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Sample records for acetylated histones h3

  1. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

  2. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Aberrant levels of histone H3 acetylation induce spermatid anomaly in mouse testis.

    PubMed

    Dai, Lei; Endo, Daisuke; Akiyama, Naotaro; Yamamoto-Fukuda, Tomomi; Koji, Takehiko

    2015-02-01

    Histone acetylation is involved in the regulation of chromatin structure and gene function. We reported previously that histone H3 acetylation pattern is subject to dynamic changes and limited to certain stages of germ cell differentiation during murine spermatogenesis, suggesting a crucial role for acetylation in the process. In the present study, we investigated the effects of hyper- and hypo-acetylation on spermatogenesis. Changes in acetylation level were induced by either in vivo administration of sodium phenylbutyrate, a histone deacetylase inhibitor, or by knockdown of histone acetyltransferases using short hairpin RNA plasmids transfection. Administration of sodium phenylbutyrate induced accumulation of acetylated histone H3 at lysine 9 and lysine 18 in round spermatids, together with spermatid morphological abnormalities and induction of apoptosis through a Bax-related pathway. Knockdown of steroid receptor coactivator 1, a member of histone acetyltransferases, but not general control of amino acid synthesis 5 nor elongator protein 3 by in vivo electroporation of shRNA plasmids, reduced acetylated histone H3 at lysine 9 in round spermatids, and induced morphological abnormalities. We concluded that the proper regulation of histone H3 acetylation levels is important for spermatid differentiation and complex chromatin remodeling during spermiogenesis.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2013-04-08

    Dynamic variations in the structure of chromatin influence virtually all DNA-related processes in eukaryotes and are controlled in part by post-translational modifications of histones. One such modification, the acetylation of lysine 56 (H3K56ac) in the amino-terminal α-helix (αN) of histone H3, has been implicated in the regulation of nucleosome assembly during DNA replication and repair, and nucleosome disassembly during gene transcription. In Saccharomyces cerevisiae, the histone chaperone Rtt106 contributes to the deposition of newly synthesized H3K56ac-carrying H3-H4 complex on replicating DNA, but it is unclear how Rtt106 binds H3-H4 and specifically recognizes H3K56ac as there is no apparent acetylated lysine reader domain in Rtt106. Here, we show that two domains of Rtt106 are involved in a combinatorial recognition of H3-H4. An N-terminal domain homodimerizes and interacts with H3-H4 independently of acetylation while a double pleckstrin-homology (PH) domain binds the K56-containing region of H3. Affinity is markedly enhanced upon acetylation of K56, an effect that is probably due to increased conformational entropy of the αN helix of H3. Our data support a mode of interaction where the N-terminal homodimeric domain of Rtt106 intercalates between the two H3-H4 components of the (H3-H4)2 tetramer while two double PH domains in the Rtt106 dimer interact with each of the two H3K56ac sites in (H3-H4)2. We show that the Rtt106-(H3-H4)2 interaction is important for gene silencing and the DNA damage response.

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2011-07-01

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

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

    NASA Astrophysics Data System (ADS)

    Zhang, Kangling

    2008-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-03-25

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

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

    PubMed

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

    2017-02-23

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2014-11-21

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

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

    PubMed Central

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

    2009-01-01

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

  16. Hypoacetylation of acetyl-histone H3 (H3K9ac) as marker of poor prognosis in oral cancer.

    PubMed

    Webber, Liana P; Wagner, Vivian P; Curra, Marina; Vargas, Pablo A; Meurer, Luise; Carrard, Vinícius C; Squarize, Cristiane H; Castilho, Rogério M; Martins, Manoela D

    2017-08-01

    Epigenetics refers to changes in cell characteristics that occur independently of modifications to the DNA sequence. Oral carcinogenesis is influenced by modifications in epigenetic mechanisms, including changes in histones, which are proteins that support chromatin remodelling for the dynamic regulation of gene expression and silencing. The dysregulation of histone acetylation can lead to the uncontrolled activity of different genes, thereby triggering events associated with malignant transformation. The aim of this study was to analyse the expression of acetyl-histone H3 at lys9 (H3K9ac) in oral leucoplakia (OL) and oral squamous cell carcinoma (OSCC) in addition to its association with cell proliferation, epithelial-mesenchymal transition (EMT) and clinical-pathological findings. Samples of normal oral mucosa (NOM), OL and OSCC were submitted to immunohistochemical analysis using anti-H3K9ac, Ki67 and vimentin. Slides were submitted to quantitative analysis regarding the percentage of positive cells. OSCC presented less expression of H3K9ac in comparison to OL (P < 0.01), whereas Ki67 and vimentin levels increased from OL to OSCC (P < 0.001 and P = 0.03, respectively). OSCC patients with poor prognosis had less H3K9ac expression (P = 0.04). The Kaplan-Meier cumulative survival curves also revealed lower survival rates in patients with less H3K9ac expression (P < 0.01). The present findings suggest that changes in H3K9ac occur during the process of oral carcinogenesis along with an increase in cell proliferation and EMT. The results demonstrate that H3K9ac may be a useful novel prognostic marker for OSCC. © 2017 John Wiley & Sons Ltd.

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-09

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

    Bian, Chuanbing; Xu, Chao; Ruan, Jianbin; Lee, Kenneth K; Burke, Tara L; Tempel, Wolfram; Barsyte, Dalia; Li, Jing; Wu, Minhao; Zhou, Bo O; Fleharty, Brian E; Paulson, Ariel; Allali-Hassani, Abdellah; Zhou, Jin-Qiu; Mer, Georges; Grant, Patrick A; Workman, Jerry L; Zang, Jianye; Min, Jinrong

    2011-01-01

    The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation. PMID:21685874

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

    SciTech Connect

    Bian, Chuanbing; Xu, Chao; Ruan, Jianbin; Lee, Kenneth K.; Burke, Tara L.; Tempel, Wolfram; Barsyte, Dalia; Li, Jing; Wu, Minhao; Zhou, Bo O.; Fleharty, Brian E.; Paulson, Ariel; Allali-Hassani, Abdellah; Zhou, Jin-Qiu; Mer, Georges; Grant, Patrick A.; Workman, Jerry L.; Zang, Jianye; Min, Jinrong

    2011-09-28

    The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline; Lalonde, Marie-Eve; González-Aguilera, Cristina; Lambert, Jean-Philippe; Lee, Sung-Bau; Zhao, Xiaobei; Alabert, Constance; Johansen, Jens V; Paquet, Eric; Yang, Xiang-Jiao; Gingras, Anne-Claude; Côté, Jacques; Groth, Anja

    2016-01-18

    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 implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 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 replication origins. © 2015 The Authors.

  9. Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

    PubMed

    Bergmann, Jan H; Jakubsche, Julia N; Martins, Nuno M; Kagansky, Alexander; Nakano, Megumi; Kimura, Hiroshi; Kelly, David A; Turner, Bryan M; Masumoto, Hiroshi; Larionov, Vladimir; Earnshaw, William C

    2012-01-15

    Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

  10. p300-mediated Acetylation of Histone H3 Lysine 56 Functions in DNA Damage Response in Mammals*

    PubMed Central

    Vempati, Rahul K.; Jayani, Ranveer S.; Notani, Dimple; Sengupta, Amrita; Galande, Sanjeev; Haldar, Devyani

    2010-01-01

    The packaging of newly replicated and repaired DNA into chromatin is crucial for the maintenance of genomic integrity. Acetylation of histone H3 core domain lysine 56 (H3K56ac) has been shown to play a crucial role in compaction of DNA into chromatin following replication and repair in Saccharomyces cerevisiae. However, the occurrence and function of such acetylation has not been reported in mammals. Here we show that H3K56 is acetylated and that this modification is regulated in a cell cycle-dependent manner in mammalian cells. We also demonstrate that the histone acetyltransferase p300 acetylates H3K56 in vitro and in vivo, whereas hSIRT2 and hSIRT3 deacetylate H3K56ac in vivo. Further we show that following DNA damage H3K56 acetylation levels increased, and acetylated H3K56, which is localized at the sites of DNA repair. It also colocalized with other proteins involved in DNA damage signaling pathways such as phospho-ATM, CHK2, and p53. Interestingly, analysis of occurrence of H3K56 acetylation using ChIP-on-chip revealed its genome-wide spread, affecting genes involved in several pathways that are implicated in tumorigenesis such as cell cycle, DNA damage response, DNA repair, and apoptosis. PMID:20587414

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2012-04-17

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

  13. Replisome Function During Replicative Stress Is Modulated by Histone H3 Lysine 56 Acetylation Through Ctf4

    PubMed Central

    Luciano, Pierre; Dehé, Pierre-Marie; Audebert, Stéphane; Géli, Vincent; Corda, Yves

    2015-01-01

    Histone H3 lysine 56 acetylation in Saccharomyces cerevisiae is required for the maintenance of genome stability under normal conditions and upon DNA replication stress. Here we show that in the absence of H3 lysine 56 acetylation replisome components become deleterious when replication forks collapse at natural replication block sites. This lethality is not a direct consequence of chromatin assembly defects during replication fork progression. Rather, our genetic analyses suggest that in the presence of replicative stress H3 lysine 56 acetylation uncouples the Cdc45–Mcm2-7–GINS DNA helicase complex and DNA polymerases through the replisome component Ctf4. In addition, we discovered that the N-terminal domain of Ctf4, necessary for the interaction of Ctf4 with Mms22, an adaptor protein of the Rtt101-Mms1 E3 ubiquitin ligase, is required for the function of the H3 lysine 56 acetylation pathway, suggesting that replicative stress promotes the interaction between Ctf4 and Mms22. Taken together, our results indicate that Ctf4 is an essential member of the H3 lysine 56 acetylation pathway and provide novel mechanistic insights into understanding the role of H3 lysine 56 acetylation in maintaining genome stability upon replication stress. PMID:25697176

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

    PubMed Central

    Ikebe, Jinzen; Sakuraba, Shun; Kono, Hidetoshi

    2016-01-01

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

  15. The convergent chemical synthesis of histone H3 protein for site-specific acetylation at Lys56 and ubiquitination at Lys122.

    PubMed

    Qi, Yun-Kun; He, Qiao-Qiao; Ai, Hua-Song; Guo, Jing; Li, Jia-Bin

    2017-03-29

    Deposition of (H3-H4)2 tetramers is believed to be the critical step in nucleosome assembly. Site-specific acetylation and ubiquitination of histone H3 have been speculated to synergistically facilitate the formation and deposition of (H3-H4)2 tetramers. Here we report our endeavors toward the first chemical synthesis of homogenous histone H3, which bears Lys56 acetylation and Lys122 ubiquitination, for in vitro biochemical and biophysical studies.

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

    PubMed Central

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

    2015-01-01

    Brown adipocytes function to dissipate energy as heat through adaptive thermogenesis. Understanding the molecular mechanisms underlying the brown fat thermogenic program may provide insights for the development of therapeutic approaches in the treatment of obesity. Most studies investigating the mechanisms underlying brown fat development focus on genetic mechanisms; little is known about the epigenetic mechanisms in this process. We have discovered that ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase for di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), plays a potential role in regulating brown adipocyte thermogenic program. We found that UTX is up-regulated during brown adipocyte differentiation and by cold exposure in both brown adipose tissue (BAT) and white adipose tissue (WAT) of mice, suggesting a potential role in thermogenesis. Inactivation of UTX down-regulates brown fat specific gene expression, while overexpression of UTX does the opposite. Notably, activation of β adrenergic signaling recruits UTX to the UCP1 and PGC1α promoters, leading to decreased H3K27me3, a histone transcriptional repressive mark. UTX demethylates H3K27me3 and subsequently interacts with the histone acetyltransferase (HAT) protein CBP, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulate brown adipocyte thermogenic program through coordinated control of demethylating H3K27me3 and acetylating H3K27, switching the transcriptional repressive state to the transcriptional active state at the promoters of UCP1 and PGC1α. We conclude that UTX may play a potential role in regulation of brown adipocyte gene expression and may mediate β adrenergic activation of brown fat function. PMID:26306033

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

    PubMed

    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.

  18. Immunohistochemical analysis of histone H3 acetylation and methylation—Evidence for altered epigenetic signaling following traumatic brain injury in immature rats☆

    PubMed Central

    Gao, Wei-Min; Chadha, Mandeep S.; Kline, Anthony E.; Clark, Robert S.B.; Kochanek, Patrick M.; Dixon, C. Edward; Jenkins, Larry W.

    2009-01-01

    Posttranslational modifications (PTMs) of histone proteins may result in altered epigenetic signaling after pediatric traumatic brain injury (TBI). Hippocampal histone H3 acetylation and methylation in immature rats after moderate TBI were measured and decreased only in CA3 at 6 h and 24 h with persistent methylation decreases up to 72 h after injury. Decreased histone H3 acetylation and methylation suggest altered hippocampal CA3 epigenetic signaling during the first hours to days after TBI. PMID:16406269

  19. Histone H3K56 Acetylation, CAF1, and Rtt106 Coordinate Nucleosome Assembly and Stability of Advancing Replication Forks

    PubMed Central

    Clemente-Ruiz, Marta; González-Prieto, Román; Prado, Félix

    2011-01-01

    Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage. PMID:22102830

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  2. Failure of long-term memory formation in juvenile snails is determined by acetylation status of histone H3 and can be improved by NaB treatment.

    PubMed

    Danilova, Alexandra B; Grinkevich, Larisa N

    2012-01-01

    Animals' capacities for different forms of learning do not mature simultaneously during ontogenesis but the molecular mechanisms behind the delayed development of specific types of memory are not fully understood. Mollusks are considered to be among the best models to study memory formation at the molecular level. Chromatin remodeling in developmental processes, as well as in long-term memory formation, was recently shown to play a major role. Histone acetylation is a key process in the chromatin remodeling and is regulated through the signaling cascades, for example MAPK/ERK. Previously, we found that MAPK/ERK is a key pathway in the formation of the food aversion reflex in Helix. Pretreatment with upstream ERK kinase inhibitor PD98059 prevented food avoidance learning in adult Helix. In contrast to adult snails, juveniles possess immature plasticity mechanisms of the avoidance reflex until the age of 2-3 months while the MAPK/ERK cascade is not activated after aversive learning. In the present study, we focused on the potential MAPK/ERK target--histone H3. Here we found that a significant increase in histone H3 acetylation occurs in adult animals after learning, whereas no corresponding increase was observed in juveniles. The acetylation of histone H3 is regulated by ERK kinase, since the upstream ERK kinase inhibitor PD98059 prevented the increase of histone H3 acetylation upon learning. We found that the injection of histone deacetylase inhibitor sodium butyrate (NaB) prior to training led to induction in histone H3 acetylation and significantly ameliorated long-term memory formation in juvenile snails. Thus, MAPK/ERK-dependent histone H3 acetylation plays an essential role in the formation of food aversion in Helix. Dysfunction of the MAPK/ERK dependent histone H3 acetylation might determine the deficiency of avoidance behavior and long-term plasticity in juvenile animals. Stimulation of histone H3 acetylation in juvenile animals by NaB promoted avoidance

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2016-11-01

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

  5. Reduced histone H3K9 acetylation of clock genes and abnormal glucose metabolism in ob/ob mice.

    PubMed

    Ishikawa-Kobayashi, Eiko; Ushijima, Kentarou; Ando, Hitoshi; Maekawa, Tomohiro; Takuma, Masashi; Furukawa, Yusuke; Fujimura, Akio

    2012-10-01

    Recent chronobiological studies found significant correlation between lack of clock function and metabolic abnormalities. We previously showed that clock gene expressions were dampened in the peripheral tissues of obese and diabetic ob/ob mice. However, the molecular mechanism of the disturbance remained to be determined. In this study, we demonstrated for the first time that acetylation levels of histone H3 lysine 9 (H3K9) at the promoter regions of clock genes, such as Dbp, Per2, and Bmal1, in the adipose tissue of ob/ob mice were significantly reduced compared with those of its control C57BL/6J mice. Treatment with histone deacetylase (HDAC) inhibitors increased Dbp, but not Per2 or Bmal1, mRNA expression in adipose tissue, and it decreased blood glucose in these animals. In addition, 2-deoxyglucose uptake activity was significantly suppressed by silencing Dbp expression in cultured adipocytes. These results suggest that reduced H3K9 acetylation and subsequent decreased mRNA expression of the Dbp gene in adipose tissue are involved in the mechanism of development of abnormal glucose metabolism in ob/ob mice.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Liang, Gangning; Lin, Joy C. Y.; Wei, Vivian; Yoo, Christine; Cheng, Jonathan C.; Nguyen, Carvell T.; Weisenberger, Daniel J.; Egger, Gerda; Takai, Daiya; Gonzales, Felicidad A.; Jones, Peter A.

    2004-01-01

    Almost 1-2% of the human genome is located within 500 bp of either side of a transcription initiation site, whereas a far larger proportion (≈25%) is potentially transcribable by elongating RNA polymerases. This observation raises the question of how the genome is packaged into chromatin to allow start sites to be recognized by the regulatory machinery at the same time as transcription initiation, but not elongation, is blocked in the 25% of intragenic DNA. We developed a chromatin scanning technique called ChAP, coupling the chromatin immunoprecipitation assay with arbitrarily primed PCR, which allows for the rapid and unbiased comparison of histone modification patterns within the eukaryotic nucleus. Methylated lysine 4 (K4) and acetylated K9/14 of histone H3 were both highly localized to the 5′ regions of transcriptionally active human genes but were greatly decreased downstream of the start sites. Our results suggest that the large transcribed regions of human genes are maintained in a deacetylated conformation in regions read by elongating polymerase. Common models depicting widespread histone acetylation and K4 methylation throughout the transcribed unit do not therefore apply to the majority of human genes. PMID:15123803

  8. Histone Acetyl Transferase 1 Is Essential for Mammalian Development, Genome Stability, and the Processing of Newly Synthesized Histones H3 and H4

    PubMed Central

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

    2013-01-01

    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. PMID:23754951

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

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

    Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung; Lee, Daeyoup

    2016-08-05

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

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

    PubMed

    Yang, Hanna; Kwon, Chang Seob; Choi, Yoonjung; Lee, Daeyoup

    2016-08-05

    Nucleosome dynamics facilitated by histone turnover is required for transcription as well as DNA replication and repair. Histone turnover is often associated with various histone modifications such as H3K56 acetylation (H3K56Ac), H3K36 methylation (H3K36me), and H4K20 methylation (H4K20me). In order to correlate histone modifications and transcription-dependent histone turnover, we performed genome wide analyses for euchromatic regions in G2/M-arrested fission yeast. The results show that transcription-dependent histone turnover at 5' promoter and 3' termination regions is directly correlated with the occurrence of H3K56Ac and H4K20 mono-methylation (H4K20me1) in actively transcribed genes. Furthermore, the increase of H3K56Ac and H4K20me1 and antisense RNA production was observed in the absence of the histone H3K36 methyltransferase Set2 and histone deacetylase complex (HDAC) that are involved in the suppression of histone turnover within the coding regions. These results together indicate that H4K20me1 as well as H3K56Ac are bona fide marks for transcription-dependent histone turnover in fission yeast. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2014-03-21

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

  14. Failure of Long-Term Memory Formation in Juvenile Snails Is Determined by Acetylation Status of Histone H3 and Can Be Improved by NaB Treatment

    PubMed Central

    Danilova, Alexandra B.; Grinkevich, Larisa N.

    2012-01-01

    Background Animals’ capacities for different forms of learning do not mature simultaneously during ontogenesis but the molecular mechanisms behind the delayed development of specific types of memory are not fully understood. Mollusks are considered to be among the best models to study memory formation at the molecular level. Chromatin remodeling in developmental processes, as well as in long-term memory formation, was recently shown to play a major role. Histone acetylation is a key process in the chromatin remodeling and is regulated through the signaling cascades, for example MAPK/ERK. Previously, we found that MAPK/ERK is a key pathway in the formation of the food aversion reflex in Helix. Pretreatment with upstream ERK kinase inhibitor PD98059 prevented food avoidance learning in adult Helix. In contrast to adult snails, juveniles possess immature plasticity mechanisms of the avoidance reflex until the age of 2–3 months while the MAPK/ERK cascade is not activated after aversive learning. In the present study, we focused on the potential MAPK/ERK target - histone H3. Methodology/Principal Findings Here we found that a significant increase in histone H3 acetylation occurs in adult animals after learning, whereas no corresponding increase was observed in juveniles. The acetylation of histone H3 is regulated by ERK kinase, since the upstream ERK kinase inhibitor PD98059 prevented the increase of histone H3 acetylation upon learning. We found that the injection of histone deacetylase inhibitor sodium butyrate (NaB) prior to training led to induction in histone H3 acetylation and significantly ameliorated long-term memory formation in juvenile snails. Conclusions/Significance Thus, MAPK/ERK-dependent histone H3 acetylation plays an essential role in the formation of food aversion in Helix. Dysfunction of the MAPK/ERK dependent histone H3 acetylation might determine the deficiency of avoidance behavior and long-term plasticity in juvenile animals. Stimulation of

  15. Histone acetylation in heterochromatin assembly

    PubMed Central

    Kim, Jeong-Hoon; Workman, Jerry L.

    2010-01-01

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

  16. Histone H3K56 Acetylation, Rad52, and Non-DNA Repair Factors Control Double-Strand Break Repair Choice with the Sister Chromatid

    PubMed Central

    Rothstein, Rodney; Aguilera, Andrés

    2013-01-01

    DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation. PMID:23357952

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

    PubMed

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

    2012-08-01

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

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

    PubMed

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

    2011-02-01

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

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

    PubMed

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

    2016-12-01

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

  20. High levels of histone H3 acetylation at the CMV promoter are predictive of stable expression in Chinese hamster ovary cells.

    PubMed

    Moritz, Benjamin; Woltering, Laura; Becker, Peter B; Göpfert, Ulrich

    2016-05-01

    Chinese hamster ovary cells (CHO) are widely used in the production of glycosylated therapeutic proteins such as antibodies. During expansion and maintenance, CHO cell lines are prone to production instability, which may be caused by promoter silencing, loss of transgene copies, or post-transcriptional effects. Silencing of recombinant genes may be accompanied by DNA methylation and histone modification. Previously, we demonstrated that cytosine methylation of the human cytomegalovirus major immediate early promoter/enhancer (hCMV-MIE) can be used to predict instability of antibody-producing CHO cell lines. However, the high rate of false prediction motivates the search for further markers of stable promoter activity. To this end, we correlated a variety of histone modifications in the vicinity of hCMV-MIE with production stability over time. Our results suggest that acetylation of histone H3 (H3ac) is a more effective indicator of production stability than DNA methylation. Selecting cell lines with highest CMV promoter H3ac levels enriches stable expressors and improves the average stability of production cell lines. For histone H3 acetylation measurement we employed a method based on chromatin immunoprecipitation (ChIP). In its current form, the method is suitable to evaluate 10-20 cell lines within a few days. We propose to determine H3 acetylation once the number of candidate cell lines has been narrowed based on productivity and product quality. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:776-786, 2016. © 2016 American Institute of Chemical Engineers.

  1. Acetylation of H4 suppresses the repressive effects of the N-termini of histones H3/H4 and facilitates the formation of positively coiled DNA.

    PubMed

    Peterson, Sharon; Jackson, Vaughn

    2008-07-08

    We have studied the role of the N-termini of histones H3/H4 in the regulation of the conformational changes that occur in H3/H4 during their deposition on DNA by NAP1 (nucleosome assembly protein 1). Removal of the N-termini extensively increased the right-handed conformation of H3/H4 as assayed by the increased levels of positive coils that were formed on DNA. The osmolytes, TMAO, betaine, sarcosine, alanine, glycine, and proline to varying degrees, facilitated the formation of positive coils. The denaturant, urea (0.6 M), blocked the osmolyte effects, causing a preference of H3/H4 to form negative coils (the left-handed conformation). Acetylated H3/H4 also formed high levels of positive coils, and it is proposed that both the osmolytes and acetylation promote the formation of an alpha-helix in the N-termini. This structural change may ultimately explain a unique feature of transcription through nucleosomes, i.e., that H2A/H2B tends to be more mobile than H3/H4. By using combinations of H3 and H4 that were either acetylated or the N-termini removed, it was also determined that the N-terminus of H4 is primarily responsible for repressing the formation of positive coils. Additional gradient analyses indicate that NAP1 establishes an equilibrium with the H3/H4-DNA complexes. This equilibrium facilitates a histone saturation of the DNA, a unique state that promotes the right-handed conformation. NAP1 persists in the binding of the complexes through interaction with the N-terminus of H3, which may be a mechanism for subsequent remodeling of the nucleosome during transcription and replication.

  2. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells.

    PubMed

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-07-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTOR(high), aH3/aH4(low)) and tumour-suppressing signals (Akt/mTOR(low) , aH3/aH4(high)) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTOR(high)) or mTOR inhibitor (aH3/aH4(low)) alone.

  3. Cross-communication between histone H3 and H4 acetylation and Akt-mTOR signalling in prostate cancer cells

    PubMed Central

    Makarević, Jasmina; Tawanaie, Nassim; Juengel, Eva; Reiter, Michael; Mani, Jens; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A

    2014-01-01

    Molecular tumour targeting has significantly improved anti-cancer protocols. Still, the addition of molecular targeting to the treatment regime has not led to a curative breakthrough. Combined mammalian target of Rapamycin (mTOR) and histone deacetylase (HDAC) inhibition has been shown not only to enhance anti-tumour potential, but also to prevent resistance development seen under mono-drug therapy. This investigation was designed to evaluate whether cross-communication exists between mTOR signalling and epigenetic events regulated by HDAC. DU-145 prostate cancer cells were treated with insulin-like growth factor (IGF) to activate the Akt-mTOR cascade or with the HDAC-inhibitor valproic acid (VPA) to induce histone H3 and H4 acetylation (aH3, aH4). Subsequently, mTOR, Rictor, Raptor, p70s6k, Akt (all: total and phosphorylated), H3 and H4 (total and acetylated) were analysed by western blotting. Both techniques revealed a link between mTOR and the epigenetic machinery. IGF activated mTOR, Rictor, Raptor, p70s6k and Akt, but also enhanced aH3 and aH4. Inversely, IGFr blockade and knock-down blocked the Akt-mTOR axis, but simultaneously diminished aH3 and aH4. VPA treatment up-regulated histone acetylation, but also activated mTOR-Akt signalling. HDAC1 and 2 knock-down revealed that the interaction with the mTOR system is initiated by histone H3 acetylation. HDAC-mTOR communication, therefore, is apparent whereby tumour-promoting (Akt/mTORhigh, aH3/aH4low) and tumour-suppressing signals (Akt/mTORlow, aH3/aH4high) are activated in parallel. Combined use of an HDAC- and mTOR inhibitor might then diminish pro-tumour effects triggered by the HDAC- (Akt/mTORhigh) or mTOR inhibitor (aH3/aH4low) alone. PMID:24779401

  4. Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions.

