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Sample records for chromosome-specific histone h3

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

    KAUST Repository

    Chauhan, Sakshi

    2016-09-02

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

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

    Science.gov (United States)

    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.

  3. Methylation of histone H3 lysine 9 occurs during translation

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  5. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    OpenAIRE

    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...

  6. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    Science.gov (United States)

    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

  7. 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.

  8. 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.

  9. 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...

  10. 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

  11. 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

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

    Science.gov (United States)

    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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

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    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.

  20. 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…

  1. 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.

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

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    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.

  3. 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.

  4. 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.

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. 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

  13. 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.

  14. 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.

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

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    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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

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

    Science.gov (United States)

    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.

  2. 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.

  3. 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...

  4. 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.

  5. 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.

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

    DEFF Research Database (Denmark)

    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......me3. ChIP-on-chip profiling of H3, H3K4me3, H3K9me3, and H3K9ac from asynchronous parasites revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA. Remarkably, the vast majority...... of the genome shows an unexpected pattern of enrichment of H3K4me3 and H3K9ac. Analysis of synchronized parasites revealed significant developmental stage specificity of the epigenome. In rings, H3K4me3 and H3K9ac are homogenous across the genes marking active and inactive genes equally, whereas in schizonts...

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

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

    Science.gov (United States)

    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.

  13. 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.

  14. 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.

  15. 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...

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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...

  1. 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...

  2. 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.

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

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    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.

  4. 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.

  5. 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.

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

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    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.

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  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. 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.

  16. 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. Histo...

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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......-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......, 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....

  3. 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

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

    Institute of Scientific and Technical Information of China (English)

    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.

  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. 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.

  7. 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

  8. 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.

  9. 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....

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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 ...

  17. 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.

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

    DEFF Research Database (Denmark)

    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...

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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

  11. 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.

  12. 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....

  13. 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

    Acute Myeloid Leukemia (AML) is commonly associated with alterations in transcription factors due to altered expression or gene mutations. These changes might induce leukemia- specific patterns of histone modifications. We used ChIP-Chip to analyze histone H3 Lysine 9 trimethylation (H3K9me3......) patterns in primary AML (n=109), ALL (n=28), CD34+ cells (n=21) and white blood cells (n=15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions....... 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...

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

    Directory of Open Access Journals (Sweden)

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

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    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.

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

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    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

  17. A reversible histone H3 acetylation cooperates with mismatch repair and replicative polymerases in maintaining genome stability.

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    Lyudmila Y Kadyrova

    2013-10-01

    Full Text Available Mutations are a major driving force of evolution and genetic disease. In eukaryotes, mutations are produced in the chromatin environment, but the impact of chromatin on mutagenesis is poorly understood. Previous studies have determined that in yeast Saccharomyces cerevisiae, Rtt109-dependent acetylation of histone H3 on K56 is an abundant modification that is introduced in chromatin in S phase and removed by Hst3 and Hst4 in G2/M. We show here that the chromatin deacetylation on histone H3 K56 by Hst3 and Hst4 is required for the suppression of spontaneous gross chromosomal rearrangements, base substitutions, 1-bp insertions/deletions, and complex mutations. The rate of base substitutions in hst3Δ hst4Δ is similar to that in isogenic mismatch repair-deficient msh2Δ mutant. We also provide evidence that H3 K56 acetylation by Rtt109 is important for safeguarding DNA from small insertions/deletions and complex mutations. Furthermore, we reveal that both the deacetylation and acetylation on histone H3 K56 are involved in mutation avoidance mechanisms that cooperate with mismatch repair and the proofreading activities of replicative DNA polymerases in suppressing spontaneous mutagenesis. Our results suggest that cyclic acetylation and deacetylation of chromatin contribute to replication fidelity and play important roles in the protection of nuclear DNA from diverse spontaneous mutations.

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

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    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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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

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    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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

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

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    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.

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

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    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.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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).

  7. 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.

  8. PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum

    DEFF Research Database (Denmark)

    Jiang, Lubin; Mu, Jianbing; Zhang, Qingfeng;

    2013-01-01

    The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 p...... orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine....... protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown...... genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which...

  9. 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.

  10. 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.

  11. 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.

  12. Vibrio chromosome-specific families

    DEFF Research Database (Denmark)

    Lukjancenko, Oksana; Ussery, David

    2014-01-01

    We have compared chromosome-specific genes in a set of 18 finished Vibrio genomes, and, in addition, also calculated the pan- and core-genomes from a data set of more than 250 draft Vibrio genome sequences. These genomes come from 9 known species and 2 unknown species. Within the finished...

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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 ...... growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc–induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2...

  18. 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...

  19. 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

  20. 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...

  1. 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

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

    Science.gov (United States)

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

    2016-01-01

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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    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...... analysis of the dynamics of coexisting post-translational modifications in proteins....... of Suz12 deficiency on H3.2 and H3.3 from mouse ESCs. Using high mass accuracy MS combined with CID or electron transfer dissociation (ETD) tandem mass spectrometry, we identified a total of 81 unique modified peptides from H3.2 and H3.3 and assigned 46 modifications at 22 different positions, including...... distinct coexisting modifications. In certain cases, high mass accuracy LTQ-Orbitrap MS/MS allowed precise localization of near isobaric coexisting PTMs such as trimethylation and acetylation within individual peptides. ETD MS/MS facilitated sequencing and annotation of phosphorylated histone peptides...

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

    Science.gov (United States)

    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.

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. 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.

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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...

  19. 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...

  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. 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.

  2. 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.

  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. 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.

  5. 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.

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

    DEFF Research Database (Denmark)

    Rasmussen, Louise; Herzog, Marielle; Romer, 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......3K9Me3 measurements were performed using enzyme-linked immunosorbent assays. RESULTS: Stasis, white-cell and platelet contamination, within-day variations, varying storage time before centrifugation, colonoscopy, and surgical trauma had no significant influence on levels of 5mC or H3K9Me3. Day......-to-day variations of 12.7% and 11.5% (intra-individual) and 98.1% and 60.8% (inter-individual) were shown for 5mC and H3K9Me3, respectively. Levels of 5mC or H3K9Me3 were significantly higher in samples stored at room temperature until centrifugation compared to samples stored on ice. Patients with cancer had...

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. 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.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  1. 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).

  2. 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.

  3. 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

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    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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. 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.

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

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    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.

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

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    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. 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.

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

    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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Chromosome-specific families in Vibrio genomes

    Directory of Open Access Journals (Sweden)

    Oksana eLukjancenko

    2014-03-01

    Full Text Available We have compared chromosome-specific genes in a set of 18 finished Vibrio genomes, and, in addition, also calculated the pan- and core-genomes from a data set of more than 250 draft Vibrio genome sequences. These genomes come from 9 known species and 2 unknown species. Within the finished chromosomes, we find a core set of 1269 encoded protein families for chromosome 1, and a core of 252 encoded protein families for chromosome 2. Many of these core proteins are also found in the draft genomes (although which chromosome they are located on is unknown. Of the chromosome specific core protein families, 1169 and 153 are uniquely found in chromosomes 1 and 2, respectively. Gene ontology (GO terms for each of the protein families were determined, and the different sets for each chromosome were compared. A total of 363 different `Molecular Function` GO categories were found for chromosome 1 specific protein families, and these include several broad activities: pyridoxine 5' phosphate synthetase, glucosylceramidase, heme transport, DNA ligase, amino acid binding, and ribosomal components; in contrast, chromosome 2 specific protein families have only 66 Molecular Function GO terms and include many membrane-associated activities, such as ion channels, transmembrane transporters, and electron transport chain proteins. Thus, it appears that whilst there are many 'housekeeping systems' encoded in chromosome 1, there are far fewer core functions found in chromosome 2. However, the presence of many membrane-associated encoded proteins in chromosome 2 is surprising.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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...

  9. 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.

  10. 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.

  11. 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

  12. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

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

    Science.gov (United States)

    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.

  2. Chromosome-specific painting in Cucumis species using bulked oligonucleotides

    Science.gov (United States)

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a sing...

  3. 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.

  4. 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.

  5. The molecular characterization of maize B chromosome specific AFLPs

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The origin and evolution of B chromosomes could be explained by the specific DNA sequence on them.But the specific sequences known were quite limited. To investigate maize B chromosome sqicific DNA sequeces, maize genomes with and without B chromosomes were analyzed by AFLP. Only 5 markers were found specific to genomes with B chromosomes among about 2000 AFLP markers. Southern hybridization and sequence analysis revealed that only the sequence of M8-2D was a B chromosome specific sequence.This sequence contained the telomeric repeat unit AGGGTTT conserved in plant chromosome telomeres.In addition, the sequence of M8-2D shared low homology to clones from maize chromosome 4 centromere as well. M8-2D were localized to B chromosome centromeric and telomeric regions.

  6. 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...

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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...

  13. 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.

  14. 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

  15. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases.

    Science.gov (United States)

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies.

  16. 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

  17. 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...

  18. 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.

  19. Writers and readers of histone H3 methylation

    NARCIS (Netherlands)

    Warrier, R.A.

    2010-01-01

    The synthesis of RNA from a DNA template in eukaryotic cells is a complex process which is regulated at several stages. This is accomplished by RNA polymerase II and a horde of other transcription factors which together comprise the transcription machinery. Accessing information from DNA in the nucl

  20. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms.

    Science.gov (United States)

    Wang, Huanyu; Ge, Zhongqi; Walsh, Scott T R; Parthun, Mark R

    2012-01-01

    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

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

    Science.gov (United States)

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

    2015-12-18

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

  2. Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone.

    Science.gov (United States)

    Behjati, Sam; Tarpey, Patrick S; Presneau, Nadège; Scheipl, Susanne; Pillay, Nischalan; Van Loo, Peter; Wedge, David C; Cooke, Susanna L; Gundem, Gunes; Davies, Helen; Nik-Zainal, Serena; Martin, Sancha; McLaren, Stuart; Goody, Victoria; Goodie, Victoria; Robinson, Ben; Butler, Adam; Teague, Jon W; Halai, Dina; Khatri, Bhavisha; Myklebost, Ola; Baumhoer, Daniel; Jundt, Gernot; Hamoudi, Rifat; Tirabosco, Roberto; Amary, M Fernanda; Futreal, P Andrew; Stratton, Michael R; Campbell, Peter J; Flanagan, Adrienne M

    2013-12-01

    It is recognized that some mutated cancer genes contribute to the development of many cancer types, whereas others are cancer type specific. For genes that are mutated in multiple cancer classes, mutations are usually similar in the different affected cancer types. Here, however, we report exquisite tumor type specificity for different histone H3.3 driver alterations. In 73 of 77 cases of chondroblastoma (95%), we found p.Lys36Met alterations predominantly encoded in H3F3B, which is one of two genes for histone H3.3. In contrast, in 92% (49/53) of giant cell tumors of bone, we found histone H3.3 alterations exclusively in H3F3A, leading to p.Gly34Trp or, in one case, p.Gly34Leu alterations. The mutations were restricted to the stromal cell population and were not detected in osteoclasts or their precursors. In the context of previously reported H3F3A mutations encoding p.Lys27Met and p.Gly34Arg or p.Gly34Val alterations in childhood brain tumors, a remarkable picture of tumor type specificity for histone H3.3 driver alterations emerges, indicating that histone H3.3 residues, mutations and genes have distinct functions.

  3. Dangerous R loops form in the absence of H3K9 methylation

    DEFF Research Database (Denmark)

    Salcini, Anna Elisabetta

    2016-01-01

    Methylation of histone H3 on lysine 9 (H3K9) is a hallmark of transcriptionally inactive heterochromatin that is deregulated in pathological conditions. A new study shows that complete loss of H3K9 methylation in Caenorhabditis elegans leads to derepression of repetitive elements and formation...

  4. File list: His.PSC.50.H3ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.50.H3ac.AllCell mm9 Histone H3ac Pluripotent stem cell SRX882861,SRX882864,...SRX882865,SRX882862,SRX882860,SRX882863 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.50.H3ac.AllCell.bed ...

  5. File list: His.Brs.20.H3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Brs.20.H3.AllCell hg19 Histone H3 Breast SRX387610,SRX647708,SRX387614,SRX64770...9,SRX387607,SRX329008,SRX518255,SRX518248 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Brs.20.H3.AllCell.bed ...

  6. File list: His.Brs.50.H3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Brs.50.H3.AllCell hg19 Histone H3 Breast SRX387610,SRX387614,SRX647708,SRX38760...7,SRX647709,SRX329008,SRX518248,SRX518255 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Brs.50.H3.AllCell.bed ...

  7. File list: His.Brs.05.H3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Brs.05.H3.AllCell hg19 Histone H3 Breast SRX387610,SRX387607,SRX387614,SRX64770...9,SRX647708,SRX329008,SRX518255,SRX518248 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Brs.05.H3.AllCell.bed ...

  8. File list: His.ALL.10.H3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.10.H3.AllCell dm3 Histone H3 All cell types SRX367040,SRX367039,SRX215500,S...RX215499,SRX367041,SRX287784,SRX287775,SRX287783,SRX287776 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/His.ALL.10.H3.AllCell.bed ...

