and persists through late G(2) and mitosis. Thus, O-GlcNAcylation is a novel histone post-translational modification regulating chromatin conformation during transcription and cell cycle progression.

  4. The MTA family proteins as novel histone H3 binding proteins

    Directory of Open Access Journals (Sweden)

    Wu Meng

    2013-01-01

    Full Text Available Abstract Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

  5. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

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    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  6. Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1.

    Science.gov (United States)

    Zhang, Wei; Tyl, Marek; Ward, Richard; Sobott, Frank; Maman, Joseph; Murthy, Andal S; Watson, Aleksandra A; Fedorov, Oleg; Bowman, Andrew; Owen-Hughes, Tom; El Mkami, Hassane; Murzina, Natalia V; Norman, David G; Laue, Ernest D

    2013-01-01

    The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of the histone H3-H4 complex with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3-H4 between these two histone chaperones has a central role in the assembly of new nucleosomes, and we show here that the H3-H4 complex has an unexpected structural plasticity, which is important for this exchange.

  7. Mitotic accumulation of dimethylated lysine 79 of histone H3 is important for maintaining genome integrity during mitosis in human cells.

    Science.gov (United States)

    Guppy, Brent J; McManus, Kirk J

    2015-02-01

    The loss of genome stability is an early event that drives the development and progression of virtually all tumor types. Recent studies have revealed that certain histone post-translational modifications exhibit dynamic and global increases in abundance that coincide with mitosis and exhibit essential roles in maintaining genomic stability. Histone H2B ubiquitination at lysine 120 (H2Bub1) is regulated by RNF20, an E3 ubiquitin ligase that is altered in many tumor types. Through an evolutionarily conserved trans-histone pathway, H2Bub1 is an essential prerequisite for subsequent downstream dimethylation events at lysines 4 (H3K4me2) and 79 (H3K79me2) of histone H3. Although the role that RNF20 plays in tumorigenesis has garnered much attention, the downstream components of the trans-histone pathway, H3K4me2 and H3K79me2, and their potential contributions to genome stability remain largely overlooked. In this study, we employ single-cell imaging and biochemical approaches to investigate the spatial and temporal patterning of RNF20, H2Bub1, H3K4me2, and H3K79me2 throughout the cell cycle, with a particular focus on mitosis. We show that H2Bub1, H3K4me2, and H3K79me2 exhibit distinct temporal progression patterns throughout the cell cycle. Most notably, we demonstrate that H3K79me2 is a highly dynamic histone post-translational modification that reaches maximal abundance during mitosis in an H2Bub1-independent manner. Using RNAi and chemical genetic approaches, we identify DOT1L as a histone methyltransferase required for the mitotic-associated increases in H3K79me2. We also demonstrate that the loss of mitotic H3K79me2 levels correlates with increases in chromosome numbers and increases in mitotic defects. Collectively, these data suggest that H3K79me2 dynamics during mitosis are normally required to maintain genome stability and further implicate the loss of H3K79me2 during mitosis as a pathogenic event that contributes to the development and progression of tumors.

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

    Science.gov (United States)

    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.

  9. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases

    DEFF Research Database (Denmark)

    Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath;

    2011-01-01

    Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model...

  10. Altered global histone-trimethylation code and H3F3A-ATRX mutation in pediatric GBM.

    Science.gov (United States)

    Pathak, Pankaj; Jha, Prerana; Purkait, Suvendu; Sharma, Vikas; Suri, Vaishali; Sharma, Mehar C; Faruq, Mohammed; Suri, Ashish; Sarkar, Chitra

    2015-02-01

    Mutations in H3.3-ATRX-DAXX chromatin remodeling pathway have been reported in pediatric GBMs. H3.3 (H3F3A) mutations may affect transcriptional regulation by altered global histone-methylation. Therefore, we analyzed yet partly understood global histone code (H3K-4/9/27/36) trimethylation pattern in H3F3A-ATRX mutants and wild-type. H3F3A, HIST1H3B, IDH1, ATRX, DAXX and Tp53 mutations were identified by sequencing/immunohistochemistry in 27 pediatric GBMs. Global histone-methylation H3K-4/9/27/36me3 and Polycomb-protein EZH2 expression were evaluated by immunohistochemistry. H3F3A-ATRX mutation was observed in 66.7 % (18/27) of pediatric GBMs. K27M and G34R-H3F3A mutations were found in 37 % (10/27) and 14.8 % (4/27) patients respectively. G34V-H3F3A, HIST1H3B and IDH1 mutations were absent. Notably, commonest global histone-methylation mark lost was H3K27me3 (17/25, 68 %) followed by H3K4me3 (45.5 %, 10/22) and H3K9me3 (18.2 %, 4/22). Global H3K36me3 showed no loss. Most significant observation was loss of one or more histone-trimethylation mark in 80 % (20/25) pediatric GBMs. Notably, simultaneous loss of H3K27me3 and H3K4me3 were present in 7/22 (31.8 %) of pediatric GBMs. Low expression of EZH2 was found in 12/24 (50 %) of cases. However no significant correlation of loss of histone-marks or EZH2 expression with H3F3A-ATRX mutants (loss of at least one histone-marks in 87.5 % (14/16) cases) versus wild-types (loss of at least one histone-marks in 75 % (6/8) cases) was seen. The present study highlights for the first time combinatorial loss of one or more histone-trimethylation marks associated with majority of pediatric GBMs and the finding suggests significant role of histone-code in the molecular biology that underlies pediatric GBMs. Hence therapies for patients with particular combinations of histone modifications present opportunity to design innovative patient-tailored treatment protocols.

  11. K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase.

    Science.gov (United States)

    Kobza, Keyna; Camporeale, Gabriela; Rueckert, Brian; Kueh, Alice; Griffin, Jacob B; Sarath, Gautam; Zempleni, Janos

    2005-08-01

    Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues. These modifications regulate processes such as gene expression, DNA repair, and mitosis and meiosis. Recently, evidence has been provided that histones are also modified by covalent binding of the vitamin biotin. The aims of this study were to identify biotinylation sites in histone H3, and to investigate the crosstalk among histone biotinylation, methylation and phosphorylation. Synthetic peptides based on the sequence of human histone H3 were used as substrates for enzymatic biotinylation by biotinidase; biotin in peptides was probed using streptavidin peroxidase. These studies provided evidence that K4, K9 and K18 in histone H3 are good targets for biotinylation; K14 and K23 are relatively poor targets. Antibodies were generated to histone H3, biotinylated either at K4, K9 or K18. These antibodies localized to nuclei in human placental cells in immunocytochemistry and immunoblotting experiments, suggesting that lysines in histone H3 are biotinylated in vivo. Dimethylation of R2, R8 and R17 increased biotinylation of K4, K9 and K18, respectively, by biotinidase; phosphorylation of S10 abolished biotinylation of K9. These observations are consistent with crosstalk between biotinylation of histones and other known modifications of histones. We speculate that this crosstalk provides a link to known roles for biotin in gene expression and cell proliferation.

  12. Association Between Phosphorylated Histone H3 and Oncotype DX Recurrence Scores in Breast Cancer

    Science.gov (United States)

    Lee, Lik Hang; Swanson, Paul E.; Tang, Patricia A.; Bigras, Gilbert

    2017-01-01

    We investigate the association between phosphorylated histone H3 (PhH3) and Oncotype DX recurrence score (RS). All invasive breast carcinoma with RS results from our city between 2007 and 2010 (n=47) were reviewed. Whole-tumor sections were stained for PhH3. Mitotic and PhH3 counts were performed and clinical charts reviewed. PhH3 correlated well with RS (r=0.69, P<0.001). Other correlations were: PhH3 versus mitotic count (r=0.87, P<0.001), PhH3 versus mitotic score (r=0.71, P<0.001), PhH3 versus modified Bloom-Richardson-Elston (MBR) grade (r=0.65, P<0.001), RS versus mitotic count (r=0.62, P<0.001), RS versus mitotic score (r=0.44, P=0.002), and RS versus MBR grade (r=0.49, P=0.001). Significant correlation between PhH3 and RS remained after controlling for mitotic count (r=0.39, P=0.007), mitotic score (r=0.60, P<0.001), MBR grade (r=0.56, P<0.001), and all 3 (r=0.37, P=0.014) by partial correlation. Two patients died of metastasis at 12 and 38 months after diagnosis. One had intermediate RS, and 1 high RS; both were in the top-third of PhH3 count. All other patients are alive and recurrence free. Correlation between PhH3 and RS was statistically significant in our cohort, and remained significant after controlling for traditional measures of proliferation. Given that RS has an established strong relationship with prognosis and therapy responsiveness, PhH3 may thus also be an important prognostic/predictive marker in breast cancer. PMID:26371428

  13. Chromosomal protein HMGN1 enhances the acetylation of lysine 14 in histone H3

    OpenAIRE

    Lim, Jae-Hwan; West, Katherine L.; Rubinstein, Yaffa; Bergel, Michael; Postnikov, Yuri V.; Bustin, Michael

    2005-01-01

    The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1−/− cells are lower than in Hmgn1+/+ cells. Induced expressio...

  14. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3

    DEFF Research Database (Denmark)

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

    2006-01-01

    Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation...... and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing...... 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression...

  15. Citrullination of histone H3 interferes with HP1-mediated transcriptional repression.

    Directory of Open Access Journals (Sweden)

    Priyanka Sharma

    2012-09-01

    Full Text Available Multiple Sclerosis (MS is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs. We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4, an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing.

  16. Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics

    Directory of Open Access Journals (Sweden)

    Kaufman Paul D

    2006-12-01

    Full Text Available Abstract Background The histone H3/H4 chaperone Asf1 (anti-silencing function 1 is required for the establishment and maintenance of proper chromatin structure, as well as for genome stability in eukaryotes. Asf1 participates in both DNA replication-coupled (RC and replication-independent (RI histone deposition reactions in vitro and interacts with complexes responsible for both pathways in vivo. Asf1 is known to directly bind histone H3, however, high-resolution structural information about the geometry of this interaction was previously unknown. Results Here we report the structure of a histone/histone chaperone interaction. We have solved the 2.2 Å crystal structure of the conserved N-terminal immunoglobulin fold domain of yeast Asf1 (residues 2–155 bound to the C-terminal helix of yeast histone H3 (residues 121–134. The structure defines a histone-binding patch on Asf1 consisting of both conserved and yeast-specific residues; mutation of these residues abrogates H3/H4 binding affinity. The geometry of the interaction indicates that Asf1 binds to histones H3/H4 in a manner that likely blocks sterically the H3/H3 interface of the nucleosomal four-helix bundle. Conclusion These data clarify how Asf1 regulates histone stoichiometry to modulate epigenetic inheritance. The structure further suggests a physical model in which Asf1 contributes to interpretation of a "histone H3 barcode" for sorting H3 isoforms into different deposition pathways.

  17. Comprehensive histone phosphorylation analysis and identification of Pf14-3-3 protein as a histone H3 phosphorylation reader in malaria parasites.

    Directory of Open Access Journals (Sweden)

    Eeshita G Dastidar

    Full Text Available The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

  18. Comprehensive histone phosphorylation analysis and identification of Pf14-3-3 protein as a histone H3 phosphorylation reader in malaria parasites.

    Science.gov (United States)

    Dastidar, Eeshita G; Dzeyk, Kristina; Krijgsveld, Jeroen; Malmquist, Nicholas A; Doerig, Christian; Scherf, Artur; Lopez-Rubio, Jose-Juan

    2013-01-01

    The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.

  19. An H3-H4 histone gene pair in the marine copepod Tigriopus californicus, contains an intergenic dyad symmetry element.

    Science.gov (United States)

    Porter, D; Brown, D; Wells, D

    1991-01-01

    Histone genes are one of the most widely studied multigene families in eucaryotes. Over 200 histone genes have been sequenced, primarily in vertebrates, echinoderms, fungi and plants. We present here the structure and genomic orientation of an H3-H4 histone gene pair from the marine copepod, Tigriopus californicus. These histone gene sequences are the first to be determined for the class Crustacea and among the first to be determined for protostomes. The H4 and H3 genes in Tigriopus are shown to be adjacent, to have opposite polarity, and to contain a 26 bp region of dyad symmetry centrally located within the spacer region between the two genes. A similarly located dyad element has been found in yeast which contributes to the coordinated cell cycle control of the adjacent histone genes. The Tigriopus H3-H4 histone gene pair is clustered with one H2A and two H2B histone genes on a 15 kb genomic Bam H1 fragment. The H4 gene sequence predicts an H4 protein with an unusual serine to threonine substitution at the amino terminal residue. The H3 gene sequence predicts an H3 protein which is identical to the vertebrate H3.2 histone.

  20. Acetylation of Chromatin-Associated Histone H3 Lysine 56 Inhibits the Development of Encysted Artemia Embryos.

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    Rong Zhou

    Full Text Available As a response to harsh environments, the crustacean artemia produces diapause gastrula embryos (cysts, in which cell division and embryonic development are totally arrested. This dormant state can last for very long periods but be terminated by specific environmental stimuli. Thus, artemia is an ideal model organism in which to study cell cycle arrest and embryonic development.Our study focuses on the roles of H3K56ac in the arrest of cell cycle and development during artemia diapause formation and termination. We found that the level of H3K56ac on chromatin increased during diapause formation, and decreased upon diapause termination, remaining basal level throughout subsequent embryonic development. In both HeLa cells and artemia, blocking the deacetylation with nicotinamide, a histone deacetylase inhibitor, increased the level of H3K56ac on chromatin and induced an artificial cell cycle arrest. Furthermore, we found that this arrest of the cell cycle and development was induced by H3K56ac and dephosphorylation of the checkpoint protein, retinoblastoma protein.These results have revealed the dynamic change in H3K56ac on chromatin during artemia diapause formation and termination. Thus, our findings provide insight into the regulation of cell division during arrest of artemia embryonic development and provide further insight into the functions of H3K56ac.

  1. Drosophila Kismet regulates histone H3 lysine 27 methylation and early elongation by RNA polymerase II.

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    Shrividhya Srinivasan

    2008-10-01

    Full Text Available Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II. Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation-a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin.

  2. Histone H3 Interacts and Colocalizes with the Nuclear Shuttle Protein and the Movement Protein of a Geminivirus ▿ †

    Science.gov (United States)

    Zhou, Yanchen; Rojas, Maria R.; Park, Mi-Ri; Seo, Young-Su; Lucas, William J.; Gilbertson, Robert L.

    2011-01-01

    Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata. PMID:21900168

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

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    Yin-Ming Kuo

    Full Text Available 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.

  4. PRB1 is required for clipping of the histone H3 N terminal tail in Saccharomyces cerevisiae.

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    Yong Xue

    Full Text Available Cathepsin L, a lysosomal protein in mouse embryonic stem cells has been shown to clip the histone H3 N- terminus, an activity associated with gene activity during mouse cell development. Glutamate dehydrogenase (GDH was also identified as histone H3 specific protease in chicken liver, which has been connected to gene expression during aging. In baker's yeast, Saccharomyces cerevisiae, clipping the histone H3 N-terminus has been associated with gene activation in stationary phase but the protease responsible for the yeast histone H3 endopeptidase activity had not been identified. In searching for a yeast histone H3 endopeptidase, we found that yeast vacuolar protein Prb1 is present in the cellular fraction enriched for the H3 N-terminus endopeptidase activity and this endopeptidase activity is lost in the PRB1 deletion mutant (prb1Δ. In addition, like Cathepsin L and GDH, purified Prb1 from yeast cleaves H3 between Lys23 and Ala24 in the N-terminus in vitro as shown by Edman degradation. In conclusion, our data argue that PRB1 is required for clipping of the histone H3 N-terminal tail in Saccharomyces cerevisiae.

  5. PRB1 is required for clipping of the histone H3 N terminal tail in Saccharomyces cerevisiae.

    Science.gov (United States)

    Xue, Yong; Vashisht, Ajay A; Tan, Yuliang; Su, Trent; Wohlschlegel, James A

    2014-01-01

    Cathepsin L, a lysosomal protein in mouse embryonic stem cells has been shown to clip the histone H3 N- terminus, an activity associated with gene activity during mouse cell development. Glutamate dehydrogenase (GDH) was also identified as histone H3 specific protease in chicken liver, which has been connected to gene expression during aging. In baker's yeast, Saccharomyces cerevisiae, clipping the histone H3 N-terminus has been associated with gene activation in stationary phase but the protease responsible for the yeast histone H3 endopeptidase activity had not been identified. In searching for a yeast histone H3 endopeptidase, we found that yeast vacuolar protein Prb1 is present in the cellular fraction enriched for the H3 N-terminus endopeptidase activity and this endopeptidase activity is lost in the PRB1 deletion mutant (prb1Δ). In addition, like Cathepsin L and GDH, purified Prb1 from yeast cleaves H3 between Lys23 and Ala24 in the N-terminus in vitro as shown by Edman degradation. In conclusion, our data argue that PRB1 is required for clipping of the histone H3 N-terminal tail in Saccharomyces cerevisiae.

  6. Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1.

    Science.gov (United States)

    Arita, Kyohei; Isogai, Shin; Oda, Takashi; Unoki, Motoko; Sugita, Kazuya; Sekiyama, Naotaka; Kuwata, Keiko; Hamamoto, Ryuji; Tochio, Hidehito; Sato, Mamoru; Ariyoshi, Mariko; Shirakawa, Masahiro

    2012-08-07

    Multiple covalent modifications on a histone tail are often recognized by linked histone reader modules. UHRF1 [ubiquitin-like, containing plant homeodomain (PHD) and really interesting new gene (RING) finger domains 1], an essential factor for maintenance of DNA methylation, contains linked two-histone reader modules, a tandem Tudor domain and a PHD finger, tethered by a 17-aa linker, and has been implicated to link histone modifications and DNA methylation. Here, we present the crystal structure of the linked histone reader modules of UHRF1 in complex with the amino-terminal tail of histone H3. Our structural and biochemical data provide the basis for combinatorial readout of unmodified Arg-2 (H3-R2) and methylated Lys-9 (H3-K9) by the tandem tudor domain and the PHD finger. The structure reveals that the intermodule linker plays an essential role in the formation of a histone H3-binding hole between the reader modules by making extended contacts with the tandem tudor domain. The histone H3 tail fits into the hole by adopting a compact fold harboring a central helix, which allows both of the reader modules to simultaneously recognize the modification states at H3-R2 and H3-K9. Our data also suggest that phosphorylation of a linker residue can modulate the relative position of the reader modules, thereby altering the histone H3-binding mode. This finding implies that the linker region plays a role as a functional switch of UHRF1 involved in multiple regulatory pathways such as maintenance of DNA methylation and transcriptional repression.

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

    Science.gov (United States)

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

    2009-01-09

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

  8. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    Science.gov (United States)

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.

  9. Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3 predominantly mark the pericentromeric chromatin during mitosis in monokinetic plants

    Indian Academy of Sciences (India)

    SANTOSH KUMAR SHARMA; MAKI YAMAMOTO; YASUHIKO MUKAI

    2016-12-01

    Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and threemonocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomal distribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinatorial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromeric chromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immunolabelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages of mitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the whole chromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion’s CENH3 antibody on mitoticchromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3 detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromericchromatin might have attributed through ‘phospho-acetyl’ cross talk that ultimately facilitate the sister chromatid cohesion at pericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14ac histone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. The plausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogenetics with special reference to chromatin dynamics have been discussed.

  10. Dual modified antiphospho (Ser10)-acetyl (Lys14)-histone H3 predominantly mark the pericentromeric chromatin during mitosis in monokinetic plants.

    Science.gov (United States)

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2016-12-01

    Epigenetic regulatory posttranslational histone modification marks not only function individually but also capable to act in combination as a unique pattern. A total of 16 plant species belonging to 11 genera of eight families (five dicots and three monocots) including land plants, epiphytes (orchids) and the holokinetic taxa (Drosera spp.) were analysed for chromosomal distribution of dual modified antiphospho (Ser10)-acetyl (K14)-histone H3 (H3S10phK14ac) to understand the combinatorial chromatin dynamics during mitotic cell division in plants. The anti-H3S10phK14ac evidently mark the pericentromeric chromatin on mitotic chromosomes of the plants excluding the holokinetic Drosera species, which revealed the immunolabelling of whole chromosomes all along the arms. The dual modified immunosignals were absent during early stages of mitosis, appeared intensively at metaphase and remained visible until late-anaphase/telophase however, labelled the whole chromosomes during meiotic metaphase I. Colocalization of anti-H3S10phK14ac with an onion's CENH3 antibody on mitotic chromosomes of Allium revealed the chromosomal location of anti-H3S10phK14ac in the region between signals for CENH3 detection. Overall analysis suggests that the unique localization of combinatorial histone modification mark at pericentromeric chromatin might have attributed through 'phospho-acetyl' cross talk that ultimately facilitate the sister chromatid cohesion at pericentromeres following condensation events in mitotic chromosomes. Here, we propose that dual modified H3S10phK14ac histone may serve as an additional cytogenetic landmark to identify pericentromeric chromatin during mitosis in plants. The plausible role of histone cross talk and future perspectives of combinatorial histone modification marks in plant cytogenetics with special reference to chromatin dynamics have been discussed.

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

    Science.gov (United States)

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

    2011-02-01

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

  12. The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Silje V Veiseth

    2011-03-01

    Full Text Available Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3 is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

  13. Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing.

    Science.gov (United States)

    Tie, Feng; Banerjee, Rakhee; Saiakhova, Alina R; Howard, Benny; Monteith, Kelsey E; Scacheri, Peter C; Cosgrove, Michael S; Harte, Peter J

    2014-03-01

    Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a 'cellular memory' of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. We report that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trx(Z11) missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trx(Z11) also suppresses the impaired silencing phenotypes of the Pc(3) mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). We show that perturbation of Polycomb silencing by TRX overexpression requires CBP. We also show that TRX and TRR are each physically associated with CBP in vivo, that TRX binds directly to the CBP KIX domain, and that the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory.

  14. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

    DEFF Research Database (Denmark)

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline

    2016-01-01

    During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators...... recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby...

  15. Reduced Histone H3 Acetylation in CD4+ T Lymphocytes: Potential Mechanism of Latent Autoimmune Diabetes in Adults

    Directory of Open Access Journals (Sweden)

    Xi-yu Liu

    2015-01-01

    Full Text Available Aims. Latent autoimmune diabetes in adults (LADA is the result of gene-environment interactions. Histone acetylation regulates gene expression and maybe interpret how environmental factors modify LADA. Hence, we studied the histone acetylation patterns in CD4+ T lymphocytes from LADA patients. Methods. Blood CD4+ T lymphocytes from 28 patients with LADA and 28 healthy controls were obtained to detect histone H3 acetylation and H4 acetylation. The gene expression of histone acetyltransferases (P300 and CREBBP and histone deacetylases (HDAC1, HDAC2, and HDAC7 was measured by real-time polymerase chain reaction (RT-PCR. Results. Compared to healthy controls, reduced global H3 acetylation was observed in LADA patients’ CD4+ T lymphocytes (P<0.05. Global level of H4 acetylation was not statistically different. Among LADA, CD4+ T lymphocytes H3 acetylation was associated with glycosylated hemoglobin (HbA1c and GADA titer. Compared to healthy controls, the expression of histone acetyltransferases CREBBP in LADA patients was downregulated, and the expression of histone deacetylases HDAC1 and HDAC7 was upregulated. Conclusion. A concerted downregulation of histone H3 acetylation was found in CD4+ T lymphocytes of LADA patients, and this might provide evidence of a novel epigenetic explanation for the pathogenesis of LADA and its complications.

  16. Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36.

    Science.gov (United States)

    Poulin, Myles B; Schneck, Jessica L; Matico, Rosalie E; McDevitt, Patrick J; Huddleston, Michael J; Hou, Wangfang; Johnson, Neil W; Thrall, Sara H; Meek, Thomas D; Schramm, Vern L

    2016-02-02

    Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

  17. New players in heterochromatin silencing: histone variant H3.3 and the ATRX/DAXX chaperone.

    Science.gov (United States)

    Voon, Hsiao P J; Wong, Lee H

    2016-02-29

    A number of studies have demonstrated that various components of the ATRX/DAXX/Histone H3.3 complex are important for heterochromatin silencing at multiple genomic regions. We provide an overview of the individual components (ATRX, DAXX and/or H3.3) tested in each study and propose a model where the ATRX/DAXX chaperone complex deposits H3.3 to maintain the H3K9me3 modification at heterochromatin throughout the genome.

  18. Timing of transcriptional quiescence during gametogenesis is controlled by global histone H3K4 demethylation.

    Science.gov (United States)

    Xu, Mengshu; Soloveychik, Maria; Ranger, Mathieu; Schertzberg, Michael; Shah, Zarna; Raisner, Ryan; Venkatasubrahmanyan, Shivkumar; Tsui, Kyle; Gebbia, Marinella; Hughes, Tim; van Bakel, Harm; Nislow, Corey; Madhani, Hiten D; Meneghini, Marc D

    2012-11-13

    Gametes are among the most highly specialized cells produced during development. Although gametogenesis culminates in transcriptional quiescence in plants and animals, regulatory mechanisms controlling this are unknown. Here, we confirm that gamete differentiation in the single-celled yeast Saccharomyces cerevisiae is accompanied by global transcriptional shutoff following the completion of meiosis. We show that Jhd2, a highly conserved JARID1-family histone H3K4 demethylase, activates protein-coding gene transcription in opposition to this programmed transcriptional shutoff, sustaining the period of productive transcription during spore differentiation. Moreover, using genome-wide nucleosome, H3K4me, and transcript mapping experiments, we demonstrate that JHD2 globally represses intergenic noncoding transcription during this period. The widespread transcriptional defects of JHD2 mutants are associated with precocious differentiation and the production of stress-sensitive spores, demonstrating that Jhd2 regulation of the global postmeiotic transcriptional program is critical for the production of healthy meiotic progeny.

  19. Hinge and chromoshadow of HP1α participate in recognition of K9 methylated histone H3 in nucleosomes.

    Science.gov (United States)

    Mishima, Yuichi; Watanabe, Makoto; Kawakami, Toru; Jayasinghe, Chanika D; Otani, Junji; Kikugawa, Yusuke; Shirakawa, Masahiro; Kimura, Hiroshi; Nishimura, Osamu; Aimoto, Saburo; Tajima, Shoji; Suetake, Isao

    2013-01-09

    The majority of the genome in eukaryotes is packaged into transcriptionally inactive chromatin. Heterochromatin protein 1 (HP1) is a major player in the establishment and maintenance of heterochromatin. HP1 specifically recognizes a methylated lysine residue at position 9 in histone H3 through its N-terminal chromo domain (CD). To elucidate the binding properties of HP1α to nucleosomes in vitro, we reconstituted nucleosomes containing histone H3 trimethylated at lysine 9. HP1α exhibited high-affinity binding to nucleosomes containing methylated histone H3 in a nucleosome core-number-dependent manner. The hinge region (HR) connecting the CD and C-terminal chromoshadow domain (CSD), and the CSD contributed to the selective binding of HP1α to histone H3 with trimethylated lysine 9 through weak DNA binding and by suppressing the DNA binding, respectively. We propose that not only the specific recognition of lysine 9 methylation of histone H3 by the CD but also the HR and the CSD cooperatively contribute to the selective binding of HP1α to histone H3 lysine 9 methylated nucleosomes.

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

    Science.gov (United States)

    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.

  1. The Histone H3K27 Demethylase UTX Regulates Synaptic Plasticity and Cognitive Behaviors in Mice

    Directory of Open Access Journals (Sweden)

    Gang-Bin Tang

    2017-08-01

    Full Text Available Histone demethylase UTX mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3 to establish a mechanistic switch to activate large sets of genes. Mutation of Utx has recently been shown to be associated with Kabuki syndrome, a rare congenital anomaly syndrome with dementia. However, its biological function in the brain is largely unknown. Here, we observe that deletion of Utx results in increased anxiety-like behaviors and impaired spatial learning and memory in mice. Loss of Utx in the hippocampus leads to reduced long-term potentiation and amplitude of miniature excitatory postsynaptic current, aberrant dendrite development and defective synapse formation. Transcriptional profiling reveals that Utx regulates a subset of genes that are involved in the regulation of dendritic morphology, synaptic transmission, and cognition. Specifically, Utx deletion disrupts expression of neurotransmitter 5-hydroxytryptamine receptor 5B (Htr5b. Restoration of Htr5b expression in newborn hippocampal neurons rescues the defects of neuronal morphology by Utx ablation. Therefore, we provide evidence that Utx plays a critical role in modulating synaptic transmission and cognitive behaviors. Utx cKO mouse models like ours provide a valuable means to study the underlying mechanisms of the etiology of Kabuki syndrome.

  2. Quantification of histone H3 Lys27 trimethylation (H3K27me3) by high-throughput microscopy enables cellular large-scale screening for small-molecule EZH2 inhibitors.

    Science.gov (United States)

    Luense, Svenja; Denner, Philip; Fernández-Montalván, Amaury; Hartung, Ingo; Husemann, Manfred; Stresemann, Carlo; Prechtl, Stefan

    2015-02-01

    EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery. © 2014 Society for Laboratory Automation and Screening.

  3. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic

    Energy Technology Data Exchange (ETDEWEB)

    Avdic, Vanja; Zhang, Pamela; Lanouette, Sylvain; Voronova, Anastassia; Skerjanc, Ilona; Couture, Jean-Francois (Ottawa)

    2011-08-24

    The SET1 family of methyltransferases carries out the bulk of histone H3 Lys-4 methylation in vivo. One of the common features of this family is the regulation of their methyltransferase activity by a tripartite complex composed of WDR5, RbBP5, and Ash2L. To selectively probe the role of the SET1 family of methyltransferases, we have developed a library of histone H3 peptide mimetics and report herein the characterization of an N{alpha} acetylated form of histone H3 peptide (N{alpha}H3). Binding and inhibition studies reveal that the addition of an acetyl moiety to the N terminus of histone H3 significantly enhances its binding to WDR5 and prevents the stimulation of MLL1 methyltransferase activity by the WDR5-RbBP5-Ash2L complex. The crystal structure of N{alpha}H3 in complex with WDR5 reveals that a high-affinity hydrophobic pocket accommodates the binding of the acetyl moiety. These results provide the structural basis to control WDR5-RbBP5-Ash2L-MLL1 activity and a tool to manipulate stem cell differentiation programs.-Avdic, V., Zhang, P., Lanouette, S., Voronova, A., Skerjanc, I., Couture, J.-F. Fine-tuning the stimulation of MLL1 methyltransferase activity by a histone H3-based peptide mimetic.