    PubMed

    Law, R David; Suttle, Jeffrey C

    2005-06-01

    Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato (Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 33% decrease in genome-wide 5-methylcytosine (5mC) content and a transient threefold increase in transcription rates that were most evident at 6 and 9 days after subculture, respectively. In contrast to the global reduction in 5mC content, subculture resulted in a transient twofold increase in 5mC levels within 5'-CCGG-3' sequences and no detectable change in 5'-CG-3' methylation. Multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by approximately 25% at day 9, then decreased 5'-mCmCGG-3' by 30-50% and increased H3 and H4 multi-acetylation by 30-60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was approximately twofold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension cultures, rapid, reversible changes in 5mC levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-01-01

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

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

    PubMed

    Yamada, Shintaro; Ohta, Kunihiro; Yamada, Takatomi

    2013-04-01

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

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

    PubMed

    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.

  10. Hat2p recognizes the histone H3 tail to specify the acetylation of the newly synthesized H3/H4 heterodimer by the Hat1p/Hat2p complex

    PubMed Central

    Li, Yang; Zhang, Li; Liu, Tingting; Chai, Chengliang; Fang, Qianglin; Wu, Han; Agudelo Garcia, Paula A.; Han, Zhifu; Zong, Shuai; Yu, You; Zhang, Xinyue; Parthun, Mark R.; Chai, Jijie; Xu, Rui-Ming; Yang, Maojun

    2014-01-01

    Post-translational modifications of histones are significant regulators of replication, transcription, and DNA repair. Particularly, newly synthesized histone H4 in H3/H4 heterodimers becomes acetylated on N-terminal lysine residues prior to its incorporation into chromatin. Previous studies have established that the histone acetyltransferase (HAT) complex Hat1p/Hat2p medicates this modification. However, the mechanism of how Hat1p/Hat2p recognizes and facilitates the enzymatic activities on the newly assembled H3/H4 heterodimer remains unknown. Furthermore, Hat2p is a WD40 repeat protein, which is found in many histone modifier complexes. However, how the WD40 repeat proteins facilitate enzymatic activities of histone modification enzymes is unclear. In this study, we first solved the high-resolution crystal structure of a Hat1p/Hat2p/CoA/H4 peptide complex and found that the H4 tail interacts with both Hat1p and Hat2p, by which substrate recruitment is facilitated. We further discovered that H3 N-terminal peptides can bind to the Hat2p WD40 domain and solved the structure of the Hat1p/Hat2p/CoA/H4/H3 peptide complex. Moreover, the interaction with Hat2p requires unmodified Arg2/Lys4 and Lys9 on the H3 tail, suggesting a novel model to specify the activity of Hat1p/Hat2p toward newly synthesized H3/H4 heterodimers. Together, our study demonstrated the substrate recognition mechanism by the Hat1p/Hat2p complex, which is critical for DNA replication and other chromatin remodeling processes. PMID:24835250

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

    PubMed Central

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

    2013-01-01

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

  12. Histone acetylation in neurodevelopment.

    PubMed

    Contestabile, Antonio; Sintoni, Silvia

    2013-01-01

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

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

    PubMed

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

    2014-01-31

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

  14. Epigenetic regulation of condensin-mediated genome organization during the cell cycle and upon DNA damage through histone H3 lysine 56 acetylation.

    PubMed

    Tanaka, Atsunari; Tanizawa, Hideki; Sriswasdi, Sira; Iwasaki, Osamu; Chatterjee, Atreyi G; Speicher, David W; Levin, Henry L; Noguchi, Eishi; Noma, Ken-Ichi

    2012-11-30

    Complex genome organizations participate in various nuclear processes including transcription, DNA replication, and repair. However, the mechanisms that generate and regulate these functional genome structures remain largely unknown. Here, we describe how the Ku heterodimer complex, which functions in nonhomologous end joining, mediates clustering of long terminal repeat retrotransposons at centromeres in fission yeast. We demonstrate that the CENP-B subunit, Abp1, functions as a recruiter of the Ku complex, which in turn loads the genome-organizing machinery condensin to retrotransposons. Intriguingly, histone H3 lysine 56 (H3K56) acetylation, which functions in DNA replication and repair, interferes with Ku localization at retrotransposons without disrupting Abp1 localization and, as a consequence, dissociates condensin from retrotransposons. This dissociation releases condensin-mediated genomic associations during S phase and upon DNA damage. ATR (ATM- and Rad3-related) kinase mediates the DNA damage response of condensin-mediated genome organization. Our study describes a function of H3K56 acetylation that neutralizes condensin-mediated genome organization.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    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.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Ferrari, Paolo; Strubin, Michel

    2015-04-30

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

  20. The adenovirus E1A oncoprotein N-terminal transcriptional repression domain enhances p300 autoacetylation and inhibits histone H3 Lys18 acetylation

    PubMed Central

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

    2015-01-01

    Expression of the adenovirus E1A N-terminal transcription repression domain alone (E1A 1-80) represses transcription from specific promoters such as HER2 [1] and from reconstituted chromatin [2]. Significantly, E1A 1-80 can induce the death of human breast cancer cells over-expressing the HER2 oncogene [1] as well as other cancer cells. Here, we report that E1A 1-80 alone is sufficient to inhibit H3K18 acetylation in vivo and p300-mediated H3K18 acetylation in reconstituted chromatin. Of interest, hypoacetylation of H3K18 has been correlated with the survival of tumor cells and the poor prognosis of many cancers [3, 4]. E1A 1-80 enhances p300 autoacetylation and concurrently inhibits H3K18 acetylation in chromatin in a dose-dependent manner. Pre-acetylation of p300 by incubation with acetyl-CoA alone reduces p300's ability to acetylate H3K18 in chromatin. Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation. These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18. The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6] PMID:25821559

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

    PubMed Central

    2014-01-01

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

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

    PubMed

    Modak, Rahul; Das Mitra, Susweta; Vasudevan, Madavan; Krishnamoorthy, Paramanandhan; Kumar, Manoj; Bhat, Akshay V; Bhuvana, Mani; Ghosh, Sankar K; Shome, Bibek R; Kundu, Tapas K

    2014-01-01

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

  3. Histone acetylation in insect chromosomes.

    PubMed

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

    1968-01-19

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

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

    SciTech Connect

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

    2014-07-18

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

  5. H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2007-01-01

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

  8. 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.

    PubMed

    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.

  9. Structural cooperativity in histone H3 tail modifications.

    PubMed

    Sanli, Deniz; Keskin, Ozlem; Gursoy, Attila; Erman, Burak

    2011-12-01

    Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.

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

    PubMed Central

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

    2017-01-01

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

  11. H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

    PubMed Central

    Guillemette, Benoit; Drogaris, Paul; Lin, Hsiu-Hsu Sophia; Armstrong, Harry; Hiragami-Hamada, Kyoko; Imhof, Axel; Bonneil, Éric; Thibault, Pierre; Verreault, Alain; Festenstein, Richard J.

    2011-01-01

    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). PMID:21483810

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

    PubMed

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

    2015-12-01

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

  13. 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-{alpha} by decreasing levels of acetylated histone H3 and H4 at its promoter

    SciTech Connect

    Engdahl, Ryan . E-mail: rengdahl@temple.edu; Monroy, M. Alexandra; Daly, John M.

    2007-07-20

    Prostaglandin metabolite 15-Deoxy-{delta}{sup 12,14}-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPAR{gamma}. We investigated the ability of 15d-PGJ2 to inhibit TNF-{alpha} gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 {mu}M) inhibited LPS-stimulated TNF-{alpha} mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPAR{gamma} ligand, GW1929, failed to inhibit LPS-induced TNF-{alpha} mRNA expression nor did a PPAR{gamma} antagonist, GW9662, alter the repression of TNF-{alpha} mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPAR{gamma}-independent inhibition of TNF-{alpha} mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-{alpha} promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-{alpha} promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-{alpha} promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-{alpha} promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-{alpha} promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-{alpha} transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.

  14. Structure of the histone chaperone ASF1 bound to the histone H3 C-terminal helix and functional insights.

    PubMed

    Agez, Morgane; Chen, Jun; Guerois, Raphaël; van Heijenoort, Carine; Thuret, Jean-Yves; Mann, Carl; Ochsenbein, Francoise

    2007-02-01

    Asf1 is a histone chaperone that favors histone H3/H4 assembly and disassembly. We solved the structure of the conserved domain of human ASF1A in complex with the C-terminal helix of histone H3 using nuclear magnetic resonance spectroscopy. This structure is fully compatible with an association of ASF1 with the heterodimeric form of histones H3/H4. In our model, ASF1 substitutes for the second H3/H4 heterodimer that is normally found in heterotetrameric H3/H4 complexes. This result constitutes an essential step in the fundamental understanding of the mechanisms of nucleosome assembly by histone chaperones. Point mutations that perturb the Asf1/histone interface were designed from the structure. The decreased binding affinity of the Asf1-H3/H4 complex correlates with decreased levels of H3-K56 acetylation and phenotypic defects in vivo.

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

    USDA-ARS?s Scientific Manuscript database

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

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

    PubMed

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

    2008-05-01

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

  17. Causal role of histone acetylations in enhancer function

    PubMed Central

    Pradeepa, Madapura M.

    2017-01-01

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

  18. H3K36ac Is an Evolutionary Conserved Plant Histone Modification That Marks Active Genes.

    PubMed

    Mahrez, Walid; Arellano, Minerva Susana Trejo; Moreno-Romero, Jordi; Nakamura, Miyuki; Shu, Huan; Nanni, Paolo; Köhler, Claudia; Gruissem, Wilhelm; Hennig, Lars

    2016-03-01

    In eukaryotic cells, histones are subject to a large number of posttranslational modifications whose sequential or combinatorial action affects chromatin structure and genome function. We identified acetylation at Lys-36 in histone H3 (H3K36ac) as a new chromatin modification in plants. The H3K36ac modification is evolutionary conserved in seed plants, including the gymnosperm Norway spruce (Picea abies) and the angiosperms rice (Oryza sativa), tobacco (Nicotiana tabacum), and Arabidopsis (Arabidopsis thaliana). In Arabidopsis, H3K36ac is highly enriched in euchromatin but not in heterochromatin. Genome-wide chromatin immunoprecipitation sequencing experiments revealed that H3K36ac peaks at the 5' end of genes, mainly on the two nucleosomes immediately distal to the transcription start site, independently of gene length. H3K36ac overlaps with H3K4me3 and the H2A.Z histone variant. The histone acetyl transferase GCN5 and the histone deacetylase HDA19 are required for H3K36ac homeostasis. H3K36ac and H3K36me3 show negative crosstalk, which is mediated by GCN5 and the histone methyl transferase SDG8. Although H3K36ac is associated with gene activity, we did not find a linear relationship between H3K36ac and transcript levels, suggesting that H3K36ac is a binary indicator of transcription. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. The Histone Chaperones Nap1 and Vps75 Bind Histones H3 and H4 in a Tetrameric Conformation

    PubMed Central

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

    2011-01-01

    Summary 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. PMID:21329878

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

    PubMed

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

    2011-02-18

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

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

    SciTech Connect

    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.

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

    SciTech Connect

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

    1987-05-25

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

  3. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

    PubMed Central

    Seto, Edward; Yoshida, Minoru

    2014-01-01

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

  4. Histone H3 specific acetyltransferases are essential for cell cycle progression

    PubMed Central

    Howe, LeAnn; Auston, Darryl; Grant, Patrick; John, Sam; Cook, Richard G.; Workman, Jerry L.; Pillus, Lorraine

    2001-01-01

    Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G2/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities. PMID:11731478

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

    PubMed

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

    2016-01-15

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2005-11-01

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

  8. Histone H3K4 and H3K36 Methylation Independently Recruit the NuA3 Histone Acetyltransferase in Saccharomyces cerevisiae.

    PubMed

    Martin, Benjamin J E; McBurney, Kristina L; Maltby, Vicki E; Jensen, Kristoffer N; Brind'Amour, Julie; Howe, LeAnn J

    2017-03-01

    Histone post-translational modifications (PTMs) alter chromatin structure by promoting the interaction of chromatin-modifying complexes with nucleosomes. The majority of chromatin-modifying complexes contain multiple domains that preferentially interact with modified histones, leading to speculation that these domains function in concert to target nucleosomes with distinct combinations of histone PTMs. In Saccharomyces cerevisiae, the NuA3 histone acetyltransferase complex contains three domains, the PHD finger in Yng1, the PWWP domain in Pdp3, and the YEATS domain in Taf14; which in vitro bind to H3K4 methylation, H3K36 methylation, and acetylated and crotonylated H3K9, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone PTMs in targeting NuA3 is unknown. Here, through genome-wide colocalization and by mutational interrogation, we demonstrate that the PHD finger of Yng1, and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. In contrast, we find no evidence to support the YEATS domain of Taf14 functioning in NuA3 recruitment. Collectively our results suggest that the presence of multiple histone PTM binding domains within NuA3, rather than restricting it to nucleosomes containing distinct combinations of histone PTMs, can serve to increase the range of nucleosomes bound by the complex. Interestingly, however, the simple presence of NuA3 is insufficient to ensure acetylation of the associated nucleosomes, suggesting a secondary level of acetylation regulation that does not involve control of HAT-nucleosome interactions. Copyright © 2017 by the Genetics Society of America.

  9. RAPID SEMISYNTHESIS OF ACETYLATED AND SUMOYLATED HISTONE ANALOGS

    PubMed Central

    Dhall, Abhinav; Weller, Caroline E.

    2016-01-01

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

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

    PubMed

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

    2013-04-09

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Chang, Le; Takada, Shoji

    2016-01-01

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

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

    PubMed Central

    Mengel, Alexander; Ageeva, Alexandra; Durner, Jörg

    2017-01-01

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

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

    PubMed

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

    2015-12-18

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

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

    PubMed

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

    2007-03-15

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

  16. Interplay between histone H3 lysine 56 deacetylation and chromatin modifiers in response to DNA damage.

    PubMed

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

    2015-05-01

    In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage. Copyright © 2015 by the Genetics Society of America.

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

    PubMed Central

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

    2015-01-01

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

  18. Histone H3 Serine 57 and Lysine 56 Interplay in Transcription Elongation and Recovery from S-Phase Stress

    PubMed Central

    Aslam, Aamir; Logie, Colin

    2010-01-01

    Background Acetylation of lysine 56 of histone H3 plays an important role in the DNA damage response and it has been postulated to play an as yet undefined role in transcription, both in yeast and in higher eukaryotes. Because phosphorylated human histone H3 serine 57 peptides have been detected by mass spectrometry we examined whether H3-S57 phosphorylation interplays with H3-K56 acetylation in vivo. Methodology/Principal Findings To explore the physiological role of H3-S57, H3-K56 was mutated to mimic constitutively (un)acetylated forms of H3-K56 and these were combined with constitutively (un)phosphorylated mimics of H3-S57, in yeast. A phosphorylated serine mimic at position 57 lessened sensitivities to a DNA replication fork inhibitor and to a transcription elongation inhibitor that were caused by an acetylated lysine mimic at position 56, while the same substitution exacerbated sensitivities due to mimicking a constitutive non-acetylated lysine at position 56. Strikingly, opposite results were obtained in the context of a serine to alanine substitution at position 57 of histone H3. Conclusions/Significance The phenotypes elicited and the context-dependent interplay of the H3-K56 and -S57 point mutations that mimic their respective modification states suggest that serine 57 phosphorylation promotes a nucleosomal transaction when lysine 56 is acetylated. We speculate that histone H3-S57 couples H3-K56 acetylation to histone quaternary structures involving arginine 40 on histone H4 helix 1. PMID:20520775

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

    PubMed Central

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

    2016-01-01

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

  20. Sequential establishment of marks on soluble histones H3 and H4.

    PubMed

    Alvarez, Francisca; Muñoz, Francisca; Schilcher, Pierre; Imhof, Axel; Almouzni, Geneviève; Loyola, Alejandra

    2011-05-20

    Much progress has been made concerning histone function in the nucleus; however, following their synthesis, how their marking and subcellular trafficking are regulated remains to be explored. To gain an insight into these issues, we focused on soluble histones and analyzed endogenous and tagged H3 histones in parallel. We distinguished six complexes that we could place to account for maturation events occurring on histones H3 and H4 from their synthesis onward. In each complex, a different set of chaperones is involved, and we found specific post-translational modifications. Interestingly, we revealed that histones H3 and H4 are transiently poly(ADP-ribosylated). The impact of these marks in histone metabolism proved to be important as we found that acetylation of lysines 5 and 12 on histone H4 stimulated its nuclear translocation. Furthermore, we showed that, depending on particular histone H3 modifications, the balance in the presence of the different translocation complexes changes. Therefore, our results enabled us to propose a regulatory means of these marks for controlling cytoplasmic/nuclear shuttling and the establishment of early modification patterns.

  1. Methylation of histone H3 lysine 9 occurs during translation.

    PubMed

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

    2015-10-30

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

  2. Methylation of histone H3 lysine 9 occurs during translation

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27016736

  5. Transcriptional regulation of 15-lipoxygenase expression by histone h3 lysine 4 methylation/demethylation.

    PubMed

    Liu, Cheng; Xu, Dawei; Han, Hongya; Fan, Yidong; Schain, Frida; Xu, Zhonghua; Claesson, Hans-Erik; Björkholm, Magnus; Sjöberg, Jan

    2012-01-01

    15-Lipoxygenase-1 (15-LOX-1) oxidizes polyunsaturated fatty acids to a rich spectrum of biologically active metabolites and is implicated in physiological membrane remodelling, inflammation and apoptosis. Its deregulation is involved in the pathogenesis of diverse cancer and immune diseases. Recent experimental evidence reveals that dynamic histone methylation/demethylation mediated by histone methyltransferases and demethylases plays a critical role in regulation of chromatin remodelling and gene expression. In the present study, we compared the histone 3 lysine 4 (H3-K4) methylation status of the 15-LOX-1 promoter region of the two Hodgkin lymphoma (HL) cell lines L1236 and L428 with abundant and undetectable 15-LOX-1 expression, respectively. We identified a potential role of H3-K4 methylation in positive regulation of 15-LOX-1 transcription. Furthermore, we found that histone methyltransferase SMYD3 inhibition reduced 15-LOX-1 expression by decreasing promoter activity in L1236 cells. SMYD3 knock down in these cells abolished di-/trimethylation of H3-K4, attenuated the occupancy by the transactivator STAT6, and led to diminished histone H3 acetylation at the 15-LOX-1 promoter. In contrast, inhibition of SMCX, a JmjC-domain-containing H3-K4 tri-demethylase, upregulated 15-LOX-1 expression through induction of H3-K4 trimethylation, histone acetylation and STAT6 recruitment at the 15-LOX-1 promoter in L428 cells. In addition, we observed strong SMYD3 expression in the prostate cancer cell line LNCaP and its inhibition led to decreased 15-LOX-1 expression. Taken together, our data suggest that regulation of histone methylation/demethylation at the 15-LOX-1 promoter is important in 15-LOX-1 expression.

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

    SciTech Connect

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

    2007-03-02

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

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

    PubMed

    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.

  8. Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  10. Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences.

    PubMed Central

    Vidali, G; Boffa, L C; Bradbury, E M; Allfrey, V G

    1978-01-01

    Exposure of HeLa cells to Na butyrate leads to an accumulation of multiacetylated forms of histones H3 and H4. Our studies of histone acetylation in HeLa S-3 cells show that 7 mM butyrate suppresses the deacetylation of histones without influencing the rate of radioactive acetate incorporation. An alteration in nucleosome structure in highly acetylated chromatin is indicated by an increased rate of DNA degradation by DNase I. A close association of acetylated histones with the DNase I-sensitive sequences is confirmed by the finding that histones remaining after limited DNase I digestion are depleted in the multiacetylated forms of histones H3 and H4. DNase I treatment has also been found to selectively release [3H]acetyl-labeled H3 and H4 from avian erythrocyte nuclei under conditions previously shown to preferentially degrade the globlin genes in erthyrocyte chromatin. Our results are consistent with the view that histone acetylation provides a key to the mechanism for altering chromatin structure at the nucleosomal level, and that this may explain the selective DNase I sensitivity of transcriptionally active DNA sequences in different cell types. PMID:276864

  11. Characterization of histone H3K27 modifications in the {beta}-globin locus

    SciTech Connect

    Kim, Yea Woon; Kim, AeRi

    2011-02-11

    Research highlights: {yields} The {beta}-globin locus control region is hyperacetylated and monomethylated at histone H3K27. {yields} Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. {yields} Association of PRC2 subunits is comparable with H3K27me3 pattern. {yields} Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human {beta}-globin locus using the ChIP assay. The LCR of the human {beta}-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the {beta}-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  12. Low Proteolytic Clipping of Histone H3 in Cervical Cancer.

    PubMed

    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.

  13. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    PubMed Central

    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

  14. Opposing roles of H3- and H4-acetylation in the regulation of nucleosome structure––a FRET study.

    PubMed

    Gansen, Alexander; Tóth, Katalin; Schwarz, Nathalie; Langowski, Jörg

    2015-02-18

    Using FRET in bulk and on single molecules, we assessed the structural role of histone acetylation in nucleosomes reconstituted on the 170 bp long Widom 601 sequence. We followed salt-induced nucleosome disassembly, using donor–acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on H2B histone for measuring H2A/H2B dimer exchange. This allowed us to distinguish the influence of acetylation on salt-induced DNA unwrapping at the entry–exit site from its effect on nucleosome core dissociation. The effect of lysine acetylation is not simply cumulative, but showed distinct histone-specificity. Both H3- and H4-acetylation enhance DNA unwrapping above physiological ionic strength; however, while H3-acetylation renders the nucleosome core more sensitive to salt-induced dissociation and to dimer exchange, H4-acetylation counteracts these effects. Thus, our data suggest, that H3- and H4-acetylation have partially opposing roles in regulating nucleosome architecture and that distinct aspects of nucleosome dynamics might be independently controlled by individual histones.

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

    PubMed

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

    2005-04-01

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

  16. Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes

    PubMed Central

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

    2012-01-01

    Summary 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 segments ready for rearrangement. However, this double modification is much more general; it is conserved throughout eukaryotic evolution. In mouse, H3R2me2s is tightly correlated with H3K4me3 at active promoters throughout the genome. Mutational analysis in S. cerevisiae reveals that deposition of H3R2me2s requires the same Set1 complex that deposits H3K4me3. Our work suggests that H3R2me2sK4me3, not simply H3K4me3 alone, is the mark of active promoters, and that factors that recognize H3K4me3 will have their binding modulated by their preference for H3R2me2s. PMID:22720264

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

    PubMed

    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.

  18. Phosphorylation of histone H3 is functionally linked to retinoic acid receptor β promoter activation

    PubMed Central

    Lefebvre, Bruno; Ozato, Keiko; Lefebvre, Philippe

    2002-01-01

    Ligand-dependent transcriptional activation of retinoic acid receptors (RARs) is a multistep process culminating in the formation of a multimeric co-activator complex on regulated promoters. Several co-activator complexes harbor an acetyl transferase activity, which is required for retinoid-induced transcription of reporter genes. Using murine P19 embryonal carcinoma cells, we examined the relationship between histone post-translational modifications and activation of the endogenous RARβ2 promoter, which is under the control of a canonical retinoic acid response element and rapidly induced upon retinoid treatment. While histones H3 and H4 were constitutively acetylated at this promoter, retinoid agonists induced a rapid phosphorylation at Ser10 of histone H3. A retinoid antagonist, whose activity was independent of co-repressor binding to RAR, could oppose this agonist-induced H3 phosphorylation. Since such post-translational modifications were not observed at several other promoters, we conclude that histone H3 phosphorylation may be a molecular signature of the activated, retinoid-controlled mRARβ2 gene promoter. PMID:11897660

  19. ENL links histone acetylation to oncogenic gene expression in AML

    PubMed Central

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

    2017-01-01

    Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs1. Recognition of modified histones by “reader” proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of BET bromodomain inhibitors2, 3. We recently identified the YEATS domain as a novel acetyllysine-binding module4, yet its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralog AF9, is required for disease maintenance in acute myeloid leukaemia (AML). CRISPR-Cas9 mediated depletion of ENL led to anti-leukemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and ChIP-seq analyses revealed that ENL binds to acetylated histone H3, and colocalizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemias. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced RNA polymerase II recruitment to ENL target genes, leading to suppression of oncogenic gene expression programs. Importantly, disruption of ENL’s functionality further sensitized leukaemia cells to BET inhibitors. Together, our study identifies ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in AML and suggests that displacement of ENL from chromatin may be a promising epigenetic therapy alone or in combination with BET inhibitors for AML. PMID:28241141

  20. Molecular dynamics for computational proteomics of methylated histone H3.

    PubMed

    Grauffel, Cédric; Stote, Roland H; Dejaegere, Annick

    2015-05-01

    Post-translational modifications of histones, and in particular of their disordered N-terminal tails, play a major role in epigenetic regulation. The identification of proteins and proteic domains that specifically bind modified histones is therefore of paramount importance to understand the molecular mechanisms of epigenetics. We performed an energetic analysis using the MM/PBSA method in order to study known complexes between methylated histone H3 and effector domains of the PHD family. We then developed a simple molecular dynamics based predictive model based on our analysis. We present a thorough validation of our procedure, followed by the computational predictions of new PHD domains specific for binding histone H3 methylated on lysine 4 (K4). PHD domains recognize methylated K4 on histone H3 in the context of a linear interaction motif (LIM) formed by the first four amino acids of histone H3 as opposed to recognition of a single methylated site. PHD domains with different sequences find chemically equivalent solutions for stabilizing the histone LIM and these can be identified from energetic analysis. This analysis, in turn, allows for the identification of new PHD domains that bind methylated H3K4 using information that cannot be retrieved from sequence comparison alone. Molecular dynamics simulations can be used to devise computational proteomics protocols that are both easy to implement and interpret, and that yield reliable predictions that compare favorably to and complement experimental proteomics methods. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Transcription in the absence of histone H3.2 and H3K4 methylation.