  9. Computational inference of H3K4me3 and H3K27ac domain length

    Directory of Open Access Journals (Sweden)

    Julian Zubek

    2016-03-01

    Full Text Available Background. Recent epigenomic studies have shown that the length of a DNA region covered by an epigenetic mark is not just a byproduct of the assaying technologies and has functional implications for that locus. For example, expanded regions of DNA sequences that are marked by enhancer-specific histone modifications, such as acetylation of histone H3 lysine 27 (H3K27ac domains coincide with cell-specific enhancers, known as super or stretch enhancers. Similarly, promoters of genes critical for cell-specific functions are marked by expanded H3K4me3 domains in the cognate cell type, and these can span DNA regions from 4–5kb up to 40–50kb in length. These expanded H3K4me3 domains are known as buffer domains or super promoters. Methods. To ask what correlates with—and potentially regulates—the length of loci marked with these two important histone marks, H3K4me3 and H3K27ac, we built Random Forest regression models. With these models, we computationally identified genomic and epigenomic patterns that are predictive for the length of these marks in seven ENCODE cell lines. Results. We found that certain epigenetic marks and transcription factors explain the variability of the length of H3K4me3 and H3K27ac marks across different cell types, which implies that the lengths of these two epigenetic marks are tightly regulated in a given cell type. Our source code for the regression models and data can be found at our GitHub page: https://github.com/zubekj/broad_peaks. Discussion. Our Random Forest based regression models enabled us to estimate the individual contribution of different epigenetic marks and protein binding patterns to the length of H3K4me3 and H3K27ac deposition patterns, therefore potentially revealing genomic signatures at cell specific regulatory elements.

  10. H3K23me2 is a new heterochromatic mark in Caenorhabditis elegans.

    Science.gov (United States)

    Vandamme, Julien; Sidoli, Simone; Mariani, Luca; Friis, Carsten; Christensen, Jesper; Helin, Kristian; Jensen, Ole N; Salcini, Anna Elisabetta

    2015-11-16

    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 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) is extensively modified by methylation and that tri-methylated H3K9 (H3K9me3) is exclusively detected on histone tails with di-methylated H3K23 (H3K23me2). Chromatin immunoprecipitation approaches revealed a positive correlation between H3K23me2 and repressive marks. By immunofluorescence analyses, H3K23me2 appears differentially regulated in germ and somatic cells, in part by the action of the histone demethylase JMJD-1.2. H3K23me2 is enriched in heterochromatic regions, localizing in H3K9me3 and heterochromatin protein like-1 (HPL-1)-positive foci. Biochemical analyses indicated that HPL-1 binds to H3K23me2 and interacts with a conserved CoREST repressive complex. Thus, our study suggests that H3K23me2 defines repressive domains and contributes to organizing the genome in distinct heterochromatic regions during embryogenesis.

  11. 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

    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...

  12. Lack of sex chromosome specific meiotic silencing in platypus reveals origin of MSCI in therian mammals

    OpenAIRE

    Daish, Tasman J.; Casey, Aaron E.; Grutzner, Frank

    2015-01-01

    Background In therian mammals heteromorphic sex chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during meiotic prophase I while the autosomes maintain transcriptional activity. The evolution of this sex chromosome silencing is thought to result in retroposition of genes required in spermatogenesis from the sex chromosomes to autosomes. In birds sex chromosome specific silencing appears to be absent and global transcriptional reductions occur through pachytene and sex chr...

  13. A PHD in histone language

    Science.gov (United States)

    Chandrika, Nulu Naga Prafulla; Sundaravelpandian, Kalaipandian; Schmidt, Wolfgang

    2013-01-01

    Post-translational modifications of core histones are important for various DNA-templated processes such as transcription and repair. We recently reported that the ALFIN LIKE 6 (AL6) gene, identified in a forward genetic screen, is critical for phosphate deficiency-induced root hair formation and several other processes associated with the regulation of cellular phosphate homeostasis. AL6 contains a Plant Homeo Domain (PHD) finger that can bind to trimethylated lysine 4 of histone H3 (H3K4me3). Homozygous mutants defective in AL6 expression form very short root hairs under phosphate-deficient conditions, presumably caused by altered expression of putative primary and secondary down-stream targets of AL6. In this Addendum, we speculate about possible roles of AL6, H3K4 trimethylation and other chromatin modifications in the adaptation of plants to low phosphate availability. PMID:23531693

  14. Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

    Directory of Open Access Journals (Sweden)

    Tommy Kaplan

    2008-11-01

    Full Text Available Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

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

    Science.gov (United States)

    Jasencakova, Zuzana; Scharf, Annette N D; Ask, Katrine; Corpet, Armelle; Imhof, Axel; Almouzni, Geneviève; Groth, Anja

    2010-03-12

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

  16. Histamine H3-receptor isoforms.

    Science.gov (United States)

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  17. 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.

  18. File list: His.PSC.05.H3S10ph.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.05.H3S10ph.AllCell mm9 Histone H3S10ph Pluripotent stem cell SRX963273,SRX9...63272 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.05.H3S10ph.AllCell.bed ...

  19. File list: His.ALL.20.H3PERIOD3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.20.H3PERIOD3.AllCell hg19 Histone H3.3 All cell types SRX1304823,SRX502804,...SRX1304824,SRX502806,SRX006760,SRX828376,SRX828377 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.20.H3PERIOD3.AllCell.bed ...

  20. File list: His.ALL.50.H3PERIOD3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.50.H3PERIOD3.AllCell hg19 Histone H3.3 All cell types SRX1304823,SRX502804,...SRX1304824,SRX502806,SRX828376,SRX828377,SRX006760 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.50.H3PERIOD3.AllCell.bed ...

  1. File list: His.ALL.05.H3PERIOD3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.05.H3PERIOD3.AllCell hg19 Histone H3.3 All cell types SRX1304823,SRX502804,...SRX1304824,SRX502806,SRX006760,SRX828376,SRX828377 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.ALL.05.H3PERIOD3.AllCell.bed ...

  2. File list: His.ALL.20.H3PERIOD1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.20.H3PERIOD1.AllCell mm9 Histone H3.1 All cell types SRX1057035,SRX1057067,...SRX1057033,SRX1057072,SRX1057069,SRX647891,SRX1057038,SRX647890 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.ALL.20.H3PERIOD1.AllCell.bed ...

  3. File list: His.PSC.50.H3K9K14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.50.H3K9K14ac.AllCell mm9 Histone H3K9K14ac Pluripotent stem cell ERX245530,...1 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.50.H3K9K14ac.AllCell.bed ...

  4. File list: His.PSC.10.H3K122ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.10.H3K122ac.AllCell mm9 Histone H3K122ac Pluripotent stem cell ERX631826,ER...X631814 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.10.H3K122ac.AllCell.bed ...

  5. File list: His.PSC.05.H3K122ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.05.H3K122ac.AllCell mm9 Histone H3K122ac Pluripotent stem cell ERX631826,ER...X631814 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.05.H3K122ac.AllCell.bed ...

  6. File list: His.PSC.20.H3K9ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.20.H3K9ac.AllCell hg19 Histone H3K9ac Pluripotent stem cell SRX067524,SRX03...8537,SRX951984,SRX951983,SRX038538,SRX095381 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.PSC.20.H3K9ac.AllCell.bed ...

  7. File list: His.Neu.20.H3PERIOD1.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Neu.20.H3PERIOD1.AllCell mm9 Histone H3.1 Neural SRX1057035,SRX1057067,SRX10570...33,SRX1057072,SRX1057069,SRX1057038 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Neu.20.H3PERIOD1.AllCell.bed ...

  8. File list: His.PSC.10.H3PERIOD3.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.10.H3PERIOD3.AllCell mm9 Histone H3.3 Pluripotent stem cell SRX868180,SRX64...108,SRX1022124,SRX1022120,SRX1022087 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.10.H3PERIOD3.AllCell.bed ...

  9. H3 trimethyl K9 and H3 acetyl K9 chromatin modifications are associated with class switch recombination.

    Science.gov (United States)

    Kuang, Fei Li; Luo, Zhonghui; Scharff, Matthew D

    2009-03-31

    Class switch recombination (CSR) involves a DNA rearrangement in the Ig heavy chain (IgH) gene that allows the same variable (V) region to be expressed with any one of the downstream constant region (C) genes to encode antibodies with many different effector functions. One hypothesis for how CSR is targeted to different C region genes is that histone modifications increase accessibility and/or recruit activation-induced cytosine deaminase (AID) and its associated processes to particular donor and recipient switch regions. In this work, we identified H3 acetyl K9 and H3 trimethyl K9 as histone modifications that correlate with the recombining pair of donor and recipient switch regions. The appearance of H3 trimethyl K9 is surprising because usually it is thought to mark silent genes and heterochromatin. Nevertheless, the time course of appearance of these histone modifications, the regions in IgH they associate with, and their appearance independent of AID damage suggest that both modifications play a role in targeting CSR.

  10. Bivalent histone modifications during tooth development

    Institute of Scientific and Technical Information of China (English)

    Li-Wei Zheng; Bin-Peng Zhang; Ruo-Shi Xu; Xin Xu; Ling Ye; Xue-Dong Zhou

    2014-01-01

    Histone methylation is one of the most widely studied post-transcriptional modifications. It is thought to be an important epigenetic event that is closely associated with cell fate determination and differentiation. To explore the spatiotemporal expression of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) epigenetic marks and methylation or demethylation transferases in tooth organ development, we measured the expression of SET7, EZH2, KDM5B and JMJD3 via immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis in the first molar of BALB/c mice embryos at E13.5, E15.5, E17.5, P0 and P3, respectively. We also measured the expression of H3K4me3 and H3K27me3 with immunofluorescence staining. During murine tooth germ development, methylation or demethylation transferases were expressed in a spatial–temporal manner. The bivalent modification characterized by H3K4me3 and H3K27me3 can be found during the tooth germ development, as shown by immunofluorescence. The expression of SET7, EZH2 as methylation transferases and KDM5B and JMJD3 as demethylation transferases indicated accordingly with the expression of H3K4me3 and H3K27me3 respectively to some extent. The bivalent histone may play a critical role in tooth organ development via the regulation of cell differentiation.

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

    Science.gov (United States)

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

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

    OpenAIRE

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

    2002-01-01

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

  13. Selection and fine mapping of chromosome-specific cDNAs: application to human chromosome 1.

    Science.gov (United States)

    Mancini, M; Sala, C; Rivella, S; Toniolo, D

    1996-12-01

    We have developed a methodology for identification and fine mapping of chromosome-specific transcripts. Combining digestion of DNA with different restriction enzymes, ligation to "bubble" linkers, and PCR amplification from Alu and "bubble" primers, we have synthesized human chromosome 1-specific sequences from DNA of a somatic cell hybrid, A9Neol. After hybridization to human fetal brain cDNA, we could efficiently capture chromosome 1-specific cDNAs. The cDNAs were sequenced and used as probes in hybridizations to high-density filters containing the arrayed CEPH Mega-YAC library and to the arrayed cDNA library from infant brain made by B. Soares, which has been extensively sequenced. By this approach we have been able to select chromosome 1-specific cDNAs, to map them to chromosome 1 YAC contigs, and to identify and map corresponding longer cDNAs and ESTs.

  14. Structural insights into yeast histone chaperone Hif1: a scaffold protein recruiting protein complexes to core histones.

    Science.gov (United States)

    Liu, Hejun; Zhang, Mengying; He, Wei; Zhu, Zhongliang; Teng, Maikun; Gao, Yongxiang; Niu, Liwen

    2014-09-15

    Yeast Hif1 [Hat1 (histone acetyltransferase 1)-interacting factor], a homologue of human NASP (nuclear autoantigenic sperm protein), is a histone chaperone that is involved in various protein complexes which modify histones during telomeric silencing and chromatin reassembly. For elucidating the structural basis of Hif1, in the present paper we demonstrate the crystal structure of Hif1 consisting of a superhelixed TPR (tetratricopeptide repeat) domain and an extended acid loop covering the rear of TPR domain, which represent typical characteristics of SHNi-TPR [Sim3 (start independent of mitosis 3)-Hif1-NASP interrupted TPR] proteins. Our binding assay indicates that Hif1 could bind to the histone octamer via histones H3 and H4. The acid loop is shown to be crucial for the binding of histones and may also change the conformation of the TPR groove. By binding to the core histone complex Hif1 may recruit functional protein complexes to modify histones during chromatin reassembly.

  15. Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

    Indian Academy of Sciences (India)

    Chuan-Liang Deng; Wei-Li Zhang; Ying Cao; Shao-Jing Wang; Shu-Fen Li; Wu-Jun Gao; Long-Dou Lu

    2015-12-01

    The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related , m and . Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

  16. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Science.gov (United States)

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  17. Chromosome-specific segmentation revealed by structural analysis of individually isolated chromosomes.

    Science.gov (United States)

    Kitada, Kunio; Taima, Akira; Ogasawara, Kiyomoto; Metsugi, Shouichi; Aikawa, Satoko

    2011-04-01

    Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis.

  18. Data Mining Empowers the Generation of a Novel Class of Chromosome-specific DNA Probes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Hui; Weier, Heinz-Ulrich G.; Kwan, Johnson; Wang, Mei; O' Brien, Benjamin

    2011-03-08

    Probes that allow accurate delineation of chromosome-specific DNA sequences in interphase or metaphase cell nuclei have become important clinical tools that deliver life-saving information about the gender or chromosomal make-up of a product of conception or the probability of an embryo to implant, as well as the definition of tumor-specific genetic signatures. Often such highly specific DNA probes are proprietary in nature and have been the result of extensive probe selection and optimization procedures. We describe a novel approach that eliminates costly and time consuming probe selection and testing by applying data mining and common bioinformatics tools. Similar to a rational drug design process in which drug-protein interactions are modeled in the computer, the rational probe design described here uses a set of criteria and publicly available bioinformatics software to select the desired probe molecules from libraries comprised of hundreds of thousands of probe molecules. Examples describe the selection of DNA probes for the human X and Y chromosomes, both with unprecedented performance, but in a similar fashion, this approach can be applied to other chromosomes or species.

