Science.gov

Sample records for human histone modification

  1. HHMD: the human histone modification database.

    PubMed

    Zhang, Yan; Lv, Jie; Liu, Hongbo; Zhu, Jiang; Su, Jianzhong; Wu, Qiong; Qi, Yunfeng; Wang, Fang; Li, Xia

    2010-01-01

    Histone modifications play important roles in chromatin remodeling, gene transcriptional regulation, stem cell maintenance and differentiation. Alterations in histone modifications may be linked to human diseases especially cancer. Histone modifications including methylation, acetylation and ubiquitylation probed by ChIP-seq, ChIP-chip and qChIP have become widely available. Mining and integration of histone modification data can be beneficial to novel biological discoveries. There has been no comprehensive data repository that is exclusive for human histone modifications. Therefore, we developed a relatively comprehensive database for human histone modifications. Human Histone Modification Database (HHMD, http://bioinfo.hrbmu.edu.cn/hhmd) focuses on the storage and integration of histone modification datasets that were obtained from laboratory experiments. The latest release of HHMD incorporates 43 location-specific histone modifications in human. To facilitate data extraction, flexible search options are built in HHMD. It can be searched by histone modification, gene ID, functional categories, chromosome location and cancer name. HHMD also includes a user-friendly visualization tool named HisModView, by which genome-wide histone modification map can be shown. HisModView facilitates the acquisition and visualization of histone modifications. The database also has manually curated information of histone modification dysregulation in nine human cancers.

  2. Histone modifications for human epigenome analysis.

    PubMed

    Kimura, Hiroshi

    2013-07-01

    Histones function both positively and negatively in the regulation of gene expression, mainly governed by post-translational modifications on specific amino acid residues. Although histone modifications are not necessarily prerequisite codes, they may still serve as good epigenetic indicators of chromatin state associated with gene activation or repression. In particular, six emerging classes of histone H3 modifications are subjected for epigenome profiling by the International Human Epigenome Consortium. In general, transcription start sites of actively transcribed genes are marked by trimethylated H3K4 (H3K4me3) and acetylated H3K27 (H3K27ac), and active enhancers can be identified by enrichments of both monomethylated H3K4 (H3K4me1) and H3K27ac. Gene bodies of actively transcribed genes are associated with trimethylated H3K36 (H3K36me3). Gene repression can be mediated through two distinct mechanisms involving trimethylated H3K9 (H3K9me3) and trimethylated H3K27 (H3K27me3). Enrichments of these histone modifications on specific loci, or in genome wide, in given cells can be analyzed by chromatin immunoprecipitation (ChIP)-based methods using an antibody directed against the site-specific modification. When performing ChIP experiments, one should be careful about the specificity of antibody, as this affects the data interpretation. If cell samples with preserved histone-DNA contacts are available, evaluation of histone modifications, in addition to DNA methylaion, at specific gene loci would be useful for deciphering the epigenome state for human genetics studies.

  3. Biotinylation is a natural, albeit rare, modification of human histones

    PubMed Central

    Kuroishi, Toshinobu; Rios-Avila, Luisa; Pestinger, Valerie; Wijeratne, Subhashinee S. K.; Zempleni, Janos

    2011-01-01

    Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites. PMID:21930408

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

    PubMed

    Krejčí, Jana; Stixová, Lenka; Pagáčová, Eva; Legartová, Soňa; Kozubek, Stanislav; Lochmanová, Gabriela; Zdráhal, Zbyněk; Sehnalová, Petra; Dabravolski, Siarhei; Hejátko, Jan; Bártová, Eva

    2015-10-01

    We examined the levels and distribution of post-translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM-FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM-FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non-integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC-MS/MS and MALDI-TOF MS analysis. Taken together, these data indicate that particular post-translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual.

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

    PubMed Central

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

    2006-01-01

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

  6. Exercise-induced histone modifications in human skeletal muscle.

    PubMed

    McGee, Sean L; Fairlie, Erin; Garnham, Andrew P; Hargreaves, Mark

    2009-12-15

    Skeletal muscle adaptations to exercise confer many of the health benefits of physical activity and occur partly through alterations in skeletal muscle gene expression. The exact mechanisms mediating altered skeletal muscle gene expression in response to exercise are unknown. However, in recent years, chromatin remodelling through epigenetic histone modifications has emerged as a key regulatory mechanism controlling gene expression in general. The purpose of this study was to examine the effect of exercise on global histone modifications that mediate chromatin remodelling and transcriptional activation in human skeletal muscle in response to exercise. In addition, we sought to examine the signalling mechanisms regulating these processes. Following 60 min of cycling, global histone 3 acetylation at lysine 9 and 14, a modification associated with transcriptional initiation, was unchanged from basal levels, but was increased at lysine 36, a site associated with transcriptional elongation. We examined the regulation of the class IIa histone deacetylases (HDACs), which are enzymes that suppress histone acetylation and have been implicated in the adaptations to exercise. While we found no evidence of proteasomal degradation of the class IIa HDACs, we found that HDAC4 and 5 were exported from the nucleus during exercise, thereby removing their transcriptional repressive function. We also observed activation of the AMP-activated protein kinase (AMPK) and the calcium-calmodulin-dependent protein kinase II (CaMKII) in response to exercise, which are two kinases that induce phosphorylation-dependent class IIa HDAC nuclear export. These data delineate a signalling pathway that might mediate skeletal muscle adaptations in response to exercise.

  7. Transcription factor binding predicts histone modifications in human cell lines

    PubMed Central

    Benveniste, Dan; Sonntag, Hans-Joachim; Sanguinetti, Guido; Sproul, Duncan

    2014-01-01

    Gene expression in higher organisms is thought to be regulated by a complex network of transcription factor binding and chromatin modifications, yet the relative importance of these two factors remains a matter of debate. Here, we show that a computational approach allows surprisingly accurate prediction of histone modifications solely from knowledge of transcription factor binding both at promoters and at potential distal regulatory elements. This accuracy significantly and substantially exceeds what could be achieved by using DNA sequence as an input feature. Remarkably, we show that transcription factor binding enables strikingly accurate predictions across different cell lines. Analysis of the relative importance of specific transcription factors as predictors of specific histone marks recapitulated known interactions between transcription factors and histone modifiers. Our results demonstrate that reported associations between histone marks and gene expression may be indirect effects caused by interactions between transcription factors and histone-modifying complexes. PMID:25187560

  8. Readers of histone modifications

    PubMed Central

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

    2011-01-01

    Histone modifications not only play important roles in regulating chromatin structure and nuclear processes but also can be passed to daughter cells as epigenetic marks. Accumulating evidence suggests that the key function of histone modifications is to signal for recruitment or activity of downstream effectors. Here, we discuss the latest discovery of histone-modification readers and how the modification language is interpreted. PMID:21423274

  9. The landscape of histone modifications across 1% of the human genome in five human cell lines

    PubMed Central

    Koch, Christoph M.; Andrews, Robert M.; Flicek, Paul; Dillon, Shane C.; Karaöz, Ulaş; Clelland, Gayle K.; Wilcox, Sarah; Beare, David M.; Fowler, Joanna C.; Couttet, Phillippe; James, Keith D.; Lefebvre, Gregory C.; Bruce, Alexander W.; Dovey, Oliver M.; Ellis, Peter D.; Dhami, Pawandeep; Langford, Cordelia F.; Weng, Zhiping; Birney, Ewan; Carter, Nigel P.; Vetrie, David; Dunham, Ian

    2007-01-01

    We generated high-resolution maps of histone H3 lysine 9/14 acetylation (H3ac), histone H4 lysine 5/8/12/16 acetylation (H4ac), and histone H3 at lysine 4 mono-, di-, and trimethylation (H3K4me1, H3K4me2, H3K4me3, respectively) across the ENCODE regions. Studying each modification in five human cell lines including the ENCODE Consortium common cell lines GM06990 (lymphoblastoid) and HeLa-S3, as well as K562, HFL-1, and MOLT4, we identified clear patterns of histone modification profiles with respect to genomic features. H3K4me3, H3K4me2, and H3ac modifications are tightly associated with the transcriptional start sites (TSSs) of genes, while H3K4me1 and H4ac have more widespread distributions. TSSs reveal characteristic patterns of both types of modification present and the position relative to TSSs. These patterns differ between active and inactive genes and in particular the state of H3K4me3 and H3ac modifications is highly predictive of gene activity. Away from TSSs, modification sites are enriched in H3K4me1 and relatively depleted in H3K4me3 and H3ac. Comparison between cell lines identified differences in the histone modification profiles associated with transcriptional differences between the cell lines. These results provide an overview of the functional relationship among histone modifications and gene expression in human cells. PMID:17567990

  10. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    SciTech Connect

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D.

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  11. Association analysis between the distributions of histone modifications and gene expression in the human embryonic stem cell.

    PubMed

    Su, Wen-Xia; Li, Qian-Zhong; Zuo, Yong-Chun; Zhang, Lu-Qiang

    2016-01-01

    It is well known that histone modifications are associated with gene expression. In order to further study this relationship, 16 kinds of Chip-seq histone modification data and mRNA-seq data of the human embryonic stem cell H1 are chosen. The distributions of histone modifications in the regions flanking transcription start sites (TSSs) for highly expressed and lowly expressed genes are computed, respectively. And four types of distributions of histone modifications in regions flanking TSSs and the spatial patterning of the correlations between histone modifications and gene expression are detected. Our results suggest that the correlations between the regions overlapped by peaks are higher than the non-overlapped ones for each histone modification. In addition, to obtain the effect of the cooperative action of histone modification on gene expression, five histone modification clusters are found in highly expressed and lowly expressed genes, histone modification and gene expression interaction network is constructed. To further explore which region is the main target region for the specific histone modification, the human genes are divided into five functional regions. The results indicate that histone modifications are mostly located in the promoters of highly expressed genes versus the exons of lowly expressed genes, and exons have a smaller range of normalized tag counts than other gene elements in the two groups of genes. Finally, the type specificity and regional bias of histone modifications for 11 key transcription factor genes regulating the stem cell renewal are analyzed.

  12. LinkNMF: identification of histone modification modules in the human genome using nonnegative matrix factorization.

    PubMed

    Jung, Inkyung; Kim, Dongsup

    2013-04-10

    Histone modifications are ubiquitous processes involved in various cellular mechanisms. Systemic analysis of multiple chromatin modifications has been used to characterize various chromatin states associated with functional DNA elements, gene expression, and specific biological functions. However, identification of modular modification patterns is still required to understand the functional associations between histone modification patterns and specific chromatin/DNA binding factors. To recognize modular modification patterns, we developed a novel algorithm that combines nonnegative matrix factorization (NMF) and a clique-detection algorithm. We applied it, called LinkNMF, to generate a comprehensive modification map in human CD4+ T cell promoter regions. Initially, we identified 11 modules not recognized by conventional approaches. The modules were grouped into two major classes: gene activation and repression. We found that genes targeted by each module were enriched with distinguishable biological functions, suggesting that each modular pattern plays a unique functional role. To explain the formation of modular patterns, we investigated the module-specific binding patterns of chromatin regulators. Application of LinkNMF to histone modification maps of diverse cells and developmental stages will be helpful for understanding how histone modifications regulate gene expression. The algorithm is available on our website at biodb.kaist.ac.kr/LinkNMF. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Genome-wide analysis of histone modifications in human pancreatic islets.

    PubMed

    Bhandare, Reena; Schug, Jonathan; Le Lay, John; Fox, Alan; Smirnova, Olga; Liu, Chengyang; Naji, Ali; Kaestner, Klaus H

    2010-04-01

    The global diabetes epidemic poses a major challenge. Epigenetic events contribute to the etiology of diabetes; however, the lack of epigenomic analysis has limited the elucidation of the mechanistic basis for this link. To determine the epigenetic architecture of human pancreatic islets we mapped the genome-wide locations of four histone marks: three associated with gene activation-H3K4me1, H3K4me2, and H3K4me3-and one associated with gene repression, H3K27me3. Interestingly, the promoters of the highly transcribed insulin and glucagon genes are occupied only sparsely by H3K4me2 and H3K4me3. Globally, we identified important relationships between promoter structure, histone modification, and gene expression. We demonstrated co-occurrences of histone modifications including bivalent marks in mature islets. Furthermore, we found a set of promoters that is differentially modified between islets and other cell types. We also use our histone marks to determine which of the known diabetes-associated single-nucleotide polymorphisms are likely to be part of regulatory elements. Our global map of histone marks will serve as an important resource for understanding the epigenetic basis of type 2 diabetes.

  14. Distinct Histone Modifications Modulate DEFB1 Expression in Human Vaginal Keratinocytes in Response to Lactobacillus spp.

    PubMed

    Lee, Jaehyouk; Jang, Ara; Kim, Jin Wook; Han, Jun Hyun; Chun, Byung Hee; Jung, Hye Su; Jeon, Che Ok; Myung, Soon Chul

    2017-05-15

    Vaginal commensal lactobacilli are considered to contribute significantly to the control of vaginal microbiota by competing with other microflora for adherence to the vaginal epithelium and by producing antimicrobial compounds. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic communication in the vaginal ecosystem remain poorly understood. Here, we showed that both DNA methylation and histone modifications were associated with expression of the DEFB1 gene, which encodes the antimicrobial peptide human β-defensin-1, in vaginal keratinocyte VK2/E6E7 cells. We investigated whether exposure to Lactobacillus gasseri and Lactobacillus reuteri would trigger the epigenetic modulation of DEFB1 expression in VK2/E6E7 cells in a bacterial species-dependent manner. While enhanced expression of DEFB1 was observed when VK2/E6E7 cells were exposed to L. gasseri, treatment with L. reuteri resulted in reduced DEFB1 expression. Moreover, L. gasseri stimulated the recruitment of active histone marks and, in contrast, L. reuteri led to the decrease of active histone marks at the DEFB1 promoter. It was remarkable that distinct histone modifications within the same promoter region of DEFB1 were mediated by L. gasseri and L. reuteri. Therefore, our study suggested that one of the underlying mechanisms of DEFB1 expression in the vaginal ecosystem might be associated with the epigenetic crosstalk between individual Lactobacillus spp. and vaginal keratinocytes.

  15. DNA methylation-histone modification relationships across the desmin locus in human primary cells.

    PubMed

    Lindahl Allen, Marianne; Koch, Christoph M; Clelland, Gayle K; Dunham, Ian; Antoniou, Michael

    2009-05-27

    We present here an extensive epigenetic analysis of a 500 kb region, which encompasses the human desmin gene (DES) and its 5' locus control region (LCR), the only muscle-specific transcriptional regulatory element of this type described to date. These data complement and extend Encyclopaedia of DNA Elements (ENCODE) studies on region ENr133. We analysed histone modifications and underlying DNA methylation patterns in physiologically relevant DES expressing (myoblast/myotube) and non-expressing (peripheral blood mononuclear) primary human cells. We found that in expressing myoblast/myotube but not peripheral blood mononuclear cell (PBMC) cultures, histone H4 acetylation displays a broadly distributed enrichment across a gene rich 200 kb region whereas H3 acetylation localizes at the transcriptional start site (TSS) of genes. We show that the DES LCR and TSS of DES are enriched with hyperacetylated domains of acetylated histone H3, with H3 lysine 4 di- and tri-methylation (H3K4me2 and me3) exhibiting a different distribution pattern across this locus. The CpG island that extends into the first intron of DES is methylation-free regardless of the gene's expression status and in non-expressing PBMCs is marked with histone H3 lysine 27 tri-methylation (H3K27me3). Overall, our results constitute the first study correlating patterns of histone modifications and underlying DNA methylation of a muscle-specific LCR and its associated downstream gene region whilst additionally placing this within a much broader genomic context. Our results clearly show that there are distinct patterns of histone H3 and H4 acetylation and H3 methylation at the DES LCR, promoter and intragenic region. In addition, the presence of H3K27me3 at the DES methylation-free CpG only in non-expressing PBMCs may serve to silence this gene in non-muscle tissues. Generally, our work demonstrates the importance of using multiple, physiologically relevant tissue types that represent different expressing

  16. Estimating the effects of transcription factors binding and histone modifications on gene expression levels in human cells

    PubMed Central

    Zhang, Lu-Qiang; Li, Qian-Zhong

    2017-01-01

    Transcription factors and histone modifications are vital for the regulation of gene expression. Hence, to estimate the effects of transcription factors binding and histone modifications on gene expression, we construct a statistical model for the genome-wide 15 transcription factors binding data, 10 histone modifications profiles and DNase-I hypersensitivity data in three mammalian. Remarkably, our results show POLR2A and H3K36me3 can highly and consistently predict gene expression in three cell lines. And H3K4me3, H3K27me3 and H3K9ac are more reliable predictors than other histone modifications in human embryonic stem cells. Moreover, genome-wide statistical redundancies exist within and between transcription factors and histone modifications, and these phenomena may be caused by the regulation mechanism. In further study, we find that even though transcription factors and histone modifications offer similar effects on expression levels of genome-wide genes, the effects of transcription factors and histone modifications on predictive abilities are different for genes in independent biological processes. PMID:28454114

  17. Histone modifications in cancer biology and prognosis.

    PubMed

    Kurdistani, Siavash K

    2011-01-01

    Cancer is a disease of genome sequence alterations as well as epigenetic changes. Epigenetics refers in part to the mechanisms by which histones affect various DNA-based processes, such as gene regulation. Histones are proteins around which the DNA wraps itself to form chromatin--the physiologically relevant form of the human genome. Histones are modified extensively by posttranslational modifications that alter chromatin structure and serve to recruit to or exclude protein complexes from DNA. Aberrations in histone modifications occur frequently in cancer including changes in their levels and distribution at gene promoters, gene coding regions, repetitive DNA sequences, and other genomic elements. Locus-specific alterations in histone modifications may have adverse effects on expression of nearby genes but so far have not been shown to have clinical utility. Cancer cells also exhibit alterations in global levels of specific histone modifications, generating an additional layer of epigenetic heterogeneity at the cellular level in tumor tissues. Unlike locus-specific changes, the cellular epigenetic heterogeneity can be used to define previously unrecognized subsets of cancer patients with distinct clinical outcomes. In general, increased prevalence of cells with lower global levels of histone modifications is prognostic of poorer clinical outcome such as increased risk of tumor recurrence and/or decreased survival probability. Prognostic utility of histone modifications has been demonstrated independently for multiple cancers including those of prostate, lung, kidney, breast, ovary, and pancreas, suggesting a fundamental association between global histone modification levels and tumor aggressiveness, regardless of cancer tissue of origin. Cellular levels of histone modifications may also predict response to certain chemotherapeutic agents, serving as predictive biomarkers that could inform clinical decisions on choice and course of therapy. The challenge before us

  18. Chatting histone modifications in mammals

    PubMed Central

    Izzo, Annalisa

    2010-01-01

    Eukaryotic chromatin can be highly dynamic and can continuously exchange between an open transcriptionally active conformation and a compacted silenced one. Post-translational modifications of histones have a pivotal role in regulating chromatin states, thus influencing all chromatin dependent processes. Methylation is currently one of the best characterized histone modification and occurs on arginine and lysine residues. Histone methylation can regulate other modifications (e.g. acetylation, phosphorylation and ubiquitination) in order to define a precise functional chromatin environment. In this review we focus on histone methylation and demethylation, as well as on the enzymes responsible for setting these marks. In particular we are describing novel concepts on the interdependence of histone modifications marks and discussing the molecular mechanisms governing this cross-talks. PMID:21266346

  19. Histone code or not? Combinatorial pattern analyses of histone modifications

    NASA Astrophysics Data System (ADS)

    Zang, Chongzhi; Peng, Weiqun; Wang, Zhibin; Schones, Dustin E.; Barski, Artem; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Zhao, Keji; Rosenfeld, Jeffrey; Zhang, Michael

    2008-03-01

    Eukaryotic genomes are organized into chromatin, the structure of which plays critical role in the program of gene expression. Chromatin structure and function is regulated by a myriad of posttranslational modifications on histone tails of the nucleosomes, the fundamental unit of chromatin. It remains unclear how different modifications interact. Based on high- resolution genomic maps of close to 40 histone methylations and acetylations in human T-cells obtained experimentally by ChIP- Seq technique, we investigated the combinatorial patterns of histone modifications at gene promoter regions. We found that a very limited number of patterns dominate. Modifications within a pattern are strongly correlated and each pattern is associated with a distinct gene expression distribution. Our results suggest that it is the patterns rather than the individual modifications that affect the downstream readout.

  20. Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array

    PubMed Central

    Partolina, Marina; Thoms, Hazel C; MacLeod, Kenneth G; Rodriguez-Blanco, Giovanny; Clarke, Matthew N; Venkatasubramani, Anuroop V; Beesoo, Rima; Larionov, Vladimir; Neergheen-Bhujun, Vidushi S; Serrels, Bryan; Kimura, Hiroshi; Carragher, Neil O; Kagansky, Alexander

    2017-01-01

    The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2–24 h after inhibition of HDACs in different cancer cell lines

  1. Epigenetic Modifications of Histones in Periodontal Disease.

    PubMed

    Martins, M D; Jiao, Y; Larsson, L; Almeida, L O; Garaicoa-Pazmino, C; Le, J M; Squarize, C H; Inohara, N; Giannobile, W V; Castilho, R M

    2016-02-01

    Periodontitis is a chronic infectious disease driven by dysbiosis, an imbalance between commensal bacteria and the host organism. Periodontitis is a leading cause of tooth loss in adults and occurs in about 50% of the US population. In addition to the clinical challenges associated with treating periodontitis, the progression and chronic nature of this disease seriously affect human health. Emerging evidence suggests that periodontitis is associated with mechanisms beyond bacteria-induced protein and tissue degradation. Here, we hypothesize that bacteria are able to induce epigenetic modifications in oral epithelial cells mediated by histone modifications. In this study, we found that dysbiosis in vivo led to epigenetic modifications, including acetylation of histones and downregulation of DNA methyltransferase 1. In addition, in vitro exposure of oral epithelial cells to lipopolysaccharides resulted in histone modifications, activation of transcriptional coactivators, such as p300/CBP, and accumulation of nuclear factor-κB (NF-κB). Given that oral epithelial cells are the first line of defense for the periodontium against bacteria, we also evaluated whether activation of pathogen recognition receptors induced histone modifications. We found that activation of the Toll-like receptors 1, 2, and 4 and the nucleotide-binding oligomerization domain protein 1 induced histone acetylation in oral epithelial cells. Our findings corroborate the emerging concept that epigenetic modifications play a role in the development of periodontitis. © International & American Associations for Dental Research 2015.

  2. Histone modifications in zebrafish development.

    PubMed

    Cunliffe, V T

    2016-01-01

    Reversible covalent histone modifications are known to influence spatiotemporal patterns of gene transcription during development. Here I review recent advances in the development and use of methods to analyze the distribution and functions of histone modifications in zebrafish chromatin. I discuss the roles of dynamic histone modification patterns at the promoters and enhancers of genes during the process of zygotic gene activation at blastula stages and the interplay between the molecular machinery responsible for histone modifications, chromatin remodeling and DNA methylation. Interactions are also described between developmentally regulated enhancer sequences and modified histones. A detailed method for chromatin immunoprecipitation using antibodies is provided, and I describe the use of high-throughput whole genome sequencing technology to generate DNA sequence data from chromatin immunoprecipitates. I also discuss computational approaches to integrating DNA sequence data obtained from chromatin immunoprecipitates with annotated reference genome sequences, transcriptome and methylome sequence data, transcription factor binding motif databases, and gene ontologies and describe the types of software tools currently available for visualizing the results. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Exposure of Human Prostaspheres to Bisphenol A Epigenetically Regulates SNORD Family Noncoding RNAs via Histone Modification

    PubMed Central

    Cheong, Ana; Lam, Hung-Ming; Hu, Wen-Yang; Shi, Guang-Bin; Zhu, Xuegong; Chen, Jing; Zhang, Xiang; Medvedovic, Mario; Leung, Yuet-Kin; Prins, Gail S.

    2015-01-01

    Bisphenol A (BPA) is a ubiquitous endocrine disruptor exerting lifelong effects on gene expression in rodent prostate cancer (PCa) models. Here, we aimed to determine whether epigenetic events mediating the action of BPA on human prostaspheres enriched in epithelial stem-like/progenitor cells is linked to PCa. We performed genome-wide transcriptome and methylome analyses to identify changes in prostaspheres treated with BPA (10nM, 200nM, and 1000nM) or estradiol-17β (E2) (0.1nM) for 7 days and validated changes in expression, methylation, and histone marks in parallel-treated prostaspheres. BPA/E2-treatment altered expression of 91 genes but not the methylation status of 485 000 CpG sites in BPA/E2-treated prostaspheres. A panel of 26 genes was found repressed in all treatment groups. Fifteen of them were small nucleolar RNAs with C/D motif (SNORDs), which are noncoding, small nucleolar RNAs known to regulate ribosomal RNA assembly and function. Ten of the most down-regulated SNORDs were further studied. All 10 were confirmed repressed by BPA, but only 3 ratified as E2-repressed. SNORD suppression showed no correlation with methylation status changes in CpG sites in gene regulatory regions. Instead, BPA-induced gene silencing was found to associate with altered recruitments of H3K9me3, H3K4me3, and H3K27me3 to 5′-regulatory/exonic sequences of 5 SNORDs. Expression of 4 out of these 5 SNORDs (SNORD59A, SNORD82, SNORD116, and SNORD117) was shown to be reduced in PCa compared with adjacent normal tissue. This study reveals a novel and unique action of BPA in disrupting expression of PCa-associated SNORDs and a putative mechanism for reprogramming the prostasphere epigenome via histone modification. PMID:26248216

  4. Dynamics of Post-Translational Modifications on Human Histone H4 Through the Cell Cycle

    DTIC Science & Technology

    2006-08-11

    Pesavento – my mentor, teacher, and go-to guy. Jim, because of you I now know the ins and outs of ECD, manuscript writing, and vegetarian food in CU. We...JJ., Bullock, CR., Mizzen, CA., Kelleher, NL. (2006) Top Down Mass Spectrometric Analyses of Human Histone H4 in Human Cancer Cells and

  5. Histone modifications and traditional Chinese medicinals

    PubMed Central

    2013-01-01

    Background Chromatin, residing in the nuclei of eukaryotic cells, comprises DNA and histones to make up chromosomes. Chromatin condenses to compact the chromosomes and loosens to facilitate gene transcription and DNA replication/repair. Chemical modifications to the histones mediate changes in chromatin structure. Histone-modifying enzymes are potential drug targets. How herbs affect phenotypes through histone modifications is interesting. Methods Two public traditional Chinese medicine (TCM) databases were accessed to retrieve the chemical constituents and TCM natures of 3,294 TCM medicinals. NCBI taxonomy database was accessed to build the phylogenetic tree of the TCM medicinals. Statistical test was used to test if TCM natures of the medicinals cluster in the phylogenetic tree. A public chemical-protein interaction database was accessed to identify TCM medicinals whose constituent chemicals interact with human histone-modifying enzymes. For each histone modification, a correlation coefficient was calculated between the medicinals’ TCM natures and modification modulabilities. Information of the ingredient medicinals of 200 classical TCM formulas was accessed from a public database. Results It was found that 1,170 or 36% of the 3,294 TCM medicinals interact with human histone-modifying enzymes. Among the histone-modifying medicinals, 56% of them promote chromatin condensation. The cold-hot natures of TCM medicinals were found to be phylogenetically correlated. Furthermore, cold (hot) TCM medicinals were found to be associated with heterochromatinization (euchromatinization) through mainly H3K9 methylation and H3K4 demethylation. The associations were weak yet statistically significant. On the other hand, analysis of TCM formulas, the major form of TCM prescriptions in clinical practice, found that 99% of 200 government approved TCM formulas are histone-modifying. Furthermore, in formula formation, heterochromatic medicinals were found to team up with other

  6. Control of human cytomegalovirus gene expression by differential histone modifications during lytic and latent infection of a monocytic cell line.

    PubMed

    Ioudinkova, Elena; Arcangeletti, Maria Cristina; Rynditch, Alla; De Conto, Flora; Motta, Federica; Covan, Silvia; Pinardi, Federica; Razin, Sergey V; Chezzi, Carlo

    2006-12-15

    Non-differentiated THP-1 cells can be infected by human cytomegalovirus (HCMV) Towne strain, which persists in these cells in a non-active (latent) form without undergoing a productive cycle. The same cells become permissive for HCMV lytic infection after induction of cell differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. We used this cellular model to study the possible role of histone modifications in the control of HCMV latency. Using chromatin immunoprecipitation with antibodies against histone H3 acetylated or dimethylated in position K9, we demonstrated that in lytically infected cells the HCMV enhancer was associated with heavy acetylated but not dimethylated H3. In the case of latent infection, the HCMV enhancer was associated with neither acetylated nor dimethylated H3. HCMV genes encoding DNA polymerase (early), pp65 (early-late) and pp150 (late) proteins were associated preferentially with acetylated H3 in lytically infected cells and with dimethylated H3 in latently infected cells. These data strongly suggest that K9 methylation of H3 is involved in HCMV gene repression, while association of the above genes with acetylated histones is likely to be necessary for active transcription. It can be postulated that the same histone modifications are used to mark active and repressed genes in both cellular and viral chromatin.

  7. The patterns of histone modifications in the vicinity of transcription factor binding sites in human lymphoblastoid cell lines.

    PubMed

    Nie, Yumin; Liu, Hongde; Sun, Xiao

    2013-01-01

    Transcription factor (TF) binding at specific DNA sequences is the fundamental step in transcriptional regulation and is highly dependent on the chromatin structure context, which may be affected by specific histone modifications and variants, known as histone marks. The lack of a global binding map for hundreds of TFs means that previous studies have focused mainly on histone marks at binding sites for several specific TFs. We therefore studied 11 histone marks around computationally-inferred and experimentally-determined TF binding sites (TFBSs), based on 164 and 34 TFs, respectively, in human lymphoblastoid cell lines. For H2A.Z, methylation of H3K4, and acetylation of H3K27 and H3K9, the mark patterns exhibited bimodal distributions and strong pairwise correlations in the 600-bp region around enriched TFBSs, suggesting that these marks mainly coexist within the two nucleosomes proximal to the TF sites. TFs competing with nucleosomes to access DNA at most binding sites, contributes to the bimodal distribution, which is a common feature of histone marks for TF binding. Mark H3K79me2 showed a unimodal distribution on one side of TFBSs and the signals extended up to 4000 bp, indicating a longer-distance pattern. Interestingly, H4K20me1, H3K27me3, H3K36me3 and H3K9me3, which were more diffuse and less enriched surrounding TFBSs, showed unimodal distributions around the enriched TFBSs, suggesting that some TFs may bind to nucleosomal DNA. Besides, asymmetrical distributions of H3K36me3 and H3K9me3 indicated that repressors might establish a repressive chromatin structure in one direction to repress gene expression. In conclusion, this study demonstrated the ranges of histone marks associated with TF binding, and the common features of these marks around the binding sites. These findings have epigenetic implications for future analysis of regulatory elements.

  8. Combinations of Histone Modifications for Pattern Genes.

    PubMed

    Cui, Xiang-Jun; Shi, Chen-Xia

    2016-06-01

    Histone post-translational modifications play important roles in transcriptional regulation. It is known that multiple histone modifications can act in a combinatorial manner. In this study, we investigated the effects of multiple histone modifications on expression levels of five gene categories (four kinds of pattern genes and non-pattern genes) in coding regions. The combinatorial patterns of modifications for the five gene categories were also studied in the regions. Our results indicated that the differences in the expression levels between any two gene categories were significant. There were some corresponding differences in multiple histone modification levels among the five gene categories. Multiple histone modifications jointly impacted expression levels of every gene category. Four mutual combinations of histone modifications were found and analyzed.

  9. Human linker histones: interplay between phosphorylation and O-β-GlcNAc to mediate chromatin structural modifications

    PubMed Central

    2011-01-01

    Eukaryotic chromatin is a combination of DNA and histone proteins. It is established fact that epigenetic mechanisms are associated with DNA and histones. Initial studies emphasize on core histones association with DNA, however later studies prove the importance of linker histone H1 epigenetic. There are many types of linker histone H1 found in mammals. These subtypes are cell specific and their amount in different types of cells varies as the cell functions. Many types of post-translational modifications which occur on different residues in each subtype of linker histone H1 induce conformational changes and allow the different subtypes of linker histone H1 to interact with chromatin at different stages during cell cycle which results in the regulation of transcription and gene expression. Proposed O-glycosylation of linker histone H1 promotes condensation of chromatin while phosphorylation of linker histone H1 is known to activate transcription and gene regulation by decondensation of chromatin. Interplay between phosphorylation and O-β-GlcNAc modification on Ser and Thr residues in each subtype of linker histone H1 in Homo sapiens during cell cycle may result in diverse functional regulation of proteins. This in silico study describes the potential phosphorylation, o-glycosylation and their possible interplay sites on conserved Ser/Thr residues in various subtypes of linker histone H1 in Homo sapiens. PMID:21749719

  10. Coordination of cell signaling, chromatin remodeling, histone modifications, and regulator recruitment in human matrix metalloproteinase 9 gene transcription.

    PubMed

    Ma, Zhendong; Shah, Reesha C; Chang, Mi Jung; Benveniste, Etty N

    2004-06-01

    Transcriptional activation of eukaryotic genes depends on the precise and ordered recruitment of activators, chromatin modifiers/remodelers, coactivators, and general transcription factors to the promoters of target genes. Using the human matrix metalloproteinase 9 (MMP-9) gene as a model system, we investigated the sequential assembly and dynamic formation of transcription complexes on a human promoter under the influence of mitogen signaling. We find that, coincident with activation of the MMP-9 gene, activators, chromatin remodeling complexes, and coactivators are recruited to the preassembled MMP-9 promoter in a stepwise and coordinated order, which is dependent on activation of MEK-1/extracellular signal-regulated kinase and NF-kappa B signaling pathways. Conversely, corepressor complexes are released from the MMP-9 promoter after transcriptional activation. Histone modifications shift from repressive to permissive modifications concurrent with activation of the MMP-9 gene. Chromatin remodeling induced by Brg-1 is required for MMP-9 gene transcription, which is concomitant with initiation of transcription. Therefore, coordination of cell signaling, chromatin remodeling, histone modifications, and stepwise recruitment of transcription regulators is critical to precisely regulate MMP-9 gene transcription in a temporally and spatially dependent manner. Given the important role of MMP-9 in both normal development and pathological conditions, understanding MMP-9 gene regulation is of great relevance.

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

    PubMed Central

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

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  12. Modeling exon expression using histone modifications.

    PubMed

    Zhu, Shijia; Wang, Guohua; Liu, Bo; Wang, Yadong

    2013-01-01

    Histones undergo numerous covalent modifications that play important roles in regulating gene expression. Previous investigations have focused on the effects of histone modifications on gene promoters, whereas efforts to unravel their effects on transcribed regions have lagged behind. To elucidate the effects of histone modification on transcribed regions, we constructed a quantitative model, which we suggest can predict the variation of gene expression more faithfully than the model constructed on promoters. Moreover, motivated by the fact that exon spicing is functionally coupled to transcription, we also devised a quantitative model to predict alternative exon expression using histone modifications on exons. This model was found to be general across different exon types and even cell types. Furthermore, an interaction network linking histone modifications to alternative exon expression was constructed using partial correlations. The network indicated that gene expression and specific histone modifications (H3K36me3 and H4K20me1) could directly influence the exon expression, while other modifications could act in an additive way to account for the stability and robustness. In addition, our results suggest that combinations of histone modifications contribute to exon splicing in a redundant and cumulative fashion. To conclude, this study provides a better understanding of the effects of histone modifications on gene transcribed regions.

  13. Modeling Exon Expression Using Histone Modifications

    PubMed Central

    Zhu, Shijia; Wang, Guohua; Liu, Bo; Wang, Yadong

    2013-01-01

    Histones undergo numerous covalent modifications that play important roles in regulating gene expression. Previous investigations have focused on the effects of histone modifications on gene promoters, whereas efforts to unravel their effects on transcribed regions have lagged behind. To elucidate the effects of histone modification on transcribed regions, we constructed a quantitative model, which we suggest can predict the variation of gene expression more faithfully than the model constructed on promoters. Moreover, motivated by the fact that exon spicing is functionally coupled to transcription, we also devised a quantitative model to predict alternative exon expression using histone modifications on exons. This model was found to be general across different exon types and even cell types. Furthermore, an interaction network linking histone modifications to alternative exon expression was constructed using partial correlations. The network indicated that gene expression and specific histone modifications (H3K36me3 and H4K20me1) could directly influence the exon expression, while other modifications could act in an additive way to account for the stability and robustness. In addition, our results suggest that combinations of histone modifications contribute to exon splicing in a redundant and cumulative fashion. To conclude, this study provides a better understanding of the effects of histone modifications on gene transcribed regions. PMID:23825663

  14. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

    PubMed Central

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J.; Rogowska-Wrzesinska, Adelina; Jensen, Ole N.

    2015-01-01

    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3. PMID:26424599

  15. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns.