    PubMed

    Hödl, Martina; Basler, Konrad

    2012-12-04

    Histone H3 proteins play fundamental roles in DNA packaging, gene transcription, and the transmission of epigenetic states. In addition to posttranslational modifications of their N termini, the use of H3 variants contributes to their regulatory repertoire. Canonical histone H3.2 is expressed during S phase and differs by four amino acid residues from the variant histone H3.3, which is synthesized in a cell-cycle-independent manner. Because H3.3 is enriched within actively transcribed loci, and because di- and trimethylation of H3 lysine 4 are hallmarks of chromatin at such sites in the genome, the H3.3K4 residue is considered to serve as the major regulatory determinant for the transcriptional state of a gene. Here we use genetic approaches in Drosophila to replace all 46 gene copies of His3.2 with mutant derivatives and thereby demonstrate that canonical and variant H3 can functionally replace each other. Cells are able to divide and differentiate when H3.2 is entirely absent but replaced by S phase-expressed H3.3. Moreover, although slowed down in their proliferative capacity, cells that code for a nonmethylatable residue instead of K4 in all canonical and variant H3 genes are competent to respond to major developmental signaling pathways by activating target gene expression. Hence, the presence of different H3 protein species is not essential in Drosophila and transcriptional regulation can occur in the complete absence of H3K4 methylation.

  2. Genome-Wide Histone Acetylation Is Altered in a Transgenic Mouse Model of Huntington's Disease

    PubMed Central

    McFarland, Karen N.; Das, Sudeshna; Sun, Ting Ting; Leyfer, Dmitri; Xia, Eva; Sangrey, Gavin R.; Kuhn, Alexandre; Luthi-Carter, Ruth; Clark, Timothy W.; Sadri-Vakili, Ghazaleh; Cha, Jang-Ho J.

    2012-01-01

    In Huntington's disease (HD; MIM ID #143100), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation. PMID:22848491

  3. Maintenance of cell fates through acetylated histone and the histone variant H2A.z in C. elegans.

    PubMed

    Shibata, Yukimasa; Nishiwaki, Kiyoji

    2014-01-01

    Maintenance of cell fates is essential for the development and homeostasis of multicellular organisms and involves the preservation of the expression status of selector genes that control many target genes. Epigenetic marks have pivotal roles in the maintenance of gene expression status, as occurs with methylation on lysine 27 of histone H3 (H3K27me) for Hox gene regulation. In contrast, because the levels of histone acetylation decrease during the mitotic phase, acetylated histone has not been believed to contribute to the maintenance of cell fates. Because members of the bromodomain and extra terminal (BET) family bind to acetylated histones localized on mitotic chromosomes, it is possible that they may regulate the transcriptional status of genes throughout the cell cycle. In this commentary, we discuss the recent analyses of C. elegans BET family protein BET-1, which contributes to the maintenance of cell fates through the histone H2A variant HTZ-1/H2A.z. This mechanism represses transcription of selector genes in the genomic region where lysine 27 of histone H3 (H3K27) is demethylated by histone demethylase UTX-1. We discuss the possibility that BET-1 and HTZ-1 maintain the poised state of RNA polymerase II in the cell such that it is ready to respond to differentiation signals.

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

    PubMed

    Yorita, Satoko; Mochizuki, Kazuki; Goda, Toshinao

    2009-04-23

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

  5. Paraquat Induces Epigenetic Changes by Promoting Histone Acetylation in Cell Culture Models of Dopaminergic Degeneration

    PubMed Central

    Song, C.; Kanthasamy, A.; Jin, H.; Anantharam, V.; Kanthasamy, A. G.

    2012-01-01

    Environmental neurotoxic exposure to agrochemicals has been implicated in the etiopathogenesis of Parkinson’s disease (PD). The widely used herbicide paraquat is among the few environmental chemicals potentially linked with PD. Since epigenetic changes are beginning to emerge as key mechanisms in neurodegenerative diseases, herein we examined the effects of paraquat on histone acetylation, a major epigenetic change in chromatin that can regulate gene expression, chromatin remodeling, cell survival and cell death. Exposure of N27 dopaminergic cells to paraquat induced histone H3 acetylation in a time-dependent manner. However, paraquat did not alter acetylation of another core histone H4. Paraquat-induced histone acetylation was associated with decreased total histone deacetylase (HDAC) activity and HDAC4 and 7 protein expression levels. To determine if histone acetylation plays a role in paraquat-induced apoptosis, the novel HAT inhibitor anacardic acid was used. Anacardic acid treatment significantly attenuated paraquat-induced caspase-3 enzyme activity, suppressed proteolytic activation and kinase activity of protein kinase C delta (PKCδ) and also blocked paraquat-induced cytotoxicity. Together, these results demonstrate that the neurotoxic agent paraquat induced acetylation of core histones in cell culture models of PD and that inhibition of HAT activity by anacardic acid protects against apoptotic cell death, indicating that histone acetylation may represent key epigenetic changes in dopaminergic neuronal cells during neurotoxic insults. PMID:21777615

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

    SciTech Connect

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

    2011-08-24

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

  7. Histones associated with downregulated genes are hypo-acetylated in Huntington's disease models.

    PubMed

    Sadri-Vakili, Ghazaleh; Bouzou, Bérengère; Benn, Caroline L; Kim, Mee-Ohk; Chawla, Prianka; Overland, Ryan P; Glajch, Kelly E; Xia, Eva; Qiu, Zhihua; Hersch, Steven M; Clark, Timothy W; Yohrling, George J; Cha, Jang-Ho J

    2007-06-01

    Transcriptional dysregulation plays a major role in the pathology of Huntington's disease (HD). However, the mechanisms causing selective downregulation of genes remain unknown. Histones regulate chromatin structure and thereby control gene expression; recent studies have demonstrated a therapeutic role for histone deacetylase (HDAC) inhibitors in polyglutamine diseases. This study demonstrates that despite no change in overall acetylated histone levels, histone H3 is hypo-acetylated at promoters of downregulated genes in R6/2 mice, ST14a and STHdh cells, as demonstrated by in vivo chromatin immunoprecipitation. In addition, HDAC inhibitor treatment increases association of acetylated histones with downregulated genes and corrects mRNA abnormalities. In contrast, there is a decrease in mRNA levels in wild-type cells following treatment with a histone acetyltransferase inhibitor. Although changes in histone acetylation correlate with decreased gene expression, histone hypo-acetylation may be a late event, as no hypo-acetylation is observed in 4-week-old R6/2 mice. Nevertheless, treatment with HDAC inhibitors corrects mRNA abnormalities through modification of histone proteins and may prove to be of therapeutic value in HD.

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

    PubMed

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

    2013-01-01

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

  9. Marking histone H3 variants: how, when and why?

    PubMed

    Loyola, Alejandra; Almouzni, Geneviève

    2007-09-01

    DNA in eukaryotic cells is compacted into chromatin, a regular repeated structure in which the nucleosome represents the basic unit. The nucleosome not only serves to compact the genetic material but also provides information that affects nuclear functions including DNA replication, repair and transcription. This information is conveyed through numerous combinations of histone post-translational modifications (PTMs) and histone variants. A recent challenge has been to understand how and when these combinations of PTMs are imposed and to what extent they are determined by the choice of a specific histone variant. Here we focus on histone H3 variants and the PTMs that they carry before and after their assembly into chromatin. We review and discuss recent knowledge about how the choice and initial modifications of a specific variant might affect PTM states and eventually the final epigenetic state of a chromosomal domain.

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2013-09-01

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

  12. The dynamics of histone H3 modifications is species-specific in plant meiosis.

    PubMed

    Oliver, Cecilia; Pradillo, Mónica; Corredor, Eduardo; Cuñado, Nieves

    2013-07-01

    Different histone modifications often modify DNA-histone interactions affecting both local and global structure of chromatin, thereby providing a vast potential for functional responses. Most studies have focused on the role of several modifications in gene transcription regulation, being scarce on other aspects of eukaryotic chromosome structure during cell division, mainly in meiosis. To solve this issue we have performed a cytological analysis to determine the chromosomal distribution of several histone H3 modifications throughout all phases of both mitosis and meiosis in different plant species. We have chosen Aegilops sp. and Secale cereale (monocots) and Arabidopsis thaliana (dicots) because they differ in their phylogenetic affiliation as well as in content and distribution of constitutive heterochromatin. In the species analyzed, the patterns of H3 acetylation and methylation were held constant through mitosis, including modifications associated with "open chromatin". Likewise, the immunolabeling patterns of H3 methylation remained invariable throughout meiosis in all cases. On the contrary, there was a total loss of acetylated H3 immunosignals on condensed chromosomes in both meiotic divisions, but only in monocot species. Regarding the phosphorylation of histone H3 at Ser10, present on condensed chromosomes, although we did not observe any difference in the dynamics, we found slight differences between the chromosomal distribution of this modification between Arabidopsis and cereals (Aegilops sp. and rye). Thus far, in plants chromosome condensation throughout cell division appears to be associated with a particular combination of H3 modifications. Moreover, the distribution and dynamics of these modifications seem to be species-specific and even differ between mitosis and meiosis in the same species.

  13. Overlapping Functions between SWR1 Deletion and H3K56 Acetylation in Candida albicans

    PubMed Central

    Guan, Zhiyun

    2015-01-01

    Nucleosome destabilization by histone variants and modifications has been implicated in the epigenetic regulation of gene expression, with the histone variant H2A.Z and acetylation of H3K56 (H3K56ac) being two examples. Here we find that deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin in exchange for H2A, promotes epigenetic white-opaque switching in Candida albicans. We demonstrate through nucleosome mapping that SWR1 is required for proper nucleosome positioning on the promoter of WOR1, the master regulator of switching, and that its effects differ in white and opaque cells. Furthermore, we find that H2A.Z is enriched adjacent to nucleosome-free regions at the WOR1 promoter in white cells, suggesting a role in the stabilization of a repressive chromatin state. Deletion of YNG2, a subunit of the NuA4 H4 histone acetyltransferase (HAT) that targets SWR1 activity through histone acetylation, produces a switching phenotype similar to that of swr1, and both may act downstream of the GlcNAc signaling pathway. We further uncovered a genetic interaction between swr1 and elevated H3K56ac with the discovery that the swr1 deletion mutant is highly sensitive to nicotinamide. Our results suggest that the interaction of H2A.Z and H3K56ac regulates epigenetic switching at the nucleosome level, as well as having global effects. PMID:25862154

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

    PubMed

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

    2004-05-11

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

  15. Cadmium Induces Histone H3 Lysine Methylation by Inhibiting Histone Demethylase Activity

    PubMed Central

    Xiao, Chunlian; Liu, Yin; Xie, Chengfeng; Tu, Wei; Xia, Yujie; Costa, Max; Zhou, Xue

    2015-01-01

    Cadmium is an established human lung carcinogen with weak mutagenicity. However, the mechanisms underlying cadmium-induced carcinogenesis remain obscure. It has been suggested that epigenetic mechanisms may play a role in cadmium-induced carcinogenesis. In this study, we investigated the effects of cadmium on histone methylation and histone demethylases, and the role of histone methylation in transformation of immortalized normal human bronchial epithelial (BEAS-2B) cells. Exposure to 0.625, 1.25, 2.5, and 5.0 μM of cadmium for 6, 24, and 48 h increased global trimethylated histone H3 on lysine 4 (H3K4me3) and dimethylated histone H3 on lysine 9 (H3K9me2) in BEAS-2B cells compared with untreated cells, and most of these changes remained after the removal of cadmium (P < .05 or P < .01 for most modifications). Meanwhile, cadmium inhibited the activities of histone H3 on lysine 4 (H3K4) and histone H3 on lysine 9 (H3K9) demethylases which were detected by histone demethylation assay. However, there was no significant change in the protein levels of the H3K4 demethylase lysine-specific demethylase 5A (KDM5A) and the H3K9 demethylase lysine-specific demethylase 3A (KDM3A). Interestingly, during transformation of BEAS-2B cells by 20 weeks of exposure to 2.0 μM cadmium as assessed by anchorage-independent growth in soft agar, global H3K4me3, and H3K9me2 were significantly increased at 4 weeks (P < .05 or P < .01), whereas no significant change was observed at 8, 12, 16, and 20 weeks compared with control. Our study suggests that cadmium increases global H3K4me3 and H3K9me2 by inhibiting the activities of histone demethylases, and aberrant histone methylation that occurs early (48 h) and at 4 weeks is associated with cadmium-induced transformation of BEAS-2B cells at the early stage. PMID:25673502

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    1989-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    SciTech Connect

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

    2013-01-24

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2007-01-01

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

  2. Sites of acetylation on newly synthesized histone H4 are required for chromatin assembly and DNA damage response signaling.

    PubMed

    Ge, Zhongqi; Nair, Devi; Guan, Xiaoyan; Rastogi, Neha; Freitas, Michael A; Parthun, Mark R

    2013-08-01

    The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.

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

    PubMed

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

    2009-10-01

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

  4. Conformational changes in the H3. H4 histone complex

    SciTech Connect

    Feldman, L.; Beaudette, N.V.; Stollar, B.D.; Fasman, G.D.

    1980-08-10

    The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO/sub 4/, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the ..cap alpha.. helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

  5. H4K12 and H3K18 Acetylation Associates With Poor Prognosis in Pancreatic Cancer.

    PubMed

    Juliano, Camila N; Izetti, Patricia; Pereira, Marcia P; Dos Santos, Alex P; Bravosi, Charles P; Abujamra, Ana L; Prolla, Patricia A; Osvaldt, Alessandro B; Edelweiss, Maria I A

    2016-01-01

    Epigenetic deregulation may be involved in tumor cell biology, including differentiation, tumor progression, and cell death, and histone acetylation is a major regulatory mechanism of gene transcription. Patterns of global histone modifications have been recently suggested as outcome predictors in cancer patients, but few studies have been conducted on pancreatic ductal adenocarcinomas (PDACs). This study was designed to investigate the predictive value of histone acetylation modifications on PDAC. A retrospective clinicopathologic analysis was undertaken in 119 patients diagnosed with PDAC between 2005 and 2011, and immunohistochemistry performed with polyclonal antibodies against H4K12ac, H3K9ac, and H3K18ac. Positive nuclear staining for each histone was measured as the intensity and expression, being classified into low-staining or high-staining groups. Results were analyzed in relation to patients' clinicopathologic parameters. There was a positive relationship between tumor differentiation and H4K12ac high scores (P<0.05) and staining with the 3 markers correlated positively with tumor stage (P<0.01). Univariate analysis showed worse survival in patients with high detection levels of H4K12ac (P=0.038) and H3K18Ac (P=0.033). A backwards Cox proportional hazards model analysis revealed the independent prognostic effect of high H4K12ac and H3K18ac levels (hazard ratios of 1.6 and 1.7, respectively, P<0.05), especially for patients at early stages of disease. We propose that acetylation of H4K12 and H3K18 may be considered valuable prognostic factors for pancreatic cancer, although the mechanism involved needs further investigation. Increasing insights into histone acetylation modifications can ultimately generate new ideas for rational and molecularly based diagnostic and therapeutic approaches.

  6. Acupuncture promotes angiogenesis after myocardial ischemia through H3K9 acetylation regulation at VEGF gene.

    PubMed

    Fu, Shu-Ping; He, Su-Yun; Xu, Bin; Hu, Chen-Jun; Lu, Sheng-Feng; Shen, Wei-Xing; Huang, Yan; Hong, Hao; Li, Qian; Wang, Ning; Liu, Xuan-Liang; Liang, Fanrong; Zhu, Bing-Mei

    2014-01-01

    Acupuncture exerts cardioprotective effects on several types of cardiac injuries, especially myocardial ischemia (MI), but the mechanisms have not yet been well elucidated. Angiogenesis mediated by VEGF gene expression and its modification through histone acetylation has been considered a target in treating myocardial ischemia. This study aims to exam whether modulation of angiogenesis through H3K9 acetylation regulation at VEGF gene is one possible cardioprotective mechanism of acupuncture. We generated rat MI models by ligating the left anterior descending coronary artery and applied electroacupuncture (EA) treatment at the Neiguan (PC6) acupoint. Our results showed that acupuncture reversed the S-T segment change, reduced Q-wave area, decreased CK, CK-MB, LDH levels, mitigated myocardial remodeling, and promoted microvessel formation in the MI heart. RNA-seq analysis showed that VEGF-induced angiogenesis signaling was involved in the modulation of EA. Western blot results verified that the protein expressions of VEGF, Ras, phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-SAPK/JNK and Akt, were all elevated significantly by EA treatment in the MI heart. Furthermore, increased H3K9 acetylation was also observed according with the VEGF. ChIP assay confirmed that EA treatment could notably stimulate the recruitment of H3K9ace at the VEGF promoter. Our study demonstrates for the first time that acupuncture can effectively up-regulate VEGF expression through H3K9 acetylation modification directly at the VEGF promoter and hence activate VEGF-induced angiogenesis in rat MI models. We employed high throughput sequencing in this study and, for the first time, generated genome-wide gene expression profiles both in the rat MI model and in acupuncture treatment.

  7. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline.

    PubMed

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-11-05

    A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously, we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here, we show that phosphorylation at threonine 3 of H3 (H3T3P) distinguishes pre-existing versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects, including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. These studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3 in order to coordinate asymmetric segregation of "old" H3 into GSCs and that tight regulation of H3T3 phosphorylation is required for male germline activity.

  8. Histone H3 Threonine Phosphorylation Regulates Asymmetric Histone Inheritance in the Drosophila Male Germline

    PubMed Central

    Xie, Jing; Wooten, Matthew; Tran, Vuong; Chen, Bi-Chang; Pozmanter, Caitlin; Simbolon, Christine; Betzig, Eric; Chen, Xin

    2015-01-01

    SUMMARY A long-standing question concerns how stem cells maintain their identity through multiple divisions. Previously we reported that pre-existing and newly synthesized histone H3 are asymmetrically distributed during Drosophila male germline stem cell (GSC) asymmetric division. Here we show that phosphorylation at Threonine 3 of H3 (H3T3P) distinguishes preexisting versus newly synthesized H3. Converting T3 to the unphosphorylatable residue alanine (H3T3A) or to the phosphomimetic aspartate (H3T3D) disrupts asymmetric H3 inheritance. Expression of H3T3A or H3T3D specifically in early-stage germline also leads to cellular defects including GSC loss and germline tumors. Finally, compromising the activity of the H3T3 kinase Haspin enhances the H3T3A but suppresses the H3T3D phenotypes. Together these studies demonstrate that H3T3P distinguishes sister chromatids enriched with distinct pools of H3, coordinating asymmetric segregation of “old” H3 into GSCs, and that a tight regulation of H3T3 phosphorylation is required for male germline activity. PMID:26522592

  9. Butyrate, an HDAC inhibitor, stimulates interplay between different posttranslational modifications of histone H3 and differently alters G1-specific cell cycle proteins in vascular smooth muscle cells.

    PubMed

    Mathew, Omana P; Ranganna, Kasturi; Yatsu, Frank M

    2010-12-01

    HDACs and HATs regulate histone acetylation, an epigenetic modification that controls chromatin structure and through it, gene expression. Butyrate, a dietary HDAC inhibitor, inhibits VSMC proliferation, a crucial factor in atherogenesis, and the principle mechanism in arterial and in-stent restenosis. Here, the link between antiproliferation action of butyrate and the portraits of global covalent modifications of histone H3 that it induces are characterized to understand the mechanics of butyrate-arrested VSMC proliferation. Analysis of histone H3 modifications specific to butyrate arrested VSMC proliferation display induction of histone H3-Lysine9 acetylation, inhibition of histone H3-Serine10 phosphorylation, reduction of histone H3-Lysine9 dimethylation and stimulation of histone H3-Lysine4 di-methylation, which is linked to transcriptional activation, cell cycle/mitosis, transcriptional suppression and activation, respectively. Conversely, untreated VSMCs exhibit inhibition of H3-Lysine9 acetylation, induction of H3-Serine10 phosphorylation, stimulation of H3-Lysine9 di-methylation and reduction in H3-Lysine4 di-methylation. Butyrate's cooperative effects on H3-Lysine9 acetylation and H3-Serine10 phosphorylation, and contrasting effects on di-methylation of H3-Lysine9 and H3-Lysine4 suggests that the interplay between these site-specific modifications cause distinct chromatin alterations that allow cyclin D1 and D3 induction, G1-specific cdk4, cdk6 and cdk2 downregulation, and upregulation of cdk inhibitors, p15INK4b and p21Cip1. Regardless of butyrate's effect on D-type cyclins, downregulation of G1-specific cdks and upregulation of cdk inhibitors by butyrate prevents cell cycle progression by failing to inactivate Rb. Overall, through chromatin remodeling, butyrate appears to differentially alter G1-specific cell cycle proteins to ensure proliferation arrest of VSMCs, a crucial cellular component of blood vessel wall.

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

    PubMed Central

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

    2010-01-01

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

  11. MMP-9 facilitates selective proteolysis of the histone H3 tail at genes necessary for proficient osteoclastogenesis

    PubMed Central

    Kim, Kyunghwan; Punj, Vasu; Kim, Jin-Man; Lee, Sunyoung; Ulmer, Tobias S.; Lu, Wange; Rice, Judd C.; An, Woojin

    2016-01-01

    Although limited proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during mammalian differentiation, the specific genomic sites targeted for H3NT proteolysis and the functional significance of H3NT cleavage remain largely unknown. Here we report the first method to identify and examine H3NT-cleaved regions in mammals, called chromatin immunoprecipitation (ChIP) of acetylated chromatin (ChIPac). By applying ChIPac combined with deep sequencing (ChIPac-seq) to an established cell model of osteoclast differentiation, we discovered that H3NT proteolysis is selectively targeted near transcription start sites of a small group of genes and that most H3NT-cleaved genes displayed significant expression changes during osteoclastogenesis. We also discovered that the principal H3NT protease of osteoclastogenesis is matrix metalloproteinase 9 (MMP-9). In contrast to other known H3NT proteases, MMP-9 primarily cleaved H3K18-Q19 in vitro and in cells. Furthermore, our results support CBP/p300-mediated acetylation of H3K18 as a central regulator of MMP-9 H3NT protease activity both in vitro and at H3NT cleavage sites during osteoclastogenesis. Importantly, we found that abrogation of H3NT proteolysis impaired osteoclastogenic gene activation concomitant with defective osteoclast differentiation. Our collective results support the necessity of MMP-9-dependent H3NT proteolysis in regulating gene pathways required for proficient osteoclastogenesis. PMID:26744418

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2017-02-01

    Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined "hot spots." In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots.

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

    PubMed Central

    Wu, Zhen; Fallahi, Mohammad; Ouizem, Souad; Liu, Qin; Li, Weimin; Costi, Roberta; Roush, William R.; Bois, Philippe R. J.

    2016-01-01

    ABSTRACT Meiotic recombination initiates following the formation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease early in prophase I, at discrete regions in the genome coined “hot spots.” In mammals, meiotic DSB site selection is directed in part by sequence-specific binding of PRDM9, a polymorphic histone H3 (H3K4Me3) methyltransferase. However, other chromatin features needed for meiotic hot spot specification are largely unknown. Here we show that the recombinogenic cores of active hot spots in mice harbor several histone H3 and H4 acetylation and methylation marks that are typical of open, active chromatin. Further, deposition of these open chromatin-associated histone marks is dynamic and is manifest at spermatogonia and/or pre-leptotene-stage cells, which facilitates PRDM9 binding and access for Spo11 to direct the formation of DSBs, which are initiated at the leptotene stage. Importantly, manipulating histone acetylase and deacetylase activities established that histone acetylation marks are necessary for both hot spot activity and crossover resolution. We conclude that there are functional roles for histone acetylation marks at mammalian meiotic recombination hot spots. PMID:27821479

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

    PubMed

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

    2008-11-28

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

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

    PubMed Central

    Hammond, Colin M.; Sundaramoorthy, Ramasubramanian; Larance, Mark; Lamond, Angus; Stevens, Michael A.; El-Mkami, Hassane; Norman, David G.; Owen-Hughes, Tom

    2016-01-01

    Vps75 is a histone chaperone that has been historically characterized as homodimer by X-ray crystallography. In this study, we present a crystal structure containing two related tetrameric forms of Vps75 within the crystal lattice. We show Vps75 associates with histones in multiple oligomers. In the presence of equimolar H3–H4 and Vps75, the major species is a reconfigured Vps75 tetramer bound to a histone H3–H4 tetramer. However, in the presence of excess histones, a Vps75 dimer bound to a histone H3–H4 tetramer predominates. We show the Vps75–H3–H4 interaction is compatible with the histone chaperone Asf1 and deduce a structural model of the Vps75–Asf1-H3–H4 (VAH) co-chaperone complex using the Pulsed Electron-electron Double Resonance (PELDOR) technique and cross-linking MS/MS distance restraints. The model provides a molecular basis for the involvement of both Vps75 and Asf1 in Rtt109 catalysed histone H3 K9 acetylation. In the absence of Asf1 this model can be used to generate a complex consisting of a reconfigured Vps75 tetramer bound to a H3–H4 tetramer. This provides a structural explanation for many of the complexes detected biochemically and illustrates the ability of Vps75 to interact with dimeric or tetrameric H3–H4 using the same interaction surface. PMID:27036862

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

    PubMed Central

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

    2016-01-01

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

  19. Histone H3G34R mutation causes replication stress, homologous recombination defects and genomic instability in S. pombe

    PubMed Central

    Yadav, Rajesh K; Jablonowski, Carolyn M; Fernandez, Alfonso G; Lowe, Brandon R; Henry, Ryan A; Finkelstein, David; Barnum, Kevin J; Pidoux, Alison L; Kuo, Yin-Ming; Huang, Jie; O’Connell, Matthew J; Andrews, Andrew J; Onar-Thomas, Arzu; Allshire, Robin C; Partridge, Janet F

    2017-01-01

    Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma. DOI: http://dx.doi.org/10.7554/eLife.27406.001 PMID:28718400

  20. Insulin induced alteration in post-translational modifications of histone H3 under a hyperglycemic condition in L6 skeletal muscle myoblasts.