  19. Chromosome Specific Substitution Lines of Aegilops geniculata Alter Parameters of Bread Making Quality of Wheat

    Science.gov (United States)

    Tsujimoto, Hisashi; Gupta, Raj Kumar; Kumar, Aman; Kaur, Navneet; Kumar, Rohit; Chunduri, Venkatesh; Sharma, Nand Kishor; Chawla, Meenakshi; Sharma, Saloni; Mundey, Jaspreet Kaur

    2016-01-01

    Wheat cultivars with wide introgression have strongly impacted global wheat production. Aegilops geniculata (MgUg) is an important wild relative with several useful traits that can be exploited for wheat improvement. Screening of Ae. geniculata addition lines indicated a negative effect of 1Ug and the positive effect of 1Mg chromosome on wheat dough strength. Negative effect of 1Ug is probably associated with variation in number and position of the tripeptide repeat motif in the high molecular weight glutenin (HMW-G) gene. To utilize the positive potential of 1Mg chromosome, three disomic substitution lines (DSLs) 1Mg(1A), 1Mg(1B) and 1Mg(1D) were created. These lines were characterized for morphological, cytogenetic properties and biochemical signatures using FISH, 1D-, 2D-PAGE and RP-HPLC. Contribution of wheat 1A, 1B and 1D chromosomes towards dough mixing and baking parameters, chapatti quality, Fe/Zn content and glume color were identified. Observed order of variation in the dough mixing and baking parameters {1Mg(1D) ≤wheat ≤1Mg(1B) ≤1Mg(1A)} indicated that chromosome specific introgression is desirable for best utilization of wild species’ potential. PMID:27755540

  20. Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1

    OpenAIRE

    Musselman, Catherine A.; Avvakumov, Nikita; Watanabe, Reiko; Abraham, Christopher G.; Lalonde, Marie-Eve; Hong, Zehui; Allen, Christopher; Roy, Siddhartha; Nuñez, James K.; Nickoloff, Jac; Kulesza, Caroline A.; Yasui, Akira; Côté, Jacques; Kutateladze, Tatiana G.

    2012-01-01

    The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here, we demonstrate that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9 Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR analysis. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate H3K27 in vitro and in vivo....

  1. H3 internationalization to Brazil

    OpenAIRE

    2012-01-01

    A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics h3 is a Portuguese firm competing in the fast food market with a hamburger gourmet concept. The firm is now a strong player in Portugal (39 stores); Poland, Warsaw (1 store); Spain, Madrid (1 store) and Brazil, São Paulo (2 stores). As a result of their successes in the domestic market, internationalization was a natural step. One o...

  2. TALE-directed local modulation of H3K9 methylation shapes exon recognition.

    Science.gov (United States)

    Bieberstein, Nicole I; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K; Krchňáková, Zuzana; Krausová, Michaela; Carrillo Oesterreich, Fernando; Staněk, David

    2016-07-21

    In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

  3. Histone deacetylation is required for progression through mitosis in tobacco cells.

    Science.gov (United States)

    Li, Yan; Butenko, Yana; Grafi, Gideon

    2005-02-01

    Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.

  4. A histone demethylase is necessary for regeneration in zebrafish.

    Science.gov (United States)

    Stewart, Scott; Tsun, Zhi-Yang; Izpisua Belmonte, Juan Carlos

    2009-11-24

    Urodele amphibians and teleost fish regenerate amputated body parts via a process called epimorphic regeneration. A hallmark of this phenomenon is the reactivation of silenced developmental regulatory genes that previously functioned during embryonic patterning. We demonstrate that histone modifications silence promoters of numerous genes involved in zebrafish caudal fin regeneration. Silenced developmental regulatory genes contain bivalent me(3)K4/me(3)K27 H3 histone modifications created by the concerted action of Polycomb (PcG) and Trithorax histone methyltransferases. During regeneration, this silent, bivalent chromatin is converted to an active state by loss of repressive me(3)K27 H3 modifications, occurring at numerous genes that appear to function during regeneration. Loss-of-function studies demonstrate a requirement for a me(3)K27 H3 demethylase during fin regeneration. These results indicate that histone modifications at discreet genomic positions may serve as a crucial regulatory event in the initiation of fin regeneration.

  5. Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

    Science.gov (United States)

    Pickard, Amanda J.; Diaz, Anthony Joseph; Mura, Hugo; Nyuwen, Lila; Coello, Daniel; Sheva, Saif; Maria, Nava; Gallo, James M.; Wang, Tieli

    2017-01-01

    Background/Aim The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas –known as glioblastoma multiforme (GBM)– an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Materials and Methods Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Results Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. Conclusion The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment. PMID:27354585

  6. Mechanism of histone survival during transcription by RNA polymerase II.

    Science.gov (United States)

    Kulaeva, Olga I; Studitsky, Vasily M

    2010-01-01

    This work is related to and stems from our recent NSMB paper, "Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II" (December 2009). Synopsis. Recent genomic studies from many laboratories have suggested that nucleosomes are not displaced from moderately transcribed genes. Furthermore, histones H3/H4 carrying the primary epigenetic marks are not displaced or exchanged (in contrast to H2A/H2B histones) during moderate transcription by RNA polymerase II (Pol II) in vivo. These exciting observations suggest that the large molecule of Pol II passes through chromatin structure without even transient displacement of H3/H4 histones. The most recent analysis of the RNA polymerase II (Pol II)-type mechanism of chromatin remodeling in vitro (described in our NSMB 2009 paper) suggests that nucleosome survival is tightly coupled with formation of a novel intermediate: a very small intranucleosomal DNA loop (Ø-loop) containing transcribing Pol II. In the submitted manuscript we critically evaluate one of the key predictions of this model: the lack of even transient displacement of histones H3/H4 during Pol II transcription in vitro. The data suggest that, indeed, histones H3/H4 are not displaced during Pol II transcription in vitro. These studies are directly connected with the observation in vivo on the lack of exchange of histones H3/H4 during Pol II transcription.

  7. Polycomb Group Protein Displacement and Gene Activation through MSK-Dependent H3K27me3S28 Phosphorylation

    DEFF Research Database (Denmark)

    Gehani, Simmi Suman; Agrawal-Singh, Shuchi; Dietrich, Nikolaj;

    2010-01-01

    Epigenetic regulation of chromatin structure is essential for the expression of genes determining cellular specification and function. The Polycomb repressive complex 2 (PRC2) di- and trimethylates histone H3 on lysine 27 (H3K27me2/me3) to establish repression of specific genes in embryonic stem ...

  8. The Histone Deacetylase Complex 1 Protein of Arabidopsis Has the Capacity to Interact with Multiple Proteins Including Histone 3-Binding Proteins and Histone 1 Variants.

    Science.gov (United States)

    Perrella, Giorgio; Carr, Craig; Asensi-Fabado, Maria A; Donald, Naomi A; Páldi, Katalin; Hannah, Matthew A; Amtmann, Anna

    2016-05-01

    Intrinsically disordered proteins can adopt multiple conformations, thereby enabling interaction with a wide variety of partners. They often serve as hubs in protein interaction networks. We have previously shown that the Histone Deacetylase Complex 1 (HDC1) protein from Arabidopsis (Arabidopsis thaliana) interacts with histone deacetylases and quantitatively determines histone acetylation levels, transcriptional activity, and several phenotypes, including abscisic acid sensitivity during germination, vegetative growth rate, and flowering time. HDC1-type proteins are ubiquitous in plants, but they contain no known structural or functional domains. Here, we explored the protein interaction spectrum of HDC1 using a quantitative bimolecular fluorescence complementation assay in tobacco (Nicotiana benthamiana) epidermal cells. In addition to binding histone deacetylases, HDC1 directly interacted with histone H3-binding proteins and corepressor-associated proteins but not with H3 or the corepressors themselves. Surprisingly, HDC1 also was able to interact with variants of the linker histone H1. Truncation of HDC1 to the ancestral core sequence narrowed the spectrum of interactions and of phenotypic outputs but maintained binding to a H3-binding protein and to H1. Thus, HDC1 provides a potential link between H1 and histone-modifying complexes.

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

    Science.gov (United States)

    Ng, Marlee K; Cheung, Peter

    2016-02-01

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

  10. Drosophila KDM2 is a H3K4me3 demethylase regulating nucleolar organization

    Directory of Open Access Journals (Sweden)

    Birchler James A

    2009-10-01

    Full Text Available Abstract Background CG11033 (dKDM2 is the Drosophila homolog of the gene KDM2B. dKDM2 has been known to possess histone lysine demethylase activity towards H3K36me2 in cell lines and it regulates H2A ubiquitination. The human homolog of the gene has dual activity towards H3K36me2 as well as H3K4me3, and plays an important role in cellular senescence. Findings We have used transgenic flies bearing an RNAi construct for the dKDM2 gene. The knockdown of dKDM2 gene was performed by crossing UAS-RNAi-dKDM2 flies with actin-Gal4 flies. Western blots of acid extracted histones and immunofluoresence analysis of polytene chromosome showed the activity of the enzyme dKDM2 to be specific for H3K4me3 in adult flies. Immunofluoresence analysis of polytene chromosome also revealed the presence of multiple nucleoli in RNAi knockdown mutants of dKDM2 and decreased H3-acetylation marks associated with active transcription. Conclusion Our findings indicate that dKDM2 is a histone lysine demethylase with specificity for H3K4me3 and regulates nucleolar organization.

  11. File list: His.Bld.50.H3S10phK14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.50.H3S10phK14ac.AllCell mm9 Histone H3S10phK14ac Blood SRX759801,SRX759800 ...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.50.H3S10phK14ac.AllCell.bed ...

  12. Structural biology of human H3K9 methyltransferases.

    Directory of Open Access Journals (Sweden)

    Hong Wu

    Full Text Available UNLABELLED: SET domain methyltransferases deposit methyl marks on specific histone tail lysine residues and play a major role in epigenetic regulation of gene transcription. We solved the structures of the catalytic domains of GLP, G9a, Suv39H2 and PRDM2, four of the eight known human H3K9 methyltransferases in their apo conformation or in complex with the methyl donating cofactor, and peptide substrates. We analyzed the structural determinants for methylation state specificity, and designed a G9a mutant able to tri-methylate H3K9. We show that the I-SET domain acts as a rigid docking platform, while induced-fit of the Post-SET domain is necessary to achieve a catalytically competent conformation. We also propose a model where long-range electrostatics bring enzyme and histone substrate together, while the presence of an arginine upstream of the target lysine is critical for binding and specificity. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  13. HAMLET interacts with histones and chromatin in tumor cell nuclei.

    Science.gov (United States)

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

    2003-10-24

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

  14. File list: His.PSC.20.H3K9ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.PSC.20.H3K9ac.AllCell mm9 Histone H3K9ac Pluripotent stem cell SRX185844,SRX185...848,SRX873351,SRX873349,SRX873345,SRX097641,SRX130021,SRX873347,SRX187620 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.PSC.20.H3K9ac.AllCell.bed ...

  15. H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis.

    Science.gov (United States)

    Wang, Qian; Wei, Haojie; Du, Juan; Cao, Yan; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Chen, Dandan; Ma, Wei

    2016-01-01

    Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.

  16. Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

    Science.gov (United States)

    Ejlassi-Lassallette, Aïda; Thiriet, Christophe

    2012-02-01

    The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

  17. Quantitative proteomic analysis of post-translational modifications of human histones

    DEFF Research Database (Denmark)

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

    2006-01-01

    software for qualitative and quantitative proteomic analysis of histones extracted from human small cell lung cancer cells. A total of 32 acetylations, methylations, and ubiquitinations were located in the human histones H2A, H2B, H3, and H4, including seven novel modifications. An LC-MSMS-based method...... was applied in a quantitative proteomic study of the dose-response effect of the histone deacetylase inhibitor (HDACi) PXD101 on histone acetylation in human cell cultures. Triplicate LC-MSMS runs at six different HDACi concentrations demonstrated that PXD101 affects acetylation of histones H2A, H2B, H3...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post...

  18. 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.

  19. Readers of histone modifications

    Institute of Scientific and Technical Information of China (English)

    Miyong Yun; Jun Wu; Jerry L Workman; Bing Li

    2011-01-01

    Histone modifications not only play important roles in regulating chromatin structure and nuclear processes but also can be passed to daughter cells as epigenetic marks.Accumulating evidence suggests that the key function of histone modifications is to signal for recruitment or activity of downstream effectors. Here, we discuss the latest discovery of histone-modification readers and how the modification language is interpreted.