  4. KdmA, a histone H3 demethylase with bipartite function, differentially regulates primary and secondary metabolism in Aspergillus nidulans.

    Science.gov (United States)

    Gacek-Matthews, Agnieszka; Noble, Luke M; Gruber, Clemens; Berger, Harald; Sulyok, Michael; Marcos, Ana T; Strauss, Joseph; Andrianopoulos, Alex

    2015-05-01

    Aspergillus nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity towards lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that - in submerged early and late cultures - between 25% and 30% of the genome is under KdmA influence respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low-density visible light on solid medium. Although KdmA acts as transcriptional co-repressor of primary metabolism genes, it is required for full expression of several genes involved in biosynthesis of secondary metabolites. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2004-03-01

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

  6. Why are histones dynamic?

    OpenAIRE

    Ferrari, Paolo

    2015-01-01

    Afin de pouvoir être contenu dans le noyau des cellules eucaryotes, l’ADN doit être compacté en un ensemble très structuré. L’ADN eucaryote est généralement associé à des protéines, principalement à des histones, pour former cette structure très compact appelée chromatine. La chromatine régule de nombreux processus cellulaires de bases reposant sur les interactions ADN-protéines, y compris la transcription des gènes. La liaison des facteurs de transcription aux séquences régulatrices des gène...

  7. Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

    Science.gov (United States)

    Aida, Masatoshi; Hamad, Nesreen; Stanlie, Andre; Begum, Nasim A.; Honjo, Tasuku

    2013-01-01

    Somatic hypermutation (SHM) requires not only the expression of activation-induced cytidine deaminase, but also transcription in the target regions. However, how transcription guides activation-induced cytidine deaminase in targeting SHM to the Ig genes is not fully understood. Here, we found that the “facilitates chromatin transcription” (FACT) complex promotes SHM by RNAi screening of transcription elongation factors. Furthermore, FACT and histone H3.3, a hallmark of transcription-coupled histone turnover, are enriched at the V(D)J region, 5′ flanking sequence of the Sμ switch region and the light chain Jκ 5 segment region in the Ig loci. The regions with the most abundant deposition of FACT and H3.3 were also the most efficient targets of SHM. These results demonstrate the importance of histone-exchanging dynamics at the chromatin of SHM targets, especially in Ig genes. PMID:23610419

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

    Science.gov (United States)

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

    2016-01-01

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

  9. The PHD finger of human UHRF1 reveals a new subgroup of unmethylated histone H3 tail readers.

    Directory of Open Access Journals (Sweden)

    Nada Lallous

    Full Text Available The human UHRF1 protein (ubiquitin-like containing PHD and RING finger domains 1 has emerged as a potential cancer target due to its implication in cell cycle regulation, maintenance of DNA methylation after replication and heterochromatin formation. UHRF1 functions as an adaptor protein that binds to histones and recruits histone modifying enzymes, like HDAC1 or G9a, which exert their action on chromatin. In this work, we show the binding specificity of the PHD finger of human UHRF1 (huUHRF1-PHD towards unmodified histone H3 N-terminal tail using native gel electrophoresis and isothermal titration calorimetry. We report the molecular basis of this interaction by determining the crystal structure of huUHRF1-PHD in complex with the histone H3 N-terminal tail. The structure reveals a new mode of histone recognition involving an extra conserved zinc finger preceding the conventional PHD finger region. This additional zinc finger forms part of a large surface cavity that accommodates the side chain of the histone H3 lysine K4 (H3K4 regardless of its methylation state. Mutation of Q330, which specifically interacts with H3K4, to alanine has no effect on the binding, suggesting a loose interaction between huUHRF1-PHD and H3K4. On the other hand, the recognition appears to rely on histone H3R2, which fits snugly into a groove on the protein and makes tight interactions with the conserved aspartates D334 and D337. Indeed, a mutation of the former aspartate disrupts the formation of the complex, while mutating the latter decreases the binding affinity nine-fold.

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

    Science.gov (United States)

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

    2010-01-01

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

  11. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development

    DEFF Research Database (Denmark)

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

    2007-01-01

    -methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages...... of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads...... associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation....

  12. Characterization of centromeric histone H3 (CENH3 variants in cultivated and wild carrots (Daucus sp..

    Directory of Open Access Journals (Sweden)

    Frank Dunemann

    Full Text Available In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR marker, and the CENH3 locus was mapped on the carrot chromosome 9.

  13. ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons.

    Science.gov (United States)

    Noh, Kyung-Min; Maze, Ian; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W; Shen, Li; Li, Haitao; Allis, C David

    2015-06-02

    ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

  14. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases

    DEFF Research Database (Denmark)

    Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath

    2011-01-01

    Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model...... organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well...... at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation...

  15. A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin*

    Science.gov (United States)

    Sankaran, Saumya M.; Wilkinson, Alex W.; Elias, Joshua E.; Gozani, Or

    2016-01-01

    The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2. PMID:26912663

  16. Distribution pattern of histone H3 phosphorylation at serine 10 during mitosis and meiosis in Brachiaria species

    Indian Academy of Sciences (India)

    C. M. P. Paula; V. H. Techio; F. Souza Sobrinho; A. S. Freitas

    2013-08-01

    Histones are the major eukaryotic DNA-binding proteins. Posttranslational modifications on N-terminal tails of histones that form nucleosomes are often associated with distinct biological functions. Some theories suggest that one of these modifications, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays a role in both chromosome condensation and sister chromatid cohesion. Although histones and some of their modifications are highly conserved, studies have shown that role and distribution of H3S10ph may differ between species. We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated during meiosis I, whereas phosphorylation was restricted to the pericentromeric region in both meiosis II and mitosis. There was no variation in phosphorylation patterns between Brachiaria species and diploid and tetraploid genotypes. Regarding spatiotemporal coordination in the Brachiaria species evaluated, H3S10ph is related to maintenance of sister chromatid cohesion during cell divisions.

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

    Science.gov (United States)

    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. Copyright © 2011 The Protein Society.

  18. EFFECTS OF CURCUMIN ON PROLIFERATION OF NB4 CELLS AND ACETYLATION OF HISTONE H3 AND P53

    Institute of Scientific and Technical Information of China (English)

    陈燕; 李新刚; 吴青

    2004-01-01

    Objective: To investigate the effects of curcumin on the proliferation of NB4 cells and the acetylation of histone H3 and no-histone p53. Methods: NB4 cells were cultured and treated with or without curcumin at different concentration (50 (mol/L, 25(mol/L, 12.5(mol/L, 6.25(mol/L, 3.125(mol/L) at various time points (oh, 4h, 8h, 12h, 24h). Western blot analysis was performed to determine the level of acetylated histone H3, p53 and acetylated p53, MTT assay was performed to examine the proliferation of NB4 cells. Results: the proliferation of NB4 cells was inhibited by curcumin in a time- and dose-dependent manner, the IC50 at 24h and 36h were 40(mol/L and 25(mol/L respectively. The levels of acetylated histone H3 and acetylated p53 were increased obviously. Conclusion: curcumin possessed the founction of deacetylases inhibitor, increased the level of acetylated histone H3 and the expression of tumor suppressor p53, enhanced later's activity, and inhibited the proliferation of NB4 cell.

  19. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in histone H3.

    Science.gov (United States)

    Kobza, Keyna; Sarath, Gautam; Zempleni, Janos

    2008-04-30

    BirA ligase is a prokaryotic ortholog of holocarboxylase synthetase (HCS) that can biotinylate proteins. This study tested the hypothesis that BirA ligase catalyzes the biotinylation of eukaryotic histones. If so, this would mean that recombinant BirA ligase is a useful surrogate for HCS in studies of histone biotinylation. The biological activity of recombinant BirA ligase was confirmed by enzymatic biotinylation of p67. In particular, it was found that BirA ligase biotinylated both calf thymus histone H1 and human bulk histone extracts. Incubation of recombinant BirA ligase with H3-based synthetic peptides showed that lysines 4, 9, 18, and 23 in histone H3 are the targets for the biotinylation by BirA ligase. Modification of the peptides (e.g., serine phosphorylation) affected the subsequent biotinylation by BirA ligase, suggesting crosstalk between modifications. In conclusion, this study suggests that prokaryotic BirA ligase is a promiscuous enzyme and biotinylates eukaryotic histones. Moreover the biotinylation of histones by BirA ligase is consistent with the proposed role of human HCS in chromatin.

  20. An RNA Polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair

    OpenAIRE

    Jha, Deepak Kumar; Brian D Strahl

    2014-01-01

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered ...

  1. Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids.

    Science.gov (United States)

    Sharma, Santosh Kumar; Yamamoto, Maki; Mukai, Yasuhiko

    2017-01-01

    Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique "meiosis-mitosis shift" at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.

  2. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

    Science.gov (United States)

    Faucher, David; Wellinger, Raymund J

    2010-08-26

    Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3), both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB) repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

  3. The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9

    Directory of Open Access Journals (Sweden)

    Smith Kristina M

    2008-11-01

    Full Text Available Abstract Background Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized. Results The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1 mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased. Conclusion We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.

  4. Methylated H3K4, a transcription-associated histone modification, is involved in the DNA damage response pathway.

    Directory of Open Access Journals (Sweden)

    David Faucher

    2010-08-01

    Full Text Available Eukaryotic genomes are associated with a number of proteins such as histones that constitute chromatin. Post-translational histone modifications are associated with regulatory aspects executed by chromatin and all transactions on genomic DNA are dependent on them. Thus, it will be relevant to understand how histone modifications affect genome functions. Here we show that the mono ubiquitylation of histone H2B and the tri-methylation of histone H3 on lysine 4 (H3K4me3, both known for their involvement in transcription, are also important for a proper response of budding yeast cells to DNA damaging agents and the passage through S-phase. Cells that cannot methylate H3K4 display a defect in double-strand break (DSB repair by non-homologous end joining. Furthermore, if such cells incur DNA damage or encounter a stress during replication, they very rapidly lose viability, underscoring the functional importance of the modification. Remarkably, the Set1p methyltransferase as well as the H3K4me3 mark become detectable on a newly created DSB. This recruitment of Set1p to the DSB is dependent on the presence of the RSC complex, arguing for a contribution in the ensuing DNA damage repair process. Taken together, our results demonstrate that Set1p and its substrate H3K4me3, which has been reported to be important for the transcription of active genes, also plays an important role in genome stability of yeast cells. Given the high degree of conservation for the methyltransferase and the histone mark in a broad variety of organisms, these results could have similar implications for genome stability mechanisms in vertebrate and mammalian cells.

  5. Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian

    2016-01-01

    . We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks...

  6. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis.

    Science.gov (United States)

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-02-16

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.

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

    Science.gov (United States)

    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. © 2015 Chen et al.; Published by Cold Spring Harbor Laboratory Press.

  8. Squamocin modulates histone H3 phosphorylation levels and induces G1 phase arrest and apoptosis in cancer cells

    Directory of Open Access Journals (Sweden)

    Wu Yang-Chang

    2011-02-01

    Full Text Available Abstract Background Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. Increased phosphorylation levels of histone H3 as a result of aurora B and pMSK1 overexpression were observed in various tumors. We selected aurora B and MSK1 as representatives for testing various compounds and drugs, and found that squamocin, a bis-tetrahydrofuran annonaceous acetogenin, exerted a potent effect on histone H3 phosphorylation. Methods GBM8401, Huh-7, and SW620 cells were incubated with 15, 30, and 60 μM squamocin for 24 h. The expressions of mRNA and proteins were analyzed by qRT-PCR and Western blotting, respectively. The cell viability was determined by an MTT assay. Cell cycle distribution and apoptotic cells were analyzed by flow cytometry. Results Our results showed that squamocin inhibited the proliferation of GBM8401, Huh-7, and SW620 cells, arrested the cell cycle at the G1 phase, and activated both intrinsic and extrinsic pathways to apoptosis. In addition, we demonstrated that squamocin had the ability to modulate the phosphorylation levels of H3S10 (H3S10p and H3S28 (H3S28p in association with the downregulation of aurora B and pMSK1 expressions. Conclusions This study is the first to show that squamocin affects epigenetic alterations by modulating histone H3 phosphorylation at S10 and S28, providing a novel view of the antitumor mechanism of squamocin.

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

    Science.gov (United States)

    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.

  10. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases.

    Science.gov (United States)

    Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath; Watt, Stephen; Verhein-Hansen, Janne; Bähler, Jürg; Martienssen, Robert A; Partridge, Janet F; Cohen, Amikam; Thon, Geneviève

    2011-01-06

    Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

  11. H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases.

    Directory of Open Access Journals (Sweden)

    Klavs R Hansen

    Full Text Available Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

  12. Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation.

    Science.gov (United States)

    Zhang, Xuesen; Bolt, Michael; Guertin, Michael J; Chen, Wei; Zhang, Sheng; Cherrington, Brian D; Slade, Daniel J; Dreyton, Christina J; Subramanian, Venkataraman; Bicker, Kevin L; Thompson, Paul R; Mancini, Michael A; Lis, John T; Coonrod, Scott A

    2012-08-14

    Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.

  13. SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability

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    Sophia X. Pfister

    2014-06-01

    Full Text Available Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP and promotes DSB resection, allowing Replication Protein A (RPA and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.

  14. The histone H3K79 methyltransferase Dot1L is essential for mammalian development and heterochromatin structure.

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    Brendan Jones

    Full Text Available Dot1 is an evolutionarily conserved histone methyltransferase specific for lysine 79 of histone H3 (H3K79. In Saccharomyces cerevisiae, Dot1-mediated H3K79 methylation is associated with telomere silencing, meiotic checkpoint control, and DNA damage response. The biological function of H3K79 methylation in mammals, however, remains poorly understood. Using gene targeting, we generated mice deficient for Dot1L, the murine Dot1 homologue. Dot1L-deficient embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. To gain insights into the cellular function of Dot1L, we derived embryonic stem (ES cells from Dot1L mutant blastocysts. Dot1L-deficient ES cells show global loss of H3K79 methylation as well as reduced levels of heterochromatic marks (H3K9 di-methylation and H4K20 tri-methylation at centromeres and telomeres. These changes are accompanied by aneuploidy, telomere elongation, and proliferation defects. Taken together, these results indicate that Dot1L and H3K79 methylation play important roles in heterochromatin formation and in embryonic development.

  15. Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome

    Science.gov (United States)

    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

  16. HDAC inhibitor increases histone H3 acetylation and reduces microglia inflammatory response following traumatic brain injury in rats

    Science.gov (United States)

    Zhang, Bin; West, Eric J.; Van, Ken C.; Gurkoff, Gene G.; Zhou, Jia; Zhang, Xiu-Mei; Kozikowski, Alan P.; Lyeth, Bruce G.

    2008-01-01

    Traumatic brain injury (TBI) produces a rapid and robust inflammatory response in the brain characterized in part by activation of microglia. A novel histone deacetylase (HDAC) inhibitor, 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB), was administered (0, 0.25, 2.5, 25 mg/kg) systemically immediately after lateral fluid percussion TBI in rats. Hippocampal CA2/3 tissue was processed for acetyl-histone H3 immunolocalization, OX-42 immunolocalization (for microglia), and Fluoro-Jade B histofluorescence (for degenerating neurons) at 24 h after injury. Vehicle-treated TBI rats exhibited a significant reduction in acetyl-histone H3 immunostaining in the ipsilateral CA2/3 hippocampus compared to the sham TBI group (p<0.05). The reduction in acetyl-histone H3 immunostaining was attenuated by each of the DMA-PB dosage treatment groups. Vehicle-treated TBI rats exhibited a high density of phagocytic microglia in the ipsilateral CA2/3 hippocampus compared to sham TBI in which none were observed. All doses of DMA-PB significantly reduced the density of phagocytic microglia (p<0.05). There was a trend for DMA-PB to reduce the number of degenerating neurons in the ipsilateral CA2/3 hippocampus (p = 0.076). We conclude that the HDAC inhibitor DMA-PB is a potential novel therapeutic for inhibiting neuroinflammation associated with TBI. PMID:18582446

  17. The Nrde Pathway Mediates Small-RNA-Directed Histone H3 Lysine 27 Trimethylation in Caenorhabditis elegans.

    Science.gov (United States)

    Mao, Hui; Zhu, Chengming; Zong, Dandan; Weng, Chenchun; Yang, Xiangwei; Huang, Hui; Liu, Dun; Feng, Xuezhu; Guang, Shouhong

    2015-09-21

    Small-RNA-mediated chromatin modifications have been widely studied in plants and S. pombe. However, direct evidence of small-RNA-guided sequence-specific chromatin alterations is scarce in animals. In C. elegans, the nuclear RNAi defective (Nrde) pathway functions to transport siRNA from the cytoplasm to the nucleus, modulate transcription elongation, induce histone H3 lysine 9 (H3K9) trimethylation, and mediate transgenerational inheritance of RNAi. Here, we show that both exogenous RNAi and NRDE-bound endogenous 22G RNAs can direct sequence-specific histone H3 lysine 27 (H3K27) trimethylation at targeted loci through the Nrde pathway. The resulting H3K27me3 status can be inherited by progeny for multiple generations. piRNAs and WAGO-1-associated siRNAs induce H3K27 methylation as well. Interestingly, CSR-1-associated endogenous siRNAs fail to trigger H3K27 methylation, whereas exogenous provision of dsRNAs can induce H3K27 methylation at the CSR-1-targeted loci via the Nrde pathway. We further observed distinct genetic requirements of H3K9 and H3K27 trimethylation. Whereas set-25 and met-2 are required for K9 methylation, mes-2 is required for K27 methylation. The depletion of mes-2 leads to a nuclear RNAi defective phenotype. These results indicate that dsRNA-triggered chromatin modification is a sequence-specific response that engages the Nrde pathway in C. elegans. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

    DEFF Research Database (Denmark)

    Tariq, M.; Saze, H.; Probst, A.;

    2003-01-01

    DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation...... in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation....

  19. Histone H3 Phosphorylation in Human Skin Histoculture as a Tool to Evaluate Patient’s Response to Antiproliferative Drugs

    Science.gov (United States)

    Ugarte, Fernando; Porth, Katherine; Sadekova, Svetlana

    2015-01-01

    Evaluation of patient’s response to chemotherapeutic drugs is often difficult and time consuming. Skin punch biopsies are easily accessible material that can be used for the evaluation of surrogate biomarkers of a patient’s response to a drug. In this study, we hypothesized that assessment of phosphorylated histone H3 in human skin punch biopsies could be used as a pharmacodynamics biomarker of patient’s response to the kinesin spindle protein inhibitor SCH2047069. To test this hypothesis, we used a human skin histoculture technique that allows culturing intact human skin in the presence of the drug. Human melanoma and skin histocultures were treated with SCH2047069, and the effect of the drug was assessed by increasing histone H3 phosphorylation using immunohistochemistry. Our results demonstrate that SCH2047069 has a significant effect on cell proliferation in human melanoma and skin histoculture and justify using human skin punch biopsies for evaluation of the pharmacodynamic changes induced by SCH2047069. ACRONYMS Histone subunit H3 (H3), Kinesin spindle protein (KSP), 5-ethynyl-2′-deoxyuridine (EDU), Dimethyl sulfoxide (DMSO), Formalin-fixed paraffin embedded (FFPE). PMID:26917945

  20. PHF20 Readers Link Methylation of Histone H3K4 and p53 with H4K16 Acetylation

    Directory of Open Access Journals (Sweden)

    Brianna J. Klein

    2016-10-01

    Full Text Available PHF20 is a core component of the lysine acetyltransferase complex MOF (male absent on the first-NSL (non-specific lethal that generates the major epigenetic mark H4K16ac and is necessary for transcriptional regulation and DNA repair. The role of PHF20 in the complex remains elusive. Here, we report on functional coupling between methylation readers in PHF20. We show that the plant homeodomain (PHD finger of PHF20 recognizes dimethylated lysine 4 of histone H3 (H3K4me2 and represents an example of a native reader that selects for this modification. Biochemical and structural analyses help to explain this selectivity and the preference of Tudor2, another reader in PHF20, for dimethylated p53. Binding of the PHD finger to H3K4me2 is required for histone acetylation, accumulation of PHF20 at target genes, and transcriptional activation. Together, our findings establish a unique PHF20-mediated link between MOF histone acetyltransferase (HAT, p53, and H3K4me2, and suggest a model for rapid spreading of H4K16ac-enriched open chromatin.

  1. Dynamic fuzziness during linker histone action.

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    McBryant, Steven J; Hansen, Jeffrey C

    2012-01-01

    Linker histones are multi-domain nucleosome binding proteins that stabilize higher order chromatin structures and engage in specific protein-protein interactions. Here we emphasize the structural and functional properties of the linker histone C-terminal domain (CTD), focusing on its intrinsic disorder, interaction-induced secondary structure formation and dynamic fuzziness. We argue that the fuzziness inherent in the CTD is a primary molecular mechanism underlying linker histone function in the nucleus.

  2. Histone H3 lysine 23 acetylation is associated with oncogene TRIM24 expression and a poor prognosis in breast cancer.

    Science.gov (United States)

    Ma, Li; Yuan, Lili; An, Jing; Barton, Michelle C; Zhang, Qingyuan; Liu, Zhaoliang

    2016-11-01

    Acetylated H3 lysine 23 (H3K23ac) is a specific histone post-translational modification recognized by oncoprotein TRIM24. However, it is not clear whether H3K23ac levels are correlated with TRIM24 expression and what role H3K23ac may have in cancer. In this study, we collected breast carcinoma samples from 121 patients and conducted immunohistochemistry to determine the levels of TRIM24 and H3K23ac in breast cancer. Our results demonstrated that TRIM24 expression is positively correlated with H3K23ac levels, and high levels of both TRIM24 and H3K23ac predict shorter overall survival of breast cancer patients. We also showed that both TRIM24 and H3K23ac are higher in HER2-positive patients, and their levels were positively correlated with HER2 levels in breast cancer. Moreover, TRIM24 expression is associated with estrogen receptor (ER) and progesterone receptor (PR) statuses in both our cohort and The Cancer Genome Atlas (TCGA) breast carcinoma. In summary, our results revealed an important role of TRIM24 and H3K23ac in breast cancer and provided further evidence that TRIM24 small-molecule inhibitors may benefit ER- and PR-negative or HER2-positive breast cancer patients.

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

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    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 (pgenes 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.

  4. Slowly on, Slowly off: Bisubstrate-Analogue Conjugates of 5-Iodotubercidin and Histone H3 Peptide Targeting Protein Kinase Haspin.

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    Kestav, Katrin; Viht, Kaido; Konovalov, Anton; Enkvist, Erki; Uri, Asko; Lavogina, Darja

    2017-02-09

    The atypical protein kinase Haspin serves as one of a key players in mitosis by catalysing phosphorylation of Thr3 in histone H3, and thus sustaining the normal functioning of the chromosomal passenger complex. Here, we report the development of bisubstrate-analogue inhibitors targeting Haspin. The compounds were constructed by linking 5-iodotubercidin moiety to the N-terminal sequence of histone H3. The new conjugates possessed high affinity (KD in the subnanomolar range) towards Haspin as well as slow kinetics of association and dissociation (residence time on the scale of several hours), which reflected their unique binding mode and translated into improved selectivity. The latter was confirmed in a biochemical binding/displacement assay with a panel of 10 protein kinases, in thermal shift assay with off-targets of 5-iodotubercidin represented by adenosine kinase and the Cdc2-like kinase family, as well as in assay with spiked lysates of HeLa cells.

  5. Protein phosphatase 1γ is responsible for dephosphorylation of histone H3 at Thr 11 after DNA damage.

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    Shimada, Midori; Haruta, Mayumi; Niida, Hiroyuki; Sawamoto, Kazunobu; Nakanishi, Makoto

    2010-11-01

    The DNA-damage-induced transcriptional suppression of cell cycle regulatory genes correlates with a reduction in histone H3-Thr 11 phosphorylation (H3-pThr 11) on their promoters that is partly mediated by the dissociation of Chk1 from chromatin. In this study, we identify protein phosphatase 1γ (PP1γ) as a phosphatase responsible for DNA-damage-induced H3-pThr 11 dephosphorylation. PP1γ is activated after DNA damage, which is mainly mediated by a reduction in Cdk-dependent phosphorylation of PP1γ at Thr 311. The depletion of PP1γ sensitizes HCT116 cells to DNA damage. Our results suggest that the ataxia telangiectasia, mutated and Rad3-related-Chk1 axis regulates H3-pThr 11 dephosphorylation on DNA damage, at least in part by the activation of PP1γ through Chk1-dependent inhibition of Cdks.

  6. Structure and activity of the peptidyl-prolyl isomerase domain from the histone chaperone Fpr4 toward histone H3 proline isomerization.

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    Monneau, Yoan R; Soufari, Heddy; Nelson, Christopher J; Mackereth, Cameron D

    2013-09-06

    The FK506-binding protein (FKBP) family of peptidyl-prolyl isomerases (PPIases) is characterized by a common catalytic domain that binds to the inhibitors FK506 and rapamycin. As one of four FKBPs within the yeast Saccharomyces cerevisiae, Fpr4 has been described as a histone chaperone, and is in addition implicated in epigenetic function in part due to its mediation of cis-trans conversion of proline residues within histone tails. To better understand the molecular details of this activity, we have determined the solution structure of the Fpr4 C-terminal PPIase domain by using NMR spectroscopy. This canonical FKBP domain actively increases the rate of isomerization of three decapeptides derived from the N terminus of yeast histone H3, whereas maintaining intrinsic cis and trans populations. Observation of the uncatalyzed and Fpr4-catalyzed isomerization rates at equilibrium demonstrate Pro(16) and Pro(30) of histone H3 as the major proline targets of Fpr4, with little activity shown against Pro(38). This alternate ranking of the three target prolines, as compared with affinity determination or the classical chymotrypsin-based fluorescent assay, reveals the mechanistic importance of substrate residues C-terminal to the peptidyl-prolyl bond.

  7. Arabidopsis COMPASS-like complexes mediate histone H3 lysine-4 trimethylation to control floral transition and plant development.

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    Danhua Jiang

    2011-03-01

    Full Text Available Histone H3 lysine-4 (H3K4 methylation is associated with transcribed genes in eukaryotes. In Drosophila and mammals, both di- and tri-methylation of H3K4 are associated with gene activation. In contrast to animals, in Arabidopsis H3K4 trimethylation, but not mono- or di-methylation of H3K4, has been implicated in transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS or COMPASS-like in yeast and mammals. Here, we report that Arabidopsis homologs of the COMPASS and COMPASS-like complex core components known as Ash2, RbBP5, and WDR5 in humans form a nuclear subcomplex during vegetative and reproductive development, which can associate with multiple putative H3K4 methyltransferases. Loss of function of ARABIDOPSIS Ash2 RELATIVE (ASH2R causes a great decrease in genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Knockdown of ASH2R or the RbBP5 homolog suppresses the expression of a crucial Arabidopsis floral repressor, FLOWERING LOCUS C (FLC, and FLC homologs resulting in accelerated floral transition. ASH2R binds to the chromatin of FLC and FLC homologs in vivo and is required for H3K4 trimethylation, but not for H3K4 dimethylation in these loci; overexpression of ASH2R causes elevated H3K4 trimethylation, but not H3K4 dimethylation, in its target genes FLC and FLC homologs, resulting in activation of these gene expression and consequent late flowering. These results strongly suggest that H3K4 trimethylation in FLC and its homologs can activate their expression, providing concrete evidence that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings suggest that there are multiple COMPASS-like complexes in Arabidopsis and that these complexes deposit trimethyl but not di- or mono-methyl H3K4 in target genes to promote their expression, providing a molecular explanation for the observed coupling of H3K4 trimethylation (but not H3

  8. Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing.

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    Hitomi Miyazaki

    2013-11-01

    Full Text Available Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII, thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l, a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, we found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. We also found that Brg1 is another key player involved in the response. Our results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing.

  9. Structural basis for specific binding of human MPP8 chromodomain to histone H3 methylated at lysine 9.

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    Jing Li

    Full Text Available BACKGROUND: M-phase phosphoprotein 8 (MPP8 was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. METHODOLOGY/PRINCIPAL FINDINGS: Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15. The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits. CONCLUSIONS/SIGNIFICANCE: Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

  10. Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

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    Li, Jing; Li, Zhihong; Ruan, Jianbin; Xu, Chao; Tong, Yufeng; Pan, Patricia W.; Tempel, Wolfram; Crombet, Lissete; Min, Jinrong; Zang, Jianye (Toronto); (Chinese Aca. Sci.)

    2012-02-27

    M-phase phosphoprotein 8 (MPP8) was initially identified to be a component of the RanBPM-containing large protein complex, and has recently been shown to bind to methylated H3K9 both in vivo and in vitro. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. Here, we reported the crystal structures of human MPP8 chromodomain alone and in complex with the trimethylated histone H3K9 peptide (residue 1-15). The complex structure unveils that the human MPP8 chromodomain binds methylated H3K9 through a conserved recognition mechanism, which was also observed in Drosophila HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two {beta} strands from the two protomer subunits. Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore.

  11. GmPHD5 acts as an important regulator for crosstalk between histone H3K4 di-methylation and H3K14 acetylation in response to salinity stress in soybean

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    Wu Tao

    2011-12-01

    Full Text Available Abstract Background Accumulated evidence suggest that specific patterns of histone posttranslational modifications (PTMs and their crosstalks may determine transcriptional outcomes. However, the regulatory mechanisms of these "histone codes" in plants remain largely unknown. Results In this study, we demonstrate for the first time that a salinity stress inducible PHD (plant homeodomain finger domain containing protein GmPHD5 can read the "histone code" underlying the methylated H3K4. GmPHD5 interacts with other DNA binding proteins, including GmGNAT1 (an acetyl transferase, GmElongin A (a transcription elongation factor and GmISWI (a chromatin remodeling protein. Our results suggest that GmPHD5 can recognize specific histone methylated H3K4, with preference to di-methylated H3K4. Here, we illustrate that the interaction between GmPHD5 and GmGNAT1 is regulated by the self-acetylation of GmGNAT1, which can also acetylate histone H3. GmGNAT1 exhibits a preference toward acetylated histone H3K14. These results suggest a histone crosstalk between methylated H3K4 and acetylated H3K14. Consistent to its putative roles in gene regulation under salinity stress, we showed that GmPHD5 can bind to the promoters of some confirmed salinity inducible genes in soybean. Conclusion Here, we propose a model suggesting that the nuclear protein GmPHD5 is capable of regulating the crosstalk between histone methylation and histone acetylation of different lysine residues. Nevertheless, GmPHD5 could also recruit chromatin remodeling factors and transcription factors of salt stress inducible genes to regulate their expression in response to salinity stress.