    PubMed

    Kabra, Dhiraj G; Gupta, Jeena; Tikoo, Kulbhushan

    2009-06-01

    Chromatin remodelling events, especially histone modifications are proposed to form the mainstay for most of the biological processes. However, the role of these histone modifications in the progression of diabetes is still unknown. Hyperglycemia plays a major role in diabetes and its complications. The present study was undertaken to check the effect of insulin on alterations in post-translational modifications of histone H3 in L6 myoblasts under a hyperglycemic condition. We provide first evidence that insulin under hyperglycemic condition alters multiple histone modifications by enhanced production of reactive oxygen species. Insulin induces dose dependent changes in Lysine 4 and 9 methylation, Ser 10 phosphorylation and acetylation of histone H3. Interestingly, insulin induced generation of reactive oxygen species induces dephosphorylation and deacetylation of histone H3. Preincubation with catalase and DPI prevents these changes in post-translational modifications of histone H3. Furthermore, changes in histone H3 phosphorylation was found to be independent of ERK, p38, RSK2 and MSK1. Moreover, serine/threonine phosphatase inhibitor, okadaic acid attenuates insulin induced dephosphorylation and deacetylation of histone H3, suggesting a role of serine/threonine phosphatases in altering modifications of histone H3. These changes in epigenetic modifications can provide new insights into pathogenesis of diabetes.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  3. Toxoplasma histone acetylation remodelers as novel drug targets

    PubMed Central

    Vanagas, Laura; Jeffers, Victoria; Bogado, Silvina S; Dalmasso, Maria C; Sullivan, William J; Angel, Sergio O

    2013-01-01

    Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite. PMID:23199404

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

    PubMed

    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.

  5. The Differential Mobilization of Histones H3.1 and H3.3 by Herpes Simplex Virus 1 Relates Histone Dynamics to the Assembly of Viral Chromatin

    PubMed Central

    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. PMID:24130491

  6. NIRF, a Novel Ubiquitin Ligase, Inhibits Hepatitis B Virus Replication Through Effect on HBV Core Protein and H3 Histones.

    PubMed

    Qian, Guanhua; Hu, Bin; Zhou, Danlin; Xuan, Yanyan; Bai, Lu; Duan, Changzhu

    2015-05-01

    Np95/ICBP90-like RING finger protein (NIRF), a novel E3 ubiquitin ligase, has been shown to interact with HBc and promote its degradation. This study investigated the effects of NIRF on replication of hepatitis B virus (HBV) and the mechanisms. We have shown that NIRF inhibits replication of HBV DNA and secretion of HBsAg and HBeAg in HepG2 cells transfected with pAAV-HBV1.3. NIRF also inhibits the replication and secretion of HBV in a mouse model that expressed HBV. NIRF reduces acetylation of HBV cccDNA-bound H3 histones. These results showed that NIRF is involved in the HBV replication cycle not only through direct interaction with HBc but also reduces acetylation of HBV cccDNA-bound H3 histones.

  7. Histone H3 K56 Hyperacetylation Perturbs Replisomes and Causes DNA Damage

    PubMed Central

    Celic, Ivana; Verreault, Alain; Boeke, Jef D.

    2008-01-01

    Deacetylation of histone H3 K56, regulated by the sirtuins Hst3p and Hst4p, is critical for maintenance of genomic stability. However, the physiological consequences of a lack of H3 K56 deacetylation are poorly understood. Here we show that cells lacking Hst3p and Hst4p, in which H3 K56 is constitutively hyperacetylated, exhibit hallmarks of spontaneous DNA damage, such as activation of the checkpoint kinase Rad53p and upregulation of DNA-damage inducible genes. Consistently, hst3 hst4 cells display synthetic lethality interactions with mutations that cripple genes involved in DNA replication and DNA double-strand break (DSB) repair. In most cases, synthetic lethality depends upon hyperacetylation of H3 K56 because it can be suppressed by mutation of K56 to arginine, which mimics the nonacetylated state. We also show that hst3 hst4 phenotypes can be suppressed by overexpression of the PCNA clamp loader large subunit, Rfc1p, and by inactivation of the alternative clamp loaders CTF18, RAD24, and ELG1. Loss of CTF4, encoding a replisome component involved in sister chromatid cohesion, also suppresses hst3 hst4 phenotypes. Genetic analysis suggests that CTF4 is a part of the K56 acetylation pathway that converges on and modulates replisome function. This pathway represents an important mechanism for maintenance of genomic stability and depends upon proper regulation of H3 K56 acetylation by Hst3p and Hst4p. Our data also suggest the existence of a precarious balance between Rfc1p and the other RFC complexes and that the nonreplicative forms of RFC are strongly deleterious to cells that have genomewide and constitutive H3 K56 hyperacetylation. PMID:18579506

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

    PubMed Central

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

    2013-01-01

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

  9. New functions for an old variant: no substitute for histone H3.3

    PubMed Central

    Elsaesser, Simon J; Goldberg, Aaron D; Allis, C David

    2010-01-01

    Histone proteins often come in different variants serving specialized functions in addition to their fundamental role in packaging DNA. The metazoan histone H3.3 has been most closely associated with active transcription. Its role in histone replacement at active genes and promoters is conserved to the single histone H3 in yeast. However, recent genetic studies in flies have challenged its importance as a mark of active chromatin, and revealed unexpected insights into essential functions of H3.3 in the germline. With strikingly little amino acid sequence difference to the canonical H3, H3.3 therefore accomplishes a surprising variety of cellular and developmental processes. PMID:20153629

  10. The Histone H3 Acetylase dGcn5 Is a Key Player in Drosophila melanogaster Metamorphosis

    PubMed Central

    Carré, Clément; Szymczak, Dimitri; Pidoux, Josette; Antoniewski, Christophe

    2005-01-01

    Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure and gene transcription, there is only little information on their developmental role in higher organisms. Gcn5 was the first transcription factor with HAT activity identified in eukaryotes. Here we report the isolation and characterization of Drosophila melanogaster dGcn5 mutants. Null dGcn5 alleles block the onset of both oogenesis and metamorphosis, while hypomorphic dGcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and K14 lysine residues of histone H3 in dGcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are observed. In vivo complementation experiments revealed that dGcn5 integrates specific functions in addition to chromosome binding and acetylation. Surprisingly, a dGcn5 variant protein with a deletion of the bromodomain, which has been shown to recognize acetylated histones, appears to be fully functional. Our results establish dGcn5 as a major histone H3 acetylase in Drosophila which plays a key role in the control of specific morphogenetic cascades during developmental transitions. PMID:16135811

  11. Proportions of acetyl-histone-positive hepatocytes indicate the functional status and prognosis of cirrhotic patients

    PubMed Central

    Zhou, Ping; Xia, Jie; Zhou, Yong-Jie; Wan, Jun; Li, Li; Bao, Ji; Shi, Yu-Jun; Bu, Hong

    2015-01-01

    AIM: To investigate whether the proportions of acetyl-histone-positive hepatocytes could be used as markers of deteriorating liver function. METHODS: In total, 611 cirrhotic cases from 3701 patients who were diagnosed during the past 15 years were screened, and 152 follow-up cases were selected. Paraffin tissue microarray was prepared for immunohistochemistry to examine acetyl-histone expression. The proportions of positive hepatocytes were recorded, and their correlations to clinical and laboratory indicators were analyzed statistically. RESULTS: The proportions of H2AK5ac+, H3K9/K14ac+ and H3K27ac+ hepatocytes gradually increased with deteriorating liver function and with increasing levels of serum markers of liver injury. In the follow-up cases, patients with > 70% H2AK5ac+, H3K9/K14ac+ or H3K27ac+ hepatocytes had statistically lower survival rates (P < 0.05). Furthermore, > 70% H2AK5ac+ or H3K27ac+ hepatocytes were strong independent predictors of overall survival (P < 0.05). CONCLUSION: The proportions of acetyl-histone-positive hepatocytes are closely associated with the liver function and prognosis of cirrhotic patients. PMID:26074705

  12. Proportions of acetyl-histone-positive hepatocytes indicate the functional status and prognosis of cirrhotic patients.

    PubMed

    Zhou, Ping; Xia, Jie; Zhou, Yong-Jie; Wan, Jun; Li, Li; Bao, Ji; Shi, Yu-Jun; Bu, Hong

    2015-06-07

    To investigate whether the proportions of acetyl-histone-positive hepatocytes could be used as markers of deteriorating liver function. In total, 611 cirrhotic cases from 3701 patients who were diagnosed during the past 15 years were screened, and 152 follow-up cases were selected. Paraffin tissue microarray was prepared for immunohistochemistry to examine acetyl-histone expression. The proportions of positive hepatocytes were recorded, and their correlations to clinical and laboratory indicators were analyzed statistically. The proportions of H2AK5ac(+), H3K9/K14ac(+) and H3K27ac(+) hepatocytes gradually increased with deteriorating liver function and with increasing levels of serum markers of liver injury. In the follow-up cases, patients with > 70% H2AK5ac(+), H3K9/K14ac(+) or H3K27ac(+) hepatocytes had statistically lower survival rates (P < 0.05). Furthermore, > 70% H2AK5ac(+) or H3K27ac(+) hepatocytes were strong independent predictors of overall survival (P < 0.05). The proportions of acetyl-histone-positive hepatocytes are closely associated with the liver function and prognosis of cirrhotic patients.

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

    PubMed Central

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

    2015-01-01

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

  14. Histone H1 binding is inhibited by histone variant H3.3.

    PubMed

    Braunschweig, Ulrich; Hogan, Greg J; Pagie, Ludo; van Steensel, Bas

    2009-12-02

    Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

  15. Environmental Neurotoxic Pesticide Increases Histone Acetylation to Promote Apoptosis in Dopaminergic Neuronal Cells: Relevance to Epigenetic Mechanisms of Neurodegeneration

    PubMed Central

    Song, C.; Kanthasamy, A.; Anantharam, V.; Sun, F.

    2010-01-01

    Pesticide exposure has been implicated in the etiopathogenesis of Parkinson's disease (PD); in particular, the organochlorine insecticide dieldrin is believed to be associated with PD. Emerging evidence indicates that histone modifications play a critical role in cell death. In this study, we examined the effects of dieldrin treatment on histone acetylation and its role in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. In mesencephalic dopaminergic neuronal cells, dieldrin induced a time-dependent increase in the acetylation of core histones H3 and H4. Histone acetylation occurred within 10 min of dieldrin exposure indicating that acetylation is an early event in dieldrin neurotoxicity. The hyperacetylation was attributed to dieldrin-induced proteasomal dysfunction, resulting in accumulation of a key histone acetyltransferase (HAT), cAMP response element-binding protein. The novel HAT inhibitor anacardic acid significantly attenuated dieldrin-induced histone acetylation, Protein kinase C δ proteolytic activation and DNA fragmentation in dopaminergic cells protected against dopaminergic neuronal degeneration in primary mesencephalic neuronal cultures. Furthermore, 30-day exposure of dieldrin in mouse models induced histone hyperacetylation in the striatum and substantia nigra. For the first time, our results collectively demonstrate that exposure to the neurotoxic pesticide dieldrin induces acetylation of core histones because of proteasomal dysfunction and that hyperacetylation plays a key role in dopaminergic neuronal degeneration after exposure of dieldrin. PMID:20097775

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2008-05-01

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

  18. WDR5 Intearcts with Mixed Lineage Leukemia (MLL) Protein via the Histone H3-binding Pocket

    SciTech Connect

    Song, J.; Kingston, R

    2008-01-01

    WDR5 is a component of the mixed lineage leukemia (MLL) complex, which methylates lysine 4 of histone H3, and was identified as a methylated Lys-4 histone H3-binding protein. Here, we present a crystal structure of WDR5 bound to an MLL peptide. Surprisingly, we find that WDR5 utilizes the same pocket shown to bind histone H3 for this MLL interaction. Furthermore, the WDR5-MLL interaction is disrupted preferentially by mono- and di-methylated Lys-4 histone H3 over unmodified and tri-methylated Lys-4 histone H3. These data implicate a delicate interplay between the effector, WDR5, the catalytic subunit, MLL, and the substrate, histone H3, of the MLL complex. We suggest that the activity of the MLL complex might be regulated through this interplay.

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

    PubMed Central

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

    2015-01-01

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

  20. Histone h3 glutathionylation in proliferating mammalian cells destabilizes nucleosomal structure.

    PubMed

    García-Giménez, José Luis; Òlaso, Gloria; Hake, Sandra B; Bönisch, Clemens; Wiedemann, Sonja M; Markovic, Jelena; Dasí, Francisco; Gimeno, Amparo; Pérez-Quilis, Carme; Palacios, Oscar; Capdevila, Mercè; Viña, José; Pallardó, Federico V

    2013-10-20

    Here we report that chromatin, the complex and dynamic eukaryotic DNA packaging structure, is able to sense cellular redox changes. Histone H3, the only nucleosomal protein that possesses cysteine(s), can be modified by glutathione (GSH). Using Biotin labeled glutathione ethyl ester (BioGEE) treatment of nucleosomes in vitro, we show that GSH, the most abundant antioxidant in mammals, binds to histone H3. BioGEE treatment of NIH3T3 cells indicates that glutathionylation of H3 is maximal in fast proliferating cells, correlating well with enhanced levels of H3 glutathionylation in different tumor cell lines. Furthermore, glutathionylation of H3 in vivo decreases in livers from aged SAMP8 and C57BL/6J mice. We demonstrate biochemically and by mass spectrometry that histone variants H3.2/H3.3 are glutathionylated on their cysteine residue 110. Furthermore, circular dichroism, thermal denaturation of reconstituted nucleosomes, and molecular modeling indicate that glutathionylation of histone H3 produces structural changes affecting nucleosomal stability. We characterize the implications of histone H3 glutathionylation in cell physiology and the modulation of core histone proteins structure affected by this modification. Histone H3 senses cellular redox changes through glutathionylation of Cys, which increases during cell proliferation and decreases during aging. Glutathionylation of histone H3 affects nucleosome stability structure leading to a more open chromatin structure.

  1. Histone H3 Glutathionylation in Proliferating Mammalian Cells Destabilizes Nucleosomal Structure

    PubMed Central

    Olaso, Gloria; Hake, Sandra B.; Bönisch, Clemens; Wiedemann, Sonja M.; Markovic, Jelena; Dasí, Francisco; Gimeno, Amparo; Pérez-Quilis, Carme; Palacios, Òscar; Capdevila, Mercè; Viña, José

    2013-01-01

    Abstract Aims: Here we report that chromatin, the complex and dynamic eukaryotic DNA packaging structure, is able to sense cellular redox changes. Histone H3, the only nucleosomal protein that possesses cysteine(s), can be modified by glutathione (GSH). Results: Using Biotin labeled glutathione ethyl ester (BioGEE) treatment of nucleosomes in vitro, we show that GSH, the most abundant antioxidant in mammals, binds to histone H3. BioGEE treatment of NIH3T3 cells indicates that glutathionylation of H3 is maximal in fast proliferating cells, correlating well with enhanced levels of H3 glutathionylation in different tumor cell lines. Furthermore, glutathionylation of H3 in vivo decreases in livers from aged SAMP8 and C57BL/6J mice. We demonstrate biochemically and by mass spectrometry that histone variants H3.2/H3.3 are glutathionylated on their cysteine residue 110. Furthermore, circular dichroism, thermal denaturation of reconstituted nucleosomes, and molecular modeling indicate that glutathionylation of histone H3 produces structural changes affecting nucleosomal stability. Innovation: We characterize the implications of histone H3 glutathionylation in cell physiology and the modulation of core histone proteins structure affected by this modification. Conclusion: Histone H3 senses cellular redox changes through glutathionylation of Cys, which increases during cell proliferation and decreases during aging. Glutathionylation of histone H3 affects nucleosome stability structure leading to a more open chromatin structure. Antioxid. Redox Signal. 19, 1305–1320. PMID:23541030

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

    PubMed

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

    2011-09-01

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

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

    PubMed

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

    2014-12-11

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

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2010-10-28

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

  6. Differential Expression of Histone H3 Gene Variants during Cell Cycle and Somatic Embryogenesis in Alfalfa

    PubMed Central

    Kapros, Tamás; Bögre, László; Németh, Kinga; Bakó, László; Györgyey, János; Wu, Sheng Cheng; Dudits, Dénes

    1992-01-01

    Northern analysis has revealed substantial differences in mRNA accumulation of the two histone H3 gene variants represented by pH3c-1 and pH3c-11 cDNA clones. Both in partially synchronized cell suspension cultures and in protoplast-derived cells from alfalfa, Medicago varia, the maximal level of the histone H3-1 gene transcript coincided with the peak in [3H]thymidine incorporation. Histone H3-11 mRNA was detectable in cells throughout the period of the cell cycle studied. Various stress factors such as medium replacement, enzyme digestion of the cell wall, osmotic shock, and auxin treatment considerably increased the level of the histone H3-11 transcript. In alfalfa (Medicago sativa), the presence of H3-11 mRNA in unorganized tissues of microcallus suspension and in somatic embryos induced by auxin treatment supports the idea that this H3 variant exists in a continously active state of transcription. During embryo development, the early globular stage embryos showed increased accumulation of histone H3-11 mRNA in comparison with the later stages. The highest level of the histone H3-1 transcript was detectable 1 day after treatment of callus tissues with 2,4-dichlorophenoxyacetic acid. Somatic embryos contained appreciable levels of histone H3-1 transcripts at all stages of somatic embryo development. These observations suggest that the histone H3-1 gene belòngs to the class of replication-dependent histone genes. The histone H3-11 gene showed characteristics of a constitutively expressed replacement-type histone gene, with a specific characteristic that external factors can influence the level of gene transcription. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668686

  7. Combinatorial readout of unmodified H3R2 and acetylated H3K14 by the tandem PHD finger of MOZ reveals a regulatory mechanism for HOXA9 transcription.

    PubMed

    Qiu, Yu; Liu, Lei; Zhao, Chen; Han, Chuanchun; Li, Fudong; Zhang, Jiahai; Wang, Yan; Li, Guohong; Mei, Yide; Wu, Mian; Wu, Jihui; Shi, Yunyu

    2012-06-15

    Histone acetylation is a hallmark for gene transcription. As a histone acetyltransferase, MOZ (monocytic leukemia zinc finger protein) is important for HOX gene expression as well as embryo and postnatal development. In vivo, MOZ forms a tetrameric complex with other subunits, including several chromatin-binding modules with regulatory functions. Here we report the solution structure of the tandem PHD (plant homeodomain) finger (PHD12) of human MOZ in a free state and the 1.47 Å crystal structure in complex with H3K14ac peptide, which reveals the structural basis for the recognition of unmodified R2 and acetylated K14 on histone H3. Moreover, the results of chromatin immunoprecipitation (ChIP) and RT-PCR assays indicate that PHD12 facilitates the localization of MOZ onto the promoter locus of the HOXA9 gene, thereby promoting the H3 acetylation around the promoter region and further up-regulating the HOXA9 mRNA level. Taken together, our findings suggest that the combinatorial readout of the H3R2/K14ac by PHD12 might represent an important epigenetic regulatory mechanism that governs transcription and also provide a clue of cross-talk between the MOZ complex and histone H3 modifications.

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

    PubMed

    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.

  9. A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair.

    PubMed

    Lee, Han-Sae; Park, Ji-Hye; Kim, So-Jung; Kwon, Su-Jung; Kwon, Jongbum

    2010-04-21

    Although recent studies highlight the importance of histone modifications and ATP-dependent chromatin remodelling in DNA double-strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser-139 (S139ph) after DNA damage, binds to gamma-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to gamma-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with gamma-H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to gamma-H2AX nucleosomes. S139ph stimulates the H3 acetylation on gamma-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on gamma-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, gamma-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

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

    PubMed

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

    2012-02-15

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

  11. Structural basis for the recognition and cleavage of histone H3 by cathepsin L

    PubMed Central

    Adams-Cioaba, Melanie A.; Krupa, Joanne C.; Xu, Chao; Mort, John S.; Min, Jinrong

    2011-01-01

    Proteolysis of eukaryotic histone tails has emerged as an important factor in the modulation of cell-cycle progression and cellular differentiation. The recruitment of lysosomal cathepsin L to the nucleus where it mediates proteolysis of the mouse histone H3 tail has been described recently. Here, we report the three-dimensional crystal structures of a mature, inactive mutant of human cathepsin L alone and in complex with a peptide derived from histone H3. Canonical substrate–cathepsin L interactions are observed in the complex between the protease and the histone H3 peptide. Systematic analysis of the impact of posttranslational modifications at histone H3 on substrate selectivity suggests cathepsin L to be highly accommodating of all modified peptides. This is the first report of cathepsin L–histone H3 interaction and the first structural description of cathepsin L in complex with a substrate. PMID:21326229

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

    PubMed

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

    2016-01-21

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

  13. The C Terminus of the Histone Chaperone Asf1 Cross-Links to Histone H3 in Yeast and Promotes Interaction with Histones H3 and H4

    PubMed Central

    Dennehey, Briana K.; Noone, Seth; Liu, Wallace H.; Smith, Luke

    2013-01-01

    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. PMID:23184661

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

    PubMed

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

    2013-02-01

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

  15. Quantitative Histone Mass Spectrometry Identifies Elevated Histone H3 Lysine 27 (Lys27) Trimethylation in Melanoma*

    PubMed Central

    Sengupta, Deepanwita; Byrum, Stephanie D.; Avaritt, Nathan L.; Davis, Lauren; Shields, Bradley; Mahmoud, Fade; Reynolds, Matthew; Orr, Lisa M.; Mackintosh, Samuel G.; Shalin, Sara C.; Tackett, Alan J.

    2016-01-01

    Normal cell growth is characterized by a regulated epigenetic program that drives cellular activities such as gene transcription, DNA replication, and DNA damage repair. Perturbation of this epigenetic program can lead to events such as mis-regulation of gene transcription and diseases such as cancer. To begin to understand the epigenetic program correlated to the development of melanoma, we performed a novel quantitative mass spectrometric analysis of histone post-translational modifications mis-regulated in melanoma cell culture as well as patient tumors. Aggressive melanoma cell lines as well as metastatic melanoma were found to have elevated histone H3 Lys27 trimethylation (H3K27me3) accompanied by overexpressed methyltransferase EZH2 that adds the specific modification. The altered epigenetic program that led to elevated H3K27me3 in melanoma cell culture was found to directly silence transcription of the tumor suppressor genes RUNX3 and E-cadherin. The EZH2-mediated silencing of RUNX3 and E-cadherin transcription was also validated in advanced stage human melanoma tissues. This is the first study focusing on the detailed epigenetic mechanisms leading to EZH2-mediated silencing of RUNX3 and E-cadherin tumor suppressors in melanoma. This study underscores the utility of using high resolution mass spectrometry to identify mis-regulated epigenetic programs in diseases such as cancer, which could ultimately lead to the identification of biological markers for diagnostic and prognostic applications. PMID:26621846

  16. Histone chaperone ASF1B promotes human β-cell proliferation via recruitment of histone H3.3.

    PubMed

    Paul, Pradyut K; Rabaglia, Mary E; Wang, Chen-Yu; Stapleton, Donald S; Leng, Ning; Kendziorski, Christina; Lewis, Peter W; Keller, Mark P; Attie, Alan D

    2016-12-01

    Anti-silencing function 1 (ASF1) is a histone H3-H4 chaperone involved in DNA replication and repair, and transcriptional regulation. Here, we identify ASF1B, the mammalian paralog to ASF1, as a proliferation-inducing histone chaperone in human β-cells. Overexpression of ASF1B led to distinct transcriptional signatures consistent with increased cellular proliferation and reduced cellular death. Using multiple methods of monitoring proliferation and mitotic progression, we show that overexpression of ASF1B is sufficient to induce human β-cell proliferation. Co-expression of histone H3.3 further augmented β-cell proliferation, whereas suppression of endogenous H3.3 attenuated the stimulatory effect of ASF1B. Using the histone binding-deficient mutant of ASF1B (V94R), we show that histone binding to ASF1B is required for the induction of β-cell proliferation. In contrast to H3.3, overexpression of histone H3 variants H3.1 and H3.2 did not have an impact on ASF1B-mediated induction of proliferation. Our findings reveal a novel role of ASF1B in human β-cell replication and show that ASF1B and histone H3.3A synergistically stimulate human β-cell proliferation.

  17. Histone H3 N-terminal mutations allow hyperactivation of the yeast GAL1 gene in vivo.

    PubMed Central

    Mann, R K; Grunstein, M

    1992-01-01

    Recent work has shown that the yeast histone H4 N-terminus, while not essential for viability, is required for repression of the silent mating loci and activation of GAL1 and PHO5 promoters. Because histone H3 shares many structural features with histone H4 and is intimately associated with H4 in the assembled nucleosome, we asked whether H3 has similar functions. While the basic N-terminal domain of H3 is found to be non-essential (deletion of residues 4-40 of this 135 amino acid protein allows viability), its removal has only a minor effect on mating. Surprisingly, both deletions (of residues 4-15) and acetylation site substitutions (at residues 9, 14 and 18) within the N-terminus of H3 allow hyperactivation of the GAL1 promoter as well as a number of other GAL4-regulated genes including GAL2, GAL7 and GAL10. To a limited extent glucose repression is also alleviated by H3 N-terminal deletions. Expression of another inducible promoter, PHO5, is shown to be relatively unaffected. We conclude that the H3 and H4 N-termini have different functions in both the repression of the silent mating loci and in the regulation of GAL1. Images PMID:1505519

  18. Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain

    PubMed Central

    Tie, Feng; Banerjee, Rakhee; Fu, Chen; Stratton, Carl A.; Fang, Ming; Harte, Peter J.