    PubMed

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J; Rogowska-Wrzesinska, Adelina; Jensen, Ole N

    2015-12-01

    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

  16. Histone Modifications and Nuclear Architecture: A Review

    PubMed Central

    Bártová, Eva; Krejčí, Jana; Harničarová, Andrea; Galiová, Gabriela; Kozubek, Stanislav

    2008-01-01

    Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei. (J Histochem Cytochem 56:711–721, 2008) PMID:18474937

  17. Bivalent histone modifications in stem cells poise miRNA loci for CpG island hypermethylation in human cancer.

    PubMed

    Iliou, Maria S; Lujambio, Amaia; Portela, Anna; Brüstle, Oliver; Koch, Philipp; Andersson-Vincent, Per Henrik; Sundström, Erik; Hovatta, Outi; Esteller, Manel

    2011-11-01

    It has been proposed that the existence of stem cell epigenetic patterns confer a greater likelihood of CpG island hypermethylation on tumor suppressor-coding genes in cancer. The suggested mechanism is based on the Polycomb-mediated methylation of K27 of histone H3 and the recruitment of DNA methyltransferases on the promoters of tumor suppressor genes in cancer cells, when those genes are preferentially pre-marked in embryonic stem cells (ESCs) with bivalent chromatin domains. On the other hand, miRNAs appear to be dysregulated in cancer, with many studies reporting silencing of miRNA genes due to aberrant hypermethylation of their promoter regions. We wondered whether a pre-existing histone modification profile in stem cells might also contribute to the DNA methylation-associated silencing of miRNA genes in cancer. To address this, we examined a group of tumor suppressor miRNA genes previously reported to become hypermethylated and inactivated specifically in cancer cells. We analyzed the epigenetic events that take place along their promoters in human embryonic stem cells and in transformed cells. Our results suggest that there is a positive correlation between the existence of bivalent chromatin domains on miRNA promoters in ESCs and the hypermethylation of those genes in cancer, leading us to conclude that this epigenetic mark could be a mechanism that prepares miRNA promoters for further DNA hypermethylation in human tumors.

  18. Histone modifications in DNA damage response.

    PubMed

    Cao, Lin-Lin; Shen, Changchun; Zhu, Wei-Guo

    2016-03-01

    DNA damage is a relatively common event in eukaryotic cell and may lead to genetic mutation and even cancer. DNA damage induces cellular responses that enable the cell either to repair the damaged DNA or cope with the damage in an appropriate way. Histone proteins are also the fundamental building blocks of eukaryotic chromatin besides DNA, and many types of post-translational modifications often occur on tails of histones. Although the function of these modifications has remained elusive, there is ever-growing studies suggest that histone modifications play vital roles in several chromatin-based processes, such as DNA damage response. In this review, we will discuss the main histone modifications, and their functions in DNA damage response.

  19. Alterations of histone modifications by cobalt compounds

    PubMed Central

    Li, Qin; Ke, Qingdong; Costa, Max

    2009-01-01

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

  20. Balancing chromatin remodeling and histone modifications in transcription

    PubMed Central

    Petty, Emily; Pillus, Lorraine

    2013-01-01

    Chromatin remodelers use the energy of ATP hydrolysis to reposition or evict nucleosomes or to replace canonical histones with histone variants. By regulating nucleosome dynamics, remodelers gate access to the underlying DNA for replication, repair, and transcription. Nucleosomes are subject to extensive post-translational modifications that can recruit regulatory proteins or alter the local chromatin structure. Just as extensive cross-talk has been observed between different histone post-translational modifications, there is growing evidence for both coordinated and antagonistic functional relationships between nucleosome remodeling and modifying machineries. Defining the combined functions of the complexes that alter nucleosome interactions, position, and stability is key to understanding processes that require access to DNA, particularly with growing appreciation of their contributions to human health and disease. Here, we highlight recent advances in the interactions between histone modifications and the ISWI and CHD1 chromatin remodelers from studies in budding yeast, fission yeast, flies, and mammalian cells, with a focus on yeast. PMID:23870137

  1. Detection of histone modifications in plant leaves.

    PubMed

    Jaskiewicz, Michal; Peterhansel, Christoph; Conrath, Uwe

    2011-09-23

    Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles(1-2). H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues(1-2). These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)(3-7). Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde(8,9), extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies(9,10), de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C(4;) photosynthesis in maize(5,11) and systemic immunity in Arabidopsis(3).

  2. Histone Post-Translation Modifications Influence Chromatin Mechanical Stability

    NASA Astrophysics Data System (ADS)

    Poirier, Michael

    2011-03-01

    Histone proteins organize the human genome into chromatin fibers while their post-translation modification (PTM) regulates genome replication, expression and repair. The mechanistic connections between histone PTMs and biological functions remain enigmatic. We find with a combination of magnetic tweezers mechanical measurements and biochemical studies that a number of histone PTMs influence the DNA mismatch repair process by mechanically destabilizing chromatin. The location of the PTM within the chromatin structure appears to determine the mechanism by which it alters the mechanical stability. These findings have direct implications for understanding the repair of the human genome.

  3. Bivalent histone modifications in early embryogenesis.

    PubMed

    Vastenhouw, Nadine L; Schier, Alexander F

    2012-06-01

    Histone modifications influence the interactions of transcriptional regulators with chromatin. Studies in embryos and embryonic stem (ES) cells have uncovered histone modification patterns that are diagnostic for different cell types and developmental stages. For example, bivalent domains consisting of regions of H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 4 trimethylation (H3K4me3) mark lineage control genes in ES cells and zebrafish blastomeres. Such bivalent domains have garnered attention because the H3K27me3 mark might help repress lineage-regulatory genes during pluripotency while the H3K4me3 mark could poise genes for activation upon differentiation. Despite the prominence of the bivalent domain concept, studies in other model organisms have questioned its universal nature, and the function of bivalent domains has remained unclear. Histone marks are also associated with developmental regulatory genes in sperm. These observations have raised the possibility that specific histone modification patterns might persist from parent to offspring, but it is unclear whether histone marks are inherited or formed de novo. Here, we review the potential roles of H3K4me3 and H3K27me3 marks in embryos and ES cells and discuss how histone marks might be established, maintained and resolved during embryonic development.

  4. DNA methylation and histone modifications cause silencing of Wnt antagonist gene in human renal cell carcinoma cell lines.

    PubMed

    Kawamoto, Ken; Hirata, Hiroshi; Kikuno, Nobuyuki; Tanaka, Yuichiro; Nakagawa, Masayuki; Dahiya, Rajvir

    2008-08-01

    Secreted frizzled-related protein 2 (sFRP2) is a negative modulator of the Wingless-type (Wnt) signaling pathway, and shown to be inactivated in renal cell carcinoma (RCC). However, the molecular mechanism of silencing of sFRP2 is not fully understood. Our study was designed to elucidate the silencing mechanism of sFRP2 in RCC. Expression of sFRP2 was examined in 20 pairs of primary cancers by immunohistochemistry. Kidney cell lines (HK-2, Caki-1, Caki-2, A-498 and ACHN) were analyzed for sFRP2 expression using real-time RT-PCR and Western blotting. The methylation status at 46 CpG sites of the 2 CpG islands in the sFRP2 promoter was characterized by bisulfite DNA sequencing. Histone modifications were assessed by chromatin immunoprecipitation (ChIP) assay using antibodies against AcH3, AcH4, H3K4 and H3K9. sFRP2 was frequently repressed in primary cancers and in RCC cells. The majority of sFRP2 negative cells had a methylated promoter. Meanwhile, sFRP2 expression was repressed by a hypomethylated promoter in Caki-1 cells, and these cells had a repressive histone modification at the promoter. In Caki-1 cells, sFRP2 was reactivated by trichostatin A (TSA). Repressive histone modifications were also observed in RCC cells with hypermethylated promoters, but sFRP2 was reactivated only by 5-aza-2'-deoxycytidine (DAC) and not by TSA. However, the activation of the silenced sFRP2 gene could be achieved in all cells using a combination of DAC and TSA. This is the first report indicating that aberrant DNA methylation and histone modifications work together to silence the sFRP2 gene in RCC cells.

  5. CR Cistrome: a ChIP-Seq database for chromatin regulators and histone modification linkages in human and mouse.

    PubMed

    Wang, Qixuan; Huang, Jinyan; Sun, Hanfei; Liu, Jing; Wang, Juan; Wang, Qian; Qin, Qian; Mei, Shenglin; Zhao, Chengchen; Yang, Xiaoqin; Liu, X Shirley; Zhang, Yong

    2014-01-01

    Diversified histone modifications (HMs) are essential epigenetic features. They play important roles in fundamental biological processes including transcription, DNA repair and DNA replication. Chromatin regulators (CRs), which are indispensable in epigenetics, can mediate HMs to adjust chromatin structures and functions. With the development of ChIP-Seq technology, there is an opportunity to study CR and HM profiles at the whole-genome scale. However, no specific resource for the integration of CR ChIP-Seq data or CR-HM ChIP-Seq linkage pairs is currently available. Therefore, we constructed the CR Cistrome database, available online at http://compbio.tongji.edu.cn/cr and http://cistrome.org/cr/, to further elucidate CR functions and CR-HM linkages. Within this database, we collected all publicly available ChIP-Seq data on CRs in human and mouse and categorized the data into four cohorts: the reader, writer, eraser and remodeler cohorts, together with curated introductions and ChIP-Seq data analysis results. For the HM readers, writers and erasers, we provided further ChIP-Seq analysis data for the targeted HMs and schematized the relationships between them. We believe CR Cistrome is a valuable resource for the epigenetics community.

  6. Modeling the dynamics of bivalent histone modifications.

    PubMed

    Ku, Wai Lim; Girvan, Michelle; Yuan, Guo-Cheng; Sorrentino, Francesco; Ott, Edward

    2013-01-01

    Epigenetic modifications to histones may promote either activation or repression of the transcription of nearby genes. Recent experimental studies show that the promoters of many lineage-control genes in stem cells have "bivalent domains" in which the nucleosomes contain both active (H3K4me3) and repressive (H3K27me3) marks. It is generally agreed that bivalent domains play an important role in stem cell differentiation, but the underlying mechanisms remain unclear. Here we formulate a mathematical model to investigate the dynamic properties of histone modification patterns. We then illustrate that our modeling framework can be used to capture key features of experimentally observed combinatorial chromatin states.

  7. Genistein affects histone modifications on Dickkopf-related protein 1 (DKK1) gene in SW480 human colon cancer cell line.

    PubMed

    Wang, Huan; Li, Qian; Chen, Hong

    2012-01-01

    Genistein (GEN) is a plant-derived isoflavone and can block uncontrolled cell growth in colon cancer by inhibiting the WNT signaling pathway. This study aimed to test the hypothesis that the enhanced gene expression of the WNT signaling pathway antagonist, DKK1 by genistein treatment is associated with epigenetic modifications of the gene in colon cancer cells. Genistein treatment induced a concentration-dependent G2 phase arrest in the human colon cancer cell line SW480 and reduced cell proliferation. Results from several other human colon cancer cell lines confirmed the growth inhibitory effects of genistein. Overexpression of DKK1 confirmed its involvement in growth inhibition. Knockdown of DKK1 expression by siRNA slightly induced cell growth. DKK1 gene expression was increased by genistein in SW480 and HCT15 cells. DNA methylation at the DKK1 promoter was not affected by genistein treatment in all the cell lines tested. On the other hand, genistein induced histone H3 acetylation of the DKK1 promoter region in SW480 and HCT15 cells. This indicates that increased histone acetylation is associated with the genistein-induced DKK1 expression. The association between histone acetylation and DKK1 gene expression is confirmed by the histone deacetylase inhibitor trichostatin A (TSA) treatment. In conclusion, genistein treatment decreases cell growth and proliferation in colon cancer cell lines. The effect is associated with the increased DKK1 expression through the induction of histone acetylation at the DKK1 promoter region.

  8. Comprehensive Catalog of Currently Documented Histone Modifications.

    PubMed

    Zhao, Yingming; Garcia, Benjamin A

    2015-09-01

    Modern techniques in molecular biology, genomics, and mass spectrometry-based proteomics have identified a large number of novel histone posttranslational modifications (PTMs), many of whose functions are still under intense investigation. Here, we catalog histone PTMs under two classes: first, those whose functions have been fairly well studied and, second, those PTMs that have been more recently identified but whose functions remain unclear. We hope that this will be a useful resource for researchers from all biological or technical backgrounds, aiding in their chromatin and epigenetic pursuits. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  9. EGFR promoter exhibits dynamic histone modifications and binding of ASH2L and P300 in human germinal matrix and gliomas.

    PubMed

    Erfani, Parsa; Tome-Garcia, Jessica; Canoll, Peter; Doetsch, Fiona; Tsankova, Nadejda M

    2015-01-01

    Several signaling pathways important for the proliferation and growth of brain cells are pathologically dysregulated in gliomas, including the epidermal growth factor receptor (EGFR). Expression of EGFR is high in neural progenitors during development and in gliomas but decreases significantly in most adult brain regions. Here we show that EGFR expression is maintained in the astrocyte ribbon of the adult human subventricular zone. The transcriptional regulation of EGFR expression is poorly understood. To investigate the role of epigenetics on EGFR regulation in the contexts of neural development and gliomagenesis, we measured levels of DNA methylation and histone H3 modifications at the EGFR promoter in human brain tissues, glioma specimens, and EGFR-expressing neural cells, acutely isolated from their native niche. While DNA was constitutively hypomethylated in non-neoplastic and glioma samples, regardless of their EGFR-expression status, the activating histone modifications H3K27ac and H3K4me3 were enriched only when EGFR is highly expressed (developing germinal matrix and gliomas). Conversely, repressive H3K27me3 marks predominated in adult white matter where EGFR is repressed. Furthermore, the histone methyltransferase core enzyme ASH2L was bound at EGFR in the germinal matrix and in gliomas where levels of H3K4me3 are high, and the histone acetyltransferase P300 was bound in samples with H3K27ac enrichment. Our studies use human cells and tissues undisturbed by cell-culture artifact, and point to an important, locus-specific role for chromatin remodeling in EGFR expression in human neural development that may be dysregulated during gliomagenesis, unraveling potential novel targets for future drug therapy.

  10. EGFR promoter exhibits dynamic histone modifications and binding of ASH2L and P300 in human germinal matrix and gliomas

    PubMed Central

    Erfani, Parsa; Tome-Garcia, Jessica; Canoll, Peter; Doetsch, Fiona; Tsankova, Nadejda M

    2015-01-01

    Several signaling pathways important for the proliferation and growth of brain cells are pathologically dysregulated in gliomas, including the epidermal growth factor receptor (EGFR). Expression of EGFR is high in neural progenitors during development and in gliomas but decreases significantly in most adult brain regions. Here we show that EGFR expression is maintained in the astrocyte ribbon of the adult human subventricular zone. The transcriptional regulation of EGFR expression is poorly understood. To investigate the role of epigenetics on EGFR regulation in the contexts of neural development and gliomagenesis, we measured levels of DNA methylation and histone H3 modifications at the EGFR promoter in human brain tissues, glioma specimens, and EGFR-expressing neural cells, acutely isolated from their native niche. While DNA was constitutively hypomethylated in non-neoplastic and glioma samples, regardless of their EGFR-expression status, the activating histone modifications H3K27ac and H3K4me3 were enriched only when EGFR is highly expressed (developing germinal matrix and gliomas). Conversely, repressive H3K27me3 marks predominated in adult white matter where EGFR is repressed. Furthermore, the histone methyltransferase core enzyme ASH2L was bound at EGFR in the germinal matrix and in gliomas where levels of H3K4me3 are high, and the histone acetyltransferase P300 was bound in samples with H3K27ac enrichment. Our studies use human cells and tissues undisturbed by cell-culture artifact, and point to an important, locus-specific role for chromatin remodeling in EGFR expression in human neural development that may be dysregulated during gliomagenesis, unraveling potential novel targets for future drug therapy. PMID:25996283

  11. Histone modification profiling reveals differential signatures associated with human embryonic stem cell self-renewal and differentiation

    PubMed Central

    Bhanu, Natarajan V.; Sidoli, Simone; Garcia, Benjamin A.

    2016-01-01

    In this study, we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy, qPCR and flow cytometry, we classified the treatment outcome as inducing pluripotency (hESC, flurbiprofen and gatifloxacin), mesendoderm (sinomenine), differentiation (cyamarin, digoxin, digitoxin, selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions, the above classification was reassorted. Hyperacetylation at H3K4, 9, 14, 18, 56 and 122 as well as H4K5, 8, 12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9, K20, K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to “differentiation initiators”, consistent with flow cytometry where it induced mesendoderm, along with cyamarin and possibly selegnine. Neurectoderm, induced by RA and theanine manifested methylations on H3 shifts to H3.3. By both flow cytometry and histone PTM clustering, it appears that cells treated with gatifloxacin, flurbiprofen, digitoxin and digoxin were not yet lineage-committed or mixed cell types. Taken together, our moderate-throughput histone PTM profiling approach highlighted subtle epigenetic signatures that permitted us to predict divergent lineage progression even in differentiating cells with similar phenotype and gene expression. PMID:26631989

  12. Bivalent histone modifications during tooth development.

    PubMed

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

    2014-12-01

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

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

    PubMed

    Hassan, Yousef I; Zempleni, Janos

    2008-12-01

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

  14. 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.

  15. The apolipoprotein CIII enhancer regulates both extensive histone modification and intergenic transcription of human apolipoprotein AI/CIII/AIV genes but not apolipoprotein AV.

    PubMed

    Li, Ya-Jun; Wei, Yu-Sheng; Fu, Xiang-Hui; Hao, De-Long; Xue, Zheng; Gong, Huan; Zhang, Zhu-Qin; Liu, De-Pei; Liang, Chih-Chuan

    2008-10-17

    The apolipoprotein (apo) AI/CIII/AIV/AV cluster genes are expressed at different levels in the liver and intestine. The apoCIII enhancer, a common regulatory element, regulates the tissue-specific expression of apoAI, apoCIII, and apoAIV but not apoAV. To study this regulation at the chromatin level, the histone modifications and intergenic transcription in the human apoAI/CIII/AIV/AV cluster were investigated in HepG2 and Caco-2 cells and in the livers of transgenic mice carrying the human gene cluster constructs with or without the apoCIII enhancer. We found that both the promoters and the intergenic regions of the apoAI/CIII/AIV genes were hyperacetylated and formed an open subdomain that did not include the apoAV gene. Hepatic and intestinal intergenic transcripts were identified to transcribe bidirectionally with strand preferences along the cluster. The deletion of the apoCIII enhancer influenced both histone modification and intergenic transcription in the apoAI/CIII/AIV gene region. These results demonstrate that the apoCIII enhancer contributes to the maintenance of an active chromatin subdomain of the apoAI/CIII/AIV genes, but not apoAV.

  16. Epigenetic Control of Reprogramming and Transdifferentiation by Histone Modifications.

    PubMed

    Qin, Hua; Zhao, Andong; Zhang, Cuiping; Fu, Xiaobing

    2016-12-01

    Somatic cells can be reprogrammed to pluripotent stem cells or transdifferentiate to another lineage cell type. Much efforts have been made to unravel the epigenetic mechanisms underlying the cell fate conversion. Histone modifications as the major epigenetic regulator are implicated in various aspects of reprogramming and transdifferentiation. Here, we discuss the roles of histone modifications on reprogramming and transdifferentiation and hopefully provide new insights into induction and promotion of the cell fate conversion by modulating histone modifications.

  17. HIstome--a relational knowledgebase of human histone proteins and histone modifying enzymes.

    PubMed

    Khare, Satyajeet P; Habib, Farhat; Sharma, Rahul; Gadewal, Nikhil; Gupta, Sanjay; Galande, Sanjeev

    2012-01-01

    Histones are abundant nuclear proteins that are essential for the packaging of eukaryotic DNA into chromosomes. Different histone variants, in combination with their modification 'code', control regulation of gene expression in diverse cellular processes. Several enzymes that catalyze the addition and removal of multiple histone modifications have been discovered in the past decade, enabling investigations of their role(s) in normal cellular processes and diverse pathological conditions. This sudden influx of data, however, has resulted in need of an updated knowledgebase that compiles, organizes and presents curated scientific information to the user in an easily accessible format. Here, we present HIstome, a browsable, manually curated, relational database that provides information about human histone proteins, their sites of modifications, variants and modifying enzymes. HIstome is a knowledgebase of 55 human histone proteins, 106 distinct sites of their post-translational modifications (PTMs) and 152 histone-modifying enzymes. Entries have been grouped into 5 types of histones, 8 types of post-translational modifications and 14 types of enzymes that catalyze addition and removal of these modifications. The resource will be useful for epigeneticists, pharmacologists and clinicians. HIstome: The Histone Infobase is available online at http://www.iiserpune.ac.in/∼coee/histome/ and http://www.actrec.gov.in/histome/.

  18. Metabolic regulation of histone post-translational modifications

    PubMed Central

    Fan, Jing; Krautkramer, Kimberly A.; Feldman, Jessica L.; Denu, John M.

    2015-01-01

    Histone post-translational modifications regulate transcription and other DNA-templated functions. This process is dynamically regulated by specific modifying enzymes whose activities require metabolites that either serve as co-substrates or act as activators/inhibitors. Therefore, metabolism can influence histone modification by changing local concentrations of key metabolites. Physiologically, the epigenetic response to metabolism is important for nutrient sensing and environment adaption. In pathologic states, the connection between metabolism and histone modification mediates epigenetic abnormality in complex disease. In this review, we summarize recent studies of the molecular mechanisms involved in metabolic regulation of histone modifications and discuss their biological significance. PMID:25562692

  19. Specific histone modification responds to arsenic-induced oxidative stress.

    PubMed

    Ma, Lu; Li, Jun; Zhan, Zhengbao; Chen, Liping; Li, Daochuan; Bai, Qing; Gao, Chen; Li, Jie; Zeng, Xiaowen; He, Zhini; Wang, Shan; Xiao, Yongmei; Chen, Wen; Zhang, Aihua

    2016-07-01

    To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P<0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showed that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β=0.16; P=0.042, H3K18ac: β=-0.24; P=0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO2 treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Histone modifications in the male germ line of Drosophilaa

    PubMed Central

    2013-01-01

    Background In the male germ line of Drosophila chromatin remains decondensed and highly transcribed during meiotic prophase until it is rapidly compacted. A large proportion of the cell cycle-regulated histone H3.1 is replaced by H3.3, a histone variant encoded outside the histone repeat cluster and not subject to cell cycle controlled expression. Results We investigated histone modification patterns in testes of D. melanogaster and D. hydei. In somatic cells of the testis envelope and in germ cells these modification patterns differ from those typically seen in eu- and heterochromatin of other somatic cells. During the meiotic prophase some modifications expected in active chromatin are not found or are found at low level. The absence of H4K16ac suggests that dosage compensation does not take place. Certain histone modifications correspond to either the cell cycle-regulated histone H3.1 or to the testis-specific variant H3.3. In spermatogonia we found H3K9 methylation in cytoplasmic histones, most likely corresponding to the H3.3 histone variant. Most histone modifications persist throughout the meiotic divisions. The majority of modifications persist until the early spermatid nuclei, and only a minority further persist until the final chromatin compaction stages before individualization of the spermatozoa. Conclusion Histone modification patterns in the male germ line differ from expected patterns. They are consistent with an absence of dosage compensation of the X chromosome during the male meiotic prophase. The cell cycle-regulated histone variant H3.1 and H3.3, expressed throughout the cell cycle, also vary in their modification patterns. Postmeiotically, we observed a highly complex pattern of the histone modifications until late spermatid nuclear elongation stages. This may be in part due to postmeiotic transcription and in part to differential histone replacement during chromatin condensation. PMID:23433182

  1. Theoretical framework for the histone modification network: modifications in the unstructured histone tails form a robust scale-free network.

    PubMed

    Hayashi, Yohei; Senda, Toshiya; Sano, Norihiko; Horikoshi, Masami

    2009-07-01

    A rapid increase in research on the relationship between histone modifications and their subsequent reactions in the nucleus has revealed that the histone modification system is complex, and robust against point mutations. The prevailing theoretical framework (the histone code hypothesis) is inadequate to explain either the complexity or robustness, making the formulation of a new theoretical framework both necessary and desirable. Here, we develop a model of the regulatory network of histone modifications in which we encode histone modifications as nodes and regulatory interactions between histone modifications as links. This network has scale-free properties and subnetworks with a pseudo-mirror symmetry structure, which supports the robustness of the histone modification network. In addition, we show that the unstructured tail regions of histones are suitable for the acquisition of this scale-free property. Our model and related insights provide the first framework for an overall architecture of a histone modification network system, particularly with regard to the structural and functional roles of the unstructured histone tail region. In general, the post-translational "modification webs" of natively unfolded regions (proteins) may function as signal routers for the robust processing of the large amounts of signaling information.

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

    PubMed

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

    2010-05-01

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

  3. Production and Purification of Antibodies Against Histone Modifications.

    PubMed

    Guillemette, Benoit; Hammond-Martel, Ian; Wurtele, Hugo; Verreault, Alain

    2017-01-01

    Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

  4. Targeting post-translational modifications of histones for cancer therapy.

    PubMed

    Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H

    2015-10-30

    Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment.

  5. Genetically encoding lysine modifications on histone H4.

    PubMed

    Wilkins, Bryan J; Hahn, Liljan E; Heitmüller, Svenja; Frauendorf, Holm; Valerius, Oliver; Braus, Gerhard H; Neumann, Heinz

    2015-04-17

    Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.

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

    PubMed

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

    2015-10-06

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

  7. Genome-Wide Profiling of Histone Modifications and Histone Variants in Arabidopsis thaliana and Marchantia polymorpha.

    PubMed

    Yelagandula, Ramesh; Osakabe, Akihisa; Axelsson, Elin; Berger, Frederic; Kawashima, Tomokazu

    2017-01-01

    Histone modifications and histone variants barcode the genome and play major roles in epigenetic regulations. Chromatin immunoprecipitation (ChIP) coupled with next-generation sequencing (NGS) is a well-established method to investigate the landscape of epigenetic marks at a genomic level. Here, we describe procedures for conducting ChIP, subsequent NGS library construction, and data analysis on histone modifications and histone variants in Arabidopsis thaliana. We also describe an optimized nuclear isolation procedure to prepare chromatin for ChIP in the liverwort, Marchantia polymorpha, which is the emerging model plant ideal for evolutionary studies.

  8. Quantification of SAHA-Dependent Changes in Histone Modifications Using Data-Independent Acquisition Mass Spectrometry

    PubMed Central

    Krautkramer, Kimberly A.; Reiter, Lukas; Denu, John M.; Dowell, James A.

    2015-01-01

    Histone post-translational modifications (PTMs) are important regulators of chromatin structure and gene expression. Quantitative analysis of histone PTMs by mass spectrometry remains extremely challenging due to the complex and combinatorial nature of histone PTMs. The most commonly used mass spectrometry-based method for high-throughput histone PTM analysis is data-dependent acquisition (DDA). However, stochastic precursor selection and dependence on MS1 ions for quantification impede comprehensive interrogation of histone PTM states using DDA methods. To overcome these limitations, we utilized a data-independent acquisition (DIA) workflow that provides superior run-to-run consistency and post-acquisition flexibility in comparison to DDA methods. In addition, we developed a novel DIA-based methodology to quantify isobaric, co-eluting histone peptides that lack unique MS2 transitions. Our method enabled deconvolution and quantification of histone PTMs that are otherwise refractory to quantitation, including the heavily acetylated tail of histone H4. Using this workflow, we investigated the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) on the global histone PTM state of human breast cancer MCF7 cells. A total of 62 unique histone PTMs were quantified, revealing novel SAHA-induced changes in acetylation and methylation of histones H3 and H4. PMID:26120868

  9. Examining Histone Posttranslational Modification Patterns by High Resolution Mass Spectrometry

    PubMed Central

    Lin, Shu; Garcia, Benjamin A.

    2014-01-01

    Histone variants and posttranslational modifications (PTMs) are essential for epigenetic regulation of transcriptional expression. Single and/or combinatorial PTMs of histones play important roles in development and disease formation. Mass spectrometry (MS) has been a powerful tool to study histone variants and PTMs as it not only can identify novel PTMs but also can provide quantitative measurement of a spectrum of histone variants and PTMs in the same sample. In this chapter, we employ a combination of chemical derivation and high resolution mass spectrometry to identify and quantify multiple histone variants and PTMs. Histones are acid extracted and modified with propionyl groups, and subsequently produces suitable sizes of fragments for MS analysis by trypsin digestion. The newly generated N-termini of histone peptides can be differentially marked by stable isotope labeling in a second reaction of propionylation, which enables direct comparison between two different samples in the following MS analysis. PMID:22910200

  10. Lateral Thinking: How Histone Modifications Regulate Gene Expression.

    PubMed

    Lawrence, Moyra; Daujat, Sylvain; Schneider, Robert

    2016-01-01

    The DNA of each cell is wrapped around histone octamers, forming so-called 'nucleosomal core particles'. These histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing all DNA-based processes, including chromatin compaction, nucleosome dynamics, and transcription. In contrast to those present in histone tails, modifications in the core regions of the histones had remained largely uncharacterised until recently, when some of these modifications began to be analysed in detail. Overall, recent work has shown that histone core modifications can not only directly regulate transcription, but also influence processes such as DNA repair, replication, stemness, and changes in cell state. In this review, we focus on the most recent developments in our understanding of histone modifications, particularly those on the lateral surface of the nucleosome. This region is in direct contact with the DNA and is formed by the histone cores. We suggest that these lateral surface modifications represent a key insight into chromatin regulation in the cell. Therefore, lateral surface modifications form a key area of interest and a focal point of ongoing study in epigenetics. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Cancer Chemoprotection Through Nutrient-mediated Histone Modifications

    PubMed Central

    Gao, Yifeng; Tollefsbol, Trygve O.

    2016-01-01

    Epigenetics, the study of heritable changes in gene expression without modifying the nucleotide sequence, is among the most important topics in medicinal chemistry and cancer chemoprotection. Among those changes, DNA methylation and histone modification have been shown to be associated with various types of cancers in a number of ways, many of which are regulated by dietary components that are mostly found in plants. Although, mechanisms of nutrient components affecting histone acetylation/deacetylation in cancer are widely studied, how those natural compounds affect cancer through other histone modifications, such as methylation, phosphorylation and ubiquitylation, is rarely reviewed. Thus, this review article discusses impacts recently studied on histone acetylation as well as other histone modifications by dietary components, such as genistein, resveratrol, curcumin, epigallocatechin-3-gallate (EGCG), 3,3′-diindolylmethane (DIM), diallyl disulfide, garcinol, procyanidin B3, quercetin, sulforaphane and other isothiocyanates, in various types of cancer. PMID:25891109

  12. Histone tail modifications and noncanonical functions of histones: perspectives in cancer epigenetics.

    PubMed

    Hadnagy, Annamaria; Beaulieu, Raymond; Balicki, Danuta

    2008-04-01

    Over the past few years, the histone deacetylase (HDAC) inhibitors have occupied an important place in the effort to develop novel, but less toxic, anticancer therapy. HDAC inhibitors block HDACs, which are the enzymes responsible for histone deacetylation, and therefore they modulate gene expression. The cellular effects of HDAC inhibitors include growth arrest and the induction of differentiation. Early successes in cancer therapeutics obtained using these drugs alone or in combination with other anticancer drugs emphasize the important place of posttranslational modifications of histones in cancer therapy. Histone tail modifications along with DNA methylation are the most studied epigenetic events related to cancer progression. Moreover, extranuclear functions of histones have also been described. Because HDAC inhibitors block HDACs and thereby increase histone acetylation, we propose a model wherein exogenous acetylated histones or other related acetylated proteins that are introduced into the nucleus become HDAC substrates and thereby compete with endogenous histones for HDACs. This competition may lead to the increased acetylation of the endogenous histones, as in the case of HDAC inhibitor therapy. Moreover, other mechanisms of action, such as binding to chromatin and modulating gene expression, are also possible for exogenously introduced histones.

  13. Histone deacetylases: salesmen and customers in the post-translational modification market.

    PubMed

    Brandl, André; Heinzel, Thorsten; Krämer, Oliver H

    2009-04-01

    HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N-epsilon-acetylated lysine residues in histones and non-histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post-translational modification. Such structural alterations can determine the stability, localization, activity and protein-protein interactions of HDACs. This subsequently affects the modification of their substrates and the co-ordination of cellular signalling networks. Intriguingly, physiologically relevant non-histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post-translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post-translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.

  14. Complex Exon-Intron Marking by Histone Modifications Is Not Determined Solely by Nucleosome Distribution

    PubMed Central

    Chiang, Kelly; Bonhoure, Nicolas; Koch, Christoph M.; Bye, Jackie; James, Keith; Foad, Nicola S.; Ellis, Peter; Watkins, Nicholas A.; Ouwehand, Willem H.; Langford, Cordelia; Andrews, Robert M.; Dunham, Ian; Vetrie, David

    2010-01-01

    It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing. PMID:20808788

  15. Regulation of human papillomavirus type 31 gene expression during the differentiation-dependent life cycle through histone modifications and transcription factor binding.

    PubMed

    Wooldridge, Tonia R; Laimins, Laimonis A

    2008-05-10

    The life cycle of high-risk human papillomaviruses is linked to epithelial differentiation with virion production restricted to highly differentiated suprabasal cells. Two major viral promoters direct high-risk HPV gene expression and their activities are dependent upon differentiation. The early promoter controls initiation of transcripts at sites upstream of the E6 open reading frame and is active in both undifferentiated as well as differentiated cells. The late viral promoter directs transcription from a series of heterogeneous start sites in E7 and is activated upon differentiation. In this study, the state of histones as well as the spectrum of transcription factors bound to the two major HPV 31 viral promoters in undifferentiated and differentiated cells were examined using chromatin immunoprecipitation assays. Our studies indicate that, in undifferentiated cells, the chromatin surrounding both promoter regions is in an open, transcriptionally active state as indicated by the presence of dimethylated forms of histone H3 K4 as well as acetylated H3 and acetylated H4. Upon differentiation, there was an increase of four to six fold in the levels of dimethylated H3K4 and acetylated H3 respectively around both promoter regions as well as an increase of approximately nine fold in acetylated H4 at the early promoter. This suggests that nucleosomes of both promoter regions are further activated through histone modifications during differentiation. Chromatin immunoprecipitation assays were also used to examine the binding of transcription factors to the keratinocyte enhancer (KE)/early promoter region in the upstream regulatory region (URR) and late promoter sequences throughout differentiation. Our results suggest that a dynamic change in transcription factor binding occurs in both regions upon differentiation; most notably a significant increase in C/EBP-beta binding to the KE/early promoter region as well as C/EBP-alpha binding to the late promoter region upon

  16. Clusterin gene is predominantly regulated by histone modifications in human colon cancer and ectopic expression of the nuclear isoform induces cell death.

    PubMed

    Deb, Moonmoon; Sengupta, Dipta; Rath, Sandip Kumar; Kar, Swayamsiddha; Parbin, Sabnam; Shilpi, Arunima; Pradhan, Nibedita; Bhutia, Sujit Kumar; Roy, Subhendu; Patra, Samir Kumar

    2015-08-01

    Clusterin (CLU) is an important glycoprotein involved in various cellular functions. Different reports have mentioned that the two isoforms of CLU; secretary (sCLU) and nuclear (nCLU) have opposite (paradoxical) roles in cancer development. sCLU provides pro-survival signal, whereas nCLU is involved in pro-apoptotic signaling. However, the molecular mechanism of CLU gene regulation is not clear as of yet. We hypothesize that CLU gene is regulated by DNA methylation and histone modifications and clusterin plays an important role in colon cancer. To evaluate the hypothesis, we investigated CLU expression in colon cancer tissues and DNA methylation and histone modification status of CLU gene promoter. It is apparent from immonohistology data that both benign and cancerous (primary and metastasis) formalin fixed paraffin embedded (FFPE) tissue samples exhibit CLU expression. However and interestingly only noncancerous tissue samples show nCLU expression. Ectopic expression of nCLU either by epigenetic modulators or by nCLU transfection is responsible for colon cancer cell death. To clarify the molecular mechanisms for regulation of expression of CLU isoforms, we have analyzed DNA methylation and histone modifications, such as histone H3K9me3, H3K27me3, H3K4me3, and H3K9AcS10P patterns around the CLU promoter. There is no remarkable change in the DNA methylation status upon treatment of the cells by AZA, TSA and SAM. Our findings clearly show that promoter histone H3K9me3 and H3K27me3 marks are elevated in comparison to H3K4me3 and H3K9AcS10P marks in colon cancer cell lines. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Physicochemical modifications of histones and their impact on epigenomics.

    PubMed

    Andreoli, Federico; Del Rio, Alberto

    2014-09-01

    The study of histone post-translational modifications (PTMs) has made extraordinary progress over the past few years and many epigenetic modifications have been identified and found to be associated with fundamental biological processes and pathological conditions. Most histone-modifying enzymes produce specific covalent modifications on histone tails that, taken together, elicit complex and concerted processes. An even higher level of complexity is generated by the action of small molecules that are able to modulate pharmacologically epigenetic enzymes and interfere with these biochemical mechanisms. In this article, we provide an overview of histone PTMs by reviewing and discussing them in terms of their physicochemical properties, emphasizing these concepts in view of recent research efforts to elucidate epigenetic mechanisms and devise future epigenetic drugs.

  18. Long Noncoding RNA as Modular Scaffold of Histone Modification Complexes

    PubMed Central

    Tsai, Miao-Chih; Manor, Ohad; Wan, Yue; Mosammaparast, Nima; Wang, Jordon K.; Lan, Fei; Shi, Yang; Segal, Eran; Chang, Howard Y.

    2010-01-01

    Long intergenic noncoding RNAs (lincRNAs) regulate chromatin states and epigenetic inheritance. Here we show that the lincRNA HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5′ domain of HOTAIR binds Polycomb Repressive Complex 2 (PRC2) while a 3′ domain of HOTAIR binds the LSD1/CoREST/REST complex. The ability to tether two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1, and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation. Our results suggest that lincRNAs may serve as scaffolds by providing binding surfaces to assemble select histone modification enzymes, and thereby specify the pattern of histone modifications on target genes. PMID:20616235

  19. Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

    PubMed

    Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-15

    establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes.

    PubMed

    Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2010-10-01

    Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone-replacement system provides a well-defined and direct cellular assay system for histone function with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism.

  1. The relationship between gene transcription and combinations of histone modifications

    NASA Astrophysics Data System (ADS)

    Cui, Xiangjun; Li, Hong; Luo, Liaofu

    2012-09-01

    Histone modification is an important subject of epigenetics which plays an intrinsic role in transcriptional regulation. It is known that multiple histone modifications act in a combinatorial fashion. In this study, we demonstrated that the pathways within constructed Bayesian networks can give an indication for the combinations among 12 histone modifications which have been studied in the TSS+1kb region in S. cerevisiae. After Bayesian networks for the genes with high transcript levels (H-network) and low transcript levels (L-network) were constructed, the combinations of modifications within the two networks were analyzed from the view of transcript level. The results showed that different combinations played dissimilar roles in the regulation of gene transcription when there exist differences for gene expression at transcription level.