  12. An RNA polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair.

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    Jha, Deepak Kumar; Strahl, Brian D

    2014-06-09

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered chromatin architecture and inappropriate resection during G1 near break sites. Surprisingly, Set2 and RNA polymerase II are programmed for destruction after DSBs in a temporal manner--resulting in H3K36me3 to H3K36me2 transition that may be linked to DSB repair. Finally, we show a requirement of Set2 in DSB repair in transcription units--thus underscoring the importance of transcription-dependent H3K36me in DSB repair.

  13. Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18.

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    Bao, Baolong; Pestinger, Valerie; Hassan, Yousef I; Borgstahl, Gloria E O; Kolar, Carol; Zempleni, Janos

    2011-05-01

    Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [(3)H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.

  14. Thermal response of proteins (histone H2AX, H3.1) by a coarse-grained Monte Carlo simulation with a knowledge-based phenomenological potential

    Science.gov (United States)

    Fritsche, Miriam; Heermann, Dieter; Pandey, Ras; Farmer, Barry

    2012-02-01

    Using a coarse-grained bond fluctuating model, we investigate structure and dynamics of two histones, H2AX (143 residues) and H3.1 (136 residues) as a function of temperature (T). A knowledged based contact matrix is used as an input for a phenomenological residue-residue interaction in a generalized Lennard-Jones potential. Metropolis algorithm is used to execute stochastic movement of each residue. A number of local and global physical quantities are analyzed. Despite unique energy and mobility profiles of its residues in a specific sequence, the histone H3.1 appears to undergo a structural transformation from a random coil to a globular conformation on reducing the temperature. The radius of gyration of the histone H2AX, in contrast, exhibits a non-monotonic dependence on temperature with a maximum at a characteristic temperature (Tc) where crossover occurs from a positive (stretching below Tc) to negative (contraction above Tc) thermal response on increasing T. Multi-scale structures of the proteins are examined by a detailed analysis of their structure functions.

  15. Different Patterns of Acetylation and Dimethylation of Histone H3 between Young and Aged Cases with Chronic Tonsillitis: Influences of Inflammation and Aging.

    Science.gov (United States)

    Saito, Akihiko; Watanabe, Ken-Ichi; Egawa, Seiko; Okubo, Kimihiro

    2016-01-01

    Epigenetics is now considered to be crucially involved in normal genetics and differentiation and in pathological conditions, such as cancer, aging, and inflammation. Epigenetic mechanisms involve DNA methylation and histone modifications. The purpose of this study was to investigate the effects of inflammation on epigenetics in young subjects and the effect of aging. The palatine tonsils were extracted from child and adult patients with chronic tonsillitis. Hematoxylin-eosin staining was performed to examine the morphology of the palatine tonsils. A fluorescence immunological examination was also performed to detect acetyl-histone H3 or dimethyl-histone H3. Confocal scanning microscopy was used for observations. Acetylated histone H3 was detected in tonsils from child patients but not from adult patients. Dimethylated histone H3 was not detected in tonsils from either group of patients. Degeneration of the tonsillar structures was apparent in tonsils from adult patients. The differential expression of acetylated histone H3 Lys9 may reflect immunological differences between young and aged tonsils. The decrease observed in the activity of histone methyltransferase induced the down-regulated expression of methylated histone H3. Our results suggest that epigenetic changes participate in chronic inflammation and aging in the palatine tonsils. Although the results do not lead to a direct treatment, the epigenetic pathogenesis of chronic inflammation, such as immunoglobulin A nephropathy, by focal infections will be described in greater detail in future studies, which will lead to new treatments being developed.

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

    Science.gov (United States)

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

    2011-04-01

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

  17. Histone H3.3K27M Represses p16 to Accelerate Gliomagenesis in a Murine Model of DIPG.

    Science.gov (United States)

    Cordero, Francisco J; Huang, Zhiqing; Grenier, Carole; He, Xingyao; Hu, Guo; McLendon, Roger E; Murphy, Susan K; Hashizume, Rintaro; Becher, Oren J

    2017-09-01

    Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor genetically distinguished from adult GBM by the high prevalence of the K27M mutation in the histone H3 variant H3.3 (H3F3A). This mutation reprograms the H3K27me3 epigenetic landscape of DIPG by inhibiting the H3K27-specific histone methyltransferase EZH2. This globally reduces H3K27me2/3, critical repressive marks responsible for cell fate decisions, and also causes focal gain of H3K27me3 throughout the epigenome. To date, the tumor-driving effects of H3.3K27M remain largely unknown. Here, it is demonstrated that H3.3K27M cooperates with PDGF-B in vivo, enhancing gliomagenesis and reducing survival of p53 wild-type (WT) and knockout murine models of DIPG. H3.3K27M expression drives increased proliferation of tumor-derived murine neurospheres, suggesting that cell-cycle deregulation contributes to increased malignancy in mutant tumors. RNA sequencing on tumor tissue from H3.3K27M-expressing mice indicated global upregulation of PRC2 target genes, and a subset of newly repressed genes enriched in regulators of development and cell proliferation. Strikingly, H3.3K27M induced targeted repression of the p16/ink4a (CDKN2A) locus, a critical regulator of the G0-G1 to S-phase transition. Increased levels of H3K27me3 were observed at the p16 promoter; however, pharmacologic reduction of methylation at this promoter did not rescue p16 expression. Although DNA methylation is also present at this promoter, it is not K27M dependent. Intriguingly, inhibition of DNA methylation restores p16 levels and is cytotoxic against murine tumor cells. Importantly, these data reveal that H3.3K27M-mediated p16 repression is an important mechanism underlying the proliferation of H3.3K27M tumor cells, as in vivo cdkn2a knockout eliminates the survival difference between H3.3K27M and H3.3WT tumor-bearing mice.Implications: This study shows that H3.3K27M mutation and PDGF signaling act in concert to

  18. Substitutions in the amino-terminal tail of neurospora histone H3 have varied effects on DNA methylation.

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    Keyur K Adhvaryu

    2011-12-01

    Full Text Available Eukaryotic genomes are partitioned into active and inactive domains called euchromatin and heterochromatin, respectively. In Neurospora crassa, heterochromatin formation requires methylation of histone H3 at lysine 9 (H3K9 by the SET domain protein DIM-5. Heterochromatin protein 1 (HP1 reads this mark and directly recruits the DNA methyltransferase, DIM-2. An ectopic H3 gene carrying a substitution at K9 (hH3(K9L or hH3(K9R causes global loss of DNA methylation in the presence of wild-type hH3 (hH3(WT. We investigated whether other residues in the N-terminal tail of H3 are important for methylation of DNA and of H3K9. Mutations in the N-terminal tail of H3 were generated and tested for effects in vitro and in vivo, in the presence or absence of the wild-type allele. Substitutions at K4, K9, T11, G12, G13, K14, K27, S28, and K36 were lethal in the absence of a wild-type allele. In contrast, mutants bearing substitutions of R2, A7, R8, S10, A15, P16, R17, K18, and K23 were viable. The effect of substitutions on DNA methylation were variable; some were recessive and others caused a semi-dominant loss of DNA methylation. Substitutions of R2, A7, R8, S10, T11, G12, G13, K14, and P16 caused partial or complete loss of DNA methylation in vivo. Only residues R8-G12 were required for DIM-5 activity in vitro. DIM-5 activity was inhibited by dimethylation of H3K4 and by phosphorylation of H3S10, but not by acetylation of H3K14. We conclude that the H3 tail acts as an integrating platform for signals that influence DNA methylation, in part through methylation of H3K9.

  19. Over-expression of histone H3K4 demethylase gene JMJ15 enhances salt tolerance in Arabidopsis

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    Yuan eShen

    2014-06-01

    Full Text Available Histone H3 lysine 4 trimethylation (H3K4me3 has been shown to be involved in stress-responsive gene expression and gene priming in plants. However, the role of H3K4me3 resetting in the processes is not clear. In this work we studied the expression and function of Arabidopsis H3K4 demethylase gene JMJ15. We show that the expression of JMJ15 was relatively low and was limited to a number of tissues during vegetative growth but was higher in young floral organs. Over-expression of the gene in gain-of-function mutants reduced the plant height with accumulation of lignin in stems, while the loss-of-function mutation did not produce any visible phenotype. The gain-of-function mutants showed enhanced salt tolerance, whereas the loss-of-function mutant was more sensitive to salt compared to the wild type. Transcriptomic analysis revealed that over-expression of JMJ15 down-regulated many genes which are preferentially marked by H3K4me3 and H3K4me2. Many of the down-regulated genes encode transcription regulators involved in stress responses. The data suggest that increased JMJ15 levels may regulate the gene expression program that enhances stress tolerance.

  20. Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger

    Institute of Scientific and Technical Information of China (English)

    Chengkun Wang; Jie Shen; Zhongzheng Yang; Ping Chen; Bin Zhao; Wei Hu; Wenxian Lan

    2011-01-01

    Dear Editor,We report two NMR complex structures of PHDUHRF1 binding to unmodified or K9 trimethylated histone tails,which clarify a controversy regarding how the binding of UHRF1 to H3 tails is mediated.Based on our structures,H3R2,not H3K9,mediates PHD binding.

  1. Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem

    Science.gov (United States)

    Steven G Hussey; Eshchar Mizrachi; Andrew Groover; Dave K Berger; Alexander A Myburg

    2015-01-01

    Background: Histone modifications play an integral role in plant development, but have been poorly studied inwoody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood...

  2. Saturated fatty acid palmitate induces extracellular release of histone H3: A possible mechanistic basis for high-fat diet-induced inflammation and thrombosis

    Energy Technology Data Exchange (ETDEWEB)

    Shrestha, Chandan [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Ito, Takashi [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Kawahara, Ko-ichi [Department of Biomedical Engineering, Osaka Institute of Technology, Osaka (Japan); Shrestha, Binita; Yamakuchi, Munekazu; Hashiguchi, Teruto [Department of Laboratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan); Maruyama, Ikuro, E-mail: rinken@m3.kufm.kagoshima-u.ac.jp [Department of Systems Biology in Thromboregulation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima (Japan)

    2013-08-09

    Highlights: •High-fat diet feeding and palmitate induces the release of nuclear protein histone H3. •ROS production and JNK signaling mediates the release of histone H3. •Extracellular histones induces proinflammatory and procoagulant response. -- Abstract: Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells toexpress the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis.

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

    Directory of Open Access Journals (Sweden)

    Jiang-Li Wu

    Full Text Available 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.

  4. Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition

    DEFF Research Database (Denmark)

    Dahl, John Arne; Jung, Inkyung; Aanes, Håvard;

    2016-01-01

    Maternal-to-zygotic transition (MZT) is essential for the formation of a new individual, but is still poorly understood despite recent progress in analysis of gene expression and DNA methylation in early embryogenesis. Dynamic histone modifications may have important roles in MZT, but direct meas...

  5. Dynamics of Histone Tails within Chromatin

    Science.gov (United States)

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

    2012-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Donglin; Qu, Xiying; Li, Lin; Zhou, Xin; Liu, Sijie; Lin, Shiguan; Wang, Pengfei; Liu, Shaohui; Kong, Chuijin; Wang, Xiaohui; Liu, Lin; Zhu, Huanzhang, E-mail: hzzhu@fudan.edu.cn

    2013-06-05

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

  7. KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation.

    Science.gov (United States)

    Ang, Siang-Yun; Uebersohn, Alec; Spencer, C Ian; Huang, Yu; Lee, Ji-Eun; Ge, Kai; Bruneau, Benoit G

    2016-03-01

    KMT2D, which encodes a histone H3K4 methyltransferase, has been implicated in human congenital heart disease in the context of Kabuki syndrome. However, its role in heart development is not understood. Here, we demonstrate a requirement for KMT2D in cardiac precursors and cardiomyocytes during cardiogenesis in mice. Gene expression analysis revealed downregulation of ion transport and cell cycle genes, leading to altered calcium handling and cell cycle defects. We further determined that myocardial Kmt2d deletion led to decreased H3K4me1 and H3K4me2 at enhancers and promoters. Finally, we identified KMT2D-bound regions in cardiomyocytes, of which a subset was associated with decreased gene expression and decreased H3K4me2 in mutant hearts. This subset included genes related to ion transport, hypoxia-reoxygenation and cell cycle regulation, suggesting that KMT2D is important for these processes. Our findings indicate that KMT2D is essential for regulating cardiac gene expression during heart development primarily via H3K4 di-methylation.

  8. An Autoregulatory Mechanism Imposes Allosteric Control on the V(DJ Recombinase by Histone H3 Methylation

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    Chao Lu

    2015-01-01

    Full Text Available V(DJ recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(DJ recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3. Engagement of H3K4me3 by a plant homeodomain (PHD in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.

  9. Genome-wide distribution of histone H3 acetylation in all-trans retinoic acid induced neuronal differentiation of SH-SY5Y cells

    Institute of Scientific and Technical Information of China (English)

    FANG HongBo; MI Yang; WU NingHua; ZHANG Ye; SHEN YuFei

    2009-01-01

    With chromatin immunoprecipitation (CHIP) and promoter DNA microarray analyses (ChiP-on-chip), we analyzed the variations of acetylation on histone H3 in all-trans retinoic acid (RA) induced neuronal cell differentiation. Neuroblastoma SH-SY5Y cells were treated with RA for 24 h and the acetylation on histone H3 in the promoter region of the genes was detected. Results showed that, after treatment, the level of acetylation on histone H3 elevated in 597 genes in the genome, and reduced in the other 647 genes compared with those of the control. In summary, we have successfully adopted a high throughput technique to detect and analyze variations of acetylation of histone H3 in human genome at the early phage of RA induced neuronal differentiation of the SH-SY5Y cells.

  10. Quantitative Proteomics Reveals That the Specific Methyltransferases Txr1p and Ezl2p Differentially Affect the Mono-, Di- and Trimethylation States of Histone H3 Lysine 27 (H3K27)*

    OpenAIRE

    Zhang, Chunchao; Molascon, Anthony J.; Gao, Shan; Liu, Yifan; Andrews, Philip C.

    2012-01-01

    Nuclear DNA in eukaryotic cells is assembled into the hierarchical chromatin structure via a process that is dynamically affected by the combinatorial set of post-translational modifications (PTMs) of histones in a dynamic manner responsive to physiological and environmental changes. The precise quantification of these complex modifications is challenging. Here we present a robust MS-based quantitative proteomics method for studying histone PTMs using 15N metabolically labeled histones as the...

  11. Saccharomyces cerevisiae MHF complex structurally resembles the histones (H3-H4)₂ heterotetramer and functions as a heterotetramer.

    Science.gov (United States)

    Yang, Hui; Zhang, Tianlong; Tao, Ye; Wu, Lijing; Li, Hong-Tao; Zhou, Jin-Qiu; Zhong, Chen; Ding, Jianping

    2012-02-08

    Fanconi anemia (FA) is a chromosomal instability disorder associated with deficiencies in the Fanconi anemia complementation group (FANC) network. A complex consisting of FANCM-associated histone-fold proteins 1 and 2 (MHF1 and MHF2) has been shown to act cooperatively with FANCM in DNA damage repair in the FA pathway. Here we report the structure of Saccharomyces cerevisiae MHF complex in which MHF1 and MHF2 assume a typical histone fold, and the complex has a heterotetrameric architecture similar to that of the histones (H3-H4)₂ heterotetramer. Loop L2 of MHF1 is probably involved in DNA binding, and loop L3 and helices α2 and α3 of one MHF1 subunit interact with those of the other to form two heterotetramer interfaces. Further genetic data demonstrate that the heterotetramer assembly is essential for the function of the complex in DNA repair. These results provide, to the best of our knowledge, new mechanistic insights into the function of the MHF complex. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Replication-Independent Histone Variant H3.3 Controls Animal Lifespan through the Regulation of Pro-longevity Transcriptional Programs

    Directory of Open Access Journals (Sweden)

    Antonia Piazzesi

    2016-10-01

    Full Text Available Chromatin structure orchestrates the accessibility to the genetic material. Replication-independent histone variants control transcriptional plasticity in postmitotic cells. The life-long accumulation of these histones has been described, yet the implications on organismal aging remain elusive. Here, we study the importance of the histone variant H3.3 in Caenorhabditis elegans longevity pathways. We show that H3.3-deficient nematodes have negligible lifespan differences compared to wild-type animals. However, H3.3 is essential for the lifespan extension of C. elegans mutants in which pronounced transcriptional changes control longevity programs. Notably, H3.3 loss critically affects the expression of a very large number of genes in long-lived nematodes, resulting in transcriptional profiles similar to wild-type animals. We conclude that H3.3 positively contributes to diverse lifespan-extending signaling pathways, with potential implications on age-related processes in multicellular organisms.

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

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    Jamy C Peng

    2009-03-01

    Full Text Available Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability.

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

    Science.gov (United States)

    Peng, Jamy C; Karpen, Gary H

    2009-03-01

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

  15. Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

    DEFF Research Database (Denmark)

    Rasmussen, Louise; Herzog, Marielle; Rømer, Eva

    2016-01-01

    Aim: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3). Materials and methods: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis...

  16. Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening.

    Science.gov (United States)

    Khoury, Laure; Zalko, Daniel; Audebert, Marc

    2016-08-01

    The in vitro micronucleus assay is broadly used, but is not per se able to discriminate aneugenic from clastogenic compounds, and cytotoxicity can be a confounding factor. In vitro genotoxicity assays generally rely on cell lines with limited metabolic capabilities. Recently, the use of histone H2AX and H3 phosphorylation markers (γH2AX and p-H3) was proposed to discriminate aneugenic from clastogenic chemicals. The aim of the present study was to develop a new genotoxic screening strategy based on the use of the γH2AX and p-H3 biomarkers in combination with cell lines with distinct biotransformation properties. First, we tested a training set of 20 model chemicals comprised of 10 aneugens, five clastogens and five cytotoxics on three human cell lines (HepG2, LS-174T and ACHN). Our data confirm the robustness of these two biomarkers to discriminate efficiently clastogens, aneugens and misleading cytotoxic chemicals in HepG2 cells. Aneugenic compounds induced either an increase or a decrease in p-H3 depending on their mode of action. Clastogens induced γH2AX, and cytotoxic compounds generated a marked decrease in these two biomarkers. Moreover, the use of different cell lines permits to discriminate direct from bioactivated genotoxins without the need of an exogenous metabolic activation system. Finally, we further evaluated this strategy using a test set of 13 chemicals with controversial genotoxic potential. The resulting data demonstrate that the combined analysis of γH2AX and p-H3 is an efficient strategy. Notably, we demonstrated that three compounds (fisetin, hydroquinone and okadaic acid) display both aneugenic and clastogenic properties.

  17. The ASH1 HOMOLOG 2 (ASHH2 histone H3 methyltransferase is required for ovule and anther development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Paul E Grini

    Full Text Available BACKGROUND: SET-domain proteins are histone lysine (K methyltransferases (HMTase implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2 protein (also called SDG8, EFS and CCR1 has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99 we observed a reduction of H3K36 trimethylation (me3, but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to

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

    Science.gov (United States)

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

    2008-06-15

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

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

    Science.gov (United States)

    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.

  20. Kinetochore function and chromosome segregation rely on critical residues in histones H3 and H4 in budding yeast.

    Science.gov (United States)

    Ng, Tessie M; Lenstra, Tineke L; Duggan, Nicole; Jiang, Shuangying; Ceto, Steven; Holstege, Frank C P; Dai, Junbiao; Boeke, Jef D; Biggins, Sue

    2013-11-01

    Accurate chromosome segregation requires that sister kinetochores biorient and attach to microtubules from opposite poles. Kinetochore biorientation relies on the underlying centromeric chromatin, which provides a platform to assemble the kinetochore and to recruit the regulatory factors that ensure the high fidelity of this process. To identify the centromeric chromatin determinants that contribute to chromosome segregation, we performed two complementary unbiased genetic screens using a library of budding yeast mutants in every residue of histone H3 and H4. In one screen, we identified mutants that lead to increased loss of a nonessential chromosome. In the second screen, we isolated mutants whose viability depends on a key regulator of biorientation, the Aurora B protein kinase. Nine mutants were common to both screens and exhibited kinetochore biorientation defects. Four of the mutants map near the unstructured nucleosome entry site, and their genetic interaction with reduced IPL1 can be suppressed by increasing the dosage of SGO1, a key regulator of biorientation. In addition, the composition of purified kinetochores was altered in six of the mutants. Together, this work identifies previously unknown histone residues involved in chromosome segregation and lays the foundation for future studies on the role of the underlying chromatin structure in chromosome segregation.

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

    Directory of Open Access Journals (Sweden)

    Patterton Hugh G

    2011-07-01

    Full Text Available Abstract 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.

  2. The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers.

    Science.gov (United States)

    Klein, Brianna J; Piao, Lianhua; Xi, Yuanxin; Rincon-Arano, Hector; Rothbart, Scott B; Peng, Danni; Wen, Hong; Larson, Connie; Zhang, Xi; Zheng, Xia; Cortazar, Michael A; Peña, Pedro V; Mangan, Anthony; Bentley, David L; Strahl, Brian D; Groudine, Mark; Li, Wei; Shi, Xiaobing; Kutateladze, Tatiana G

    2014-01-30

    The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  3. The Histone-H3K4-Specific Demethylase KDM5B Binds to Its Substrate and Product through Distinct PHD Fingers

    Directory of Open Access Journals (Sweden)

    Brianna J. Klein

    2014-01-01

    Full Text Available The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0, whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD complex and may cooperate with the histone deacetylase 1 (HDAC1 in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.

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

    Science.gov (United States)

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

    2009-11-01

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

  5. Cigarette sidestream smoke induces histone H3 phosphorylation via JNK and PI3K/Akt pathways, leading to the expression of proto-oncogenes.

    Science.gov (United States)

    Ibuki, Yuko; Toyooka, Tatsushi; Zhao, Xiaoxu; Yoshida, Ikuma

    2014-06-01

    Post-translational modifications in histones have been associated with cancer. Although cigarette sidestream smoke (CSS) as well as mainstream smoke are carcinogens, the relationship between carcinogenicity and histone modifications has not yet been clarified. Here, we demonstrated that CSS induced phosphorylation of histones, involving a carcinogenic process. Treatment with CSS markedly induced the phosphorylation of histone H3 at serine 10 and 28 residues (H3S10 and H3S28), which was independent from the cell cycle, in the human pulmonary epithelial cell model, A549 and normal human lung fibroblasts, MRC-5 and WI-38. Using specific inhibitors and small interfering RNA, the phosphorylation of H3S10 was found to be mediated by c-jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. These pathways were different from that of the CSS-induced phosphorylation of histone H2AX (γ-H2AX) mediated by Ataxia telangiectasia-mutated (ATM) and ATM-Rad3-related (ATR) protein kinases. A chromatin immunoprecipitation assay revealed that the phosphorylation of H3S10 was increased in the promoter sites of the proto-oncogenes, c-fos and c-jun, which indicated that CSS plays a role in tumor promotion. Because the phosphorylation of H3S10 was decreased in the aldehyde-removed CSS and was significantly induced by treatment with formaldehyde, aldehydes are suspected to partially contribute to this phosphorylation. These findings suggested that any chemicals in CSS, including aldehydes, phosphorylate H3S10 via JNK and PI3K/Akt pathways, which is different from the DNA damage response, resulting in tumor promotion.

  6. mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression.

    Science.gov (United States)

    Wang, Hengbin; An, Woojin; Cao, Ru; Xia, Li; Erdjument-Bromage, Hediye; Chatton, Bruno; Tempst, Paul; Roeder, Robert G; Zhang, Yi

    2003-08-01

    Methylation of histone tails plays an important role in chromatin structure and function. Previously, we reported that ESET/SETDB1 is a histone methyltransferase (HMTase). Here, we show that SETDB1 tightly associates with the human homolog of mAM, a murine ATFa-associated factor. Although recombinant ESET can methylate lysine 9 of histone H3 (H3-K9), its activity is severely compromised when compared to that of the ESET/mAM complex. mAM stimulates ESET enzymatic activity by increasing the Vmax and decreasing the Km. Importantly, mAM facilitates the ESET-dependent conversion of dimethyl H3-K9 to the trimethyl state both in vitro and in vivo. Chromatin-based transcription and ChIP analyses demonstrate that mAM enhances ESET-mediated transcriptional repression in a SAM-dependent manner, and this repression correlates with H3-K9 trimethylation at the promoter. Thus, our studies establish that promoter H3-K9 trimethylation is the cause of transcriptional repression and that mAM/hAM facilitates conversion of H3-K9 dimethyl to trimethyl by ESET/SETDB1.

  7. Kaposi΄s sarcoma-associated herpesvirus ORF36 protein induces chromosome condensation and phosphorylation of histone H3.

    Science.gov (United States)

    Kim, Sunmi; Cha, Seho; Jang, Jun Hyeong; Kim, Yejin; Seo, Taegun

    2013-01-01

    Kaposi΄s sarcoma-associated herpesvirus (KSHV) has been known as an agent causing Kaposi΄s sarcoma, primary effusion lymphoma, and multicentric Castleman΄s disease. In the lytic phase of the virus cycle, various viral genes are expressed, which causes host cell dysregulation. Among the lytic genes, viral protein kinase (vPK) encoded by ORF36 is a member of serine/threonine protein kinase (CHPK) family, which is involved in viral gene expression, viral DNA replication and encapsidation, and nuclear egress of virions. Recent studies have shown that the BGLF4 protein of Epstein-Barr virus (EBV), a member of the CHPK family, alters the host cell chromatin structure through phosphorylation of its key regulators. The role of KSHV ORF36 in cellular mitotic events, however, is not yet understood. In the current study, we showed that KSHV ORF36 induced chromosome condensation and phosphorylation of histone H3 on Ser 10, which are known as cellular mitosis markers. These processes have occurred in a kinase activity-dependent manner.

  8. Cloning of rainbow trout (Oncorhynchus mykiss ) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

    Institute of Scientific and Technical Information of China (English)

    MAO Weifeng; SUN Yonghua; WANG Yaping; WU Gang; CHEN Shangping; ZHU Zuoyan

    2004-01-01

    Rainbow trout histone H3(RH3)promoter was cloned via high fidelity PCR.The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1,resulting in an expression vector pRH3EGFP-1.The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope.Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage.The results indi cate that RH3 promoter,as a piscine promoter,could serve in producing transgenic Cyprinoid such as rare minnow.Promoter activity of RH3,CMV and common carp β-actin(CA)were compared in rare minnow by the expression of respective recombinant EGFP vectors.The expression of pCMVEGFP occurred earlier than the following one,pRH3EGFP-1,and then pCAEGFP during the embryogenesis of the transgenics.Their expression activities demonstrated that the CMV promoter is the strongest one,followed by the CA and then the RH3.

  9. Structural Insights into the Association of Hif1 with Histones H2A-H2B Dimer and H3-H4 Tetramer.

    Science.gov (United States)

    Zhang, Mengying; Liu, Hejun; Gao, Yongxiang; Zhu, Zhongliang; Chen, Zijun; Zheng, Peiyi; Xue, Lu; Li, Jixi; Teng, Maikun; Niu, Liwen

    2016-10-04

    Histone chaperones are critical for guiding specific post-transcriptional modifications of histones, safeguarding the histone deposition (or disassociation) of nucleosome (dis)assembly, and regulating chromatin structures to change gene activities. HAT1-interacting factor 1 (Hif1) has been reported to be an H3-H4 chaperone and to be involved in telomeric silencing and nucleosome (dis)assembly. However, the structural basis for the interaction of Hif1 with histones remains unknown. Here, we report the complex structure of Hif1 binding to H2A-H2B for uncovering the chaperone specificities of Hif1 on binding to both the H2A-H2B dimer and the H3-H4 tetramer. Our findings reveal that Hif1 interacts with the H2A-H2B dimer and the H3-H4 tetramer via distinct mechanisms, suggesting that Hif1 is a pivotal scaffold on alternate binding of H2A-H2B and H3-H4. These specificities are conserved features of the Sim3-Hif1-NASP interrupted tetratricopeptide repeat proteins, which provide clues for investigating their potential roles in nucleosome (dis)assembly. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-15

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

  11. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

    Science.gov (United States)

    Foda, Bardees M.; Singh, Upinder

    2015-01-01

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

  12. Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway.

    Science.gov (United States)

    Foda, Bardees M; Singh, Upinder

    2015-08-21

    RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5'-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.

    Directory of Open Access Journals (Sweden)

    Agnieszka Gacek-Matthews

    2016-08-01

    Full Text Available Histone posttranslational modifications (HPTMs are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS, genome-wide chromatin immunoprecipitation (ChIP-seq and transcriptional network analysis (RNA-seq that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3 are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism and nutrient-limiting (secondary metabolism conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.

  14. 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.

    Science.gov (United States)

    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.

  15. Exploring PHD fingers and H3K4me0 interactions with molecular dynamics simulations and binding free energy calculations: AIRE-PHD1, a comparative study.

    Directory of Open Access Journals (Sweden)

    Dimitrios Spiliotopoulos

    Full Text Available PHD fingers represent one of the largest families of epigenetic readers capable of decoding post-translationally modified or unmodified histone H3 tails. Because of their direct involvement in human pathologies they are increasingly considered as a potential therapeutic target. Several PHD/histone-peptide structures have been determined, however relatively little information is available on their dynamics. Studies aiming to characterize the dynamic and energetic determinants driving histone peptide recognition by epigenetic readers would strongly benefit from computational studies. Herein we focus on the dynamic and energetic characterization of the PHD finger subclass specialized in the recognition of histone H3 peptides unmodified in position K4 (H3K4me0. As a case study we focused on the first PHD finger of autoimmune regulator protein (AIRE-PHD1 in complex with H3K4me0. PCA analysis of the covariance matrix of free AIRE-PHD1 highlights the presence of a "flapping" movement, which is blocked in an open conformation upon binding to H3K4me0. Moreover, binding free energy calculations obtained through Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA methodology are in good qualitative agreement with experiments and allow dissection of the energetic terms associated with native and alanine mutants of AIRE-PHD1/H3K4me0 complexes. MM/PBSA calculations have also been applied to the energetic analysis of other PHD fingers recognizing H3K4me0. In this case we observe excellent correlation between computed and experimental binding free energies. Overall calculations show that H3K4me0 recognition by PHD fingers relies on compensation of the electrostatic and polar solvation energy terms and is stabilized by non-polar interactions.