    2016-01-01

    Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. Here, we identify a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). We show that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. We identify a conserved PC motif adjacent to the chromodomain required for CBP binding and show that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. We find that PC is physically associated with the initiating form of RNA polymerase II (Pol II) and that many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes. PMID:26802126

  19. Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes

    DOE PAGES

    Miao, Jiamin; Frazier, Taylor; Huang, Linkai; ...

    2016-07-12

    Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1more » genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Lastly, our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.« less

  20. Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes

    PubMed Central

    Miao, Jiamin; Frazier, Taylor; Huang, Linkai; Zhang, Xinquan; Zhao, Bingyu

    2016-01-01

    Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass. PMID:27462323

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

    PubMed

    Krajewski, W A; Luchnik, A N

    1991-12-01

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

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

    ERIC Educational Resources Information Center

    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…

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

    ERIC Educational Resources Information Center

    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…

  4. Social exclusion changes histone modifications H3K4me3 and H3K27ac in liver tissue of wild house mice.

    PubMed

    Krause, Linda; Haubold, Bernhard; Börsch-Haubold, Angelika G

    2015-01-01

    Wild house mice form social hierarchies with aggressive males defending territories, in which females, young mice and submissive adult males share nests. In contrast, socially excluded males are barred from breeding groups, have numerous bite wounds and patches of thinning fur. Since their feeding times are often disrupted, we investigated whether social exclusion leads to changes in epigenetic marks of metabolic genes in liver tissue. We used chromatin immunoprecipitation and quantitative PCR to measure enrichment of two activating histone marks at 15 candidate loci. The epigenetic profiles of healthy males sampled from nest boxes differed significantly from the profiles of ostracized males caught outside of nests and showing bite wounds indicative of social exclusion. Enrichment of histone-3 lysine-4 trimethylation (H3K4me3) changed significantly at genes Cyp4a14, Gapdh, Nr3c1, Pck1, Ppara, and Sqle. Changes at histone-3 lysine-27 acetylation (H3K27ac) marks were detected at genes Fasn, Nr3c1, and Plin5. A principal components analysis separated the socialized from the ostracized mice. This was independent of body weight for the H3K4me3 mark, and partially dependent for H3K27ac. There was no separation, however, between healthy males that had been sampled from two different nests. A hierarchical cluster analysis also separated the two phenotypes, which was independent of body weight for both markers. Our study shows that a period of social exclusion during adult life leads to quantitative changes in histone modification patterns in mouse liver tissue. Similar epigenetic changes might occur during the development of stress-induced metabolic disorders in humans.

  5. Modulation of histone acetylation by garlic sulfur compounds.

    PubMed

    Druesne-Pecollo, Nathalie; Latino-Martel, Paule

    2011-03-01

    Preclinical studies have shown that fresh garlic extracts, aged garlic, garlic oil and specific organosulfur compounds generated by processing garlic could alter carcinogen metabolism, inhibit tumor cell growth through induction of cell cycle arrest or apoptosis, or angiogenesis. In particular, recent studies have suggested that anticarcinogenic effects of certain garlic compounds may implicate at least in part a modulation of histone acetylation, a process involved in the regulation of gene expression, resulting from the inhibition of histone deacetylase activity. The aim of this review is to describe available data on sulfur compounds from garlic and histone acetylation and to discuss their potential for cancer prevention. Available data indicate that garlic compounds could inhibit histone deacetylase activity and induce histone hyperacetylation in vitro as well as in vivo. Sparse studies provide evidence of an involvement of histone acetylation in modulation of gene expression by diallyl disulfide and allyl mercaptan. These effects were observed at high concentrations. Further investigations are needed to determine if the HDAC inhibitory effects of garlic organosulfur compounds might play a role in primary cancer prevention at doses achievable by human diet.

  6. Possible involvement of histone acetylation in the development of emotional resistance to stress stimuli in mice.

    PubMed

    Miyagawa, K; Tsuji, M; Takeda, H

    2012-12-01

    Recent reports have implied that aberrant biochemical processes in the brain frequently accompany subtle shifts in the cellular epigenetic profile that might underlie the pathogenic progression of psychiatric disorders. Furthermore, certain antidepressants or mood stabilizers have been reported to have the ability to modulate epigenetic parameters. We previously reported that pretreatment of mice with 5-HT(1A) receptor agonists 24 h before testing suppressed the decrease in emotional behaviors induced by exposure to acute restraint stress. Based on this finding, the aim of the present study was to examine the association between the development of emotional resistance to stress stimuli and the modulation of an epigenetic parameter, particularly histone acetylation. We found that acetylated histone H3 was increased in the hippocampus of mice that had developed resistance to emotional stress by pretreatment with flesinoxan (1 mg/kg, i.p.) 24 h before testing. On the other hand, pretreatment with benzodiazepine anxiolytic diazepam (1 mg/kg, i.p.) did not have similar effects. Interestingly, similar to flesinoxan, the histone deacetylase inhibitor trichostatin A also protected against the emotional changes induced by acute restraint stress, as well as histone H3 acetylation. The present findings suggest that the epigenetic mechanisms of gene regulation may play an important role in the development of emotional resistance to stress stimuli. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Quantitative analysis of histone H3 and H4 post-translational modifications in doxorubicin-resistant leukemia cells.

    PubMed

    Liu, Tao; Guo, Qingcheng; Guo, Huaizu; Hou, Sheng; Li, Jing; Wang, Hao

    2016-04-01

    The epigenetic remodeling of chromatin through histone modifications has been widely implicated in drug resistance of cancer cells. However, whether epigenetic mechanisms contribute specifically to doxorubicin resistance in leukemia has not been carefully examined. Using a stable and sensitive workflow based on LC-MS, we quantitatively compared the extents of methylation and acetylation of histone H3 and H4 in acute leukemia cell line HL60 and its doxorubicin-resistant derivative, HL60/ADR, as well as the chronic leukemia cell line K562 and its doxorubicin-resistant derivative, K562/ADR. We found that increased levels of H3K9 methylation, H3K14, H3K18 and H3K23 acetylation, and potentially H4K20 methylation, are associated with drug resistance in both cells. Our results demonstrated that the doxorubicin-resistant acute and chronic leukemia cell lines may share a common epigenetic mechanism that involves a combination of transcriptional activation and silencing.

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

    PubMed

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

    2012-01-01

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

  9. Histone H3.3 and its proteolytically processed form drive a cellular senescence program

    PubMed Central

    Duarte, Luis F.; Young, Andrew R. J.; Wang, Zichen; Wu, Hsan-Au; Panda, Taniya; Kou, Yan; Kapoor, Avnish; Hasson, Dan; Mills, Nicholas R.; Ma’ayan, Avi; Narita, Masashi; Bernstein, Emily

    2014-01-01

    The process of cellular senescence generates a repressive chromatin environment, however, the role of histone variants and histone proteolytic cleavage in senescence remains unclear. Using models of oncogene-induced and replicative senescence, here we report novel histone H3 tail cleavage events mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and the histone variant H3.3 is the preferred cleaved form of H3. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Further, H3.3cs1 chromatin incorporation is mediated by the HUCA histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our study identifies histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. PMID:25394905

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

    PubMed

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

    2010-08-06

    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. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  11. Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex

    PubMed Central

    Daniel Ricketts, M; Frederick, Brian; Hoff, Henry; Tang, Yong; Schultz, David C.; Singh Rai, Taranjit; Grazia Vizioli, Maria; Adams, Peter D.; Marmorstein, Ronen

    2015-01-01

    Histone chaperones bind specific histones to mediate their storage, eviction or deposition from/or into chromatin. The HIRA histone chaperone complex, composed of HIRA, ubinuclein-1 (UBN1) and CABIN1, cooperates with the histone chaperone ASF1a to mediate H3.3-specific binding and chromatin deposition. Here we demonstrate that the conserved UBN1 Hpc2-related domain (HRD) is a novel H3.3-specific-binding domain. Biochemical and biophysical studies show the UBN1-HRD preferentially binds H3.3/H4 over H3.1/H4. X-ray crystallographic and mutational studies reveal that conserved residues within the UBN1-HRD and H3.3 G90 as key determinants of UBN1–H3.3-binding specificity. Comparison of the structure with the unrelated H3.3-specific chaperone DAXX reveals nearly identical points of contact between the chaperone and histone in the proximity of H3.3 G90, although the mechanism for H3.3 G90 recognition appears to be distinct. This study points to UBN1 as the determinant of H3.3-specific binding and deposition by the HIRA complex. PMID:26159857

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

    PubMed Central

    Kootstra, A

    1982-01-01

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

  13. The Commercial Antibodies Widely Used to Measure H3 K56 Acetylation Are Non-Specific in Human and Drosophila Cells

    PubMed Central

    Pal, Sangita; Graves, Hillary; Ohsawa, Ryosuke; Huang, Ting-hsiang; Wang, Pingping; Harmacek, Laura; Tyler, Jessica

    2016-01-01

    Much of our understanding of the function of histone post-translational modifications in metazoans is inferred from their genomic localization and / or extrapolated from yeast studies. For example, acetylation of histone H3 lysine 56 (H3 K56Ac) is assumed to be important for transcriptional regulation in metazoan cells based on its occurrence at promoters and its function in yeast. Here we directly assess the function of H3 K56Ac during chromatin disassembly from gene regulatory regions during transcriptional induction in human cells by using mutations that either mimic or prevent H3 K56Ac. Although there is rapid histone H3 disassembly during induction of some estrogen receptor responsive genes, depletion of the histone chaperone ASF1A/B, which is required for H3 K56 acetylation, has no effect on chromatin disassembly at these regions. During the course of this work, we found that all the commercially available antibodies to H3 K56Ac are non-specific in human cells and in Drosophila. We used H3-YFP fusions to show that the H3 K56Q mutation can promote chromatin disassembly from regulatory regions of some estrogen responsive genes in the context of transcriptional induction. However, neither the H3 K56R nor K56Q mutation significantly altered chromatin disassembly dynamics by FRAP analysis. These results indicate that unlike the situation in yeast, human cells do not use H3 K56Ac to promote chromatin disassembly from regulatory regions or from the genome in general. Furthermore, our work highlights the need for rigorous characterization of the specificity of antibodies to histone post-translational modifications in vivo. PMID:27187594

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    2016-02-12

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2017-02-01

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

  19. The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase.

    PubMed

    Lee, Nara; Zhang, Junyu; Klose, Robert J; Erdjument-Bromage, Hediye; Tempst, Paul; Jones, Richard S; Zhang, Yi

    2007-04-01

    Recent studies have demonstrated that histone methylation can be dynamically regulated through active demethylation. However, no demethylase specific to histone H3 trimethyl-Lys4 (H3K4me3) has been identified. Here we report that the Drosophila melanogaster protein 'little imaginal discs' (Lid), a JmjC domain-containing trithorax group protein, can demethylate H3K4me3. Consistent with its genetic classification, Lid positively regulates Hox gene expression in S2 cells.

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

    PubMed

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

    2017-03-09

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2008-08-01

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

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

    PubMed

    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. Human histone acetyltransferase 1 (Hat1) acetylates lysine 5 of histone H2A in vivo.

    PubMed

    Tafrova, Juliana I; Tafrov, Stefan T

    2014-07-01

    The primary structure of Histone Acetyltransferase 1 (Hat1) has been conserved throughout evolution; however, despite its ubiquity, its cellular function is not well characterized. To study its in vivo acetylation pattern and function, we utilized shRNAmir against Hat1 expressed in the well-substantiated HeLa (human cervical cancer) cell line. To reduce the interference by enzymes with similar HAT specificity, we used HeLa cells expressing histone acetyltransferase Tip60 with mutated acetyl-CoA binding site that abrogates its enzyme activity (mutant HeLa-tip60). Two shRNAmir were identified that reduced the expression of the cytoplasmic and nuclear forms of Hat1. Cytosolic protein preparations from these two clones showed decreased levels of acetylation of lysine 5 (K5) and K12 on histone H4, with the concomitant loss of the acetylation of histone H2A at K5. This pattern of decreased acetylation of H2AK5 was well defined in preparations of histone protein and insoluble nuclear-protein (INP) fractions as well. Abrogating the Hat1 expression caused a 74% decrease in colony-forming efficiency of mutant HeLa-tip60 cells, reduced the size of the colonies by 50%, and decreased the amounts of proteins with molecular weights below 35 kDa in the INP fractions.

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

    PubMed Central

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

    2016-01-01

    Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of large heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation. DOI: http://dx.doi.org/10.7554/eLife.17903.001 PMID:27648579

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

    SciTech Connect

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

    2016-09-20

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

  8. Histone deacetylase inhibitor SAHA attenuates post-seizure hippocampal microglia TLR4/MYD88 signaling and inhibits TLR4 gene expression via histone acetylation.

    PubMed

    Hu, Qing-Peng; Mao, Ding-An

    2016-05-18

    Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures. Seizure-induced TLR4/MYD88 signaling plays a critical role in activating microglia and triggering neuron apoptosis. SAHA is a histone deacetylase inhibitor that regulates gene expression by increasing chromatin histone acetylation. In this study, we investigated the role of SAHA in TLR4/MYD88 signaling in a rat seizure model. Sprague-Dawley rats with kainic acid (KA)-induced seizures were treated with SAHA. The expression of TLR4, MYD88, NF-κB P65 and IL-1β in hippocampus was detected at hour 2 and 6 and day 1, 2, and 3 post seizure. SAHA pretreatment increased seizure latency and decreased seizure scores. The expression levels of TLR4, MYD88, NF-κB and IL-1β increased significantly in both activated microglia and apoptotic neurons after KA treatment. The effects were attenuated by SAHA. Chromatin immunoprecipitation assays indicated that the H3 histone acetylation levels significantly decreased while H3K9 levels significantly increased in the KA treatment group. The H3 and H3K9 acetylation levels returned to control levels after SAHA (50 mg/kg) pretreatment. There was a positive correlation between the expression of TLR4 and the acetylation levels of H3K9. Histone deacetylase inhibitor SAHA can suppress seizure-induced TLR4/MYD88 signaling and inhibit TLR4 gene expression through histone acetylation regulation. This suggests that SAHA may protect against seizure-induced brain damage.

  9. HDA18 Affects Cell Fate in Arabidopsis Root Epidermis via Histone Acetylation at Four Kinase Genes[W

    PubMed Central

    Liu, Cui; Li, Lin-Chen; Chen, Wen-Qian; Chen, Xian; Xu, Zhi-Hong; Bai, Shu-Nong

    2013-01-01

    The differentiation of hair (H) and non-hair (N) cells in the Arabidopsis thaliana root epidermis is dependent on positional relationships with underlying cortical cells. We previously found that histone acetylation relays positional information and that a mutant altered in the histone deacetylase gene family member HISTONE DEACETYLASE 18 (HDA18) exhibits altered H and N epidermal cell patterning. Here, we report that HDA18 has in vitro histone deacetylase activity and that both mutation and overexpression of HDA18 led to cells at the N position having H fate. The HDA18 protein physically interacted with histones related to a specific group of kinase genes, which are demonstrated in this study to be components of a positional information relay system. Both down- and upregulation of HDA18 increased transcription of the targeted kinase genes. Interestingly, the acetylation levels of histone 3 lysine 9 (H3K9), histone 3 lysine 14 (H3K14) and histone 3 lysine 18 (H3K18) at the kinase genes were differentially affected by down- or upregulation of HDA18, which explains why the transcription levels of the four HDA18-target kinase genes increased in all lines with altered HDA18 expression. Our results reveal the surprisingly complex mechanism by which HDA18 affects cellular patterning in Arabidopsis root epidermis. PMID:23362208

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

    PubMed Central

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

    2012-01-01

    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. PMID:23034810

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

    PubMed

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

    2012-12-01

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

  12. Molecular basis for the role of oncogenic histone mutations in modulating H3K36 methylation

    DOE PAGES

    Zhang, Yinglu; Shan, Chun -Min; Wang, Jiyong; ...

    2017-03-03

    Histone H3 lysine 36 methylation (H3K36me) is critical for epigenetic regulation and mutations at or near H3K36 are associated with distinct types of cancers. H3K36M dominantly inhibits H3K36me on wild-type histones, whereas H3G34R/V selectively affects H3K36me on the same histone tail. Here we report the crystal structures of SETD2 SET domain in complex with an H3K36M peptide and SAM or SAH. There are large conformational changes in the substrate binding regions of the SET domain, and the K36M residue interacts with the catalytic pocket of SETD2. H3G34 is surrounded by a very narrow tunnel, which excludes larger amino acid sidemore » chains. H3P38 is in the trans configuration, and the cis configuration is incompatible with SETD2 binding. Lastly, mutations of H3G34 or H3P38 alleviate the inhibitory effects of H3K36M on H3K36me, demonstrating that the stable interaction of H3K36M with SETD2 is critical for its inhibitory effects.« less

  13. Molecular basis for the role of oncogenic histone mutations in modulating H3K36 methylation

    PubMed Central

    Zhang, Yinglu; Shan, Chun-Min; Wang, Jiyong; Bao, Kehan; Tong, Liang; Jia, Songtao

    2017-01-01

    Histone H3 lysine 36 methylation (H3K36me) is critical for epigenetic regulation and mutations at or near H3K36 are associated with distinct types of cancers. H3K36M dominantly inhibits H3K36me on wild-type histones, whereas H3G34R/V selectively affects H3K36me on the same histone tail. Here we report the crystal structures of SETD2 SET domain in complex with an H3K36M peptide and SAM or SAH. There are large conformational changes in the substrate binding regions of the SET domain, and the K36M residue interacts with the catalytic pocket of SETD2. H3G34 is surrounded by a very narrow tunnel, which excludes larger amino acid side chains. H3P38 is in the trans configuration, and the cis configuration is incompatible with SETD2 binding. Finally, mutations of H3G34 or H3P38 alleviate the inhibitory effects of H3K36M on H3K36me, demonstrating that the stable interaction of H3K36M with SETD2 is critical for its inhibitory effects. PMID:28256625

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

    PubMed

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

    2015-03-01

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

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

    PubMed

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

    2009-11-01

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

  16. Cell cycle-regulated histone acetylation required for expression of the yeast HO gene

    PubMed Central

    Krebs, Jocelyn E.; Kuo, Min-Hao; Allis, C. David; Peterson, Craig L.

    1999-01-01

    Expression of the yeast HO gene in late G1 of the cell cycle requires the SWI/SNF chromatin remodeling complex, the Gcn5p histone acetyltransferase, and two different sequence-specific transcriptional activators, Swi5p and Swi4p/Swi6p. We have used chromatin immunoprecipitation assays to investigate the role of each of these trans-acting factors in establishing a cell cycle-regulated domain of histone acetylation surrounding the HO upstream regulatory region. We detect a ∼1-kb domain of H3 and H4 acetylation that is established in mid-G1, prior to and independent of HO transcription, which then declines with kinetics similar to inactivation of HO. This cell cycle burst of histone acetylation requires Gcn5p, SWI/SNF, and the Swi5p activator, but occurs in the absence of the Swi4p activator. We also find that inactivation of the Sin3p/Rpd3p deacetylase complex leads to a high level of acetylation at the HO locus throughout the cell cycle. We propose a sequential model for activation of HO in which the Swi5p-dependent recruitment of the Gcn5p acetyltransferase requires chromatin remodeling events by the SWI/SNF complex. PMID:10364158

  17. Stimulation of V(D)J recombination by histone acetylation.

    PubMed

    McBlane, F; Boyes, J

    2000-04-20

    V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.

  18. Site-Specific Acetyl Lysine Antibodies Reveal Differential Regulation of Histone Acetylation upon Kinase Inhibition.

    PubMed

    Chen, Shi; Chen, Suping; Duan, Qianqian; Xu, Guoqiang

    2017-03-01

    Lysine acetylation regulates diverse biological functions for the modified proteins. Mass spectrometry-based proteomic approaches have identified thousands of lysine acetylation sites in cells and tissues. However, functional studies of these acetylation sites were limited by the lack of antibodies recognizing the specific modification sites. Here, we generated 55 site-specific acetyl lysine antibodies for the detection of this modification in cell lysates and evaluated the quality of these antibodies. Based on the immunoblotting analyses, we found that the nature of amino acid sequences adjacent to the modification sites affected the specificity of the site-specific acetyl lysine antibodies. Amino acids with charged, hydrophilic, small, or flexible side chains adjacent to the modification sites increase the likelihood of obtaining high quality site-specific acetyl lysine antibodies. This result may provide valuable insights in fine-tuning the amino acid sequences of the epitopes for the generation of site-specific acetyl lysine antibodies. Using the site-specific acetyl lysine antibodies, we further discovered that acetylation of histone 3 at four lysine residues was differentially regulated by kinase inhibitors. This result demonstrates the potential application of these antibodies in the study of new signaling pathways that lysine acetylation may participate in.

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

    PubMed

    Jamai, Adil; Puglisi, Andrea; Strubin, Michel

    2009-08-14

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

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

    PubMed

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

    2013-01-01

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

  1. Striatal H3K27 Acetylation Linked to Glutamatergic Gene Dysregulation in Human Heroin Abusers Holds Promise as Therapeutic Target.

    PubMed

    Egervari, Gabor; Landry, Joseph; Callens, James; Fullard, John F; Roussos, Panos; Keller, Eva; Hurd, Yasmin L

    2017-04-01

    Opiate abuse and overdose reached epidemic levels in the United States. However, despite significant advances in animal and in vitro models, little knowledge has been directly accrued regarding the neurobiology of the opiate-addicted human brain. We used postmortem human brain specimens from a homogeneous European Caucasian population of heroin users for transcriptional and epigenetic profiling, as well as direct assessment of chromatin accessibility in the striatum, a brain region central to reward and emotion. A rat heroin self-administration model was used to obtain translational molecular and behavioral insights. Our transcriptome approach revealed marked impairments related to glutamatergic neurotransmission and chromatin remodeling in the human striatum. A series of biochemical experiments tracked the specific location of the epigenetic disturbances to hyperacetylation of lysine 27 of histone H3, showing dynamic correlations with heroin use history and acute opiate toxicology. Targeted investigation of GRIA1, a glutamatergic gene implicated in drug-seeking behavior, verified the increased enrichment of lysine-27 acetylated histone H3 at discrete loci, accompanied by enhanced chromatin accessibility at hyperacetylated regions in the gene body. Analogous epigenetic impairments were detected in the striatum of heroin self-administering rats. Using this translational model, we showed that bromodomain inhibitor JQ1, which blocks the functional readout of acetylated lysines, reduced heroin self-administration and cue-induced drug-seeking behavior. Overall, our data suggest that heroin-related histone H3 hyperacetylation contributes to glutamatergic transcriptional changes that underlie addiction behavior and identify JQ1 as a promising candidate for targeted clinical interventions in heroin use disorder. Copyright © 2016 Society of Biological Psychiatry. All rights reserved.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2007-01-01

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

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

    PubMed

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

    2012-07-27

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

  5. Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.

    PubMed

    Yamaguchi, Luna; Nishiyama, Atsuya; Misaki, Toshinori; Johmura, Yoshikazu; Ueda, Jun; Arita, Kyohei; Nagao, Koji; Obuse, Chikashi; Nakanishi, Makoto

    2017-03-03

    Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2014-04-01

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

  10. Tissue-Specific Expression and Posttranslational Modification of Histone H3 Variants

    PubMed Central

    Garcia, Benjamin A.; Thomas, C. Eric; Kelleher, Neil L.; Mizzen, Craig A.

    2008-01-01

    Analyses of histone H3 from ten rat tissues using a Middle Down proteomics platform revealed tissue-specific differences in their expression and global PTM abundance. ESI/FTMS with electron capture dissociation showed that, in general, these proteins were hypomodified in heart, liver and testes. H3.3 was hypermodified compared to H3.2 in some, but not all tissues. In addition, a novel rat testes-specific H3 protein was identified with this approach. PMID:18700791

  11. Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression*

    PubMed Central

    Zheng, Yupeng; Fornelli, Luca; Compton, Philip D.; Sharma, Seema; Canterbury, Jesse; Mullen, Christopher; Zabrouskov, Vlad; Fellers, Ryan T.; Thomas, Paul M.; Licht, Jonathan D.; Senko, Michael W.; Kelleher, Neil L.

    2016-01-01

    Histones, and their modifications, are critical components of cellular programming and epigenetic inheritance. Recently, cancer genome sequencing has uncovered driver mutations in chromatin modifying enzymes spurring high interest how such mutations change histone modification patterns. Here, we applied Top-Down mass spectrometry for the characterization of combinatorial modifications (i.e. methylation and acetylation) on full length histone H3 from human cell lines derived from multiple myeloma patients with overexpression of the histone methyltransferase MMSET as the result of a t(4;14) chromosomal translocation. Using the latest in Orbitrap-based technology for clean isolation of isobaric proteoforms containing up to 10 methylations and/or up to two acetylations, we provide extensive characterization of histone H3.1 and H3.3 proteoforms. Differential analysis of modifications by electron-based dissociation recapitulated antagonistic crosstalk between K27 and K36 methylation in H3.1, validating that full-length histone H3 (15 kDa) can be analyzed with site-specific assignments for multiple modifications. It also revealed K36 methylation in H3.3 was affected less by the overexpression of MMSET because of its higher methylation levels in control cells. The co-occurrence of acetylation with a minimum of three methyl groups in H3K9 and H3K27 suggested a hierarchy in the addition of certain modifications. Comparative analysis showed that high levels of MMSET in the myeloma-like cells drove the formation of hypermethyled proteoforms containing H3K36me2 co-existent with the repressive marks H3K9me2/3 and H3K27me2/3. Unique histone proteoforms with such “trivalent hypermethylation” (K9me2/3-K27me2/3-K36me2) were not discovered when H3.1 peptides were analyzed by Bottom-Up. Such disease-correlated proteoforms could link tightly to aberrant transcription programs driving cellular proliferation, and their precise description demonstrates that Top-Down mass spectrometry

  12. Hypoxia-mediated histone acetylation and expression of N-myc transcription factor dictate aggressiveness of neuroblastoma cells.