  2. Histone modifications induced by a family of bacterial toxins.

    PubMed

    Hamon, Mélanie Anne; Batsché, Eric; Régnault, Béatrice; Tham, To Nam; Seveau, Stéphanie; Muchardt, Christian; Cossart, Pascale

    2007-08-14

    Upon infection, pathogens reprogram host gene expression. In eukaryotic cells, genetic reprogramming is induced by the concerted activation/repression of transcription factors and various histone modifications that control DNA accessibility in chromatin. We report here that the bacterial pathogen Listeria monocytogenes induces a dramatic dephosphorylation of histone H3 as well as a deacetylation of histone H4 during early phases of infection. This effect is mediated by the major listerial toxin listeriolysin O in a pore-forming-independent manner. Strikingly, a similar effect also is observed with other toxins of the same family, such as Clostridium perfringens perfringolysin and Streptococcus pneumoniae pneumolysin. The decreased levels of histone modifications correlate with a reduced transcriptional activity of a subset of host genes, including key immunity genes. Thus, control of epigenetic regulation emerges here as an unsuspected function shared by several bacterial toxins, highlighting a common strategy used by intracellular and extracellular pathogens to modulate the host response early during infection.

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

    PubMed

    Swygert, Sarah G; Peterson, Craig L

    2014-08-01

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

  4. histoneHMM: Differential analysis of histone modifications with broad genomic footprints.

    PubMed

    Heinig, Matthias; Colomé-Tatché, Maria; Taudt, Aaron; Rintisch, Carola; Schafer, Sebastian; Pravenec, Michal; Hubner, Norbert; Vingron, Martin; Johannes, Frank

    2015-02-22

    ChIP-seq has become a routine method for interrogating the genome-wide distribution of various histone modifications. An important experimental goal is to compare the ChIP-seq profiles between an experimental sample and a reference sample, and to identify regions that show differential enrichment. However, comparative analysis of samples remains challenging for histone modifications with broad domains, such as heterochromatin-associated H3K27me3, as most ChIP-seq algorithms are designed to detect well defined peak-like features. To address this limitation we introduce histoneHMM, a powerful bivariate Hidden Markov Model for the differential analysis of histone modifications with broad genomic footprints. histoneHMM aggregates short-reads over larger regions and takes the resulting bivariate read counts as inputs for an unsupervised classification procedure, requiring no further tuning parameters. histoneHMM outputs probabilistic classifications of genomic regions as being either modified in both samples, unmodified in both samples or differentially modified between samples. We extensively tested histoneHMM in the context of two broad repressive marks, H3K27me3 and H3K9me3, and evaluated region calls with follow up qPCR as well as RNA-seq data. Our results show that histoneHMM outperforms competing methods in detecting functionally relevant differentially modified regions. histoneHMM is a fast algorithm written in C++ and compiled as an R package. It runs in the popular R computing environment and thus seamlessly integrates with the extensive bioinformatic tool sets available through Bioconductor. This makeshistoneHMM an attractive choice for the differential analysis of ChIP-seq data. Software is available from http://histonehmm.molgen.mpg.de .

  5. Histone modifications silence the GATA transcription factor genes in ovarian cancer.

    PubMed

    Caslini, C; Capo-chichi, C D; Roland, I H; Nicolas, E; Yeung, A T; Xu, X-X

    2006-08-31

    Altered expression of GATA factors was found and proposed as the underlying mechanism for dedifferentiation in ovarian carcinogenesis. In particular, GATA6 is lost or excluded from the nucleus in 85% of ovarian tumors and GATA4 expression is absent in majority of ovarian cancer cell lines. Here, we evaluated their DNA and histone epigenetic modifications in five ovarian epithelial and carcinoma cell lines (human 'immortalized' ovarian surface epithelium (HIO)-117, HIO-114, A2780, SKOV3 and ES2). GATA4 and GATA6 gene silencing was found to correlate with hypoacetylation of histones H3 and H4 and loss of histone H3/lysine K4 tri-methylation at their promoters in all lines. Conversely, histone H3/lysine K9 di-methylation and HP1gamma association were not observed, excluding reorganization of GATA genes into heterochromatic structures. The histone deacetylase inhibitor trichostatin A, but not the DNA methylation inhibitor 5'-aza-2'-deoxycytidine, re-established the expression of GATA4 and/or GATA6 in A2780 and HIO-114 cells, correlating with increased histone H3 and H4 acetylation, histone H3 lysine K4 methylation and DNase I sensitivity at the promoters. Therefore, altered histone modification of the promoter loci is one mechanism responsible for the silencing of GATA transcription factors and the subsequent loss of a target gene, the tumor suppressor Disabled-2, in ovarian carcinogenesis.

  6. Histone modifications associated with cancer cell migration and invasion.

    PubMed

    Hieda, Miki; Matsuura, Nariaki; Kimura, Hiroshi

    2015-01-01

    Genome-wide aberrant histone modifications are present in a wide range of cancers, and they are associated with carcinogenesis and cancer progression. Aberrant histone modification patterns affect transcriptional regulation, chromosome stability, chromatin structure, chromatin remodeling, and DNA methylation; furthermore, these patterns can predict clinical outcome in many types of cancer. The main cause of poor clinical outcome is metastasis, which is strongly associated with tissue invasion at the primary tumor site. Invasion of cancer cells into surrounding tissue and the vasculature is an important initial step in tumor metastasis, and cell migration is a critical requirement for metastasis. Here, we describe the advantages of detecting global histone modifications by immunohistochemical analysis and provide a collection of protocols for assaying cell migration, invasion, and cell-extracellular matrix adhesion in vitro.

  7. The Histone Modification Code in the Pathogenesis of Autoimmune Diseases

    PubMed Central

    2017-01-01

    Autoimmune diseases are chronic inflammatory disorders caused by a loss of self-tolerance, which is characterized by the appearance of autoantibodies and/or autoreactive lymphocytes and the impaired suppressive function of regulatory T cells. The pathogenesis of autoimmune diseases is extremely complex and remains largely unknown. Recent advances indicate that environmental factors trigger autoimmune diseases in genetically predisposed individuals. In addition, accumulating results have indicated a potential role of epigenetic mechanisms, such as histone modifications, in the development of autoimmune diseases. Histone modifications regulate the chromatin states and gene transcription without any change in the DNA sequence, possibly resulting in phenotype alteration in several different cell types. In this paper, we discuss the significant roles of histone modifications involved in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, primary biliary cirrhosis, and type 1 diabetes. PMID:28127155

  8. Histones and histone modifications in perinuclear chromatin anchoring: from yeast to man.

    PubMed

    Harr, Jennifer C; Gonzalez-Sandoval, Adriana; Gasser, Susan M

    2016-02-01

    It is striking that within a eukaryotic nucleus, the genome can assume specific spatiotemporal distributions that correlate with the cell's functional states. Cell identity itself is determined by distinct sets of genes that are expressed at a given time. On the level of the individual gene, there is a strong correlation between transcriptional activity and associated histone modifications. Histone modifications act by influencing the recruitment of non-histone proteins and by determining the level of chromatin compaction, transcription factor binding, and transcription elongation. Accumulating evidence also shows that the subnuclear position of a gene or domain correlates with its expression status. Thus, the question arises whether this spatial organization results from or determines a gene's chromatin status. Although the association of a promoter with the inner nuclear membrane (INM) is neither necessary nor sufficient for repression, the perinuclear sequestration of heterochromatin is nonetheless conserved from yeast to man. How does subnuclear localization influence gene expression? Recent work argues that the common denominator between genome organization and gene expression is the modification of histones and in some cases of histone variants. This provides an important link between local chromatin structure and long-range genome organization in interphase cells. In this review, we will evaluate how histones contribute to the latter, and discuss how this might help to regulate genes crucial for cell differentiation.

  9. Regulation of Cellular Immune Responses in Sepsis by Histone Modifications.

    PubMed

    Carson, W F; Kunkel, S L

    2017-01-01

    Severe sepsis, septic shock, and related inflammatory syndromes are driven by the aberrant expression of proinflammatory mediators by immune cells. During the acute phase of sepsis, overexpression of chemokines and cytokines drives physiological stress leading to organ failure and mortality. Following recovery from sepsis, the immune system exhibits profound immunosuppression, evidenced by an inability to produce the same proinflammatory mediators that are required for normal responses to infectious microorganisms. Gene expression in inflammatory responses is influenced by the transcriptional accessibility of the chromatin, with histone posttranslational modifications determining whether inflammatory gene loci are set to transcriptionally active, repressed, or poised states. Experimental evidence indicates that histone modifications play a central role in governing the cytokine storm of severe sepsis, and that aberrant chromatin modifications induced during the acute phase of sepsis may mediate chronic immunosuppression in sepsis survivors. This review will focus on the role of histone modifications in governing immune responses in severe sepsis, with an emphasis on specific leukocyte subsets and the histone modifications observed in these cells during chronic stages of sepsis. Additionally, the expression and function of chromatin-modifying enzymes (CMEs) will be discussed in the context of severe sepsis, as potential mediators of epigenetic regulation of gene expression in sepsis responses. In summary, this review will argue for the use of chromatin modifications and CME expression in leukocytes as potential biomarkers of immunosuppression in patients with severe sepsis.

  10. Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics.

    PubMed

    Kulej, Katarzyna; Avgousti, Daphne C; Weitzman, Matthew D; Garcia, Benjamin A

    2015-11-15

    Viruses are obligate intracellular parasites that necessarily rely on hijacking cellular resources to produce viral progeny. The success of viral infection requires manipulation of host chromatin in order to activate genes useful for production of viral proteins as well as to suppress antiviral responses. Host chromatin manipulation on a global level is likely reliant on modulation of post-translational modifications (PTMs) on histone proteins. Mass spectrometry (MS) is a powerful tool to quantify and identify novel histone PTMs, beyond the limitations of site-specific antibodies. Here, we employ MS to investigate global changes in histone PTM relative abundance in human cells during infection with adenovirus. Our method reveals several changes in histone PTM patterns during infection. We propose that this method can be used to uncover global changes in histone PTM patterns that are universally modulated by viruses to take over the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Heterochromatin, histone modifications, and nuclear architecture in disease vectors

    PubMed Central

    Sharakhov, Igor V.; Sharakhova, Maria V.

    2015-01-01

    Interactions between a pathogen and a vector are plastic and dynamic. Such interactions can be more rapidly accommodated by epigenetic changes than by genetic mutations. Gene expression can be affected by the proximity to the heterochromatin, by local histone modifications, and by the three-dimensional position within the nucleus. Recent studies of disease vectors indicate that gene regulation by these factors can be important for susceptibility to pathogens, reproduction, immunity, development, and longevity. Knowledge about heterochromatin, histone modifications, and nuclear architecture will help our understanding of epigenetic mechanisms that control gene function at traits related to vectorial capacity. PMID:26097808

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

    PubMed

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

    2004-08-01

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

  13. The proteasome and epigenetics: zooming in on histone modifications.

    PubMed

    Bach, Svitlana V; Hegde, Ashok N

    2016-08-01

    The proteasome is a structural complex of many proteins that degrades substrates marked by covalent linkage to ubiquitin. Many years of research has shown a role for ubiquitin-proteasome-mediated proteolysis in synaptic plasticity and memory mainly in degrading synaptic, cytoplasmic and nuclear proteins. Recent work indicates that the proteasome has wider proteolytic and non-proteolytic roles in processes such as histone modifications that affect synaptic plasticity and memory. In this review, we assess the evidence gathered from neuronal as well as non-neuronal cell types regarding the function of the proteasome in positive or negative regulation of posttranslational modifications of histones, such as acetylation, methylation and ubiquitination. We discuss the critical roles of the proteasome in clearing excess histone proteins in various cellular contexts and the possible non-proteolytic functions in regulating transcription of target genes. In addition, we summarize the current literature on diverse chromatin-remodeling machineries, such as histone acetyltransferases, deacetylates, methyltransferases and demethylases, as targets for proteasomal degradation across experimental models. Lastly, we provide a perspective on how proteasomal regulation of histone modifications may modulate synaptic plasticity in the nervous system.

  14. Histone Ketoamide Adduction by 4-Oxo-2-nonenal Is a Reversible Posttranslational Modification Regulated by Sirt2.

    PubMed

    Cui, Yiwen; Li, Xin; Lin, Jianwei; Hao, Quan; Li, Xiang David

    2017-01-20

    Lipid-derived electrophiles (LDEs) directly modify proteins to modulate cellular signaling pathways in response to oxidative stress. One such LDE, 4-oxo-2-nonenal (4-ONE), has recently been found to target histones and interfere with histone assembly into nucleosomes. Unlike other LDEs that preferentially modify cysteine via nucleophilic Michael addition, 4-ONE reacts with histone lysine residues to form a new histone modification, gamma-oxononanoylation (Kgon). However, it remains unclear whether Kgon can cause irreversible damage or be regulated by enzymes "erasing" this nonenzymatic modification. Here, we report that human Sirt2 catalyzes the removal of histone Kgon. Among the tested human sirtuins, Sirt2 showed robust deacylase activity toward the Kgon-carrying histone peptides in vitro. We use alkynyl-4-ONE as a chemical reporter for Kgon to demonstrate that Sirt2 is responsible for removing histone Kgon in cells. Furthermore, we develop a ketone-reactive chemical probe to detect histones modified by endogenous 4-ONE in macrophages in response to inflammatory stimulation. Using this probe, we show Sirt2 as a deacylase able to control histone Kgon in stimulated macrophages. This study unravels a new mechanism for the regulation of LDE-derived protein posttranslational modifications, as well as a novel role played by Sirt2 as a histone Kgon deacylase in cytoprotective signaling responses.

  15. Endometriosis Is Characterized by a Distinct Pattern of Histone 3 and Histone 4 Lysine Modifications

    PubMed Central

    Monteiro, Janice B.; Colón-Díaz, Maricarmen; García, Miosotis; Gutierrez, Sylvia; Colón, Mariano; Seto, Edward; Laboy, Joaquín

    2014-01-01

    Background: The histone modification patterns in endometriosis have not been fully characterized. This gap in knowledge results in a poor understanding of the epigenetic mechanisms (and potential therapeutic targets) at play. We aimed to (1) assess global acetylation status of histone 3 (H3) and histone 4 (H4), (2) measure levels of H3 and H4 lysine (K) acetylation and methylation, and (3) to identify histone acetylation patterns in promoter regions of candidate genes in tissues from patients and controls. Methods: Global and K-specific acetylation/methylation levels of histones were measured in 24 lesions, 15 endometrium from patients, and 26 endometrium from controls. Chromatin immunoprecipitation (ChIP)–polymerase chain reaction was used to determine the histone acetylation status of the promoter regions of candidate genes in tissues. Results: The lesions were globally hypoacetylated at H3 (but not H4) compared to eutopic endometrium from controls. Lesions had significantly lower levels of H3K9ac and H4K16ac compared to eutopic endometrium from patients and controls. Tissues from patients were hypermethylated at H3K4, H3K9, and H3K27 compared to endometrium from controls. The ChIP analysis showed hypoacetylation of H3/H4 within promoter regions of candidate genes known to be downregulated in endometriosis (e.g., HOXA10, ESR1, CDH1, and p21WAF1/Cip1) in lesions versus control endometrium. The stereoidogenic factor 1 (SF1) promoter region was enriched for acetylated H3 and H4 in lesions versus control tissues, correlating with its reported high expression in lesions. Conclusions: This study describes the histone code of lesions and endometrium from patients with endometriosis and provides support for a possible role of histone modification in modulation of gene expression in endometriosis. PMID:23899551

  16. 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.

  17. A facile method to synthesize histones with posttranslational modification mimics.

    PubMed

    Wang, Zhiyong U; Wang, Yane-Shih; Pai, Pei-Jing; Russell, William K; Russell, David H; Liu, Wenshe R

    2012-07-03

    Using an evolved pyrrolysyl-tRNA synthetase-tRNA(Pyl) pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.

  18. Chromatin dynamics: Interplay between remodeling enzymes and histone modifications

    PubMed Central

    Swygert, Sarah G.; Peterson, Craig L.

    2014-01-01

    Chromatin dynamics play an essential role in regulating the accessibility of genomic DNA for a variety of nuclear processes, including gene transcription and DNA repair. The posttranslational modification of the core histones and the action of ATP-dependent chromatin remodeling enzymes represent two primary mechanisms by which chromatin dynamics are controlled and linked to nuclear events. Although there are examples in which a histone modification or a remodeling enzyme may be sufficient to drive a chromatin transition, these mechanisms typically work in concert to integrate regulatory inputs, leading to a coordinated alteration in chromatin structure and function. Indeed, site-specific histone modifications can facilitate the recruitment of chromatin remodeling enzymes to particular genomic regions, or they can regulate the efficiency or the outcome of a chromatin remodeling reaction. Conversely, chromatin remodeling enzymes can also influence, and sometimes directly modulate, the modification state of histones. These functional interactions are generally complex, frequently transient, and often require the association of myriad additional factors. PMID:24583555

  19. Finding Associations among Histone Modifications Using Sparse Partial Correlation Networks

    PubMed Central

    Lasserre, Julia; Chung, Ho-Ryun; Vingron, Martin

    2013-01-01

    Histone modifications are known to play an important role in the regulation of transcription. While individual modifications have received much attention in genome-wide analyses, little is known about their relationships. Some authors have built Bayesian networks of modifications, however most often they have used discretized data, and relied on unrealistic assumptions such as the absence of feedback mechanisms or hidden confounding factors. Here, we propose to infer undirected networks based on partial correlations between histone modifications. Within the partial correlation framework, correlations among two variables are controlled for associations induced by the other variables. Partial correlation networks thus focus on direct associations of histone modifications. We apply this methodology to data in CD4+ cells. The resulting network is well supported by common knowledge. When pairs of modifications show a large difference between their correlation and their partial correlation, a potential confounding factor is identified and provided as explanation. Data from different cell types (IMR90, H1) is also exploited in the analysis to assess the stability of the networks. The results are remarkably similar across cell types. Based on this observation, the networks from the three cell types are integrated into a consensus network to increase robustness. The data and the results discussed in the manuscript can be found, together with code, on http://spcn.molgen.mpg.de/index.html. PMID:24039558

  20. Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.

    PubMed

    Sharma, Alok; Nguyen, Hieu; Geng, Cuiyu; Hinman, Melissa N; Luo, Guangbin; Lou, Hua

    2014-11-18

    In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.

  1. Glutamine methylation in Histone H2A is an RNA Polymerase I dedicated modification

    PubMed Central

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J; Nielsen, Michael L.; Kouzarides, Tony

    2013-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes1. Here, we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that Fibrillarin is the ortholog enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me specific antibody, show that this modification is exclusively enriched over the 35S rDNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (Facilitator of Transcription) complex2. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 exhibits reduced histone incorporation and increased transcription at the rDNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes. PMID:24352239

  2. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    NASA Astrophysics Data System (ADS)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  3. Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

    PubMed Central

    Verzijlbergen, Kitty F; Faber, Alex W; Stulemeijer, Iris JE; van Leeuwen, Fred

    2009-01-01

    Background Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers. Result We determined that loss of H3K79 methylation results in a partial silencing defect that could be bypassed by conditions that promote targeting of Sir proteins to heterochromatin. Furthermore, the silencing defect in strains lacking Dot1 was dependent on methylation of H3K4 by Set1 and histone acetylation by Gcn5, Elp3, and Sas2 in euchromatin. Our study shows that multiple histone modifications associated with euchromatin positively modulate the function of heterochromatin by distinct mechanisms. Genetic interactions between Set1 and Set2 suggested that the H3K36 methyltransferase Set2, unlike most other euchromatic modifiers, negatively affects gene silencing. Conclusion Our genetic dissection of Dot1's role in silencing in budding yeast showed that heterochromatin formation is modulated by multiple euchromatic histone modifiers that act by non-overlapping mechanisms. We discuss how euchromatic histone modifiers can make negative as well as positive contributions to gene silencing by competing with heterochromatin proteins within heterochromatin, within euchromatin, and at the boundary between euchromatin and heterochromatin. PMID:19638198

  4. Epigenetic modifications of histone h4 in lung neuroendocrine tumors.

    PubMed

    Li, Faqian; Ye, Bo; Hong, Longsheng; Xu, Haodong; Fishbein, Michael C

    2011-10-01

    Global profiling of histone changes in some human cancers demonstrated that loss of histone H4 acetylation at lysine16 (H4KA16) and trimethylation at lysine 20 (H4KM20) was a common hallmark of cancer. It is not clear whether these epigenetic changes also exist in neuroendocrine carcinomas. We semiquantitatively analyzed 32 cases of lung neuroendocrine tumors (LNETs) immunohistochemically stained with H4KA16, H4KM20, and Ki67 antibodies by calculating cumulative scores based on the sum of the product of nuclear stain intensity (1-3) and percentages of positive cells in each category. H4KA16 and H4KM20 levels were compared among typical carcinoid (TC, 11), atypical carcinoid (AC, 6), large cell neuroendocrine carcinoma (LCNEC, 8), and small cell lung cancer (SCLC, 7) and correlated with histologic types and Ki67 labeling. Data were presented as mean±standard error of the mean and statistically analyzed by 1-way analysis of variance and Holm-Sidak method. Normal bronchiolar epithelium had relatively uniform and strong +3 positivity of H4KM20 and H4KA16, which was considered as internal positive controls. This uniformity, however, was gradually lost from low to high grades of LNETs. Semiquantitative analysis revealed that there were significant differences in cumulative scores of H4KA16 (TC, 2.36±0.03; AC, 2.04±0.08; LCNEC, 1.58±0.07; SCLC 1.32±0.05) among LNETs. For H4KM20, significant differences were only observed between low grade (TC, 2.49±0.05 and AC, 2.24±0.09) and high grade (LCNEC, 1.58±0.10 and SCLC 1.68±0.11) LNETs, but not within low or high grade LNETs. The Ki67 cumulative scores (TC, 0.06±0.02; AC, 0.41±0.08; LCNEC, 1.29±0.09; SCLC 1.83±0.06) were inversely correlated with both cumulative H4KA16 and H4KM20 scores by Pearson correlation. We conclude that progressive loss of H4KA16 and H4KM20 from low to high grade LNETs reflects the degree of differentiation and proliferative activity. These histone modifications may serve as tumor biomarkers

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

    PubMed

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

    2012-05-01

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

  6. Histone modifications: Targeting head and neck cancer stem cells

    PubMed Central

    Le, John M; Squarize, Cristiane H; Castilho, Rogerio M

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and is responsible for a quarter of a million deaths annually. The survival rate for HNSCC patients is poor, showing only minor improvement in the last three decades. Despite new surgical techniques and chemotherapy protocols, tumor resistance to chemotherapy remains a significant challenge for HNSCC patients. Numerous mechanisms underlie chemoresistance, including genetic and epigenetic alterations in cancer cells that may be acquired during treatment and activation of mitogenic signaling pathways, such as nuclear factor kappa-light-chain-enhancer-of activated B cell, that cause reduced apoptosis. In addition to dysfunctional molecular signaling, emerging evidence reveals involvement of cancer stem cells (CSCs) in tumor development and in tumor resistance to chemotherapy and radiotherapy. These observations have sparked interest in understanding the mechanisms involved in the control of CSC function and fate. Post-translational modifications of histones dynamically influence gene expression independent of alterations to the DNA sequence. Recent findings from our group have shown that pharmacological induction of post-translational modifications of tumor histones dynamically modulates CSC plasticity. These findings suggest that a better understanding of the biology of CSCs in response to epigenetic switches and pharmacological inhibitors of histone function may directly translate to the development of a mechanism-based strategy to disrupt CSCs. In this review, we present and discuss current knowledge on epigenetic modifications of HNSCC and CSC response to DNA methylation and histone modifications. In addition, we discuss chromatin modifications and their role in tumor resistance to therapy. PMID:25426249

  7. Targeting post-translational histone modifications for the treatment of non-medullary thyroid cancer.

    PubMed

    Celano, Marilena; Mio, Catia; Sponziello, Marialuisa; Verrienti, Antonella; Bulotta, Stefania; Durante, Cosimo; Damante, Giuseppe; Russo, Diego

    2017-06-02

    Genomic and epigenetic alterations are now being exploited as molecular targets in cancer treatment. Abnormalities involving the post-translational modification of histones have been demonstrated in thyroid cancer, and they are regarded as promising molecular targets for novel drug treatment of tumors that are resistant to conventional therapies. After a brief overview of the histone modifications most commonly associated with human malignancies, we will review recently published preclinical and clinical findings regarding the use of histone-activity modulators in thyroid cancers. Particular attention will be focused on their use as re-differentiating or anti-proliferating agents, the differential effects observed when they are used alone and in combination with other targeted drugs, and current prospects for their use in the treatment of thyroid cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The interplay of histone modifications - writers that read.

    PubMed

    Zhang, Tianyi; Cooper, Sarah; Brockdorff, Neil

    2015-11-01

    Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  9. The interplay of histone modifications – writers that read

    PubMed Central

    Zhang, Tianyi; Cooper, Sarah; Brockdorff, Neil

    2015-01-01

    Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. PMID:26474904

  10. Heterochromatic histone modifications at transposons in Xenopus tropicalis embryos.

    PubMed

    van Kruijsbergen, Ila; Hontelez, Saartje; Elurbe, Dei M; van Heeringen, Simon J; Huynen, Martijn A; Veenstra, Gert Jan C

    2016-09-14

    Transposable elements are parasitic genomic elements that can be deleterious for host gene function and genome integrity. Heterochromatic histone modifications are involved in the repression of transposons. However, it remains unknown how these histone modifications mark different types of transposons during embryonic development. Here we document the variety of heterochromatic epigenetic signatures at parasitic elements during development in Xenopus tropicalis, using genome-wide ChIP-sequencing data and ChIP-qPCR analysis. We show that specific subsets of transposons in various families and subfamilies are marked by different combinations of the heterochromatic histone modifications H4K20me3, H3K9me2/3 and H3K27me3. Many DNA transposons are marked at the blastula stage already, whereas at retrotransposons the histone modifications generally accumulate at the gastrula stage or later. Furthermore, transposons marked by H3K9me3 and H4K20me3 are more prominent in gene deserts. Using intra-subfamily divergence as a proxy for age, we show that relatively young DNA transposons are preferentially marked by early embryonic H4K20me3 and H3K27me3. In contrast, relatively young retrotransposons are marked by increasing H3K9me3 and H4K20me3 during development, and are also linked to piRNA-sized small non-coding RNAs. Our results implicate distinct repression mechanisms that operate in a transposon-selective and developmental stage-specific fashion.

  11. 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.

  12. Correlating Histone Modification Patterns with Gene Expression Data During Hematopoiesis

    PubMed Central

    Hu, Gangqing; Zhao, Keji

    2014-01-01

    Hematopoietic stem cells (HSC) in mammals are an ideal system to study differentiation. While transcription factors (TFs) control the differentiation of HSCs to distinctive terminal blood cells, accumulating evidence suggests that chromatin structure and modifications constitute another critical layer of gene regulation. Recent genome-wide studies based on next-generation sequencing reveal that histone modifications are linked to gene expression and contribute to hematopoiesis. Here, we briefl y review the bioinformatics aspects for ChIP-Seq and RNA-Seq data analysis with applications to the epigenetic studies of hematopoiesis and provide a practical guide to several basic data analysis methods. PMID:24743998

  13. Combinatorial patterns of histone acetylations and methylations in the human genome

    PubMed Central

    Wang, Zhibin; Zang, Chongzhi; Rosenfeld, Jeffrey A; Schones, Dustin E; Barski, Artem; Cuddapah, Suresh; Cui, Kairong; Roh, Tae-Young; Peng, Weiqun; Zhang, Michael Q; Zhao, Keji

    2008-01-01

    Histones are characterized by numerous posttranslational modifications that influence gene transcription1,2. However, because of the lack of global distribution data in higher eukaryotic systems3, the extent to which gene-specific combinatorial patterns of histone modifications exist remains to be determined. Here, we report the patterns derived from the analysis of 39 histone modifications in human CD4+ T cells. Our data indicate that a large number of patterns are associated with promoters and enhancers. In particular, we identify a common modification module consisting of 17 modifications detected at 3,286 promoters. These modifications tend to colocalize in the genome and correlate with each other at an individual nucleosome level. Genes associated with this module tend to have higher expression, and addition of more modifications to this module is associated with further increased expression. Our data suggest that these histone modifications may act cooperatively to prepare chromatin for transcriptional activation. PMID:18552846

  14. Chromatin remodeling and bivalent histone modifications in embryonic stem cells.

    PubMed

    Harikumar, Arigela; Meshorer, Eran

    2015-12-01

    Pluripotent embryonic stem cells (ESCs) are characterized by distinct epigenetic features including a relative enrichment of histone modifications related to active chromatin. Among these is tri-methylation of lysine 4 on histone H3 (H3K4me3). Several thousands of the H3K4me3-enriched promoters in pluripotent cells also contain a repressive histone mark, namely H3K27me3, a situation referred to as "bivalency". While bivalent promoters are not unique to pluripotent cells, they are relatively enriched in these cell types, largely marking developmental and lineage-specific genes which are silent but poised for immediate action. The H3K4me3 and H3K27me3 modifications are catalyzed by lysine methyltransferases which are usually found within, although not entirely limited to, the Trithorax group (TrxG) and Polycomb group (PcG) protein complexes, respectively, but these do not provide selective bivalent specificity. Recent studies highlight the family of ATP-dependent chromatin remodeling proteins as regulators of bivalent domains. Here, we discuss bivalency in general, describe the machineries that catalyze bivalent chromatin domains, and portray the emerging connection between bivalency and the action of different families of chromatin remodelers, namely INO80, esBAF, and NuRD, in pluripotent cells. We posit that chromatin remodeling proteins may enable "bivalent specificity", often selectively acting on, or selectively depleted from, bivalent domains.

  15. Physarum polycephalum for Studying the Function of Histone Modifications In Vivo.

    PubMed

    Menil-Philippot, Vanessa; Thiriet, Christophe

    2017-01-01

    Histone modifications have been widely correlated with genetic activities. However, how these posttranslational modifications affect the dynamics and the structure of chromatin is poorly understood. Here, we describe the incorporation of the exogenous histone proteins into the slime mold Physarum polycephalum, which has been revealed to be a valuable tool for examining different facets of the function histones in chromatin dynamics like replication-coupled chromatin assembly, histone exchange, and nucleosome turnover.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. DNA methylation and histone modification in onion chromosomes.

    PubMed

    Suzuki, Go; Shiomi, Maho; Morihana, Sayuri; Yamamoto, Maki; Mukai, Yasuhiko

    2010-01-01

    Onion, Allium cepa, is a model plant for experimental observation of somatic cell division, whose mitotic chromosome is extremely large, and contains the characteristic terminal heterochromatin. Epigenetic status of the onion chromosome is a matter of deep interest from a molecular cytogenetic point of view, because epigenetic marks regulate chromatin structure and gene expression. Here we examined chromosomal distribution of DNA methylation and histone modification in A. cepa in order to reveal the chromatin structure in detail. Immunodetection of 5-methylcytosine (5mC) and in situ nick-translation analysis showed that onion genomic DNA was highly methylated, and the methylated CG dinucleotides were distributed in entire chromosomes. In addition, distributions of histone methylation codes, which occur in close association with DNA methylation, were similar to those of other large genome species. From these results, a highly heterochromatic and less euchromatic state of large onion chromosomes were demonstrated at an epigenetic level.

  19. Independent regulation of gene expression level and noise by histone modifications.

    PubMed

    Wu, Shaohuan; Li, Ke; Li, Yingshu; Zhao, Tong; Li, Ting; Yang, Yu-Fei; Qian, Wenfeng

    2017-06-01

    The inherent stochasticity generates substantial gene expression variation among isogenic cells under identical conditions, which is frequently referred to as gene expression noise or cell-to-cell expression variability. Similar to (average) expression level, expression noise is also subject to natural selection. Yet it has been observed that noise is negatively correlated with expression level, which manifests as a potential constraint for simultaneous optimization of both. Here, we studied expression noise in human embryonic cells with computational analysis on single-cell RNA-seq data and in yeast with flow cytometry experiments. We showed that this coupling is overcome, to a certain degree, by a histone modification strategy in multiple embryonic developmental stages in human, as well as in yeast. Importantly, this epigenetic strategy could fit into a burst-like gene expression model: promoter-localized histone modifications (such as H3K4 methylation) are associated with both burst size and burst frequency, which together influence expression level, while gene-body-localized ones (such as H3K79 methylation) are more associated with burst frequency, which influences both expression level and noise. We further knocked out the only "writer" of H3K79 methylation in yeast, and observed that expression noise is indeed increased. Consistently, dosage sensitive genes, such as genes in the Wnt signaling pathway, tend to be marked with gene-body-localized histone modifications, while stress responding genes, such as genes regulating autophagy, tend to be marked with promoter-localized ones. Our findings elucidate that the "division of labor" among histone modifications facilitates the independent regulation of expression level and noise, extend the "histone code" hypothesis to include expression noise, and shed light on the optimization of transcriptome in evolution.

  20. Independent regulation of gene expression level and noise by histone modifications

    PubMed Central

    Li, Yingshu; Zhao, Tong; Li, Ting; Yang, Yu-Fei

    2017-01-01

    The inherent stochasticity generates substantial gene expression variation among isogenic cells under identical conditions, which is frequently referred to as gene expression noise or cell-to-cell expression variability. Similar to (average) expression level, expression noise is also subject to natural selection. Yet it has been observed that noise is negatively correlated with expression level, which manifests as a potential constraint for simultaneous optimization of both. Here, we studied expression noise in human embryonic cells with computational analysis on single-cell RNA-seq data and in yeast with flow cytometry experiments. We showed that this coupling is overcome, to a certain degree, by a histone modification strategy in multiple embryonic developmental stages in human, as well as in yeast. Importantly, this epigenetic strategy could fit into a burst-like gene expression model: promoter-localized histone modifications (such as H3K4 methylation) are associated with both burst size and burst frequency, which together influence expression level, while gene-body-localized ones (such as H3K79 methylation) are more associated with burst frequency, which influences both expression level and noise. We further knocked out the only “writer” of H3K79 methylation in yeast, and observed that expression noise is indeed increased. Consistently, dosage sensitive genes, such as genes in the Wnt signaling pathway, tend to be marked with gene-body-localized histone modifications, while stress responding genes, such as genes regulating autophagy, tend to be marked with promoter-localized ones. Our findings elucidate that the “division of labor” among histone modifications facilitates the independent regulation of expression level and noise, extend the “histone code” hypothesis to include expression noise, and shed light on the optimization of transcriptome in evolution. PMID:28665997

  1. Tumor necrosis factor-alpha enhances neutrophil adhesiveness: induction of vascular cell adhesion molecule-1 via activation of Akt and CaM kinase II and modifications of histone acetyltransferase and histone deacetylase 4 in human tracheal smooth muscle cells.

    PubMed

    Lee, Chiang-Wen; Lin, Chih-Chung; Luo, Shue-Fen; Lee, Hui-Chun; Lee, I-Ta; Aird, William C; Hwang, Tsong-Long; Yang, Chuen-Mao

    2008-05-01

    Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-alpha-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-kappaB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-alpha-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-alpha significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R(*),9S(*),11S(*))-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl

  2. Post-Translational Modifications of Histones in Vertebrate Neurogenesis

    PubMed Central

    Mitrousis, Nikolaos; Tropepe, Vincent; Hermanson, Ola

    2015-01-01

    The process of neurogenesis, through which the entire nervous system of an organism is formed, has attracted immense scientific attention for decades. How can a single neural stem cell give rise to astrocytes, oligodendrocytes, and neurons? Furthermore, how is a neuron led to choose between the hundreds of different neuronal subtypes that the vertebrate CNS contains? Traditionally, niche signals and transcription factors have been on the spotlight. Recent research is increasingly demonstrating that the answer may partially lie in epigenetic regulation of gene expression. In this article, we comprehensively review the role of post-translational histone modifications in neurogenesis in both the embryonic and adult CNS. PMID:26733796

  3. Histone 3 modifications and blood pressure in the Beijing Truck Driver Air Pollution Study.

    PubMed

    Kresovich, Jacob K; Zhang, Zhou; Fang, Fang; Zheng, Yinan; Sanchez-Guerra, Marco; Joyce, Brian T; Zhong, Jia; Chervona, Yana; Wang, Sheng; Chang, Dou; McCracken, John P; Díaz, Anaite; Bonzini, Matteo; Carugno, Michele; Koutrakis, Petros; Kang, Choong-Min; Bian, Shurui; Gao, Tao; Byun, Hyang-Min; Schwartz, Joel; Baccarelli, Andrea A; Hou, Lifang

    2017-09-01

    Histone modifications regulate gene expression; dysregulation has been linked with cardiovascular diseases. Associations between histone modification levels and blood pressure in humans are unclear. We examine the relationship between global histone concentrations and various markers of blood pressure. Using the Beijing Truck Driver Air Pollution Study, we investigated global peripheral white blood cell histone modifications (H3K9ac, H3K9me3, H3K27me3, and H3K36me3) associations with pre- and post-work measurements of systolic (SBP) and diastolic (DBP) blood pressure, mean arterial pressure (MAP), and pulse pressure (PP) using multivariable mixed-effect models. H3K9ac was negatively associated with pre-work SBP and MAP; H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP; and H3K27me3 was negatively associated with pre-work SBP. Among office workers, H3K9me3 was negatively associated with pre-work SBP, DBP, and MAP. Among truck drivers, H3K9ac and H3K27me were negatively associated with pre-work SBP, and H3K27me3 was positively associated with post-work PP. Epigenome-wide H3K9ac, H3K9me3, and H3K27me3 were negatively associated with multiple pre-work blood pressure measures. These associations substantially changed during the day, suggesting an influence of daily activities. Blood-based histone modification biomarkers are potential candidates for studies requiring estimations of morning/pre-work blood pressure.