  16. The Histone Demethylase Jarid1b Ensures Faithful Mouse Development by Protecting Developmental Genes from Aberrant H3K4me3

    DEFF Research Database (Denmark)

    Albert, Mareike; Schmitz, Sandra U; Kooistra, Susanne M

    2013-01-01

    of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line...... with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis...... of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  18. Influence of chromatin structure, antibiotics, and endogenous histone methylation on phosphorylation of histones H1 and H3 in the presence of protein kinase A in rat liver nuclei in vitro.

    Science.gov (United States)

    Prusov, A N; Smirnova, T A; Kolomijtseva, G Ya

    2013-02-01

    In vitro phosphorylation of histones H1 and H3 by cAMP-dependent protein kinase A and endogenous phosphokinases in the presence of [γ-³²P]ATP was studied in isolated rat liver nuclei with different variants of chromatin structural organization: condensed (diameter of fibrils 100-200 nm; N-1) and partly decondensed (diameter of fibrils ~30 nm; N-2). In the N-1 state histone, H1 is phosphorylated approximately twice as much than histone H3. Upon the decondensation of the chromatin in the N-2 state, 1.5-fold decrease of total phosphorylation of H1 is observed, while that of H3 does not change, although the endogenous phosphorylation of both histones is reduced by half. Changes in histone phosphorylation in the presence of low or high concentrations of distamycin and chromomycin differ for H1 and H3 in N-1 and N-2. It was found that distamycin (DM) stimulates the phosphorylation of tightly bound H1 fraction, which is not extractable by polyglutamic acid (PG), especially in N-1. Chromomycin (CM) increases the phosphorylation of both histones in PG extracts and in the nuclear pellets, particularly in N-2. At the same time, in N-1 one can detect phosphorylation of a tightly bound fraction of histones H1 whose N-termini are located on AT-rich sites that become inaccessible for protein kinase in the process of chromatin decondensation in N-2. At the same time, in N-2 the accessibility for protein kinase A of tightly bound H1 fractions, whose N-termini are located on GC-rich sites, increases dramatically. High concentrations of both CM and DM in N-1 and N-2 stimulated phosphorylation of the non-extractable by PG fraction of H1 whose N-termini are located on sites where AT ≈ GC. CM at high concentration stimulated 4-7 times the phosphorylation of a small fraction of H3, which is extracted by PG from both types of nuclei. We detected an effect of endogenous methylation of histones H1 and H3 in the nuclei on their subsequent phosphorylation depending on the chromatin

  19. Localization of Phosphorylated Histone H3 at Mitosis and Meiosis in Wheat%磷酸化组蛋白H3在小麦有丝分裂与减数分裂中的分布

    Institute of Scientific and Technical Information of China (English)

    杨琴; 黄熙泰; 耿朝晖; 俞新大

    2002-01-01

    One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphorylation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase Ⅰ until telophase Ⅰ, whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase Ⅱ until telophase Ⅱ. The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post_translational chromatin modification might be involved in more roles besides chromosome condensation.%在细胞周期中, 与染色质凝集偶联的一类组蛋白修饰是组蛋白H3的磷酸化.运用H3-Ser 10磷酸化的特异性抗体,通过间接免疫荧光标记检测了磷酸化组蛋白H3在小麦(Triticum aestivum L.)有丝分裂与减数分裂细胞中的分布.有丝分裂时,H3磷酸化起始于早前期,消失于末期,在中期与后期,H3磷酸化主要分布在着丝粒两侧的异染色质区.减数分裂时,H3磷酸化起始于细线期向偶线期转换时,并且从前期Ⅰ到后期Ⅰ保持均一分布于整个染色体上,直到末期Ⅰ消失,而中期Ⅱ与后期Ⅱ在着丝粒两侧的异染色质区的信号略强于染色体臂,直至消失于末期Ⅱ.磷酸化组蛋白H3在两类细胞分裂中的不同分布暗示这种保守的翻译后修饰可能发挥着除参与

  20. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells.

    Science.gov (United States)

    Lee, Wei-Jiunn; Chen, Yun-Ru; Tseng, Tsui-Hwa

    2011-02-01

    Quercetin, a naturally occurring flavonoid abundant in fruits and vegetables, has been demonstrated as a multipotent bioflavonoid with great potential for the prevention and treatment of cancer. Apoptosis is thought to be an important response to most chemotherapeutic agents in leukemia cells. However, the underlying mechanism of induction of apoptosis by quercetin involving epigenetic regulation is poorly understood. In the present study, by evaluation of fragmentation of DNA, poly (ADP-ribose) polymerase (PARP) and procaspases, we found that quercetin was able to induce apoptosis of human leukemia HL-60 cells in a dose-dependent manner. Quercetin triggered the extrinsic apoptosis pathway through activation of caspase-8 and induction of Bid cleavage, Bax conformation change and cytochrome c release. Furthermore, quercetin induced Fas ligand (FasL) expression involving activation of the extracellular signal-regulated kinase (ERK) and Jun N-terminus kinase (JNK) signaling pathways. In addition to activation of c-Jun, quercetin increased histone H3 acetylation which resulted in the promotion of the expression of FasL. Quercetin exhibited potential for the activation of histone acetyltransferase (HAT) and the inhibition of histone deacetyltransferase (HADC), both of which contributed to histone acetylation. However, only the activation effect on HAT was associated with the ERK and JNK pathway. These results demonstrated that quercetin induced FasL-related apoptosis by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in HL-60 cells.

  1. Evolutionary relationships within the protostome phylum Sipuncula: a molecular analysis of ribosomal genes and histone H3 sequence data.

    Science.gov (United States)

    Maxmen, Amy B; King, Burnett F; Cutler, Edward B; Giribet, Gonzalo

    2003-06-01

    The phylogenetic relationships of the members of the phylum Sipuncula are investigated by means of DNA sequence data from three nuclear markers, two ribosomal genes (18S rRNA and the D3 expansion fragment of 28S rRNA), and one protein-coding gene, histone H3. Phylogenetic analysis via direct optimization of DNA sequence data using parsimony as optimality criterion is executed for 12 combinations of parameter sets accounting for different indel costs and transversion/transition cost ratios in a sensitivity analysis framework. Alternative outgroup analyses are also performed to test whether they affected rooting of the sipunculan topology. Nodal support is measured by parsimony jackknifing and Bremer support values. Results from the different partitions are highly congruent, and the combined analysis for the parameter set that minimizes overall incongruence supports monophyly of Sipuncula, but nonmonophyly of several higher taxa recognized for the phylum. Mostly responsible for this is the split of the family Sipunculidae in three main lineages, with the genus Sipunculus being the sister group to the remaining sipunculans, the genus Phascolopsis nesting within the Golfingiiformes, and the genus Siphonosoma being associated to the Phascolosomatidea. Other interesting results are the position of Phascolion within Golfingiidae and the position of Antillesoma within Aspidosiphonidae. These results are not affected by the loci selected or by the outgroup chosen. The position of Apionsoma is discussed, although more data would be needed to better ascertain its phylogenetic affinities. Monophyly of the genera with multiple representatives (Themiste, Aspidosiphon, and Phascolosoma) is well supported, but not the monophyly of the genera Nephasoma or Golfingia. Interesting phylogeographic questions arise from analysis of multiple representatives of a few species.

  2. Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Qianzheng; Battu, Aruna; Ray, Alo; Wani, Gulzar; Qian, Jiang; He, Jinshan; Wang, Qi-en [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Wani, Altaf A., E-mail: wani.2@osu.edu [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210 (United States); James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 (United States)

    2015-06-15

    Highlights: • HDAC1 and HDAC2 co-localize with UV radiation-induced DNA damage sites. • HDAC1 translocation to chromatin is dependent on DDB2 function. • HDAC1 and HDAC2 are involved in H3K56Ac deacetylation. • H3K56Ac deacetylation requires DDB1 and DDB2 but not XPA or XPC functions. • HDAC1/2 depletion decreases XPC ubiquitination and local γH2AX accumulation. - Abstract: Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

  3. Nocturnal activation of aurora C in rat pineal gland: its role in the norepinephrine-induced phosphorylation of histone H3 and gene expression.

    Science.gov (United States)

    Price, D M; Kanyo, R; Steinberg, N; Chik, C L; Ho, A K

    2009-05-01

    We have shown previously that Ser10 phosphorylation of histone H3 occurs in rat pinealocytes after stimulation with norepinephrine (NE) and that histone modifications such as acetylation appear to play an important role in pineal gene transcription. Here we report the nocturnal phosphorylation of a Ser10 histone H3 kinase, Aurora C, in the rat pineal gland. The time profile of this phosphorylation parallels the increase in the level of phospho-Ser10 histone H3. Studies with cultured pinealocytes indicate that Aurora C phosphorylation is induced by NE and this induction can be blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor. Moreover, only treatment with dibutyryl cAMP, but not other kinase activators, mimics the effect of NE on Aurora C phosphorylation. These results indicate that Aurora C is phosphorylated primarily by a beta-adrenergic/protein kinase A-mediated mechanism. Treatment with an Aurora C inhibitor reduces the NE-induced histone H3 phosphorylation and suppresses the NE-stimulated induction of arylalkylamine N-acetyltransferase (AA-NAT), the rhythm-controlling enzyme of melatonin synthesis, and melatonin production. The effects of Aurora C inhibitors on adrenergic-induced genes in rat pinealocytes are gene specific: inhibitory for Aa-nat and inducible cAMP repressor but stimulatory for c-fos. Together our results support a role for the NE-stimulated phosphorylation of Aurora C and the subsequent remodeling of chromatin in NE-stimulated Aa-nat transcription. This phenomenon suggests that activation of this mitotic kinase can be induced by extracellular signals to participate in the transcriptional induction of a subset of genes in the rat pineal gland.

  4. Role of the structural domains of linker histones and histone H3 in the chromatin fiber structure at low-ionic strength: scanning force microscopy (SFM) studies on partially trypsinized chromatin

    Science.gov (United States)

    Zlatanova, Jordanka; Leuba, Sanford S.; Bustamante, Carlos J.; van Holde, Kensal

    1995-03-01

    Chromatin fibers have been hydrolyzed by trypsin and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and scanning force microscopy (SFM). At early points of hydrolysis, when mostly only the tails of the linker histones have been cleaved, nucleosomes appear to pile upon each other within a fiber. Later, once significant hydrolysis of the N-terminal tail of histone H3 has occurred, fibers exhibit an open, three-dimensional arrangement of nucleosomes. Linker DNA between adjacent nucleosomes is observed for the first time. Adjacent nucleosomes appear to have a 'zig-zag' arrangement. Finally, when all the tails of the linker histones and the N-terminal tails of H3 have been cleaved, then the fibers exhibit (i) a flat two- dimensional arrangement of nucleosomes, (ii) linker DNA between nearly all nucleosomes, and (iii) a zig-zag arrangement among some nucleosomes. We suggest that (i) the linker histone globular domains help fix the angle of the DNA entering and exiting the nucleosome, (ii) the angle, however, is not sufficient to maintain the three-dimensionality of the fiber, and (iii) the N-terminal tails of histone H3 ares necessary for the three-dimensional conformation of the fiber at low ionic strength.

  5. H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3.

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    Richárd Bártfai

    Full Text Available Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes.

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

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

    2016-01-01

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

  7. Preferential recruitment of the maternal centromere-specific histone H3 (CENH3) in oat (Avena sativa L.) × pearl millet (Pennisetum glaucum L.) hybrid embryos.

    Science.gov (United States)

    Ishii, Takayoshi; Sunamura, Naohiro; Matsumoto, Ayaka; Eltayeb, Amin Elsadig; Tsujimoto, Hisashi

    2015-12-01

    Chromosome elimination occurs frequently in interspecific hybrids between distantly related species in Poaceae. However, chromosomes from both parents behave stably in a hybrid of female oat (Avena sativa L.) pollinated by pearl millet (Pennisetum glaucum L.). To analyze the chromosome behavior in this hybrid, we cloned the centromere-specific histone H3 (CENH3) genes of oat and pearl millet and produced a pearl millet-specific anti-CENH3 antibody. Application of this antibody together with a grass species common anti-CENH3 antibody revealed the dynamic CENH3 composition of the hybrid cells before and after fertilization. Despite co-expression of CENH3 genes encoded by oat and pearl millet, only an oat-type CENH3 was incorporated into the centromeres of both species in the hybrid embryo. Oat CENH3 enables a functional centromere in pearl millet chromosomes in an oat genetic background. Comparison of CENH3 genes among Poaceae species that show chromosome elimination in interspecific hybrids revealed that the loop 1 regions of oat and pearl millet CENH3 exhibit exceptionally high similarity.

  8. High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin.

    Science.gov (United States)

    Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J; Shanker, Sreenath; Kuscu, Canan; Joshua-Tor, Leemor; Partridge, Janet F

    2009-04-10

    In fission yeast, assembly of centromeric heterochromatin requires the RITS complex, which consists of Ago1, Tas3, Chp1, and siRNAs derived from centromeric repeats. Recruitment of RITS to centromeres has been proposed to depend on siRNA-dependent targeting of Ago1 to centromeric sequences. Previously, we demonstrated that methylated lysine 9 of histone H3 (H3K9me) acts upstream of siRNAs during heterochromatin establishment. Our crystal structure of Chp1's chromodomain in complex with a trimethylated lysine 9 H3 peptide reveals extensive sites of contact that contribute to Chp1's high-affinity binding. We found that this high-affinity binding is critical for the efficient establishment of centromeric heterochromatin, but preassembled heterochromatin can be maintained when Chp1's affinity for H3K9me is greatly reduced.

  9. Specific promoter deacetylation of histone H3 is conserved across mouse models of Huntington's disease in the absence of bulk changes.

    Science.gov (United States)

    Guiretti, Deisy; Sempere, Ana; Lopez-Atalaya, Jose P; Ferrer-Montiel, Antonio; Barco, Angel; Valor, Luis M

    2016-05-01

    Defective epigenetic regulation has been postulated as a possible cause for the extensive and premature transcriptional dysregulation observed in experimental models of Huntington's disease (HD). In this study, we extended our observations in the N171-82Q mouse strain relating to the limited impact of polyQ pathology on the global histone acetylation to other animal and cellular models of HD, namely the R6/1 and YAC128 strains, striatal-electroporated mice, primary neuronal cultures and stably transfected PC12 cells. In the absence of bulk chromatin changes, we nonetheless documented histone deacetylation events at the transcription start sites (TSS) of genes relevant to neuronal functions (e.g., Rin1, Plk5, Igfbp5, Eomes, and Fos). In some instances, these local deficits were associated with an increased susceptibility to transcriptional dysregulation (e.g., Camk1g and Rasl11b) and the defective trimethylation of histone H3 at lysine 4 (H3K4me3), another covalent modification of histone tails that is related to active transcription and is also altered in HD. Overall, this study provides further insight into the nature and extent of epigenetic dysregulation in HD pathology.

  10. Global Decrease of Histone H3K27 Acetylation in ZEB1-Induced Epithelial to Mesenchymal Transition in Lung Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Joëlle, E-mail: joelle.roche@univ-poitiers.fr [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States); CNRS FRE 3511, University of Poitiers, 1 rue Georges Bonnet, F-86022 Poitiers Cédex (France); Nasarre, Patrick; Gemmill, Robert [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States); Baldys, Aleksander [Department of Medicine, Nephrology Division, MUSC, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29425 (United States); Pontis, Julien [Epigénétique & Destin Cellulaire, CNRS UMR 7216, University of Paris Diderot, Sorbonne Paris Cité, F-75013 Paris (France); Korch, Christopher [CU DNA Sequencing and Analysis Core, University of Colorado, School of Medicine, Anschutz Medical Campus, 12801 E. 17th Ave., Aurora, CO 80045 (United States); Guilhot, Joëlle [INSERM, CIC 0802, CHU de Poitiers, F-86021 (France); Ait-Si-Ali, Slimane [Epigénétique & Destin Cellulaire, CNRS UMR 7216, University of Paris Diderot, Sorbonne Paris Cité, F-75013 Paris (France); Drabkin, Harry [Department of Medicine, Hematology Oncology Division, MUSC, 96 Jonathan Lucas St., Charleston, SC 29425 (United States)

    2013-04-03

    The epithelial to mesenchymal transition (EMT) enables epithelial cells with a migratory mesenchymal phenotype. It is activated in cancer cells and is involved in invasion, metastasis and stem-like properties. ZEB1, an E-box binding transcription factor, is a major suppressor of epithelial genes in lung cancer. In the present study, we show that in H358 non-small cell lung cancer cells, ZEB1 downregulates EpCAM (coding for an epithelial cell adhesion molecule), ESRP1 (epithelial splicing regulatory protein), ST14 (a membrane associated serine protease involved in HGF processing) and RAB25 (a small G-protein) by direct binding to these genes. Following ZEB1 induction, acetylation of histone H4 and histone H3 on lysine 9 (H3K9) and 27 (H3K27) was decreased on ZEB1 binding sites on these genes as demonstrated by chromatin immunoprecipitation. Of note, decreased H3K27 acetylation could be also detected by western blot and immunocytochemistry in ZEB1 induced cells. In lung cancers, H3K27 acetylation level was higher in the tumor compartment than in the corresponding stroma where ZEB1 was more often expressed. Since HDAC and DNA methylation inhibitors increased expression of ZEB1 target genes, targeting these epigenetic modifications would be expected to reduce metastasis.

  11. Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    David Reynolds

    2016-01-01

    Full Text Available Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In Trypanosoma brucei, J is enriched, along with histone H3 variant (H3.V, at sites involved in RNA Polymerase (RNAP II termination and telomeric sites involved in regulating variant surface glycoprotein gene (VSG transcription by RNAP I. Reduction of J in T. brucei indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the H3.V knockout. mRNA-seq indicates gene derepression includes VSG genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in T. brucei, depending on chromatin context (and transcribing polymerase. As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.

  12. Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex

    Directory of Open Access Journals (Sweden)

    Gottlieb Paul D

    2006-06-01

    Full Text Available Abstract Background Disrupting the balance of histone lysine methylation alters the expression of genes involved in tumorigenesis including proto-oncogenes and cell cycle regulators. Methylation of lysine residues is commonly catalyzed by a family of proteins that contain the SET domain. Here, we report the identification and characterization of the SET domain-containing protein, Smyd2. Results Smyd2 mRNA is most highly expressed in heart and brain tissue, as demonstrated by northern analysis and in situ hybridization. Over-expressed Smyd2 localizes to the cytoplasm and the nucleus in 293T cells. Although accumulating evidence suggests that methylation of histone 3, lysine 36 (H3K36 is associated with actively transcribed genes, we show that the SET domain of Smyd2 mediates H3K36 dimethylation and that Smyd2 represses transcription from an SV40-luciferase reporter. Smyd2 associates specifically with the Sin3A histone deacetylase complex, which was recently linked to H3K36 methylation within the coding regions of active genes in yeast. Finally, we report that exogenous expression of Smyd2 suppresses cell proliferation. Conclusion We propose that Sin3A-mediated deacetylation within the coding regions of active genes is directly linked to the histone methyltransferase activity of Smyd2. Moreover, Smyd2 appears to restrain cell proliferation, likely through direct modulation of chromatin structure.

  13. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

    Science.gov (United States)

    Rodriguez, Pedro; Rojas, Juan

    2016-03-01

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

  14. Identification and characterization of the Spodoptera Su(var 3-9 histone H3K9 trimethyltransferase and its effect in AcMNPV infection.

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    Binbin Li

    Full Text Available Histone H3-lysine(9 (H3K9 trimethyltransferase gene Su(var 3-9 was cloned and identified in three Spodoptera insects, Spodopterafrugiperda (S. frugiperda, S. exigua and S. litura. Sequence analysis showed that Spodoptera Su(var 3-9 is highly conserved evolutionarily. Su(var 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a and HP1b. A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C. Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation. In contrast, overexpression of Su(var 3-9 caused increased histone methylation level and cellular genome compaction. In AcMNV-infected Sf9 cells, the transcription of Su(var 3-9 increased at late time of infection, although the mRNA levels of most cellular genes decreased. Pre-treatment of Sf9 cells with chaetocin speeded up viral DNA replication, and increased the transcription level of a variety of virus genes, whereas in Sf9 cells pre-transformed with Su(var 3-9 expression vector, viral DNA replication slow down slightly. These findings suggest that Su(var 3-9 might participate in the viral genes expression an genome replication repression during AcMNPV infection. It provided a new insight for the understanding virus-host interaction mechanism.

  15. Archaeal histones: dynamic and versatile genome architects

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    Bram Henneman

    2015-12-01

    Full Text Available Genome organization and compaction in Archaea involves different chromatin proteins, among which homologues of eukaryotic histones. Archaeal histones are considered the ancestors of their eukaryotic counterparts, which isreflected in the way they position along the genome and wrap DNA. Evolution from the archaeal modes of action to the prototypical eukaryotic nucleosome may be attributed to altered histone-histone interactions and DNA sequence determinants cooperating to yield stable multimeric structures. The identification of a new candidate phylum, proposed to be a missing link between archaea and eukaryotes, Lokiarchaeaota, may be instrumental in addressing this hypothesis.

  16. Signalling couples hair follicle stem cell quiescence with reduced histone H3 K4/K9/K27me3 for proper tissue homeostasis.

    Science.gov (United States)

    Lee, Jayhun; Kang, Sangjo; Lilja, Karin C; Colletier, Keegan J; Scheitz, Cornelia Johanna Franziska; Zhang, Ying V; Tumbar, Tudorita

    2016-04-15

    Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

  17. H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing.

    Science.gov (United States)

    Rondinelli, Beatrice; Schwerer, Hélène; Antonini, Elena; Gaviraghi, Marco; Lupi, Alessio; Frenquelli, Michela; Cittaro, Davide; Segalla, Simona; Lemaitre, Jean-Marc; Tonon, Giovanni

    2015-03-11

    DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.

  18. Mutant versions of the S. cerevisiae transcription elongation factor Spt16 define regions of Spt16 that functionally interact with histone H3.

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    Catherine N Myers

    Full Text Available In eukaryotic cells, the highly conserved FACT (FAcilitates Chromatin Transcription complex plays important roles in several chromatin-based processes including transcription initiation and elongation. During transcription elongation, the FACT complex interacts directly with nucleosomes to facilitate histone removal upon RNA polymerase II (Pol II passage and assists in the reconstitution of nucleosomes following Pol II passage. Although the contribution of the FACT complex to the process of transcription elongation has been well established, the mechanisms that govern interactions between FACT and chromatin still remain to be fully elucidated. Using the budding yeast Saccharomyces cerevisiae as a model system, we provide evidence that the middle domain of the FACT subunit Spt16--the Spt16-M domain--is involved in functional interactions with histone H3. Our results show that the Spt16-M domain plays a role in the prevention of cryptic intragenic transcription during transcription elongation and also suggest that the Spt16-M domain has a function in regulating dissociation of Spt16 from chromatin at the end of the transcription process. We also provide evidence for a role for the extreme carboxy terminus of Spt16 in functional interactions with histone H3. Taken together, our studies point to previously undescribed roles for the Spt16 M-domain and extreme carboxy terminus in regulating interactions between Spt16 and chromatin during the process of transcription elongation.

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

    Science.gov (United States)

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

    2005-04-01

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

  20. Systems Level Analysis of Histone H3 Post-translational Modifications (PTMs) Reveals Features of PTM Crosstalk in Chromatin Regulation

    DEFF Research Database (Denmark)

    Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian

    2016-01-01

    molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse...

  1. Prokaryotic BirA ligase biotinylates K4, K9, K18 and K23 in eukaryotic histone H3

    Science.gov (United States)

    BirA ligase, a prokaryotic ortholog of human holocarboxylase synthetase (HCS), is known to biotinylate proteins. Here, we tested the hypothesis that BirA ligase may also catalyze biotinylation of eukaryotic histones. If so, this would render recombinant BirA ligase a useful surrogate for HCS in stud...

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

    Science.gov (United States)

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

    2002-08-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  4. Physical mapping of 18S and 5S rDNA loci and histone H3 gene in grasshopper species of the subfamily Gomphocerinae (Acrididae).

    Science.gov (United States)

    Silva-Neto, L C; Bernardino, A C S; Loreto, V; Moura, R C

    2015-11-25

    In this study, fluorescence in situ hybridization (FISH) analysis was used to determine and compare the numbers and chromosomal locations of two multigene families (rDNA and histone H3) in four Neotropical species of gomphocerine grasshoppers. FISH using the 18S rDNA probe identified a single site on the S9 chromosome of Amblytropidia sp and Cauratettix borelli, a single site on chromosome M6 of Compsacris pulcher, and two sites (chromosomes L1 and L2) in Orphulella punctata. By contrast, FISH with a 5S rDNA probe identified dispersion of this sequence in the genomes of the four species, with evidence of intraspecific variations. Amblytropidia sp had six to eight FISH signals on autosomal chromosomes, while C. pulcher exhibited a signal only on the M5 bivalent. The histone H3 gene was less variable and was restricted to a single pair in all species. The conservation of the numbers and locations of 18S rDNA and H3 genes in conjunction with data from the literature was useful for evaluating karyotype evolution in this subfamily. The variation in the number and sizes of 5S rDNA sites indicates a process of recent dispersion that might have been mediated by transposition.

  5. The histone H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers

    Science.gov (United States)

    Klein, Brianna J.; Piao, Lianhua; Xi, Yuanxin; Rincon-Arano, Hector; Rothbart, Scott B.; Peng, Danni; Wen, Hong; Larson, Connie; Zhang, Xi; Zheng, Xia; Cortazar, Michael A.; Peña, Pedro V.; Mangan, Anthony; Bentley, David L.; Strahl, Brian D.; Groudine, Mark; Li, Wei; Shi, Xiaobing; Kutateladze, Tatiana G.

    2014-01-01

    SUMMARY The histone lysine demethylase KDM5B regulates gene transcription and cell differentiation. It contains three PHD fingers, the biological roles of which remain elusive. Here, we show that the first PHD1 finger of KDM5B binds unmodified histone H3, whereas the third PHD3 finger prefers the trimethylated mark, H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for a set of genes. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with HDAC1 in gene repression. Compared with the estrogen receptor positive breast cancers, KDM5B is downregulated in the triple-negative breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion ability, and the PHD1-H3K4me0 interaction is important for inhibition of migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a novel multivalent mechanism for KDM5B-mediated transcriptional regulation. PMID:24412361

  6. Expression of p21WAF1 is related to acetylation of histone H3 in total chromatin in human colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Ying-Xuan Chen; Jing-Yuan Fang; Rong Lu; De-Kai Qiu

    2007-01-01

    AIM: To explore the relationship between acetylation of histone in total chromatin and p21WAF1 expression regulation in human colorectal carcinoma.METHODS: We analyzed the expression of tumor suppressor gene p21WAF1 mRNA by RT-PCR or realtime PCR in 33 samples of colorectal cancerous tissue,corresponding para-cancerous tissue and normal colorectal mucosa, and also examined the level of acetylated histone H3 in total chromatin using Western blotting.RESULTS: The expression level of p21WAF1 mRNA was significantly lower in colorectal cancerous tissue from 33 patients than in para-cancerous tissue and normal colorectal mucosa (2377.95 ± 865.80 vs 3216.58 ±1149.42 and 3541.61 ± 1433.17 respectively, P <0.01). In addition, when p21WAF1 mRNA expression was undectectable or at very low level (50% less than that in adjacent tissue and normal colorectal mucosa) in all tissues, the level of acetylated histone H3 in colorectal cancerous tissue was significantly lower than that in corresponding para-cancerous tissue and normal colorectal mucosa in five of seven (71.43%) cases. The transcriptional level of p21WAF1 in colorectal carcinoma might not be associated with its biological behaviors.CONCLUSION: The down-regulation of p21WAF1 transcription is involved in the tumorigenesis and development of colorectal carcinoma. The down-expression of p21WAF1 mRNA in colorectal carcinoma might be associated with histone hypoacetylation in chromatin but not with biological behaviors.

  7. Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

    NARCIS (Netherlands)

    Vlaming, Hanneke; van Welsem, Tibor; de Graaf, Erik L; Ontoso, David; Altelaar, A F Maarten; San-Segundo, Pedro A; Heck, Albert J R; van Leeuwen, Fred

    2014-01-01

    Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 a

  8. Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo (Corrigendum)

    NARCIS (Netherlands)

    Vlaming, Hanneke; van Welsem, Tibor; de Graaf, Erik L; Ontoso, David; Altelaar, A F Maarten; San-Segundo, Pedro A; Heck, Albert J R; van Leeuwen, Fred

    2014-01-01

    Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 a

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Jamy C.; Karpen, Gary H.

    2006-06-15

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

  11. Chromosomal mapping of rDNAs and H3 histone sequences in the grasshopper rhammatocerus brasiliensis (acrididae, gomphocerinae: extensive chromosomal dispersion and co-localization of 5S rDNA/H3 histone clusters in the A complement and B chromosome

    Directory of Open Access Journals (Sweden)

    Oliveira Nathalia L

    2011-11-01

    Full Text Available Abstract Background Supernumerary B chromosomes occur in addition to standard karyotype and have been described in about 15% of eukaryotes, being the repetitive DNAs the major component of these chromosomes, including in some cases the presence of multigene families. To advance in the understanding of chromosomal organization of multigene families and B chromosome structure and evolution, the distribution of rRNA and H3 histone genes were analyzed in the standard karyotype and B chromosome of three populations of the grasshopper Rhammatocerus brasiliensis. Results The location of major rDNA was coincident with the previous analysis for this species. On the other hand, the 5S rDNA mapped in almost all chromosomes of the standard complement (except in the pair 11 and in the B chromosome, showing a distinct result from other populations previously analyzed. Besides the spreading of 5S rDNA in the genome of R. brasiliensis it was also observed multiple sites for H3 histone genes, being located in the same chromosomal regions of 5S rDNAs, including the presence of the H3 gene in the B chromosome. Conclusions Due to the intense spreading of 5S rRNA and H3 histone genes in the genome of R. brasiliensis, their chromosomal distribution was not informative in the clarification of the origin of B elements. Our results indicate a linked organization for the 5S rRNA and H3 histone multigene families investigated in R. brasiliensis, reinforcing previous data concerning the association of both genes in some insect groups. The present findings contribute to understanding the organization/evolution of multigene families in the insect genomes.