    PubMed

    Poljaková, Jitka; Groh, Tomáš; Gudino, Zaneta Omana; Hraběta, Jan; Bořek-Dohalská, Lucie; Kizek, René; Doktorová, Helena; Eckschlager, Tomáš; Stiborová, Marie

    2014-04-01

    Cells of solid malignancies generally adapt to entire lack of oxygen. Hypoxia induces the expression of several genes, which allows the cells to survive. For DNA transcription, it is necessary that DNA structure is loosened. In addition to structural characteristics of DNA, its epigenetic alterations influence a proper DNA transcription. Since histones play a key role in epigenetics, changes in expression levels of acetylated histones H3 and H4 as well as of hypoxia-inducible factor-1α (HIF-1α) in human neuroblastoma cell lines cultivated under standard or hypoxic conditions (1% O2) were investigated. Moreover, the effect of hypoxia on the expression of two transcription factors, c-Myc and N-myc, was studied. Hypoxic stress increased levels of acetylated histones H3 and H4 in UKF-NB-3 and UKF-NB-4 neuroblastoma cells with N-myc amplification, whereas almost no changes in acetylation of these histones were found in an SK-N-AS neuroblastoma cell line, the line with diploid N-myc status. An increase in histone H4 acetylation caused by hypoxia in UKF-NB-3 and UKF-NB-4 corresponds to increased levels of N-myc transcription factor in these cells.

  13. Histone H4 acetylation and the epigenetic reader Brd4 are critical regulators of pluripotency in embryonic stem cells.

    PubMed

    Gonzales-Cope, Michelle; Sidoli, Simone; Bhanu, Natarajan V; Won, Kyoung-Jae; Garcia, Benjamin A

    2016-02-04

    Pluripotent cells can be differentiated into many different cell types in vitro. Successful differentiation is guided in large part by epigenetic reprogramming and regulation of critical gene expression patterns. Recent genome-wide studies have identified the distribution of different histone-post-translational modifications (PTMs) in various conditions and during cellular differentiation. However, our understanding of the abundance of histone PTMs and their regulatory mechanisms still remain unknown. Here, we present a quantitative and comprehensive study of the abundance levels of histone PTMs during the differentiation of mouse embryonic stem cells (ESCs) using mass spectrometry (MS). We observed dynamic changes of histone PTMs including increased H3K9 methylation levels in agreement with previously reported results. More importantly, we found a global decrease of multiply acetylated histone H4 peptides. Brd4 targets acetylated H4 with a strong affinity to multiply modified H4 acetylation sites. We observed that the protein levels of Brd4 decreased upon differentiation together with global histone H4 acetylation. Inhibition of Brd4:histone H4 interaction by the BET domain inhibitor (+)-JQ1 in ESCs results in enhanced differentiation to the endodermal lineage, by disrupting the protein abundance dynamics. Genome-wide ChIP-seq mapping showed that Brd4 and H4 acetylation are co-occupied in the genome, upstream of core pluripotency genes such as Oct4 and Nanog in ESCs and lineage-specific genes in embryoid bodies (EBs). Together, our data demonstrate the fundamental role of Brd4 in monitoring cell differentiation through its interaction with acetylated histone marks and disruption of Brd4 may cause aberrant differentiation.

  14. DNA damage response induces structural alterations in histone H3–H4

    PubMed Central

    Izumi, Yudai; Fujii, Kentaro; Yamamoto, Satoshi; Matsuo, Koichi; Namatame, Hirofumi; Taniguchi, Masaki; Yokoya, Akinari

    2017-01-01

    Synchrotron-radiation circular-dichroism spectroscopy was used to reveal that the DNA damage response induces a decrement of α-helix and an increment of β-strand contents of histone H3–H4 extracted from X-ray–irradiated human HeLa cells. The trend of the structural alteration was qualitatively opposite to that of our previously reported results for histone H2A–H2B. These results strongly suggest that histones share roles in DNA damage responses, particularly in DNA repair processes and chromatin remodeling, via a specific structural alteration of each histone. PMID:27672100

  15. Genome-wide analysis of histone modifications: H3K4me2, H3K4me3, H3K9ac, and H3K27ac in Oryza sativa L. Japonica.

    PubMed

    Du, Zhou; Li, Hui; Wei, Qiang; Zhao, Xin; Wang, Chunchao; Zhu, Qilin; Yi, Xin; Xu, Wenying; Liu, X Shirley; Jin, Weiwei; Su, Zhen

    2013-09-01

    While previous studies have shown that histone modifications could influence plant growth and development by regulating gene transcription, knowledge about the relationships between these modifications and gene expression is still limited. This study used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), to investigate the genome-wide distribution of four histone modifications: di and trimethylation of H3K4 (H3K4me2 and H3K4me3) and acylation of H3K9 and H3K27 (H3K9ac and H3K27ac) in Oryza sativa L. japonica. By analyzing published DNase-Seq data, this study explored DNase-Hypersensitive (DH) sites along the rice genome. The histone marks appeared mainly in generic regions and were enriched around the transcription start sites (TSSs) of genes. This analysis demonstrated that the four histone modifications and the DH sites were all associated with active transcription. Furthermore, the four histone modifications were highly concurrent with transcript regions-a promising feature that was used to predict missing genes in the rice gene annotation. The predictions were further validated by experimentally confirming the transcription of two predicted missing genes. Moreover, a sequence motif analysis was constructed in order to identify the DH sites and many putative transcription factor binding sites.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Liu, Xiangyong; Zhang, Xiaohua; Zhang, Zhaojie

    2014-10-10

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

  18. Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila.

    PubMed

    Deng, Huai; Bao, Xiaomin; Cai, Weili; Blacketer, Melissa J; Belmont, Andrew S; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M

    2008-02-01

    Histones are subject to numerous post-translational modifications that correlate with the state of higher-order chromatin structure and gene expression. However, it is not clear whether changes in these epigenetic marks are causative regulatory factors in chromatin structure changes or whether they play a mainly reinforcing or maintenance role. In Drosophila phosphorylation of histone H3S10 in euchromatic chromatin regions by the JIL-1 tandem kinase has been implicated in counteracting heterochromatization and gene silencing. Here we show, using a LacI-tethering system, that JIL-1 mediated ectopic histone H3S10 phosphorylation is sufficient to induce a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. This effect was absent when a ;kinase dead' LacI-JIL-1 construct without histone H3S10 phosphorylation activity was expressed. Instead, the 'kinase dead' construct had a dominant-negative effect, leading to a disruption of chromatin structure that was associated with a global repression of histone H3S10 phosphorylation levels. These findings provide direct evidence that the epigenetic histone tail modification of H3S10 phosphorylation at interphase can function as a causative regulator of higher-order chromatin structure in Drosophila in vivo.

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

    PubMed

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

    2016-06-21

    The histone demethylase LSD1 facilitates epithelial-to-mesenchymal transition (EMT) and tumor progression by repressing epithelial marker expression. However, little is known about how its function may be modulated. Here, we report that LSD1 is acetylated in epithelial but not mesenchymal cells. Acetylation of LSD1 reduces its association with nucleosomes, thus increasing histone H3K4 methylation at its target genes and activating transcription. The MOF acetyltransferase interacts with LSD1 and is responsible for its acetylation. MOF is preferentially expressed in epithelial cells and is downregulated by EMT-inducing signals. Expression of exogenous MOF impedes LSD1 binding to epithelial gene promoters and histone demethylation, thereby suppressing EMT and tumor invasion. Conversely, MOF depletion enhances EMT and tumor metastasis. In human cancer, high MOF expression correlates with epithelial markers and a favorable prognosis. These findings provide insight into the regulation of LSD1 and EMT and identify MOF as a critical suppressor of EMT and tumor progression. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development.

    PubMed

    Lin, Chih-Jen; Conti, Marco; Ramalho-Santos, Miguel

    2013-09-01

    Histone variants can replace canonical histones in the nucleosome and modify chromatin structure and gene expression. The histone variant H3.3 preferentially associates with active chromatin and has been implicated in the regulation of a diverse range of developmental processes. However, the mechanisms by which H3.3 may regulate gene activity are unclear and gene duplication has hampered an analysis of H3.3 function in mouse. Here, we report that the specific knockdown of H3.3 in fertilized mouse zygotes leads to developmental arrest at the morula stage. This phenotype can be rescued by exogenous H3.3 but not by canonical H3.1 mRNA. Loss of H3.3 leads to over-condensation and mis-segregation of chromosomes as early as the two-cell stage, with corresponding high levels of aneuploidy, but does not appear to affect zygotic gene activation at the two-cell stage or lineage gene transcription at the morula stage. H3.3-deficient embryos have significantly reduced levels of markers of open chromatin, such as H3K36me2 and H4K16Ac. Importantly, a mutation in H3.3K36 that disrupts H3K36 methylation (H3.3K36R) does not rescue the H3.3 knockdown (KD) phenotype. In addition, H3.3 KD embryos have increased incorporation of linker H1. Knockdown of Mof (Kat8), an acetyltransferase specific for H4K16, similarly leads to excessive H1 incorporation. Remarkably, pan-H1 RNA interference (RNAi) partially rescues the chromosome condensation of H3.3 KD embryos and allows development to the blastocyst stage. These results reveal that H3.3 mediates a balance between open and condensed chromatin that is crucial for the fidelity of chromosome segregation during early mouse development.

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

    PubMed

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

    2011-04-01

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

  2. Genomewide analysis of nucleosome density histone acetylation and HDAC function in fission yeast.

    PubMed

    Wirén, Marianna; Silverstein, Rebecca A; Sinha, Indranil; Walfridsson, Julian; Lee, Hang-Mao; Laurenson, Patricia; Pillus, Lorraine; Robyr, Daniel; Grunstein, Michael; Ekwall, Karl

    2005-08-17

    We have conducted a genomewide investigation into the enzymatic specificity, expression profiles, and binding locations of four histone deacetylases (HDACs), representing the three different phylogenetic classes in fission yeast (Schizosaccharomyces pombe). By directly comparing nucleosome density, histone acetylation patterns and HDAC binding in both intergenic and coding regions with gene expression profiles, we found that Sir2 (class III) and Hos2 (class I) have a role in preventing histone loss; Clr6 (class I) is the principal enzyme in promoter-localized repression. Hos2 has an unexpected role in promoting high expression of growth-related genes by deacetylating H4K16Ac in their open reading frames. Clr3 (class II) acts cooperatively with Sir2 throughout the genome, including the silent regions: rDNA, centromeres, mat2/3 and telomeres. The most significant acetylation sites are H3K14Ac for Clr3 and H3K9Ac for Sir2 at their genomic targets. Clr3 also affects subtelomeric regions which contain clustered stress- and meiosis-induced genes. Thus, this combined genomic approach has uncovered different roles for fission yeast HDACs at the silent regions in repression and activation of gene expression.

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

    PubMed

    Peng, Guang-Hua; Chen, Shiming

    2007-10-15

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

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

    PubMed Central

    Peng, Guang-Hua; Chen, Shiming

    2008-01-01

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

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

    SciTech Connect

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

    2010-07-19

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

  6. The histone variant H3.3 claims its place in the crowded scene of epigenetics.

    PubMed

    Bano, Daniele; Piazzesi, Antonia; Salomoni, Paolo; Nicotera, Pierluigi

    2017-03-10

    Histones are evolutionarily conserved DNA-binding proteins. As scaffolding molecules, they significantly regulate the DNA packaging into the nucleus of all eukaryotic cells. As docking units, they influence the recruitment of the transcriptional machinery, thus establishing unique gene expression patterns that ultimately promote different biological outcomes. While canonical histones H3.1 and H3.2 are synthetized and loaded during DNA replication, the histone variant H3.3 is expressed and deposited into the chromatin throughout the cell cycle. Recent findings indicate that H3.3 replaces the majority of canonical H3 in non-dividing cells, reaching almost saturation levels in a time-dependent manner. Consequently, H3.3 incorporation and turnover represent an additional layer in the regulation of the chromatin landscape during aging. In this respect, work from our group and others suggest that H3.3 plays an important function in age-related processes throughout evolution. Here, we summarize the current knowledge on H3.3 biology and discuss the implications of its aberrant dynamics in the establishment of cellular states that may lead to human pathology. Critically, we review the importance of H3.3 turnover as part of epigenetic events that influence senescence and age-related processes. We conclude with the emerging evidence that H3.3 is required for proper neuronal function and brain plasticity.

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

    PubMed

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

    2016-08-17

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

  8. Impaired replication elongation in Tetrahymena mutants deficient in histone H3 Lys 27 monomethylation

    PubMed Central

    Gao, Shan; Xiong, Jie; Zhang, Chunchao; Berquist, Brian R.; Yang, Rendong; Zhao, Meng; Molascon, Anthony J.; Kwiatkowski, Shaina Y.; Yuan, Dongxia; Qin, Zhaohui; Wen, Jianfan; Kapler, Geoffrey M.; Andrews, Philip C.; Miao, Wei; Liu, Yifan

    2013-01-01

    Replication of nuclear DNA occurs in the context of chromatin and is influenced by histone modifications. In the ciliate Tetrahymena thermophila, we identified TXR1, encoding a histone methyltransferase. TXR1 deletion resulted in severe DNA replication stress, manifested by the accumulation of ssDNA, production of aberrant replication intermediates, and activation of robust DNA damage responses. Paired-end Illumina sequencing of ssDNA revealed intergenic regions, including replication origins, as hot spots for replication stress in ΔTXR1 cells. ΔTXR1 cells showed a deficiency in histone H3 Lys 27 monomethylation (H3K27me1), while ΔEZL2 cells, deleting a Drosophila E(z) homolog, were deficient in H3K27 di- and trimethylation, with no detectable replication stress. A point mutation in histone H3 at Lys 27 (H3 K27Q) mirrored the phenotype of ΔTXR1, corroborating H3K27me1 as a key player in DNA replication. Additionally, we demonstrated interactions between TXR1 and proliferating cell nuclear antigen (PCNA). These findings support a conserved pathway through which H3K27me1 facilitates replication elongation. PMID:23884606

  9. 17β-Estradiol regulates histone alterations associated with memory consolidation and increases Bdnf promoter acetylation in middle-aged female mice.

    PubMed

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

    2014-09-01

    Histone acetylation is essential for hippocampal memory formation in young adult rodents. Although dysfunctional histone acetylation has been associated with age-related memory decline in male rodents, little is known about whether histone acetylation is altered by aging in female rodents. In young female mice, the ability of 17β-estradiol (E2) to enhance object recognition memory consolidation requires histone H3 acetylation in the dorsal hippocampus. However, the extent to which histone acetylation is regulated by E2 in middle-aged females is unknown. The mnemonic benefits of E2 in aging females appear to be greatest in middle age, and so pinpointing the molecular mechanisms through which E2 enhances memory at this age could lead to the development of safer and more effective treatments for maintaining memory function without the side effects of current therapies. Here, we show that dorsal hippocampal infusion of E2 rapidly enhanced object recognition and spatial memory, and increased histone H3 acetylation in the dorsal hippocampus, while also significantly reducing levels of histone deacetylase (HDAC2 and HDAC3) proteins. E2 specifically increased histone H3 acetylation at Bdnf promoters pII and pIV in the dorsal hippocampus of both young and middle-aged mice, despite age-related decreases in pI and pIV acetylation. Furthermore, levels of mature BDNF and pro-BDNF proteins in the dorsal hippocampus were increased by E2 in middle-aged females. Together, these data suggest that the middle-aged female dorsal hippocampus remains epigenetically responsive to E2, and that E2 may enhance memory in middle-aged females via epigenetic regulation of Bdnf.

  10. Acetylation of histones in neocortex and hippocampus of rats exposed to different modes of hypobaric hypoxia: Implications for brain hypoxic injury and tolerance.

    PubMed

    Samoilov, Mikhail; Churilova, Anna; Gluschenko, Tatjana; Vetrovoy, Oleg; Dyuzhikova, Natalia; Rybnikova, Elena

    2016-03-01

    Acetylation of nucleosome histones results in relaxation of DNA and its availability for the transcriptional regulators, and is generally associated with the enhancement of gene expression. Although it is well known that activation of a variety of pro-adaptive genes represents a key event in the development of brain hypoxic/ischemic tolerance, the role of epigenetic mechanisms, in particular histone acetylation, in this process is still unexplored. The aim of the present study was to investigate changes in acetylation of histones in vulnerable brain neurons using original well-standardized model of hypobaric hypoxia and preconditioning-induced tolerance of the brain. Using quantitative immunohistochemistry and Western blot, effects of severe injurious hypobaric hypoxia (SH, 180mm Hg, 3h) and neuroprotective preconditioning mode (three episodes of 360mm Hg for 2h spaced at 24h) on the levels of the acetylated proteins and acetylated H3 Lys24 (H3K24ac) in the neocortex and hippocampus of rats were studied. SH caused global repression of the acetylation processes in the neocortex (layers II-III, V) and hippocampus (CA1, CA3) by 3-24h, and this effect was prevented by the preconditioning. Moreover, hypoxic preconditioning remarkably increased the acetylation of H3K24 in response to SH in the brain areas examined. The preconditioning hypoxia without subsequent SH also stimulated acetylation processes in the neocortex and hippocampus. The moderately enhanced expression of the acetylated proteins in the preconditioned rats was maintained for 24h, whereas acetylation of H3K24 was intense but transient, peaked at 3h. The novel data obtained in the present study indicate that large activation of the acetylation processes, in particular acetylation of histones might be essential for the development of brain hypoxic tolerance.

  11. Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure.

    PubMed

    North, Justin A; Šimon, Marek; Ferdinand, Michelle B; Shoffner, Matthew A; Picking, Jonathan W; Howard, Cecil J; Mooney, Alex M; van Noort, John; Poirier, Michael G; Ottesen, Jennifer J

    2014-04-01

    Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.

  12. H3.5 is a novel hominid-specific histone H3 variant that is specifically expressed in the seminiferous tubules of human testes.

    PubMed

    Schenk, Raphael; Jenke, Andreas; Zilbauer, Matthias; Wirth, Stefan; Postberg, Jan

    2011-06-01

    The incorporation of histone variants into chromatin plays an important role for the establishment of particular chromatin states. Six human histone H3 variants are known to date, not counting CenH3 variants: H3.1, H3.2, H3.3 and the testis-specific H3.1t as well as the recently described variants H3.X and H3.Y. We report the discovery of H3.5, a novel non-CenH3 histone H3 variant. H3.5 is encoded on human chromosome 12p11.21 and probably evolved in a common ancestor of all recent great apes (Hominidae) as a consequence of H3F3B gene duplication by retrotransposition. H3.5 mRNA is specifically expressed in seminiferous tubules of human testis. Interestingly, H3.5 has two exact copies of ARKST motifs adjacent to lysine-9 or lysine-27, and lysine-79 is replaced by asparagine. In the Hek293 cell line, ectopically expressed H3.5 is assembled into chromatin and targeted by PTM. H3.5 preferentially colocalizes with euchromatin, and it is associated with actively transcribed genes and can replace an essential function of RNAi-depleted H3.3 in cell growth.

  13. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni

    PubMed Central

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M.; Romier, Christophe; Pierce, Raymond

    2017-01-01

    Background Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. Methodology/Principal findings TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Conclusions/Significance Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression

  14. Histone deacetylase inhibition modulates histone acetylation at gene promoter regions and affects genome-wide gene transcription in Schistosoma mansoni.

    PubMed

    Anderson, Letícia; Gomes, Monete Rajão; daSilva, Lucas Ferreira; Pereira, Adriana da Silva Andrade; Mourão, Marina M; Romier, Christophe; Pierce, Raymond; Verjovski-Almeida, Sergio

    2017-04-01

    Schistosomiasis is a parasitic disease infecting hundreds of millions of people worldwide. Treatment depends on a single drug, praziquantel, which kills the Schistosoma spp. parasite only at the adult stage. HDAC inhibitors (HDACi) such as Trichostatin A (TSA) induce parasite mortality in vitro (schistosomula and adult worms), however the downstream effects of histone hyperacetylation on the parasite are not known. TSA treatment of adult worms in vitro increased histone acetylation at H3K9ac and H3K14ac, which are transcription activation marks, not affecting the unrelated transcription repression mark H3K27me3. We investigated the effect of TSA HDACi on schistosomula gene expression at three different time points, finding a marked genome-wide change in the transcriptome profile. Gene transcription activity was correlated with changes on the chromatin acetylation mark at gene promoter regions. Moreover, combining expression data with ChIP-Seq public data for schistosomula, we found that differentially expressed genes having the H3K4me3 mark at their promoter region in general showed transcription activation upon HDACi treatment, compared with those without the mark, which showed transcription down-regulation. Affected genes are enriched for DNA replication processes, most of them being up-regulated. Twenty out of 22 genes encoding proteins involved in reducing reactive oxygen species accumulation were down-regulated. Dozens of genes encoding proteins with histone reader motifs were changed, including SmEED from the PRC2 complex. We targeted SmEZH2 methyltransferase PRC2 component with a new EZH2 inhibitor (GSK343) and showed a synergistic effect with TSA, significantly increasing schistosomula mortality. Genome-wide gene expression analyses have identified important pathways and cellular functions that were affected and may explain the schistosomicidal effect of TSA HDACi. The change in expression of dozens of histone reader genes involved in regulation of the

  15. Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y.

    PubMed

    Kujirai, Tomoya; Horikoshi, Naoki; Xie, Yan; Taguchi, Hiroyuki; Kurumizaka, Hitoshi

    2017-05-04

    Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

  16. Differential Subnuclear Localization and Replication Timing of Histone H3 Lysine 9 Methylation States

    PubMed Central

    Wu, Rong; Terry, Anna V.; Singh, Prim B.; Gilbert, David M.

    2005-01-01

    Mono-, di-, and trimethylation of specific histone residues adds an additional level of complexity to the range of histone modifications that may contribute to a histone code. However, it has not been clear whether different methylated states reside stably at different chromatin sites or whether they represent dynamic intermediates at the same chromatin sites. Here, we have used recently developed antibodies that are highly specific for mono-, di-, and trimethylated lysine 9 of histone H3 (MeK9H3) to examine the subnuclear localization and replication timing of chromatin containing these epigenetic marks in mammalian cells. Me1K9H3 was largely restricted to early replicating, small punctate domains in the nuclear interior. Me2K9H3 was the predominant MeK9 epitope at the nuclear and nucleolar periphery and colocalized with sites of DNA synthesis primarily in mid-S phase. Me3K9H3 decorated late-replicating pericentric heterochromatin in mouse cells and sites of DAPI-dense intranuclear heterochromatin in human and hamster cells that replicated throughout S phase. Disruption of the Suv39h1,2 or G9a methyltransferases in murine embryonic stem cells resulted in a redistribution of methyl epitopes, but did not alter the overall spatiotemporal replication program. These results demonstrate that mono-, di-, and trimethylated states of K9H3 largely occupy distinct chromosome domains. PMID:15788566

  17. A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions*

    PubMed Central

    Gilbert, Tonya M.; McDaniel, Stephen L.; Byrum, Stephanie D.; Cades, Jessica A.; Dancy, Blair C. R.; Wade, Herschel; Tackett, Alan J.; Strahl, Brian D.; Taverna, Sean D.

    2014-01-01

    Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with

  18. Trimethylation of histone H3 lysine 36 by human methyltransferase PRDM9 protein.

    PubMed

    Eram, Mohammad S; Bustos, Susan P; Lima-Fernandes, Evelyne; Siarheyeva, Alena; Senisterra, Guillermo; Hajian, Taraneh; Chau, Irene; Duan, Shili; Wu, Hong; Dombrovski, Ludmila; Schapira, Matthieu; Arrowsmith, Cheryl H; Vedadi, Masoud

    2014-04-25

    PRDM9 (PR domain-containing protein 9) is a meiosis-specific protein that trimethylates H3K4 and controls the activation of recombination hot spots. It is an essential enzyme in the progression of early meiotic prophase. Disruption of the PRDM9 gene results in sterility in mice. In human, several PRDM9 SNPs have been implicated in sterility as well. Here we report on kinetic studies of H3K4 methylation by PRDM9 in vitro indicating that PRDM9 is a highly active histone methyltransferase catalyzing mono-, di-, and trimethylation of the H3K4 mark. Screening for other potential histone marks, we identified H3K36 as a second histone residue that could also be mono-, di-, and trimethylated by PRDM9 as efficiently as H3K4. Overexpression of PRDM9 in HEK293 cells also resulted in a significant increase in trimethylated H3K36 and H3K4 further confirming our in vitro observations. Our findings indicate that PRDM9 may play critical roles through H3K36 trimethylation in cells.

  19. Histone H3.3 regulates dynamic chromatin states during spermatogenesis

    PubMed Central

    Yuen, Benjamin T. K.; Bush, Kelly M.; Barrilleaux, Bonnie L.; Cotterman, Rebecca; Knoepfler, Paul S.