  4. Recognition of a Mononucleosomal Histone Modification Pattern by BPTF via Multivalent Interactions

    SciTech Connect

    Ruthenburg, Alexander J.; Li, Haitao; Milne, Thomas A.; Dewell, Scott; McGinty, Robert K.; Yuen, Melanie; Ueberheide, Beatrix; Dou, Yali; Muir, Tom W.; Patel, Dinshaw J.; Allis, C. David

    2011-09-20

    Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.

  5. Recognition of a Mononucleosomal Histone Modification Pattern by BPTF via Multivalent Interactions

    SciTech Connect

    A Ruthenburg; H Li; T Milne; S Dewell; R McGinty; M Yuen; B Ueberheide; Y Dou; T Muir; et al.

    2011-12-31

    Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.

  6. Compendium of aberrant DNA methylation and histone modifications in cancer.

    PubMed

    Hattori, Naoko; Ushijima, Toshikazu

    2014-12-05

    Epigenetics now refers to the study or research field related to DNA methylation and histone modifications. Historically, global DNA hypomethylation was first revealed in 1983, and, after a decade, silencing of a tumor suppressor gene by regional DNA hypermethylation was reported. After the proposal of the histone code in the 2000s, alterations of histone methylation were also identified in cancers. Now, it is established that aberrant epigenetic alterations are involved in cancer development and progression, along with mutations and chromosomal losses. Recent cancer genome analyses have revealed a large number of mutations of epigenetic modifiers, supporting their important roles in cancer pathogenesis. Taking advantage of the reversibility of epigenetic alterations, drugs targeting epigenetic regulators and readers have been developed for restoration of normal pattern of the epigenome, and some have already demonstrated clinical benefits. In addition, DNA methylation of specific marker genes can be used as a biomarker for cancer diagnosis, including risk diagnosis, detection of cancers, and pathophysiological diagnosis. In this paper, we will summarize the major concepts of cancer epigenetics, placing emphasis on history.

  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. Global histone post-translational modifications and cancer: Biomarkers for diagnosis, prognosis and treatment?

    PubMed Central

    Khan, Shafqat Ali; Reddy, Divya; Gupta, Sanjay

    2015-01-01

    Global alterations in epigenetic landscape are now recognized as a hallmark of cancer. Epigenetic mechanisms such as DNA methylation, histone modifications, nucleosome positioning and non-coding RNAs are proven to have strong association with cancer. In particular, covalent post-translational modifications of histone proteins are known to play an important role in chromatin remodeling and thereby in regulation of gene expression. Further, histone modifications have also been associated with different aspects of carcinogenesis and have been studied for their role in the better management of cancer patients. In this review, we will explore and discuss how histone modifications are involved in cancer diagnosis, prognosis and treatment. PMID:26629316

  9. Emerging roles for histone modifications in DNA excision repair.

    PubMed

    Mao, Peng; Wyrick, John J

    2016-11-01

    DNA repair is critical to maintain genome stability. In eukaryotic cells, DNA repair is complicated by the packaging of the DNA 'substrate' into chromatin. DNA repair pathways utilize different mechanisms to overcome the barrier presented by chromatin to efficiently locate and remove DNA lesions in the genome. DNA excision repair pathways are responsible for repairing a majority of DNA lesions arising in the genome. Excision repair pathways include nucleotide excision repair (NER) and base excision repair (BER), which repair bulky and non-bulky DNA lesions, respectively. Numerous studies have suggested that chromatin inhibits both NER and BER in vitro and in vivo Growing evidence demonstrates that histone modifications have important roles in regulating the activity of NER and BER enzymes in chromatin. Here, we will discuss the roles of different histone modifications and the corresponding modifying enzymes in DNA excision repair, highlighting the role of yeast as a model organism for many of these studies. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction.

    PubMed

    Akai, Yusuke; Adachi, Naruhiko; Hayashi, Yohei; Eitoku, Masamitsu; Sano, Norihiko; Natsume, Ryo; Kudo, Norio; Tanokura, Masaru; Senda, Toshiya; Horikoshi, Masami

    2010-05-04

    Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 A resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).

  11. Structure of the histone chaperone CIA/ASF1–double bromodomain complex linking histone modifications and site-specific histone eviction

    PubMed Central

    Akai, Yusuke; Adachi, Naruhiko; Hayashi, Yohei; Eitoku, Masamitsu; Sano, Norihiko; Natsume, Ryo; Kudo, Norio; Tanokura, Masaru; Senda, Toshiya; Horikoshi, Masami

    2010-01-01

    Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 Å resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model). PMID:20393127

  12. Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

    PubMed Central

    Su, Xiaodan; Ren, Chen; Freitas, Michael A

    2008-01-01

    Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. PMID:17425457

  13. Tracking epigenetic histone modifications in single cells using Fab-based live endogenous modification labeling.

    PubMed

    Hayashi-Takanaka, Yoko; Yamagata, Kazuo; Wakayama, Teruhiko; Stasevich, Timothy J; Kainuma, Takashi; Tsurimoto, Toshiki; Tachibana, Makoto; Shinkai, Yoichi; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

    2011-08-01

    Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

  14. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-03

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism.

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

    PubMed Central

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

    2012-01-01

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

  16. Histone Posttranslational Modifications of CD4+ T Cell in Autoimmune Diseases

    PubMed Central

    Wang, Zijun; Yin, Heng; Lau, Chak Sing; Lu, Qianjin

    2016-01-01

    The complexity of immune system is tempered by precise regulation to maintain stabilization when exposed to various conditions. A subtle change in gene expression may be magnified when drastic changes are brought about in cellular development and function. Posttranslational modifications (PTMs) timely alter the functional activity of immune system, and work proceeded in these years has begun to throw light upon it. Posttranslational modifications of histone tails have been mentioned in a large scale of biological developments and disease progression, thereby making them a central field to investigate. Conventional assessments of these changes are centered on the transcription factors and cytokines in T cells regulated by variable histone codes to achieve chromatin remodeling, as well as involved in many human diseases, especially autoimmune diseases. We here put forward an essential review of core posttranslational modulations that regulate T cell function and differentiation in the immune system, with a special emphasis on histone modifications in different T helper cell subsets as well as in autoimmune diseases. PMID:27669210

  17. Influences of the Gut Microbiota on DNA Methylation and Histone Modification.

    PubMed

    Ye, Jianzhong; Wu, Wenrui; Li, Yating; Li, Lanjuan

    2017-05-01

    The gut microbiota is a vast ensemble of microorganisms inhabiting the mammalian gastrointestinal tract that can impact physiologic and pathologic processes. However, our understanding of the underlying mechanism for the dynamic interaction between host and gut microbiota is still in its infancy. The highly evolved epigenetic modifications allow hosts to reprogram the genome in response to environmental stimuli, which may play a key role in triggering multiple human diseases. In spite of increasing studies in gut microbiota and epigenetic modifications, the correlation between them has not been well elaborated. Here, we review current knowledge of gut microbiota impacts on epigenetic modifications, the major evidence of which centers on DNA methylation and histone modification of the immune system.

  18. Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

    PubMed

    Swalm, Brooke M; Hallenbeck, Kenneth K; Majer, Christina R; Jin, Lei; Scott, Margaret Porter; Moyer, Mikel P; Copeland, Robert A; Wigle, Tim J

    2013-07-15

    H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

  19. Histone Modifications in Senescence-Associated Resistance to Apoptosis by Oxidative Stress☆☆☆

    PubMed Central

    Sanders, Yan Y.; Liu, Hui; Zhang, Xiangyu; Hecker, Louise; Bernard, Karen; Desai, Leena; Liu, Gang; Thannickal, Victor J.

    2013-01-01

    Aging and age-related diseases are associated with cellular senescence that results in variable apoptosis susceptibility to oxidative stress. Although fibroblast senescence has been associated with apoptosis resistance, mechanisms for this have not been well defined. In this report, we studied epigenetic mechanisms involving histone modifications that confer apoptosis resistance to senescent human diploid fibroblasts (HDFs). HDFs that undergo replicative senescence display typical morphological features, express senescence-associated β-galactosidase, and increased levels of the tumor suppressor genes, p16, p21, and caveolin-1. Senescent HDFs are more resistant to oxidative stress (exogenous H2O2)-induced apoptosis in comparison to non-senescent (control) HDFs; this is associated with constitutively high levels of the anti-apoptotic gene, Bcl-2, and low expression of the pro-apoptotic gene, Bax. Cellular senescence is characterized by global increases in H4K20 trimethylation and decreases in H4K16 acetylation in association with increased activity of Suv420h2 histone methyltransferase (which targets H4K20), decreased activity of the histone acetyltransferase, Mof (which targets H4K16), as well as decreased total histone acetyltransferase activity. In contrast to Bax gene, chromatin immunoprecipitation studies demonstrate marked enrichment of the Bcl-2 gene with H4K16Ac, and depletion with H4K20Me3, predicting active transcription of this gene in senescent HDFs. These data indicate that both global and locus-specific histone modifications of chromatin regulate altered Bcl-2:Bax gene expression in senescent fibroblasts, contributing to its apoptosis-resistant phenotype. PMID:24024133

  20. Histone modifications in senescence-associated resistance to apoptosis by oxidative stress.

    PubMed

    Sanders, Yan Y; Liu, Hui; Zhang, Xiangyu; Hecker, Louise; Bernard, Karen; Desai, Leena; Liu, Gang; Thannickal, Victor J

    2013-01-01

    Aging and age-related diseases are associated with cellular senescence that results in variable apoptosis susceptibility to oxidative stress. Although fibroblast senescence has been associated with apoptosis resistance, mechanisms for this have not been well defined. In this report, we studied epigenetic mechanisms involving histone modifications that confer apoptosis resistance to senescent human diploid fibroblasts (HDFs). HDFs that undergo replicative senescence display typical morphological features, express senescence-associated β-galactosidase, and increased levels of the tumor suppressor genes, p16, p21, and caveolin-1. Senescent HDFs are more resistant to oxidative stress (exogenous H2O2)-induced apoptosis in comparison to non-senescent (control) HDFs; this is associated with constitutively high levels of the anti-apoptotic gene, Bcl-2, and low expression of the pro-apoptotic gene, Bax. Cellular senescence is characterized by global increases in H4K20 trimethylation and decreases in H4K16 acetylation in association with increased activity of Suv420h2 histone methyltransferase (which targets H4K20), decreased activity of the histone acetyltransferase, Mof (which targets H4K16), as well as decreased total histone acetyltransferase activity. In contrast to Bax gene, chromatin immunoprecipitation studies demonstrate marked enrichment of the Bcl-2 gene with H4K16Ac, and depletion with H4K20Me3, predicting active transcription of this gene in senescent HDFs. These data indicate that both global and locus-specific histone modifications of chromatin regulate altered Bcl-2:Bax gene expression in senescent fibroblasts, contributing to its apoptosis-resistant phenotype.

  1. Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies.

    PubMed

    Liu, Chih Long; Tangsombatvisit, Stephanie; Rosenberg, Jacob M; Mandelbaum, Gil; Gillespie, Emily C; Gozani, Or P; Alizadeh, Ash A; Utz, Paul J

    2012-02-02

    Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.

  2. Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies

    PubMed Central

    2012-01-01

    Introduction Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones. Methods We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen. Results We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE. Conclusions Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified. PMID:22300536

  3. Application of histone modification-specific interaction domains as an alternative to antibodies

    PubMed Central

    Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z.; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul

    2014-01-01

    Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. PMID:25301795

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

    PubMed Central

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

    2011-01-01

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

  5. Gene Expression Differences Among Primates Are Associated With Changes in a Histone Epigenetic Modification

    PubMed Central

    Cain, Carolyn E.; Blekhman, Ran; Marioni, John C.; Gilad, Yoav

    2011-01-01

    Changes in gene regulation are thought to play an important role in speciation and adaptation, especially in primates. However, we still know relatively little about the mechanisms underlying regulatory evolution. In particular, the extent to which epigenetic modifications underlie gene expression differences between primates is not yet known. Our study focuses on an epigenetic histone modification, H3K4me3, which is thought to promote transcription. To investigate the contribution of H3K4me3 to regulatory differences between species, we collected gene expression data and identified H3K4me3-associated genomic regions in lymphoblastoid cell lines (LCLs) from humans, chimpanzees, and rhesus macaques, using three cell lines from each species. We found strong evidence for conservation of H3K4me3 localization in primates. Moreover, regardless of species, H3K4me3 is consistently enriched near annotated transcription start sites (TSS), and highly expressed genes are more likely than lowly expressed genes to have the histone modification near their TSS. Interestingly, we observed an enrichment of interspecies differences in H3K4me3 at the TSS of genes that are differentially expressed between species. We estimate that as much as 7% of gene expression differences between the LCLs of humans, chimpanzees, and rhesus macaques may be explained, at least in part, by changes in the status of H3K4me3 histone modifications. Our results suggest a modest, yet important role for epigenetic changes in gene expression differences between primates. PMID:21321133

  6. 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.

  7. ChromClust: A semi-supervised chromatin clustering toolkit for mining histone modifications interplay.

    PubMed

    Noureen, Nighat; Touseef, Muhammad; Fazal, Sahar; Qadir, Muhammad Abdul

    2015-12-01

    Mining patterns of histone modifications interplay from epigenomic profiles are one of the leading research areas these days. Various methods based on clustering approaches and hidden Markov models have been presented so far with some limitations. Here we present ChromClust, a semi-supervised clustering tool for mining commonly occurring histone modifications at various locations of the genome. Applying our method to 11 chromatin marks in nine human cell types recovered 11 clusters based on distinct chromatin signatures mapping to various elements of the genome. Our approach is efficient in respect to time and space usage along with the added facility of maintaining database at the backend. It outperforms the existing methods with respect to mining patterns in a semi-supervised fashion mapping to various functional elements of the genome. It will aid in future by saving the resources of time and space along with efficiently retrieving the hidden interplay of histone combinations. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Evaluation of Proteomic Search Engines for the Analysis of Histone Modifications

    PubMed Central

    2015-01-01

    Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118. PMID:25167464

  9. Evaluation of proteomic search engines for the analysis of histone modifications.

    PubMed

    Yuan, Zuo-Fei; Lin, Shu; Molden, Rosalynn C; Garcia, Benjamin A

    2014-10-03

    Identification of histone post-translational modifications (PTMs) is challenging for proteomics search engines. Including many histone PTMs in one search increases the number of candidate peptides dramatically, leading to low search speed and fewer identified spectra. To evaluate database search engines on identifying histone PTMs, we present a method in which one kind of modification is searched each time, for example, unmodified, individually modified, and multimodified, each search result is filtered with false discovery rate less than 1%, and the identifications of multiple search engines are combined to obtain confident results. We apply this method for eight search engines on histone data sets. We find that two search engines, pFind and Mascot, identify most of the confident results at a reasonable speed, so we recommend using them to identify histone modifications. During the evaluation, we also find some important aspects for the analysis of histone modifications. Our evaluation of different search engines on identifying histone modifications will hopefully help those who are hoping to enter the histone proteomics field. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001118.

  10. "Hunt"-ing for post-translational modifications that underlie the histone code

    NASA Astrophysics Data System (ADS)

    Taverna, Sean D.; David Allis, C.; Hake, Sandra B.

    2007-01-01

    Eukaryotic cells package their DNA with histone proteins to form chromatin that can be regulated to enable transcription, DNA repair and replication in response to cellular needs and external stimuli. A wealth of recent studies of post-translational histone modifications and histone variants have led to an explosion of insights into and more questions about how these processes might be regulated. Work from Donald Hunt and colleagues contributed greatly to our understanding of the "histone code" by developing novel methods to study and identify histone modifications in both generic and specialized variant histone proteins. Without his expertise, the field of chromatin biology would not be where it is today. In recognition, we are pleased to contribute to a special issue of the International Journal of Mass Spectrometry dedicated to the many advances pioneered by the Hunt laboratory, which have enhanced the science of many fields and the careers of many scientists.

  11. The Role of Histone Protein Modifications and Mutations in Histone Modifiers in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia

    PubMed Central

    Janczar, Szymon; Janczar, Karolina; Pastorczak, Agata; Harb, Hani; Paige, Adam J. W.; Zalewska-Szewczyk, Beata; Danilewicz, Marian; Mlynarski, Wojciech

    2017-01-01

    While cancer has been long recognized as a disease of the genome, the importance of epigenetic mechanisms in neoplasia was acknowledged more recently. The most active epigenetic marks are DNA methylation and histone protein modifications and they are involved in basic biological phenomena in every cell. Their role in tumorigenesis is stressed by recent unbiased large-scale studies providing evidence that several epigenetic modifiers are recurrently mutated or frequently dysregulated in multiple cancers. The interest in epigenetic marks is especially due to the fact that they are potentially reversible and thus druggable. In B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) there is a relative paucity of reports on the role of histone protein modifications (acetylation, methylation, phosphorylation) as compared to acute myeloid leukemia, T-cell ALL, or other hematologic cancers, and in this setting chromatin modifications are relatively less well studied and reviewed than DNA methylation. In this paper, we discuss the biomarker associations and evidence for a driver role of dysregulated global and loci-specific histone marks, as well as mutations in epigenetic modifiers in BCP-ALL. Examples of chromatin modifiers recurrently mutated/disrupted in BCP-ALL and associated with disease outcomes include MLL1, CREBBP, NSD2, and SETD2. Altered histone marks and histone modifiers and readers may play a particular role in disease chemoresistance and relapse. We also suggest that epigenetic regulation of B-cell differentiation may have parallel roles in leukemogenesis. PMID:28054944

  12. Regulation of alternative splicing by local histone modifications: potential roles for RNA-guided mechanisms

    PubMed Central

    Zhou, Hua-Lin; Luo, Guangbin; Wise, Jo Ann; Lou, Hua

    2014-01-01

    The molecular mechanisms through which alternative splicing and histone modifications regulate gene expression are now understood in considerable detail. Here, we discuss recent studies that connect these two previously separate avenues of investigation, beginning with the unexpected discoveries that nucleosomes are preferentially positioned over exons and DNA methylation and certain histone modifications also show exonic enrichment. These findings have profound implications linking chromatin structure, histone modification and splicing regulation. Complementary single gene studies provided insight into the mechanisms through which DNA methylation and histones modifications modulate alternative splicing patterns. Here, we review an emerging theme resulting from these studies: RNA-guided mechanisms integrating chromatin modification and splicing. Several groundbreaking papers reported that small noncoding RNAs affect alternative exon usage by targeting histone methyltransferase complexes to form localized facultative heterochromatin. More recent studies provided evidence that pre-messenger RNA itself can serve as a guide to enable precise alternative splicing regulation via local recruitment of histone-modifying enzymes, and emerging evidence points to a similar role for long noncoding RNAs. An exciting challenge for the future is to understand the impact of local modulation of transcription elongation rates on the dynamic interplay between histone modifications, alternative splicing and other processes occurring on chromatin. PMID:24081581

  13. [Effect of DNA methylation and histone modification during the development of cloned animals.].

    PubMed

    Li, Hui; Han, Zhi-Ming

    2010-08-01

    Somatic cell nuclear transfer (SCNT) has great potential for agricultural applications, generation of medical model animals, transgenic farm animals or generating human embryonic stem cells for treatment of human diseases. Cloned animals derived from somatic cells have been generated in several mammal species, but there are still some unsolved problems with current cloning technology, for example, the low efficiency of animal cloning and the abnormal development of cloned animals. One critical factor of these developmental failures of cloned embryos is the aberrant epigenetic reprogramming. This review focuses on DNA methylation and histone modifications and the relationship between these two epigenetic modifications and the development of cloned embryos. Understanding the mechanisms of epigenetic regulation will be useful to solve the technical problems of SCNT and enable better applications of this technology.

  14. Novel types and sites of histone modifications emerge as players in the transcriptional regulation contest.

    PubMed

    Kebede, Adam F; Schneider, Robert; Daujat, Sylvain

    2015-05-01

    N-terminal tails of histones are easily accessible outside of the nucleosomal core particle and post-translational modifications (PTMs) of these tails have been the focus of attention in the past 15-20 years. By recruiting (or excluding) specific readers, histone modifications can regulate chromatin dynamics and, by extension, DNA-dependent processes. However, until very recently, the direct impact of histone PTMs on nucleosome structure and thus on chromatin function has remained somewhat elusive. Recent findings of novel sites and types of histone PTMs located within the globular domain of histones and, in particular, on the lateral surface of the histone octamer have changed this. As a result of their structurally important location in close proximity to the DNA molecule, this new class of histone PTMs can have a direct impact on chromatin function. Depending on their precise position at the nucleosome lateral surface (e.g. near the DNA entry/exit sites or in the dyad region), histone PTMs can regulate nucleosome structure and/or stability differently. We review recent progress on how histone PTMs can influence DNA unwrapping and/or nucleosome disassembly and shed light on how these types of novel modifications contribute mechanistically to the regulation of transcriptional activity.

  15. Histone modifications and regeneration in the planarian Schmidtea mediterranea.

    PubMed

    Robb, Sofia M C; Sánchez Alvarado, Alejandro

    2014-01-01

    The freshwater planarian Schmidtea mediterranea has emerged as a powerful model system for studying regeneration and adult stem cell (ASC) biology. This is largely due to the developmental plasticity of these organisms and the abundant distribution and experimental accessibility of their ASCs. Techniques such as whole mount in situ hybridization, dsRNA-mediated interference, halogenated thymidine analogs for defining cell lineages, and fluorescence-activated cell sorting among other methods, have allowed researchers to interrogate the biology and attendant pluripotent stem cells of these animals in great detail. Therefore, it has now become possible to interrogate and define the roles that epigenetic states may play in regulating ASCs, and by extension, regeneration proper. Here, we provide a primer on the types and number of histone families found in S. mediterranea, known as epigenetic marks of these molecules and a survey of epigenetic modifying enzymes encoded by the planarian genome. We also review experimental evidence indicating that such modifications may in fact play key roles in determining the activities of planarian stem cells. © 2014 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  17. Histone Modifications Depict an Aberrantly Heterochromatinized FMR1 Gene in Fragile X Syndrome

    PubMed Central

    Coffee, Bradford; Zhang, Fuping; Ceman, Stephanie; Warren, Stephen T.; Reines, Daniel

    2002-01-01

    Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation but not with chromatin condensation. We also identify alterations in patient cells of two recently identified histone H3 modifications: methylation of histone H3 at lysine 4 and methylation of histone H3 at lysine 9, which are marks for euchromatin and heterochromatin, respectively. In fragile X cells, there is a decrease in methylation of histone H3 at lysine 4 with a large increase in methylation at lysine 9, a change that is consistent with the model of FMR1's switch from euchromatin to heterochromatin in the disease state. The high level of histone H3 methylation at lysine 9 may account for the failure of H3 to be acetylated after treatment of fragile X cells with inhibitors of histone deacetylases, a treatment that fully restores acetylation to histone H4. Using 5-aza-2′-deoxycytidine, we show that DNA methylation is tightly coupled to the histone modifications associated with euchromatin but not to the heterochromatic mark of methylation of histone H3 at lysine 9, consistent with recent findings that this histone modification may direct DNA methylation. Despite the drug-induced accumulation of mRNA in patient cells to 35% of the wild-type level, FMR1 protein remained undetectable. The identification of intermediates in the heterochromatinization of FMR1 has enabled us to begin to dissect the epigenetics of silencing of a disease-related gene in its natural chromosomal context. PMID:12232854

  18. Using ChIP-Seq Technology to Generate High-Resolution Profiles of Histone Modifications

    PubMed Central

    O’Geen, Henriette; Echipare, Lorigail; Farnham, Peggy J.

    2014-01-01

    The dynamic modification of DNA and histones plays a key role in transcriptional regulation through altering the packaging of DNA and modifying the nucleosome surface. These chromatin states, also referred to as the epigenome, are distinctive for different tissues, developmental stages, and disease states and can also be altered by environmental influences. New technologies allow the genome-wide visualization of the information encoded in the epigenome. For example, the chromatin immunoprecipitation (ChIP) assay allows investigators to characterize DNA–protein interactions in vivo. ChIP followed by hybridization to microarrays (ChIP-chip) or by high-throughput sequencing (ChIP-seq) are both powerful tools to identify genome-wide profiles of transcription factors, histone modifications, DNA methylation, and nucleosome positioning. ChIP-seq technology, which can now interrogate the entire human genome at high resolution with only one lane of sequencing, has recently surpassed ChIP-chip technology for epigenomic analyses. Importantly, for the study of primary cells and tissues, epigenetic profiles can be generated using as little as 1 μg of chromatin. In this chapter, we describe in detail the steps involved in performing ChIP assays (with a focus on characterizing histone modifications in primary cells) either manually or using the IP-Star ChIP robot, followed by a detailed protocol to prepare successful libraries for Illumina sequencing. Critical quality control checkpoints are discussed. Although not a focus of this chapter, we also point the reader to several methods by which massive ChIP-seq data sets can be analyzed to extract the tremendous information contained within. PMID:21913086

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

    USDA-ARS?s Scientific Manuscript database

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

  20. RNF8-dependent histone modifications regulate nucleosome removal during spermatogenesis.

    PubMed

    Lu, Lin-Yu; Wu, Jiaxue; Ye, Lin; Gavrilina, Galina B; Saunders, Thomas L; Yu, Xiaochun

    2010-03-16

    During spermatogenesis, global nucleosome removal occurs where histones are initially replaced by transition proteins and subsequently by protamines. This chromatin reorganization is thought to facilitate the compaction of the paternal genome into the sperm head and to protect the DNA from damaging agents. Histone ubiquitination has been suggested to be important for sex chromosome inactivation during meiotic prophase and nucleosome removal at postmeiotic stages. However, the mechanisms regulating these ubiquitin-mediated processes are unknown. In this study, we investigate the role of the ubiquitin ligase RNF8 during spermatogenesis and find that RNF8-deficient mice are proficient in meiotic sex chromosome inactivation (MSCI) but deficient in global nucleosome removal. Moreover, we show that RNF8-dependent histone ubiquitination induces H4K16 acetylation, which may be an initial step in nucleosome removal. Thus, our results show that RNF8 plays an important role during spermatogenesis through histone ubiquitination, resulting in trans-histone acetylation and global nucleosome removal.

  1. Histones and Their Modifications in Ovarian Cancer – Drivers of Disease and Therapeutic Targets

    PubMed Central

    Marsh, Deborah J.; Shah, Jaynish S.; Cole, Alexander J.

    2014-01-01

    Epithelial ovarian cancer has the highest mortality of the gynecological malignancies. High grade serous epithelial ovarian cancer (SEOC) is the most common subtype, with the majority of women presenting with advanced disease where 5-year survival is around 25%. Platinum-based chemotherapy in combination with paclitaxel remains the most effective treatment despite platinum therapies being introduced almost 40 years ago. Advances in molecular medicine are underpinning new strategies for the treatment of cancer. Major advances have been made by international initiatives to sequence cancer genomes. For SEOC, with the exception of TP53 that is mutated in virtually 100% of these tumors, there is no other gene mutated at high frequency. There is extensive copy number variation, as well as changes in methylation patterns that will influence gene expression. To date, the role of histones and their post-translational modifications in ovarian cancer is a relatively understudied field. Post-translational histone modifications play major roles in gene expression as they direct the configuration of chromatin and so access by transcription factors. Histone modifications include methylation, acetylation, and monoubiquitination, with involvement of enzymes including histone methyltransferases, histone acetyltransferases/deacetylases, and ubiquitin ligases/deubiquitinases, respectively. Complexes such as the Polycomb repressive complex also play roles in the control of histone modifications and more recently roles for long non-coding RNA and microRNAs are emerging. Epigenomic-based therapies targeting histone modifications are being developed and offer new approaches for the treatment of ovarian cancer. Here, we discuss histone modifications and their aberrant regulation in malignancy and specifically in ovarian cancer. We review current and upcoming histone-based therapies that have the potential to inform and improve treatment strategies for women with ovarian cancer. PMID

  2. Characterization of antimicrobial histone sequences and posttranslational modifications by mass spectrometry.

    PubMed

    Ouvry-Patat, Séverine A; Schey, Kevin L

    2007-05-01

    Histones typically play a role in DNA packaging and transcription regulation. These proteins are heavily modified by acetylation, methylation, phosphorylation and/or ubiquitination, and various combinations of these modifications alter histone functions and form the basis of the histone code. Furthermore, histones, including those found in shrimp, have recently been found to possess antimicrobial properties; however, the sequences and posttranslational modifications of shrimp histones are largely unknown. In this study mass spectrometry was used to characterize the primary structure of the shrimp antimicrobial histone. A combination of in-solution digestion and in-gel propionylation/digestion followed by LC-MS-MS and MALDI-TOF-TOF analysis was used. Over 80% of each histone sequence was obtained by in-solution digestion; however, none of the N-terminal domains was sequenced with this method. An in-gel propionylation method was optimized to recover and sequence the extremely hydrophilic histone N-termini. This method was then applied to shrimp hemocyte lysates separated on a 1-D SDS-PAGE gel. Overall, 95% coverage was obtained for the histone sequences as well as the identification of posttranslational sites such as acetylation, methylation and phosphorylation.

  3. Synergistic Modification Induced Specific Recognition between Histone and TRIM24 via Fluctuation Correlation Network Analysis

    PubMed Central

    Zhang, Jinmai; Luo, Huajie; Liu, Hao; Ye, Wei; Luo, Ray; Chen, Hai-Feng

    2016-01-01

    Histone modification plays a key role in gene regulation and gene expression. TRIM24 as a histone reader can recognize histone modification. However the specific recognition mechanism between TRIM24 and histone modification is unsolved. Here, systems biology method of dynamics correlation network based on molecular dynamics simulation was used to answer the question. Our network analysis shows that the dynamics correlation network of H3K23ac is distinctly different from that of wild type and other modifications. A hypothesis of “synergistic modification induced recognition” is then proposed to link histone modification and TRIM24 binding. These observations were further confirmed from community analysis of networks with mutation and network perturbation. Finally, a possible recognition pathway is also identified based on the shortest path search for H3K23ac. Significant difference of recognition pathway was found among different systems due to methylation and acetylation modifications. The analysis presented here and other studies show that the dynamic network-based analysis might be a useful general strategy to study the biology of protein post-translational modification and associated recognition. PMID:27079666

  4. Synergistic Modification Induced Specific Recognition between Histone and TRIM24 via Fluctuation Correlation Network Analysis

    NASA Astrophysics Data System (ADS)

    Zhang, Jinmai; Luo, Huajie; Liu, Hao; Ye, Wei; Luo, Ray; Chen, Hai-Feng

    2016-04-01

    Histone modification plays a key role in gene regulation and gene expression. TRIM24 as a histone reader can recognize histone modification. However the specific recognition mechanism between TRIM24 and histone modification is unsolved. Here, systems biology method of dynamics correlation network based on molecular dynamics simulation was used to answer the question. Our network analysis shows that the dynamics correlation network of H3K23ac is distinctly different from that of wild type and other modifications. A hypothesis of “synergistic modification induced recognition” is then proposed to link histone modification and TRIM24 binding. These observations were further confirmed from community analysis of networks with mutation and network perturbation. Finally, a possible recognition pathway is also identified based on the shortest path search for H3K23ac. Significant difference of recognition pathway was found among different systems due to methylation and acetylation modifications. The analysis presented here and other studies show that the dynamic network-based analysis might be a useful general strategy to study the biology of protein post-translational modification and associated recognition.

  5. Profiling Changes in Histone Post-translational Modifications by Top-Down Mass Spectrometry

    SciTech Connect

    Zhou, Mowei; Wu, Si; Stenoien, David L.; Zhang, Zhaorui; Connolly, Lanelle; Freitag, Michael; Pasa-Tolic, Ljiljana

    2016-11-11

    Top-down mass spectrometry is a valuable tool for charactering post-translational modifications on histones for understanding of gene control and expression. In this protocol, we describe a top-down workflow using liquid chromatography coupled to mass spectrometry for fast global profiling of changes in histone proteoforms between a wild-type and a mutant of a fungal species. The proteoforms exhibiting different abundances can be subjected to further targeted studies by other mass spectrometric or biochemical assays. This method can be generally adapted for preliminary screening for changes in histone modifications between samples such as wild-type vs. mutant, and control vs. disease.

  6. Measuring dynamic changes in histone modifications and nucleosome density during activated transcription in budding yeast.

    PubMed

    Govind, Chhabi K; Ginsburg, Daniel; Hinnebusch, Alan G

    2012-01-01

    Chromatin immunoprecipitation is widely utilized to determine the in vivo binding of factors that regulate transcription. This procedure entails formaldehyde-mediated cross-linking of proteins and isolation of soluble chromatin followed by shearing. The fragmented chromatin is subjected to immunoprecipitation using antibodies against the protein of interest and the associated DNA is identified using quantitative PCR. Since histones are posttranslationally modified during transcription, this technique can be effectively used to determine the changes in histone modifications that occur during transcription. In this paper, we describe a detailed methodology to determine changes in histone modifications in budding yeast that takes into account reductions in nucleosome.

  7. Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets – “Sand out and Gold Stays”

    PubMed Central

    Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y.; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T.; Wang, Hong; Yang, Xiao-feng

    2016-01-01

    To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: 1) Histone enzymes are differentially expressed in cardiovascular, immune and other tissues; 2) Our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, histone methylation/demethylation are in the highest varieties; and 3) Histone enzymes are more downregulated than upregulated in metabolic diseases and Treg polarization/differentiation, but not in tumors. These results have demonstrated a new working model of “sand out and gold stays,” where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity. PMID:26746407

  8. Metabolic Diseases Downregulate the Majority of Histone Modification Enzymes, Making a Few Upregulated Enzymes Novel Therapeutic Targets--"Sand Out and Gold Stays".

    PubMed

    Shao, Ying; Chernaya, Valeria; Johnson, Candice; Yang, William Y; Cueto, Ramon; Sha, Xiaojin; Zhang, Yi; Qin, Xuebin; Sun, Jianxin; Choi, Eric T; Wang, Hong; Yang, Xiao-feng

    2016-02-01

    To determine whether the expression of histone modification enzymes is regulated in physiological and pathological conditions, we took an experimental database mining approach pioneered in our labs to determine a panoramic expression profile of 164 enzymes in 19 human and 17 murine tissues. We have made the following significant findings: (1) Histone enzymes are differentially expressed in cardiovascular, immune, and other tissues; (2) our new pyramid model showed that heart and T cells are among a few tissues in which histone acetylation/deacetylation, and histone methylation/demethylation are in the highest varieties; and (3) histone enzymes are more downregulated than upregulated in metabolic diseases and regulatory T cell (Treg) polarization/ differentiation, but not in tumors. These results have demonstrated a new working model of "Sand out and Gold stays," where more downregulation than upregulation of histone enzymes in metabolic diseases makes a few upregulated enzymes the potential novel therapeutic targets in metabolic diseases and Treg activity.

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones. PMID:21965532

  11. Human CRP defends against the toxicity of circulating histones.

    PubMed

    Abrams, Simon T; Zhang, Nan; Dart, Caroline; Wang, Susan Siyu; Thachil, Jecko; Guan, Yunyan; Wang, Guozheng; Toh, Cheng-Hock

    2013-09-01

    C-reactive protein (CRP) is an acute-phase protein that plays an important defensive role in innate immunity against bacterial infection, but it is also upregulated in many noninfectious diseases. The generic function of this highly conserved molecule in diseases that range from infection, inflammation, trauma, and malignancy is not well understood. In this article, we demonstrate that CRP defends the human body against the toxicity of histones released into the circulation after extensive cell death. In vitro, CRP significantly alleviates histone-induced endothelial cell damage, permeability increase, and platelet aggregation. In vivo, CRP rescues mice challenged with lethal doses of histones by inhibiting endothelial damage, vascular permeability, and coagulation activation, as reflected by significant reductions in lung edema, hemorrhage, and thrombosis. In patients, elevation of CRP significantly increases the capacity to neutralize extracellular histones in the circulation. We have also confirmed that CRP interacts with individual histones in vitro and forms CRP-histone complexes in serum from patients with both elevated CRP and histones. CRP is able to compete with phospholipid-containing liposomes for the binding to histones. This explains how CRP prevents histones from integrating into cell membranes, which would otherwise induce calcium influx as the major mechanism of cytotoxicity caused by extracellular histones. Because histone elevation occurs in the acute phase of numerous critical illnesses associated with extensive cell death, CRP detoxification of circulating histones would be a generic host defense mechanism in humans.

  12. Dynamic and distinct histone modifications modulate the expression of key adipogenesis regulatory genes.

    PubMed

    Zhang, Qiongyi; Ramlee, Muhammad Khairul; Brunmeir, Reinhard; Villanueva, Claudio J; Halperin, Daniel; Xu, Feng

    2012-12-01

    Histone modifications and their modifying enzymes are fundamentally involved in the epigenetic regulation of adipogenesis. This study aimed to define the roles of various histone modifications and their "division of labor" in fat cell differentiation. To achieve these goals, we examined the distribution patterns of eight core histone modifications at five key adipogenic regulatory genes, Pref-1, C/EBPβ, C/EBPα, PPARγ2 and aP2, during the adipogenesis of C3H 10T1/2 mouse mesenchymal stem cells (MSCs) and 3T3-L1 preadipocytes. We found that the examined histone modifications are globally stable throughout adipogenesis but show distinct and highly dynamic distribution patterns at specific genes. For example, the Pref-1 gene has lower levels of active chromatin markers and significantly higher H3 K27 tri-methylation in MSCs compared with committed preadipocytes; the C/EBPβ gene is enriched in active chromatin markers at its 3'-UTR; the C/EBPα gene is predominantly marked by H3 K27 tri-methylation in adipogenic precursor cells, and this repressive marker decreases dramatically upon induction; the PPARγ2 and aP2 genes show increased histone acetylation on both H3 and H4 tails during adipogenesis. Further functional studies revealed that the decreased level of H3 K27 tri-methylation leads to de-repression of Pref-1 gene, while the increased level of histone acetylation activates the transcription of PPARγ2 and aP2 genes. Moreover, the active histone modification-marked 3'-UTR of C/EBPβ gene was demonstrated as a strong enhancer element by luciferase assay. Our results indicate that histone modifications are gene-specific at adipogenic regulator genes, and they play distinct roles in regulating the transcriptional network during adipogenesis.