  12. Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

    Science.gov (United States)

    Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

    2016-01-01

    A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico-identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

  13. Modeling cis-regulation with a compendium of genome-wide histone H3K27ac profiles.

    Science.gov (United States)

    Wang, Su; Zang, Chongzhi; Xiao, Tengfei; Fan, Jingyu; Mei, Shenglin; Qin, Qian; Wu, Qiu; Li, Xujuan; Xu, Kexin; He, Housheng Hansen; Brown, Myles; Meyer, Clifford A; Liu, X Shirley

    2016-10-01

    Model-based analysis of regulation of gene expression (MARGE) is a framework for interpreting the relationship between the H3K27ac chromatin environment and differentially expressed gene sets. The framework has three main functions: MARGE-potential, MARGE-express, and MARGE-cistrome. MARGE-potential defines a regulatory potential (RP) for each gene as the sum of H3K27ac ChIP-seq signals weighted by a function of genomic distance from the transcription start site. The MARGE framework includes a compendium of RPs derived from 365 human and 267 mouse H3K27ac ChIP-seq data sets. Relative RPs, scaled using this compendium, are superior to superenhancers in predicting BET (bromodomain and extraterminal domain) -inhibitor repressed genes. MARGE-express, which uses logistic regression to retrieve relevant H3K27ac profiles from the compendium to accurately model a query set of differentially expressed genes, was tested on 671 diverse gene sets from MSigDB. MARGE-cistrome adopts a novel semisupervised learning approach to identify cis-regulatory elements regulating a gene set. MARGE-cistrome exploits information from H3K27ac signal at DNase I hypersensitive sites identified from published human and mouse DNase-seq data. We tested the framework on newly generated RNA-seq and H3K27ac ChIP-seq profiles upon siRNA silencing of multiple transcriptional and epigenetic regulators in a prostate cancer cell line, LNCaP-abl. MARGE-cistrome can predict the binding sites of silenced transcription factors without matched H3K27ac ChIP-seq data. Even when the matching H3K27ac ChIP-seq profiles are available, MARGE leverages public H3K27ac profiles to enhance these data. This study demonstrates the advantage of integrating a large compendium of historical epigenetic data for genomic studies of transcriptional regulation. © 2016 Wang et al.; Published by Cold Spring Harbor Laboratory Press.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Genome-wide analysis of histone H3 acetylation patterns in AML identifies PRDX2 as an epigenetically silenced tumor suppressor gene

    DEFF Research Database (Denmark)

    Agrawal-Singh, Shuchi; Isken, Fabienne; Agelopoulos, Konstantin

    2012-01-01

    With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34+ progenitor cells. Importantly, core promoter regions tended......, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell...... as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML....

  16. ChIP-seq analysis of histone H3K9 trimethylation in peripheral blood mononuclear cells of membranous nephropathy patients

    Energy Technology Data Exchange (ETDEWEB)

    Sui, W.G. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); He, H.Y. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Yan, Q.; Chen, J.J. [Guangxi Key Laboratory of Metabolic Diseases Research, Nephrology Department, 181st Hospital, Guilin, Guangxi (China); Zhang, R.H. [The Life Science College, Guangxi Normal University, Guilin, Guangxi (China); Dai, Y. [Clinical Medical Research Center, The Second Clinical Medical College, Shenzhen People’s Hospital, Jinan University, Shenzhen, Guangdong (China)

    2013-12-12

    Membranous nephropathy (MN), characterized by the presence of diffuse thickening of the glomerular basement membrane and subepithelial in situ immune complex disposition, is the most common cause of idiopathic nephrotic syndrome in adults, with an incidence of 5-10 per million per year. A number of studies have confirmed the relevance of several experimental insights to the pathogenesis of human MN, but the specific biomarkers of MN have not been fully elucidated. As a result, our knowledge of the alterations in histone methylation in MN is unclear. We used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyze the variations in a methylated histone (H3K9me3) in peripheral blood mononuclear cells from 10 MN patients and 10 healthy subjects. There were 108 genes with significantly different expression in the MN patients compared with the normal controls. In MN patients, significantly increased activity was seen in 75 H3K9me3 genes, and decreased activity was seen in 33, compared with healthy subjects. Five positive genes, DiGeorge syndrome critical region gene 6 (DGCR6), sorting nexin 16 (SNX16), contactin 4 (CNTN4), baculoviral IAP repeat containing 3 (BIRC3), and baculoviral IAP repeat containing 2 (BIRC2), were selected and quantified. There were alterations of H3K9me3 in MN patients. These may be candidates to help explain pathogenesis in MN patients. Such novel findings show that H3K9me3 may be a potential biomarker or promising target for epigenetic-based MN therapies.

  17. MoSET1 (Histone H3K4 Methyltransferase in Magnaporthe oryzae Regulates Global Gene Expression during Infection-Related Morphogenesis.

    Directory of Open Access Journals (Sweden)

    Kieu Thi Minh Pham

    2015-07-01

    Full Text Available Here we report the genetic analyses of histone lysine methyltransferase (KMT genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1 impaired in histone H3 lysine 4 methylation (H3K4me showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3 and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1 Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2 In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3 Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes, and during infection-related morphogenesis (1,385 genes, indicating that MoSET1 has a role in gene repression either

  18. Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje

    2010-01-01

    . The altered genomic regions showed an overrepresentation of cis-binding sites for ets and c-AMP response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML...

  19. Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading.

    Directory of Open Access Journals (Sweden)

    Danilo Bueno

    Full Text Available Supernumerary chromosomes (B chromosomes occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C0t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.

  20. The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Mesenchymal stem cells (MSCs) of nonembryortic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem ceils, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide historic H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways,cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.

  1. Histone H3.3 promotes IgV gene diversification by enhancing formation of AID-accessible single-stranded DNA.

    Science.gov (United States)

    Romanello, Marina; Schiavone, Davide; Frey, Alexander; Sale, Julian E

    2016-07-01

    Immunoglobulin diversification is driven by activation-induced deaminase (AID), which converts cytidine to uracil within the Ig variable (IgV) regions. Central to the recruitment of AID to the IgV genes are factors that regulate the generation of single-stranded DNA (ssDNA), the enzymatic substrate of AID Here, we report that chicken DT40 cells lacking variant histone H3.3 exhibit reduced IgV sequence diversification. We show that this results from impairment of the ability of AID to access the IgV genes due to reduced formation of ssDNA during IgV transcription. Loss of H3.3 also diminishes IgV R-loop formation. However, reducing IgV R-loops by RNase HI overexpression in wild-type cells does not affect IgV diversification, showing that these structures are not necessary intermediates for AID access. Importantly, the reduction in the formation of AID-accessible ssDNA in cells lacking H3.3 is independent of any effect on the level of transcription or the kinetics of RNAPII elongation, suggesting the presence of H3.3 in the nucleosomes of the IgV genes increases the chances of the IgV DNA becoming single-stranded, thereby creating an effective AID substrate.

  2. Polycomb CBX7 directly controls trimethylation of histone H3 at lysine 9 at the p16 locus.

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    Qiang Li

    Full Text Available BACKGROUND: H3K9 trimethylation (H3K9me3 and binding of PcG repressor complex-1 (PRC1 may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD. CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC. Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter.

  3. Polycomb CBX7 directly controls trimethylation of histone H3 at lysine 9 at the p16 locus.

    Science.gov (United States)

    Li, Qiang; Wang, Xiuhong; Lu, Zheming; Zhang, Baozhen; Guan, Zhenpo; Liu, Zhaojun; Zhong, Qiming; Gu, Liankun; Zhou, Jing; Zhu, Budong; Ji, Jiafu; Deng, Dajun

    2010-10-29

    H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter.

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

    Science.gov (United States)

    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.

  5. Interaction of HP1 and Brg1/Brm with the globular domain of histone H3 is required for HP1-mediated repression.

    Directory of Open Access Journals (Sweden)

    Marc Lavigne

    2009-12-01

    Full Text Available The heterochromatin-enriched HP1 proteins play a critical role in regulation of transcription. These proteins contain two related domains known as the chromo- and the chromoshadow-domain. The chromo-domain binds histone H3 tails methylated on lysine 9. However, in vivo and in vitro experiments have shown that the affinity of HP1 proteins to native methylated chromatin is relatively poor and that the opening of chromatin occurring during DNA replication facilitates their binding to nucleosomes. These observations prompted us to investigate whether HP1 proteins have additional histone binding activities, envisioning also affinity for regions potentially occluded by the nucleosome structure. We find that the chromoshadow-domain interacts with histone H3 in a region located partially inside the nucleosomal barrel at the entry/exit point of the nucleosome. Interestingly, this region is also contacted by the catalytic subunits of the human SWI/SNF complex. In vitro, efficient SWI/SNF remodeling requires this contact and is inhibited in the presence of HP1 proteins. The antagonism between SWI/SNF and HP1 proteins is also observed in vivo on a series of interferon-regulated genes. Finally, we show that SWI/SNF activity favors loading of HP1 proteins to chromatin both in vivo and in vitro. Altogether, our data suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin and that HP1 proteins use this property to detect and arrest unwanted chromatin remodeling.

  6. Altered expression of BRG1 and histone demethylases, and aberrant H3K4 methylation in less developmentally competent embryos at the time of embryonic genome activation.

    Science.gov (United States)

    Glanzner, Werner G; Wachter, Audrey; Coutinho, Ana Rita S; Albornoz, Marcelo S; Duggavathi, Raj; GonÇAlves, Paulo B D; Bordignon, Vilceu

    2017-01-01

    Epigenetics is a fundamental regulator underlying many biological functions, such as development and cell differentiation. Epigenetic modifications affect key chromatin regulation, including transcription and DNA repair, which are critical for normal embryo development. In this study, we profiled the expression of epigenetic modifiers and patterns of epigenetic changes in porcine embryos around the period of embryonic genome activation (EGA). We observed that Brahma-related gene 1 (BRG1) and Lysine demethylase 1A (KDM1A), which can alter the methylation status of lysine 4 in histone 3 (H3K4), localize to the nucleus at Day 3-4 of development. We then compared the abundance of epigenetic modifiers between early- and late-cleaving embryos, which were classified based on the time to the first cell cleavage, to investigate if their nuclear localization contributes to developmental competence. The mRNA abundance of BRG1, KDM1A, as well as other lysine demethylases (KDM1B, KDM5A, KDM5B, and KDM5C), were significantly higher in late- compared to early-cleaving embryos near the EGA period, although these difference disappeared at the blastocyst stage. The abundance of H3K4 mono- (H3K4me) and di-methylation (H3K4me2) during the EGA period was reduced in late-cleaving and less developmentally competent embryos. By contrast, BRG1, KDM1A, and H3K4me2 abundance was greater in embryos with more than eight cells at Day 3-4 of development compared to those with fewer than four cells. These findings suggest that altered epigenetic modifications of H3K4 around the EGA period may affect the developmental capacity of porcine embryos to reach the blastocyst stage. Mol. Reprod. Dev. 84: 19-29, 2017. © 2016 Wiley Periodicals, Inc.

  7. Histone H3 Methyltransferase Suv39h1 Prevents Myogenic Terminal Differentiation by Repressing MEF2 Activity in Muscle Cells

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    Wei Jin

    2016-11-01

    Full Text Available The myogenic regulatory factors (MRFs and myocyte enhancer factor 2 (MEF2 transcription factors have been extensively studied as key transcription factors that regulate myogenic gene expression. However, few reports on the molecular mechanism that modulates chromatin remodeling during skeletal muscle differentiation are available. We reported here that the expression of the H3-K9 methyltransferase Suv39h1 was decreased during myoblast differentiation. Ectopic expression of Suv39h1 could inhibit myoblast differentiation, increasing H3-K9 methylation levels, whereas knockdown of Suv39h1 stimulated myoblast differentiation. Furthermore, Suv39h1 interacted with MEF2C directly and inhibited MEF2 transcription activity in a dose-dependent manner. Together, our studies revealed a molecular mechanism wherein Suv39h1 modulated myogenic gene expression and activation during skeletal muscle differentiation.

  8. Chromosomal mapping of H3 histone and 5S rRNA genes in eight species of Astyanax (Pisces, Characiformes) with different diploid numbers: syntenic conservation of repetitive genes.

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    Piscor, Diovani; Parise-Maltempi, Patricia Pasquali

    2016-03-01

    The genus Astyanax is widely distributed from the southern United States to northern Patagonia, Argentina. While cytogenetic studies have been performed for this genus, little is known about the histone gene families. The aim of this study was to examine the chromosomal relationships among the different species of Astyanax. The chromosomal locations of the 5S rRNA and H3 histone genes were determined in A. abramis, A. asuncionensis, A. altiparanae, A. bockmanni, A. eigenmanniorum, A. mexicanus (all 2n = 50), A. fasciatus (2n = 46), and A. schubarti (2n = 36). All eight species exhibited H3 histone clusters on two chromosome pairs. In six species (A. abramis, A. asuncionensis, A. altiparanae, A. bockmanni, A. eigenmanniorum, and A. fasciatus), syntenic clusters of H3 histone and 5S rDNA were observed on metacentric (m) or submetacentric (sm) chromosomes. In seven species, clusters of 5S rDNA sequences were located on one or two chromosome pairs. In A. mexicanus, 5S rDNA clusters were located on four chromosome pairs. This study demonstrates that H3 histone clusters are conserved on two chromosome pairs in the genus Astyanax, and specific chromosomal features may contribute to the genomic organization of the H3 histone and 5S rRNA genes.

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

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

    2002-03-01

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

  10. The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

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    Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

    2014-08-01

    Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast.

  11. The relationship between lysine 4 on histone H3 methylation levels of alcohol tolerance genes and changes of ethanol tolerance in Saccharomyces cerevisiae.

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    Wang, Hang; Ji, Binfeng; Ren, Hongzhen; Meng, Chun

    2014-07-01

    We evaluated whether epigenetic changes contributed to improve ethanol tolerance in mutant populations of Saccharomyces cerevisiae (S. cerevisiae). Two ethanol-tolerant variants of S. cerevisiae were used to evaluate the genetic stability in the process of stress-free passage cultures. We found that acquired ethanol tolerance was lost and transcription level of some genes (HSP104, PRO1, TPS1, and SOD1) closely related to ethanol tolerance decreased significantly after the 10th passage in ethanol-free medium. Tri-methylation of lysine 4 on histone H3 (H3K4) enhanced at the promoter of HSP104, PRO1, TPS1 and SOD1 in ethanol-tolerant variants of S. cerevisiae was also diminished after tenth passage in stress-free cultures. The ethanol tolerance was reacquired when exogenous SOD1 transferred in some tolerance-lost strains. This showed that H3K4 methylation is involved in phenotypic variation with regard to ethanol tolerance with respect to classic breeding methods used in yeast. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  12. Spp1, a member of the Set1 Complex, promotes meiotic DSB formation in promoters by tethering histone H3K4 methylation sites to chromosome axes.

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    Sommermeyer, Vérane; Béneut, Claire; Chaplais, Emmanuel; Serrentino, Maria Elisabetta; Borde, Valérie

    2013-01-10

    Meiotic chromosomes are organized into arrays of loops that are anchored to the chromosome axis structure. Programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination, catalyzed by Spo11 and accessory DSB proteins, form in loop sequences in promoters, whereas the DSB proteins are located on chromosome axes. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes during meiosis, where it physically interacts with the Mer2 DSB protein. The PHD finger module of Spp1, which reads H3K4 methylation close to promoters, promotes DSB formation by tethering these regions to chromosome axes and activating cleavage by the DSB proteins. This paper provides the molecular mechanism linking DSB sequences to chromosome axes and explains why H3K4 methylation is important for meiotic recombination.

  13. Increased expression of the histone H3 lysine 4 methyltransferase MLL4 and the histone H3 lysine 27 demethylase UTX prolonging the overall survival of patients with glioblastoma and a methylated MGMT promoter.

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    Kim, Jinho; Lee, Sung-Hun; Jang, Ji Hwan; Kim, Mee-Seon; Lee, Eun Hee; Kim, Young Zoon

    2016-07-01

    OBJECTIVE The purpose of the present study was to investigate the epigenetic and prognostic roles of an H3K4 methyltransferase (mixed lineage leukemia 4 [MLL4]) and H3K27 demethylase (ubiquitously transcribed tetratricopeptide repeat gene on X chromosome [UTX]) in progression-free survival (PFS) and overall survival (OS) of patients with glioblastoma (GBM) who were treated with radiotherapy, chemotherapy, or both after resection. In addition, the authors examined methylation at the promoter of the O-6-methylguanine-DNA methyltransferase (MGMT) gene and other prognostic factors predicting length of PFS and OS in these patients. METHODS The medical records of 76 patients having a new diagnosis of histologically ascertained GBM in the period of January 2002 to December 2013 at the authors' institution were retrospectively reviewed. Immunohistochemical staining for MLL4 and UTX was performed on archived paraffin-embedded tissues obtained by biopsy or resection. The methylation status of the MGMT promoter in these tissues was determined by methylation-specific PCR analysis. RESULTS During the follow-up period (mean length 18.1 months, range 4.1-43.5 months), 68 (89.5%) of the patients died. The MGMT promoter was methylated in 49 patients (64.5%) and unmethylated in 27 (35.5%). The immunoreactivity pattern of UTX was identical to that of MLL4; increased expression of these 2 proteins was observed in samples from 34 patients (44.7%) and decreased expression in 42 patients (55.3%). The mean length of PFS was 9.2 months (95% CI 6.8-11.6 months). Extent of surgery, recursive partitioning analysis (RPA) class, and methylation status of the MGMT promoter were all associated with increased PFS in the multivariate analysis of factors predicting PFS. The mean length of OS was 18.6 months (95% CI 14.3-22.9 months). Patient age (p = 0.004), WHO performance status score (p = 0.019), extent of surgery (p = 0.007), RPA class (p = 0.036), methylation status of the MGMT promoter (p = 0

  14. Chromatin-modifying complex component Nurf55/p55 associates with histones H3 and H4 and polycomb repressive complex 2 subunit Su(z)12 through partially overlapping binding sites.

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    Nowak, Agnieszka J; Alfieri, Claudio; Stirnimann, Christian U; Rybin, Vladimir; Baudin, Florence; Ly-Hartig, Nga; Lindner, Doris; Müller, Christoph W

    2011-07-01

    Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

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

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

    2011-11-01

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

  16. Structure and carboxyl-terminal domain (CTD) binding of the Set2 SRI domain that couples histone H3 Lys36 methylation to transcription.

    Science.gov (United States)

    Vojnic, Erika; Simon, Bernd; Strahl, Brian D; Sattler, Michael; Cramer, Patrick

    2006-01-06

    During mRNA elongation, the SRI domain of the histone H3 methyltransferase Set2 binds to the phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. The solution structure of the yeast Set2 SRI domain reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. NMR titration shows that the SRI domain binds an Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues, whereas a single CTD repeat is insufficient for binding. Residues that show strong chemical shift perturbations upon CTD binding cluster in two regions. Both CTD tyrosine side chains contact the SRI domain. One of the tyrosines binds in the region with the strongest chemical shift perturbations, formed by the two NH2-terminal helices. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in bacterial sigma factors (domain sigma2 in sigma70).

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-04-26

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

  18. Genome-wide quantitative analysis of histone H3 lysine 4 trimethylation in wild house mouse liver: environmental change causes epigenetic plasticity.

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    Angelika G Börsch-Haubold

    Full Text Available In mammals, exposure to toxic or disease-causing environments can change epigenetic marks that are inherited independently of the intrauterine environment. Such inheritance of molecular phenotypes may be adaptive. However, studies demonstrating molecular evidence for epigenetic inheritance have so far relied on extreme treatments, and are confined to inbred animals. We therefore investigated whether epigenomic changes could be detected after a non-drastic change in the environment of an outbred organism. We kept two populations of wild-caught house mice (Mus musculus domesticus for several generations in semi-natural enclosures on either standard diet and light cycle, or on an energy-enriched diet with longer daylight to simulate summer. As epigenetic marker for active chromatin we quantified genome-wide histone-3 lysine-4 trimethylation (H3K4me3 from liver samples by chromatin immunoprecipitation and high-throughput sequencing as well as by quantitative polymerase chain reaction. The treatment caused a significant increase of H3K4me3 at metabolic genes such as lipid and cholesterol regulators, monooxygenases, and a bile acid transporter. In addition, genes involved in immune processes, cell cycle, and transcription and translation processes were also differently marked. When we transferred young mice of both populations to cages and bred them under standard conditions, most of the H3K4me3 differences were lost. The few loci with stable H3K4me3 changes did not cluster in metabolic functional categories. This is, to our knowledge, the first quantitative study of an epigenetic marker in an outbred mammalian organism. We demonstrate genome-wide epigenetic plasticity in response to a realistic environmental stimulus. In contrast to disease models, the bulk of the epigenomic changes we observed were not heritable.

  19. Alcohol-induced histone H3K9 hyperacetylation and cardiac hypertrophy are reversed by a histone acetylases inhibitor anacardic acid in developing murine hearts.

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    Peng, Chang; Zhang, Weihua; Zhao, Weian; Zhu, Jing; Huang, Xupei; Tian, Jie

    2015-06-01

    The expression of cardiac genes is precisely regulated, and any perturbation may cause developmental defects. In a previous study, we demonstrated that alcohol consumption during pregnancy could lead to uncontrolled expressions of cardiac genes and eventually result in cardiac dysplasia. However, the underlying mechanisms remain unclear. In the present study, we have investigated the alcohol-induced cardiac hypertrophy and its potential mechanisms. Furthermore, the protective effect of anacardic acid against the alcohol-induced cardiac hypertrophy has been explored in experimental mice. C57BL/6 pregnant mice were gavaged with 56% ethanol or saline and the hearts of their fetus were collected for analysis. Binding of p300, CBP, PCAF, SRC1, except GCN5, were increased to the NKX2.5 promoter in fetal mouse hearts exposed to alcohol. Increased acetylation of H3K9 and increased mRNA expression of NKX2.5, β-MHC and Cx43 were observed in the same samples. Treatment with a pan-acetylase inhibitor, anacardic acid, reduced the binding affinity of p300 and PCAF to the NKX2.5, β-MHC, Cx43 promoters and attenuated H3K9 hyperacetylation. Interestingly, anacardic acid down-regulated over-expression of these cardiac genes induced by alcohol and ultimately attenuated ethanol-induced cardiac hypertrophy in fetal mice. Our results indicate that alcohol exposure during pregnancy could lead to fetal cardiac hypertrophy. The over-expression of NKX2.5, β-MHC, Cx43 mediated by p300 and PCAF may be critical mechanisms of alcohol-induced cardiac hypertrophy. Anacardic acid can down-regulate the over-expression of cardiac genes and reverse cardiac hypertrophy caused by alcohol treatment in pregnant mice, suggesting it could be a potential therapeutic agent for the treatment of cardiac hypertrophy. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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

    Science.gov (United States)

    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

  1. Chromatin dynamics: interplay between remodeling enzymes and histone modifications.

    Science.gov (United States)

    Swygert, Sarah G; Peterson, Craig L

    2014-08-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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    Pavel Burda

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation.

    Science.gov (United States)

    Hacker, Kathryn E; Fahey, Catherine C; Shinsky, Stephen A; Chiang, Yun-Chen J; DiFiore, Julia V; Jha, Deepak Kumar; Vo, Andy H; Shavit, Jordan A; Davis, Ian J; Strahl, Brian D; Rathmell, W Kimryn

    2016-09-30

    The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

  5. Specific loss of histone H3 lysine 9 trimethylation and HP1gamma/cohesin binding at D4Z4 repeats is associated with facioscapulohumeral dystrophy (FSHD.

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    Weihua Zeng

    2009-07-01

    Full Text Available Facioscapulohumeral dystrophy (FSHD is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed "phenotypic" FSHD, and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4-specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA-treated cells. We found that SUV39H1-mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1gamma and cohesin are co-recruited to D4Z4 in an H3K9me3-dependent and cell type-specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type-specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1gamma/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.

  6. Evolutionarily conserved histone methylation dynamics during seed life-cycle transitions.

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    Kerstin Müller

    Full Text Available Plants have a remarkable ability to react to seasonal changes by synchronizing life-cycle transitions with environmental conditions. We addressed the question of how transcriptional re-programming occurs in response to an environmental cue that triggers the major life cycle transition from seed dormancy to germination and seedling growth. We elucidated an important mechanistic aspect of this process by following the chromatin dynamics of key regulatory genes with a focus on the two antagonistic marks, H3K4me3 and H3K27me3. Histone methylation patterns of major dormancy regulators changed during the transition to germination and seedling growth. We observed a switch from H3K4me3 and high transcription levels to silencing by the repressive H3K27me3 mark when dormancy was broken through exposure to moist chilling, underscoring that a functional PRC2 complex is necessary for this transition. Moreover, this reciprocal regulation by H3K4me3 and H3K27me3 is evolutionarily conserved from gymnosperms to angiosperms.

  7. The expression patterns and correlations of claudin-6, methy-CpG binding protein 2, DNA methyltransferase 1, histone deacetylase 1, acetyl-histone H3 and acetyl-histone H4 and their clinicopathological significance in breast invasive ductal carcinomas

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    Xu Xiaoming

    2012-03-01

    Full Text Available Abstract Background Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2 and DNA methyltransferase 1 (DNMT1 and histone modification associated proteins (histone deacetylase 1 (HDAC1, acetyl-histone H3 (H3Ac and acetyl- histone H4 (H4Ac. Methods We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry. Results Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P P P = 0.001, HDAC1 (P P = 0.004 expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021. Increased expression of HDAC1 was correlated with histological grade (P P = 0.004, clinical stage (P = 0.007 and lymph node metastasis (P = 0.001. H3Ac expression was associated with tumor size (P = 0.044 and clinical stage of cancers (P = 0.034. MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05. We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression. Conclusions Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1 or histone modification associated proteins (HDAC1, H3Ac, H4Ac. Interestingly, the

  8. REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation.

    Science.gov (United States)

    Baiula, Monica; Carbonari, Gioia; Dattoli, Samantha D; Calienni, Maria; Bedini, Andrea; Spampinato, Santi

    2012-08-01

    REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-X(L) whereas in HeLa cells overexpressing REST, the reduction of Bcl-X(L) mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.

  9. The histone H3K36 methyltransferase MES-4 acts epigenetically to transmit the memory of germline gene expression to progeny.

    Directory of Open Access Journals (Sweden)

    Andreas Rechtsteiner

    2010-09-01

    Full Text Available Methylation of histone H3K36 in higher eukaryotes is mediated by multiple methyltransferases. Set2-related H3K36 methyltransferases are targeted to genes by association with RNA Polymerase II and are involved in preventing aberrant transcription initiation within the body of genes. The targeting and roles of the NSD family of mammalian H3K36 methyltransferases, known to be involved in human developmental disorders and oncogenesis, are not known. We used genome-wide chromatin immunoprecipitation (ChIP to investigate the targeting and roles of the Caenorhabditis elegans NSD homolog MES-4, which is maternally provided to progeny and is required for the survival of nascent germ cells. ChIP analysis in early C. elegans embryos revealed that, consistent with immunostaining results, MES-4 binding sites are concentrated on the autosomes and the leftmost approximately 2% (300 kb of the X chromosome. MES-4 overlies the coding regions of approximately 5,000 genes, with a modest elevation in the 5' regions of gene bodies. Although MES-4 is generally found over Pol II-bound genes, analysis of gene sets with different temporal-spatial patterns of expression revealed that Pol II association with genes is neither necessary nor sufficient to recruit MES-4. In early embryos, MES-4 associates with genes that were previously expressed in the maternal germ line, an interaction that does not require continued association of Pol II with those loci. Conversely, Pol II association with genes newly expressed in embryos does not lead to recruitment of MES-4 to those genes. These and other findings suggest that MES-4, and perhaps the related mammalian NSD proteins, provide an epigenetic function for H3K36 methylation that is novel and likely to be unrelated to ongoing transcription. We propose that MES-4 transmits the memory of gene expression in the parental germ line to offspring and that this memory role is critical for the PGCs to execute a proper germline program.

  10. The histone H3K36 methyltransferase MES-4 acts epigenetically to transmit the memory of germline gene expression to progeny.

    Directory of Open Access Journals (Sweden)

    Andreas Rechtsteiner

    2010-09-01

    Full Text Available Methylation of histone H3K36 in higher eukaryotes is mediated by multiple methyltransferases. Set2-related H3K36 methyltransferases are targeted to genes by association with RNA Polymerase II and are involved in preventing aberrant transcription initiation within the body of genes. The targeting and roles of the NSD family of mammalian H3K36 methyltransferases, known to be involved in human developmental disorders and oncogenesis, are not known. We used genome-wide chromatin immunoprecipitation (ChIP to investigate the targeting and roles of the Caenorhabditis elegans NSD homolog MES-4, which is maternally provided to progeny and is required for the survival of nascent germ cells. ChIP analysis in early C. elegans embryos revealed that, consistent with immunostaining results, MES-4 binding sites are concentrated on the autosomes and the leftmost approximately 2% (300 kb of the X chromosome. MES-4 overlies the coding regions of approximately 5,000 genes, with a modest elevation in the 5' regions of gene bodies. Although MES-4 is generally found over Pol II-bound genes, analysis of gene sets with different temporal-spatial patterns of expression revealed that Pol II association with genes is neither necessary nor sufficient to recruit MES-4. In early embryos, MES-4 associates with genes that were previously expressed in the maternal germ line, an interaction that does not require continued association of Pol II with those loci. Conversely, Pol II association with genes newly expressed in embryos does not lead to recruitment of MES-4 to those genes. These and other findings suggest that MES-4, and perhaps the related mammalian NSD proteins, provide an epigenetic function for H3K36 methylation that is novel and likely to be unrelated to ongoing transcription. We propose that MES-4 transmits the memory of gene expression in the parental germ line to offspring and that this memory role is critical for the PGCs to execute a proper germline program.