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

    Science.gov (United States)

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

    2011-07-13

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

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

    Directory of Open Access Journals (Sweden)

    Kan Rui

    2012-06-01

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

  2. Brd3 promotes IL-6 production via enhancing acetylase CBP recruitment and histone 3 acetylation within IL6 promoter%Brd3通过促进IL6基因启动子区乙酰基转移酶CBP的募集和组蛋白H3乙酰化修饰促进IL-6产生

    Institute of Scientific and Technical Information of China (English)

    任文汇; 孙东豪; 王春梅; 李楠

    2016-01-01

    目的 探索巨噬细胞中含bromodomain蛋白3(Brd3)对脂多糖(LPS)诱导的白细胞介素6(IL-6)产生的调控作用.方法 利用CRISPR-Cas9技术筛选出Brd3敲除的细胞,以Brd3敲除细胞为实验组,正常表达Brd3的细胞为对照组.100 ng/mL LPS刺激后ELISA检测细胞培养上清中IL-6的水平;采用Western blot法检测核因子κB(NF-κB)和丝裂原激活蛋白激酶(MAPK)信号通路的表达活化情况;染色质免疫沉淀实验检测IL6基因启动子区乙酰基转移酶CREB结合蛋白(CBP)的募集和组蛋白H3乙酰化修饰水平.结果 小鼠腹腔巨噬细胞中Brd3 mRNA和蛋白表达水平在LPS刺激后显著降低.与对照组相比,Brd3敲除细胞中LPS诱导产生的IL-6水平显著降低.NF-κB和MAPK信号通路相关分子的表达无差异;Brd3敲除细胞IL6启动子区乙酰基转移酶CBP的募集显著下降,组蛋白H3的乙酰化水平显著降低.结论 Brd3通过促进IL6启动子区乙酰基转移酶CBP的结合和组蛋白H3的乙酰化修饰,促进巨噬细胞中LPS触发的IL-6的产生.

  3. Jmjd3基因慢病毒干扰载体的构建及其去H3K27三甲基化作用研究%Construction of a Lentiviral Vector Targeting Jmjd3 Gene and Its Effect as a Histone Demethylase

    Institute of Scientific and Technical Information of China (English)

    吕娟; 孙荷; 张帆; 董学君

    2013-01-01

    目的 构建针对小鼠Jmjd3基因的RNAi慢病毒载体,感染小鼠骨髓间充质干细胞(mouse bone marrow mesenchymal stem cells,mBM-MSCs)后观察其对Jmjd3基因表达的影响及对组蛋白H3K27me3的去甲基化作用.方法 根据Jmjd3mRNA序列设计并合成3对shRNA寡核苷酸片段,退火形成双链并连接入PLL3.7慢病毒干扰载体,双酶切和DNA测序鉴定重组克隆,重组质粒与其他3种辅助包装质粒(pRsv-REV,pMDlg-pRRE,pMD2G)共转染293T细胞生产病毒液,病毒液感染mBM-MSCs,荧光定量PCR和Western blot检测目的基因Jmjd3的表达,筛选出最有效的干扰序列.Western blot检测组蛋白H3 K27 me3的表达量,分析Jmjd3的生物学功能.结果 双酶切和测序结果证实Jmjd3基因shRNA序列正确插入慢病毒载体.荧光定量PCR和Western blot结果显示,感染后Jmjd3表达显著下调,与空载体对照组相比,Jmjd3-shRNA1组、Jmjd3-shRNA2组和Jmjd3-shRNA3组分别下调88.9% (P <0.001)、67.9% (P <0.001)和72.4% (P <0.001),Jmjd3-shRNA1组为最佳干扰组.Western blot结果显示干扰Jmjd3之后,H3K27me3的表达较空载体对照组升高.结论 成功构建了针对小鼠Jmjd3基因的慢病毒干扰载体,并能有效抑制mBM-MSCs中Jmjd3基因的表达,Jmjd3具有组蛋白H3K27me3的去甲基化作用.

  4. Analysis of the Genes Encoding the Histones of Microsporidia Nosema bombycis

    Directory of Open Access Journals (Sweden)

    Liu Yang

    2013-02-01

    Full Text Available Histone proteins are essential components of eukaryotic chromosomes, the objective of the study is to provide some new insights into its evolution through analysis of N. bombycis Histone genes at genomic level. In the study, genes encoding core Histone H2A, H2B, H3 and H4 from Nosema bombycis were analyzed by multiple sequence alignments. Analysis showed that: each type of the core Histone genes, sharing high similarity with each other in both coding and non-coding regions, has low copy number. Multiple sequence alignments showed N. bombycis core Histones diverge obviously, relative-rate test revealed Histone proteins have accelerated in the evolutionary rate of amino acid substitution. The distance between the stop codon and consensus poly (A signal is compacted, no conserved hair-pin element was found in 3'-untranslated regions of Histone mRNAs and overlapping gene transcription was observed in the downstream region of Histone variant H3_3, that implies there maybe have only single class of core Histone genes encoding replication-independent Histones in N. bombycis. Surveying the upstream of the coding region of all core Histone genes, there were no canonical TATA or CAAT boxes except that a common Histone motif (TTTCCCTCC was discovered. Moreover, no similar Histone motif mentioned above existed in Encephalitozoon cuniculi, the closely related organisms. That means that similar Histone motif maybe exists in microsporidian last common ancestor, N. bombycis retained Histone motif, while E. cuniculi have lost Histone motif after the differentiation from the common ancestor with the change of the host. Therefore the analysis of the genes encoding the Histones ofN. bombycis revealed that there maybe have two evolution directions in microsporidia, that is, genome extreme compact and mild compact, during the course of evolution. It contributes us to have the knowledge of that there have different genome size in microsporidia and provide useful

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

    Science.gov (United States)

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

    2015-01-01

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

  6. Loss of the DNA methyltransferase MET1 Induces H3K9 hypermethylation at PcG target genes and redistribution of H3K27 trimethylation to transposons in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Angelique Deleris

    Full Text Available Dimethylation of histone H3 lysine 9 (H3K9m2 and trimethylation of histone H3 lysine 27 (H3K27m3 are two hallmarks of transcriptional repression in many organisms. In Arabidopsis thaliana, H3K27m3 is targeted by Polycomb Group (PcG proteins and is associated with silent protein-coding genes, while H3K9m2 is correlated with DNA methylation and is associated with transposons and repetitive sequences. Recently, ectopic genic DNA methylation in the CHG context (where H is any base except G has been observed in globally DNA hypomethylated mutants such as met1, but neither the nature of the hypermethylated loci nor the biological significance of this epigenetic phenomenon have been investigated. Here, we generated high-resolution, genome-wide maps of both H3K9m2 and H3K27m3 in wild-type and met1 plants, which we integrated with transcriptional data, to explore the relationships between these two marks. We found that ectopic H3K9m2 observed in met1 can be due to defects in IBM1-mediated H3K9m2 demethylation at some sites, but most importantly targets H3K27m3-marked genes, suggesting an interplay between these two silencing marks. Furthermore, H3K9m2/DNA-hypermethylation at these PcG targets in met1 is coupled with a decrease in H3K27m3 marks, whereas CG/H3K9m2 hypomethylated transposons become ectopically H3K27m3 hypermethylated. Our results bear interesting similarities with cancer cells, which show global losses of DNA methylation but ectopic hypermethylation of genes previously marked by H3K27m3.

  7. Relationship between gene body DNA methylation and intragenic H3K9me3 and H3K36me3 chromatin marks.

    Directory of Open Access Journals (Sweden)

    Maria A Hahn

    Full Text Available To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO. Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i genes with low CpG density and H3K9me3 in the gene body or (ii genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively.

  8. High-resolution genome-wide mapping of histone modifications.

    Science.gov (United States)

    Roh, Tae-young; Ngau, Wing Chi; Cui, Kairong; Landsman, David; Zhao, Keji

    2004-08-01

    The expression patterns of eukaryotic genomes are controlled by their chromatin structure, consisting of nucleosome subunits in which DNA of approximately 146 bp is wrapped around a core of 8 histone molecules. Post-translational histone modifications play an essential role in modifying chromatin structure. Here we apply a combination of SAGE and chromatin immunoprecipitation (ChIP) protocols to determine the distribution of hyperacetylated histones H3 and H4 in the Saccharomyces cerevisiae genome. We call this approach genome-wide mapping technique (GMAT). Using GMAT, we find that the highest acetylation levels are detected in the 5' end of a gene's coding region, but not in the promoter. Furthermore, we show that the histone acetyltransferase, GCN5p, regulates H3 acetylation in the promoter and 5' end of the coding regions. These findings indicate that GMAT should find valuable applications in mapping target sites of chromatin-modifying enzymes.

  9. Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides.

    Science.gov (United States)

    Fedoreyeva, L I; Smirnova, T A; Kolomijtseva, G Ya; Khavinson, V Kh; Vanyushin, B F

    2013-02-01

    Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2B, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind predominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.

  10. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    Science.gov (United States)

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  11. A model for transmission of the H3K27me3 epigenetic mark

    DEFF Research Database (Denmark)

    Hansen, Klaus H; Bracken, Adrian P; Pasini, Diego;

    2008-01-01

    Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division...... during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure...

  12. 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

  13. Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation.

    Science.gov (United States)

    Meiklejohn, Colin D; Landeen, Emily L; Cook, Jodi M; Kingan, Sarah B; Presgraves, Daven C

    2011-08-01

    The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females) has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation--the equalization of X chromosome gene expression in males and females--and meiotic sex chromosome inactivation (MSCI)--the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female) germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.

  14. Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Warburton, P.E.; Gosden, J.; Lawson, D. [Western General Hospital, Edinburgh (United Kingdom)] [and others

    1996-04-15

    Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.

  15. Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation.

    Directory of Open Access Journals (Sweden)

    Colin D Meiklejohn

    2011-08-01

    Full Text Available The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation--the equalization of X chromosome gene expression in males and females--and meiotic sex chromosome inactivation (MSCI--the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.

  16. Structural insight into histone recognition by the ING PHD fingers.

    Science.gov (United States)

    Champagne, Karen S; Kutateladze, Tatiana G

    2009-05-01

    The Inhibitor of Growth (ING) tumor suppressors are implicated in oncogenesis, control of DNA damage repair, cellular senescence and apoptosis. All members of the ING family contain unique amino-terminal regions and a carboxy-terminal plant homeodomain (PHD) finger. While the amino-terminal domains associate with a number of protein effectors including distinct components of histone deacetylase (HDAC) and histone acetyltransferase (HAT) complexes, the PHD finger binds strongly and specifically to histone H3 trimethylated at lysine 4 (H3K4me3). In this review we describe the molecular mechanism of H3K4me3 recognition by the ING1-5 PHD fingers, analyze the determinants of the histone specificity and compare the biological activities and structures within subsets of PHD fingers. The atomic-resolution structures of the ING PHD fingers in complex with a H3K4me3 peptide reveal that the histone tail is bound in a large and deep binding site encompassing nearly one-third of the protein surface. An extensive network of intermolecular hydrogen bonds, hydrophobic and cation-pi contacts, and complementary surface interactions coordinate the first six residues of the H3K4me3 peptide. The trimethylated Lys4 occupies an elongated groove, formed by the highly conserved aromatic and hydrophobic residues of the PHD finger, whereas the adjacent groove accommodates Arg2. The two grooves are connected by a narrow channel, the small size of which defines the PHD finger's specificity, excluding interactions with other modified histone peptides. Binding of the ING PHD fingers to H3K4me3 plays a critical role in regulating chromatin acetylation. The ING proteins function as tethering molecules that physically link the HDAC and HAT enzymatic complexes to chromatin. In this review we also highlight progress recently made in understanding the molecular basis underlying biological and tumorigenic activities of the ING tumor suppressors.

  17. 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.

  18. Curcumin-induced Histone Acetylation in Malignant Hematologic Cells

    Institute of Scientific and Technical Information of China (English)

    Junbin HU; Yan WANG; Yan CHEN

    2009-01-01

    This study investigated the inhibitory effects of curcumin on proliferation of hemato-logical malignant cells in vitro and the anti-tumor mechanism at histone acetylation/histone deacety-lation levels.The effects of curcumin and histone deacetylase inhibitor trichostatin A (TSA) on the growth of Raji cells were tested by MTT assay.The expression of acetylated histone-3 (H3) in Raji,HL60 and K562 cells,and peripheral blood mononuclear cells (PBMCs) treated with curcumin or TSA was detected by immunohistochemistry and FACS.The results showed curcumin inhibited pro-liferation of Raji cells significantly in a time- and dose-dependent fashion,while exhibited low toxic-ity in PBMCs.Curcumin induced up-regulation of the expression of acetylated H3 dose-dependently in all malignant cell lines tested.In conclusion,curcumin inhibited proliferation of Raji cells selec-tively,enhanced the level of acetylated H3 in Raji,HL60,and K562 cells,which acted as a histone deacetylase inhibitor like TSA.Furthermore,up-regulation of H3 acetylation may play an important role in regulating the proliferation of Raji cells.

  19. Histone chaperone-mediated nucleosome assembly process.

    Science.gov (United States)

    Fan, Hsiu-Fang; Liu, Zi-Ning; Chow, Sih-Yao; Lu, Yi-Han; Li, Hsin

    2015-01-01

    A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1's specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.

  20. Histone variants of the insect Plodia interpunctella during metamorphosis.

    Science.gov (United States)

    Pataryas, T A; Sekeri-Pataryas, K T; Bonner, W M; Marinou, V A

    1984-01-01

    The pattern of histone variants from the meal moth Plodia interpunctella was compared to the mouse histone variant pattern. Plodia contains histones which comigrate on two dimensional gels with H3.2, H3.3, H4 and H2A.Z in mouse. Plodia H2A.1 and H2B.1 migrate somewhat differently from the respective mouse histones. Comparison of the iodinated tryptic peptides of H2A.1 and H2A.Z from mouse and Plodia showed that the H2A.Z proteins have two iodinated peptides that comigrate in the two species and three more that are different. The H2A.1 proteins in the two species have one iodinated peptide which comigrates and two more which migrate very close to each other. The histone variants from three developmental stages, larval, pupal and adult of Plodia interpunctella were also identified and compared. The same histone variant pattern is found through all stages of development. It is concluded that histone gene expression does not change during metamorphosis in Plodia .