  13. Oxidative stress alters global histone modification and DNA methylation.

    PubMed

    Niu, Yingmei; DesMarais, Thomas L; Tong, Zhaohui; Yao, Yixin; Costa, Max

    2015-05-01

    The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H2O2) for 3h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2',7'-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H2O2 inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.

  14. Therapeutic Targeting of Histone Modifications in Adult and Pediatric High-Grade Glioma

    PubMed Central

    Williams, Maria J.; Singleton, Will G. B.; Lowis, Stephen P.; Malik, Karim; Kurian, Kathreena M.

    2017-01-01

    Recent exciting work partly through The Cancer Genome Atlas has implicated epigenetic mechanisms including histone modifications in the development of both pediatric and adult high-grade glioma (HGG). Histone lysine methylation has emerged as an important player in regulating gene expression and chromatin function. Lysine (K) 27 (K27) is a critical residue in all seven histone 3 variants and the subject of posttranslational histone modifications, as it can be both methylated and acetylated. In pediatric HGG, two critical single-point mutations occur in the H3F3A gene encoding the regulatory histone variant H3.3. These mutations occur at lysine (K) 27 (K27M) and glycine (G) 34 (G34R/V), both of which are involved with key regulatory posttranscriptional modifications. Therefore, these mutations effect gene expression, cell differentiation, and telomere maintenance. In recent years, alterations in histone acetylation have provided novel opportunities to explore new pharmacological targeting, with histone deacetylase (HDAC) overexpression reported in high-grade, late-stage proliferative tumors. HDAC inhibitors have shown promising therapeutic potential in many malignancies. This review focuses on the epigenetic mechanisms propagating pediatric and adult HGGs, as well as summarizing the current advances in clinical trials using HDAC inhibitors. PMID:28401060

  15. Genome-wide analysis of histone modifications in latently HIV-1 infected T cells.

    PubMed

    Park, Jihwan; Lim, Chae Hyun; Ham, Seokjin; Kim, Sung Soon; Choi, Byeong-Sun; Roh, Tae-Young

    2014-07-31

    The transcriptional silencing of HIV type 1 (HIV-1) provirus in latently infected cells is a major hurdle on the pathway to HIV-1 elimination. The epigenetic mechanisms established by histone modifications may affect the transcriptional silencing of HIV-1 and viral latency. A systematic epigenome profiling could be applicable to develop new epigenetic diagnostic markers for detecting HIV-1 latency. The HIV-1 latency cell lines (NCHA1, NCHA2 and ACH2] were compared with CD4⁺ T-cell line (A3.01). The histone modification profiles obtained from chromatin immunoprecipiation followed by sequencing (ChIP-Seq) for histone H3K4me3 and H3K9ac were systematically examined and differential gene expression patterns along with levels of histone modifications were used for network analysis. The HIV-1 latency gave rise to downregulation of histone H3K4me3 and H3K9ac levels in 387 and 493 regions and upregulation in 451 and 962 sites, respectively. By network analysis, five gene clusters were associated with downregulated histone modifications and six gene clusters came up with upregulated histone modifications. Integration of gene expression with epigenetic information revealed that the cell cycle regulatory genes such as CDKN1A (p21) and cyclin D2 (CCND2) identified by differentially modified histones might play an important role in maintaining the HIV-1 latency. The transcriptional regulation by epigenetic memory should play a key role in the evolution and maintenance of HIV-1 latency accompanied by modulation of signalling molecules in the host cells.

  16. Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications.

    PubMed

    Arnaudo, Anna M; Link, A James; Garcia, Benjamin A

    2016-03-18

    The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers. Here, we present a method that merges bioorthogonal chemistry with mass spectrometry for the study of modifications on newly synthesized histones in mammalian cells. HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine and heavy (13)C6,(15)N4 isotope labeled arginine. Heavy amino acid labeling marks newly synthesized histones while azidohomoalanine incorporation allows for their isolation using bioorthogonal ligation. Labeled mononucleosomes were covalently linked via a copper catalyzed reaction to a FLAG-GGR-alkyne peptide, immunoprecipitated, and subjected to mass spectrometry for quantitative modification analysis. Mononucleosomes containing new histones were successfully isolated using this approach. Additionally, the development of this method highlights the potential deleterious effects of azidohomoalanine labeling on protein PTMs and cell cycle progression, which should be considered for future studies utilizing bioorthogonal labeling strategies in mammalian cells.

  17. Antibodies from the sera of HIV-infected patients efficiently hydrolyze all human histones.

    PubMed

    Baranova, Svetlana V; Buneva, Valentina N; Nevinsky, Georgy A

    2016-08-01

    Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Here, using ELISA it was shown that sera of HIV-infected patients and healthy donors contain autoantibodies against histones. Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether antibodies from sera of HIV-infected patients can hydrolyze human histones. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of HIV-infected patients by chromatography on several affinity sorbents. We present first evidence showing that 100% of IgGs purified from the sera of 32 HIV-infected patients efficiently hydrolyze from one to five human histones. Several rigid criteria have been applied to show that the histone-hydrolyzing activity is an intrinsic property of IgGs of HIV-infected patients. The relative efficiency of hydrolysis of histones (H1, H2a, H2b, H3, and H4) significantly varied for IgGs of different patients. IgGs from the sera of 40% of healthy donors also hydrolyze histones but with an average efficiency approximately 16-fold lower than that of HIV-infected patients. Similar to proteolytic abzymes from the sera of patients with several autoimmune diseases, histone-hydrolyzing IgGs from HIV-infected patients were inhibited by specific inhibitors of serine and of metal-dependent proteases, but an unexpected significant inhibition of the activity by specific inhibitor of thiol-like proteases was also observed. Because IgGs can efficiently hydrolyze histones, a negative role of abzymes in development of acquired immune deficiency syndrome cannot be

  18. Brownian dynamics simulation of the effect of histone modification on nucleosome structure

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2007-05-01

    Using Brownian dynamics we simulate the effect of histone modification, such as phosphorylation, acetylation, and methylation, on nucleosome structure by varying the interaction force between DNA and the histone octamer. The simulation shows that the structural stability of nucleosome is very sensitive to the interaction force, and the DNA unwrapping from the modified histone octamer usually occurs turn by turn. Furthermore, the effects of temperature and DNA break as well as the competition between modified and normal histone octamers are investigated, with the simulation results being in agreement with the experimental observation that phosphorylated nucleosomes near DNA breaks are more easily depleted. Though the simulation study may only give a coarse grained view of the DNA unwrapping process for the modified histone octamer, it may provide insight into the mechanism of DNA repair.

  19. Histone Modifications, Modifiers and Readers in Melanoma Resistance to Targeted and Immune Therapy

    PubMed Central

    Gallagher, Stuart J; Tiffen, Jessamy C; Hersey, Peter

    2015-01-01

    The treatment of melanoma has been revolutionized by new therapies targeting MAPK signaling or the immune system. Unfortunately these therapies are hindered by either primary resistance or the development of acquired resistance. Resistance mechanisms involving somatic mutations in genes associated with resistance have been identified in some cases of melanoma, however, the cause of resistance remains largely unexplained in other cases. The importance of epigenetic factors targeting histones and histone modifiers in driving the behavior of melanoma is only starting to be unraveled and provides significant opportunity to combat the problems of therapy resistance. There is also an increasing ability to target these epigenetic changes with new drugs that inhibit these modifications to either prevent or overcome resistance to both MAPK inhibitors and immunotherapy. This review focuses on changes in histones, histone reader proteins and histone positioning, which can mediate resistance to new therapeutics and that can be targeted for future therapies. PMID:26426052

  20. Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

    PubMed Central

    Brunmeir, Reinhard; Lagger, Sabine; Simboeck, Elisabeth; Sawicka, Anna; Egger, Gerda; Hagelkruys, Astrid; Zhang, Yu; Matthias, Patrick

    2010-01-01

    Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model

  1. Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

    PubMed Central

    Sidoli, Simone; Bhanu, Natarajan V.; Karch, Kelly R.; Wang, Xiaoshi; Garcia, Benjamin A.

    2016-01-01

    Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude. PMID:27286567

  2. Apparent microRNA-Target-specific Histone Modification in Mammalian Spermatogenesis

    PubMed Central

    Taguchi, Y-H

    2015-01-01

    BACKGROUND Epigenetics is an important mRNA expression regulator. However, how distinct epigenetic factors, such as microRNAs (miRNAs) and promoter methylation, cooperatively regulate mRNA expression is rarely discussed. Recently, apparent miRNA regulation of promoter methylation was identified by bioinformatic analysis; however, it has not yet been experimentally confirmed. If miRNA regulation of other epigenetic factors were identified, it would reveal another layer of epigenetic regulation. In this paper, histone modifications (H3K4me1, H3K4me3, H3K27me3, H3K27ac, H3K9ac, and H2AZ) during mammalian spermatogenesis were studied and the apparent miRNA-target-specific histone modification was investigated by bioinformatic analyses of publicly available datasets. RESULTS We identified several miRNAs’ target genes that are significantly associated with histone modification during mammalian spermatogenesis. MiRNAs that target genes associated with the most significant histone modifications are expressed before or during spermatogenesis; thus the results were convincing. CONCLUSIONS In this paper, we identified apparent miRNA regulation of histone modifications using a bioinformatics approach. The biological mechanisms of this effect should be further experimentally investigated. PMID:25780334

  3. 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

  4. 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.

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

    PubMed

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

    2014-01-01

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

  6. Enhanced top-down characterization of histone post-translational modifications

    SciTech Connect

    Tian, Zhixin; Tolić, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Paša-Tolić, Ljiljana

    2012-01-01

    Background: Multiple post-translational modifications (PTMs) on core histones often work synergistically to fine tune chromatin structure and functions, generating a “histone code” that can be interpreted by a variety of chromatin interacting proteins. Although previous bottom-up and middle-down proteomic approaches have been developed for limited characterization of PTMs on histone N-terminal tails, high-throughput methods for comprehensive identification of PTMs distributed along the entire primary amino acid sequence are yet to be implemented. Results: Here we report a novel online two-dimensional liquid chromatography - tandem mass spectrometry (2D LC–MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The metal-free LC system with reverse phase separation followed by weak cation exchange – hydrophilic interaction chromatography (WCX-HILIC) and online Orbitrap Velos tandem mass spectrometry allowed for unambiguous identification of over 700 histone isoforms from a single 2D LC–MS/MS analysis of 7.5 µg of purified core histones. In comparison with previous offline top-down analysis of H4, this online study identified 100 additional isoforms from 100-fold less sample. This platform enabled comprehensive characterization of histone modifications, including those beyond tail regions, with dramatically improved throughput and sensitivity compared to more traditional platforms. Isoforms identified included those with combinatorial PTMs extending well beyond the N-terminal tail regions as well as a large number of phosphorylated isoforms.

  7. 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.

  8. An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.

    PubMed

    Veluchamy, Alaguraj; Rastogi, Achal; Lin, Xin; Lombard, Bérangère; Murik, Omer; Thomas, Yann; Dingli, Florent; Rivarola, Maximo; Ott, Sandra; Liu, Xinyue; Sun, Yezhou; Rabinowicz, Pablo D; McCarthy, James; Allen, Andrew E; Loew, Damarys; Bowler, Chris; Tirichine, Leïla

    2015-05-20

    Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.

  9. Statistical mechanics model for the dynamics of collective epigenetic histone modification.

    PubMed

    Zhang, Hang; Tian, Xiao-Jun; Mukhopadhyay, Abhishek; Kim, K S; Xing, Jianhua

    2014-02-14

    Epigenetic histone modifications play an important role in the maintenance of different cell phenotypes. The exact molecular mechanism for inheritance of the modification patterns over cell generations remains elusive. We construct a Potts-type model based on experimentally observed nearest-neighbor enzyme lateral interactions and nucleosome covalent modification state biased enzyme recruitment. The model can lead to effective nonlocal interactions among nucleosomes suggested in previous theoretical studies, and epigenetic memory is robustly inheritable against stochastic cellular processes.

  10. Statistical Mechanics Model for the Dynamics of Collective Epigenetic Histone Modification

    NASA Astrophysics Data System (ADS)

    Zhang, Hang; Tian, Xiao-Jun; Mukhopadhyay, Abhishek; Kim, K. S.; Xing, Jianhua

    2014-02-01

    Epigenetic histone modifications play an important role in the maintenance of different cell phenotypes. The exact molecular mechanism for inheritance of the modification patterns over cell generations remains elusive. We construct a Potts-type model based on experimentally observed nearest-neighbor enzyme lateral interactions and nucleosome covalent modification state biased enzyme recruitment. The model can lead to effective nonlocal interactions among nucleosomes suggested in previous theoretical studies, and epigenetic memory is robustly inheritable against stochastic cellular processes.

  11. Changes to histone modifications following prenatal alcohol exposure: An emerging picture.

    PubMed

    Chater-Diehl, Eric J; Laufer, Benjamin I; Singh, Shiva M

    2017-05-01

    Epigenetic mechanisms are important for facilitating gene-environment interactions in many disease etiologies, including Fetal Alcohol Spectrum Disorders (FASD). Extensive research into the role of DNA methylation and miRNAs in animal models has illuminated the complex role of these mechanisms in FASD. In contrast, histone modifications have not been as well researched, due in part to being less stable than DNA methylation and less well-characterized in disease. It is now apparent that even changes in transient marks can have profound effects if they alter developmental trajectories. In addition, many histone methylations are now known to be relatively stable and can propagate themselves. As technologies and knowledge have advanced, a small group has investigated the role of histone modifications in FASD. Here, we synthesize the data on the effects of prenatal alcohol exposure (PAE) on histone modifications. Several key points are evident. AS with most alcohol-induced outcomes, timing and dosage differences yield variable effects. Nevertheless, these studies consistently find enrichment of H3K9ac, H3K27me2,3, and H3K9me2, and increased expression of histone acetyltransferases and methyltransferases. The consistency of these alterations may implicate them as key mechanisms underlying FASD. Histone modification changes do not often correlate with gene expression changes, though some important examples exist. Encouragingly, attempts to reproduce specific histone modification changes are very often successful. We comment on possible directions for future studies, focusing on further exploration of current trends, expansion of time-point and dosage regimes, and evaluation of biomarker potential. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Critical roles of non-histone protein lysine methylation in human tumorigenesis.

    PubMed

    Hamamoto, Ryuji; Saloura, Vassiliki; Nakamura, Yusuke

    2015-02-01

    Several protein lysine methyltransferases and demethylases have been identified to have critical roles in histone modification. A large body of evidence has indicated that their dysregulation is involved in the development and progression of various diseases, including cancer, and these enzymes are now considered to be potential therapeutic targets. Although most studies have focused on histone methylation, many reports have revealed that these enzymes also regulate the methylation dynamics of non-histone proteins such as p53, RB1 and STAT3 (signal transducer and activator of transcription 3), which have important roles in human tumorigenesis. In this Review, we summarize the molecular functions of protein lysine methylation and its involvement in human cancer, with a particular focus on lysine methylation of non-histone proteins.

  13. Effects of low-level laser therapy on M1-related cytokine expression in monocytes via histone modification.

    PubMed

    Chen, Chia-Hsin; Wang, Chau-Zen; Wang, Yan-Hsiung; Liao, Wei-Ting; Chen, Yi-Jen; Kuo, Chang-Hung; Kuo, Hsuan-Fu; Hung, Chih-Hsing

    2014-01-01

    Low-level laser therapy (LLLT) has been used in the treatment of radiotherapy-induced oral mucositis and allergic rhinitis. However, the effects of LLLT on human monocyte polarization into M1 macrophages are unknown. To evaluate the effects of LLLT on M1-related cytokine and chemokine production and elucidate the mechanism, the human monocyte cell line THP-1 was treated with different doses of LLLT. The expression of M1-related cytokines and chemokines (CCL2, CXCL10, and TNF-α) was determined by ELISA and real-time PCR. LLLT-associated histone modifications were examined by chromatin immunoprecipitation (ChIP) assays. Mitochondrial involvement in the LLLT-induced M1-related cytokine expression was evaluated by quantitative real-time PCR. Flow cytometry was used to detect the cell surface markers for monocyte polarization. The results showed that LLLT (660 nm) significantly enhanced M1-related cytokine and chemokine expression in mRNA and protein levels. Mitochondrial copy number and mRNA levels of complex I-V protein were increased by LLLT (1 J/cm(2)). Activation of M1 polarization was concomitant with histone modification at TNF-α gene locus and IP-10 gene promoter area. This study indicates that LLLT (660 nm) enhanced M1-related cytokine and chemokine expression via mitochondrial biogenesis and histone modification, which may be a potent immune-enhancing agent for the treatment of allergic diseases.

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

    PubMed Central

    Montes de Oca, Rocío; Andreassen, Paul R.

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

  16. Proteolytic histone modification by mast cell tryptase, a serglycin proteoglycan-dependent secretory granule protease.

    PubMed

    Melo, Fabio R; Vita, Francesca; Berent-Maoz, Beata; Levi-Schaffer, Francesca; Zabucchi, Giuliano; Pejler, Gunnar

    2014-03-14

    A hallmark feature of mast cells is their high content of cytoplasmic secretory granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a secretory granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.

  17. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

    PubMed Central

    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David

    2015-01-01

    Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. PMID:25680960

  18. Epigenetic histone modification regulates developmental lead exposure induced hyperactivity in rats.

    PubMed

    Luo, Man; Xu, Yi; Cai, Rong; Tang, Yuqing; Ge, Meng-Meng; Liu, Zhi-Hua; Xu, Li; Hu, Fan; Ruan, Di-Yun; Wang, Hui-Li

    2014-02-10

    Lead (Pb) exposure was commonly considered as a high environmental risk factor for the development of attention-deficit/hyperactivity disorder (ADHD). However, the molecular basis of this pathological process still remains elusive. In light of the role of epigenetics in modulating the neurological disease and the causative environment, the alterations of histone modifications in the hippocampus of rats exposed by various doses of lead, along with concomitant behavioral deficits, were investigated in this study. According to the free and forced open field test, there showed that in a dosage-dependent manner, lead exposure could result in the increased locomotor activity of rats, that is, hyperactivity: a subtype of ADHD. Western blotting assays revealed that the levels of histone acetylation increased significantly in the hippocampus by chronic lead exposure, while no dramatic changes were detected in terms of expression yields of ADHD-related dopaminergic proteins, indicating that histone acetylation plays essential roles in this toxicant-involved pathogenesis. In addition, the increased level of histone acetylation might be attributed to the enzymatic activity of p300, a typical histone acetyltransferase, as the transcriptional level of p300 was significantly increased upon higher-dose Pb exposure. In summary, this study first discovered the epigenetic mechanism bridging the environmental influence (Pb) and the disease itself (ADHD) in the histone modification level, paving the way for the comprehensive understanding of ADHD's etiology and in further steps, establishing the therapy strategy of this widespread neurological disorder.

  19. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction.

    PubMed

    Gilbert, Nick; Thomson, Inga; Boyle, Shelagh; Allan, James; Ramsahoye, Bernard; Bickmore, Wendy A

    2007-05-07

    DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

  20. Global profiling of histone modifications in the polyomavirus BK virion minichromosome.

    PubMed

    Fang, Chiung-Yao; Shen, Cheng-Huang; Wang, Meilin; Chen, Pei-Lain; Chan, Michael W Y; Hsu, Pang-Hung; Chang, Deching

    2015-09-01

    During polyomavirus infection, the viral DNA adopts histones from host cells and forms minichromosomes as an important part of the viral life cycle. However, the detailed mechanisms of this histone incorporation remain unclear. Here, we profiled the histone posttranslational modifications (PTMs) in BKPyV minichromosomes and in the chromatin of BKPyV host cells. Through Triton-acetic acid-urea (TAU)-PAGE separation followed by nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis, we identified different kinds of PTMs on histones from BKPyV minichromosomes and from host cells. We observed not only the common PTMs on histones such as acetylation, methylation, phosphorylation, ubiquitination, and formylation but also several novel PTM sites. Our results also confirmed that the BKPyV minichromosome is hyperacetylated. Our detailed histone PTM profiles for the BKPyV minichromosome provide insights for future exploration of the underlying mechanisms and biological relevance of these histone PTMs. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. The Role of DNA Methylation and Histone Modifications in Neurodegenerative Diseases: A Systematic Review

    PubMed Central

    El-Khodor, Bassem; Dhana, Klodian; Nano, Jana; Pulido, Tammy; Kraja, Bledar; Zaciragic, Asija; Bramer, Wichor M.; Troup, John; Chowdhury, Rajiv; Ikram, M. Arfam; Dehghan, Abbas; Muka, Taulant; Franco, Oscar H.

    2016-01-01

    Importance Epigenetic modifications of the genome, such as DNA methylation and histone modifications, have been reported to play a role in neurodegenerative diseases (ND) such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Objective To systematically review studies investigating epigenetic marks in AD or PD. Methods Eleven bibliographic databases (Embase.com, Medline (Ovid), Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central, ProQuest, Lilacs, Scielo and Google Scholar) were searched until July 11th 2016 to identify relevant articles. We included all randomized controlled trials, cohort, case-control and cross-sectional studies in humans that examined associations between epigenetic marks and ND. Two independent reviewers, with a third reviewer available for disagreements, performed the abstract and full text selection. Data was extracted using a pre-designed data collection form. Results Of 6,927 searched references, 73 unique case-control studies met our inclusion criteria. Overall, 11,453 individuals were included in this systematic review (2,640 AD and 2,368 PD outcomes). There was no consistent association between global DNA methylation pattern and any ND. Studies reported epigenetic regulation of 31 genes (including cell communication, apoptosis, and neurogenesis genes in blood and brain tissue) in relation to AD and PD. Methylation at the BDNF, SORBS3 and APP genes in AD were the most consistently reported associations. Methylation of α-synuclein gene (SNCA) was also found to be associated with PD. Seven studies reported histone protein alterations in AD and PD. Conclusion Many studies have investigated epigenetics and ND. Further research should include larger cohort or longitudinal studies, in order to identify clinically significant epigenetic changes. Identifying relevant epigenetic changes could lead to interventional strategies in ND. PMID:27973581

  2. In situ detection of histone variants and modifications in mouse brain using imaging mass spectrometry.

    PubMed

    Lahiri, Shibojyoti; Sun, Na; Solis-Mezarino, Victor; Fedisch, Andreas; Ninkovic, Jovica; Feuchtinger, Annette; Götz, Magdalena; Walch, Axel; Imhof, Axel

    2016-02-01

    Histone posttranslational modifications and histone variants control the epigenetic regulation of gene expression and affect a wide variety of biological processes. A complex pattern of such modifications and variants defines the identity of cells within complex organ systems and can therefore be used to characterize cells at a molecular level. However, their detection and identification in situ has been limited so far due to lack of specificity, selectivity, and availability of antihistone antibodies. Here, we describe a novel MALDI imaging MS based workflow, which enables us to detect and characterize histones by their intact mass and their correlation with cytological properties of the tissue using novel statistical and image analysis tools. The workflow allows us to characterize the in situ distribution of the major histone variants and their modification in the mouse brain. This new analysis tool is particularly useful for the investigation of expression patterns of the linker histone H1 variants for which suitable antibodies are so far not available. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. The role of global histone post-translational modifications during mammalian hibernation.

    PubMed

    Tessier, Shannon N; Luu, Bryan E; Smith, Jeffrey C; Storey, Kenneth B

    2017-04-01

    Mammalian hibernators must cope with hypothermia, ischemia-reperfusion, and finite fuel reserves during days or weeks of continuous torpor. One means of lowering ATP demands during hibernation involves substantial transcriptional controls. The present research analyzed epigenetic regulatory factors as a means of achieving transcriptional control over cycles of torpor-arousal. This study analyzes differential regulation of select histone modifications (e.g. phosphorylation, acetylation, methylation), and identifies post-translational modifications on purified histones using mass spectrometry from thirteen-lined ground squirrels (Ictidomys tridecemlineatus). Post-translational modifications on histone proteins were responsive to torpor-arousal, suggesting a potential mechanism to dynamically alter chromatin structure. Furthermore, proteomic sequencing data of ground squirrel histones identified lysine 19 and 24 acetylation on histone H3, while acetylation sites identified on H2B were lysine 6, 47, 110, and 117. The present study provides a new glimpse into the epigenetic mechanisms which may play a role in transcriptional regulation during mammalian hibernation. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

    Keck, Kristin M; Pemberton, Lucy F

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  7. Butyrate induced IGF2 activation correlated with distinct chromatin landscapes due to histone modification

    USDA-ARS?s Scientific Manuscript database

    Histone modification has emerged as a very important mechanism regulating the transcriptional status of the genome. Insulin-like growth factor 2 (IGF2) is a peptide hormone controlling various cellular processes such as proliferation and apoptosis. IGF2 and H19 are reciprocally regulated imprinted ...

  8. Epigenomic landscape modified by histone modification correlated with activation of IGF2 gene

    USDA-ARS?s Scientific Manuscript database

    The links of histone post-translational modifications and chromatin structure to cell cycle progression, DNA replication, and overall chromosome functions are very clear. The modulation of genome expression as a consequence of chromatin structural changes is most likely a basic mechanism. The epige...

  9. Comprehensive profiling of histone modifications using a label-free approach and its applications in determining structure-function relationships.

    PubMed

    Drogaris, Paul; Wurtele, Hugo; Masumoto, Hiroshi; Verreault, Alain; Thibault, Pierre

    2008-09-01

    A two-pronged approach using specialized peptide detection and clustering tools was developed to profile changes in histone post-translational modifications (PTMs). The extent and nature of modification was inferred by comparing the mass profiles of intact core histones from nano LC-MS experiments. Histones displaying changes in their intact mass profiles were fractionated, derivatized with propionic anhydride, and digested with trypsin prior to nano LC-MS analyses. Our methodology was validated by comparing the abundance of histone PTMs in wild type and mutant strains of Saccharomyces cerevisiae lacking the histone acetyltransferase Rtt109 and a nucleosome assembly factor known as Asf1. Both Rtt109 and Asf1 were previously found to be essential for acetylation of histone H3 lysine 56 (H3K56ac), a modification that plays an important role in the response to genotoxic agents that interfere with DNA replication. The generation of ion abundance distribution plots enabled a rapid and comprehensive profiling of histone peptides. Our analytical methodology and data mining approach showed that most common histone PTMs were unaffected in mutant yeast cells lacking Rtt109 and Asf1. However, a subpopulation representing 17% of all H3 histones in wild type cells showed an acetylated K56 residue that was significantly reduced in both mutant strains. Our generic strategy for histone PTM profiling can be applied to assess global changes in histone PTMs across sample sets and to establish structure-function relationships.

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

    PubMed

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

    2015-07-30

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

  11. Heterologous expression of human H1 histones in yeast.

    PubMed

    Albig, W; Runge, D M; Kratzmeier, M; Doenecke, D

    1998-09-18

    The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast. Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones. For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid. The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively. This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

  12. Acute ethanol alters multiple histone modifications at model gene promoters in the cerebral cortex.

    PubMed

    Finegersh, Andrey; Homanics, Gregg E

    2014-07-01

    Ethanol (EtOH) exposure alters gene expression in the cerebral cortex (CCx); however, mechanisms of EtOH-induced gene regulation are not well understood. We hypothesized that EtOH regulates gene expression by differentially altering histone modifications at gene promoters that are up- and down-regulated by EtOH. Such epigenetic mechanisms may ultimately contribute to EtOH-induced neuro-adaptations that underlie tolerance, dependence, and EtOH-use disorders. Eight-week-old, male C57BL/6J mice were treated with 3 g/kg EtOH (intraperitoneally) or saline and sacrificed 6 hours after injection; the CCx and hippocampus (HC) were immediately removed and flash frozen. Chromatin immunoprecipitation was used to study the association of model gene promoters with histone modifications. Western blot was used to detect global changes in the histone modifications studied. We also used a polymerase chain reaction (PCR) array to identify changes in expression of chromatin-modifying enzymes. In CCx, acute EtOH decreased expression of Gad1, Hdac2, and Hdac11, which was associated with decreased histone acetylation at the Gad1 and Hdac2 promoters; we also identified increased expression of Mt1, Mt2, Egr1, which was associated with increased H3K4me3 levels at the Mt2 promoter and decreased H3K27me3 levels at the Mt1 promoter. We identified an increase in global levels of H3K4me3 in CCx as well as a global increase in H3K9ac and H3K14ac in HC. The PCR array identified decreased expression of Csrp2 bp, Hdac2, and Hdac11 as well as increased expression of Kat2b in CCx. Acute EtOH induces chromatin remodeling at model up- and down-regulated genes in CCx. Different patterns of histone modifications at these gene promoters indicate that EtOH may be acting through multiple histone-modifying enzymes to alter gene expression; in particular, differential expression of Kat2b, Hdac2, Hdac11, and Csrp2 bp in CCx may mediate EtOH-induced chromatin remodeling. Additional studies are necessary to

  13. Immunostaining of modified histones defines high-level features of the human metaphase epigenome

    PubMed Central

    2010-01-01

    Background Immunolabeling of metaphase chromosome spreads can map components of the human epigenome at the single cell level. Previously, there has been no systematic attempt to explore the potential of this approach for epigenomic mapping and thereby to complement approaches based on chromatin immunoprecipitation (ChIP) and sequencing technologies. Results By immunostaining and immunofluorescence microscopy, we have defined the distribution of selected histone modifications across metaphase chromosomes from normal human lymphoblastoid cells and constructed immunostained karyotypes. Histone modifications H3K9ac, H3K27ac and H3K4me3 are all located in the same set of sharply defined immunofluorescent bands, corresponding to 10- to 50-Mb genomic segments. Primary fibroblasts gave broadly the same banding pattern. Bands co-localize with regions relatively rich in genes and CpG islands. Staining intensity usually correlates with gene/CpG island content, but occasional exceptions suggest that other factors, such as transcription or SINE density, also contribute. H3K27me3, a mark associated with gene silencing, defines a set of bands that only occasionally overlap with gene-rich regions. Comparison of metaphase bands with histone modification levels across the interphase genome (ENCODE, ChIP-seq) shows a close correspondence for H3K4me3 and H3K27ac, but major differences for H3K27me3. Conclusions At metaphase the human genome is packaged as chromatin in which combinations of histone modifications distinguish distinct regions along the euchromatic chromosome arms. These regions reflect the high-level interphase distributions of some histone modifications, and may be involved in heritability of epigenetic states, but we also find evidence for extensive remodeling of the epigenome at mitosis. PMID:21078160

  14. 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

  15. SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple Biological Samples.

    PubMed

    Cuomo, Alessandro; Soldi, Monica; Bonaldi, Tiziana

    2017-01-01

    Histone posttranslational modifications (hPTMs) play a key role in regulating chromatin dynamics and fine-tuning DNA-based processes. Mass spectrometry (MS) has emerged as a versatile technology for the analysis of histones, contributing to the dissection of hPTMs, with special strength in the identification of novel marks and in the assessment of modification cross talks. Stable isotope labeling by amino acid in cell culture (SILAC), when adapted to histones, permits the accurate quantification of PTM changes among distinct functional states; however, its application has been mainly confined to actively dividing cell lines. A spike-in strategy based on SILAC can be used to overcome this limitation and profile hPTMs across multiple samples. We describe here the adaptation of SILAC to the analysis of histones, in both standard and spike-in setups. We also illustrate its coupling to an implemented "shotgun" workflow, by which heavy arginine-labeled histone peptides, produced upon Arg-C digestion, are qualitatively and quantitatively analyzed in an LC-MS/MS system that combines ultrahigh-pressure liquid chromatography (UHPLC) with new-generation Orbitrap high-resolution instrument.

  16. Nitric oxide regulates gene expression in cancers by controlling histone posttranslational modifications

    PubMed Central

    Vasudevan, Divya; Hickok, Jason R.; Bovee, Rhea C.; Pham, Vy; Mantell, Lin L.; Bahroos, Neil; Kanabar, Pinal; Cao, Xing-Jun; Maienschein-Cline, Mark; Garcia, Benjamin A.; Thomas, Douglas D.

    2015-01-01

    Altered nitric oxide (•NO) metabolism underlies cancer pathology, but mechanisms explaining many •NO-associated phenotypes remain unclear. We have found that cellular exposure to •NO changes histone posttranslational modifications (PTMs) by directly inhibiting the catalytic activity of JmjC-domain containing histone demethylases. Herein, we describe how •NO exposure links modulation of histone PTMs to gene expression changes that promote oncogenesis. Through high-resolution mass spectrometry, we generated an extensive map of •NO-mediated histone PTM changes at 15 critical lysine residues on the core histones H3 and H4. Concomitant microarray analysis demonstrated that exposure to physiologic •NO resulted in the differential expression of over 6,500 genes in breast cancer cells. Measurements of the association of H3K9me2 and H3K9ac across genomic loci revealed that differential distribution of these particular PTMs correlated with changes in the level of expression of numerous oncogenes, consistent with epigenetic code. Our results establish that •NO functions as an epigenetic regulator of gene expression mediated by changes in histone PTMs. PMID:26542213

  17. 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.

  18. Engineering chromatin states: chemical and synthetic biology approaches to investigate histone modification function.

    PubMed

    Pick, Horst; Kilic, Sinan; Fierz, Beat

    2014-08-01

    Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Effect of adenovirus infection on expression of human histone genes.

    PubMed Central

    Flint, S J; Plumb, M A; Yang, U C; Stein, G S; Stein, J L

    1984-01-01

    The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA

  20. Post-translational Modifications of Trypanosoma cruzi Canonical and Variant Histones.

    PubMed

    Picchi, Gisele F A; Zulkievicz, Vanessa; Krieger, Marco A; Zanchin, Nilson T; Goldenberg, Samuel; de Godoy, Lyris M F

    2017-03-03

    Chagas disease, caused by Trypanosoma cruzi, still affects millions of people around the world. No vaccines nor treatment for chronic Chagas disease are available, and chemotherapy for the acute phase is hindered by limited efficacy and severe side effects. The processes by which the parasite acquires infectivity and survives in different hosts involve tight regulation of gene expression, mainly post-transcriptionally. Nevertheless, chromatin structure/organization of trypanosomatids is similar to other eukaryotes, including histone variants and post-translational modifications. Emerging evidence suggests that epigenetic mechanisms also play an important role in the biology/pathogenesis of these parasites, making epigenetic targets suitable candidates to drug discovery. Here, we present the first comprehensive map of post-translational modifications of T. cruzi canonical and variant histones and show that its histone code can be as sophisticated as that of other eukaryotes. A total of 13 distinct modification types were identified, including rather novel and unusual ones such as alternative lysine acylations, serine/threonine acetylation, and N-terminal methylation. Some histone marks correlate to those described for other organisms, suggesting that similar regulatory mechanisms may be in place. Others, however, are unique to T. cruzi or to trypanosomatids as a group and might represent good candidates for the development of antiparasitic drugs.

  1. Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.

    PubMed

    Stewart, Kathleen R; Veselovska, Lenka; Kim, Jeesun; Huang, Jiahao; Saadeh, Heba; Tomizawa, Shin-ichi; Smallwood, Sébastien A; Chen, Taiping; Kelsey, Gavin

    2015-12-01

    Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

  2. The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism to affect cell senescence.

    PubMed

    Feng, Yunpeng; Wang, Xiuli; Xu, Liang; Pan, Hong; Zhu, Shan; Liang, Qian; Huang, Baiqu; Lu, Jun

    2009-08-01

    The transcription factor ZBP-89 has been implicated in the induction of growth arrest and apoptosis. In this article, we demonstrate that ZBP-89 was able to restrain senescence in NCI-H460 human lung cancer cells, through epigenetically regulating p(16INK4a) expression. Specifically, our results indicate that knockdown of ZBP-89 by RNA interference stimulated cellular senescence in NCI-H460 cells, as judged by the senescence-associated beta-galactosidase activity assay and senescence-associated heterochromatin foci assay, and this process could be reversed by RNA interference-mediated p16(INK4a) silencing. We also show that histone deacetylase (HDAC) 3 and HDAC4 inhibited p16(INK4a) promoter activity in a dose-dependent manner. Furthermore, chromatin immunoprecipitation assays verified that HDAC3 was recruited to the p16(INK4a) promoter by ZBP-89 through an epigenetic mechanism involving histone acetylation modification. Moreover, immunofluorescence and coimmunoprecipitation assays revealed that ZBP-89 and HDAC3 formed a complex. These data suggest that ZBP-89 and HDAC3, but not HDAC4, can work coordinately to restrain cell senescence by downregulating p16(INK4a) expression through an epigenetic modification of histones.

  3. Genome-wide analyses of four major histone modifications in Arabidopsis hybrids at the germinating seed stage.

    PubMed

    Zhu, Anyu; Greaves, Ian K; Dennis, Elizabeth S; Peacock, W James

    2017-02-07

    Hybrid vigour (heterosis) has been used for decades in cropping agriculture, especially in the production of maize and rice, because hybrid varieties exceed their parents in plant biomass and seed yield. The molecular basis of hybrid vigour is not fully understood. Previous studies have suggested that epigenetic systems could play a role in heterosis. In this project, we investigated genome-wide patterns of four histone modifications in Arabidopsis hybrids in germinating seeds. We found that although hybrids have similar histone modification patterns to the parents in most regions of the genome, they have altered patterns at specific loci. A small subset of genes show changes in histone modifications in the hybrids that correlate with changes in gene expression. Our results also show that genome-wide patterns of histone modifications in geminating seeds parallel those at later developmental stages of seedlings. Ler/C24 hybrids showed similar genome-wide patterns of histone modifications as the parents at an early germination stage. However, a small subset of genes, such as FLC, showed correlated changes in histone modification and in gene expression in the hybrids. The altered patterns of histone modifications for those genes in hybrids could be related to some heterotic traits in Arabidopsis, such as flowering time, and could play a role in hybrid vigour establishment.