  11. Histone Methylation Dynamics and Gene Regulation Occur through the Sensing of One-Carbon Metabolism.

    Science.gov (United States)

    Mentch, Samantha J; Mehrmohamadi, Mahya; Huang, Lei; Liu, Xiaojing; Gupta, Diwakar; Mattocks, Dwight; Gómez Padilla, Paola; Ables, Gene; Bamman, Marcas M; Thalacker-Mercer, Anna E; Nichenametla, Sailendra N; Locasale, Jason W

    2015-11-01

    S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) link one-carbon metabolism to methylation status. However, it is unknown whether regulation of SAM and SAH by nutrient availability can be directly sensed to alter the kinetics of key histone methylation marks. We provide evidence that the status of methionine metabolism is sufficient to determine levels of histone methylation by modulating SAM and SAH. This dynamic interaction led to rapid changes in H3K4me3, altered gene transcription, provided feedback regulation to one-carbon metabolism, and could be fully recovered upon restoration of methionine. Modulation of methionine in diet led to changes in metabolism and histone methylation in the liver. In humans, methionine variability in fasting serum was commensurate with concentrations needed for these dynamics and could be partly explained by diet. Together these findings demonstrate that flux through methionine metabolism and the sensing of methionine availability may allow direct communication to the chromatin state in cells.

  12. Rbm15-Mkl1 interacts with the Setd1b histone H3-Lys4 methyltransferase via a SPOC domain that is required for cytokine-independent proliferation.

    Directory of Open Access Journals (Sweden)

    Jeong-Heon Lee

    Full Text Available The Rbm15-Mkl1 fusion protein is associated with acute megakaryoblastic leukemia (AMKL, although little is known regarding the molecular mechanism(s whereby this fusion protein contributes to leukemogenesis. Here, we show that both Rbm15 and the leukemogenic Rbm15-Mkl1 fusion protein interact with the Setd1b histone H3-Lys4 methyltransferase (also known as KMT2G. This interaction is direct and requires the Rbm15 SPOC domain and the Setd1b LSD motif. Over-expression of Rbm15-Mkl1 in the 6133 megakaryoblastic leukemia cell line, previously established by expression of the Rbm15-Mkl1 fusion protein in mice (Mercher et al., [2009] J. Clin. Invest. 119, 852-864, leads to decreased levels of endogenous Rbm15 and increased levels of endogenous Mkl1. These cells exhibit enhanced proliferation and cytokine-independent cell growth, which requires an intact Rbm15 SPOC domain that mediates interaction between the Rbm15-Mkl1 fusion protein and the Setd1b methyltransferase. These results reveal altered Setd1b complex function and consequent altered epigenetic regulation as a possible molecular mechanism that mediates the leukemogenic activity of the Rbm15-Mkl1 fusion protein in AMKL.

  13. Rbm15-Mkl1 interacts with the Setd1b histone H3-Lys4 methyltransferase via a SPOC domain that is required for cytokine-independent proliferation.

    Science.gov (United States)

    Lee, Jeong-Heon; Skalnik, David G

    2012-01-01

    The Rbm15-Mkl1 fusion protein is associated with acute megakaryoblastic leukemia (AMKL), although little is known regarding the molecular mechanism(s) whereby this fusion protein contributes to leukemogenesis. Here, we show that both Rbm15 and the leukemogenic Rbm15-Mkl1 fusion protein interact with the Setd1b histone H3-Lys4 methyltransferase (also known as KMT2G). This interaction is direct and requires the Rbm15 SPOC domain and the Setd1b LSD motif. Over-expression of Rbm15-Mkl1 in the 6133 megakaryoblastic leukemia cell line, previously established by expression of the Rbm15-Mkl1 fusion protein in mice (Mercher et al., [2009] J. Clin. Invest. 119, 852-864), leads to decreased levels of endogenous Rbm15 and increased levels of endogenous Mkl1. These cells exhibit enhanced proliferation and cytokine-independent cell growth, which requires an intact Rbm15 SPOC domain that mediates interaction between the Rbm15-Mkl1 fusion protein and the Setd1b methyltransferase. These results reveal altered Setd1b complex function and consequent altered epigenetic regulation as a possible molecular mechanism that mediates the leukemogenic activity of the Rbm15-Mkl1 fusion protein in AMKL.

  14. Recent relaxation of purifying selection on the tandem-repetitive early-stage histone H3 gene in brooding sea stars.

    Science.gov (United States)

    Foltz, David W; Mah, Christopher L

    2009-06-01

    Patterns of nucleotide substitution differ between marine species that have a pelagic feeding (planktotrophic) larval stage and related species that lack such a stage, for both adaptive and non-adaptive reasons. Here, patterns of nucleotide and inferred amino acid substitution are analyzed for the tandem-repetitive early-stage histone H3 gene in 36 sea star species of the order Forcipulatida with documented larval habitat. The relative rate of nonsynonymous substitution (expressed as ω=d(N)/d(S)) was significantly higher in lineages with a brooded non-feeding (lecithotrophic) larval form than in lineages with a planktotrophic larval form. There was also a significant excess of conservative over radical substitutions. The increase in ω for brooders as compared to non-brooders was much greater than for previously analyzed mitochondrial sequences in echinoderms. These data are consistent with the hypothesis that purifying selection on this gene has been relaxed in brooding lineages compared to non-brooding lineages. The hypotheses of adaptive or neutral evolution are less plausible, although recent pseudogenization following a period of relaxed purifying selection could also explain the results.

  15. 5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

    Science.gov (United States)

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

    2007-01-04

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Si, Lina; Shi, Jin; Gao, Wenqun [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Zheng, Min [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Liu, Lingjuan; Zhu, Jing [Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China); Tian, Jie, E-mail: jietian@cqmu.edu.cn [Heart Centre, Children’s Hospital of Chongqing Medical University, 136 Zhongshan 2nd Road, Yu Zhong District, Chongqing 400014 (China)

    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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Dynamic Regulation and Function of Histone Monoubiquitination in Plants

    Directory of Open Access Journals (Sweden)

    Jing eFeng

    2014-03-01

    Full Text Available Polyubiquitin chain deposition on a target protein frequently leads to proteasome-mediated degradation whereas monoubiquitination modifies target protein property and function independent of proteolysis. Histone monoubiquitination occurs in chromatin and is in nowadays recognized as one critical type of epigenetic marks in eukaryotes. While H2A monoubiquitination (H2Aub1 is generally associated with transcription repression mediated by the Polycomb pathway, H2Bub1 is involved in transcription activation. H2Aub1 and H2Bub1 levels are dynamically regulated via deposition and removal by specific enzymes. We review knows and unknowns of dynamic regulation of H2Aub1 and H2Bub1 deposition and removal in plants and highlight the underlying crucial functions in gene transcription, cell proliferation/differentiation, and plant growth and development. We also discuss crosstalks existing between H2Aub1 or H2Bub1 and different histone methylations for an ample mechanistic understanding.

  20. Epigenetic regulation: methylation of histone and non-histone proteins

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Histone methylation is believed to play important roles in epigenetic memory in various biological processes. However, questions like whether the methylation marks themselves are faithfully transmit- ted into daughter cells and through what mechanisms are currently under active investigation. Previ- ously, methylation was considered to be irreversible, but the recent discovery of histone lysine de- methylases revealed a dynamic nature of histone methylation regulation on four of the main sites of methylation on histone H3 and H4 tails (H3K4, H3K9, H3K27 and H3K36). Even so, it is still unclear whether demethylases specific for the remaining two sites, H3K79 and H4K20, exist. Furthermore, be- sides histone proteins, the lysine methylation and demethylation also occur on non-histone proteins, which are probably subjected to similar regulation as histones. This review discusses recent pro- gresses in protein lysine methylation regulation focusing on the above topics, while referring readers to a number of recent reviews for the biochemistry and biology of these enzymes.

  1. Vanadate triggers the transition from chromosome condensation to decondensation in a mitotic mutant (tsTM13) inactivation of p34cdc2/H1 kinase and dephosphorylation of mitosis-specific histone H3.

    Science.gov (United States)

    Ajiro, K; Yasuda, H; Tsuji, H

    1996-11-01

    At the nonpermissive temperature (39 degrees C), chromosomes remain condensed in a temperature-sensitive cell mutant (tsTM13) arrested in the late stage of mitosis. Highly increased activity of histone H1 kinase, hyperphosphorylation of histone H1, and mitosis-specific histone H3 phosphorylation are maintained, even in telophase. In the present study, the defect of chromosome decondensation in tsTM13 cells was found to be partially normalized by a tyrosine phosphatase inhibitor, vanadate, with induction of chromosome decondensation and the formation of multinucleated cells. In the presence of vanadate, the H1 kinase activity dropped to near normal levels and the amount of the inactive from of p34cdc2 protein phosphorylated at a tyrosine residue was increased. H1 and H3 were also extensively de- phosphorylated, the latter being tightly associated with chromosome decondensation. Serine/threonine-protein phosphatase in late mitosis of the mutant works normally at 39 degrees C. The results indicate that (a) the genetic defect in the mutant may be involved in the control mechanism of the p34cdc2/H1 kinase activity in the late M phase rather than the phosphatase, (b) normalization of the defect of the mutant by vanadate results from inactivation of H1 kinase, and (c) late mitosis-specific events (p34cdc2/H1 kinase inactivation, mitosis-specific dephosphorylation of histone H1 and H3) are closely operating with chromosome decondensation.

  2. Histone H3 lysine 27 and 9 hypermethylation within the Bad promoter region mediates 5-Aza-2'-deoxycytidine-induced Leydig cell apoptosis: implications of 5-Aza-2'-deoxycytidine toxicity to male reproduction.

    Science.gov (United States)

    Choi, Ji-Young; Lee, Sangmi; Hwang, Soojin; Jo, Sangmee Ahn; Kim, Miji; Kim, Young Ju; Pang, Myung-Geol; Jo, Inho

    2013-01-01

    5-Aza-2'-deoxycitidine (5-Aza), an anticancer agent, results in substantial toxicity to male reproduction, causing a decline in sperm quality associated with reduced testosterone. Here, we report that 5-Aza increased the apoptotic protein Bad epigenetically in the testosterone-producing mouse TM3 Leydig cell line. 5-Aza decreased cell viability in a dose- and time-dependent manner with concomitant increase in Bad protein. This increase is accompanied by increased cleavages of both poly ADP ribose polymerase and caspase-3. Flow cytometric analysis further supported 5-Aza-derived apoptosis in TM3 cells. Bisulfite sequencing analysis failed to identify putative methylcytosine site(s) in CpG islands of the Bad promoter. A chromatin immunoprecipitation assay revealed decreased levels of trimethylation at lysine 27 of histone H3 (H3K27-3me) and H3K9-3me in the Bad promoter region in response to 5-Aza treatment. Knock-down by siRNA of enhancer of zeste homologue 2 (EZH2), a histone methyltransferase responsible for H3K27-3me, or demethylation of H3K9-3me by BIX-01294 showed significantly increased levels in Bad expression and consequent Leydig cell apoptosis. In conclusion, our results demonstrate for the first time that Bad expression is regulated at least by EZH2-mediated H3K27-3me or G9a-like protein/euchromatic histone methyltransferase 1 (GLP/Eu-HMTase1)-mediated H3K9-3me in mouse TM3 Leydig cells, which may be implicated in 5-Aza-derived toxicity to male reproduction.

  3. Epigenetic regulation: methylation of histone and non-histone proteins

    Institute of Scientific and Technical Information of China (English)

    LAN Fei; SHI Yang

    2009-01-01

    Histone methylation is believed to play important roles In epigenetic memory in various biological processes. However, questions like whether the methylation marks themselves are faithfully transmit-ted into daughter cells and through what mechanisms are currently under active investigation. Previ-ously, methylation was considered to be irreversible, but the recent discovery of histone lysine de-methylases revealed a dynamic nature of histone methylation regulation on four of the main sites of methylation on histone H3 and H4 tails (H3K4, H3K9, H3K27 and H3K36). Even so, it is stlll unclear whether demethylases specific for the remaining two sites, H3K79 and H4K20, exist. Furthermore, be-sides hlstone proteins, the lysine methylation and demethylation also occur on non-histone proteins,which are probably subjected to similar regulation as histones. This review discusses recent pro-gresses In protein lysine methylation regulation focusing on the above topics, while referring readers to a number of recent reviews for the biochemistry and biology of these enzymes.

  4. Drosophila p53 is required to increase the levels of the dKDM4B demethylase after UV-induced DNA damage to demethylate histone H3 lysine 9.

    Science.gov (United States)

    Palomera-Sanchez, Zoraya; Bucio-Mendez, Alyeri; Valadez-Graham, Viviana; Reynaud, Enrique; Zurita, Mario

    2010-10-01

    Chromatin undergoes a variety of changes in response to UV-induced DNA damage, including histone acetylation. In human and Drosophila cells, this response is affected by mutations in the tumor suppressor p53. In this work, we report that there is a global decrease in trimethylated Lys-9 in histone H3 (H3K9me3) in salivary gland cells in wild type flies in response to UV irradiation. In contrast, flies with mutations in the Dmp53 gene have reduced basal levels of H3K9me3, which are then increased after UV irradiation. The reduction of H3K9me3 in response to DNA damage occurs preferentially in heterochromatin. Our experiments demonstrate that UV irradiation enhances the levels of Lys-9 demethylase (dKDM4B) transcript and protein in wild type flies, but not in Dmp53 mutant flies. Dmp53 binds to a DNA element in the dKdm4B gene as a response to UV irradiation. Furthermore, heterozygous mutants for the dKdm4B gene are more sensitive to UV irradiation; they are deficient in the removal of cyclobutane-pyrimidine dimers, and the decrease of H3K9me3 levels following DNA damage is not observed in dKdm4B mutant flies. We propose that in response to UV irradiation, Dmp53 enhances the expression of the dKDM4B histone demethylase, which demethylates H3K9me3 preferentially in heterochromatin regions. This mechanism appears to be essential for the proper function of the nucleotide excision repair system.

  5. Histone variants: key players of chromatin.

    Science.gov (United States)

    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.

  6. The methyltransferase NSD3 has chromatin-binding motifs, PHD5-C5HCH, that are distinct from other NSD (nuclear receptor SET domain) family members in their histone H3 recognition.

    Science.gov (United States)

    He, Chao; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Shi, Yunyu

    2013-02-15

    The NSD (nuclear receptor SET domain-containing) family members, consisting of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1), are SET domain-containing methyltransferases and aberrant expression of each member has been implicated in multiple diseases. They have specific mono- and dimethylase activities for H3K36, whereas play nonredundant roles during development. Aside from the well characterized catalytic SET domain, NSD proteins have multiple potential chromatin-binding motifs that are clinically relevant, including the fifth plant homeodomain (PHD5) and the adjacent Cys-His-rich domain (C5HCH) located at the C terminus. Herein, we report the crystal structures of the PHD5-C5HCH module of NSD3, in the free state and in complex with H3(1-7) (H3 residues 1-7), H3(1-15) (H3 residues 1-15), and H3(1-15)K9me3 (H3 residues 1-15 with trimethylation on K9) peptides. These structures reveal that the PHD5 and C5HCH domains fold into a novel integrated PHD-PHD-like structural module with H3 peptide bound only on the surface of PHD5 and provide the molecular basis for the recognition of unmodified H3K4 and trimethylated H3K9 by NSD3 PHD5. Structural studies and binding assays show that differences exist in histone binding specificity of the PHD5 domain between three members of the NSD family. For NSD2, the PHD5-C5HCH:H3 N terminus interaction is largely conserved, although with a stronger preference for unmethylated H3K9 (H3K9me0) than trimethylated H3K9 (H3K9me3), and NSD1 PHD5-C5HCH does not bind to H3 peptides. Our results shed light on how NSD proteins that mediate H3K36 methylation are localized to specific genomic sites and provide implications for the mechanism of functional diversity of NSD proteins.

  7. 喉癌细胞系中抑癌基因MGMT表达与DNA甲基化及组蛋白H3-K9甲基化的关系%Correlation between histone H3-K9 methylation, DNA methylation and expression of gene MGMT in Hep-2 cell line

    Institute of Scientific and Technical Information of China (English)

    杨静; 何丽霞; 季文樾; 金明珠; 赵旭东

    2012-01-01

    To explore the correlation between histone H3-K9 methylation, DNA methylation and expression of carcinoma suppressor gene MGMT in laryngeal carcinoma Hep-2 cell line. Method: 5-Aza-dC was used to deal with Hep-2 cell cultured in vitro. ChIP, MSP and Realtime- PCR were used to detect H3-K9 methylation, DNA methylation, of MGMT gene promoter region and MGMT gene expression before and after treatment with drugs. Result: In Hep-2 cell line,gene MGMT was characterized by DNA methylation and histone H3-K9 hypermethylation. 5-Aza-dC was able to reduce H3-K9 methylation of MGMT gene histone in Hep-2 cell line, 5-Aza-dC was able to reverse DNA methylation of MGMT gene histone in Hep-2 cell line,5-Aza-dC was able to up-regulate the down-regulated gene expression of tumor suppressor genes MGMT. Conclusion: Promoter methylation of cancer suppressor gene MGMT may induce the gene inactivity. DNA methylation may increase H3-K9 methylation. 5-Aza-dC can reduce H3-K9 methylation of tumor suppressor gene MGMT histone by reversing DNA methylation of tumor suppressor gene MGMT, and then the expression of tumor suppressor genes is increased and tumor development is inhibited.%目的:探讨喉癌Hep-2细胞中组蛋白H3-K9甲基化与DNA甲基化及抑癌基因MGMT表达的关系.方法:应用去甲基化制剂5-氮杂-2'-脱氧胞苷(5-Aza-dC)处理体外培养的Hep-2细胞.应用染色质免疫沉淀技术、甲基化特异性聚合酶链反应和实时定量逆转录聚合酶链反应分析药物作用前后MGMT基因启动子区组蛋白H3-K9甲基化、DNA甲基化和MGMT表达情况.结果:①在Hep-2细胞未经药物干预前,MGMT表现为DNA甲基化,组蛋白H3-K9高甲基化,MGMT低表达.②在5-Aza-dC的作用下,Hep-2细胞中MGMT的组蛋白H3-K9甲基化状态被降低;MGMT的DNA甲基化状态得到了逆转;原来低表达的MGMT表达上调.结论:喉癌细胞系中MGMT启动子区甲基化可能是导致其基因失活的主要原因.DNA甲基化可能导致组蛋白H

  8. Global migration dynamics underlie evolution and persistence of human influenza A (H3N2).

    Science.gov (United States)

    Bedford, Trevor; Cobey, Sarah; Beerli, Peter; Pascual, Mercedes

    2010-05-27

    The global migration patterns of influenza viruses have profound implications for the evolutionary and epidemiological dynamics of the disease. We developed a novel approach to reconstruct the genetic history of human influenza A (H3N2) collected worldwide over 1998 to 2009 and used it to infer the global network of influenza transmission. Consistent with previous models, we find that China and Southeast Asia lie at the center of this global network. However, we also find that strains of influenza circulate outside of Asia for multiple seasons, persisting through dynamic migration between northern and southern regions. The USA acts as the primary hub of temperate transmission and, together with China and Southeast Asia, forms the trunk of influenza's evolutionary tree. These findings suggest that antiviral use outside of China and Southeast Asia may lead to the evolution of long-term local and potentially global antiviral resistance. Our results might also aid the design of surveillance efforts and of vaccines better tailored to different geographic regions.

  9. Histone H3 lysine 9 methylation is associated with the expression of hMLH1 and DNA methylation in gastric cancer cells%胃癌细胞系中组蛋白H3-K9甲基化与错配修复基因hMLH1表达及DNA甲基化的关系

    Institute of Scientific and Technical Information of China (English)

    孟春风; 朱新江; 彭过; 戴冬秋

    2008-01-01

    Objective To identify the association of his tone H3 lysine 9 (H3-K9) methylafion with DNA methylation and the expression of the mismatch repair gene hMLH1 in human gastric cancer cells. Methods Gastric cancer cells of the lines BGC-823 and MGC-803 were cultured and treated with 5- Aza-2'-deoxycytidine (5-Aza-dC), a demethylation agent, for 72 hour. Chromatin immunoprecipitation (CHIP) assay was used to assess the status of histone H3 lysine 9 methylation in the promoter regions of hMLH1 gene. Methylation-specific PCR (MSP) was used to evaluate the effect of 5-Aza-dC on DNA methylation status. RT-PCR was used to examine the hMLH1 gene expression. Results In the MGC-803 cells, silenced hMLH1 gene was characterized by DNA methylation and histone H3-K9 hypermethylation; 5-Aza-dC demethylated the DNA and reduced the histone H3-K9 methylation at silenced loci and resulted in reactivation of hMLHI gene therein. Contrary to the MGC-803 ceils, BGC-823 cells expressed hMLH1 gene with DNA demethylation and histone H3-K9 hypomethylation; and 5-Aza-dC had no effects on the gene expression, DNA methylation, and histone H3-K9 methylation therein. Conclusion Hypermethylation of DNA in the promoter region is related to transcriptional silencing of hMLH1 gene. Histone H3-K9 methylation in different regions of the promoter studied correlates with DNA methylatian status of hMLH1 gene in gastric cancer cells. Alteration of DNA methylation affects histone H3-K9 methylation. 5-Aza-dC can control hMLHI expression, DNA methylation, and histone H3-K9 methylation in the promoter.%目的 探讨胃癌细胞系中组蛋白H3-K9甲基化与DNA甲基化及错配修复基因hMLHI表达的关系.方法 应用去甲基化制剂5-氮杂-2'-脱氧胞苷(5-Aza-dC)作用于2个胃癌细胞系,MGC-803和BGC-823.应用染色质免疫沉淀(CHIP)、甲基化特异性聚合酶链反应(MSP)和逆转录聚合酶链反应(RT-PCR)分析药物作用前后hMLH1基因启动子区域组蛋白H3-K9

  10. Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21(WAF1/CIP1) expression.

    Science.gov (United States)

    Tseng, Tsui-Hwa; Chien, Ming-Hsien; Lin, Wea-Lung; Wen, Yu-Ching; Chow, Jyh-Ming; Chen, Chi-Kuan; Kuo, Tsang-Chih; Lee, Wei-Jiunn

    2017-02-01

    Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21(WAF1/CIP1) and increased the interaction of p21(WAF1/CIP1) with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21(WAF1/CIP1) promoter region, resulting in the increase of p21(WAF1/CIP1) transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21(WAF1/CIP1) and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017.

  11. Sulfyhydryl-reactive site-directed cross-linking as a method for probing the tetrameric structure of histones H3 and H4.

    Science.gov (United States)

    Bowman, Andrew; Owen-Hughes, Tom

    2012-01-01

    The structural characterisation of protein-protein interactions is often challenging. Where interactions are not amenable to high-resolution approaches, alternatives providing lower resolution information are often of value. One such approach is site-directed cross-linking. Here, through the introduction of cysteine residues at strategic locations in histone proteins, we use site-directed cross-linking to monitor the association of chromatin subunits. This approach is informative for the study of both recombinant and native chromatin complexes consisting either of histone subunits alone or in association with accessory proteins, in this case histone chaperones. The approaches described may be generally applicable for monitoring the interactions of a diverse range of multi-protein complexes.

  12. 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

    Science.gov (United States)

    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

  13. Conformational response to solvent interaction and temperature of a protein (Histone h3.1) by a multi-grained monte carlo simulation.

    Science.gov (United States)

    Pandey, Ras B; Farmer, Barry L

    2013-01-01

    Interaction with the solvent plays a critical role in modulating the structure and dynamics of a protein. Because of the heterogeneity of the interaction strength, it is difficult to identify multi-scale structural response. Using a coarse-grained Monte Carlo approach, we study the structure and dynamics of a protein (H3.1) in effective solvent media. The structural response is examined as a function of the solvent-residue interaction strength (based on hydropathy index) in a range of temperatures (spanning low to high) involving a knowledge-based (Miyazawa-Jernigan(MJ)) residue-residue interaction. The protein relaxes rapidly from an initial random configuration into a quasi-static structure at low temperatures while it continues to diffuse at high temperatures with fluctuating conformation. The radius of gyration (Rg ) of the protein responds non-monotonically to solvent interaction, i.e., on increasing the residue-solvent interaction strength (fs ), the increase in Rg (fs ≤fsc ) is followed by decay (fs ≥fsc ) with a maximum at a characteristic value (fsc ) of the interaction. Raising the temperature leads to wider spread of the distribution of the radius of gyration with higher magnitude of fsc . The effect of solvent on the multi-scale (λ: residue to Rg ) structures of the protein is examined by analyzing the structure factor (S( q ),|q| = 2π/λ is the wave vector of wavelength, λ) in detail. Random-coil to globular transition with temperature of unsolvated protein (H3.1) is dramatically altered by the solvent at low temperature while a systematic change in structure and scale is observed on increasing the temperature. The interaction energy profile of the residues is not sufficient to predict its mobility in the solvent. Fine-grain representation of protein with two-node and three-node residue enhances the structural resolution; results of the fine-grained simulations are consistent with the finding described above of the coarse-grained description

  14. Epigenetics and sex differences in the brain: A genome-wide comparison of histone-3 lysine-4 trimethylation (H3K4me3) in male and female mice.

    Science.gov (United States)

    Shen, Erica Y; Ahern, Todd H; Cheung, Iris; Straubhaar, Juerg; Dincer, Aslihan; Houston, Isaac; de Vries, Geert J; Akbarian, Schahram; Forger, Nancy G

    2015-06-01

    Many neurological and psychiatric disorders exhibit gender disparities, and sex differences in the brain likely explain some of these effects. Recent work in rodents points to a role for epigenetics in the development or maintenance of neural sex differences, although genome-wide studies have so far been lacking. Here we review the existing literature on epigenetics and brain sexual differentiation and present preliminary analyses on the genome-wide distribution of histone-3 lysine-4 trimethylation in a sexually dimorphic brain region in male and female mice. H3K4me3 is a histone mark primarily organized as 'peaks' surrounding the transcription start site of active genes. We microdissected the bed nucleus of the stria terminalis and preoptic area (BNST/POA) in adult male and female mice and used ChIP-Seq to compare the distribution of H3K4me3 throughout the genome. We found 248 genes and loci with a significant sex difference in H3K4me3. Of these, the majority (71%) had larger H3K4me3 peaks in females. Comparisons with existing databases indicate that genes and loci with increased H3K4me3 in females are associated with synaptic function and with expression atlases from related brain areas. Based on RT-PCR, only a minority of genes with a sex difference in H3K4me3 has detectable sex differences in expression at baseline conditions. Together with previous findings, our data suggest that there may be sex biases in the use of epigenetic marks. Such biases could underlie sex differences in vulnerabilities to drugs or diseases that disrupt specific epigenetic processes. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-07-18

    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 GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

  16. Regulation of replication fork progression through histone supply and demand

    DEFF Research Database (Denmark)

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

    2007-01-01

    DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone...... chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf......1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork...

  17. The H3K4me3/2 histone demethylase RBR-2 controls axon guidance by repressing the actin-remodeling gene wsp-1

    DEFF Research Database (Denmark)

    Mariani, Luca; Lussi, Yvonne C.; Vandamme, Julien;

    2016-01-01

    . Here, we show that RBR-2, the sole homolog of the KDM5 family of H3K4me3/2 demethylases in Caenorhabditis elegans, ensures correct axon guidance by controlling the expression of the actin regulator wsp-1. Loss of rbr-2 results in increased levels of H3K4me3 at the transcriptional start site of wsp-1...

  18. Interactions between DNA and histones- a dynamic process of nucleosome formation

    Institute of Scientific and Technical Information of China (English)

    李伟; 王鹏业; 窦硕星; 童培庆

    2003-01-01

    We have studied the dynamic process of interactions between a DNA chain and a histone octamer by numerical simulations. It is found that DNA indeed may wrap around the histone octaner about two turns as in the actual situations. The simulation shows that the interaction potential between DNA and histone is a key factor for the wrapping of DNA, and the temperature is also an important parameter in the process.

  19. Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

    Science.gov (United States)

    Swalm, Brooke M; Hallenbeck, Kenneth K; Majer, Christina R; Jin, Lei; Scott, Margaret Porter; Moyer, Mikel P; Copeland, Robert A; Wigle, Tim J

    2013-07-15

    H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

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

    Science.gov (United States)

    2015-09-01

    Approved for public release; distribution unlimited NUMBER(S) 13. SUPPLEMENTARY NOTES 14. ABSTRACT Here we identify a conserved feedback mechanism that...a. Publications, conference papers, and presentations Journal publications: 1. Hinchcliffe, E.H. (2015). “Video microscopy”. eLS . DOI...conserved feedback mechanism that monitors the relative position of lagging chromosomes during anaphase via the differential phosphorylation of histone

  1. Hepatitis B virus X protein induces the histone H3 lysine 9 trimethylation on the promoter of p16 gene in hepatocarcinogenesis.

    Science.gov (United States)

    Wang, Di-Yi; Zou, Li-Ping; Liu, Xiao-Jia; Zhu, Hong-Guang; Zhu, Rong

    2015-12-01

    Our previous study showed hepatitis B virus X protein (HBx) suppresses the p16 expression in hepatocarcinogenesis. In this study we explored the relationship between HBx and trimethylation of H3K9 (H3K9me3), and elucidated the underlying mechanisms in HBx inducing the tumor suppressor p16 gene silence. SMMC-7721 and HepG2 hepatoma cell lines were transfected with HBx-expressing plasmid. Immunohistochemistry, Western blotting and real-time polymerase chain reaction, were performed to detect the expressions of HBx, H3K9me3, and jumonji domain-containing protein 2B (JMJd2B). H3K9me3 enrichment on the p16 promoter was measured by immunoprecipitation-PCR (ChIP-PCR) analyses, and 39 cases of hepatitis B virus (HBV) associated-hepatocellular carcinoma (HCC) and corresponding noncancerous liver tissues were also examined. We demonstrated that HBx was able to upregulate H3K9me3 and suppress JMJd2B mRNA and protein levels in SMMC-7721 and HepG2 hepatoma cell lines. JMJd2B, as a specific target of H3K9me3 for demethylation, was inversely correlated with the levels of H3K9me3 in SMMC-7721 (r=-0.666, Pp16 promoter region. Immunohistochemistry analysis showed that H3K9me3 expression in HBx positive HCC samples were significantly higher than that in HBx negative HCC tissues and were associated with decreased levels of JMJd2B expression. JMJd2B immunoreactivity was also remarkably inversed to that of HBx in HCC tissues (r=-0.630, Pp16 promoter via the decrease of demethylase JMJd2B expression and thus promote the repression of p16 gene expression to enhance hepatocarcinogenesis.