    2014-01-01

    The histone variant H3.3 is involved in diverse biological processes, including development, transcriptional memory and transcriptional reprogramming, as well as diseases, including most notably malignant brain tumors. Recently, we developed a knockout mouse model for the H3f3b gene, one of two genes encoding H3.3. Here, we show that targeted disruption of H3f3b results in a number of phenotypic abnormalities, including a reduction in H3.3 histone levels, leading to male infertility, as well as abnormal sperm and testes morphology. Additionally, null germ cell populations at specific stages in spermatogenesis, in particular spermatocytes and spermatogonia, exhibited increased rates of apoptosis. Disruption of H3f3b also altered histone post-translational modifications and gene expression in the testes, with the most prominent changes occurring at genes involved in spermatogenesis. Finally, H3f3b null testes also exhibited abnormal germ cell chromatin reorganization and reduced protamine incorporation. Taken together, our studies indicate a major role for H3.3 in spermatogenesis through regulation of chromatin dynamics. PMID:25142466

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

    PubMed Central

    2010-01-01

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

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

    PubMed

    Postberg, Jan; Forcob, Sakeh; Chang, Wei-Jen; Lipps, Hans J

    2010-08-25

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

  2. Regulation of histone H3 lysine 9 methylation in oocytes and early pre-implantation embryos.

    PubMed

    Liu, Honglin; Kim, Jin-Moon; Aoki, Fugaku

    2004-05-01

    Epigenetic modifications of the genome, such as covalent modification of histone residues, ensure appropriate gene activation during pre-implantation development, and are probably involved in the asymmetric reprogramming of the parental genomes after fertilization. We investigated the methylation patterns of histone H3 at lysine 9 (H3/K9), and the regulatory mechanism involved in the asymmetric remodeling of parental genomes during early preimplantation development in mice. Immunocytochemistry with an antibody that specifically recognizes methylated H3/K9 showed a very weak or absent methylation signal in the male pronucleus, whereas a distinct methylation signal was detected in the female pronucleus. This asymmetric H3/K9 methylation pattern in the different parental genomes persisted until the two-cell stage. However, de novo methylation of H3/K9 occurred and the asymmetry was lost during the four-cell stage. The unmethylated male pronucleus underwent de novo methylation when it was transferred into enucleated GV- or MII-stage oocytes, which suggests that histone H3 methylase is active before fertilization, but not afterwards, and that the asymmetric methylation pattern is generated by this change in methylase activity in the cytoplasm after fertilization. Thus, histone H3 is methylated only in the maternal chromosomes, which are present in the oocytes before fertilization, and is not methylated in the paternal chromosomes, which are absent. The maintenance of asymmetric H3/K9 methylation patterns in early embryos is an active process that depends on protein synthesis and zygotic transcription, as de novo methylation in the male pronucleus occurred when either protein synthesis or gene expression was inhibited by cycloheximide or alpha-amanitin, respectively. In addition, corresponding de novo methylation of H3/K9 and DNA occurred when the male pronucleus was transferred to an enucleated GV oocyte. Our results suggest that H3/K9 methylation is an epigenetic

  3. A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation*

    PubMed Central

    Darwanto, Agus; Curtis, Matthew P.; Schrag, Matthew; Kirsch, Wolff; Liu, Peng; Xu, Guoliang; Neidigh, Jonathan W.; Zhang, Kangling

    2010-01-01

    Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation. PMID:20442396

  4. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

    PubMed Central

    Yoshida, Makoto M.; Ting, Lily; Gygi, Steven P.

    2016-01-01

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  5. Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

    PubMed

    Kurihara, Daisuke; Matsunaga, Sachihiro; Omura, Tomohiro; Higashiyama, Tetsuya; Fukui, Kiichi

    2011-04-28

    Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear. Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth. Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

  6. H3 and H4 Lysine Acetylation Correlates with Developmental and Experimentally Induced Adult Experience-Dependent Plasticity in the Mouse Visual Cortex

    PubMed Central

    Vierci, Gabriela; Pannunzio, Bruno; Bornia, Natalia; Rossi, Francesco M.

    2016-01-01

    Histone posttranslational modifications play a fundamental role in orchestrating gene expression. In this work, we analyzed the acetylation of H3 and H4 histones (AcH3–AcH4) and its modulation by visual experience in the mouse visual cortex (VC) during normal development and in two experimental conditions that restore juvenile-like plasticity levels in adults (fluoxetine treatment and enriched environment). We found that AcH3–AcH4 declines with age and is upregulated by treatments restoring plasticity in the adult. We also found that visual experience modulates AcH3–AcH4 in young and adult plasticity-restored mice but not in untreated ones. Finally, we showed that the transporter vGAT is downregulated in adult plasticity-restored models. In summary, we identified a dynamic regulation of AcH3–AcH4, which is associated with high plasticity levels and enhanced by visual experience. These data, along with recent ones, indicate H3–H4 acetylation as a central hub in the control of experience-dependent plasticity in the VC. PMID:27891053

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

    PubMed

    Honma, Kazue; Mochizuki, Kazuki; Goda, Toshinao

    2007-06-15

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

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

    PubMed

    Yu, Jingwen; Wu, Yanqing; Yang, Peixin

    2016-05-01

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

  9. Histone methyltransferase TXR1 is required for both H3 and H3.3 lysine 27 methylation in the well-known ciliated protist Tetrahymena thermophila.

    PubMed

    Zhao, Xiaolu; Wang, Yuanyuan; Wang, Yurui; Liu, Yifan; Gao, Shan

    2017-03-01

    DNA replication elongation is tightly controlled by histone-modifying enzymes. Our previous studies showed that the histone methytransferase TXR1 (Tetrahymena Trithorax related protein 1) specifically catalyzes H3K27 monomethylation and affects DNA replication elongation in Tetrahymena thermophila. In this study, we investigated whether TXR1 has a substrate preference to the canonical H3 over the replacement variant H3.3. We demonstrated by histone mutagenesis that K27Q mutation in H3.3 further aggravated the replication stress phenotype of K27Q mutation in canonical H3, supporting H3.3 as a physiologically relevant substrate of TXR1. This result is in apparent contrast to the strong preference for canonical H3 recently reported in Arabidopsis homologues ATXR5 and ATXR6, and further corroborates the role of TXR1 in DNA replication.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Histone H3 Recogntion and Presentation by the WDR5 Module of the MLL1 Complex

    SciTech Connect

    Ruthenburg,A.; Wang, W.; Graybosch, D.; Li, H.; Allis, D.; Patel, D.; Verdine, G.

    2006-01-01

    WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nzeta of Lys4 (H3K4). The role of WDR5 in the MLL1 complex has recently been described as specific recognition of dimethyl-K4 in the context of a histone H3 amino terminus; WDR5 is essential for vertebrate development, Hox gene activation and global H3K4 trimethylation. We report the high-resolution X-ray structures of WDR5 in the unliganded form and complexed with histone H3 peptides having unmodified and mono-, di- and trimethylated K4, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40-repeat fold. Contrary to predictions, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes.

  12. Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event

    PubMed Central

    Campos, Eric I.; Smits, Arne H.; Kang, Young-Hoon; Landry, Sébastien; Escobar, Thelma M.; Nayak, Shruti; Ueberheide, Beatrix M.; Durocher, Daniel; Vermeulen, Michiel; Hurwitz, Jerard; Reinberg, Danny

    2015-01-01

    SUMMARY Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Though histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones, but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase. PMID:26527279

  13. The N-terminal domains of histones H3 and H4 are not necessary for chromatin assembly factor-1- mediated nucleosome assembly onto replicated DNA in vitro

    PubMed Central

    Shibahara, Kei-ichi; Verreault, Alain; Stillman, Bruce

    2000-01-01

    An in vitro reconstitution system for the analysis of replication-coupled nucleosome assembly is described. In this “two-step system,” nucleosome assembly is performed in a separate reaction from DNA replication, wherein purified newly replicated DNA remains noncovalently marked for subsequent chromatin assembly factor-1 (CAF-1)-dependent nucleosome assembly. Because the nucleosome assembly is performed separately from the DNA replication step, this system is more versatile and biochemically tractable when compared with nucleosome assembly during simian virus 40 (SV40) DNA replication. The N-terminal domains of histones H3 and H4 play an important but redundant function in nucleosome assembly in the budding yeast, Saccharomyces cerevisiae. It had been proposed that at least one tail of histone H3 or H4 is required for replication-coupled nucleosome assembly. However, we demonstrate that the N-terminal domains of both histone H3 and H4 are dispensable for CAF-1-mediated formation of nucleosome cores onto newly replicated DNA in vitro. CAF-1 and each of its individual subunits stably bound to recombinant (H3.H4)2 tetramers lacking the N-terminal domains of both H3 and H4. Therefore, the N-terminal tails of histone H3 and H4 that contain the specific acetylation sites are not necessary for CAF-1-dependent nucleosome assembly onto replicated DNA. We suggest that the histone acetylation may be required for a CAF-1 independent pathway or function after deposition, by marking of newly replicated chromatin. PMID:10884407

  14. Substitutions in the Amino-Terminal Tail of Neurospora Histone H3 Have Varied Effects on DNA Methylation

    PubMed Central

    Adhvaryu, Keyur K.; Berge, Emanuela; Tamaru, Hisashi; Freitag, Michael; Selker, Eric U.

    2011-01-01

    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 (hH3K9L or hH3K9R) causes global loss of DNA methylation in the presence of wild-type hH3 (hH3WT). 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. PMID:22242002

  15. Histone acetylation regulates orphan nuclear receptor NR4A1 expression in hypercholesterolaemia.

    PubMed

    Xie, Xina; Song, Xuhong; Yuan, Song; Cai, Haitao; Chen, Yequn; Chang, Xiaolan; Liang, Bin; Huang, Dongyang

    2015-12-01

    Hypercholesterolaemia and inflammation are correlated with atherogenesis. Orphan nuclear receptor NR4A1, as a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the mechanism by which lipids regulate NR4A1 expression remains unknown. We aimed to elucidate the underlying mechanism of NR4A1 expression in monocytes during hypercholesterolaemia, and reveal the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. Circulating leucocytes were collected from blood samples of 139 patients with hypercholesterolaemia and 139 sex- and age-matched healthy subjects. We found that there was a low-grade inflammatory state and higher expression of NR4A1 in patients. Both total cholesterol and low-density lipoprotein cholesterol levels in plasma were positively correlated with NR4A1 mRNA level. ChIP revealed that acetylation of histone H3 was enriched in the NR4A1 promoter region in patients. Human mononuclear cell lines THP-1 and U937 were treated with cholesterol. Supporting our clinical observations, cholesterol enhanced p300 acetyltransferase and decreased HDAC7 (histone deacetylase 7) recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation and further contributing to NR4A1 up-regulation in monocytes. Moreover, cytosporone B, an NR4A1 agonist, completely reversed cholesterol-induced IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1) expression to below basal levels, and knockdown of NR4A1 expression by siRNA not only mimicked, but also exaggerated the effects of cholesterol on inflammatory biomarker up-regulation. Thus we conclude that histone acetylation contributes to the regulation of NR4A1 expression in hypercholesterolaemia, and that NR4A1 expression reduces hypercholesterolaemia-induced inflammation. © 2015 Authors; published by Portland Press Limited.

  16. Loss of histone H3 methylation at lysine 4 triggers apoptosis in Saccharomyces cerevisiae.

    PubMed

    Walter, David; Matter, Anja; Fahrenkrog, Birthe

    2014-01-01

    Monoubiquitination of histone H2B lysine 123 regulates methylation of histone H3 lysine 4 (H3K4) and 79 (H3K79) and the lack of H2B ubiquitination in Saccharomyces cerevisiae coincides with metacaspase-dependent apoptosis. Here, we discovered that loss of H3K4 methylation due to depletion of the methyltransferase Set1p (or the two COMPASS subunits Spp1p and Bre2p, respectively) leads to enhanced cell death during chronological aging and increased sensitivity to apoptosis induction. In contrast, loss of H3K79 methylation due to DOT1 disruption only slightly affects yeast survival. SET1 depleted cells accumulate DNA damage and co-disruption of Dot1p, the DNA damage adaptor protein Rad9p, the endonuclease Nuc1p, and the metacaspase Yca1p, respectively, impedes their early death. Furthermore, aged and dying wild-type cells lose H3K4 methylation, whereas depletion of the H3K4 demethylase Jhd2p improves survival, indicating that loss of H3K4 methylation is an important trigger for cell death in S. cerevisiae. Given the evolutionary conservation of H3K4 methylation this likely plays a role in apoptosis regulation in a wide range of organisms.

  17. Methylation of histone H3 in euchromatin of plant chromosomes depends on basic nuclear DNA content.

    PubMed

    Houben, Andreas; Demidov, Dmitri; Gernand, Dorota; Meister, Armin; Leach, Carolyn R; Schubert, Ingo

    2003-03-01

    Strong methylation of lysine 4 (K4) and low methylation of lysine 9 (K9) have been proposed as modifications of histone H3, typical for transcriptionally active euchromatin and the opposite for inactive heterochromatin. We have analysed the correlation between the global distribution of histone H3, methylated at either lysine 4 or lysine 9, and of microscopically detectable euchromatic or heterochromatic regions in relation to genome size for 24 plant species. Two different distribution patterns of methylated (K9)H3 (Met(K9)H3) were found that depend on genome size. For most species with small genomes (1C <500 Mbp), including Arabidopsis thaliana, strong methylation of (K9)H3 was restricted to constitutive heterochromatin. Species with larger genomes showed a uniform distribution of Met(K9)H3. Contrary to this and regardless of the genome size, methylated (K4)H3 (Met(K4)H3) was found to be enriched within the euchromatin of all species. Transcriptionally less active B chromosomes showed the same patterns as basic A chromosomes. We thus propose that large genomes with high amounts of dispersed repetitive sequences (mainly retroelements) have to silence these sequences and therefore display epigenetic modifications such as methylation of DNA and (K9)H3 also within euchromatic regions.

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

    PubMed

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

    2016-10-03

    Social defeat (SD) induces a long-lasting increase in the rewarding effects of psychostimulants measured using the self-administration and conditioned place procedures (CPP). However, little is known about the epigenetic changes induced by social stress and about their role in the increased response to the rewarding effects of psychostimulants. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced behavioral changes, we addressed the hypothesis that SD induces transcriptional changes by histone modifications associated with the acquisition of place conditioning. After a fourth defeat, H3(K9) acetylation was decreased in the hippocampus, while there was an increase of HAT and a decrease of HDAC levels in the cortex. Three weeks after the last defeat, mice displayed an increase in histone H4(K12) acetylation and an upregulation of histone acetyl transferase (HAT) activity in the hippocampus. In addition, H3(K4)me3, which is closely associated with transcriptional initiation, was also augmented in the hippocampus three weeks after the last defeat. Inhibition of HAT by curcumin (100mg/kg) before each SD blocked the increase in the conditioned reinforcing effects of 1mg/kg of cocaine, while inhibition of HDAC by valproic acid (500mg/kg) before social stress potentiated cocaine-induced CPP. Preference was reinstated when animals received a priming dose of 0.5mg/kg of cocaine, an effect that was absent in untreated defeated mice. These results suggest that the experience of SD induces chromatin remodeling, alters histone acetylation and methylation, and modifies the effects of cocaine on place conditioning. They also point to epigenetic mechanisms as potential avenues leading to new treatments for the long-term effects of social stress on drug addiction. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites.

    PubMed

    Borde, Valérie; Robine, Nicolas; Lin, Waka; Bonfils, Sandrine; Géli, Vincent; Nicolas, Alain

    2009-01-21

    The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

  20. Histone H3 lysine 36 methylation affects temperature-induced alternative splicing and flowering in plants.

    PubMed

    Pajoro, A; Severing, E; Angenent, G C; Immink, R G H

    2017-06-01

    Global warming severely affects flowering time and reproductive success of plants. Alternative splicing of pre-messenger RNA (mRNA) is an important mechanism underlying ambient temperature-controlled responses in plants, yet its regulation is poorly understood. An increase in temperature promotes changes in plant morphology as well as the transition from the vegetative to the reproductive phase in Arabidopsis thaliana via changes in splicing of key regulatory genes. Here we investigate whether a particular histone modification affects ambient temperature-induced alternative splicing and flowering time. We use a genome-wide approach and perform RNA-sequencing (RNA-seq) analyses and histone H3 lysine 36 tri-methylation (H3K36me3) chromatin immunoprecipitation sequencing (ChIP-seq) in plants exposed to different ambient temperatures. Analysis and comparison of these datasets reveal that temperature-induced differentially spliced genes are enriched in H3K36me3. Moreover, we find that reduction of H3K36me3 deposition causes alteration in temperature-induced alternative splicing. We also show that plants with mutations in H3K36me3 writers, eraser, or readers have altered high ambient temperature-induced flowering. Our results show a key role for the histone mark H3K36me3 in splicing regulation and plant plasticity to fluctuating ambient temperature. Our findings open new perspectives for the breeding of crops that can better cope with environmental changes due to climate change.

  1. Functional antagonism between histone H3K4 demethylases in vivo

    PubMed Central

    Di Stefano, Luisa; Walker, James A.; Burgio, Giosalba; Corona, Davide F.V.; Mulligan, Peter; Näär, Anders M.; Dyson, Nicholas J.

    2011-01-01

    Dynamic regulation of histone modifications is critical during development, and aberrant activity of chromatin-modifying enzymes has been associated with diseases such as cancer. Histone demethylases have been shown to play a key role in eukaryotic gene transcription; however, little is known about how their activities are coordinated in vivo to regulate specific biological processes. In Drosophila, two enzymes, dLsd1 (Drosophila ortholog of lysine-specific demethylase 1) and Lid (little imaginal discs), demethylate histone H3 at Lys 4 (H3K4), a residue whose methylation is associated with actively transcribed genes. Our studies show that compound mutation of Lid and dLsd1 results in increased H3K4 methylation levels. However, unexpectedly, Lid mutations strongly suppress dLsd1 mutant phenotypes. Investigation of the basis for this antagonism revealed that Lid opposes the functions of dLsd1 and the histone methyltransferase Su(var)3–9 in promoting heterochromatin spreading at heterochromatin–euchromatin boundaries. Moreover, our data reveal a novel role for dLsd1 in Notch signaling in Drosophila, and a complex network of interactions between dLsd1, Lid, and Notch signaling at euchromatic genes. These findings illustrate the complexity of functional interplay between histone demethylases in vivo, providing insights into the epigenetic regulation of heterochromatin/euchromatin boundaries by Lid and dLsd1 and showing their involvement in Notch pathway-specific control of gene expression in euchromatin. PMID:21205864

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

    PubMed

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

    2006-12-12

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

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

    PubMed

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

    2015-11-01

    The cereal aleurone layer plays an important role in seed germination, and reactive oxygen species (ROS) in aleurone layers act as crucial signal molecules in this progression. Recent studies have revealed that epigenetic modification is involved in plant development and seed germination. However, little is known about a possible relationship between histone modification and the ROS signaling pathway in cereal aleurone layers during seed germination. Here, we found that the expression of both histone acetyltransferases (HATs) and histone deacetylases (HDACs) was increased gradually during seed germination, accompanied by an increase in global acetylation levels of histones H3 and H4 in maize aleurone layers. The acetylation was found to be promoted by GA(3) and suppressed by ABA. However, when the HDAC inhibitor trichostatin A (TSA) was used, the increased H3K9ac and H4K5ac level correlated with an inhibition of the germination. These results indicated that the overall histone acetylation in the aleurone layers is not required for germination. Similarly these two hormones, GA(3) and ABA, exerted opposed effects on the expression of the ROS-related gene sodCp. Furthermore, chromatin immunoprecipitation experiments showed that the promoter region of the sodCp gene was hyperacetylated during germination, and this acetylation was promoted by GA(3) and inhibited by both ABA and TSA. These results suggested that GA(3)-mediated expression of the sodCp gene in aleurone layers is associated with histone hyperacetylation on the promoter and coding region of this gene, consequently leading to an accumulation of H(2)O(2) which regulated production of α-amylase during seed germination. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast.

    PubMed

    Shankaranarayana, Gurumurthy D; Motamedi, Mohammad R; Moazed, Danesh; Grewal, Shiv I S

    2003-07-15

    Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.

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

    SciTech Connect

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

    2007-02-28

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

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

    PubMed

    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.

  7. Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy.

    PubMed

    Awad, Salma; Kunhi, Muhammad; Little, Gillian H; Bai, Yan; An, Woojin; Bers, Donald; Kedes, Larry; Poizat, Coralie

    2013-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in pathological cardiac hypertrophy, but the mechanisms by which it modulates gene activity in the nucleus to mediate hypertrophic signaling remain unclear. Here, we report that nuclear CaMKII activates cardiac transcription by directly binding to chromatin and regulating the phosphorylation of histone H3 at serine-10. These specific activities are demonstrated both in vitro and in primary neonatal rat cardiomyocytes. Activation of CaMKII signaling by hypertrophic agonists increases H3 phosphorylation in primary cardiac cells and is accompanied by concomitant cellular hypertrophy. Conversely, specific silencing of nuclear CaMKII using RNA interference reduces both H3 phosphorylation and cellular hypertrophy. The hyper-phosphorylation of H3 associated with increased chromatin binding of CaMKII occurs at specific gene loci reactivated during cardiac hypertrophy. Importantly, H3 Ser-10 phosphorylation and CaMKII recruitment are associated with increased chromatin accessibility and are required for chromatin-mediated transcription of the Mef2 transcription factor. Unlike phosphorylation of H3 by other kinases, which regulates cellular proliferation and immediate early gene activation, CaMKII-mediated signaling to H3 is associated with hypertrophic growth. These observations reveal a previously unrecognized function of CaMKII as a kinase signaling to histone H3 and remodeling chromatin. They suggest a new epigenetic mechanism controlling cardiac hypertrophy.

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

    PubMed

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

    2014-12-01

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

  9. Methamphetamine Causes Differential Alterations in Gene Expression and Patterns of Histone Acetylation/Hypoacetylation in the Rat Nucleus Accumbens

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2011-06-01

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

  12. Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers.

    PubMed

    Chirinos, M; Hernández, F; Palacián, E

    1999-10-15

    To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers. Under conditions promoting a low transcription rate, it is unambiguously shown with pGEMEX-1 that the block to initiation due to the presence of core histone octamers is substantially removed when (H3 x H4)(2) is substituted for the whole octamer. With T7-207-18, under assay conditions allowing transcription of the whole coding region of the naked DNA, analysis of the transcription products indicates that RNA elongation on the template containing (H3 x H4)(2) tetramers takes place as easily as on free DNA, in contrast with the significant inhibition observed in the presence of whole histone octamers. Copyright 1999 Academic Press.

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

    PubMed

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

    2015-05-10

    Histone modifications play an integral role in plant development, but have been poorly studied in woody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood formation) and identify novel functional regions in plant genomes. However, woody tissue poses unique challenges for using high-throughput chromatin immunoprecipitation (ChIP) techniques for studying genome-wide histone modifications in vivo. We investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene expression during the early stages of wood formation using ChIP-seq in Eucalyptus grandis, a woody biomass model. Plant chromatin fixation and isolation protocols were optimized for developing xylem tissue collected from field-grown E. grandis trees. A "nano-ChIP-seq" procedure was employed for ChIP DNA amplification. Over 9 million H3K4me3 ChIP-seq and 18 million control paired-end reads were mapped to the E. grandis reference genome for peak-calling using Model-based Analysis of ChIP-Seq. The 12,177 significant H3K4me3 peaks identified covered ~1.5% of the genome and overlapped some 9,623 protein-coding genes and 38 noncoding RNAs. H3K4me3 library coverage, peaking ~600 - 700 bp downstream of the transcription start site, was highly correlated with gene expression levels measured with RNA-seq. Overall, H3K4me3-enriched genes tended to be less tissue-specific than unenriched genes and were overrepresented for general cellular metabolism and development gene ontology terms. Relative expression of H3K4me3-enriched genes in developing secondary xylem was higher than unenriched genes, however, and highly expressed secondary cell wall-related genes were enriched for H3K4me3 as validated using ChIP-qPCR. In this first genome-wide analysis of a modified histone in a woody tissue, we optimized a ChIP-seq procedure suitable

  14. RNase P protein subunit Rpp29 represses histone H3.3 nucleosome deposition

    PubMed Central

    Newhart, Alyshia; Powers, Sara Lawrence; Shastrula, Prashanth Krishna; Sierra, Isabel; Joo, Lucy M.; Hayden, James E.; Cohen, Andrew R.; Janicki, Susan M.

    2016-01-01

    In mammals, histone H3.3 is a critical regulator of transcription state change and heritability at both euchromatin and heterochromatin. The H3.3-specific chaperone, DAXX, together with the chromatin-remodeling factor, ATRX, regulates H3.3 deposition and transcriptional silencing at repetitive DNA, including pericentromeres and telomeres. However, the events that precede H3.3 nucleosome incorporation have not been fully elucidated. We previously showed that the DAXX-ATRX-H3.3 pathway regulates a multi-copy array of an inducible transgene that can be visualized in single living cells. When this pathway is impaired, the array can be robustly activated. H3.3 is strongly recruited to the site during activation where it accumulates in a complex with transcribed sense and antisense RNA, which is distinct from the DNA/chromatin. This suggests that transcriptional events regulate H3.3 recruited to its incorporation sites. Here we report that the nucleolar RNA proteins Rpp29, fibrillarin, and RPL23a are also components of this H3.3/RNA complex. Rpp29 is a protein subunit of RNase P. Of the other subunits, POP1 and Rpp21 are similarly recruited suggesting that a variant of RNase P regulates H3.3 chromatin assembly. Rpp29 knockdown increases H3.3 chromatin incorporation, which suggests that Rpp29 represses H3.3 nucleosome deposition, a finding with implications for epigenetic regulation. PMID:26842893

  15. Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

    PubMed

    Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

    2010-02-01

    The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

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

    PubMed Central

    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

  17. Histone H3.3 regulates mitotic progression in mouse embryonic fibroblasts.

    PubMed

    Ors, Aysegul; Papin, Christophe; Favier, Bertrand; Roulland, Yohan; Dalkara, Defne; Ozturk, Mehmet; Hamiche, Ali; Dimitrov, Stefan; Padmanabhan, Kiran

    2017-08-01

    H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity.

  18. Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae

    PubMed Central

    Sorenson, Matthew R.; Jha, Deepak K.; Ucles, Stefanie A.; Flood, Danielle M.; Strahl, Brian D.; Stevens, Scott W.; Kress, Tracy L.