  4. 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

  5. Profiling Analysis of Histone Modifications and Gene Expression in Lewis Lung Carcinoma Murine Cells Resistant to Anti-VEGF Treatment

    PubMed Central

    Du, Yanhua; Chen, Kaiming; Liu, Zhenping; Li, Bing; Li, Jie; Tao, Fei; Gu, Hua; Jiang, Cizhong; Fang, Jianmin

    2016-01-01

    Tumor cells become resistant after long-term use of anti-VEGF (vascular endothelial growth factor) agents. Our previous study shows that treatment with a VEGF inhibitor (VEGF-Trap) facilitates to develop tumor resistance through regulating angiogenesis-related genes. However, the underlying molecular mechanisms remain elusive. Histone modifications as a key epigenetic factor play a critical role in regulation of gene expression. Here, we explore the potential epigenetic gene regulatory functions of key histone modifications during tumor resistance in a mouse Lewis lung carcinoma (LLC) cell line. We generated high resolution genome-wide maps of key histone modifications in sensitive tumor sample (LLC-NR) and resistant tumor sample (LLC-R) after VEGF-Trap treatment. Profiling analysis of histone modifications shows that histone modification levels are effectively predictive for gene expression. Composition of promoters classified by histone modification state is different between LLC-NR and LLC-R cell lines regardless of CpG content. Histone modification state change between LLC-NR and LLC-R cell lines shows different patterns in CpG-rich and CpG-poor promoters. As a consequence, genes with different level of CpG content whose gene expression level are altered are enriched in distinct functions. Notably, histone modification state change in promoters of angiogenesis-related genes consists with their expression alteration. Taken together, our findings suggest that treatment with anti-VEGF therapy results in extensive histone modification state change in promoters with multiple functions, particularly, biological processes related to angiogenesis, likely contributing to tumor resistance development. PMID:27362259

  6. Profiling Analysis of Histone Modifications and Gene Expression in Lewis Lung Carcinoma Murine Cells Resistant to Anti-VEGF Treatment.

    PubMed

    Li, Dong; Shi, Jiejun; Du, Yanhua; Chen, Kaiming; Liu, Zhenping; Li, Bing; Li, Jie; Tao, Fei; Gu, Hua; Jiang, Cizhong; Fang, Jianmin

    2016-01-01

    Tumor cells become resistant after long-term use of anti-VEGF (vascular endothelial growth factor) agents. Our previous study shows that treatment with a VEGF inhibitor (VEGF-Trap) facilitates to develop tumor resistance through regulating angiogenesis-related genes. However, the underlying molecular mechanisms remain elusive. Histone modifications as a key epigenetic factor play a critical role in regulation of gene expression. Here, we explore the potential epigenetic gene regulatory functions of key histone modifications during tumor resistance in a mouse Lewis lung carcinoma (LLC) cell line. We generated high resolution genome-wide maps of key histone modifications in sensitive tumor sample (LLC-NR) and resistant tumor sample (LLC-R) after VEGF-Trap treatment. Profiling analysis of histone modifications shows that histone modification levels are effectively predictive for gene expression. Composition of promoters classified by histone modification state is different between LLC-NR and LLC-R cell lines regardless of CpG content. Histone modification state change between LLC-NR and LLC-R cell lines shows different patterns in CpG-rich and CpG-poor promoters. As a consequence, genes with different level of CpG content whose gene expression level are altered are enriched in distinct functions. Notably, histone modification state change in promoters of angiogenesis-related genes consists with their expression alteration. Taken together, our findings suggest that treatment with anti-VEGF therapy results in extensive histone modification state change in promoters with multiple functions, particularly, biological processes related to angiogenesis, likely contributing to tumor resistance development.

  7. cChIP-seq: a robust small-scale method for investigation of histone modifications.

    PubMed

    Valensisi, Cristina; Liao, Jo Ling; Andrus, Colin; Battle, Stephanie L; Hawkins, R David

    2015-12-21

    ChIP-seq is highly utilized for mapping histone modifications that are informative about gene regulation and genome annotations. For example, applying ChIP-seq to histone modifications such as H3K4me1 has facilitated generating epigenomic maps of putative enhancers. This powerful technology, however, is limited in its application by the large number of cells required. ChIP-seq involves extensive manipulation of sample material and multiple reactions with limited quality control at each step, therefore, scaling down the number of cells required has proven challenging. Recently, several methods have been proposed to overcome this limit but most of these methods require extensive optimization to tailor the protocol to the specific antibody used or number of cells being profiled. Here we describe a robust, yet facile method, which we named carrier ChIP-seq (cChIP-seq), for use on limited cell amounts. cChIP-seq employs a DNA-free histone carrier in order to maintain the working ChIP reaction scale, removing the need to tailor reactions to specific amounts of cells or histone modifications to be assayed. We have applied our method to three different histone modifications, H3K4me3, H3K4me1 and H3K27me3 in the K562 cell line, and H3K4me1 in H1 hESCs. We successfully obtained epigenomic maps for these histone modifications starting with as few as 10,000 cells. We compared cChIP-seq data to data generated as part of the ENCODE project. ENCODE data are the reference standard in the field and have been generated starting from tens of million of cells. Our results show that cChIP-seq successfully recapitulates bulk data. Furthermore, we showed that the differences observed between small-scale ChIP-seq data and ENCODE data are largely to be due to lab-to-lab variability rather than operating on a reduced scale. Data generated using cChIP-seq are equivalent to reference epigenomic maps from three orders of magnitude more cells. Our method offers a robust and straightforward

  8. Histone modifications and skeletal muscle metabolic gene expression.

    PubMed

    McGee, Sean L; Hargreaves, Mark

    2010-03-01

    1. Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases. 2. Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression. 3. Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response. 4. The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified. 5. Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases. 6. Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

  9. Comparison of peptide mass mapping and electron capture dissociation as assays for histone posttranslational modifications

    NASA Astrophysics Data System (ADS)

    Zhang, Liwen; Freitas, Michael A.

    2004-05-01

    Posttranslational modifications of core histones play a critical role in the structure of chromatin and the regulation of gene activities. Improved techniques for determining these modification sites may lead to a better understanding of histone regulation at the molecular level. LC-MS peptide mass mapping was performed on pepsin, trypsin and Glu-C digests of bovine thymus H4 using a QqTOF instrument. The well established modification sites of H4 (acetylation of K8, 12, 16 and methylation of K20) were observed in addition to several recently discovered modifications including: methylation of K31, 44, 59 and acetylation of K20, 77, 79. For comparison, electron capture dissociation (ECD) was performed on intact H4 along with several peptides from enzymatic digestion. The results from the ECD experiments of histone H4 indicated the acetylation of K5, 12, 16, 31, 91 and the methylation of K20 and 59 in good agreement with the result from peptide mapping. The work is dedicated to Alan G. Marshall on his 60th birthday. His endeavors in the advancement of FT-ICR facilitated experiments reported herein.

  10. Post-translational modifications of linker histone H1 variants in mammals

    NASA Astrophysics Data System (ADS)

    Starkova, T. Yu; Polyanichko, A. M.; Artamonova, T. O.; Khodorkovskii, M. A.; Kostyleva, E. I.; Chikhirzhina, E. V.; Tomilin, A. N.

    2017-02-01

    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

  11. Post-translational modifications of linker histone H1 variants in mammals.

    PubMed

    Starkova, T Yu; Polyanichko, A M; Artamonova, T O; Khodorkovskii, M A; Kostyleva, E I; Chikhirzhina, E V; Tomilin, A N

    2017-02-16

    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

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

    PubMed Central

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

    2017-01-01

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

  13. Regulation of chromatin structure via histone post-translational modification and the link to carcinogenesis.

    PubMed

    Thompson, Laura L; Guppy, Brent J; Sawchuk, Laryssa; Davie, James R; McManus, Kirk J

    2013-12-01

    The loss of genome integrity contributes to the development of tumors. Although genome instability is associated with virtually all tumor types including both solid and liquid tumors, the aberrant molecular origins that drive this instability are poorly understood. It is now becoming clear that epigenetics and specific histone post-translational modifications (PTMs) have essential roles in maintaining genome stability under normal conditions. A strong relationship exists between aberrant histone PTMs, genome instability, and tumorigenesis. Changes in the genomic location of specific histone PTMs or alterations in the steady-state levels of the PTM are the consequence of imbalances in the enzymes and their activities catalyzing the addition of PTMs ("writers") or removal of PTMs ("erasers"). This review focuses on the misregulation of three specific types of histone PTMs: histone H3 phosphorylation at serines 10 and 28, H4 mono-methylation at lysine 20, and H2B ubiquitination at lysine 120. We discuss the normal regulation of these PTMs by the respective "writers" and "erasers" and the impact of their misregulation on genome stability.

  14. 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.

  15. Histone modifications patterns in tissues and tumours from acute promyelocytic leukemia xenograft model in response to combined epigenetic therapy.

    PubMed

    Valiulienė, Giedrė; Treigytė, Gražina; Savickienė, Jūratė; Matuzevičius, Dalius; Alksnė, Milda; Jarašienė-Burinskaja, Rasa; Bukelskienė, Virginija; Navakauskas, Dalius; Navakauskienė, Rūta

    2016-04-01

    Xenograft models are suitable for in vivo study of leukemia's pathogenesis and the preclinical development of anti-leukemia agents but understanding of epigenetic regulatory mechanisms linking to adult cell functions in pathological conditions during different in vivo treatments is yet unknown. In this study, for the first time epigenetic chromatin modifications were characterized in tissues and tumours from murine xenograft model generated using the human acute promyelocytic leukemia (APL) NB4 cells engrafted in immunodeficient NOG mice. Xenografts were subjected to combined epigenetic treatment by histone deacetylase inhibitor Belinostat, histone methyltransferase inhibitor 3-DZNeaplanocin A and all-trans-retinoic acid based on in vitro model, where such combination inhibited NB4 cell growth and enhanced retinoic acid-induced differentiation to granulocytes. Xenotransplantation was assessed by peripheral blood cells counts, the analysis of cell surface markers (CD15, CD33, CD45) and the expression of certain genes (PML-RAR alpha, CSF3, G-CSFR, WT1). The combined treatment prolonged APL xenograft mice survival and prevented tumour formation. The analysis of the expression of histone marks such as acetylation of H4, trimethylation of H3K4, H3K9 and H3K27 in APL xenograft mice tumours and tissues demonstrated tissue-specific changes in the level of histone modifications and the APL prognostic mark, WT1 protein. In summary, the effects of epigenetic agents used in this study were positive for leukemia prevention and linked to a modulation of the chromatin epigenetic environment in adult tissues of malignant organism.

  16. Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

    PubMed

    Delporte, Annelies; De Zaeytijd, Jeroen; De Storme, Nico; Azmi, Abdelkrim; Geelen, Danny; Smagghe, Guy; Guisez, Yves; Van Damme, Els J M

    2014-10-01

    The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones.

  17. Early histone modifications in the ventromedial hypothalamus and preoptic area following oestradiol administration.

    PubMed

    Gagnidze, K; Weil, Z M; Faustino, L C; Schaafsma, S M; Pfaff, D W

    2013-10-01

    Expression of the primary female sex behaviour, lordosis, in laboratory animals depends on oestrogen-induced expression of progesterone receptor (PgR) within a defined cell group in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMH). The minimal latency from oestradiol administration to lordosis is 18 h. During that time, ligand-bound oestrogen receptors (ER), members of a nuclear receptor superfamily, recruit transcriptional coregulators, which induce covalent modifications of histone proteins, thus leading to transcriptional activation or repression of target genes. The present study aimed to investigate the early molecular epigenetic events underlying oestrogen-regulated transcriptional activation of the Pgr gene in the VMH of female mice. Oestradiol (E₂) administration induced rapid and transient global histone modifications in the VMH of ovariectomised female mice. Histone H3 N-terminus phosphorylation (H3S10phK14Ac), acetylation (H3Ac) and methylation (H3K4me3) exhibited distinct temporal patterns facilitative to the induction of transcription. A transcriptional repressive (H3K9me3) modification showed a different temporal pattern. Collectively, this should create a permissive environment for the transcriptional activity necessary for lordosis, within 3-6 h after E₂ treatment. In the VMH, changes in the H3Ac and H3K4me3 levels of histone H3 were also detected at the promoter region of the Pgr gene within the same time window, although they were delayed in the preoptic area. Moreover, examination of histone modifications associated with the promoter of another ER-target gene, oxytocin receptor (Oxtr), revealed gene- and brain-region specific effects of E₂ treatment. In the VMH of female mice, E₂ treatment resulted in the recruitment of ERα to the oestrogen-response-elements-containing putative enhancer site of Pgr gene, approximately 200 kb upstream of the transcription start site, although it failed to increase ER

  18. Mass Spectrometry-Based Methodology for Identification of Native Histone Variant Modifications From Mammalian Tissues and Solid Tumors.

    PubMed

    Nuccio, A G; Bui, M; Dalal, Y; Nita-Lazar, A

    2017-01-01

    Histone posttranslational modifications (PTMs) are key epigenetic marks involved in gene silencing or activation. Histone modifications impact chromatin organization and transcriptional processes through the changes in charge density between histones and DNA. They also serve as recognition and binding sites for specific binding proteins. Histone tails and globular cores contain many basic amino acid residues, which are subject to various dynamic modifications, making the modification repertoire extremely diverse. Consequently, determination of histone PTM identity and quantity has been a challenging task. In recent years, mass spectrometry-based methods have proven useful in histone PTM characterization. This chapter provides a brief overview of these methods and describes the approach to analyze the PTMs of the histone variant CENP-A, essential for the cell cycle progression, when present in minute amounts from tumor and mammalian tissues. Because this method does not rely on antibody-based immunopurification, we anticipate that these tools could be readily adaptable to the investigation to other histone variants in a range of mammalian tissues and solid tumors. Published by Elsevier Inc.

  19. Landscape of histone modifications in a sponge reveals the origin of animal cis-regulatory complexity.

    PubMed

    Gaiti, Federico; Jindrich, Katia; Fernandez-Valverde, Selene L; Roper, Kathrein E; Degnan, Bernard M; Tanurdžić, Miloš

    2017-04-11

    Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear when this regulatory system evolved. By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedon queenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity.

  20. Landscape of histone modifications in a sponge reveals the origin of animal cis-regulatory complexity

    PubMed Central

    Gaiti, Federico; Jindrich, Katia; Fernandez-Valverde, Selene L; Roper, Kathrein E; Degnan, Bernard M; Tanurdžić, Miloš

    2017-01-01

    Combinatorial patterns of histone modifications regulate developmental and cell type-specific gene expression and underpin animal complexity, but it is unclear when this regulatory system evolved. By analysing histone modifications in a morphologically-simple, early branching animal, the sponge Amphimedonqueenslandica, we show that the regulatory landscape used by complex bilaterians was already in place at the dawn of animal multicellularity. This includes distal enhancers, repressive chromatin and transcriptional units marked by H3K4me3 that vary with levels of developmental regulation. Strikingly, Amphimedon enhancers are enriched in metazoan-specific microsyntenic units, suggesting that their genomic location is extremely ancient and likely to place constraints on the evolution of surrounding genes. These results suggest that the regulatory foundation for spatiotemporal gene expression evolved prior to the divergence of sponges and eumetazoans, and was necessary for the evolution of animal multicellularity. DOI: http://dx.doi.org/10.7554/eLife.22194.001 PMID:28395144

  1. Histone modifications and alcohol-induced liver disease: are altered nutrients the missing link?

    PubMed

    Moghe, Akshata; Joshi-Barve, Swati; Ghare, Smita; Gobejishvili, Leila; Kirpich, Irina; McClain, Craig J; Barve, Shirish

    2011-05-28

    Alcoholism is a major health problem in the United States and worldwide, and alcohol remains the single most significant cause of liver-related diseases and deaths. Alcohol is known to influence nutritional status at many levels including nutrient intake, absorption, utilization, and excretion, and can lead to many nutritional disturbances and deficiencies. Nutrients can dramatically affect gene expression and alcohol-induced nutrient imbalance may be a major contributor to pathogenic gene expression in alcohol-induced liver disease (ALD). There is growing interest regarding epigenetic changes, including histone modifications that regulate gene expression during disease pathogenesis. Notably, modifications of core histones in the nucleosome regulate chromatin structure and DNA methylation, and control gene transcription. This review highlights the role of nutrient disturbances brought about during alcohol metabolism and their impact on epigenetic histone modifications that may contribute to ALD. The review is focused on four critical metabolites, namely, acetate, S-adenosylmethionine, nicotinamide adenine dinucleotide and zinc that are particularly relevant to alcohol metabolism and ALD.

  2. Histone posttranslational modifications predict specific alternative exon subtypes in mammalian brain.

    PubMed

    Hu, Qiwen; Kim, Eun Ji; Feng, Jian; Grant, Gregory R; Heller, Elizabeth A

    2017-06-01

    A compelling body of literature, based on next generation chromatin immunoprecipitation and RNA sequencing of reward brain regions indicates that the regulation of the epigenetic landscape likely underlies chronic drug abuse and addiction. It is now critical to develop highly innovative computational strategies to reveal the relevant regulatory transcriptional mechanisms that may underlie neuropsychiatric disease. We have analyzed chromatin regulation of alternative splicing, which is implicated in cocaine exposure in mice. Recent literature has described chromatin-regulated alternative splicing, suggesting a novel function for drug-induced neuroepigenetic remodeling. However, the extent of the genome-wide association between particular histone modifications and alternative splicing remains unexplored. To address this, we have developed novel computational approaches to model the association between alternative splicing and histone posttranslational modifications in the nucleus accumbens (NAc), a brain reward region. Using classical statistical methods and machine learning to combine ChIP-Seq and RNA-Seq data, we found that specific histone modifications are strongly associated with various aspects of differential splicing. H3K36me3 and H3K4me1 have the strongest association with splicing indicating they play a significant role in alternative splicing in brain reward tissue.

  3. Detection of active transcription factor binding sites with the combination of DNase hypersensitivity and histone modifications.

    PubMed

    Gusmao, Eduardo G; Dieterich, Christoph; Zenke, Martin; Costa, Ivan G

    2014-11-15

    The identification of active transcriptional regulatory elements is crucial to understand regulatory networks driving cellular processes such as cell development and the onset of diseases. It has recently been shown that chromatin structure information, such as DNase I hypersensitivity (DHS) or histone modifications, significantly improves cell-specific predictions of transcription factor binding sites. However, no method has so far successfully combined both DHS and histone modification data to perform active binding site prediction. We propose here a method based on hidden Markov models to integrate DHS and histone modifications occupancy for the detection of open chromatin regions and active binding sites. We have created a framework that includes treatment of genomic signals, model training and genome-wide application. In a comparative analysis, our method obtained a good trade-off between sensitivity versus specificity and superior area under the curve statistics than competing methods. Moreover, our technique does not require further training or sequence information to generate binding location predictions. Therefore, the method can be easily applied on new cell types and allow flexible downstream analysis such as de novo motif finding. Our framework is available as part of the Regulatory Genomics Toolbox. The software information and all benchmarking data are available at http://costalab.org/wp/dh-hmm. ivan.costa@rwth-aachen.de or eduardo.gusmao@rwth-aachen.de Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  4. Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo.

    PubMed

    Riu, Efren; Chen, Zhi-Ying; Xu, Hui; He, Chen-Yi; Kay, Mark A

    2007-07-01

    One of the major obstacles to success in non-viral gene therapy is transcriptional silencing of the DNA vector. The mechanisms underlying gene silencing/repression in mammalian cells are complex and remain unclear. Because changes in chromatin structure and, in particular, histone modifications are involved in transcriptional regulation of endogenous genes, we hypothesized that changes in the pattern of histone modifications were related to the observed transcriptional silencing of exogenous DNA vectors. We used antibodies against specific modified histones to perform chromatin immunoprecipitation (ChIP) analyses on liver lysates from mice transfected with two types of plasmids: (i) DNA minicircles (MCs) devoid of bacterial plasmid backbone DNA, which showed marked persistence of transgene expression, and (ii) their parental plasmids, which were silenced over time. Silencing of the transgene from the parental vectors was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the MC DNA was consistent with euchromatin. Our data indicates that (i) episomal vectors undergo chromatinization in vivo, and (ii) both persistence and silencing of transgene expression are associated with specific histone modifications.

  5. Histone modification of embryonic stem cells produced by somatic cell nuclear transfer and fertilized blastocysts.

    PubMed

    Farifteh, Fattaneh; Salehi, Mohammad; Bandehpour, Mojgan; Nariman, Mosaffa; Ghafari Novin, Marefat; Hosseini, Taher; Nematollahi, Sedigheh; Noroozian, Mohsen; Keshavarzi, Somayeh; Hosseini, Ahmad

    2014-01-01

    Nuclear transfer-embryonic stem cells (NT-ESCs) are genetically identical to the donor's cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A (TSA) as a histone deacetylase in- hibitor (HDACi) plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer (SCNT) can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines. In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6×DBA/2) F 1 mice] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were ac- tivated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts (MEF), so ESCs lines were estab- lished. ESCs markers were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Histone modifications were analyzed by enzyme linked immunosorbent assay (ELISA). Result of this study showed that TSA treatment after SCNT can improve devel- opmental rate of embryos (21.12 ± 3.56 vs. 8.08 ± 7.92), as well as establishment rate of NT-ESCs line (25 vs. 12.5). We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells (ESCs) lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs. TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a man- ner similar to that of ESCs established from normal blastocysts.

  6. Histone Modification of Embryonic Stem Cells Produced by Somatic Cell Nuclear Transfer and Fertilized Blastocysts

    PubMed Central

    Farifteh, Fattaneh; Salehi, Mohammad; Bandehpour, Mojgan; Nariman, Mosaffa; Ghafari Novin, Marefat; Hosseini, Taher; Nematollahi, Sedigheh; Noroozian, Mohsen; Keshavarzi, Somayeh; Hosseini, Ahmad

    2014-01-01

    Objective Nuclear transfer-embryonic stem cells (NT-ESCs) are genetically identical to the donor’s cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A (TSA) as a histone deacetylase in- hibitor (HDACi) plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer (SCNT) can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines. Materials and Methods In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6×DBA/2) F 1 mice] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were ac- tivated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts (MEF), so ESCs lines were estab- lished. ESCs markers were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Histone modifications were analyzed by enzyme linked immunosorbent assay (ELISA). Results Result of this study showed that TSA treatment after SCNT can improve devel- opmental rate of embryos (21.12 ± 3.56 vs. 8.08 ± 7.92), as well as establishment rate of NT-ESCs line (25 vs. 12.5). We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells (ESCs) lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs. Conclusion TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a man- ner similar to that of ESCs established from normal blastocysts. PMID:24381856

  7. WaveSeq: A Novel Data-Driven Method of Detecting Histone Modification Enrichments Using Wavelets

    PubMed Central

    Mitra, Apratim; Song, Jiuzhou

    2012-01-01

    Background Chromatin immunoprecipitation followed by next-generation sequencing is a genome-wide analysis technique that can be used to detect various epigenetic phenomena such as, transcription factor binding sites and histone modifications. Histone modification profiles can be either punctate or diffuse which makes it difficult to distinguish regions of enrichment from background noise. With the discovery of histone marks having a wide variety of enrichment patterns, there is an urgent need for analysis methods that are robust to various data characteristics and capable of detecting a broad range of enrichment patterns. Results To address these challenges we propose WaveSeq, a novel data-driven method of detecting regions of significant enrichment in ChIP-Seq data. Our approach utilizes the wavelet transform, is free of distributional assumptions and is robust to diverse data characteristics such as low signal-to-noise ratios and broad enrichment patterns. Using publicly available datasets we showed that WaveSeq compares favorably with other published methods, exhibiting high sensitivity and precision for both punctate and diffuse enrichment regions even in the absence of a control data set. The application of our algorithm to a complex histone modification data set helped make novel functional discoveries which further underlined its utility in such an experimental setup. Conclusions WaveSeq is a highly sensitive method capable of accurate identification of enriched regions in a broad range of data sets. WaveSeq can detect both narrow and broad peaks with a high degree of accuracy even in low signal-to-noise ratio data sets. WaveSeq is also suited for application in complex experimental scenarios, helping make biologically relevant functional discoveries. PMID:23029045

  8. Maize Histone Deacetylase hda101 Is Involved in Plant Development, Gene Transcription, and Sequence-Specific Modulation of Histone Modification of Genes and Repeats[W

    PubMed Central

    Rossi, Vincenzo; Locatelli, Sabrina; Varotto, Serena; Donn, Guenter; Pirona, Raul; Henderson, David A.; Hartings, Hans; Motto, Mario

    2007-01-01

    Enzymes catalyzing histone acetylation and deacetylation contribute to the modulation of chromatin structure, thus playing an important role in regulating gene and genome activity. We showed that downregulation and overexpression of the maize (Zea mays) Rpd3-type hda101 histone deacetylase gene induced morphological and developmental defects. Total levels of acetylated histones and histone acetylation of both repetitive and nonrepetitive sequences were affected in hda101 transgenic mutants. However, only transcript levels of genes but not repeats were altered. In particular, hda101 transgenic mutants showed differential expression of genes involved in vegetative-to-reproductive transition, such as liguleless2 and knotted-like genes and their repressor rough sheath2, which are required for meristem initiation and maintenance. Perturbation of hda101 expression also affected histone modifications other than acetylation, including histone H3 dimethylation at Lys-4 and Lys-9 and phosphorylation at Ser-10. Our results indicate that hda101 affects gene transcription and provide evidence of its involvement in setting the histone code, thus mediating developmental programs. Possible functional differences between maize hda101 and its Arabidopsis thaliana ortholog HDA19 are discussed. PMID:17468264

  9. Maize histone deacetylase hda101 is involved in plant development, gene transcription, and sequence-specific modulation of histone modification of genes and repeats.

    PubMed

    Rossi, Vincenzo; Locatelli, Sabrina; Varotto, Serena; Donn, Guenter; Pirona, Raul; Henderson, David A; Hartings, Hans; Motto, Mario

    2007-04-01

    Enzymes catalyzing histone acetylation and deacetylation contribute to the modulation of chromatin structure, thus playing an important role in regulating gene and genome activity. We showed that downregulation and overexpression of the maize (Zea mays) Rpd3-type hda101 histone deacetylase gene induced morphological and developmental defects. Total levels of acetylated histones and histone acetylation of both repetitive and nonrepetitive sequences were affected in hda101 transgenic mutants. However, only transcript levels of genes but not repeats were altered. In particular, hda101 transgenic mutants showed differential expression of genes involved in vegetative-to-reproductive transition, such as liguleless2 and knotted-like genes and their repressor rough sheath2, which are required for meristem initiation and maintenance. Perturbation of hda101 expression also affected histone modifications other than acetylation, including histone H3 dimethylation at Lys-4 and Lys-9 and phosphorylation at Ser-10. Our results indicate that hda101 affects gene transcription and provide evidence of its involvement in setting the histone code, thus mediating developmental programs. Possible functional differences between maize hda101 and its Arabidopsis thaliana ortholog HDA19 are discussed.

  10. Radiation-induced alterations in histone modification patterns and their potential impact on short-term radiation effects

    PubMed Central

    Friedl, Anna A.; Mazurek, Belinda; Seiler, Doris M.

    2012-01-01

    Detection and repair of radiation-induced DNA damage occur in the context of chromatin. An intricate network of mechanisms defines chromatin structure, including DNA methylation, incorporation of histone variants, histone modifications, and chromatin remodeling. In the last years it became clear that the cellular response to radiation-induced DNA damage involves all of these mechanisms. Here we focus on the current knowledge on radiation-induced alterations in post-translational histone modification patterns and their effect on the chromatin accessibility, transcriptional regulation and chromosomal stability. PMID:23050241

  11. Molecular insights into the recognition of N-terminal histone modifications by the BRPF1 bromodomain

    PubMed Central

    Poplawski, Amanda; Hu, Kaifeng; Lee, Woonghee; Natesan, Senthil; Peng, Danni; Carlson, Samuel; Shi, Xiaobing; Balaz, Stefan; Markley, John L.; Glass, Karen C.

    2014-01-01

    The monocytic leukemic zinc-finger (MOZ) histone acetyltransferase (HAT) acetylates free histones H3, H4, H2A, and H2B in vitro and is associated with up-regulation of gene transcription. The MOZ HAT functions as a quaternary complex with the bromodomain-PHD finger protein 1 (BRPF1), inhibitor of growth 5 (ING5), and hEaf6 subunits. BRPF1 links the MOZ catalytic subunit to the ING5 and hEaf6 subunits, thereby promoting MOZ HAT activity. Human BRPF1 contains multiple effector domains with known roles in gene transcription, and chromatin binding and remodeling. However, the biological function of the BRPF1 bromodomain remains unknown. Our findings reveal novel interactions of the BRPF1 bromodomain with multiple acetyllysine residues on the N-terminus of histones, and show it preferentially selects for H2AK5ac, H4K12ac and H3K14ac. We used chemical shift perturbation data from NMR titration experiments to map the BRPF1 bromodomain ligand binding pocket and identified key residues responsible for coordination of the post-translationally modified histones. Extensive molecular dynamics simulations were used to generate structural models of bromodomain-histone ligand complexes, to analyze H-bonding and other interactions, and to calculate the binding free energies. Our results outline the molecular mechanism driving binding specificity of the BRPF1 bromodomain for discrete acetyllysine residues on the N-terminal histone tails. Together these data provide insights on how histone recognition by the bromodomain directs the biological function of BRPF1, ultimately targeting the MOZ HAT complex to chromatin substrates. PMID:24333487

  12. Flagging False Positives Following Untargeted LC-MS Characterization of Histone Post-Translational Modification Combinations.

    PubMed

    Willems, Sander; Dhaenens, Maarten; Govaert, Elisabeth; De Clerck, Laura; Meert, Paulien; Van Neste, Christophe; Van Nieuwerburgh, Filip; Deforce, Dieter

    2017-02-03

    Epigenetic changes can be studied with an untargeted characterization of histone post-translational modifications (PTMs) by liquid chromatography-mass spectrometry (LC-MS). While prior information about more than 20 types of histone PTMs exists, little is known about histone PTM combinations (PTMCs). Because of the combinatorial explosion it is intrinsically impossible to consider all potential PTMCs in a database search. Consequentially, high-scoring false positives with unconsidered but correct alternative isobaric PTMCs can occur. Current quality controls can neither estimate the amount of unconsidered alternatives nor flag potential false positives. Here, we propose a conceptual workflow that provides such options. In this workflow, an in silico modeling of all candidate isoforms with known-to-exist PTMs is made. The most frequently occurring PTM sets of these candidate isoforms are determined and used in several database searches. This suppresses the combinatorial explosion while considering as many candidate isoforms as possible. Finally, annotations can be classified as unique or ambiguous, the latter implying false positives. This workflow was evaluated on an LC-MS data set containing 44 histone extracts. We were able to consider 60% of all candidate isoforms. Importantly, 40% of all annotations were classified as ambiguous. This highlights the need for a more thorough evaluation of modified peptide annotations.

  13. A Common histone modification code on C4 genes in maize and its conservation in Sorghum and Setaria italica.

    PubMed

    Heimann, Louisa; Horst, Ina; Perduns, Renke; Dreesen, Björn; Offermann, Sascha; Peterhansel, Christoph

    2013-05-01

    C4 photosynthesis evolved more than 60 times independently in different plant lineages. Each time, multiple genes were recruited into C4 metabolism. The corresponding promoters acquired new regulatory features such as high expression, light induction, or cell type-specific expression in mesophyll or bundle sheath cells. We have previously shown that histone modifications contribute to the regulation of the model C4 phosphoenolpyruvate carboxylase (C4-Pepc) promoter in maize (Zea mays). We here tested the light- and cell type-specific responses of three selected histone acetylations and two histone methylations on five additional C4 genes (C4-Ca, C4-Ppdk, C4-Me, C4-Pepck, and C4-RbcS2) in maize. Histone acetylation and nucleosome occupancy assays indicated extended promoter regions with regulatory upstream regions more than 1,000 bp from the transcription initiation site for most of these genes. Despite any detectable homology of the promoters on the primary sequence level, histone modification patterns were highly coregulated. Specifically, H3K9ac was regulated by illumination, whereas H3K4me3 was regulated in a cell type-specific manner. We further compared histone modifications on the C4-Pepc and C4-Me genes from maize and the homologous genes from sorghum (Sorghum bicolor) and Setaria italica. Whereas sorghum and maize share a common C4 origin, C4 metabolism evolved independently in S. italica. The distribution of histone modifications over the promoters differed between the species, but differential regulation of light-induced histone acetylation and cell type-specific histone methylation were evident in all three species. We propose that a preexisting histone code was recruited into C4 promoter control during the evolution of C4 metabolism.

  14. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote

    PubMed Central

    Schübeler, Dirk; MacAlpine, David M.; Scalzo, David; Wirbelauer, Christiane; Kooperberg, Charles; van Leeuwen, Fred; Gottschling, Daniel E.; O'Neill, Laura P.; Turner, Bryan M.; Delrow, Jeffrey; Bell, Stephen P.; Groudine, Mark

    2004-01-01

    The covalent modification of nucleosomal histones has emerged as a major determinant of chromatin structure and gene activity. To understand the interplay between various histone modifications, including acetylation and methylation, we performed a genome-wide chromatin structure analysis in a higher eukaryote. We found a binary pattern of histone modifications among euchromatic genes, with active genes being hyperacetylated for H3 and H4 and hypermethylated at Lys 4 and Lys 79 of H3, and inactive genes being hypomethylated and deacetylated at the same residues. Furthermore, the degree of modification correlates with the level of transcription, and modifications are largely restricted to transcribed regions, suggesting that their regulation is tightly linked to polymerase activity. PMID:15175259

  15. Histone Modifications Define Expression Bias of Homoeologous Genomes in Allotetraploid Cotton1[OPEN

    PubMed Central

    Ye, Wenxue; Song, Qingxin; Zhang, Tianzhen

    2016-01-01

    Histone modifications regulate gene expression in eukaryotes, but their roles in gene expression changes in interspecific hybrids or allotetraploids are poorly understood. Histone modifications can be mapped by immunostaining of metaphase chromosomes at the single cell level and/or by chromatin immunoprecipitation-sequencing (ChIP-seq) for analyzing individual genes. Here, we comparatively analyzed immunostained metaphase chromosomes and ChIP-seq of individual genes, which revealed a chromatin basis for biased homoeologous gene expression in polyploids. We examined H3K4me3 density and transcriptome maps in root-tip cells of allotetraploid cotton (Gossypium hirsutum). The overall H3K4me3 levels were relatively equal between A and D chromosomes, which were consistent with equal numbers of expressed genes between the two subgenomes. However, intensities per chromosomal area were nearly twice as high in the D homeologs as in the A homeologs. Consistent with the cytological observation, ChIP-seq analysis showed that more D homeologs with biased H3K4me3 levels than A homeologs with biased modifications correlated with the greater number of the genes with D-biased expression than that with A-biased expression in most homeologous chromosome pairs. Two chromosomes displayed different expression levels compared with other chromosomes, which correlate with known translocations and may affect the local chromatin structure and expression levels for the genes involved. This example of genome-wide histone modifications that determine expression bias of homeologous genes in allopolyploids provides a molecular basis for the evolution and domestication of polyploid species, including many important crops. PMID:27637746

  16. Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase.

    PubMed

    Chew, Yap Ching; Camporeale, Gabriela; Kothapalli, Nagarama; Sarath, Gautam; Zempleni, Janos

    2006-04-01

    In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.

  17. Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells.

    PubMed

    Lin, Shu; Yuan, Zuo-Fei; Han, Yumiao; Marchione, Dylan M; Garcia, Benjamin A

    2016-07-15

    How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

    PubMed

    Noubouossie, Denis F; Whelihan, Matthew F; Yu, Yuan-Bin; Sparkenbaugh, Erica; Pawlinski, Rafal; Monroe, Dougald M; Key, Nigel S

    2017-02-23

    NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins. © 2017 by The American Society of Hematology.

  19. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.

    PubMed

    Rodriguez-Collazo, Pedro; Leuba, Sanford H; Zlatanova, Jordanka

    2009-06-01

    Post-translational modifications (PTMs) of histones play a role in modifying chromatin structure for DNA-templated processes in the eukaryotic nucleus, such as transcription, replication, recombination and repair; thus, histone PTMs are considered major players in the epigenetic control of these processes. Linking specific histone PTMs to gene expression is an arduous task requiring large amounts of highly purified and natively modified histones to be analyzed by various techniques. We have developed robust and complementary procedures, which use strong protein denaturing conditions and yield highly purified core and linker histones from unsynchronized proliferating, M-phase arrested and butyrate-treated cells, fully preserving their native PTMs without using enzyme inhibitors. Cell hypotonic swelling and lysis, nuclei isolation/washing and chromatin solubilization under mild conditions are bypassed to avoid compromising the integrity of histone native PTMs. As controls for our procedures, we tested the most widely used conventional methodologies and demonstrated that they indeed lead to drastic histone dephosphorylation. Additionally, we have developed methods for preserving acid-labile histone modifications by performing non-acid extractions to obtain highly purified H3 and H4. Importantly, isolation of histones H3, H4 and H2A/H2B is achieved without the use of HPLC. Functional supercoiling assays reveal that both hyper- and hypo-phosphorylated histones can be efficiently assembled into polynucleosomes. Notably, the preservation of fully phosphorylated mitotic histones and their assembly into polynucleosomes should open new avenues to investigate an important but overlooked question: the impact of mitotic phosphorylation in chromatin structure and function.