  1. NICKEL COMPOUNDS INDUCE HISTONE UBIQUITINATION BY INHIBITING HISTONE DEUBIQUITINATING ENZYME ACTIVITY

    OpenAIRE

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

    2007-01-01

    Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of c...

  2. 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.

  3. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation.

    Science.gov (United States)

    Karagianni, Panagiota; Amazit, Larbi; Qin, Jun; Wong, Jiemin

    2008-01-01

    Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

  4. 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.

  5. H3N2v and You

    Science.gov (United States)

    ... H3N2)v Virus Infection Interim Guidance on Case Definitions to be Used For Investigations of Influenza A (H3N2)v Virus Cases Guidance for Public Health Other Guidance Documents Supplemental Interim Guidance for School Administrators Issues for Fair Organizers to Consider When ...

  6. Detection of extragalactic H3O+

    NARCIS (Netherlands)

    van der Tak, F. F. S.; Aalto, S.; Meijerink, R.

    2008-01-01

    Context. The H3O+ molecule probes the oxygen chemistry and the ionization rate of dense circumnuclear gas in galaxies. Aims. Recent H3O+ observations show variations in the cosmic-ray ionization rate, by factors of >10 within our Galaxy. Methods. Using the JCMT, we have observed the 364 GHz line of

  7. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Jensen, Michael R

    2004-01-01

    SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show tha...

  8. Development of one set of chromosome-specific microsatellite-containing BACs and their physical mapping in Gossypium hirsutum L.

    Science.gov (United States)

    Wang, Kai; Guo, Wangzhen; Zhang, Tianzhen

    2007-09-01

    Fluorescence in situ hybridization (FISH), using bacterial artificial chromosome (BAC) clone as probe, is a reliable cytological technique for chromosome identification. It has been used in many plants, especially in those containing numerous small chromosomes. We previously developed eight chromosome-specific BAC clones from tetraploid cotton, which were used as excellent cytological markers for chromosomes identification. Here, we isolated the other chromosome-specific BAC clones to make a complete set for the identification of all 26 chromosome-pairs by this technology in tetraploid cotton (Gossypium hirsutum L.). This set of BAC markers was demonstrated to be useful to assign each chromosome to a genetic linkage group unambiguously. In addition, these BAC clones also served as convenient and reliable landmarks for establishing physical linkage with unknown targeted sequences. Moreover, one BAC containing an EST, with high sequence similarity to a G. hirsutum ethylene-responsive element-binding factor was located physically on the long arm of chromosome A7 with the help of a chromosome-A7-specific BAC FISH marker. Comparative analysis of physical marker positions in the chromosomes by BAC-FISH and genetic linkage maps demonstrated that most of the 26 BAC clones were localized close to or at the ends of their respective chromosomes, and indicated that the recombination active regions of cotton chromosomes are primarily located in the distal regions. This technology also enables us to make associations between chromosomes and their genetic linkage groups and re-assign each chromosome according to the corresponding genetic linkage group. This BAC clones and BAC-FISH technology will be useful for us to evaluate grossly the degree to which a linkage map provides adequate coverage for developing a saturated genetic map, and provides a powerful resource for cotton genomic researches.

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

    Science.gov (United States)

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

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

  10. Heterochromatin marks HP1γ, HP1α and H3K9me3, and DNA damage response activation in human testis development and germ cell tumours

    DEFF Research Database (Denmark)

    Bartkova, J; Moudry, P; Hodny, Z;

    2011-01-01

    -stage-associated patterns of key heterochromatin marks: histone H3 trimethylated at lysine 9 (H3K9me3) and heterochromatic adaptor proteins HP1α and HP1γ, compared with the γH2AX marker of endogenously activated DNA damage response (DDR) and proliferation markers in normal human foetal (n=4) and adult (n=29) testes, pre...

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

    Directory of Open Access Journals (Sweden)

    Zee Barry M

    2010-12-01

    peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants. Conclusions Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.

  12. Overexpression of several Arabidopsis histone genes increases agrobacterium-mediated transformation and transgene expression in plants.

    Science.gov (United States)

    Tenea, Gabriela N; Spantzel, Joerg; Lee, Lan-Ying; Zhu, Yanmin; Lin, Kui; Johnson, Susan J; Gelvin, Stanton B

    2009-10-01

    The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double- or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation.

  13. Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism.

    Directory of Open Access Journals (Sweden)

    Mayumi Nakahara

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

  14. Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

    Science.gov (United States)

    Ishikawa, Katsuyuki; Ohsumi, Tatsuya; Tada, Shusuke; Natsume, Ryo; Kundu, Lena Rani; Nozaki, Naohito; Senda, Toshiya; Enomoto, Takemi; Horikoshi, Masami; Seki, Masayuki

    2011-10-01

    The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.

  15. A model for transmission of the H3K27me3 epigenetic mark.

    Science.gov (United States)

    Hansen, Klaus H; Bracken, Adrian P; Pasini, Diego; Dietrich, Nikolaj; Gehani, Simmi S; Monrad, Astrid; Rappsilber, Juri; Lerdrup, Mads; Helin, Kristian

    2008-11-01

    Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Financing exponential growth at H3

    OpenAIRE

    2012-01-01

    H3 is a fast-food chain that introduced the concept of gourmet hamburgers in the Portuguese market. This case-study illustrates its financing strategy that supported an exponential growth represented by opening 33 restaurants within approximately 3 years of its inception. H3 is now faced with the challenge of structuring its foreign ventures and change its financial approach. The main covered topics are the options an entrepreneur has for financing a new venture and how it evolves along th...

  18. Histone 3 s10 phosphorylation: "caught in the R loop!".

    Science.gov (United States)

    Skourti-Stathaki, Konstantina; Proudfoot, Nicholas J

    2013-11-21

    In this issue of Molecular Cell, Castellano-Pozo et al. (2013) describe a connection between R loop structures and histone 3 S10 phosphorylation (H3S10P), a mark of chromatin compaction. Their results constitute a significant advance in our understanding of the role of R loops in genomic instability.

  19. Histone Variants in Development and Diseases

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Jicheng Zhao; Guohong Li

    2013-01-01

    Eukaryotic genomic DNA is highly packaged into chromatin by histones to fit inside the nucleus.Other than the bulk packaging role of canonical histones with an expression peak at S phase and replication-coupled deposition,different histone variants have evolved distinct regulatory mechanisms for their expression,deposition and functional implications.The diversity of histone variants results in structural plasticity of chromatin and highlights functionally distinct chromosomal domain,which plays critical roles in development from a fertilized egg into a complex organism,as well as in aging and diseases.However,the mechanisms of this fundamental process are poorly understood so far.It is of particular interest to investigate how the variants are incorporated into chromatin and mark specific chromatin states to regulate gene expression,and how they are involved in development and diseases.In this review,we focus on recent progress in studies of epigenetic regulation of three extensively investigated variants including H2A.Z,macroH2A and H3.3,and their functional implications in development and diseases.

  20. File list: His.ALL.20.H3S10phK14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.20.H3S10phK14ac.AllCell mm9 Histone H3S10phK14ac All cell types SRX759801,S...RX759800 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.ALL.20.H3S10phK14ac.AllCell.bed ...

  1. File list: His.ALL.50.H3S10phK14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.50.H3S10phK14ac.AllCell mm9 Histone H3S10phK14ac All cell types SRX759801,S...RX759800 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.ALL.50.H3S10phK14ac.AllCell.bed ...

  2. File list: His.ALL.05.H3S10phK14ac.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.ALL.05.H3S10phK14ac.AllCell mm9 Histone H3S10phK14ac All cell types SRX759801,S...RX759800 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.ALL.05.H3S10phK14ac.AllCell.bed ...

  3. The conformation alteration of mouse hepatic histones after reacting with nicotine in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    UV differential spectroscopy, fluorescence spectroscopy and circular dichroism (CD) spectroscopy assays have been applied to studying the conformation alteration of mouse hepatic histones H1 and H3 after reacting with nicotine in vitro. The results indicate that their conformation changes from regular form to random form with the increasing reaction dose of nicotine. The adduction of nicotine or its metabolites with histones H1 and H3 accounts for the conformation alteration. Nicotine may affect the structure, function and expression of genes of chromosome by changing the conformation of histones.

  4. 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

  5. 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

  6. 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...

  7. Prepatterning of developmental gene expression by modified histones before zygotic genome activation

    DEFF Research Database (Denmark)

    Lindeman, Leif C.; Andersen, Ingrid S.; Reiner, Andrew H.

    2011-01-01

    A hallmark of anamniote vertebrate development is a window of embryonic transcription-independent cell divisions before onset of zygotic genome activation (ZGA). Chromatin determinants of ZGA are unexplored; however, marking of developmental genes by modified histones in sperm suggests a predictive...... role of histone marks for ZGA. In zebrafish, pre-ZGA development for ten cell cycles provides an opportunity to examine whether genomic enrichment in modified histones is present before initiation of transcription. By profiling histone H3 trimethylation on all zebrafish promoters before and after ZGA...

  8. Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  9. 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.

  10. Reversible Regulation of Promoter and Enhancer Histone Landscape by DNA Methylation in Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Andrew D. King

    2016-09-01

    Full Text Available DNA methylation is one of a number of modes of epigenetic gene regulation. Here, we profile the DNA methylome, transcriptome, and global occupancy of histone modifications (H3K4me1, H3K4me3, H3K27me3, and H3K27ac in a series of mouse embryonic stem cells (mESCs with varying DNA methylation levels to study the effects of DNA methylation on deposition of histone modifications. We find that genome-wide DNA demethylation alters occupancy of histone modifications at both promoters and enhancers. This is reversed upon remethylation by Dnmt expression. DNA methylation promotes H3K27me3 deposition at bivalent promoters, while opposing H3K27me3 at silent promoters. DNA methylation also reversibly regulates H3K27ac and H3K27me3 at previously identified tissue-specific enhancers. These effects require DNMT catalytic activity. Collectively, our data show that DNA methylation is essential and instructive for deposition of specific histone modifications across regulatory regions, which together influences gene expression patterns in mESCs.

  11. 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.

  12. 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.

  13. A comparison of control samples for ChIP-seq of histone modifications.

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    Flensburg, Christoffer; Kinkel, Sarah A; Keniry, Andrew; Blewitt, Marnie E; Oshlack, Alicia

    2014-01-01

    The advent of high-throughput sequencing has allowed genome wide profiling of histone modifications by Chromatin ImmunoPrecipitation (ChIP) followed by sequencing (ChIP-seq). In this assay the histone mark of interest is enriched through a chromatin pull-down assay using an antibody for the mark. Due to imperfect antibodies and other factors, many of the sequenced fragments do not originate from the histone mark of interest, and are referred to as background reads. Background reads are not uniformly distributed and therefore control samples are usually used to estimate the background distribution at any given genomic position. The Encyclopedia of DNA Elements (ENCODE) Consortium guidelines suggest sequencing a whole cell extract (WCE, or "input") sample, or a mock ChIP reaction such as an IgG control, as a background sample. However, for a histone modification ChIP-seq investigation it is also possible to use a Histone H3 (H3) pull-down to map the underlying distribution of histones. In this paper we generated data from a hematopoietic stem and progenitor cell population isolated from mouse fetal liver to compare WCE and H3 ChIP-seq as control samples. The quality of the control samples is estimated by a comparison to pull-downs of histone modifications and to expression data. We find minor differences between WCE and H3 ChIP-seq, such as coverage in mitochondria and behavior close to transcription start sites. Where the two controls differ, the H3 pull-down is generally more similar to the ChIP-seq of histone modifications. However, the differences between H3 and WCE have a negligible impact on the quality of a standard analysis.

  14. A comparison of control samples for ChIP-seq of histone modifications

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    Christoffer eFlensburg

    2014-09-01

    Full Text Available The advent of high-throughput sequencing has allowed genome wide profiling of histone modifications by Chromatin ImmunoPrecipitation (ChIP followed by sequencing (ChIP-seq. In this assay the histone mark of interest is enriched through a chromatin pull-down assay using an antibody for the mark. Due to imperfect antibodies and other factors, many of the sequenced fragments do not originate from the histone mark of interest, and are referred to as background reads. Background reads are not uniformly distributed and therefore control samples are usually used to estimate the background distribution at any given genomic position. The Encyclopedia of DNA Elements (ENCODE Consortium guidelines suggest sequencing a whole cell extract (WCE, or input sample, or a mock ChIP reaction such as an IgG control, as a background sample. However, for a histone modification ChIP-seq investigation it is also possible to use a Histone H3 (H3 pull-down to map the underlying distribution of histones.In this paper we generated data from a hematopoietic stem and progenitor cell population isolated from mouse foetal liver to compare WCE and H3 ChIP-seq as control samples. The quality of the control samples is estimated by a comparison to pull-downs of histone modifications and to expression data. We find minor differences between WCE and H3 ChIP-seq, such as coverage in mitochondria and behaviour close to transcription start sites. Where the two controls differ, the H3 pull-down is generally more similar to the ChIP-seq of histone modifications. However, the differences between H3 and WCE have a negligible impact on the quality of a standard analysis.