  2. A wide reprogramming of histone H3 modifications during male meiosis I in rice is dependent on the Argonaute protein MEL1.

    Science.gov (United States)

    Liu, Hua; Nonomura, Ken-Ichi

    2016-10-01

    The roles of epigenetic mechanisms, including small-RNA-mediated silencing, in plant meiosis largely remain unclear, despite their importance in plant reproduction. This study unveiled that rice chromosomes are reprogrammed during the premeiosis-to-meiosis transition in pollen mother cells (PMCs). This large-scale meiotic chromosome reprogramming (LMR) continued throughout meiosis I, during which time H3K9 dimethylation (H3K9me2) was increased, and H3K9 acetylation and H3S10 phosphorylation were broadly decreased, with an accompanying immunostaining pattern shift of RNA polymerase II. LMR was dependent on the rice Argonaute protein, MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), which is specifically expressed in germ cells prior to meiosis, because LMR was severely diminished in mel1 mutant anthers. Pivotal meiotic events, such as pre-synaptic centromere association, DNA double-strand break initiation and synapsis of homologous chromosomes, were also disrupted in this mutant. Interestingly, and as opposed to the LMR loss in most chromosomal regions, aberrant meiotic protein loading and hypermethylation of H3K9 emerged on the nucleolar organizing region in the mel1 PMCs. These results suggest that MEL1 plays important roles in epigenetic LMR to promote faithful homologous recombination and synapsis during rice meiosis. © 2016. Published by The Company of Biologists Ltd.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

    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.

  5. Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Wei Jiang; Jinzhao Wang; Yi Zhang

    2013-01-01

    Definitive endoderm differentiation is crucial for generating respiratory and gastrointestinal organs including pancreas and liver.However,whether epigenetic regulation contributes to this process is unknown.Here,we show that the H3K27me3 demethylases KDM6A and KDM6B play an important role in endoderm differentiation from human ESCs.Knockdown of KDM6A or KDM6B impairs endoderm differentiation,which can be rescued by sequential treatment with WNT agonist and antagonist.KDM6A and KDM6B contribute to the activation of WNT3 and DKK1 at different differentiation stages when WNT3 and DKK1 are required for mesendoderm and definitive endoderm differentiation,respectively.Our study not only uncovers an important role of the H3K27me3 demethylases in definitive endoderm differentiation,but also reveals that they achieve this through modulating the WNT signalingpathway.

  6. Brownian dynamics simulation of the effect of histone modification on nucleosome structure

    Science.gov (United States)

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

    2007-05-01

    Using Brownian dynamics we simulate the effect of histone modification, such as phosphorylation, acetylation, and methylation, on nucleosome structure by varying the interaction force between DNA and the histone octamer. The simulation shows that the structural stability of nucleosome is very sensitive to the interaction force, and the DNA unwrapping from the modified histone octamer usually occurs turn by turn. Furthermore, the effects of temperature and DNA break as well as the competition between modified and normal histone octamers are investigated, with the simulation results being in agreement with the experimental observation that phosphorylated nucleosomes near DNA breaks are more easily depleted. Though the simulation study may only give a coarse grained view of the DNA unwrapping process for the modified histone octamer, it may provide insight into the mechanism of DNA repair.

  7. Density-dependent dynamical coexistence of excitons and free carriers in the organolead perovskite C H3N H3Pb I3

    Science.gov (United States)

    Wang, Wei; Li, Yu; Wang, Xiangyuan; Lv, Yanping; Wang, Shufeng; Wang, Kai; Shi, Yantao; Xiao, Lixin; Chen, Zhijian; Gong, Qihuang

    2016-10-01

    The high efficiency of perovskite solar cells benefits from the high density of photoinduced free carriers. We studied how exciton and free carriers, as the two major photoproducts, coexist inside the C H3N H3Pb I3 perovskite. A new density-resolved spectroscopic method was developed for this purpose. The density-dependent coexistence of excitons and free carriers over a wide density range was experimentally observed. The quantitative analysis on the density-resolved spectra revealed a moderate exciton binding energy of 24 ± 2 meV. The results effectively proved that the strong ionic polarization inside the perovskite has a negligible contribution to exciton formation. The spectra also efficiently uncovered the effective mass of electron-hole pairs. Our spectroscopic method and the results profoundly enrich the understanding of the photophysics in perovskite materials for photovoltaic applications.

  8. Current Proteomic Methods to Investigate the Dynamics of Histone Turnover in the Central Nervous System

    Science.gov (United States)

    Farrelly, L.A.; Dill, B.D.; Molina, H.; Birtwistle, M.R.; Maze, I.

    2016-01-01

    Characterizing the dynamic behavior of nucleosomes in the central nervous system is vital to our understanding of brain-specific chromatin-templated processes and their roles in transcriptional plasticity. Histone turnover—the complete loss of old, and replacement by new, nucleosomal histones—is one such phenomenon that has recently been shown to be critical for cell-type-specific transcription in brain, synaptic plasticity, and cognition. Such revelations that histones, long believed to static proteins in postmitotic cells, are highly dynamic in neurons were only possible owing to significant advances in analytical chemistry-based techniques, which now provide a platform for investigations of histone dynamics in both healthy and diseased tissues. Here, we discuss both past and present proteomic methods (eg, mass spectrometry, human “bomb pulse labeling”) for investigating histone turnover in brain with the hope that such information may stimulate future investigations of both adaptive and aberrant forms of “neuroepigenetic” plasticity. PMID:27423867

  9. Dynamic expression of combinatorial replication-dependent histone variant genes during mouse spermatogenesis.

    Science.gov (United States)

    Sun, Rongfang; Qi, Huayu

    2014-01-01

    Nucleosomes are basic chromatin structural units that are formed by DNA sequences wrapping around histones. Global chromatin states in different cell types are specified by combinatorial effects of post-translational modifications of histones and the expression of histone variants. During mouse spermatogenesis, spermatogonial stem cells (SSCs) self-renew while undergo differentiation, events that occur in the company of constant re-modeling of chromatin structures. Previous studies have shown that testes contain highly expressed or specific histone variants to facilitate these epigenetic modifications. However, mechanisms of regulating the epigenetic changes and the specific histone compositions of spermatogenic cells are not fully understood. Using real time quantitative RT-PCR, we examined the dynamic expression of replication-dependent histone genes in post-natal mouse testes. It was found that distinct sets of histone genes are expressed in various spermatogenic cells at different stages during spermatogenesis. While gonocyte-enriched testes from mice at 2-dpp (days post partum) express pre-dominantly thirteen histone variant genes, SSC-stage testes at 9-dpp highly express a different set of eight histone genes. During differentiation stage when testes are occupied mostly by spermatocytes and spermatids, another twenty-two histone genes are expressed much higher than the rest, including previously known testis-specific hist1h1t, hist1h2ba and hist1h4c. In addition, histone genes that are pre-dominantly expressed in gonocytes and SSCs are also highly expressed in embryonic stem cells. Several of them were changed when embryoid bodies were formed from ES cells, suggesting their roles in regulating pluripotency of the cells. Further more, differentially expressed histone genes are specifically localized in either SSCs or spermatocytes and spermatids, as demonstrated by in situ hybridization using gene specific probes. Taken together, results presented here

  10. HistoneDB 2.0: a histone database with variants--an integrated resource to explore histones and their variants.

    Science.gov (United States)

    Draizen, Eli J; Shaytan, Alexey K; Mariño-Ramírez, Leonardo; Talbert, Paul B; Landsman, David; Panchenko, Anna R

    2016-01-01

    Compaction of DNA into chromatin is a characteristic feature of eukaryotic organisms. The core (H2A, H2B, H3, H4) and linker (H1) histone proteins are responsible for this compaction through the formation of nucleosomes and higher order chromatin aggregates. Moreover, histones are intricately involved in chromatin functioning and provide a means for genome dynamic regulation through specific histone variants and histone post-translational modifications. 'HistoneDB 2.0--with variants' is a comprehensive database of histone protein sequences, classified by histone types and variants. All entries in the database are supplemented by rich sequence and structural annotations with many interactive tools to explore and compare sequences of different variants from various organisms. The core of the database is a manually curated set of histone sequences grouped into 30 different variant subsets with variant-specific annotations. The curated set is supplemented by an automatically extracted set of histone sequences from the non-redundant protein database using algorithms trained on the curated set. The interactive web site supports various searching strategies in both datasets: browsing of phylogenetic trees; on-demand generation of multiple sequence alignments with feature annotations; classification of histone-like sequences and browsing of the taxonomic diversity for every histone variant. HistoneDB 2.0 is a resource for the interactive comparative analysis of histone protein sequences and their implications for chromatin function. Database URL: http://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0.

  11. Effects of histone acetylation and H3K4 methylation on PRA/PRB in human uterine smooth muscle cells during pregnancy%组蛋白H3、H4乙酰化及H3K4甲基化对人妊娠子宫平滑肌细胞PRA/PRB的影响

    Institute of Scientific and Technical Information of China (English)

    罗慧; 陈诚; 梁志清

    2014-01-01

    目的 通过组蛋白去乙酰化酶抑制剂(trichostatin A,TSA)、H3 K4甲基化酶抑制剂(5’-deoxy-5’-methylthioadenosine,MTA)处理人妊娠子宫平滑肌细胞,干预组蛋白H3、H4乙酰化及H3K4甲基化水平,探讨组蛋白H3、H4乙酰化及H3K4甲基化对人妊娠子宫平滑肌细胞PRA/PRB的影响.方法 分离纯化人妊娠子宫平滑肌细胞(n=16),免疫组化定位孕激素受体(progesterone receptor,PR)及孕激素受体B(progesterone receptor B,PRB)在子宫平滑肌细胞核的表达.分别利用不同浓度TSA、MTA对其进行处理,Real-time PCR检测PR、PRA、PRB mRNA的表达;染色质免疫共沉淀技术(Chromatin immunoprecipitation,ChIP)比较处理前后PRA、PRB启动子区H3、H4乙酰化及H3K4三甲基化水平.结果 TSA可使PRA/PRB明显增高(P<0.05),使PRA启动子区H3、H4乙酰化水平明显上升(P<0.05).MTA可使PRA/PRB明显下降(P<0.05),PRA启动子区H3K4乙酰化水平明显下降(P<0.05).两种药物的干预主要通过对PRA的调控来调节PRA/PRB比值.结论 组蛋白H3、H4乙酰化和H3K4甲基化均可使人妊娠子宫平滑肌细胞PRA/PRB比值发生改变,可能参与“功能性孕激素撤退”机制的调节.

  12. Study on histone acetylation modification conditions of group H3 protein in I region of human glioma GDNF gene promoter%人脑胶质瘤GDNF基因启动子Ⅰ区H3组蛋白乙酰化修饰情况研究

    Institute of Scientific and Technical Information of China (English)

    戴燕燕; 陈茂华

    2015-01-01

    目的 分析GDNF基因启动子在人体胶质瘤Ⅰ区区间的H3组蛋白乙酰化(acetylization)修饰情况以及人体胶质瘤的GDNF基因启动子表达方式. 方法 选取正常脑组织20例,实施反转录(RT-PCR)的检测方法来检测GDNF基因启动子的相关表达方式,在实时定量荧光PCR(Real-time PCR)的基础上建立好染色质免疫沉淀(CHIP). 选取人体胶质瘤20例,实施反转录的检测方法来检测GDNF基因启动子的相关表达方式,在此基础上建立好CHIP方法分析GDNF基因启动子在Ⅰ区区间的H3组蛋白乙酰化的相关修饰情况. 结果 实时定量荧光PCR方法在检查人体胶质瘤GDNF基因启动子方式的表达方面, 随着RT-PCR的检测水平级别升高而升高,转录水平中的正常组以及高低级别组之间差异有统计学意义(P<0.05). 结论 GDNF基因启动子在人体胶质瘤Ⅰ区区间的H3组蛋白乙酰化修饰情况很有可能会影响到GDNF基因的相关表达方式.%Objective To analyze the H3 protein histone acetylation modification conditions in I region of human gliomas GDNF gene promoter and to explore the expression of human glioma GDNF gene promoter. Methods A total of 20 cases of patients with normal brain tissue were chosen, and the detection method of reverse transcription (RT-PCR) was used to detect the associated expression method of GDNF gene promoter. On the basis of real-time quantitative fluorescent PCR (Real-time PCR), Chromatin immunoprecipitation (CHIP) was established. 20 cases of patients with human glioma were chosen, and reverse transcription method was implemented to detect the relevant expression of GDNF gene promoter. On this basis, CHIP method was established to analyze the H3 protein histone acetylation modifi-cation conditions in I region of human gliomas GDNF gene promoter. Results By real-time quantitative fluorescent PCR method in checking human glioma GDNF gene expression, with the elevated detected levels of RT-PCR, the

  13. Global dynamics of newly constructed oligonucleosomes of conventional and variant H2A.Z histone

    Directory of Open Access Journals (Sweden)

    Ioshikhes Ilya

    2007-11-01

    Full Text Available Abstract Background Complexes of nucleosomes, which often occur in the gene promoter areas, are one of the fundamental levels of chromatin organization and thus are important for transcription regulation. Investigating the dynamic structure of a single nucleosome as well as nucleosome complexes is important for understanding transcription within chromatin. In a previous work, we highlighted the influence of histone variants on the functional dynamics of a single nucleosome using normal mode analysis developed by Bahar et al. The present work further analyzes the dynamics of nucleosome complexes (nucleosome oligomers or oligonucleosomes such as dimer, trimer and tetramer (beads on a string model with conventional core histones as well as with the H2A.Z histone variant using normal mode analysis. Results The global dynamics of oligonucleosomes reveal larger amplitude of motion within the nucleosomes that contain the H2A.Z variant with in-planar and out-of-planar fluctuations as the common mode of relaxation. The docking region of H2A.Z and the L1:L1 interactions between H2A.Z monomers of nucleosome (that are responsible for the highly stable nucleosome containing variant H2A.Z-histone are highly dynamic throughout the first two dynamic modes. Conclusion Dissection of the dynamics of oligonucleosomes discloses in-plane as well as out-of-plane fluctuations as the common mode of relaxation throughout the global motions. The dynamics of individual nucleosomes and the combination of the relaxation mechanisms expressed by the individual nucleosome are quite interesting and highly dependent on the number of nucleosome fragments present in the complexes. Distortions generated by the non-planar dynamics influence the DNA conformation, and hence the histone-DNA interactions significantly alter the dynamics of the DNA. The variant H2A.Z histone is a major source of weaker intra- and inter-molecular correlations resulting in more disordered motions.

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

    Science.gov (United States)

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

    2005-01-01

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

  15. 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.

    Science.gov (United States)

    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.

  16. The Structural Determinants behind the Epigenetic Role of Histone Variants

    Directory of Open Access Journals (Sweden)

    Manjinder S. Cheema

    2015-07-01

    Full Text Available Histone variants are an important part of the histone contribution to chromatin epigenetics. In this review, we describe how the known structural differences of these variants from their canonical histone counterparts impart a chromatin signature ultimately responsible for their epigenetic contribution. In terms of the core histones, H2A histone variants are major players while H3 variant CenH3, with a controversial role in the nucleosome conformation, remains the genuine epigenetic histone variant. Linker histone variants (histone H1 family haven’t often been studied for their role in epigenetics. However, the micro-heterogeneity of the somatic canonical forms of linker histones appears to play an important role in maintaining the cell-differentiated states, while the cell cycle independent linker histone variants are involved in development. A picture starts to emerge in which histone H2A variants, in addition to their individual specific contributions to the nucleosome structure and dynamics, globally impair the accessibility of linker histones to defined chromatin locations and may have important consequences for determining different states of chromatin metabolism.

  17. The intrinsically disordered distal face of nucleoplasmin recognizes distinct oligomerization states of histones

    Science.gov (United States)

    Ramos, Isbaal; Fernández-Rivero, Noelia; Arranz, Rocío; Aloria, Kerman; Finn, Ron; Arizmendi, Jesús M.; Ausió, Juan; Valpuesta, José María; Muga, Arturo; Prado, Adelina

    2014-01-01

    The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics. PMID:24121686

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

    Directory of Open Access Journals (Sweden)

    Buro Lauren J

    2010-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiang Yuan

    2016-10-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

  1. spFRET studies of nucleosome dynamics modulated by histone modifications, histone variants and neighboring nucleosomes

    NARCIS (Netherlands)

    Buning, Ruth

    2015-01-01

    At the basis of the regulation of the genetic code (DNA) in eukaryotes is its organization into nucleosomes. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA breathing - the transient unwrapping of DNA from the nucleosome. Single-pair Fluorescence Resonance Energy Tran

  2. Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry

    Directory of Open Access Journals (Sweden)

    M Luz Valero

    2016-03-01

    Full Text Available Tandem affinity purification method (TAP allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex by information‐dependent acquisition (IDA LC–MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: PXD002671 and http://dx.doi.org/10.6019/PXD002671. These data are discussed and interpreted in http://dx.doi.org/10.1016/j.jprot.2016.01.004. Valero et al. (2016 [1].

  3. Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-01

    Tandem affinity purification method (TAP) allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex) by information-dependent acquisition (IDA) LC-MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: PXD002671 and http://dx.doi.org/10.6019/PXD002671. These data are discussed and interpreted in http://dx.doi.org/10.1016/j.jprot.2016.01.004. Valero et al. (2016) [1].

  4. Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor

    DEFF Research Database (Denmark)

    Kristensen, Line Hyltoft; Nielsen, Anders Laerke; Helgstrand, Charlotte

    2012-01-01

    Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2 specific lysine demethylase belonging to the family of JmjC domain containin...

  5. Characterization of Chromatin Structure-associated Histone Modifications in Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Chang Pyo Hong

    2012-09-01

    Full Text Available Chromatin structure and dynamics that are influenced by epigenetic marks, such as histone modification and DNA methylation, play a crucial role in modulating gene transcription. To understand the relationship between histone modifications and regulatory elements in breast cancer cells, we compared our chromatin immunoprecipitation sequencing (ChIP-Seq histone modification patterns for histone H3K4me1, H3K4me3, H3K9/16ac, and H3K27me3 in MCF-7 cells with publicly available formaldehyde-assisted isolation of regulatory elements (FAIRE-chip signals in human chromosomes 8, 11, and 12, identified by a method called FAIRE. Active regulatory elements defined by FAIRE were highly associated with active histone modifications, like H3K4me3 and H3K9/16ac, especially near transcription start sites. The H3K9/16ac-enriched genes that overlapped with FAIRE signals (FAIRE-H3K9/14ac were moderately correlated with gene expression levels. We also identified functional sequence motifs at H3K4me1-enriched FAIRE sites upstream of putative promoters, suggesting that regulatory elements could be associated with H3K4me1 to be regarded as distal regulatory elements. Our results might provide an insight into epigenetic regulatory mechanisms explaining the association of histone modifications with open chromatin structure in breast cancer cells.

  6. Dynamic Convergent Evolution Drives the Passage Adaptation across 48 Years' History of H3N2 Influenza Evolution.

    Science.gov (United States)

    Chen, Hui; Deng, Qiang; Ng, Sock Hoon; Lee, Raphael Tze Chuen; Maurer-Stroh, Sebastian; Zhai, Weiwei

    2016-12-01

    Influenza viruses are often propagated in a diverse set of culturing media and additional substitutions known as passage adaptation can cause extra evolution in the target strain, leading to ineffective vaccines. Using 25,482 H3N2 HA1 sequences curated from Global Initiative on Sharing All Influenza Data and National Center for Biotechnology Information databases, we found that passage adaptation is a very dynamic process that changes over time and evolves in a seesaw like pattern. After crossing the species boundary from bird to human in 1968, the influenza H3N2 virus evolves to be better adapted to the human environment and passaging them in embryonated eggs (i.e., an avian environment) leads to increasingly stronger positive selection. On the contrary, passage adaptation to the mammalian cell lines changes from positive selection to negative selection. Using two statistical tests, we identified 19 codon positions around the receptor binding domain strongly contributing to passage adaptation in the embryonated egg. These sites show strong convergent evolution and overlap extensively with positively selected sites identified in humans, suggesting that passage adaptation can confound many of the earlier studies on influenza evolution. Interestingly, passage adaptation in recent years seems to target a few codon positions in antigenic surface epitopes, which makes it difficult to produce antigenically unaltered vaccines using embryonic eggs. Our study outlines another interesting scenario whereby both convergent and adaptive evolution are working in synchrony driving viral adaptation. Future studies from sequence analysis to vaccine production need to take careful consideration of passage adaptation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Effects of Collective Histone State Dynamics on Epigenetic Landscape and Kinetics of Cell Reprogramming

    Science.gov (United States)

    Ashwin, S. S.; Sasai, Masaki

    2015-11-01

    Cell reprogramming is a process of transitions from differentiated to pluripotent cell states via transient intermediate states. Within the epigenetic landscape framework, such a process is regarded as a sequence of transitions among basins on the landscape; therefore, theoretical construction of a model landscape which exhibits experimentally consistent dynamics can provide clues to understanding epigenetic mechanism of reprogramming. We propose a minimal gene-network model of the landscape, in which each gene is regulated by an integrated mechanism of transcription-factor binding/unbinding and the collective chemical modification of histones. We show that the slow collective variation of many histones around each gene locus alters topology of the landscape and significantly affects transition dynamics between basins. Differentiation and reprogramming follow different transition pathways on the calculated landscape, which should be verified experimentally via single-cell pursuit of the reprogramming process. Effects of modulation in collective histone state kinetics on transition dynamics and pathway are examined in search for an efficient protocol of reprogramming.

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

    Directory of Open Access Journals (Sweden)

    Valledor Luis

    2010-01-01

    Full Text Available Abstract 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.

  9. Characterization of H3.3K36M as a tool to study H3K36 methylation in cancer cells.

    Science.gov (United States)

    Sankaran, Saumya M; Gozani, Or

    2017-09-21

    Recurrent mutations at key lysine residues in the histone variant H3.3 are thought to play an etiologic role in the development of distinct subsets of pediatric gliomas and bone and cartilage cancers. H3.3K36M is one such mutation that was originally identified in chondroblastomas, and its expression in these tumors contributes to oncogenic reprogramming by triggering global depletion of dimethylation and trimethylation at H3K36 with a concomitant increase in the levels of H3K27 trimethylation. H3.3K36M expression can also cause epigenomic changes in cell types beyond chondrocytic cells. Here we show that expression of H3.3K36M in HT1080 fibrosarcoma cancer cells severely impairs cellular proliferation, which contrasts its role in promoting transformation of chondrocytic cells. H3.3K36M-associated cellular toxicity phenocopies the specific depletion of H3K36me2, but not loss of H3K36me3. We further find that the H3K36me2-associated toxicity is largely independent of changes in H3K27me3. Together, our findings lend support to the argument that H3K36me2 has distinct roles in cancer cells independent of H3K36me3 and H3K27me3, and highlight the use of H3.3K36M as an epigenetic tool to study H3K36 and H3K27 methylation dynamics in diverse cell types.

  10. Progressive methylation of ageing histones by Dot1 functions as a timer.

    Science.gov (United States)

    De Vos, Dirk; Frederiks, Floor; Terweij, Marit; van Welsem, Tibor; Verzijlbergen, Kitty F; Iachina, Ekaterina; de Graaf, Erik L; Altelaar, A F Maarten; Oudgenoeg, Gideon; Heck, Albert J R; Krijgsveld, Jeroen; Bakker, Barbara M; van Leeuwen, Fred

    2011-09-01

    Post-translational modifications of histone proteins have a crucial role in regulating gene expression. If efficiently re-established after chromosome duplication, histone modifications could help propagate gene expression patterns in dividing cells by epigenetic mechanisms. We used an integrated approach to investigate the dynamics of the conserved methylation of histone H3 Lys 79 (H3K79) by Dot1. Our results show that methylation of H3K79 progressively changes after histone deposition, which is incompatible with a rapid copy mechanism. Instead, methylation accumulates on ageing histones, providing the cell with a timer mechanism to directly couple cell-cycle length to changes in chromatin modification on the nucleosome core.

  11. Current Perspectives on Histone Demethylases

    Institute of Scientific and Technical Information of China (English)

    Xiaoqing TIAN; Jingyuan FANG

    2007-01-01

    The posttranslational modification of histones plays an important role in chromatin regulation.Histone methylation influences constitutive heterochromatin, genomic imprinting, X-chromosome inactivation and gene transcription. Histone demethylase catalyzes the removal of methyl groups on lysine or arginine residues of histones. Two kinds of histone lysine demethylases have been identified, including lysine specific demethylase 1 and Jumonji C (JmjC) domain family proteins. These histone demethylases are involved in the regulation of gene expression. Histone modification is a dynamic process, and the imbalance of histone methylation has been linked to cancers. Therefore, histone demethylases may represent a new target for anti-cancer therapy.

  12. Cell differentiation and development in Arabidopsis are associated with changes in histone dynamics at the single-cell level.

    Science.gov (United States)

    Rosa, Stefanie; Ntoukakis, Vardis; Ohmido, Nobuko; Pendle, Ali; Abranches, Rita; Shaw, Peter

    2014-12-01

    The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels. © 2014 American Society of Plant Biologists. All rights reserved.

  13. Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells

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    Aster H. Juan

    2016-10-01

    Full Text Available The polycomb repressive complex 2 (PRC2 methylates lysine 27 of histone H3 (H3K27 through its catalytic subunit Ezh2. PRC2-mediated di- and tri-methylation (H3K27me2/H3K27me3 have been interchangeably associated with gene repression. However, it remains unclear whether these two degrees of H3K27 methylation have different functions. In this study, we have generated isogenic mouse embryonic stem cells (ESCs with a modified H3K27me2/H3K27me3 ratio. Our findings document dynamic developmental control in the genomic distribution of H3K27me2 and H3K27me3 at regulatory regions in ESCs. They also reveal that modifying the ratio of H3K27me2 and H3K27me3 is sufficient for the acquisition and repression of defined cell lineage transcriptional programs and phenotypes and influences induction of the ESC ground state.

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

    Science.gov (United States)

    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.

  15. Exploring the dynamics of C/H and C/Cl exchanges in the C(3P) + C2H3Cl reaction

    Science.gov (United States)

    Lee, Shih-Huang; Chen, Wei-Kan; Chin, Chih-Hao; Huang, Wen-Jian

    2013-10-01

    The dynamics of the C(3P) + C2H3Cl reaction at collision energy 3.8 kcal mol-1 was investigated in a crossed molecular-beam apparatus using synchrotron vacuum-ultraviolet ionization. Time-of-flight spectra of products C3H2Cl, C3H3, and Cl were recorded at various laboratory scattering angles, from which translational-energy distributions and angular distributions of product channels C3H2Cl + H and C3H3 + Cl were derived. Cl correlates satisfactorily with C3H3 in linear momentum and angular distributions, which confirms the production of C3H3 + Cl. The H-loss (Cl-loss) channel has average translational-energy release 14.3 (8.8) kcal mol-1 corresponding to a fraction 0.30 (0.14) of available energy into the translational degrees of freedom of product HCCCHCl + H (H2CCCH + Cl). The branching ratio of channel H to channel Cl was determined approximately as 12:88. The measurements of translational-energy releases and photoionization thresholds cannot distinguish HCCCHCl from H2CCCCl because both isomers have similar enthalpy of formation and ionization energy; nevertheless, the Rice-Ramsperger-Kassel-Marcus calculation prefers HCCCHCl. The measurement of photoionization spectra identifies product C3H3 as H2CCCH (propargyl). Both products C3H2Cl + H and C3H3 + Cl might correlate to the same triplet intermediate H2CCCHCl but have distinct angular distributions; the former is nearly isotropic whereas the latter is forward biased. A comparison with the C(3P) + C2H3F reaction is stated.

  16. Molecular Epidemiology and Spatio-Temporal Dynamics of the H3N8 Equine Influenza Virus in South America

    Directory of Open Access Journals (Sweden)

    Cecilia Olguin Perglione

    2016-10-01

    Full Text Available Equine influenza virus (EIV is considered the most important respiratory pathogen of horses as outbreaks of the disease lead to substantial economic losses. The H3N8 EIV has caused respiratory disease in horses across the world, including South American countries. Nucleotide and deduced amino acid sequences for the complete haemagglutinin gene of the H3N8 EIV detected in South America since 1963 were analyzed. Phylogenetic and Bayesian coalescent analyses were carried out to study the origin, the time of the most recent common ancestors (tMRCA, the demographic and the phylogeographic patterns of the H3N8 EIV. The phylogenetic analysis demonstrated that the H3N8 EIV detected in South America grouped in 5 well-supported monophyletic clades, each associated with strains of different origins. The tMRCA estimated for each group suggested that the virus was circulating in North America at least one year before its effective circulation in the South American population. Phylogenetic and coalescent analyses revealed a polyphyletic behavior of the viruses causing the outbreaks in South America between 1963 and 2012, possibly due to the introduction of at least 4 different EIVs through the international movement of horses. In addition, phylodynamic analysis suggested South America as the starting point of the spread of the H3N8 EIV in 1963 and showed migration links from the United States to South America in the subsequent EIV irruptions. Further, an increase in the relative genetic diversity was observed between 2006 and 2007 and a subsequent decline since 2009, probably due to the co-circulation of different lineages and as a result of the incorporation of the Florida clade 2 strain in vaccines, respectively. The observed data highlight the importance of epidemiological surveillance and the implementation of appropriate quarantine procedures to prevent outbreaks of the disease.