  20. Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

    PubMed Central

    He, Shibin; Yan, Shihan; Wang, Pu; Zhu, Wei; Wang, Xiangwu; Shen, Yao; Shao, Kejia; Xin, Haiping; Li, Shaohua; Li, Lijia

    2014-01-01

    Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (Zea mays L. ssp. parviglumis and Zea nicaraguensis) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome. PMID:24819606

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

    SciTech Connect

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

    2013-09-25

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

  2. Tubulin Tyrosine Ligase Like 12, a TTLL Family Member with SET- and TTL-Like Domains and Roles in Histone and Tubulin Modifications and Mitosis

    PubMed Central

    Brants, Jan; Semenchenko, Kostyantyn; Wasylyk, Christine; Robert, Aude; Carles, Annaick; Zambrano, Alberto; Pradeau-Aubreton, Karine; Birck, Catherine; Schalken, Jack A.; Poch, Olivier; de Mey, Jan; Wasylyk, Bohdan

    2012-01-01

    hTTLL12 is a member of the tubulin tyrosine ligase (TTL) family that is highly conserved in phylogeny. It has both SET-like and TTL-like domains, suggesting that it could have histone methylation and tubulin tyrosine ligase activities. Altered expression of hTTLL12 in human cells leads to specific changes in H4K20 trimethylation, and tubulin detyrosination, hTTLL12 does not catalyse histone methylation or tubulin tyrosination in vitro, as might be expected from the lack of critical amino acids in its SET-like and TTLL-like domains. hTTLL12 misexpression increases mitotic duration and chromosome numbers. These results suggest that hTTLL12 has non-catalytic functions related to tubulin and histone modification, which could be linked to its effects on mitosis and chromosome number stability. PMID:23251473

  3. ChIPnorm: A Statistical Method for Normalizing and Identifying Differential Regions in Histone Modification ChIP-seq Libraries

    PubMed Central

    Bucher, Philipp; Moret, Bernard M. E.

    2012-01-01

    The advent of high-throughput technologies such as ChIP-seq has made possible the study of histone modifications. A problem of particular interest is the identification of regions of the genome where different cell types from the same organism exhibit different patterns of histone enrichment. This problem turns out to be surprisingly difficult, even in simple pairwise comparisons, because of the significant level of noise in ChIP-seq data. In this paper we propose a two-stage statistical method, called ChIPnorm, to normalize ChIP-seq data, and to find differential regions in the genome, given two libraries of histone modifications of different cell types. We show that the ChIPnorm method removes most of the noise and bias in the data and outperforms other normalization methods. We correlate the histone marks with gene expression data and confirm that histone modifications H3K27me3 and H3K4me3 act as respectively a repressor and an activator of genes. Compared to what was previously reported in the literature, we find that a substantially higher fraction of bivalent marks in ES cells for H3K27me3 and H3K4me3 move into a K27-only state. We find that most of the promoter regions in protein-coding genes have differential histone-modification sites. The software for this work can be downloaded from http://lcbb.epfl.ch/software.html. PMID:22870189

  4. Combined oral contraceptive synergistically activates mineralocorticoid receptor through histone code modifications.

    PubMed

    Igunnu, Adedoyin; Seok, Young-Mi; Olatunji, Lawrence A; Kang, Seol-Hee; Kim, Inkyeom

    2015-12-15

    Clinical studies have shown that the use of combined oral contraceptive in pre-menopausal women is associated with fluid retention. However, the molecular mechanism is still elusive. We hypothesized that combined oral contraceptive (COC) ethinyl estradiol (EE) and norgestrel (N) synergistically activates mineralocorticoid receptor (MR) through histone code modifications. Twelve-week-old female Sprague-Dawley rats were treated with olive oil (control), a combination of 0.1µg EE and 1.0µg N (low COC) or 1.0µg EE and 10.0µg N (high COC) as well as 0.1 or 1.0µg EE and 1.0 or 10.0µg N daily for 6 weeks. Expression of MR target genes in kidney cortex was determined by quantitative real-time polymerase chain reaction. MR was quantified by western blot. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes as well as histone code modifications was analyzed by chromatin immunoprecipitation assay. Treatment with COC increased renal cortical expression of MR target genes such as serum and glucocorticoid-regulated kinase 1 (Sgk-1), glucocorticoid-induced leucine zipper (Gilz), epithelial Na(+)channel (Enac) and Na(+)-K(+)-ATPase subunit α1 (Atp1a1). Although COC increased neither serum aldosterone nor MR expression in kidney cortex, it increased recruitment of MR and Pol II in parallel with increased H3Ac and H3K4me3 on the promoter regions of MR target genes. However, treatment with EE or N alone did not affect renal cortical expression of Sgk-1, Gilz, Enac or Atp1a1. These results indicate that COC synergistically activates MR through histone code modifications.

  5. Regulation of transcription by the Arabidopsis UVR8 photoreceptor involves a specific histone modification.

    PubMed

    Velanis, Christos N; Herzyk, Pawel; Jenkins, Gareth I

    2016-11-01

    The photoreceptor UV RESISTANCE LOCUS 8 (UVR8) specifically mediates photomorphogenic responses to UV-B wavelengths. UVR8 acts by regulating transcription of a set of genes, but the underlying mechanisms are unknown. Previous research indicated that UVR8 can associate with chromatin, but the specificity and functional significance of this interaction are not clear. Here we show, by chromatin immunoprecipitation, that UV-B exposure of Arabidopsis increases acetylation of lysines K9 and/or K14 of histone H3 at UVR8-regulated gene loci in a UVR8-dependent manner. The transcription factors HY5 and/or HYH, which mediate UVR8-regulated transcription, are also required for this chromatin modification, at least for the ELIP1 gene. Furthermore, sequencing of the immunoprecipitated DNA revealed that all UV-B-induced enrichments in H3K9,14diacetylation across the genome are UVR8-dependent, and approximately 40 % of the enriched loci contain known UVR8-regulated genes. In addition, inhibition of histone acetylation by anacardic acid reduces the UV-B induced, UVR8 mediated expression of ELIP1 and CHS. No evidence was obtained in yeast 2-hybrid assays for a direct interaction between either UVR8 or HY5 and several proteins involved in light-regulated histone modification, nor for the involvement of these proteins in UVR8-mediated responses in plants, although functional redundancy between proteins could influence the results. In summary, this study shows that UVR8 regulates a specific chromatin modification associated with transcriptional regulation of a set of UVR8-target genes.

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

    PubMed

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

    2011-02-01

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

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

    PubMed

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

    2016-03-16

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

  8. Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry.

    PubMed

    Zee, Barry M; Alekseyenko, Artyom A; McElroy, Kyle A; Kuroda, Mitzi I

    2016-02-16

    Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.

  9. Synergetic effects of DNA methylation and histone modification during mouse induced pluripotent stem cell generation

    PubMed Central

    Wang, Guiying; Weng, Rong; Lan, Yuanyuan; Guo, Xudong; Liu, Qidong; Liu, Xiaoqin; Lu, Chenqi; Kang, Jiuhong

    2017-01-01

    DNA methylation and histone methylation (H3K27me3) have been reported as major barriers to induced pluripotent stem cell (iPSC) generation using four core transcription factors (Oct4, Sox2, Klf4, and c-Myc, termed OSKM). Here, to illustrate the possibility of deriving iPSCs via demethylation, as well as the exact effects of DNA methylation and histone modification on gene expression regulation, we performed RNA sequencing to characterize the transcriptomes of ES cells and iPSCs derived by demethylation with miR-29b or shDnmt3a, and carried out integrated analyses. Results showed that OSKM + miR-29b-iPSC was more close to ES cells than the others, and up-regulated genes typically presented with methylated CpG-dense promoters and H3K27me3-enriched regions. The differentially expressed genes caused by introduction of DNA demethylation during somatic cell reprogramming mainly focus on stem cell associated GO terms and KEGG signaling pathways, which may decrease the tumorigenesis risk of iPSCs. These findings indicated that DNA methylation and histone methylation have synergetic effects on regulating gene expression during iPSC generation, and demethylation by miR-29b is better than shDnmt3a for iPSC quality. Furthermore, integrated analyses are superior for exploration of slight differences as missed by individual analysis. PMID:28155862

  10. Signal analysis for genome-wide maps of histone modifications measured by ChIP-seq.

    PubMed

    Beck, Dominik; Brandl, Miriam B; Boelen, Lies; Unnikrishnan, Ashwin; Pimanda, John E; Wong, Jason W H

    2012-04-15

    Chromatin structure, including post-translational modifications of histones, regulates gene expression, alternative splicing and cell identity. ChIP-seq is an increasingly used assay to study chromatin function. However, tools for downstream bioinformatics analysis are limited and are only based on the evaluation of signal intensities. We reasoned that new methods taking into account other signal characteristics such as peak shape, location and frequencies might reveal new insights into chromatin function, particularly in situation where differences in read intensities are subtle. We introduced an analysis pipeline, based on linear predictive coding (LPC), which allows the capture and comparison of ChIP-seq histone profiles. First, we show that the modeled signal profiles distinguish differentially expressed genes with comparable accuracy to signal intensities. The method was robust against parameter variations and performed well up to a signal-to-noise ratio of 0.55. Additionally, we show that LPC profiles of activating and repressive histone marks cluster into distinct groups and can be used to predict their function. http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP A Matlab implementation along with usage instructions and an example input file are available from: http://www.cancerresearch.unsw.edu.au/crcweb.nsf/page/LPCHP.

  11. Association Between Arsenic Exposure and Global Post-translational Histone Modifications Among Adults in Bangladesh

    PubMed Central

    Chervona, Yana; Hall, Megan N.; Arita, Adriana; Wu, Fen; Sun, Hong; Tseng, Hsiang-Chi; Ali, Eunus; Uddin, Mohammad Nasir; Liu, Xinhua; Zoroddu, Maria Antonietta; Gamble, Mary V.; Costa, Max

    2012-01-01

    Background Exposure to arsenic (As) is associated with an increased risk of several cancers, as well as, cardiovascular disease, and childhood neuro-developmental deficits. Arsenic compounds are weakly mutagenic, alter gene expression and post-translational histone modifications (PTHMs) in vitro. Methods Water and urinary As concentrations, as well as, global levels of histone 3 lysine 9 di-methylation and acetylation (H3K9me2 and H3K9ac), histone 3 lysine 27 trimethylation and acetylation (H3K27me3 and H3K27ac), histone 3 lysine 18 acetylation (H3K18ac) and histone 3 lysine 4 trimethylation (H3K4me3) were measured in peripheral blood mononuclear cells (PBMCs) from a subset of participants (N=40) of a folate clinical trial in Bangladesh (FACT study). Results Total urinary As (uAs) was positively correlated with H3K9me2 (r=0.36, p=0.02) and inversely with H3K9ac (r= -0.47, p=0.002). The associations between As and other PTHMs differed in a gender-dependent manner. Water As (wAs) was positively correlated with H3K4me3 (r=0.45, p=0.05) and H3K27me3 (r=0.50, p=0.03) among females and negatively correlated among males (H3K4me3: r= -0.44, p=0.05; H3K27me3: r= -0.34, p=0.14). Conversely, wAs was inversely associated with H3K27ac among females (r= -0.44, p=0.05) and positively associated among males (r=0.29, p=0.21). A similar pattern was observed for H3K18ac (females: r= -0.22, p=0.36; males: r=0.27, p=0.24). Conclusion Exposure to As is associated with alterations of global PTHMs; gender-specific patterns of association were observed between As exposure and several histone marks. Impact These findings contribute to the growing body of evidence linking As exposure to epigenetic dysregulation, which may play a role in the pathogenesis of As toxicity. PMID:23064002

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  15. 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.

  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. Transcriptional regulation by histone modifications: towards a theory of chromatin re-organization during stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Binder, Hans; Steiner, Lydia; Przybilla, Jens; Rohlf, Thimo; Prohaska, Sonja; Galle, Jörg

    2013-04-01

    Chromatin-related mechanisms, as e.g. histone modifications, are known to be involved in regulatory switches within the transcriptome. Only recently, mathematical models of these mechanisms have been established. So far they have not been applied to genome-wide data. We here introduce a mathematical model of transcriptional regulation by histone modifications and apply it to data of trimethylation of histone 3 at lysine 4 (H3K4me3) and 27 (H3K27me3) in mouse pluripotent and lineage-committed cells. The model describes binding of protein complexes to chromatin which are capable of reading and writing histone marks. Molecular interactions of the complexes with DNA and modified histones create a regulatory switch of transcriptional activity. The regulatory states of the switch depend on the activity of histone (de-) methylases, the strength of complex-DNA-binding and the number of nucleosomes capable of cooperatively contributing to complex-binding. Our model explains experimentally measured length distributions of modified chromatin regions. It suggests (i) that high CpG-density facilitates recruitment of the modifying complexes in embryonic stem cells and (ii) that re-organization of extended chromatin regions during lineage specification into neuronal progenitor cells requires targeted de-modification. Our approach represents a basic step towards multi-scale models of transcriptional control during development and lineage specification.

  18. Transcriptional regulation by histone modifications: towards a theory of chromatin re-organization during stem cell differentiation.

    PubMed

    Binder, Hans; Steiner, Lydia; Przybilla, Jens; Rohlf, Thimo; Prohaska, Sonja; Galle, Jörg

    2013-04-01

    Chromatin-related mechanisms, as e.g. histone modifications, are known to be involved in regulatory switches within the transcriptome. Only recently, mathematical models of these mechanisms have been established. So far they have not been applied to genome-wide data. We here introduce a mathematical model of transcriptional regulation by histone modifications and apply it to data of trimethylation of histone 3 at lysine 4 (H3K4me3) and 27 (H3K27me3) in mouse pluripotent and lineage-committed cells. The model describes binding of protein complexes to chromatin which are capable of reading and writing histone marks. Molecular interactions of the complexes with DNA and modified histones create a regulatory switch of transcriptional activity. The regulatory states of the switch depend on the activity of histone (de-) methylases, the strength of complex-DNA-binding and the number of nucleosomes capable of cooperatively contributing to complex-binding. Our model explains experimentally measured length distributions of modified chromatin regions. It suggests (i) that high CpG-density facilitates recruitment of the modifying complexes in embryonic stem cells and (ii) that re-organization of extended chromatin regions during lineage specification into neuronal progenitor cells requires targeted de-modification. Our approach represents a basic step towards multi-scale models of transcriptional control during development and lineage specification.

  19. Arsenic alters global histone modifications in lymphocytes in vitro and in vivo.

    PubMed

    Pournara, Angeliki; Kippler, Maria; Holmlund, Teresa; Ceder, Rebecca; Grafström, Roland; Vahter, Marie; Broberg, Karin; Wallberg, Annika E

    2016-08-01

    Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1-proteins that are part of a gene expression silencing complex.

  20. Modeling the Dynamics of Bivalent Histone Modifications in Embryonic Stem Cells

    NASA Astrophysics Data System (ADS)

    Ku, Wai; Yuan, Guo; Sorrentino, Francesco; Girvan, Michelle; Ott, Edward

    2013-03-01

    Epigenetic modifications to histones may either promote the activation or repression of the transcription of nearby genes. Recent experiments have discovered bivalent domains of nucleosomes in which the domain as a whole contains both active and repressive marks. These domains occur in the promoters of most lineage-control genes in embryonic stem cells. It is generally agreed that bivalent domains play an important role in stem cell differentiation, but the mechanisms remain unclear. Here we propose and study a dynamical model of histone modification which, unlike previous models, captures the general features of the bivalent domains observed in experiments. A key feature of our model is the existence of ``A/R states,'' by which we mean states in which there are a significant number of nucleosomes each of which individually has both active and repressive marks. We use our model to investigate the formation and decay of A/R states, the localization of A/R nucleosomes, and the effect of DNA replication on the stability of A/R states. The goals of our model are to help understand the underlying principles and mechanisms of bivalent domain dynamics and to suggest directions for future experiments.

  1. Early life stress triggers sustained changes in histone deacetylase expression and histone H4 modifications that alter responsiveness to adolescent antidepressant treatment

    PubMed Central

    Levine, Amir; Worrell, Trent R.; Zimnisky, Ross; Schmauss, Claudia

    2011-01-01

    Early life stress can elicit long-lasting changes in gene expression and behavior. Recent studies on rodents suggest that these lasting effects depend on the genetic background. Whether epigenetic factors also play a role remains to be investigated. Here we exposed the stress-susceptible mouse strain Balb/c and the more resilient strain C57Bl/6 to a powerful early life stress paradigm, infant maternal separation. In Balb/c mice, infant maternal separation led to decreased expression of mRNA encoding the histone deacetylases (HDACs) 1, 3, 7, 8, and 10 in the forebrain neocortex in adulthood, an effect accompanied by increased expression of acetylated histone H4 proteins, especially acetylated H4K12 protein. These changes in HDAC expression and histone modifications were not detected in C57Bl/6 mice exposed to early life stress. Moreover, a reversal of the H4K12 hyperacetylation detected in infant maternally separated Balb/c mice (achieved with chronic adolescent treatment with a low dose of theophylline that only activates HDACs) worsened the abnormal emotional phenotype resulting from this early life stress exposure. In contrast, fluoxetine, a drug with potent antidepressant efficacy in infant maternally separated Balb/c mice, potentiated all histone modifications triggered by early life stress. Moreover, in non-stressed Balb/c mice, co-administration of an HDAC inhibitor and fluoxetine, but not fluoxetine alone, elicited antidepressant effects and also triggered changes in histone H4 expression that were similar to those provoked by fluoxetine treatment of mice exposed to early life stress. These results suggest that Balb/c mice develop epigenetic modifications after early life stress exposure that, in terms of the emotive phenotype, are of adaptive nature, and that enhance the efficacy of antidepressant drugs. PMID:21964251

  2. Characterization of human papillomavirus type 16 pseudovirus containing histones.

    PubMed

    Kim, Hyoung Jin; Kwag, Hye-Lim; Kim, Hong-Jin

    2016-08-27

    Pseudoviruses (PsVs) that encapsidate a reporter plasmid DNA have been used as surrogates for native human papillomavirus (HPV), whose continuous production is technically difficult. HPV PsVs have been designed to form capsids made up of the major capsid protein L1 and the minor capsid proteins L2. HPV PsVs have been produced in 293TT cells transfected with plasmid expressing L1 and L2 protein and plasmid containing the reporter gene. Several studies have suggested that naturally occurring HPV virions contain cellular histones, and histones have also been identified in mature HPV PsVs. However, the effect of the histones on the properties of the PsVs has not been investigated. Using heparin chromatography, we separated mature HPV type 16 PsVs into three fractions (I, II, and III) according to their heparin-binding affinities. The amounts of cellular histone and cellular nucleotides per PsV were found to increase in the order fraction I, II and III. It appeared that PsVs in fraction I contains just small amount of cellular histone in Western blot analysis. The proportions of the three fractions in PsV preparations were 83.4, 7.5, and 9.1 % for fraction I, II, and III PsVs, respectively. In the electron microscope PsVs in fraction I appeared to have a more condensed structure than those in fractions II and III. Under the electron microscope fraction II and III PsVs appeared to be covered by substantial amounts of cellular histone while there was no visible histone covering PsVs of fraction I. Also the levels of reporter gene expression in infections of fraction II and III PsVs to 293TT cells were significantly lower than those in infections of fraction I PsV, and fraction II and III particles had significantly reduced immunogenicity. Our findings suggest that the involvement of large amounts of cellular histones during PsV formation interferes with the structural integrity of the PsVs and affects their immunogenicity. The fraction I particle therefore has the most

  3. Discovery of histone modification crosstalk networks by stable isotope labeling of amino acids in cell culture mass spectrometry (SILAC MS).

    PubMed

    Guan, Xiaoyan; Rastogi, Neha; Parthun, Mark R; Freitas, Michael A

    2013-08-01

    In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation.

  4. HMCan-diff: a method to detect changes in histone modifications in cells with different genetic characteristics.

    PubMed

    Ashoor, Haitham; Louis-Brennetot, Caroline; Janoueix-Lerosey, Isabelle; Bajic, Vladimir B; Boeva, Valentina

    2017-05-05

    Comparing histone modification profiles between cancer and normal states, or across different tumor samples, can provide insights into understanding cancer initiation, progression and response to therapy. ChIP-seq histone modification data of cancer samples are distorted by copy number variation innate to any cancer cell. We present HMCan-diff, the first method designed to analyze ChIP-seq data to detect changes in histone modifications between two cancer samples of different genetic backgrounds, or between a cancer sample and a normal control. HMCan-diff explicitly corrects for copy number bias, and for other biases in the ChIP-seq data, which significantly improves prediction accuracy compared to methods that do not consider such corrections. On in silico simulated ChIP-seq data generated using genomes with differences in copy number profiles, HMCan-diff shows a much better performance compared to other methods that have no correction for copy number bias. Additionally, we benchmarked HMCan-diff on four experimental datasets, characterizing two histone marks in two different scenarios. We correlated changes in histone modifications between a cancer and a normal control sample with changes in gene expression. On all experimental datasets, HMCan-diff demonstrated better performance compared to the other methods. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. HMCan-diff: a method to detect changes in histone modifications in cells with different genetic characteristics

    PubMed Central

    Ashoor, Haitham; Louis-Brennetot, Caroline; Janoueix-Lerosey, Isabelle

    2017-01-01

    Abstract Comparing histone modification profiles between cancer and normal states, or across different tumor samples, can provide insights into understanding cancer initiation, progression and response to therapy. ChIP-seq histone modification data of cancer samples are distorted by copy number variation innate to any cancer cell. We present HMCan-diff, the first method designed to analyze ChIP-seq data to detect changes in histone modifications between two cancer samples of different genetic backgrounds, or between a cancer sample and a normal control. HMCan-diff explicitly corrects for copy number bias, and for other biases in the ChIP-seq data, which significantly improves prediction accuracy compared to methods that do not consider such corrections. On in silico simulated ChIP-seq data generated using genomes with differences in copy number profiles, HMCan-diff shows a much better performance compared to other methods that have no correction for copy number bias. Additionally, we benchmarked HMCan-diff on four experimental datasets, characterizing two histone marks in two different scenarios. We correlated changes in histone modifications between a cancer and a normal control sample with changes in gene expression. On all experimental datasets, HMCan-diff demonstrated better performance compared to the other methods. PMID:28053124

  6. 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.

  7. Arabidopsis flowering locus D influences systemic-acquired-resistance- induced expression and histone modifications of WRKY genes.

    PubMed

    Singh, Vijayata; Roy, Shweta; Singh, Deepjyoti; Nandi, Ashis Kumar

    2014-03-01

    A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections--a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that flowering locus D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD's involvement in epigenetic regulation of SAR.

  8. Mapping of histone modifications in episomal HBV cccDNA uncovers an unusual chromatin organization amenable to epigenetic manipulation

    PubMed Central

    Tropberger, Philipp; Mercier, Alexandre; Robinson, Margaret; Zhong, Weidong; Ganem, Don E.; Holdorf, Meghan

    2015-01-01

    Chronic hepatitis B virus (HBV) infection affects 240 million people worldwide and is a major risk factor for liver failure and hepatocellular carcinoma. Current antiviral therapy inhibits cytoplasmic HBV genomic replication, but is not curative because it does not directly affect nuclear HBV closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence. Novel approaches that directly target cccDNA regulation would therefore be highly desirable. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications (PTMs). Here, using a new cccDNA ChIP-Seq approach, we report, to our knowledge, the first genome-wide maps of PTMs in cccDNA-containing chromatin from de novo infected HepG2 cells, primary human hepatocytes, and from HBV-infected liver tissue. We find high levels of PTMs associated with active transcription enriched at specific sites within the HBV genome and, surprisingly, very low levels of PTMs linked to transcriptional repression even at silent HBV promoters. We show that transcription and active PTMs in HBV chromatin are reduced by the activation of an innate immunity pathway, and that this effect can be recapitulated with a small molecule epigenetic modifying agent, opening the possibility that chromatin-based regulation of cccDNA transcription could be a new therapeutic approach to chronic HBV infection. PMID:26438841

  9. Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1.

    PubMed

    Bonet-Costa, Carles; Vilaseca, Marta; Diema, Claudio; Vujatovic, Olivera; Vaquero, Alejandro; Omeñaca, Núria; Castejón, Lucía; Bernués, Jordi; Giralt, Ernest; Azorín, Fernando

    2012-07-16

    Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

  10. A histone arginine methylation localizes to nucleosomes in satellite II and III DNA sequences in the human genome.

    PubMed

    Capurso, Daniel; Xiong, Hao; Segal, Mark R

    2012-11-15

    Applying supervised learning/classification techniques to epigenomic data may reveal properties that differentiate histone modifications. Previous analyses sought to classify nucleosomes containing histone H2A/H4 arginine 3 symmetric dimethylation (H2A/H4R3me2s) or H2A.Z using human CD4+ T-cell chromatin immunoprecipitation sequencing (ChIP-Seq) data. However, these efforts only achieved modest accuracy with limited biological interpretation. Here, we investigate the impact of using appropriate data pre-processing -deduplication, normalization, and position- (peak-) finding to identify stable nucleosome positions - in conjunction with advanced classification algorithms, notably discriminatory motif feature selection and random forests. Performance assessments are based on accuracy and interpretative yield. We achieved dramatically improved accuracy using histone modification features (99.0%; previous attempts, 68.3%) and DNA sequence features (94.1%; previous attempts, <60%). Furthermore, the algorithms elicited interpretable features that withstand permutation testing, including: the histone modifications H4K20me3 and H3K9me3, which are components of heterochromatin; and the motif TCCATT, which is part of the consensus sequence of satellite II and III DNA. Downstream analysis demonstrates that satellite II and III DNA in the human genome is occupied by stable nucleosomes containing H2A/H4R3me2s, H4K20me3, and/or H3K9me3, but not 18 other histone methylations. These results are consistent with the recent biochemical finding that H4R3me2s provides a binding site for the DNA methyltransferase (Dnmt3a) that methylates satellite II and III DNA. Classification algorithms applied to appropriately pre-processed ChIP-Seq data can accurately discriminate between histone modifications. Algorithms that facilitate interpretation, such as discriminatory motif feature selection, have the added potential to impart information about underlying biological mechanism.

  11. Histone Modifications in a Mouse Model of Early Adversities and Panic Disorder: Role for Asic1 and Neurodevelopmental Genes.

    PubMed

    Cittaro, Davide; Lampis, Valentina; Luchetti, Alessandra; Coccurello, Roberto; Guffanti, Alessandro; Felsani, Armando; Moles, Anna; Stupka, Elia; D' Amato, Francesca R; Battaglia, Marco

    2016-04-28

    Hyperventilation following transient, CO2-induced acidosis is ubiquitous in mammals and heritable. In humans, respiratory and emotional hypersensitivity to CO2 marks separation anxiety and panic disorders, and is enhanced by early-life adversities. Mice exposed to the repeated cross-fostering paradigm (RCF) of interference with maternal environment show heightened separation anxiety and hyperventilation to 6% CO2-enriched air. Gene-environment interactions affect CO2 hypersensitivity in both humans and mice. We therefore hypothesised that epigenetic modifications and increased expression of genes involved in pH-detection could explain these relationships. Medullae oblongata of RCF- and normally-reared female outbred mice were assessed by ChIP-seq for H3Ac, H3K4me3, H3K27me3 histone modifications, and by SAGE for differential gene expression. Integration of multiple experiments by network analysis revealed an active component of 148 genes pointing to the mTOR signalling pathway and nociception. Among these genes, Asic1 showed heightened mRNA expression, coherent with RCF-mice's respiratory hypersensitivity to CO2 and altered nociception. Functional enrichment and mRNA transcript analyses yielded a consistent picture of enhancement for several genes affecting chemoception, neurodevelopment, and emotionality. Particularly, results with Asic1 support recent human findings with panic and CO2 responses, and provide new perspectives on how early adversities and genes interplay to affect key components of panic and related disorders.

  12. Histone Modifications in a Mouse Model of Early Adversities and Panic Disorder: Role for Asic1 and Neurodevelopmental Genes

    PubMed Central

    Cittaro, Davide; Lampis, Valentina; Luchetti, Alessandra; Coccurello, Roberto; Guffanti, Alessandro; Felsani, Armando; Moles, Anna; Stupka, Elia; D’ Amato, Francesca R.; Battaglia, Marco

    2016-01-01

    Hyperventilation following transient, CO2-induced acidosis is ubiquitous in mammals and heritable. In humans, respiratory and emotional hypersensitivity to CO2 marks separation anxiety and panic disorders, and is enhanced by early-life adversities. Mice exposed to the repeated cross-fostering paradigm (RCF) of interference with maternal environment show heightened separation anxiety and hyperventilation to 6% CO2-enriched air. Gene-environment interactions affect CO2 hypersensitivity in both humans and mice. We therefore hypothesised that epigenetic modifications and increased expression of genes involved in pH-detection could explain these relationships. Medullae oblongata of RCF- and normally-reared female outbred mice were assessed by ChIP-seq for H3Ac, H3K4me3, H3K27me3 histone modifications, and by SAGE for differential gene expression. Integration of multiple experiments by network analysis revealed an active component of 148 genes pointing to the mTOR signalling pathway and nociception. Among these genes, Asic1 showed heightened mRNA expression, coherent with RCF-mice’s respiratory hypersensitivity to CO2 and altered nociception. Functional enrichment and mRNA transcript analyses yielded a consistent picture of enhancement for several genes affecting chemoception, neurodevelopment, and emotionality. Particularly, results with Asic1 support recent human findings with panic and CO2 responses, and provide new perspectives on how early adversities and genes interplay to affect key components of panic and related disorders. PMID:27121911

  13. Epigenetic regulation of GATA4 expression by histone modification in AFP-producing gastric adenocarcinoma.

    PubMed

    Yamamura, Nobuhisa; Kishimoto, Takashi

    2012-08-01

    AFP-producing adenocarcinoma is a variant of adenocarcinoma with high malignancy. Production of AFP suggests enteroblastic or hepatoid differentiation of cancer cells. GATA4 is a key molecule involved in the prenatal development of the stomach and liver. GATA4 is epigenetically silenced by hypermethylation of primer region in many types of cancers including gastric cancer. The aim of this study is to investigate the expression and epigenetic regulation of GATA4 in AFP-producing adenocarcinoma. Immunohistochemical analysis revealed that GATA4 was positive in 3/8 cases of AFP-producing gastric adenocarcinomas and in 28/30 cases of common type adenocarcinomas. Epigenetic modification of GATA4 promoter region was investigated with 3 AFP-producing and 4 common-type gastric cancer cell lines. GATA4 mRNA was detected in 1/3 of AFP-producing and 2/4 of common-type gastric cancer cell lines by RT-PCR. Methylation-specific PCR revealed no GATA4 methylation in any of the AFP-producing gastric cancers, whereas methylation was consistent with GATA4 expression in the common-type gastric cancers. Chromatin immunoprecipitation assay for AFP-producing gastric cancers revealed that histones H3 and H4 were hypoacetylated in the GATA4-negative cells, while they were hyperacetylated in the GATA4-positive cells. Treatment with trichostain A, an inhibitor for histone deacetylase, induced acetylation of histones H3 and H4, and tri-methylation of lysine 4 of histone H3, which was associated with the active transcription of GATA4 in GATA4-negative AFP-producing cells. These results indicated that histone deacetylation is a silencing mechanism for GATA4 expression in AFP-producing gastric cancer cells. Differences between AFP-producing gastric cancer and common-type gastric cancer in terms of the mechanism of GATA4 regulation may be reflected in the phenotypic deviation of AFP-producing gastric cancer from common-type gastric cancer.

  14. Divergence and Selectivity of Expression-Coupled Histone Modifications in Budding Yeasts

    PubMed Central

    Mosesson, Yaron; Voichek, Yoav; Barkai, Naama

    2014-01-01

    Various histone modifications are widely associated with gene expression, but their functional selectivity at individual genes remains to be characterized. Here, we identify widespread differences between genome-wide patterns of two prominent marks, H3K9ac and H3K4me3, in budding yeasts. As well as characteristic gene profiles, relative modification levels vary significantly amongst genes, irrespective of expression. Interestingly, we show that these differences couple to contrasting features: higher methylation to essential, periodically expressed, ‘DPN’ (Depleted Proximal Nucleosome) genes, and higher acetylation to non-essential, responsive, ‘OPN’ (Occupied Proximal Nucleosome) genes. Thus, H3K4me3 may generally associate with expression stability, and H3K9ac, with variability. To evaluate this notion, we examine their association with expression divergence between the closely related species, S. cerevisiae and S. paradoxus. Although individually well conserved at orthologous genes, changes between modifications are mostly uncorrelated, indicating largely non-overlapping regulatory mechanisms. Notably, we find that inter-species differences in methylation, but not acetylation, are well correlated with expression changes, thereby proposing H3K4me3 as a candidate regulator of expression divergence. Taken together, our results suggest distinct evolutionary roles for expression-linked modifications, wherein H3K4me3 may contribute to stabilize average expression, whilst H3K9ac associates with more indirect aspects such as responsiveness. PMID:25007273

  15. Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry.

    PubMed

    Zhou, Mowei; Paša-Tolić, Ljiljana; Stenoien, David L

    2017-02-03

    As histones play central roles in most chromosomal functions including regulation of DNA replication, DNA damage repair, and gene transcription, both their basic biology and their roles in disease development have been the subject of intense study. Because multiple post-translational modifications (PTMs) along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here we used state-of-the-art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as a search engine, and LcMsSpectator as a data visualization tool. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

  16. Profiling of Histone Post-Translational Modifications in Mouse Brain with High-Resolution Top-Down Mass Spectrometry

    SciTech Connect

    Zhou, Mowei; Paša-Tolić, Ljiljana; Stenoien, David L.

    2016-12-21

    Histones play central roles in most chromosomal functions and both their basic biology and roles in disease have been the subject of intense study. Since multiple PTMs along the entire protein sequence are potential regulators of histones, a top-down approach, where intact proteins are analyzed, is ultimately required for complete characterization of proteoforms. However, significant challenges remain for top-down histone analysis primarily because of deficiencies in separation/resolving power and effective identification algorithms. Here, we used state of the art mass spectrometry and a bioinformatics workflow for targeted data analysis and visualization. The workflow uses ProMex for intact mass deconvolution, MSPathFinder as search engine, and LcMsSpectator as a data visualization tool. ProMex sums across retention time to maximize sensitivity and accuracy for low abundance species in MS1deconvolution. MSPathFinder searches the MS2 data against protein sequence databases with user-defined modifications. LcMsSpectator presents the results from ProMex and MSPathFinder in a format that allows quick manual evaluation of critical attributes for high-confidence identifications. When complemented with the open-modification tool TopPIC, this workflow enabled identification of novel histone PTMs including tyrosine bromination on histone H4 and H2A, H3 glutathionylation, and mapping of conventional PTMs along the entire protein for many histone subunits.

  17. Associations between post translational histone modifications, myelomeningocele risk, environmental arsenic exposure, and folate deficiency among participants in a case control study in Bangladesh.

    PubMed

    Tauheed, Jannah; Sanchez-Guerra, Marco; Lee, Jane J; Paul, Ligi; Ibne Hasan, Md Omar Sharif; Quamruzzaman, Quazi; Selhub, Jacob; Wright, Robert O; Christiani, David C; Coull, Brent A; Baccarelli, Andrea A; Mazumdar, Maitreyi

    2017-06-03

    Arsenic exposure may contribute to disease risk in humans through alterations in the epigenome. Previous studies reported that arsenic exposure is associated with changes in plasma histone concentrations. Posttranslational histone modifications have been found to differ between the brain tissue of human embryos with neural tube defects and that of controls. Our objectives were to investigate the relationships between plasma histone 3 levels, history of having an infant with myelomeningocele, biomarkers of arsenic exposure, and maternal folate deficiency. These studies took place in Bangladesh, a country with high environmental arsenic exposure through contaminated drinking water. We performed ELISA assays to investigate plasma concentration of total histone 3 (H3) and the histone modification H3K27me3. The plasma samples were collected from 85 adult women as part of a case-control study of arsenic and myelomeningocele risk in Bangladesh. We found significant associations between plasma %H3K27me3 levels and risk of myelomeningocele (P<0.05). Mothers with higher %H3K27me3 in their plasma had lower risk of having an infant with myelomeningocele (odds ratio: 0.91, 95% confidence interval: 0.84, 0.98). We also found that arsenic exposure, as estimated by arsenic concentration in toenails, was associated with lower total H3 concentrations in plasma, but only among women with folate deficiency (β = -9.99, standard error = 3.91, P=0.02). Our results suggest that %H3K27me3 in maternal plasma differs between mothers of infants with myelomeningocele and mothers of infants without myelomeningocele, and may be a marker for myelomeningocele risk. Women with folate deficiency may be more susceptible to the epigenetic effects of environmental arsenic exposure.

  18. Characterization of individual histone post-translational modifications and their combinatorial patterns by mass spectrometry-based proteomics strategies

    PubMed Central

    Sidoli, Simone; Garcia, Benjamin A.

    2017-01-01

    Summary Histone post-translational modifications (PTMs) play an essential role in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Such PTMs are mostly localized on histone N-terminal tails where, as single units or in a combinatorial manner, influence chromatin reader protein binding and fine-tune the abovementioned activities. Mass spectrometry (MS) is currently the most adopted strategy to characterize proteins and protein PTMs. We hereby describe the protocols to identify and quantify histone PTMs and their patterns using either bottom-up or middle-down proteomics. In the bottom-up strategy we obtain 5–20 aa peptides by derivatization with propionylation followed by trypsin digestion. The newly generated N-termini of histone peptides can be further derivatized with light or isotopically heavy propionyl groups to increase chromatographic retention and allow multiplexed analyses. Moreover, we describe how to perform derivatization and trypsin digestion of histones loaded into a gel, which is usually the final step of immunoprecipitation experiments. In the middle-down strategy we obtain intact histone tails of 50–60 aa by digestion with the enzyme GluC. This allows characterization of combinatorial histone PTMs on N-terminal tails. PMID:27854019

  19. Quantification of histone modification ChIP-seq enrichment for data mining and machine learning applications.

    PubMed

    Hoang, Stephen A; Xu, Xiaojiang; Bekiranov, Stefan

    2011-08-11

    The advent of ChIP-seq technology has made the investigation of epigenetic regulatory networks a computationally tractable problem. Several groups have applied statistical computing methods to ChIP-seq datasets to gain insight into the epigenetic regulation of transcription. However, methods for estimating enrichment levels in ChIP-seq data for these computational studies are understudied and variable. Since the conclusions drawn from these data mining and machine learning applications strongly depend on the enrichment level inputs, a comparison of estimation methods with respect to the performance of statistical models should be made. Various methods were used to estimate the gene-wise ChIP-seq enrichment levels for 20 histone methylations and the histone variant H2A.Z. The Multivariate Adaptive Regression Splines (MARS) algorithm was applied for each estimation method using the estimation of enrichment levels as predictors and gene expression levels as responses. The methods used to estimate enrichment levels included tag counting and model-based methods that were applied to whole genes and specific gene regions. These methods were also applied to various sizes of estimation windows. The MARS model performance was assessed with the Generalized Cross-Validation Score (GCV). We determined that model-based methods of enrichment estimation that spatially weight enrichment based on average patterns provided an improvement over tag counting methods. Also, methods that included information across the entire gene body provided improvement over methods that focus on a specific sub-region of the gene (e.g., the 5' or 3' region). The performance of data mining and machine learning methods when applied to histone modification ChIP-seq data can be improved by using data across the entire gene body, and incorporating the spatial distribution of enrichment. Refinement of enrichment estimation ultimately improved accuracy of model predictions.