  15. Use of laser microdissection for the construction of Humulus japonicus Siebold et Zuccarini, 1846 (Cannabaceae sex chromosome-specific DNA library and cytogenetics analysis

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    Nickolay Yakovin

    2014-12-01

    Full Text Available Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2 of Humulus japonicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of H. japonicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR. Fast fluorescence in situ hybridization (FAST-FISH using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, H. japonicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to H. lupulus and 88% to Cannabis sativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of H. japonicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in H. japonicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction.

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

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    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.

  17. A PHD in histone language: on the role of histone methylation in plant responses to phosphate deficiency.

    Science.gov (United States)

    Chandrika, Nulu Naga Prafulla; Sundaravelpandian, Kalaipandian; Schmidt, Wolfgang

    2013-06-01

    Post-translational modifications of core histones are important for various DNA-templated processes such as transcription and repair. We recently reported that the ALFIN LIKE 6 (AL6) gene, identified in a forward genetic screen, is critical for phosphate deficiency-induced root hair formation and several other processes associated with the regulation of cellular phosphate homeostasis. AL6 contains a Plant Homeo Domain (PHD) finger that can bind to trimethylated lysine 4 of histone H3 (H3K4me3). Homozygous mutants defective in AL6 expression form very short root hairs under phosphate-deficient conditions, presumably caused by altered expression of putative primary and secondary down-stream targets of AL6. In this Addendum, we speculate about possible roles of AL6, H3K4 trimethylation and other chromatin modifications in the adaptation of plants to low phosphate availability.

  18. CenH3 evolution reflects meiotic symmetry as predicted by the centromere drive model.

    Science.gov (United States)

    Zedek, František; Bureš, Petr

    2016-09-15

    The centromere drive model explaining rapid evolution of eukaryotic centromeres predicts higher frequency of positive selection acting on centromeric histone H3 (CenH3) in clades with asymmetric meiosis compared to the clades with only symmetric meiosis. However, despite the impression one might get from the literature, this key prediction of the centromere drive model has not only never been confirmed, but it has never been tested, because all the previous studies dealt only with the presence or absence instead of the frequency of positive selection. To provide evidence for or against different frequencies of positively selected CenH3 in asymmetrics and symmetrics, we have inferred the selective pressures acting on CenH3 in seventeen eukaryotic clades, including plants, animals, fungi, ciliates and apicomplexa, using codon-substitution models, and compared the inferred frequencies between asymmetrics and symmetrics in a quantitative manner. We have found that CenH3 has been evolving adaptively much more frequently in clades with asymmetric meiosis compared with clades displaying only symmetric meiosis which confirms the prediction of centromere drive model. Our findings indicate that the evolution of asymmetric meiosis required CenH3 to evolve adaptively more often to counterbalance the negative consequences of centromere drive.

  19. Histone demethylase Jumonji D3 (JMJD3 as a tumor suppressor by regulating p53 protein nuclear stabilization.

    Directory of Open Access Journals (Sweden)

    Chibawanye I Ene

    Full Text Available Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3 resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs, where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation and chromatin independent (nuclear p53 protein stabilization mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

  20. 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.

  1. Diverse histone modifications on histone 3 lysine 9 and their relation to DNA methylation in specifying gene silencing

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    Wu Yue-Zhong

    2007-05-01

    Full Text Available Abstract Background Previous studies of individual genes have shown that in a self-enforcing way, dimethylation at histone 3 lysine 9 (dimethyl-H3K9 and DNA methylation cooperate to maintain a repressive mode of inactive genes. Less clear is whether this cooperation is generalized in mammalian genomes, such as mouse genome. Here we use epigenomic tools to simultaneously interrogate chromatin modifications and DNA methylation in a mouse leukemia cell line, L1210. Results Histone modifications on H3K9 and DNA methylation in L1210 were profiled by both global CpG island array and custom mouse promoter array analysis. We used chromatin immunoprecipitation microarray (ChIP-chip to examine acetyl-H3K9 and dimethyl-H3K9. We found that the relative level of acetyl-H3K9 at different chromatin positions has a wider range of distribution than that of dimethyl-H3K9. We then used differential methylation hybridization (DMH and the restriction landmark genome scanning (RLGS to analyze the DNA methylation status of the same targets investigated by ChIP-chip. The results of epigenomic profiling, which have been independently confirmed for individual loci, show an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing. In contrast to the previous notion, dimethyl-H3K9 seems to be less distinct in specifying silencing for the genes tested. Conclusion This study demonstrates in L1210 leukemia cells a diverse relationship between histone modifications and DNA methylation in the maintenance of gene silencing. Acetyl-H3K9 shows an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing as expected. However, dimethyl-H3K9 seems to be less distinct in relation to promoter methylation. Meanwhile, a combination of epigenomic tools is of help in understanding the heterogeneity of epigenetic regulation, which may further our vision accumulated from single-gene studies.

  2. Ash2 acts as an ecdysone receptor coactivator by stabilizing the histone methyltransferase Trr.

    Science.gov (United States)

    Carbonell, Albert; Mazo, Alexander; Serras, Florenci; Corominas, Montserrat

    2013-02-01

    The molting hormone ecdysone triggers chromatin changes via histone modifications that are important for gene regulation. On hormone activation, the ecdysone receptor (EcR) binds to the SET domain-containing histone H3 methyltransferase trithorax-related protein (Trr). Methylation of histone H3 at lysine 4 (H3K4me), which is associated with transcriptional activation, requires several cofactors, including Ash2. We find that ash2 mutants have severe defects in pupariation and metamorphosis due to a lack of activation of ecdysone-responsive genes. This transcriptional defect is caused by the absence of the H3K4me3 marks set by Trr in these genes. We present evidence that Ash2 interacts with Trr and is required for its stabilization. Thus we propose that Ash2 functions together with Trr as an ecdysone receptor coactivator.

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

    Science.gov (United States)

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

    2015-02-01

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

  4. The development of 7E chromosome-specific molecular markers for Thinopyrum elongatum based on SLAF-seq technology.

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    Shiqiang Chen

    Full Text Available Thinopyrum elongatum is an important relative of wheat, it is favored by many researchers for the disease resistant genes that exist in its E genome. Some studies have showed that the 7E chromosome of Th. elongatum contains resistance genes related to Fusarium head blight and wheat rust. Therefore, developing 7E chromosome-specific molecular markers linked to resistance genes will provide an important tool for exploring and using the resistant genes of Th. elongatum. In addition, it would greatly contribute in the effort to cultivate disease-resistant wheat varieties. Featured in high throughput, high-accuracy and low-cost, SLAF-seq technology has been widely used in molecular breeding, system evolution, and germplasm resource detection. Based on SLAF-seq, 518 specific fragments on the 7E chromosome of Th. elongatum were successfully amplified. A total of 135 primers were designed according to 135 randomly selected fragments, and 89 specific molecular markers of Th. elongatum were developed, with efficiencies up to 65.9%. These markers were all detected in a variety of materials, and they are all proved to be specific and stable. These markers can be used not only for detecting the 7E chromosome of Th. elongatum but also for providing an important theoretical and practical basis for wheat breeding by marker-assisted selection (MAS. This paper reports the first application of SLAF-seq technology with a high success rate in developing specific molecular markers for Th. elongatum, providing a strong case for the application of this new technology.

  5. Syndrome-related chromosome-specific radiation-induced break points of various inherited human metabolic disorders.

    Science.gov (United States)

    Radha, S; Marimuthu, K M

    2003-07-08

    The frequency, distribution pattern and localisation of gamma radiation-induced break points on the chromosomes of patients with various inherited metabolic disorders were studied to detect: (i) whether the break point distribution following irradiation is random and proportional to the length or the DNA content of the chromosome, or non-proportionally distributed on their length and at times clustering to form hot spots on certain region of the chromosomes; and (ii) to find whether there exists a syndrome-related chromosome-specific pattern of radiation-induced break points. Lymphocyte cultures from patients of haemophilia, ichthyosis, Duchenne muscular dystrophy, retinitis pigmentosa and alpha-thalassemia, whose defective gene loci were located by DNA probe method, were subjected to 3Gy of gamma radiation at G(0). The chromosomal break point analysis was carried out on all the 23 types of chromosomes (excluding Y chromosome) using G banding and FISH painting. The exact location of the break points on G-banded chromosomes was identified using a semi-automated microscope densitometer system (Leitz MPV2). In normal individuals in all the chromosomes except the chromosome 1, a random distribution of break points proportional to their length based on their DNA content was observed. However, in all the syndromes studied a mixture of hypersensitive chromosomes with a non-random distribution pattern of chromosomal break points invariably clustering to form hot spots, and chromosomes with random distribution of break points proportional to their length were observed. The hypersensitive chromosomes and their hot spots were syndrome-specific.

  6. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.

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    Michael Brauchle

    Full Text Available Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.

  7. Structural and Histone Binding Ability Characterizations of Human PWWP Domains

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

    2013-09-25

    The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

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

    Science.gov (United States)

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

    2012-01-01

    Methyl-CpG-binding protein 2 (MeCP2) is a chromatin-binding protein that mediates transcriptional regulation, and is highly abundant in brain. The nature of its binding to reconstituted templates has been well characterized in vitro. However, its interactions with native chromatin are less understood. Here we show that MeCP2 displays a distinct distribution within fractionated chromatin from various tissues and cell types. Artificially induced global changes in DNA methylation by 3-aminobenzamide or 5-aza-2′-deoxycytidine, do not significantly affect the distribution or amount of MeCP2 in HeLa S3 or 3T3 cells. Most MeCP2 in brain is chromatin-bound and localized within highly nuclease-accessible regions. We also show that, while in most tissues and cell lines, MeCP2 forms stable complexes with nucleosome, in brain, a fraction of it is loosely bound to chromatin, likely to nucleosome-depleted regions. Finally, we provide evidence for novel associations of MeCP2 with mononucleosomes containing histone H2A.X, H3K9me2 and H3K27me3 in different chromatin fractions from brain cortex and in vitro. We postulate that the functional compartmentalization and tissue-specific distribution of MeCP2 within different chromatin types may be directed by its association with nucleosomes containing specific histone variants, and post-translational modifications. PMID:22144686

  9. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    OpenAIRE

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been u...

  10. Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

    Science.gov (United States)

    Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

    2015-11-01

    The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified.

  11. Suite of experiments in beam H3

    CERN Multimedia

    1977-01-01

    The H3 beam runs from bottom to top, in the middle of the photo. On its left preparation is going on for WA10 in beam H5, on its right, behind the concrete wall, the S3 beam runs to BEBC. The H3 beam first meets the IKAR target of the experiment WA9 (right, bottom corner) then crosses the polarized hydrogen target of experiment WA6 sitting inside the large green magnet (centre, also visible the yellow support of the recoil chamber telescope). Finally the beam reaches WA11 and the Goliath magnet (under the white passerelle). The WA9 (left) and the WA5/11 (right) huts stand on top of the photo, the WA6 stands at the right, centre. The three experiments were running in alternance.

  12. Inhibition of H3K4me2 Demethylation Protects Auditory Hair Cells from Neomycin-Induced Apoptosis.

    Science.gov (United States)

    He, Yingzi; Yu, Huiqian; Cai, Chengfu; Sun, Shan; Chai, Renjie; Li, Huawei

    2015-08-01

    Aminoglycoside-induced hair cell loss is a major cause of hearing impairment in children and deserves more attention in medical research. Epigenetic mechanisms have been shown to protect hair cells from ototoxic drugs. In this study, we focused on the role of dimethylated histone H3K4 (H3K4me2) in hair cell survival. To investigate the effects of lysine-specific demethylase 1 (LSD1)--the histone demethylase primarily responsible for demethylating H3K4me2--on neomycin-induced hair cell loss, isolated cochleae were pretreated with LSD1 inhibitors followed by neomycin exposure. There was a severe loss of hair cells in the organ of Corti after neomycin exposure, and inhibition of LSD1 significantly protected against neomycin-induced hair cell loss. H3K4me2 expression in the nuclei of hair cells decreased after exposure to neomycin, and blocking the decreased expression of H3K4me2 with LSD1 inhibitors prevented hair cell loss. Local delivery of these inhibitors in vivo also protected hair cells from neomycin-induced ototoxicity and maintained the hearing threshold in mice as determined by auditory brain stem response. This inhibition of neomycin-induced apoptosis occurs via reduced caspase-3 activation. Together, our findings demonstrate the protective role for H3K4me2 against neomycin-induced hair cell loss and hearing loss.

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

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    Tomaru Yasuhiro

    2005-08-01

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

  14. Histone variants and lipid metabolism

    NARCIS (Netherlands)

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

    2014-01-01

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

  15. A Phosphotyrosine Switch Controls the Association of Histone Mark Readers with Methylated Proteins.

    Science.gov (United States)

    Irving-Hooper, Bronwyn Kate; Binda, Olivier

    2016-03-22

    Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information.

  16. Quantitative Assessment of Chromatin Immunoprecipitation Grade Antibodies Directed against Histone Modifications Reveals Patterns of Co-occurring Marks on Histone Protein Molecules*

    Science.gov (United States)

    Peach, Sally E.; Rudomin, Emily L.; Udeshi, Namrata D.; Carr, Steven A.; Jaffe, Jacob D.

    2012-01-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the “histone code.” We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to “canonical” chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living

  17. HP1 recruits activity-dependent neuroprotective protein to H3K9me3 marked pericentromeric heterochromatin for silencing of major satellite repeats.