    2016-01-01

    ABSTRACT Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) – a residue methylated by Set2 during transcription elongation – exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

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

    PubMed

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

    2016-03-01

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

  20. The chromosomal distribution of phosphorylated histone H3 differs between plants and animals at meiosis.

    PubMed

    Manzanero, S; Arana, P; Puertas, M J; Houben, A

    2000-01-01

    Plant (Secale cereale, Triticum aestivum) and animal (Eyprepocnemis plorans) meiocytes were analyzed by indirect immunostaining with an antibody recognizing histone H3 phosphorylated at serine 10, to study the relationship between H3 phosphorylation and chromosome condensation at meiosis. To investigate whether the dynamics of histone H3 phosphorylation differs between chromosomes with a different mode of segregation, we included in this study mitotic cells and also meiotic cells of individuals forming bivalents plus three different types of univalents (A chromosomes, B chromosomes and X chromosome). During the first meiotic division, the H3 phosphorylation of the entire chromosomes initiates at the transition from leptotene to zygotene in rye and wheat, whereas in E. plorans it does so at diplotene. In all species analyzed H3 phosphorylation terminates toward interkinesis. The immunosignals at first meiotic division are identical in bivalents and univalents of A and B chromosomes, irrespective of their equational or reductional segregation at anaphase I. The grasshopper X chromosome, which always segregates reductionally, also shows the same pattern. Remarkable differences were found at second meiotic division between plant and animal material. In E. plorans H3 phosphorylation occurred all along the chromosomes, whereas in plants only the pericentromeric regions showed strong immunosignals from prophase II until telophase II. In addition, no immunolabeling was detectable on single chromatids resulting from equational segregation of plant A or B chromosome univalents during the preceding anaphase I. Simultaneous immunostaining with anti-tubulin and anti-phosphorylated H3 antibodies demonstrated that the kinetochores of all chromosomes interact with microtubules, even in the absence of detectable phosphorylated H3 immunosignals. The different pattern of H3 phosphorylation in plant and animal meiocytes suggests that this evolutionarily conserved post

  1. Identification and Characterization of Switchgrass Histone H3 and CENH3 Genes

    SciTech Connect

    Miao, Jiamin; Frazier, Taylor; Huang, Linkai; Zhang, Xinquan; Zhao, Bingyu

    2016-07-12

    Switchgrass is one of the most promising energy crops and only recently has been employed for biofuel production. The draft genome of switchgrass was recently released; however, relatively few switchgrass genes have been functionally characterized. CENH3, the major histone protein found in centromeres, along with canonical H3 and other histones, plays an important role in maintaining genome stability and integrity. Despite their importance, the histone H3 genes of switchgrass have remained largely uninvestigated. In this study, we identified 17 putative switchgrass histone H3 genes in silico. Of these genes, 15 showed strong homology to histone H3 genes including six H3.1 genes, three H3.3 genes, four H3.3-like genes and two H3.1-like genes. The remaining two genes were found to be homologous to CENH3. RNA-seq data derived from lowland cultivar Alamo and upland cultivar Dacotah allowed us to identify SNPs in the histone H3 genes and compare their differential gene expression. Interestingly, we also found that overexpression of switchgrass histone H3 and CENH3 genes in N. benthamiana could trigger cell death of the transformed plant cells. Localization and deletion analyses of the histone H3 and CENH3 genes revealed that nuclear localization of the N-terminal tail is essential and sufficient for triggering the cell death phenotype. Lastly, our results deliver insight into the mechanisms underlying the histone-triggered cell death phenotype and provide a foundation for further studying the variations of the histone H3 and CENH3 genes in switchgrass.

  2. KDM6 Demethylase Independent Loss of Histone H3 Lysine 27 Trimethylation during Early Embryonic Development

    PubMed Central

    Shpargel, Karl B.; Starmer, Joshua; Yee, Della; Pohlers, Michael; Magnuson, Terry

    2014-01-01

    The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family. PMID:25101834

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

    PubMed

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

    2011-10-28

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

  4. Drosophila Kdm4 demethylases in histone H3 lysine 9 demethylation and ecdysteroid signaling

    PubMed Central

    Tsurumi, Amy; Dutta, Pranabanada; Yan, Shian-Jang; Sheng, Robin; Li, Willis X.

    2013-01-01

    The dynamic regulation of chromatin structure by histone post-translational modification is an essential regulatory mechanism that controls global gene transcription. The Kdm4 family of H3K9me2,3 and H3K36me2,3 dual specific histone demethylases has been implicated in development and tumorigenesis. Here we show that Drosophila Kdm4A and Kdm4B are together essential for mediating ecdysteroid hormone signaling during larval development. Loss of Kdm4 genes leads to globally elevated levels of the heterochromatin marker H3K9me2,3 and impedes transcriptional activation of ecdysone response genes, resulting in developmental arrest. We further show that Kdm4A interacts with the Ecdysone Receptor (EcR) and colocalizes with EcR at its target gene promoter. Our studies suggest that Kdm4A may function as a transcriptional co-activator by removing the repressive histone mark H3K9me2,3 from cognate promoters. PMID:24100631

  5. Comparative analysis of histone H3 and H4 post-translational modifications of esophageal squamous cell carcinoma with different invasive capabilities.

    PubMed

    Zhang, Kai; Li, Liyan; Zhu, Mengxiao; Wang, Guojuan; Xie, Jianjun; Zhao, Yunlong; Fan, Enguo; Xu, Liyan; Li, Enmin

    2015-01-01

    Eukaryotic DNA is packaged into a chromatin with the help of four core histones (H2A, H2B, H3, and H4). Diverse histone post-translational modifications (PTMs) are hence involved in the regulation of gene transcription. However, how this regulation does work is still poorly understood and lacks details. Here we used the mass spectrometry-based proteomics approach to perform a comparative analysis of histone marks at a global level in two phenotypes of esophageal squamous cell carcinoma (ESCC) with different invasiveness. We obtained a comprehensive profiling of histone H3 and H4 PTMs including lysine methylation, acetylation and novel butyrylation. The correlation between histone marks and cancer invasive capabilities was further characterized and one distinguishable PTM, H4K79me2 was discovered and verified in this study. Immunohistochemistry analysis suggests that abnormal level of H4K79me2 may be related to poor survival of ESCC patients. Our results enrich the dataset of the feature pattern of global histone PTMs in ESCC cell lines. Core histone proteins, decorated by multiple biological significant protein post-translational modifications (PTMs) such as lysine acetylation and lysine methylation, are considered to regulate gene transcription and be associated with the development of cancer. Recent studies have further shown that global level of histone modifications is the potential hallmark of cancer to predict the clinical outcomes of human cancers. However, the regulation mechanism is largely unknown. Here we used the mass spectrometry based proteomics coupled with stable isotope labeling with amino acids in cell culture (SILAC) to characterize the global levels of histone marks in two phenotypes of esophageal squamous cell carcinoma (ESCC) cell lines with different invasive capabilities. To the best of our knowledge, it is the first report about the comparative analysis for histone marks of the different invasive ESCC cell lines. A significantly

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

    PubMed

    Le, Ly-Thuy-Tram; Vu, Hong-Lien; Nguyen, Chi-Hung; Molla, Annie

    2013-04-15

    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.

  7. Identification of Tension Sensing Motif of Histone H3 in Saccharomyces cerevisiae and Its Regulation by Histone Modifying Enzymes.

    PubMed

    Luo, Jianjun; Deng, Xiexiong; Buehl, Christopher; Xu, Xinjing; Kuo, Min-Hao

    2016-11-01

    To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome biorientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif (TSM). Similar to the previously reported G44S mutant, K42A, G44A, and T45A alleles all rendered cells unable to respond to erroneous spindle attachment, a phenotype suppressed by Sgo1p overexpression. TSM functions by physically recruiting or retaining Sgo1p at pericentromeres as evidenced by chromatin immunoprecipitation and by in vitro pulldown experiments. Intriguingly, the function of TSM is likely regulated by multiple histone modifying enzymes, including the histone acetyltransferase Gcn5p, and deacetylases Rpd3p and Hos2p Defects caused by TSM mutations can be suppressed by the expression of a catalytically inactive mutant of Gcn5p Conversely, G44S mutant cells exhibit prominent chromatin instability phenotype in the absence of RPD3 Importantly, the gcn5(-) suppressor restores the tension sensing function in tsm(-) background in a fashion that bypasses the need of stably associating Sgo1p with chromatin. These results demonstrate that the TSM of histone H3 is a key component of a mechanism that ensures faithful segregation, and that interaction with chromatin modifying enzymes may be an important part of the mitotic quality control process. Copyright © 2016 by the Genetics Society of America.

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

    PubMed Central

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

    2012-01-01

    The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of histones H3–H4 with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3–H4 between these two histone chaperones plays a central role in the assembly of new nucleosomes and we show here that the H3–H4 complex has a surprising structural plasticity, which is important for this exchange. PMID:23178455

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2016-03-01

    Isoflurane-induced neurocognitive impairment in the developing rodent brain is well documented, and regular physical exercise has been demonstrated to be a viable intervention for some types of neurocognitive impairment. This study was designed to investigate the potential protective effect of swimming exercise on both neurocognitive impairment caused by repeated neonatal exposure to isoflurane and the underlying molecular mechanism. Mice received 0.75% isoflurane exposures for 4h on postnatal days 7, 8, and 9. From the third month after anesthesia, the mice were subjected to regular swimming exercise for 4weeks, followed by a contextual fear condition (CFC) trial. We found that repeated neonatal exposure to isoflurane reduced freezing behavior during CFC testing and deregulated hippocampal histone H4K12 acetylation. Conversely, mice subjected to regular swimming exercise showed enhanced hippocampal H3K9, H4K5, and H4K12 acetylation levels, increased numbers of c-Fos-positive cells 1h after CFC training, and less isoflurane-induced memory impairment. We also observed increases in histone acetylation and of cAMP-response element-binding protein (CREB)-binding protein (CBP) during the swimming exercise program. The results suggest that neonatal isoflurane exposure-induced memory impairment was associated with dysregulation of H4K12 acetylation, which may lead to less hippocampal activation following learning tasks. Swimming exercise was associated with enhanced hippocampal histone acetylation and CBP expression. Exercise most likely ameliorated isoflurane-induced memory impairment by enhancing hippocampal histone acetylation and activating more neuron cells during memory formation. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Site-specific human histone H3 methylation stability: fast K4me3 turnover

    PubMed Central

    Zheng, Yupeng; Tipton, Jeremiah D.; Thomas, Paul M.; Kelleher, Neil L.; Sweet, Steve M.M.

    2014-01-01

    We employ stable isotope labelling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half-lives of multiple cell divisions. By contrast the transcription-associated marks K4me3 and K36me3 turn over far more rapidly, with half-lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome-wide localization data and transcription-dependent nucleosome rearrangements proximal to the transcription start site. PMID:24826939

  12. Site-specific human histone H3 methylation stability: fast K4me3 turnover.

    PubMed

    Zheng, Yupeng; Tipton, Jeremiah D; Thomas, Paul M; Kelleher, Neil L; Sweet, Steve M M

    2014-10-01

    We employ stable-isotope labeling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half-lives of multiple cell divisions. By contrast, the transcription-associated marks K4me3 and K36me3 turn over far more rapidly, with half-lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome-wide localization data and transcription-dependent nucleosome rearrangements proximal to the transcription start site.

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

    PubMed

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

    2013-05-23

    Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic β subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.

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

    PubMed Central

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

    2013-01-01

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

  15. K4, K9, and K18 in Human Histone H3 are Targets for Biotinylation by Biotinidase

    PubMed Central

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

    2005-01-01

    Histones are modified posttranslationally, 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. 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. PMID:16098205

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

    PubMed

    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.

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

    PubMed Central

    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

  18. Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation.

    PubMed

    Diallo, Amadou Oury; Ali-Benali, Mohamed Ali; Badawi, Mohamed; Houde, Mario; Sarhan, Fathey

    2012-07-01

    The transition to flowering in winter wheat requires prolonged exposure to low temperature, a process called vernalization. This process is regulated by a genetic pathway that involves at least three genes, Triticum aestivum VERNALIZATION 1 (TaVRN1), Triticum aestivum VERNALIZATION 2 (TaVRN2) and Triticum aestivum FLOWERING LOCUS T-like 1 (TaFT1). These genes regulate flowering by integrating environmental and developmental cues. To determine whether the expression of these genes is associated with the chromatin methylation state during vernalization in wheat, the level of two markers of histone modifications, the activator histone H3 trimethylation of lysine 4 (H3K4me3) and the repressor histone H3 trimethylation of lysine 27 (H3K27me3) were measured at the promoter regions of these three genes. Bioinformatics analysis of these promoters demonstrates the presence of conserved cis-acting elements in the promoters of the three vernalization genes, TaVRN1, TaVRN2 and TaFT1. These elements are targeted by common transcription factors in the vernalization responsive cereals. These promoters also contain the functional "units" PRE/TRE targeted by Polycomb and Trithorax proteins that maintain repressed or active transcription states of developmentally regulated genes. These proteins are known to be associated with the regulation of H3K4me3 and H3K27me3. Expression studies indicate that TaVRN1 and TaFT1 are up-regulated by vernalization in winter wheat. This up-regulation is associated with increased level of the activator H3K4me3 with no change in the level of the repressor H3K27me3 at the promoter region. This study shows that the flowering transition induced by vernalization in winter wheat is associated with histone methylation at the promoter level of TaVRN1 and TaFT1 while the role of these markers is less evident in TaVRN2 repression. This may represent part of the cellular memory of vernalization in wheat.

  19. Regulation of histone H3K4 methylation in brain development and disease.

    PubMed

    Shen, Erica; Shulha, Hennady; Weng, Zhiping; Akbarian, Schahram

    2014-09-26

    The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders.

  20. Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

    PubMed Central

    Thornton, Janet L.; Westfield, Gerwin H.; Takahashi, Yoh-hei; Cook, Malcolm; Gao, Xin; Woodfin, Ashley R.; Lee, Jung-Shin; Morgan, Marc A.; Jackson, Jessica; Smith, Edwin R.; Couture, Jean-Francois; Skiniotis, Georgios; Shilatifard, Ali

    2014-01-01

    The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner. PMID:24402317

  1. Histone H3 modifications and Cdx-2 binding to the sucrase-isomaltase (SI) gene is involved in induction of the gene in the transition from the crypt to villus in the small intestine of rats.

    PubMed

    Suzuki, Takuji; Mochizuki, Kazuki; Goda, Toshinao

    2008-05-02

    Expression of the sucrase-isomaltase (SI) gene is induced in cells transitioning from the crypt to the villus of rat jejunum. In the present study, we revealed by ChIP assay using a cryostat sectioning technique that binding of the di-acetylated histone H3 at lysine 9/14 and the transcriptional factor Cdx-2 to the promoter region on the SI gene, as well as mRNA, increased in the transient process. Additionally, di-/tri-methylation of histone H3 at lysine 9/14 on the promoter region of the SI gene rapidly decreased with increasing mRNA. These results suggest that induction of the SI gene during the transition from the crypt to the villi is associated with changes in histone H3 modifications from methylation at lysine 9 to di-acetylation at lysine 9/14, as well as increased binding of Cdx-2 to the SI promoter region.

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

    PubMed

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

    2016-04-22

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

  3. An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.

    PubMed

    Duval, Romain; Fritsch, Lauriane; Bui, Linh-Chi; Berthelet, Jérémy; Guidez, Fabien; Mathieu, Cécile; Dupret, Jean-Marie; Chomienne, Christine; Ait-Si-Ali, Slimane; Rodrigues-Lima, Fernando

    2015-10-01

    CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

  4. Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression

    PubMed Central

    Sharma, Priyanka; Azebi, Saliha; England, Patrick; Christensen, Tove; Møller-Larsen, Anné; Petersen, Thor; Batsché, Eric; Muchardt, Christian

    2012-01-01

    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. PMID:23028349

  5. Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

    PubMed

    Schübeler, D; Lorincz, M C; Cimbora, D M; Telling, A; Feng, Y Q; Bouhassira, E E; Groudine, M

    2000-12-01

    We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

  6. Genomic Targeting of Methylated DNA: Influence of Methylation on Transcription, Replication, Chromatin Structure, and Histone Acetylation

    PubMed Central

    Schübeler, Dirk; Lorincz, Matthew C.; Cimbora, Daniel M.; Telling, Agnes; Feng, Yong-Quing; Bouhassira, Eric E.; Groudine, Mark

    2000-01-01

    We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure. PMID:11094062

  7. Histone lysine trimethylation or acetylation can be modulated by phytoestrogen, estrogen or anti-HDAC in breast cancer cell lines.

    PubMed

    Dagdemir, Aslihan; Durif, Julie; Ngollo, Marjolaine; Bignon, Yves-Jean; Bernard-Gallon, Dominique

    2013-02-01

    The isoflavones genistein, daidzein and equol (daidzein metabolite) have been reported to interact with epigenetic modifications, specifically hypermethylation of tumor suppressor genes. The objective of this study was to analyze and understand the mechanisms by which phytoestrogens act on chromatin in breast cancer cell lines. Two breast cancer cell lines, MCF-7 and MDA-MB 231, were treated with genistein (18.5 µM), daidzein (78.5 µM), equol (12.8 µM), 17β-estradiol (10 nM) and suberoylanilide hydroxamic acid (1 µM) for 48 h. A control with untreated cells was performed. 17β-estradiol and an anti-HDAC were used to compare their actions with phytoestrogens. The chromatin immunoprecipitation coupled with quantitative PCR was used to follow soy phytoestrogen effects on H3 and H4 histones on H3K27me3, H3K9me3, H3K4me3, H4K8ac and H3K4ac marks, and we selected six genes (EZH2, BRCA1, ERα, ERβ, SRC3 and P300) for analysis. Soy phytoestrogens induced a decrease in trimethylated marks and an increase in acetylating marks studied at six selected genes. We demonstrated that soy phytoestrogens tend to modify transcription through the demethylation and acetylation of histones in breast cancer cell lines.

  8. Homeobox genes gain trimethylation of histone H3 lysine 4 in glioblastoma tissue

    PubMed Central

    Luo, Kun; Luo, Donghui; Wen, Hao

    2016-01-01

    Glioblastoma multiforme (GBM) exhibits considerable heterogeneity and associates with genome-wide alterations of the repressed chromatin marks DNA methylation and H3 lysine 27 trimethylation (H3K27me3). Tri-methylation on lysine 4 of histone H3 (H3K4me3) is an activating epigenetic mark that is enriched at promoter and promotes expression. It will be helpful in GBM diagnosis and treatment to identify the alteration of H3K4me3 between human GBM and GBM-surrounding tissues. Here, we performed an analysis using next-generation sequencing techniques to identify H3K4me3 modification in a case of GBM and the GBM-surrounding tissues. The results revealed a global decrease in H3K4me3 in GBM, especially at promoters and CpG islands. In GBM, homeobox genes gain H3K4me3, whereas the cell–cell adhesion-related cadherin genes lose H3K4me3. The products of the homeobox genes are highly connected with Ras-signalling and PI3K-Akt signalling pathways. Using The Cancer Genome Atlas (TCGA) data, we inferred the homeobox-regulated genes’ expression is higher in 548 GBM cases than in 27 lower grade glioma cases giving that OLIG2 expression can be a reference. The results suggested that the H3K4me3 alteration is related to the formation and migration of GBM cells. We also found an extremely high reads count at epidermal growth factor receptor (EGFR) promoter, probably due to an amplification of copy number. Our analysis provides a case study about the change of H3K4me3 during shift to GBM. PMID:27160082

  9. The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

    PubMed Central

    de la Barre, Anne-Elisabeth; Angelov, Dimitri; Molla, Annie; Dimitrov, Stefan

    2001-01-01

    We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed ‘the ready production label’ was suggested to explain the role of histone H3 phosphorylation during cell division. PMID:11707409

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

    PubMed

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

    2011-08-23

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice.

    PubMed

    Itzhak, Yossef; Anderson, Karen L; Kelley, Jonathan B; Petkov, Martin

    2012-05-01

    Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. 2-Bromopalmitate modulates neuronal differentiation through the regulation of histone acetylation.

    PubMed

    Chen, Xueran; Du, Zhaoxia; Shi, Wei; Wang, Chen; Yang, Yang; Wang, Fen; Yao, Yao; He, Kun; Hao, Aijun

    2014-03-01

    In order to evaluate the functional significance of palmitoylation during multi-potent neural stem/progenitor cell proliferation and differentiation, retinoic acid-induced P19 cells were used in this study as a model system. Cell behaviour was monitored in the presence of the protein palmitoylation inhibitor 2-bromopalmitate (2BP). Here, we observed a significant reduction in neuronal differentiation in the 2BP-treated cell model. We further explored the underlying mechanisms and found that 2BP resulted in the decreased acetylation of histones H3 and H4 and interfered with cell cycle withdrawal and neural stem/progenitor cells' renewal. Our results established a direct link between palmitoylation and the regulation of neural cell fate specification and revealed the epigenetic regulatory mechanisms that are involved in the effects of palmitoylation during neural development. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

  17. The Catalytic Mechanism of Histone Acetyltransferase p300: From the Proton Transfer to Acetylation Reaction

    PubMed Central

    Zhang, Xinlei; Ouyang, Sisheng; Kong, Xiangqian; Liang, Zhongjie; Lu, Junyan; Zhu, Kongkai; Zhao, Dan; Zheng, Mingyue; Jiang, Hualiang; Liu, Xin; Marmorstein, Ronen; Luo, Cheng

    2014-01-01

    The transcriptional coactivator and histone acetyltransferase (HAT) p300 acetylates the four core histones and other transcription factors to regulate a plethora of fundamental biological processes including cell growth, development, oncogenesis and apoptosis. Recent structural and biochemical studies on the p300 HAT domain revealed a Theorell-Chance, or “hit-and-run”, catalytic mechanism. Nonetheless, the chemical mechanism of the entire reaction process including the proton transfer (PT) scheme and consequent acetylation reaction route remains unclear. In this study, a combined computational strategy consisting of molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation was applied to elucidate these important issues. An initial p300/H3/Ac-CoA complex structure was modeled and optimized using a 100 ns MD simulation. Residues that play important roles in substrate binding and the acetylation reaction were comprehensively investigated. For the first time, these studies reveal a plausible PT scheme consisting of Y1394, D1507 and a conserved crystallographic water molecule, with all components of the scheme being stable during the MD simulation and the energy barrier low for PT to occur. The two-dimensional potential energy surface for the nucleophilic attack process was also calculated. The comparison of potential energies for two possible elimination half-reaction mechanisms revealed that Y1467 reprotonates the coenzyme-A leaving group to form product. This study provides new insights into the detailed catalytic mechanism of p300 and has important implications for the discovery of novel small molecule regulators for p300. PMID:24521098

  18. Changes in histone acetylation in the prefrontal cortex of ethanol-exposed adolescent rats are associated with ethanol-induced place conditioning.

    PubMed

    Pascual, María; Do Couto, Bruno R; Alfonso-Loeches, Silvia; Aguilar, Maria A; Rodriguez-Arias, Marta; Guerri, Consuelo

    2012-06-01

    Alcohol drinking during adolescence can induce long-lasting effects on the motivation to consume alcohol. Abnormal plasticity in reward-related processes might contribute to the vulnerability of adolescents to drug addiction. We have shown that binge-like ethanol treatment in adolescent rats induces alterations in the dopaminergic system and causes histone modifications in brain reward regions. Considering that histone acetylation regulates transcriptional activity and contributes to drug-induced alterations in gene expression and behavior, we addressed the hypothesis that ethanol is capable of inducing transcriptional changes by histone modifications in specific gene promoters in adolescent brain reward regions, and whether these events are associated with acquisition of place conditioning. After treating juvenile and adult rats with intermittent ethanol administration, we found that ethanol treatment upregulates histone acetyl transferase (HAT) activity in adolescent prefrontal cortex and increases histone (H3 or H4) acetylation and H3(K4) dimethylation in the promoter region of cFos, Cdk5 and FosB. Inhibition of histone deacetylase by sodium butyrate before ethanol injection enhances both up-regulation of HAT activity and histone acetylation of cFos, Cdk5 and FosB. Furthermore, co-administration of sodium butyrate with ethanol prolongs the extinction of conditioned place aversion and increased the reinstatement effects of ethanol in ethanol-treated adolescents, but not in ethanol-treated adult rats. These results indicate that ethanol exposure during adolescence induces chromatin remodeling, changes histone acetylation and methylation, and modify the effects of ethanol on place conditioning. They also suggest that epigenetic mechanisms might open up avenues to new treatments for binge drinking-induced drug addiction during adolescence. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. WERAM: a database of writers, erasers and readers of histone acetylation and methylation in eukaryotes.

    PubMed

    Xu, Yang; Zhang, Shuang; Lin, Shaofeng; Guo, Yaping; Deng, Wankun; Zhang, Ying; Xue, Yu

    2017-01-04

    In this work, we developed a database WERAM (http://weram.biocuckoo.org/) for histone acetyltransferases, histone deacetylases, histone methyltransferases, histone demethylases and acetyl- or methyl-binding proteins, which catalyze, remove and recognize histone acetylation and methylation sites as 'writers', 'erasers' and 'readers', and synergistically determine the 'histone code'. From the scientific literature, we totally collected over 580 experimentally identified histone regulators from eight model organisms, including Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe and Saccharomyces cerevisiae We also collected ∼900 site-specific regulator-histone relations from the eight species. According to the experimental evidence, known histone regulators were classified into distinct families. To computationally detect more proteins in eukaryotes, we constructed hidden Markov model (HMM) profiles for histone regulator families. For families without HMM profiles, we also conducted orthologous searches. Totally, WERAM database contained more than 20 thousand non-redundant histone regulators from 148 eukaryotes. The detailed annotations and classification information of histone regulators were provided, together with site-specific histone substrates if available. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    PubMed

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

    2016-03-01

    Traffic-related air pollution (TRAP) has been acknowledged as a potential risk factor for numerous respiratory disorders including lung cancer; however, the exact mechanisms involved are still unclear. Here we investigated the effects of TRAP exposure on the H3K9 acetylation in rats. The exposure was performed in both spring and autumn with identical study procedures. In each season, 48 healthy Wistar rats were exposed to different levels of TRAP for 4 h, 7 d, 14 d, and 28 d, respectively. H3K9 acetylation levels in both the peripheral blood mononuclear cells (PBMCs) and lung tissues were quantified. Multiple linear regression was applied to assess the influence of air pollutants on H3K9 acetylation levels. The levels of PM2.5, PM10, and NO2 in the tunnel and crossroad groups were significantly higher than in the control group. The H3K9 acetylation levels were not significantly different between spring and autumn. When spring and autumn data were analyzed together, no significant association between the TRAP and H3K9 acetylation was found in 4h exposure window. However, in the 7 d exposure window, PM2.5 and PM10 exposures were associated with changes in H3K9 acetylation ranging from 0.276 (0.053, 0.498) to 0.475 (0.103, 0.848) per 1 μg/m(3) increase in the pollutant concentration. In addition, prolonged exposure of the rats in the tunnel showed that both PM2.5 and PM10 concentrations were positively associated with H3k9 acetylation in both PBMCs and lung tissues. The findings showed that 7-d and prolonged TRAP exposure could effectively increase the H3K9 acetylation level in both PBMCs and lung tissues of rats. Copyright © 2016 Elsevier B.V. All rights reserved.