  17. Molecular Epidemiology and Spatio-Temporal Dynamics of the H3N8 Equine Influenza Virus in South America

    Science.gov (United States)

    Olguin Perglione, Cecilia; Golemba, Marcelo D.; Torres, Carolina; Barrandeguy, Maria

    2016-01-01

    Equine influenza virus (EIV) is considered the most important respiratory pathogen of horses as outbreaks of the disease lead to substantial economic losses. The H3N8 EIV has caused respiratory disease in horses across the world, including South American countries. Nucleotide and deduced amino acid sequences for the complete haemagglutinin gene of the H3N8 EIV detected in South America since 1963 were analyzed. Phylogenetic and Bayesian coalescent analyses were carried out to study the origin, the time of the most recent common ancestors (tMRCA), the demographic and the phylogeographic patterns of the H3N8 EIV. The phylogenetic analysis demonstrated that the H3N8 EIV detected in South America grouped in 5 well-supported monophyletic clades, each associated with strains of different origins. The tMRCA estimated for each group suggested that the virus was circulating in North America at least one year before its effective circulation in the South American population. Phylogenetic and coalescent analyses revealed a polyphyletic behavior of the viruses causing the outbreaks in South America between 1963 and 2012, possibly due to the introduction of at least 4 different EIVs through the international movement of horses. In addition, phylodynamic analysis suggested South America as the starting point of the spread of the H3N8 EIV in 1963 and showed migration links from the United States to South America in the subsequent EIV irruptions. Further, an increase in the relative genetic diversity was observed between 2006 and 2007 and a subsequent decline since 2009, probably due to the co-circulation of different lineages and as a result of the incorporation of the Florida clade 2 strain in vaccines, respectively. The observed data highlight the importance of epidemiological surveillance and the implementation of appropriate quarantine procedures to prevent outbreaks of the disease. PMID:27754468

  18. H3 lysine 4 is acetylated at active gene promoters and is regulated by H3 lysine 4 methylation.

    Directory of Open Access Journals (Sweden)

    Benoit Guillemette

    2011-03-01

    Full Text Available Methylation of histone H3 lysine 4 (H3K4me is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP, we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3, a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS, which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me.

  19. Mechanisms of transcriptional repression by histone lysine methylation

    DEFF Research Database (Denmark)

    Hublitz, Philip; Albert, Mareike; Peters, Antoine H F M

    2009-01-01

    . In this report, we review the recent literature to deduce mechanisms underlying Polycomb and H3K9 methylation mediated repression, and describe the functional interplay with activating H3K4 methylation. We summarize recent data that indicate a close relationship between GC density of promoter sequences......, transcription factor binding and the antagonizing activities of distinct epigenetic regulators such as histone methyltransferases (HMTs) and histone demethylases (HDMs). Subsequently, we compare chromatin signatures associated with different types of transcriptional outcomes from stable repression to highly...... dynamic regulated genes, strongly suggesting that the interplay of different epigenetic pathways is essential in defining specific types of heritable chromatin and associated transcriptional states....

  20. Modeling the Dynamics of Bivalent Histone Modifications in Embryonic Stem Cells

    Science.gov (United States)

    Ku, Wai; Yuan, Guo; Sorrentino, Francesco; Girvan, Michelle; Ott, Edward

    2013-03-01

    Epigenetic modifications to histones may either promote the activation or repression of the transcription of nearby genes. Recent experiments have discovered bivalent domains of nucleosomes in which the domain as a whole contains both active and repressive marks. These domains occur in the promoters of most lineage-control genes in embryonic stem cells. It is generally agreed that bivalent domains play an important role in stem cell differentiation, but the mechanisms remain unclear. Here we propose and study a dynamical model of histone modification which, unlike previous models, captures the general features of the bivalent domains observed in experiments. A key feature of our model is the existence of ``A/R states,'' by which we mean states in which there are a significant number of nucleosomes each of which individually has both active and repressive marks. We use our model to investigate the formation and decay of A/R states, the localization of A/R nucleosomes, and the effect of DNA replication on the stability of A/R states. The goals of our model are to help understand the underlying principles and mechanisms of bivalent domain dynamics and to suggest directions for future experiments.

  1. Histone acetylation, acetyltransferases, and ataxia--alteration of histone acetylation and chromatin dynamics is implicated in the pathogenesis of polyglutamine-expansion disorders.

    Science.gov (United States)

    McCullough, Shaun D; Grant, Patrick A

    2010-01-01

    Eukaryotic chromosomal DNA is packaged into nucleosomes to form a dynamic structure known as chromatin. The compaction of DNA within chromatin poses a unique hindrance with regards to the accessibility of the DNA to enzymes involved in replication, transcriptional regulation, and repair. The physical structure and physiological activity of chromatin are regulated through a diverse set of posttranslational modifications, histone exchange, and structural remodeling. Of the covalent chromatin modifications, the acetylation of lysine residues within histone proteins by acetyltransferase enzymes, such as GCN5, is one of the most prevalent and important steps in the regulation of chromatin function. Alteration of histone acetyltransferase activity can easily result in the dysregulation of gene transcription and ultimately the onset of a disease state. Many transcription factors contain polyglutamine regions within their primary sequence. Mutations resulting in the elongation of these polyglutamine tracts are associated with a disease family known as the polyglutamine expansion disorders. Spinocerebellar ataxia type 7 (SCA7) is one of the nine diseases that are grouped in this family and is caused by polyglutamine expansion of the ataxin-7 protein, which is a component of the GCN5-containing human SAGA histone acetyltransferase complex. Mutation of ataxin-7 in this manner has been shown to disrupt the structural integrity of the SAGA complex and result in aberrant chromatin acetylation patterns at the promoters of genes involved in the normal function of tissues that are affected by the disease. The specific aspects of molecular pathology are not currently understood; however, studies carried out in laboratory systems ranging from the budding yeast Saccharomyces cerevisiae to transgenic mouse models and cultured human cells are poised to allow for the elucidation of disease mechanisms and subsequent therapeutic approaches.

  2. Single-Molecule Electronic Measurements of the Dynamic Flexibility of Histone Deacetylases

    Science.gov (United States)

    Froberg, James; You, Seungyong; Yu, Junru; Haldar, Manas; Sedigh, Abbas; Mallik, Sanku; Srivastava, D. K.; Choi, Yongki

    Due to their involvement in epigenetic regulation, histone deacetylases (HDACs) have gained considerable interest in designing drugs for treatment of a variety of human diseases including cancers. Recently, we applied a label-free, electronic single-molecule nano-circuit technique to gain insight into the contribution of the dynamic flexibility in HDACs structure during the course of substrates/ ligands binding and catalysis. We observed that HDAC8 has two major (dynamically interconvertible) conformational states, ``ground (catalytically unfavorable)'' and ``transition (catalytically favorable)''. In addition, we found that its cognate substrates/ligands reciprocally catalyze the transition of the ground to the transition state conformation of HDAC8. Thus, we propose that both enzymes and their substrates/ligands serve as ``catalysts'' in facilitating the structural changes of each other and promoting the overall chemical transformation reaction. Such new information provides the potential for designing a new class of mechanism-based inhibitors and activators of HDAC8 for treating human diseases.

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

    Science.gov (United States)

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

  4. Critical Role of Histone Turnover in Neuronal Transcription and Plasticity.

    Science.gov (United States)

    Maze, Ian; Wenderski, Wendy; Noh, Kyung-Min; Bagot, Rosemary C; Tzavaras, Nikos; Purushothaman, Immanuel; Elsässer, Simon J; Guo, Yin; Ionete, Carolina; Hurd, Yasmin L; Tamminga, Carol A; Halene, Tobias; Farrelly, Lorna; Soshnev, Alexey A; Wen, Duancheng; Rafii, Shahin; Birtwistle, Marc R; Akbarian, Schahram; Buchholz, Bruce A; Blitzer, Robert D; Nestler, Eric J; Yuan, Zuo-Fei; Garcia, Benjamin A; Shen, Li; Molina, Henrik; Allis, C David

    2015-07-01

    Turnover and exchange of nucleosomal histones and their variants, a process long believed to be static in post-replicative cells, remains largely unexplored in brain. Here, we describe a novel mechanistic role for HIRA (histone cell cycle regulator) and proteasomal degradation-associated histone dynamics in the regulation of activity-dependent transcription, synaptic connectivity, and behavior. We uncover a dramatic developmental profile of nucleosome occupancy across the lifespan of both rodents and humans, with the histone variant H3.3 accumulating to near-saturating levels throughout the neuronal genome by mid-adolescence. Despite such accumulation, H3.3-containing nucleosomes remain highly dynamic-in a modification-independent manner-to control neuronal- and glial-specific gene expression patterns throughout life. Manipulating H3.3 dynamics in both embryonic and adult neurons confirmed its essential role in neuronal plasticity and cognition. Our findings establish histone turnover as a critical and previously undocumented regulator of cell type-specific transcription and plasticity in mammalian brain. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Mean field study of a propagation-turnover lattice model for the dynamics of histone marking

    Science.gov (United States)

    Yao, Fan; Li, FangTing; Li, TieJun

    2017-02-01

    We present a mean field study of a propagation-turnover lattice model, which was proposed by Hodges and Crabtree [Proc. Nat. Acad. Sci. 109, 13296 (2012)] for understanding how posttranslational histone marks modulate gene expression in mammalian cells. The kinetics of the lattice model consists of nucleation, propagation and turnover mechanisms, and exhibits second-order phase transition for the histone marking domain. We showed rigorously that the dynamics essentially depends on a non-dimensional parameter κ = k +/ k -, the ratio between the propagation and turnover rates, which has been observed in the simulations. We then studied the lowest order mean field approximation, and observed the phase transition with an analytically obtained critical parameter. The boundary layer analysis was utilized to investigate the structure of the decay profile of the mark density. We also studied the higher order mean field approximation to achieve sharper estimate of the critical transition parameter and more detailed features. The comparison between the simulation and theoretical results shows the validity of our theory.

  6. The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

    Directory of Open Access Journals (Sweden)

    Yuguang Song

    Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

  7. Differential localization and independent acquisition of the H3K9me2 and H3K9me3 chromatin modifications in the Caenorhabditis elegans adult germ line.

    Directory of Open Access Journals (Sweden)

    Jessica B Bessler

    2010-01-01

    Full Text Available Histone methylation is a prominent feature of eukaryotic chromatin that modulates multiple aspects of chromosome function. Methyl modification can occur on several different amino acid residues and in distinct mono-, di-, and tri-methyl states. However, the interplay among these distinct modification states is not well understood. Here we investigate the relationships between dimethyl and trimethyl modifications on lysine 9 of histone H3 (H3K9me2 and H3K9me3 in the adult Caenorhabditis elegans germ line. Simultaneous immunofluorescence reveals very different temporal/spatial localization patterns for H3K9me2 and H3K9me3. While H3K9me2 is enriched on unpaired sex chromosomes and undergoes dynamic changes as germ cells progress through meiotic prophase, we demonstrate here that H3K9me3 is not enriched on unpaired sex chromosomes and localizes to all chromosomes in all germ cells in adult hermaphrodites and until the primary spermatocyte stage in males. Moreover, high-copy transgene arrays carrying somatic-cell specific promoters are highly enriched for H3K9me3 (but not H3K9me2 and correlate with DAPI-faint chromatin domains. We further demonstrate that the H3K9me2 and H3K9me3 marks are acquired independently. MET-2, a member of the SETDB histone methyltransferase (HMTase family, is required for all detectable germline H3K9me2 but is dispensable for H3K9me3 in adult germ cells. Conversely, we show that the HMTase MES-2, an E(z homolog responsible for H3K27 methylation in adult germ cells, is required for much of the germline H3K9me3 but is dispensable for H3K9me2. Phenotypic analysis of met-2 mutants indicates that MET-2 is nonessential for fertility but inhibits ectopic germ cell proliferation and contributes to the fidelity of chromosome inheritance. Our demonstration of the differential localization and independent acquisition of H3K9me2 and H3K9me3 implies that the trimethyl modification of H3K9 is not built upon the dimethyl modification in

  8. Structural and dynamic properties of linker histone H1 binding to DNA

    CERN Document Server

    Dootz, Rolf; Pfohl, Thomas

    2010-01-01

    Found in all eukaryotic cells, linker histones H1 are known to bind to and rearrange nucleosomal linker DNA. In vitro, the fundamental nature of H1/DNA interactions has attracted wide interest among research communities - for biologists from a chromatin organization deciphering point of view, and for physicists from the study of polyelectrolyte interactions point of view. Hence, H1/DNA binding processes, structural and dynamical information about these self-assemblies is of broad importance. Targeting a quantitative understanding of H1 induced DNA compaction mechanisms our strategy is based on using small angle X-ray microdiffraction in combination with microfluidics. The usage of microfluidic hydrodynamic focusing devices facilitate a microscale control of these self-assembly processes. In addition, the method enables time-resolved access to structure formation in situ, in particular to transient intermediate states. The observed time dependent structure evolution shows that the interaction of H1 with DNA ca...

  9. Gene promoters dictate histone occupancy within genes.

    Science.gov (United States)

    Perales, Roberto; Erickson, Benjamin; Zhang, Lian; Kim, Hyunmin; Valiquett, Elan; Bentley, David

    2013-10-01

    Spt6 is a transcriptional elongation factor and histone chaperone that reassembles transcribed chromatin. Genome-wide H3 mapping showed that Spt6 preferentially maintains nucleosomes within the first 500 bases of genes and helps define nucleosome-depleted regions in 5' and 3' flanking sequences. In Spt6-depleted cells, H3 loss at 5' ends correlates with reduced pol II density suggesting enhanced transcription elongation. Consistent with its 'Suppressor of Ty' (Spt) phenotype, Spt6 inactivation caused localized H3 eviction over 1-2 nucleosomes at 5' ends of Ty elements. H3 displacement differed between genes driven by promoters with 'open'/DPN and 'closed'/OPN chromatin conformations with similar pol II densities. More eviction occurred on genes with 'closed' promoters, associated with 'noisy' transcription. Moreover, swapping of 'open' and 'closed' promoters showed that they can specify distinct downstream patterns of histone eviction/deposition. These observations suggest a novel function for promoters in dictating histone dynamics within genes possibly through effects on transcriptional bursting or elongation rate.

  10. Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

    Science.gov (United States)

    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.

  11. Biological function and regulation of histone and non-histone lysine methylation in response to DNA damage

    Institute of Scientific and Technical Information of China (English)

    Yongcan Chen; Wei-Guo Zhu

    2016-01-01

    DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources.Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways.Post-translational modifications of histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway.Phosphorylation,ubiquitylation,SUMOylation,neddylation,poly(ADP-ribosyl)ation,acetylation,and methylation are all involved in the spatial-temporal regulation of DDR,among which phosphorylation and ubiquitylation are well studied.Studies in the past decade also revealed extensive roles of lysine methylation in response to DNA damage.Lysine methylation is finely regulated by plenty of lysine methyltransferases,lysine demethylases,and can be recognized by proteins with chromodomain,plant homeodomain,Tudor domain,malignant brain tumor domain,or prolinetryptophan-tryptophan-proline domain.In this review,we outline the dynamics and regulation of histone lysine methylation at canonical (H3K4,H3K9,H3K27,H3K36,H3K79,and H4K20) and non-canonical sites after DNA damage,and discuss their context-specific functions in DDR protein recruitment or extraction,chromatin environment establishment,and transcriptional regulation.We also present the emerging advances of lysine methylation in non-histone proteins during DDR.

  12. Classification of Promoters Based on the Combination of Core Promoter Elements Exhibits Different Histone Modification Patterns

    Science.gov (United States)

    Natsume-Kitatani, Yayoi; Mamitsuka, Hiroshi

    2016-01-01

    Four different histones (H2A, H2B, H3, and H4; two subunits each) constitute a histone octamer, around which DNA wraps to form histone-DNA complexes called nucleosomes. Amino acid residues in each histone are occasionally modified, resulting in several biological effects, including differential regulation of transcription. Core promoters that encompass the transcription start site have well-conserved DNA motifs, including the initiator (Inr), TATA box, and DPE, which are collectively called the core promoter elements (CPEs). In this study, we systematically studied the associations between the CPEs and histone modifications by integrating the Drosophila Core Promoter Database and time-series ChIP-seq data for histone modifications (H3K4me3, H3K27ac, and H3K27me3) during development in Drosophila melanogaster via the modENCODE project. We classified 96 core promoters into four groups based on the presence or absence of the TATA box or DPE, calculated the histone modification ratio at the core promoter region, and transcribed region for each core promoter. We found that the histone modifications in TATA-less groups were static during development and that the core promoters could be clearly divided into three types: i) core promoters with continuous active marks (H3K4me3 and H3K27ac), ii) core promoters with a continuous inactive mark (H3K27me3) and occasional active marks, and iii) core promoters with occasional histone modifications. Linear regression analysis and non-linear regression by random forest showed that the TATA-containing groups included core promoters without histone modifications, for which the measured RNA expression values were not predictable accurately from the histone modification status. DPE-containing groups had a higher relative frequency of H3K27me3 in both the core promoter region and transcribed region. In summary, our analysis showed that there was a systematic link between the existence of the CPEs and the dynamics, frequency and influence

  13. Classification of Promoters Based on the Combination of Core Promoter Elements Exhibits Different Histone Modification Patterns.

    Directory of Open Access Journals (Sweden)

    Yayoi Natsume-Kitatani

    Full Text Available Four different histones (H2A, H2B, H3, and H4; two subunits each constitute a histone octamer, around which DNA wraps to form histone-DNA complexes called nucleosomes. Amino acid residues in each histone are occasionally modified, resulting in several biological effects, including differential regulation of transcription. Core promoters that encompass the transcription start site have well-conserved DNA motifs, including the initiator (Inr, TATA box, and DPE, which are collectively called the core promoter elements (CPEs. In this study, we systematically studied the associations between the CPEs and histone modifications by integrating the Drosophila Core Promoter Database and time-series ChIP-seq data for histone modifications (H3K4me3, H3K27ac, and H3K27me3 during development in Drosophila melanogaster via the modENCODE project. We classified 96 core promoters into four groups based on the presence or absence of the TATA box or DPE, calculated the histone modification ratio at the core promoter region, and transcribed region for each core promoter. We found that the histone modifications in TATA-less groups were static during development and that the core promoters could be clearly divided into three types: i core promoters with continuous active marks (H3K4me3 and H3K27ac, ii core promoters with a continuous inactive mark (H3K27me3 and occasional active marks, and iii core promoters with occasional histone modifications. Linear regression analysis and non-linear regression by random forest showed that the TATA-containing groups included core promoters without histone modifications, for which the measured RNA expression values were not predictable accurately from the histone modification status. DPE-containing groups had a higher relative frequency of H3K27me3 in both the core promoter region and transcribed region. In summary, our analysis showed that there was a systematic link between the existence of the CPEs and the dynamics, frequency

  14. A Classical Approach in Simple Nuclear Fusion Reaction 1H2 + 1H3 using Two-Dimension Granular Molecular Dynamics Model

    CERN Document Server

    Viridi, Sparisoma; Waris, Abdul; Perkasa, Yudha Satya

    2011-01-01

    Molecular dynamics in 2-D accompanied by granular model provides an opportunity to investigate binding between nuclei particles and its properties that arises during collision in a fusion reaction. A fully classical approach is used to observe the influence of initial angle of nucleus orientation to the product yielded by the reaction. As an example, a simplest fusion reaction between 1H2 and 1H3 is observed. Several products of the fusion reaction have been obtained, even the unreported ones, including temporary 2He4 nucleus.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment...

  18. A full-dimensional quantum dynamical study of the vibrational ground state of $H_3O_2^-$ and its isotopomers

    CERN Document Server

    Yang, Yonggang

    2008-01-01

    We investigated the effect of deuteration on the vibrational ground state of the hydrated hydroxide anion using a nine-dimensional quantum dynamical model for the case of J=0. The propagation of the nuclear wave function has been performed with the multi-configuration time-dependent Hartree method which yielded zero-point energies for the normal and fully deuterated species in quantitative agreement with previous diffusion Monte Carlo calculations. According to the zero-point energy the isotopomers having the hydrogen atom in the bridging position are more stable by about 1 kJ/mol as compared to the deuterium case. This holds irrespective of the deuteration state of the two OH groups. We also report the secondary geometric H/D isotope effect on the O--O distance which amounts to an elongation of about 0.005 A for the symmetric isotopomers and 0.009 A in the asymmetric case. Finally, we explore the isotopomer sensitivity of the ground state tunneling splitting due to the torsional motion of the two OH groups.

  19. Specific and efficient N-propionylation of histones with propionic acid N-hydroxysuccinimide ester for histone marks characterization by LC-MS.

    Science.gov (United States)

    Liao, Rijing; Wu, Haiping; Deng, Haibing; Yu, Yanyan; Hu, Min; Zhai, Huili; Yang, Pengyuan; Zhou, Shaolian; Yi, Wei

    2013-02-19

    Histones participate in epigenetic regulation via a variety of dynamic posttranslational modifications (PTMs) on them. Mass spectrometry (MS) has become a powerful tool to investigate histone PTMs. With the bottom-up mass spectrometry approach, chemical derivatization of histones with propionic anhydride or deuterated acetic anhydride followed by trypsin digestion was widely used to block the hydrophilic lysine residues and generate compatible peptides for LC-MS analysis. However, certain severe side reactions (such as acylation on tyrosine or serine) caused by acid anhydrides will lead to a number of analytical issues such as reducing results accuracy and impairing the reproducibility and sensitivity of MS analysis. As an alternative approach, we report a novel derivatization method that utilizes N-hydroxysuccinimide ester to specifically and efficiently derivatize both free and monomethylated amine groups in histones. A competitive inhibiting strategy was implemented in our method to effectively prevent the side reactions. We demonstrated that our method can achieve excellent specificity and efficiency for histones derivatization in a reproducible manner. Using this derivatization method, we succeeded to quantitatively profile the histone PTMs in KMS11 cell line with selective knock out of translocated NSD2 allele (TKO) and the original parental KMS11 cell lines (PAR) (NSD2, a histone methyltransferase that catalyzes the histone H3 K36 methylation), which revealed a significant crosstalk between H3 protein K27 methylation and adjacent K36 methylation.

  20. Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

    Science.gov (United States)

    Brasacchio, Daniella; Okabe, Jun; Tikellis, Christos; Balcerczyk, Aneta; George, Prince; Baker, Emma K.; Calkin, Anna C.; Brownlee, Michael; Cooper, Mark E.; El-Osta, Assam

    2009-01-01

    OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes. PMID:19208907

  1. A role for phosphorylated Pol II CTD in modulating transcription coupled histone dynamics.

    Science.gov (United States)

    Spain, Marla M; Govind, Chhabi K

    2011-03-01

    Histone acetylation modulates histone occupancy both at promoters and in coding sequences. Based on our recent observation that HDACs in the budding yeast, Saccharomyces cerevisiae, are co-transcriptionally recruited to coding regions by elongating polymerases, we propose a model in which Pol II facilitates recruitment of chromatin remodeling complexes as well as other factors required for productive elongation.

  2. Enhancer-associated H3K4 monomethylation by trithorax-related, the drosophila homolog of mammalian MLL3/MLL4

    NARCIS (Netherlands)

    H.-M. Herz (Hans-Martin); M. Mohan (Man); A.S. Garruss (Alexander); K. Liang (Kaiwei); Y.-H. Takahashi (Yoh-hei); K. Mickey (Kristen); O. Voets (Olaf); C.P. Verrijzer (Peter); A. Shilatifard (Ali)

    2012-01-01

    textabstractMonomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are impl

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

    Science.gov (United States)

    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.

  4. Temporal dynamics of gene expression and histone marks at the Arabidopsis shoot meristem during flowering.

    Science.gov (United States)

    You, Yuan; Sawikowska, Aneta; Neumann, Manuela; Posé, David; Capovilla, Giovanna; Langenecker, Tobias; Neher, Richard A; Krajewski, Paweł; Schmid, Markus

    2017-05-17

    Plants can produce organs throughout their entire life from pluripotent stem cells located at their growing tip, the shoot apical meristem (SAM). At the time of flowering, the SAM of Arabidopsis thaliana switches fate and starts producing flowers instead of leaves. Correct timing of flowering in part determines reproductive success, and is therefore under environmental and endogenous control. How epigenetic regulation contributes to the floral transition has eluded analysis so far, mostly because of the poor accessibility of the SAM. Here we report the temporal dynamics of the chromatin modifications H3K4me3 and H3K27me3 and their correlation with transcriptional changes at the SAM in response to photoperiod-induced flowering. Emphasizing the importance of tissue-specific epigenomic analyses we detect enrichments of chromatin states in the SAM that were not apparent in whole seedlings. Furthermore, our results suggest that regulation of translation might be involved in adjusting meristem function during the induction of flowering.

  5. Lysine methylation: beyond histones

    Institute of Scientific and Technical Information of China (English)

    Xi Zhang; Hong Wen; Xiaobing Shi

    2012-01-01

    Posttranslational modifications (PTMs) of histone proteins,such as acetylation,methylation,phosphorylation,and ubiquitylation,play essential roles in regulating chromatin dynamics.Combinations of different modifications on the histone proteins,termed 'histone code' in many cases,extend the information potential of the genetic code by regulating DNA at the epigenetic level.Many PTMs occur on non-histone proteins as well as histones,regulating protein-protein interactions,stability,localization,and/or enzymatic activities of proteins involved in diverse cellular processes.Although protein phosphorylation,ubiquitylation,and acetylation have been extensively studied,only a few proteins other than histones have been reported that can be modified by lysine methylation.This review summarizes the current progress on lysine methylation of nonhistone proteins,and we propose that lysine methylation,like phosphorylation and acetylation,is a common PTM that regulates proteins in diverse cellular processes.

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

    Science.gov (United States)

    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.

  7. Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast.

    Science.gov (United States)

    Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G

    2012-01-01

    Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

  8. Chromosomal mapping of repetitive DNAs in the beetle Dichotomius geminatus provides the first evidence for an association of 5S rRNA and histone H3 genes in insects, and repetitive DNA similarity between the B chromosome and A complement.

    Science.gov (United States)

    Cabral-de-Mello, D C; Moura, R C; Martins, C

    2010-04-01

    Chromosomal banding techniques and repetitive DNA mapping are useful tools in comparative analysis and in the elucidation of genome organization of several groups of eukaryotes. In this study, we contributed to the knowledge of Coleoptera genomes by reporting the chromosomal organization of repetitive DNA sequences, as well as the presence and characteristics of a B chromosome in two natural populations of Dichotomius geminatus (Coleoptera; Scarabaeidae) using classical, chromosomal banding and molecular cytogenetic techniques. As in other coleopteran species, the heterochromatin was mainly concentrated in pericentromeric regions and the B chromosome was composed almost entirely of heterochromatin. Physical mapping using double fluorescent in situ hybridization was performed for the first time in Coleoptera; using DNA probes for 5S and 18S ribosomal RNA (rRNA) and histone H3 genes, we showed that ribosomal 18S rDNAs are located in chromosomes 3 and 4, whereas 5S rRNA and histone H3 genes are colocalized in chromosomal pair 2 and show an apparently interspersed organization. Moreover, these genes are not present in the B chromosome, suggesting that the B chromosome did not originate from chromosomal pairs 2, 3 or 4. On the other hand, mapping of the C(0)t-1 DNA fraction showed that the B chromosome is enriched in repetitive DNA elements, also present in the standard complement, indicating an intraspecific origin of this element in D. geminatus. These results will contribute to our understanding of genome organization and evolution of repetitive elements in Coleoptera and other insects regarding both A and B chromosomes.

  9. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    Science.gov (United States)

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  10. H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Vandamme, Julien; Sidoli, Simone; Mariani, Luca;

    2015-01-01

    described in this organism. We used mass spectrometry based middle-down proteomics to analyze histone H3 N-terminal tails from C. elegans embryos for the presence, the relative abundance and the potential cross-talk of co-existing PTMs. This analysis highlighted that the lysine 23 of histone H3 (H3K23......Genome-wide analyses in Caenorhabditis elegans show that post-translational modifications (PTMs) of histones are evolutionary conserved and distributed along functionally distinct genomic domains. However, a global profile of PTMs and their co-occurrence on the same histone tail has not been...

  11. Analysis of Myc-induced histone modifications on target chromatin.

    Directory of Open Access Journals (Sweden)

    Francesca Martinato

    Full Text Available The c-myc proto-oncogene is induced by mitogens and is a central regulator of cell growth and differentiation. The c-myc product, Myc, is a transcription factor that binds a multitude of genomic sites, estimated to be over 10-15% of all promoter regions. Target promoters generally pre-exist in an active or poised chromatin state that is further modified by Myc, contributing to fine transcriptional regulation (activation or repression of the afferent gene. Among other mechanisms, Myc recruits histone acetyl-transferases to target chromatin and locally promotes hyper-acetylation of multiple lysines on histones H3 and H4, although the identity and combination of the modified lysines is unknown. Whether Myc dynamically regulates other histone modifications (or marks at its binding sites also remains to be addressed. Here, we used quantitative chromatin immunoprecipitation (qChIP to profile a total of 24 lysine-acetylation and -methylation marks modulated by Myc at target promoters in a human B-cell line with a regulatable c-myc transgene. Myc binding promoted acetylation of multiple lysines, primarily of H3K9, H3K14, H3K18, H4K5 and H4K12, but significantly also of H4K8, H4K91 and H2AK5. Dimethylation of H3K79 was also selectively induced at target promoters. A majority of target promoters showed co-induction of multiple marks - in various combinations - correlating with recruitment of the two HATs tested (Tip60 and HBO1, incorporation of the histone variant H2A.Z and transcriptional activation. Based on this and previous findings, we surmise that Myc recruits the Tip60/p400 complex to achieve a coordinated histone acetylation/exchange reaction at activated promoters. Our data are also consistent with the additive and redundant role of multiple acetylation events in transcriptional activation.

  12. The conformational dynamics of H2-H3n and S2-H6 in gating ligand entry into the buried binding cavity of vitamin D receptor

    Science.gov (United States)

    Tee, Wei-Ven; Ripen, Adiratna Mat; Mohamad, Saharuddin Bin

    2016-01-01

    Crystal structures of holo vitamin D receptor (VDR) revealed a canonical conformation in which the ligand is entrapped in a hydrophobic cavity buried in the ligand-binding domain (LBD). The mousetrap model postulates that helix 12 is positioned away from the domain to expose the interior cavity. However, the extended form of helix 12 is likely due to artifacts during crystallization. In this study, we set out to investigate conformational dynamics of apo VDR using molecular dynamics simulation on microsecond timescale. Here we show the neighboring backbones of helix 2-helix 3n and beta strand 2-helix 6 of LBD, instead of the helix 12, undergo large-scale motion, possibly gating the entrance of ligand to the ligand binding domain. Docking analysis to the simulated open structure of VDR with the