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    Kerstin Mosch

    Full Text Available H3 lysine 9 trimethylation (H3K9me3 is a histone posttranslational modification (PTM that has emerged as hallmark of pericentromeric heterochromatin. This constitutive chromatin domain is composed of repetitive DNA elements, whose transcription is differentially regulated. Mammalian cells contain three HP1 proteins, HP1α, HP1β and HP1γ These have been shown to bind to H3K9me3 and are thought to mediate the effects of this histone PTM. However, the mechanisms of HP1 chromatin regulation and the exact functional role at pericentromeric heterochromatin are still unclear. Here, we identify activity-dependent neuroprotective protein (ADNP as an H3K9me3 associated factor. We show that ADNP does not bind H3K9me3 directly, but that interaction is mediated by all three HP1 isoforms in vitro. However, in cells ADNP localization to areas of pericentromeric heterochromatin is only dependent on HP1α and HP1β. Besides a PGVLL sequence patch we uncovered an ARKS motif within the ADNP homeodomain involved in HP1 dependent H3K9me3 association and localization to pericentromeric heterochromatin. While knockdown of ADNP had no effect on HP1 distribution and heterochromatic histone and DNA modifications, we found ADNP silencing major satellite repeats. Our results identify a novel factor in the translation of H3K9me3 at pericentromeric heterochromatin that regulates transcription.

  18. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

    Energy Technology Data Exchange (ETDEWEB)

    Kruidenier, Laurens; Chung, Chun-wa; Cheng, Zhongjun; Liddle, John; Che, KaHing; Joberty, Gerard; Bantscheff, Marcus; Bountra, Chas; Bridges, Angela; Diallo, Hawa; Eberhard, Dirk; Hutchinson, Sue; Jones, Emma; Katso, Roy; Leveridge, Melanie; Mander, Palwinder K.; Mosley, Julie; Ramirez-Molina, Cesar; Rowland, Paul; Schofield, Christopher J.; Sheppard, Robert J.; Smith, Julia E.; Swales, Catherine; Tanner, Robert; Thomas, Pamela; Tumber, Anthony; Drewes, Gerard; Oppermann, Udo; Patel, Dinshaw J.; Lee, Kevin; Wilson, David M. (Cellzome AG); (MSKCC); (GSK); (Oxford)

    2012-10-11

    The jumonji (JMJ) family of histone demethylases are Fe{sup 2+}- and {alpha}-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

  19. Re-patterning of H3K27me3, H3K4me3 and DNA methylation during fibroblast conversion into induced cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ziqing Liu

    2016-03-01

    Full Text Available Direct conversion of fibroblasts into induced cardiomyocytes (iCMs offers an alternative strategy for cardiac disease modeling and regeneration. During iCM reprogramming, the starting fibroblasts must overcome existing epigenetic barriers to acquire the CM-like chromatin pattern. However, epigenetic dynamics along this reprogramming process have not been studied. Here, we took advantage of our recently generated polycistronic system and determined the dynamics of two critical histone marks, H3K27me3 and H3K4me3, in parallel with gene expression at a set of carefully selected cardiac and fibroblast loci during iCM reprogramming. We observed reduced H3K27me3 and increased H3K4me3 at cardiac promoters as early as day 3, paralleled by a rapid significant increase in their mRNA expression. In contrast, H3K27me3 at loci encoding fibroblast marker genes did not increase until day 10 and H3K4me3 progressively decreased along the reprogramming process; these changes were accompanied by a gradual decrease in the mRNA expression of fibroblast marker genes. Further analyses of fibroblast-enriched transcription factors revealed a similarly late deposition of H3K27me3 and decreased mRNA expression of Sox9, Twist1 and Twist2, three important players in epithelial−mesenchymal transition. Our data suggest early rapid activation of the cardiac program and later progressive suppression of fibroblast fate at both epigenetic and transcriptional levels. Additionally, we determined the DNA methylation states of representative cardiac promoters and found that not every single CpG was equally demethylated during early stages of iCM reprogramming. Rather, there are specific CpGs, whose demethylation states correlated tightly with transcription activation, that we propose are the major contributing CpGs. Our work thus reveals a differential re-patterning of H3K27me3, H3K4me3 at cardiac and fibroblast loci during iCM reprogramming and could provide future genome

  20. Analysis of histones and histone variants in plants.

    Science.gov (United States)

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

    2012-01-01

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

  1. Inhibitors of histone deacetylase

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to compounds of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, wherein X1, X2, X3, X4, X5, W1, W2, W3, and W4 are as described. The present invention relates generally to inhibitors of histone deacetylase and to methods...

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

    Directory of Open Access Journals (Sweden)

    Tobias D Schneider

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

  3. Rapid purification of recombinant histones.

    Directory of Open Access Journals (Sweden)

    Henrike Klinker

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

  4. The chromosomal distribution of histone methylation marks in gymnosperms differs from that of angiosperms.

    Science.gov (United States)

    Fuchs, Jörg; Jovtchev, Gabriele; Schubert, Ingo

    2008-01-01

    The chromosomal distribution of seven histone methylation marks (H3K4me2, H3K9me1,2,3 and H3K27me1,2,3) was analysed in the gymnosperm species Pinus sylvestris and Picea abies. Similarly to the situation in other investigated eukaryotes, dimethylation of lysine 4 of histone H3 is restricted to euchromatin in gymnosperms. Surprisingly, also H3K9me1-a mark classified as heterochromatin-specific in angiosperms-labels the euchromatin in P. sylvestris and P. abies. The other investigated methylation marks are either equally distributed along the chromosomes, as H3K9me2 and H3K27me1 (in both species) and H3K9me3 (in P. abies), or enriched at specific types of heterochromatin, as H3K9me3 (in P. sylvestris) and H3K27me2 and H3K27me3 in both species. Although the methylation marks themselves are apparently conserved, their functional specificity within the frame of the 'epigenetic code' might have diverged during evolution.

  5. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    Science.gov (United States)

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction.

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

    Science.gov (United States)

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

    2017-01-01

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

  7. The C. elegans H3K27 Demethylase UTX-1 Is Essential for Normal Development, Independent of Its Enzymatic Activity

    DEFF Research Database (Denmark)

    Vandamme, Julien; Lettier, Gaëlle; Sidoli, Simone;

    2012-01-01

    specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required......Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase...

  8. The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

    Science.gov (United States)

    Zee, Barry M.; Dibona, Amy B.; Alekseyenko, Artyom A.; French, Christopher A.; Kuroda, Mitzi I.

    2016-01-01

    Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state. PMID:27698495

  9. Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas

    DEFF Research Database (Denmark)

    Bender, Sebastian; Tang, Yujie; Lindroth, Anders M;

    2013-01-01

    Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a glo...

  10. Drosophila Yemanuclein and HIRA cooperate for de novo assembly of H3.3-containing nucleosomes in the male pronucleus.

    Directory of Open Access Journals (Sweden)

    Guillermo A Orsi

    Full Text Available The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM, the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.

  11. Inhibition of mitotic-specific histone phophorylation by sodium arsenite

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-10-01

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

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

    Science.gov (United States)

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

    2004-05-18

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

  13. The vibration spectrum of H3

    Science.gov (United States)

    Burton, P. G.; von Nagy-Felsobuki, E.; Doherty, G.; Hamilton, M.

    The H3+ab initio potential energy surface and its analytical representation has been re-examined to promote more accurate vibration-rotation calculations. We present here a new and accurate 78 point PNO-CI grid which predicts the minimum energy geometry to be an equilateral triangle of side, r(H-H) = 1·6525 a0. and energy of -1·34188 Eh. Of the analytical representations only the sixth order Simons-Parr-Finlan (SPF) and Ogilvie force fields were found to satisfy our fitting criteria, resulting in associated errors of less than 11·1 kJ mol-1. Our calculated vibrational band origins are in good agreement with those of Carney and Porter, with the differences of the lowest-lying vibration states essentially reflecting the analytical characteristics of the potential energy surfaces used.

  14. Enhanced stability of histone octamers from plant nucleosomes: role of H2A and H2B histones.

    Science.gov (United States)

    Moehs, C P; Baxevanis, A D; Moudrianakis, E N; Spiker, S

    1992-11-10

    Gel filtration and sedimentation studies have previously established that the vertebrate animal core histone octamer is in equilibrium with an (H3-H4)2 tetramer and an H2A-H2B dimer [Eickbush, T. H., & Moudrianakis, E. N. (1978) Biochemistry 17, 4955-4964; Godfrey, J. E., Eickbush, T. H., & Moudrianakis, E. N. (1980) Biochemistry 19, 1339-1346]. We have investigated the core histone octamer of wheat (Triticum aestivum L.) and have found it to be much more stable than its vertebrate animal counterpart. When vertebrate animal histone octamers are subjected to gel filtration in 2 M NaCl, a trailing peak of H2A-H2B dimer can be clearly resolved from the main octamer peak. When the plant octamer is subjected to the identical procedure, there is no trailing peak of H2A-H2B dimer, but rather a single peak containing the octamer. A sampling across the octamer peak from leading to trailing edge shows no change in the ratio of H2A-H2B to (H3-H4)2. Surprisingly, the plant octamer shows the same stability at 0.6 M NaCl, a salt concentration in which the vertebrate animal octamer dissociates into dimers and tetramers. Equilibrium sedimentation data indicate that the assembly potential of the wheat histones in 2 M NaCl is very high at all protein concentrations above 0.1 mg mL-1. In order to disrupt the forces stabilizing the plant histone octamer at high histone concentrations, the concentration of NaCl must be lowered to approximately 0.3 M.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    Science.gov (United States)

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-12-11

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.

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

    Science.gov (United States)

    Chang, Le; Takada, Shoji

    2016-01-01

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

  17. In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana.

    Science.gov (United States)

    Chen, Jiajia; Gao, Jun; Peng, Maolin; Wang, Yi; Yu, Yanyan; Yang, Pengyuan; Jin, Hong

    2015-07-30

    Post-translational modifications (PTMs) on histone are highly correlated with genetic and epigenetic regulation of gene expression from chromatin. Mass spectrometry (MS) has developed to be an optimal tool for the identification and quantification of histone PTMs. Derivatization of histones with chemicals such as propionic anhydride, N-hydroxysuccinimide ester (NHS-propionate) has been widely used in histone PTMs analysis in bottom-up MS strategy, which requires high purity for histone samples. However, biological samples are not always prepared with high purity, containing detergents or other interferences in most cases. As an alternative approach, an adaptation of in gel derivatization method, termed In-gel NHS, is utilized for a broader application in histone PTMs analysis and it is shown to be a more time-saving preparation method. The proposed method was optimized for a better derivatization efficiency and displayed high reproducibility, indicating quantification of histone PTMs based on In-gel NHS was achievable. Without any traditional fussy histone purification procedures, we succeeded to quantitatively profile the histone PTMs from Arabidopsis with selective knock down of CLF (clf-29) and the original parental (col) with In-gel NHS method in a rapid way, which indicated the high specificity of CLF on H3K27me3 in Arabidopsis. In-gel NHS quantification results also suggest distinctive histone modification patterns in plants, which is invaluable foundation for future studies on histone modifications in plants.

  18. Histone Methylation Marks on Circulating Nucleosomes as Novel Blood-Based Biomarker in Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Ugur Gezer

    2015-12-01

    Full Text Available Circulating nucleic acids (CNAs are under investigation as a liquid biopsy in cancer as potential non-invasive biomarkers, as stable structure in circulation nucleosomes could be valuable sources for detection of cancer-specific alterations in histone modifications. Our interest is in histone methylation marks with a focus on colorectal cancer, one of the leading cancers respective the incidence and mortality. Our previous work included the analysis of trimethylations of lysine 9 on histone 3 (H3K9me3 and of lysine 20 on histone 4 (H4K20me3 by chromatin immuno- precipitation-related PCR in circulating nucleosomes. Here we asked whether global immunologic measurement of histone marks in circulation could be a suitable approach to show their potential as biomarkers. In addition to H3K9me3 and H4K20me3 we also measured H3K27me3 in plasma samples from CRC patients (n = 63 and cancer free individuals (n = 40 by ELISA-based methylation assays. Our results show that of three marks, the amounts of H3K27me3 (p = 0.04 and H4K20me3 (p < 0.001 were significantly lower in CRC patients than in healthy controls. For H3K9me3 similar amounts were measured in both groups. Areas under the curve (AUC in receiver operating characteristic (ROC curves indicating the power of CRC detection were 0.620 for H3K27me3, 0.715 for H4K20me3 and 0.769 for the combination of both markers. In conclusion, findings of this preliminary study reveal the potential of blood-based detection of CRC by quantification of histone methylation marks and the additive effect of the marker combination.

  19. Macro histone variants are critical for the differentiation of human pluripotent cells.

    Science.gov (United States)

    Barrero, María J; Sese, Borja; Martí, Mercè; Izpisua Belmonte, Juan Carlos

    2013-05-31

    We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

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

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    Xiaoru Zhang

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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Continual removal of H3K9 promoter methylation by Jmjd2 demethylases is vital for ESC self-renewal and early development

    DEFF Research Database (Denmark)

    Pedersen, Marianne Terndrup; Kooistra, Susanne Marije; Radzisheuskaya, Aliaksandra;

    2016-01-01

    Chromatin-associated proteins are essential for the specification and maintenance of cell identity. They exert these functions through modulating and maintaining transcriptional patterns. To elucidate the functions of the Jmjd2 family of H3K9/H3K36 histone demethylases, we generated conditional...... of H3K9me3 and H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2...

  5. Synthesis and characterisation of substrate-based peptides as inhibitors of histone demethylase KDM4C

    DEFF Research Database (Denmark)

    Nielsen, Simon D; Leurs, Ulrike; Bergner, Magnus;

    2016-01-01

    The design and synthesis of modified pentapeptides based on a truncated version of the substrate for KDM4C, a histone lysine demethylase (KDM), and investigation of their inhibitory activity at KDM4C is reported. By modifying the lysine residue corresponding to lysine 9 at histone 3 (H3K9), three...... different series of peptides were designed and synthesized. One series contained N-acylated H3K9 and two series introduced triazoles in this position via click chemistry to enable facile variation of headgroups. As the click reaction is compatible with free amino acids this was performed on an azido...

  6. Acetate supplementation modulates brain histone acetylation and decreases interleukin-1β expression in a rat model of neuroinflammation

    Directory of Open Access Journals (Sweden)

    Soliman Mahmoud L

    2012-03-01

    Full Text Available Abstract Background Long-term acetate supplementation reduces neuroglial activation and cholinergic cell loss in a rat model of lipopolysaccharide-induced neuroinflammation. Additionally, a single dose of glyceryl triacetate, used to induce acetate supplementation, increases histone H3 and H4 acetylation and inhibits histone deacetylase activity and histone deacetylase-2 expression in normal rat brain. Here, we propose that the therapeutic effect of acetate in reducing neuroglial activation is due to a reversal of lipopolysaccharide-induced changes in histone acetylation and pro-inflammatory cytokine expression. Methods In this study, we examined the effect of a 28-day-dosing regimen of glyceryl triacetate, to induce acetate supplementation, on brain histone acetylation and interleukin-1β expression in a rat model of lipopolysaccharide-induced neuroinflammation. The effect was analyzed using Western blot analysis, quantitative real-time polymerase chain reaction and enzymic histone deacetylase and histone acetyltransferase assays. Statistical analysis was performed using one-way analysis of variance, parametric or nonparametric when appropriate, followed by Tukey's or Dunn's post-hoc test, respectively. Results We found that long-term acetate supplementation increased the proportion of brain histone H3 acetylated at lysine 9 (H3K9, histone H4 acetylated at lysine 8 and histone H4 acetylated at lysine 16. However, unlike a single dose of glyceryl triacetate, long-term treatment increased histone acetyltransferase activity and had no effect on histone deacetylase activity, with variable effects on brain histone deacetylase class I and II expression. In agreement with this hypothesis, neuroinflammation reduced the proportion of brain H3K9 acetylation by 50%, which was effectively reversed with acetate supplementation. Further, in rats subjected to lipopolysaccharide-induced neuroinflammation, the pro-inflammatory cytokine interleukin-1β protein

  7. Recognition of Multivalent Histone States Associated with Heterochromatin by UHRF1 Protein

    Energy Technology Data Exchange (ETDEWEB)

    Nady, Nataliya; Lemak, Alexander; Walker, John R.; Avvakumov, George V.; Kareta, Michael S.; Achour, Mayada; Xue, Sheng; Duan, Shili; Allali-Hassani, Abdellah; Zuo, Xiaobing; Wang, Yun-Xing; Bronner, Christian; Chédin, Frédéric; Arrowsmith, Cheryl H.; Dhe-Paganon, Sirano (CNRS-UMR); (UCD); (NIH); (Toronto)

    2012-09-19

    Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16{sup INK4A}, when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.

  8. Histamine homologues discriminating between two functional H3 receptor assays. Evidence for H3 receptor heterogeneity?.

    Science.gov (United States)

    Leurs, R; Kathmann, M; Vollinga, R C; Menge, W M; Schlicker, E; Timmerman, H

    1996-03-01

    We studied several histamine homologues as potential ligands for the histamine H3 receptor in two binding assays ([125l]iodophenpropit and N alpha-[3H]methylhistamine binding to rat brain cortex membranes) and two functional H3 receptor models (inhibition of the neurogenic contraction in the guinea pig jejunum and of [3H]noradrenaline release in mouse brain cortex slices). The histamine homologues acted all as competitive H3 antagonists at the guinea pig jejunum. The potency in this model and/or the affinity for N alpha-[3H]methylhistamine binding was higher for the butylene (pA2 = 7.7; pKi = 9.4) and pentylene homologue (impentamine, pA2 = 8.4; pKi = 9.1) than for the propylene, hexylene and octylene homologues (pA2 = 5.9-7.8; pKi = 6.1-7.6). In the mouse brain cortex the propylene, butylene and pentylene homologues acted as partial agonists (alpha = 0.3-0.6) and the hexylene and octylene homologues acted as antagonists. [125I]Iodophenpropit binding was displaced monophasically by the propylene, hexylene and octylene homologues and biphasically by the butylene and pentylene homologues. Biphasic displacement curves were converted to monophasic ones by 10 microM guanosine-5'-O-(3-thiotriphosphate. In conclusion, the homologue of histamine with five methylene groups is a more potent H3 receptor antagonist in the guinea pig jejunum than the other homologues tested. Furthermore, the propylene, butylene and pentylene homologues can discriminate between the two functional H3 receptor models in the guinea pig jejunum and mouse brain. These data are discussed in relation to the efficiency of receptor coupling and receptor heterogeneity.

  9. Re-expression of methylation-induced tumor suppressor gene silencing is associated with the state of histone modification in gastric cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To identify the relationship between DNA hypermethylation and histone modification at a hypermethylated, silenced tumor suppressor gene promoter in human gastric cancer cell lines and to elucidate whether alteration of DNA methylation could affect histone modification.METHODS: We used chromatin immunoprecipitation(ChIP) assay to assess the status of histone acetylation and methylation in promoter regions of the p16 and mutL homolog 1(MLH1) genes in 2 gastric cancer cell lines, SGC-7901 and MGC-803. We used methylationspecific PCR (MSP) to evaluate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC), trichostatin A (TSA) or their combination treatment on DNA methylation status.We used RT-PCR to determine whether alterations of histone modification status after 5-Aza-dC and TSA treatment are reflected in gene expression.RESULTS: For the p16 and MLH1 genes in two cell lines,silenced loci associated with DNA hypermethylation were characterized by histone H3-K9 hypoacetylation and hypermethylation and histone H3-K4 hypomethylation.Treatment with TSA resulted in moderately increased histone H3-K9 acetylation at the silenced loci with no effect on histone H3-K9 methylation and minimal effects on gene expression. In contrast, treatment with 5-Aza-dC rapidly reduced histone H3-K9 methylation at the silenced loci and resulted in reactivation of the two genes. Combined treatment with 5-Aza-dC and TSA was synergistic in reactivating gene expression at the loci showing DNA hypermethylation. Similarly, histone H3-K4 methylation was not affected after TSA treatment, and increased moderately at the silenced loci after 5-Aza-dC treatment.CONCLUSION: Hypermethylation of DNA in promoter CpG islands is related to transcriptional silencing of tumor suppressor genes. Histone H3-K9 methylation in different regions of the promoters studied correlates with DNA methylation status of each gene in gastric cancer cells. However, histone H3-K9 acetylation and H3-K4 methylation inversely

  10. Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease.

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    Xi Lu

    Full Text Available Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD. While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1 and beta-site amyloid precursor protein cleaving enzyme 1(BACE1. By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP (N2a/APPswe and wild-type APP (N2a/APPwt as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected, respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT adenoviral E1A-associated 300-kDa protein (p300 is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.

  11. Histone acetyltransferase p300 mediates histone acetylation of PS1 and BACE1 in a cellular model of Alzheimer's disease.

    Science.gov (United States)

    Lu, Xi; Deng, Yushuang; Yu, Daohai; Cao, Huiming; Wang, Li; Liu, Li; Yu, Caijia; Zhang, Yuping; Guo, Xiuming; Yu, Gang

    2014-01-01

    Epigenetic modifications, particularly histone acetylation, have been implicated in Alzheimer's disease (AD). While previous studies have suggested that histone hypoacetylation may regulate the expression of genes associated with memory and learning in AD, little is known about histone regulation of AD-related genes such as Presenilin 1(PS1) and beta-site amyloid precursor protein cleaving enzyme 1(BACE1). By utilizing neuroblastoma N2a cells transfected with Swedish mutated human amyloid precursor protein (APP) (N2a/APPswe) and wild-type APP (N2a/APPwt) as cellular models of AD, we examined the alterations of histone acetylation at the promoter regions of PS1 and BACE1 in these cells. Our results revealed that histone H3 acetylation in PS1 and BACE1 promoters is markedly increased in N2a/APPswe cells when compared to N2a/APPwt cells and control cells (vector-transfected), respectively, causing the elevated expression of PS1 and BACE1. In addition, expression of histone acetyltransferase (HAT) adenoviral E1A-associated 300-kDa protein (p300) is dramatically enhanced in N2a/APPswe cells compared to N2a/APPwt and control cells. We have further demonstrated the direct binding of p300 protein to the PS1 and BACE1 promoters in N2a/APPswe cells. The expression levels of H3 acetylation of the PS1 and BACE1 promoters and p300 protein, however, were found to be not significantly different in N2a/APPwt cells when compared to controls in our studies. Furthermore, curcumin, a natural selective inhibitor of p300 in HATs, significantly suppressed the expression of PS1 and BACE1 through inhibition of H3 acetylation in their promoter regions in N2a/APPswe cells. These findings indicated that histone acetyltransferase p300 plays a critical role in controlling the expression of AD-related genes through regulating the acetylation of their promoter regions, suggesting that p300 may represent a novel potential therapeutic target for AD.

  12. Inhibition of histone methyltransferases SUV39H1 and G9a leads to neuroprotection in an in vitro model of cerebral ischemia

    OpenAIRE

    Schweizer, Sophie; Harms, Christoph; Lerch, Heike; Flynn,Jennifer; Hecht, Jochen; Yildirim, Ferah; Meisel, Andreas; Märschenz, Stefanie

    2015-01-01

    Cerebral ischemia induces a complex transcriptional response with global changes in gene expression. It is essentially regulated by transcription factors as well as epigenetic players. While it is well known that the inhibition of transcriptionally repressive histone deacetylases leads to neuroprotection, the role of histone methyltransferases in the postischemic transcriptional response remains elusive. We investigated the effects of inhibition of the repressive H3K9 histone methyltransferas...

  13. Histone methyltransferases in cancer

    DEFF Research Database (Denmark)

    Albert, Mareike; Helin, Kristian

    2009-01-01

    Cancer is perceived as a heterogeneous group of diseases that is characterized by aberrant patterns of gene expression. In the last decade, an increasing amount of data has pointed to a key role for epigenetic alterations in human cancer. In this review, we focus on a subclass of epigenetic...... regulators, namely histone methyltransferases (HMTs). Several HMTs have been linked to different types of cancer; however, in most cases we only have limited knowledge regarding the molecular mechanisms by which the HMTs contribute to disease development. We summarize the current knowledge regarding some...

  14. Histone deacetylases and atherosclerosis.

    Science.gov (United States)

    Zheng, Xia-xia; Zhou, Tian; Wang, Xin-An; Tong, Xiao-hong; Ding, Jia-wang

    2015-06-01

    Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC inhibitors(HDACi) on the progress of atherosclerosis.

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

    Science.gov (United States)

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

    1995-09-01

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

  16. Vitamin A induces inhibitory histone methylation modifications and down-regulates trained immunity in human monocytes

    DEFF Research Database (Denmark)

    Arts, Rob J W; Blok, Bastiaan A; van Crevel, Reinout;

    2015-01-01

    inhibited cytokine responses upon restimulation of monocytes, and this effect was exerted through increased expression of SUV39H2, a histone methyltransferase that induces the inhibitory mark H3K9me3. H3K9me3 at promoter sites of several cytokines was up-regulated by ATRA, and inhibition of SUV39H2 restored...... cytokine production. In addition to H3K9me3, the stimulatory histone mark H3K4me3 was down-regulated by ATRA at several promoter locations of cytokine genes. Therefore, we can conclude that ATRA inhibits cytokine production in models of direct stimulation or BCG-induced trained immunity...

  17. A selective phenelzine analogue inhibitor of histone demethylase LSD1.

    Science.gov (United States)

    Prusevich, Polina; Kalin, Jay H; Ming, Shonoi A; Basso, Manuela; Givens, Jeffrey; Li, Xin; Hu, Jianfei; Taylor, Martin S; Cieniewicz, Anne M; Hsiao, Po-Yuan; Huang, Rong; Roberson, Heather; Adejola, Nkosi; Avery, Lindsay B; Casero, Robert A; Taverna, Sean D; Qian, Jiang; Tackett, Alan J; Ratan, Rajiv R; McDonald, Oliver G; Feinberg, Andrew P; Cole, Philip A

    2014-06-20

    Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor in vitro and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1-/- cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease.

  18. Histone Deacetylases and Cardiometabolic Diseases

    OpenAIRE

    Yiew, Kan Hui; Chatterjee, Tapan K.; Hui, David Y.; Weintraub, Neal L.

    2015-01-01

    Cardiometabolic disease, emerging as a worldwide epidemic, is a combination of metabolic derangements leading to type 2 diabetes and cardiovascular disease. Genetic and environmental factors are linked through epigenetic mechanisms to the pathogenesis of cardiometabolic disease. Post-translational modifications of histone tails, including acetylation and deacetylation, epigenetically alter chromatin structure and dictate cell-specific gene expression patterns. The histone deacetylase (HDAC) f...

  19. Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression

    Directory of Open Access Journals (Sweden)

    Adam Rodney D

    2007-04-01

    Full Text Available Abstract Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him is essential for its activity. The