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

    PubMed

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

    2014-01-01

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

  1. Ornithine decarboxylase regulates M1 macrophage activation and mucosal inflammation via histone modifications

    PubMed Central

    Hardbower, Dana M.; Asim, Mohammad; Luis, Paula B.; Singh, Kshipra; Barry, Daniel P.; Yang, Chunying; Steeves, Meredith A.; Cleveland, John L.; Schneider, Claus; Piazuelo, M. Blanca; Gobert, Alain P.; Wilson, Keith T.

    2017-01-01

    Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms. PMID:28096401

  2. Cadmium affects mitotically inherited histone modification pathways in mouse embryonic stem cells.

    PubMed

    Gadhia, S R; O'Brien, D; Barile, F A

    2015-12-25

    The fetal basis of adult disease (FeBAD) theorizes that embryonic challenges initiate pathologies in adult life through epigenetic modification of gene expression. In addition, inheritance of H3K27 methylation marks, especially in vitro, is still controversial. Metals, such as Cd, are known to affect differentiation, DNA repair and epigenetic status in mES cells. We tested the premise that Cd exerts differential toxicity in mouse embryonic stem (mES) cells by targeting total histone protein (THP) production early in stem cell development, while affecting H3K27-mono-methylation (H3K27me(1)) in latter stages of differentiation. The inability of mES cells to recover from Cd insult at concentrations greater than IC50 indicates that maximum cytotoxicity occurs during initial hours of exposure. Moreover, as a measure of chromatin stability, low dose acute Cd exposure lowers THP production. The heritable effects of Cd exposure on cell proliferation, chromatin stability and transcription observed through several cell population doublings were detected only during alternate passages on days 3, 7, and 11, presumably due to slower maturation of histone methylation marks. These findings demonstrate a selective disruption of chromatin structure following acute Cd exposure, an effect not seen in developmentally mature cells. Hence, we present that acute Cd toxicity is cumulative and disrupts DNA repair, while concurrently affecting cell cycle progression, chromatin stability and transcriptional state in mES cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation.

    PubMed

    Kaufman-Szymczyk, Agnieszka; Majewski, Grzegorz; Lubecka-Pietruszewska, Katarzyna; Fabianowska-Majewska, Krystyna

    2015-12-12

    Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review.

  4. Chromatin Modulation of Herpesvirus Lytic Gene Expression: Managing Nucleosome Density and Heterochromatic Histone Modifications.

    PubMed

    Kristie, Thomas M

    2016-02-16

    Like their cellular hosts, herpesviruses are subject to the regulatory impacts of chromatin assembled on their genomes. Upon infection, these viruses are assembled into domains of chromatin with heterochromatic signatures that suppress viral gene expression or euchromatic characteristics that promote gene expression. The organization and modulation of these chromatin domains appear to be intimately linked to the coordinated expression of the different classes of viral genes and thus ultimately play an important role in the progression of productive infection or the establishment and maintenance of viral latency. A recent report from the Knipe laboratory (J. S. Lee, P. Raja, and D. M. Knipe, mBio 7:e02007-15, 2016) contributes to the understanding of the dynamic modulation of chromatin assembled on the herpes simplex virus genome by monitoring the levels of characteristic heterochromatic histone modifications (histone H3 lysine 9 and 27 methylation) associated with a model viral early gene during the progression of lytic infection. Additionally, this study builds upon previous observations that the viral immediate-early protein ICP0 plays a role in reducing the levels of heterochromatin associated with the early genes. Copyright © 2016 Kristie.

  5. Mapping and expression analyses during porcine foetal muscle development of 12 genes involved in histone modifications.

    PubMed

    Peng, Y B; Yerle, M; Liu, B

    2009-04-01

    Histone modifications (methylation and demethylation) regulate gene expression and play a role in cell proliferation and differentiation by their actions on chromatin structure. In this context, we studied the temporal expression profiles of genes acting on histone methylation and demethylation during skeletal muscle proliferation and differentiation. Quantitative real-time PCR was used to quantify the mRNA levels of CARM1, JARID1A, JMJD2A, LSD1, PRMT2, PRMT5, SMYD1, SMYD2, SMYD3, SETDB1, Suv39h2 and SUZ12 in foetal skeletal muscle. Our results showed that CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2 were differentially expressed in embryonic muscles of 33 days post-conception (dpc), 65 dpc and 90 dpc. These 12 genes were mapped to porcine chromosomes (SSC) 2q21-24, 5q25, 6q35, 6q12-21, 6p15, 7q21, 3q21-27, 9q26, 10p16, 4q15-16, 10q14-16 and 12p12 respectively. Taking into account the reported QTL mapping results, gene expression analysis and radiation hybrid mapping results, these results suggest that five genes (CARM1, JARID1A, JMJD2A, SMYD1 and SMYD2) could be good candidate genes for growth and backfat thickness traits.

  6. The Role of Sulforaphane in Epigenetic Mechanisms, Including Interdependence between Histone Modification and DNA Methylation

    PubMed Central

    Kaufman-Szymczyk, Agnieszka; Majewski, Grzegorz; Lubecka-Pietruszewska, Katarzyna; Fabianowska-Majewska, Krystyna

    2015-01-01

    Carcinogenesis as well as cancer progression result from genetic and epigenetic changes of the genome that leads to dysregulation of transcriptional activity of genes. Epigenetic mechanisms in cancer cells comprise (i) post-translation histone modification (i.e., deacetylation and methylation); (ii) DNA global hypomethylation; (iii) promoter hypermethylation of tumour suppressor genes and genes important for cell cycle regulation, cell differentiation and apoptosis; and (iv) posttranscriptional regulation of gene expression by noncoding microRNA. These epigenetic aberrations can be readily reversible and responsive to both synthetic agents and natural components of diet. A source of one of such diet components are cruciferous vegetables, which contain high levels of a number of glucosinolates and deliver, after enzymatic hydrolysis, sulforaphane and other bioactive isothiocyanates, that are involved in effective up-regulation of transcriptional activity of certain genes and also in restoration of active chromatin structure. Thus a consumption of cruciferous vegetables, treated as a source of isothiocyanates, seems to be potentially useful as an effective cancer preventive factor or as a source of nutrients improving efficacy of standard chemotherapies. In this review an attempt is made to elucidate the role of sulforaphane in regulation of gene promoter activity through a direct down-regulation of histone deacetylase activity and alteration of gene promoter methylation in indirect ways, but the sulforaphane influence on non-coding micro-RNA will not be a subject of this review. PMID:26703571

  7. Smads orchestrate specific histone modifications and chromatin remodeling to activate transcription

    PubMed Central

    Ross, Sarah; Cheung, Edwin; Petrakis, Thodoris G; Howell, Michael; Kraus, W Lee; Hill, Caroline S

    2006-01-01

    Smads are intracellular transducers for TGF-β superfamily ligands, but little is known about the mechanism by which complexes of receptor-phosphorylated Smad2 and Smad4 regulate transcription. Using an in vitro transcription system, we have discovered that, unlike most transcription factors that are sufficient to recruit the basal transcription machinery and therefore activate transcription on both naked DNA and chromatin templates, the Smads only activate transcription from chromatin templates. We demonstrate that Smad2-mediated transcription requires the histone acetyltransferase, p300. Smad2-recruited p300 exhibits an altered substrate specificity, specifically acetylating nucleosomal histone H3 at lysines 9 and 18, and these modifications are also detected on an endogenous Smad2-dependent promoter in a ligand-induced manner. Furthermore, we show that endogenous Smad2 interacts with the SWI/SNF ATPase, Brg1, in a TGF-β-dependent manner, and demonstrate that Brg1 is recruited to Smad2-dependent promoters and is specifically required for TGF-β-induced expression of endogenous Smad2 target genes. Our data indicate that the Smads define a new class of transcription factors that absolutely require chromatin to assemble the basal transcription machinery and activate transcription. PMID:16990801

  8. DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells

    PubMed Central

    Ngai, Siew Ching; Rosli, Rozita; Al Abbar, Akram

    2015-01-01

    Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV. PMID:25961011

  9. Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

    PubMed Central

    Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

    2011-01-01

    To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories. PMID:21508930

  10. Hippocampal focal knockout of CBP affects specific histone modifications, long-term potentiation, and long-term memory.

    PubMed

    Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

    2011-07-01

    To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories.

  11. New insights on chromatin modifiers and histone post-translational modifications in renal cell tumours.

    PubMed

    Vieira-Coimbra, Márcia; Henrique, Rui; Jerónimo, Carmen

    2015-01-01

    Renal cell tumours (RCTs) are the most common neoplasms affecting the kidney. They are clinically, pathologically and genetically heterogeneous, comprises four major histological subtypes [clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC) and chromophobe renal cell carcinoma (chRCC), which are malignant tumours, and oncocytoma, a benign tumour], as well as an increasing number of less common entities. Epigenetics has emerged as an important field in oncology due to the critical role it plays in neoplastic transformation and progression. Among epigenetic mechanisms, the modulation of chromatin packaging through covalent modifications is fundamental for gene transcription regulation and its deregulation is involved in carcinogenesis. Recently, deregulation of chromatin machinery in RCTs has increasingly acknowledged as an important mechanism for renal neoplastic transformation. The aim of this review is to summarize the most relevant alterations in histone post-translational modifications and chromatin modifiers, which have been implicated in renal tumorigenesis. The recognition of those modifications might provide new biomarkers for diagnosis and prognostication as well as novel targets for personalized therapeutic intervention. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

  12. Histone modifications as regulators of life and death in Saccharomyces cerevisiae

    PubMed Central

    Fahrenkrog, Birthe

    2015-01-01

    Apoptosis or programmed cell death is an integrated, genetically controlled suicide program that not only regulates tissue homeostasis of multicellular organisms, but also the fate of damaged and aged cells of lower eukaryotes, such as the yeast Saccharomyces cerevisiae. Recent years have revealed key apoptosis regulatory proteins in yeast that play similar roles in mammalian cells. Apoptosis is a process largely defined by characteristic structural rearrangements in the dying cell that include chromatin condensation and DNA fragmentation. The mechanism by which chromosomes restructure during apoptosis is still poorly understood, but it is becoming increasingly clear that altered epigenetic histone modifications are fundamental parameters that influence the chromatin state and the nuclear rearrangements within apoptotic cells. The present review will highlight recent work on the epigenetic regulation of programmed cell death in budding yeast. PMID:28357312

  13. Concerted Flexibility of Chromatin Structure, Methylome, and Histone Modifications along with Plant Stress Responses

    PubMed Central

    Santos, Ana Paula; Ferreira, Liliana J.; Oliveira, M. Margarida

    2017-01-01

    The spatial organization of chromosome structure within the interphase nucleus, as well as the patterns of methylome and histone modifications, represent intersecting layers that influence genome accessibility and function. This review is focused on the plastic nature of chromatin structure and epigenetic marks in association to stress situations. The use of chemical compounds (epigenetic drugs) or T-DNA-mediated mutagenesis affecting epigenetic regulators (epi-mutants) are discussed as being important tools for studying the impact of deregulated epigenetic backgrounds on gene function and phenotype. The inheritability of epigenetic marks and chromatin configurations along successive generations are interpreted as a way for plants to “communicate” past experiences of stress sensing. A mechanistic understanding of chromatin and epigenetics plasticity in plant response to stress, including tissue- and genotype-specific epigenetic patterns, may help to reveal the epigenetics contributions for genome and phenotype regulation. PMID:28275209

  14. Hyperglycemia impedes definitive endoderm differentiation of human embryonic stem cells by modulating histone methylation patterns.

    PubMed

    Chen, A C H; Lee, Y L; Fong, S W; Wong, C C Y; Ng, E H Y; Yeung, W S B

    2017-03-10

    Exposure to maternal diabetes during fetal growth is a risk factor for the development of type II diabetes (T2D) in later life. Discovery of the mechanisms involved in this association should provide valuable background for therapeutic treatments. Early embryogenesis involves epigenetic changes including histone modifications. The bivalent histone methylation marks H3K4me3 and H3K27me3 are important for regulating key developmental genes during early fetal pancreas specification. We hypothesized that maternal hyperglycemia disrupted early pancreas development through changes in histone bivalency. A human embryonic stem cell line (VAL3) was used as the cell model for studying the effects of hyperglycemia upon differentiation into definitive endoderm (DE), an early stage of the pancreatic lineage. Hyperglycemic conditions significantly down-regulated the expression levels of DE markers SOX17, FOXA2, CXCR4 and EOMES during differentiation. This was associated with retention of the repressive histone methylation mark H3K27me3 on their promoters under hyperglycemic conditions. The disruption of histone methylation patterns was observed as early as the mesendoderm stage, with Wnt/β-catenin signaling being suppressed during hyperglycemia. Treatment with Wnt/β-catenin signaling activator CHIR-99021 restored the expression levels and chromatin methylation status of DE markers, even in a hyperglycemic environment. The disruption of DE development was also found in mouse embryos at day 7.5 post coitum from diabetic mothers. Furthermore, disruption of DE differentiation in VAL3 cells led to subsequent impairment in pancreatic progenitor formation. Thus, early exposure to hyperglycemic conditions hinders DE development with a possible relationship to the later impairment of pancreas specification.

  15. A Histone Map of Human Chromosome 20q13.12

    PubMed Central

    Akan, Pelin; Sahlén, Martin; Deloukas, Panagiotis

    2009-01-01

    Background We present a systematic search for regulatory elements in a 3.5 Mb region on human chromosome 20q13.12, a region associated with a number of medical conditions such as type II diabetes and obesity. Methodology/Principal Findings We profiled six histone modifications alongside RNA polymerase II (PolII) and CTCF in two cell lines, HeLa S3 and NTERA-2 clone D1 (NT2/D1), by chromatin immunoprecipitation using an in-house spotted DNA array, constructed with 1.8 kb overlapping plasmid clones. In both cells, more than 90% of transcription start sites (TSSs) of expressed genes showed enrichments with PolII, di-methylated lysine 4 of histone H3 (H3K4me2), tri-methylated lysine 4 of histone H3 (H3K4me3) or acetylated H3 (H3Ac), whereas mono-methylated lysine 4 of histone H3 (H3K4me1) signals did not correlate with expression. No TSSs were enriched with tri-methylated lysine 27 of histone H3 (H3K27me3) in HeLa S3, while eight TSSs (4 expressed) showed enrichments in NT2/D1. We have also located several CTCF binding sites that are potential insulator elements. Conclusions/Significance In summary, we annotated a number of putative regulatory elements in 20q13.12 and went on to verify experimentally a subset of them using dual luciferase reporter assays. Correlating this data to sequence variation can aid identification of disease causing variants. PMID:19229332

  16. Chromatin modifiers and histone modifications in bone formation, regeneration, and therapeutic intervention for bone-related disease.

    PubMed

    Gordon, Jonathan A R; Stein, Janet L; Westendorf, Jennifer J; van Wijnen, Andre J

    2015-12-01

    Post-translational modifications of chromatin such as DNA methylation and different types of histone acetylation, methylation and phosphorylation are well-appreciated epigenetic mechanisms that confer information to progeny cells during lineage commitment. These distinct epigenetic modifications have defined roles in bone, development, tissue regeneration, cell commitment and differentiation, as well as disease etiologies. In this review, we discuss the role of these chromatin modifications and the enzymes regulating these marks (methyltransferases, demethylases, acetyltransferases, and deacetylases) in progenitor cells, osteoblasts and bone-related cells. In addition, the clinical relevance of deregulated histone modifications and enzymes as well as current and potential therapeutic interventions targeting chromatin modifiers are addressed. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Histone modifications associated with drought tolerance in the desert plant Zygophyllum dumosum Boiss.

    PubMed

    Granot, Gila; Sikron-Persi, Noga; Gaspan, Ofer; Florentin, Assa; Talwara, Susheela; Paul, Laju K; Morgenstern, Yaakov; Granot, Yigal; Grafi, Gideon

    2009-12-01

    Zygophyllum dumosum Boiss. is a perennial Saharo-Arabian phytogeographical element and a dominant shrub on the rocky limestone southeast-facing slopes of the Negev desert. The plant is highly active during the winter, and semideciduous during the dry summer, i.e., it sheds its leaflets, while leaving the thick, fleshy petiole green and rather active during the dry season. Being resistant to extreme perennial drought, Z. dumosum appears to provide an intriguing model plant for studying epigenetic mechanisms associated with drought tolerance in natural habitats. The transition from the wet to the dry season was accompanied by a significant decrease in nuclear size and with posttranslational modifications of histone H3 N-terminal tail. Dimethylation of H3 at lysine 4 (H3K4)--a modification associated with active gene expression--was found to be high during the wet season but gradually diminished on progression to the dry season. Unexpectedly, H3K9 di- and trimethylation as well as H3K27 di- and trimethylation could not be detected in Z. dumosum; H3K9 monomethylation appears to be prominent in Z. dumosum during the wet but not during the dry season. Contrary to Z. dumosum, H3K9 dimethylation was detected in other desert plants, including Artemisia sieberi, Anabasis articulata and Haloxylon scoparium. Taken together, our results demonstrate dynamic genome organization and unique pattern of histone H3 methylation displayed by Z. dumosum, which could have an adaptive value in variable environments of the Negev desert.

  18. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin

    PubMed Central

    Riddle, Nicole C.; Minoda, Aki; Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Gorchakov, Andrey A.; Jaffe, Jacob D.; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E.; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Park, Peter J.; Elgin, Sarah C.R.; Karpen, Gary H.

    2011-01-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the “silencing” marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin–heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both “activation” marks (e.g., H3K4me3 and H3K36me3) and “silencing” marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin. PMID:21177972

  19. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

    PubMed

    Riddle, Nicole C; Minoda, Aki; Kharchenko, Peter V; Alekseyenko, Artyom A; Schwartz, Yuri B; Tolstorukov, Michael Y; Gorchakov, Andrey A; Jaffe, Jacob D; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I; Pirrotta, Vincenzo; Park, Peter J; Elgin, Sarah C R; Karpen, Gary H

    2011-02-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

  20. N-formylation of lysine in histone proteins as a secondary modification arising from oxidative DNA damage.

    PubMed

    Jiang, Tao; Zhou, Xinfeng; Taghizadeh, Koli; Dong, Min; Dedon, Peter C

    2007-01-02

    The posttranslational modification of histone and other chromatin proteins has a well recognized but poorly defined role in the physiology of gene expression. With implications for interfering with these epigenetic mechanisms, we now report the existence of a relatively abundant secondary modification of chromatin proteins, the N(6)-formylation of lysine that appears to be uniquely associated with histone and other nuclear proteins. Using both radiolabeling and sensitive bioanalytical methods, we demonstrate that the formyl moiety of 3'-formylphosphate residues arising from 5'-oxidation of deoxyribose in DNA, caused by the enediyne neocarzinostatin, for example, acylate the N(6)-amino groups of lysine side chains. A liquid chromatography (LC)-tandem mass spectrometry (MS) method was developed to quantify the resulting N(6)-formyl-lysine residues, which were observed to be present in unperturbed cells and all sources of histone proteins to the extent of 0.04-0.1% of all lysines in acid-soluble chromatin proteins including histones. Cells treated with neocarzinostatin showed a clear dose-response relationship for the formation of N(6)-formyl-lysine, with this nucleosome linker-selective DNA-cleaving agent causing selective N(6)-formylation of the linker histone H1. The N(6)-formyl-lysine residue appears to represent an endogenous histone secondary modification, one that bears chemical similarity to lysine N(6)-acetylation recognized as an important determinant of gene expression in mammalian cells. The N(6)-formyl modification of lysine may interfere with the signaling functions of lysine acetylation and methylation and thus contribute to the pathophysiology of oxidative and nitrosative stress.

  1. A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.

    PubMed

    Baas, Roy; Lelieveld, Daphne; van Teeffelen, Hetty; Lijnzaad, Philip; Castelijns, Bas; van Schaik, F M; Vermeulen, Michiel; Egan, David A; Timmers, H Th Marc; de Graaf, Petra

    2014-02-01

    Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.

  2. Histone modifications involved in cassette exon inclusions: a quantitative and interpretable analysis.

    PubMed

    Liu, Hui; Jin, Ting; Guan, Jihong; Zhou, Shuigeng

    2014-12-19

    Chromatin structure and epigenetic modifications have been shown to involve in the co-transcriptional splicing of RNA precursors. In particular, some studies have suggested that some types of histone modifications (HMs) may participate in the alternative splicing and function as exon marks. However, most existing studies pay attention to the qualitative relationship between epigenetic modifications and exon inclusion. The quantitative analysis that reveals to what extent each type of epigenetic modification is responsible for exon inclusion is very helpful for us to understand the splicing process. In this paper, we focus on the quantitative analysis of HMs' influence on the inclusion of cassette exons (CEs) into mature RNAs. With the high-throughput ChIP-seq and RNA-seq data obtained from ENCODE website, we modeled the association of HMs with CE inclusions by logistic regression whose coefficients are meaningful and interpretable for us to reveal the effect of each type of HM. Three type of HMs, H3K36me3, H3K9me3 and H4K20me1, were found to play major role in CE inclusions. HMs' effect on CE inclusions is conservative across cell types, and does not depend on the expression levels of the genes hosting CEs. HMs located in the flanking regions of CEs were also taken into account in our analysis, and HMs within bounded flanking regions were shown to affect moderately CE inclusions. Moreover, we also found that HMs on CEs whose length is approximately close to nucleosomal-DNA length affect greatly on CE inclusion. We suggested that a few types of HMs correlate closely to alternative splicing and perhaps function jointly with splicing machinery to regulate the inclusion level of exons. Our findings are helpful to understand HMs' effect on exon definition, as well as the mechanism of co-transcriptional splicing.

  3. An Avidin-Based Assay for Histone Debiotinylase Activity in Human Cell Nuclei

    PubMed Central

    Chew, Yap Ching; Sarath, Gautam; Zempleni, Janos

    2006-01-01

    Covalent binding of biotin to histones participates in heterochromatin formation, cell cycle progression, and the cellular response to DNA breaks. Biotinylation of histones appears to be a reversible process but the identities of enzymes that remove the biotin mark are largely unknown. Our long-term goal is to identify histone debiotinylases in human cells. Here we developed an avidin-based plate assay to quantify histone debiotinylase activities in nuclear extracts. This assay is an essential first step in purifying and identifying histone debiotinylases from human cells. Using this assay we demonstrated that debiotinylation of histones depends on temperature and pH, consistent with enzyme catalysis. Experiments with purified histones, proteases, and protease inhibitors provide evidence that removal of the biotin mark from histones is mediated by debiotinylases rather than proteases. Activities of histone debiotinylases varied among human tissues: colon = lung > placenta = liver > lymphoid cells. The assay proved useful to monitor activities of putative histone debiotinylases during their partial purification from cells. Collectively, this assay is a useful tool for investigating histone debiotinylases in human tissues. PMID:17156993

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

    PubMed

    Faucher, David; Wellinger, Raymund J

    2010-08-26

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

  5. Role of histone deacetylase 2 and its posttranslational modifications in cardiac hypertrophy.

    PubMed

    Eom, Gwang Hyeon; Kook, Hyun

    2015-03-01

    Cardiac hypertrophy is a form of global remodeling, although the initial step seems to be an adaptation to increased hemodynamic demands. The characteristics of cardiac hypertrophy include the functional reactivation of the arrested fetal gene program, where histone deacetylases (HDACs) are closely linked in the development of the process. To date, mammalian HDACs are divided into four classes: I, II, III, and IV. By structural similarities, class II HDACs are then subdivided into IIa and IIb. Among class I and II HDACs, HDAC2, 4, 5, and 9 have been reported to be involved in hypertrophic responses; HDAC4, 5, and 9 are negative regulators, whereas HDAC2 is a pro-hypertrophic mediator. The molecular function and regulation of class IIa HDACs depend largely on the phosphorylation-mediated cytosolic redistribution, whereas those of HDAC2 take place primarily in the nucleus. In response to stresses, posttranslational modification (PTM) processes, dynamic modifications after the translation of proteins, are involved in the regulation of the activities of those hypertrophy-related HDACs. In this article, we briefly review 1) the activation of HDAC2 in the development of cardiac hypertrophy and 2) the PTM of HDAC2 and its implications in the regulation of HDAC2 activity.

  6. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes. PMID:22948226

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-11-01

    Dynamic histone modifications play an important role in controlling gene expression in response to various environmental cues. This mechanism of regulation of gene expression is important for sessile organisms, like land plants. We have previously reported consistent upregulation of various marker genes in response to gamma rays at various post-irradiation times. In the present study, we performed various chromatin modification analyses at selected loci using the standard chromatin immunoprecipitation procedure, and demonstrate that upregulation of these genes is associated with histone H3 lysine 4 tri-methylation (H3K4me3) at the gene body or transcription start sites of these loci. Further, at specific AtAgo2 loci, both H3K4me3 and histone H3 lysine 9 acetylation (H3K9ac) are important in controlling gene expression in response to gamma irradiation. There was no change in DNA methylation in these selected loci. We conclude that specific histone modification such as H3K4me3 and H3K9ac may be more important in activating gene expression in these selected loci in response to gamma irradiation than a change in DNA methylation. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

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

    SciTech Connect

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

    2014-09-15

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

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

    PubMed

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

    2016-06-01

    Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A-H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3-H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg(2+)-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin. © 2016 The Authors.

  11. Gene amplification of the histone methyltransferase SETDB1 contributes to human lung tumorigenesis

    PubMed Central

    Rodriguez-Paredes, M; Martinez de Paz, A; Simó-Riudalbas, L; Sayols, S; Moutinho, C; Moran, S; Villanueva, A; Vázquez-Cedeira, M; Lazo, P A; Carneiro, F; Moura, C S; Vieira, J; Teixeira, M R; Esteller, M

    2014-01-01

    Disruption of the histone modification patterns is one of the most common features of human tumors. However, few genetic alterations in the histone modifier genes have been described in tumorigenesis. Herein we show that the histone methyltransferase SETDB1 undergoes gene amplification in non-small and small lung cancer cell lines and primary tumors. The existence of additional copies of the SETDB1 gene in these transformed cells is associated with higher levels of the corresponding mRNA and protein. From a functional standpoint, the depletion of SETDB1 expression in amplified cells reduces cancer growth in cell culture and nude mice models, whereas its overexpression increases the tumor invasiveness. The increased gene dosage of SETDB1 is also associated with enhanced sensitivity to the growth inhibitory effect mediated by the SETDB1-interfering drug mithramycin. Overall, the findings identify SETDB1 as a bona fide oncogene undergoing gene amplification-associated activation in lung cancer and suggest its potential for new therapeutic strategies. PMID:23770855

  12. Histone deacetylase inhibition alters histone methylation associated with heat shock protein 70 promoter modifications in astrocytes and neurons

    PubMed Central

    Marinova, Zoya; Leng, Yan; Leeds, Peter; Chuang, De-Maw

    2010-01-01

    The mood-stabilizing and anticonvulsant drug valproic acid (VPA) inhibits histone deacetylases (HDACs). The aim of the present study was to determine the effect of HDAC inhibition on overall and target gene promoter-associated histone methylation in rat cortical neurons and astrocytes. We found that VPA and other HDAC inhibitors, including sodium butyrate (SB), trichostatin A (TSA), and the Class I HDAC inhibitors MS-275 and apicidin all increased levels of histone 3 lysine 4 dimethylation and trimethylation (H3K4Me2 and H3K4Me3); these processes are linked to transcriptional activation in rat cortical neurons and astrocytes. VPA, SB, TSA, MS-275, and apicidin also upregulated levels of the neuroprotective heat shock protein 70 (HSP70) in rat astrocytes. Moreover, Class I HDAC inhibition by VPA and MS-275 increased H3K4Me2 levels at the HSP70 promoter in astrocytes and neurons. We also found that VPA treatment facilitated the recruitment of acetyltransferase p300 to the HSP70 promoter and that p300 interacted with the transcription factor NF-Y in astrocytes. Taken together, the results suggest that Class I HDAC inhibition is key to upregulating overall and gene-specific H3K4 methylation in primary neuronal and astrocyte cultures. In addition, VPA-induced activation of the HSP70 promoter in astrocytes appears to involve an increase in H3K4Me2 levels and recruitment of p300. PMID:20888352

  13. Analysis of DNA methylation and histone modification profiles of liver-specific transporters.

    PubMed

    Imai, Satoki; Kikuchi, Ryota; Kusuhara, Hiroyuki; Yagi, Shintaro; Shiota, Kunio; Sugiyama, Yuichi

    2009-03-01

    Tissue-specific expression of transporters is tightly linked with their physiological functions through the regulation of the membrane transport of their substrates. We hypothesized that epigenetic regulation underlies the tissue-specific expression of mouse liver-specific transporters (Oatp1b2/Slco1b2, Ntcp/Slc10a1, Bsep/Abcb11, and Abcg5/g8). We examined their DNA methylation and histone modification profiles near the transcriptional start site (TSS) in the liver, kidney, and cerebrum. Genome-wide DNA methylation profiling with tissue-dependent differentially methylated region profiling with restriction tag-mediated amplification and subsequent bisulfite genomic sequencing demonstrated that the CpG dinucleotides around the TSS of Oatp1b2 (from -515 to +149 CpGs), Ntcp (from -481 to +495 CpGs), Bsep (from -339 to +282 CpGs), and Abcg5/g8 (from -161 to +5 CpGs for Abcg5, i.e., from -213 to -48 CpGs for Abcg8) were hypomethylated in the liver and hypermethylated in the kidney and cerebrum. The opposite pattern was observed for Pept2/Slc15a2 (from -638 to +4 CpGs), which was expressed in the kidney and cerebrum but not in the liver. These DNA methylation profiles are consistent with the tissue distribution of these transporters. A chromatin immunoprecipitation assay demonstrated that the histone H3 associated with Oatp1b2, Ntcp, Bsep, and Abcg5/g8 promoters was hyperacetylated in the liver but was acetylated very little in the kidney and cerebrum, whereas the upstream region of Pept2 was hyperacetylated only in the kidney and cerebrum. These results suggest the involvement of epigenetic systems in the tissue-specific expression of mouse transporters Oatp1b2, Ntcp, Bsep, Abcg5/g8, and Pept2.

  14. Role of the Tumor Suppressor PTEN in Antioxidant Responsive Element-mediated Transcription and Associated Histone Modifications

    PubMed Central

    Sakamoto, Kensuke; Iwasaki, Kenta; Sugiyama, Hiroyuki

    2009-01-01

    Coordinated regulation of PI3-kinase (PI3K) and the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) plays a pivotal role in various cell functions. PTEN is deficient in many cancer cells, including Jurkat human leukemia. Here, we demonstrate that the status of PTEN determines cellular susceptibility to oxidative stress through antioxidant-responsive element (ARE)-mediated transcription of detoxification genes. We found that ferritin H transcription was robustly induced in tert-butylhydroquinone (t-BHQ)-treated Jurkat cells via an ARE, and it was due to PTEN deficiency. Chromatin immunoprecipitation assays revealed that p300/CREB-binding protein (CBP) histone acetyltransferases and Nrf2 recruitment to the ARE and Bach1 release were blocked by the PI3K inhibitor LY294002, along with the partial inhibition of Nrf2 nuclear accumulation. Furthermore, acetylations of histone H3 Lys9 and Lys18, and deacetylation of Lys14 were associated with the PI3K-dependent ARE activation. Consistently, PTEN restoration in Jurkat cells inhibited t-BHQ–mediated expression of ferritin H and another ARE-regulated gene NAD(P)H:quinone oxidoreductase 1. Conversely, PTEN knockdown in K562 cells enhanced the response to t-BHQ. The PTEN status under t-BHQ treatment affected hydrogen peroxide-mediated caspase-3 cleavage. The PI3K-dependent ferritin H induction was observed by treatment with other ARE-activating agents ethoxyquin and hemin. Collectively, the status of PTEN determines chromatin modifications leading to ARE activation. PMID:19158375

  15. Human Rights and Behavior Modification

    ERIC Educational Resources Information Center

    Roos, Philip

    1974-01-01

    Criticisms of behavior modification, which charge that it violates ethical and legal principles, are discussed and reasons are presented to explain behavior modification's susceptibility to attack. (GW)

  16. H3K23me1 is an evolutionary conserved histone modification associated with CG DNA methylation in Arabidopsis.

    PubMed

    Trejo-Arellano, Minerva S; Mahrez, Walid; Nakamura, Miyuki; Moreno-Romero, Jordi; Nanni, Paolo; Köhler, Claudia; Hennig, Lars

    2017-02-09

    Amino-terminal tails of histones are targets for diverse post-translational modifications whose combinatorial action may constitute a code that will be read and interpreted by cellular proteins to define particular transcriptional states. Here, we describe monomethylation of histone H3 lysine 23 (H3K23me1) as a histone modification not previously described in plants. H3K23me1 is an evolutionary conserved mark in diverse flowering plant species. Chromatin immunoprecipitation followed by high-throughput sequencing in Arabidopsis thaliana showed that H3K23me1 was highly enriched in pericentromeric regions and depleted from chromosome arms. In transposable elements, it co-localized with CG, CHG and CHH DNA methylation as well as with the heterochromatic histone mark H3K9me2. Transposable elements are often rich in H3K23me1 but different families vary in their enrichment: LTR-Gypsy elements are most enriched and RC/Helitron elements are least enriched. The histone methyltransferase KRYPTONITE and normal DNA methylation were required for normal levels of H3K23me1 on transposable elements. Immunostaining experiments confirmed the pericentromeric localization and also showed mild enrichment in less condensed regions. Accordingly, gene bodies of protein coding genes had intermediate H3K23me1 levels, which coexisted with CG DNA methylation. Enrichment of H3K23me1 along gene bodies did not correlate with transcription levels. Together, this work establishes H3K23me1 as a not previously described component of the plant histone code. This article is protected by copyright. All rights reserved.

  17. Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification

    PubMed Central

    Mbogning, Jean; Pagé, Viviane; Burston, Jillian; Schwenger, Emily; Fisher, Robert P.; Schwer, Beate; Shuman, Stewart; Tanny, Jason C.

    2015-01-01

    Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD ‘code’ for co-transcriptional histone modifications. PMID:26275777

  18. [Histone acetylation modification of topoisomerase enzyme Ⅱα promoter regulation factors in patients with chronic benzene poisoning].

    PubMed

    Shi, Yifen; Qian, Shanhu; Li, Jiaqi; Yu, Kang

    2016-01-01

    To investigate histone acetylation modification of topoisomerase enzyme Ⅱα (TOPOⅡα) promoter regulation factors in patients with chronic benzene poisoning, to explore the possible regulatory mechanism of TOPOⅡα involved in toxicity of chronic benzene poisoning; The bone marrow samples were from 25 chronic benzene poisoning cases and 25 controls. The Chromatin Immunoprecipitation (ChIP) assay was carried out to study the possible mechanism of TOPOⅡα promoter regulation factors expression changes. TOPOⅡα promoter regulation factors mRNA were detected by RT-PCR technique. (1) Compared with the control, the histone H4 acetylation, histone H3 acetylation level of TOPOⅡα promoter regulation factors SP1, ATF-2, SP3, NF-YA, P53, C-MYB, ICBP90, NF-M in chronic benzene poisoning patients decreased, with the significant difference (P<0.05) , except for C-JUN (P>0.05) ; (2) The mRNA expression of TOPOⅡαpromoter regulation factors SP1, NF-YA, C-MYB, C-JUN and NF-M were significantly lower than in the control with the significant difference (P<0.05) , while the expression of SP3、P53 mRNA increased (P<0.05) , ATF-2、ICBP90 mRNA wasn't changed (P>0.05) . (1) Chronic benzene poisoning TOPO Ⅱα promoter regulation factors histone modification changes accompanied with mRNA level changed. (2) Histone acetylation modification of topoisomerase enzyme Ⅱα promoter regulation factors takes important role in the benezen's Hematopoietic toxicity.

  19. 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.

  20. The relationship between apoptosis, chromatin configuration, histone modification and competence of oocytes: A study using the mouse ovary-holding stress model

    PubMed Central

    Lin, Juan; Chen, Fei; Sun, Ming-Ju; Zhu, Jiang; Li, You-Wei; Pan, Liu-Zhu; Zhang, Jie; Tan, Jing-He

    2016-01-01

    The epigenetic factors causing competence differences between SN (surrounded nucleolus) and NSN (non-surrounded nucleolus) oocytes, the significance for the increased histone acetylation and methylation in SN oocytes, and whether chromatin configuration or histone modification determines oocyte competence, are unclear. This study has addressed these issues by using the ovary-holding (OH) stress models where oocyte SN configuration was uncoupled from histone modifications and developmental potential. Prepubertal mouse ovaries containing high percentages of NSN oocytes were preserved at 37 or 39 °C for 1 or 2 h before examination for oocyte chromatin configuration, developmental competence, histone modification and apoptosis. Whereas 1-h OH at 37 °C caused a moderate apoptosis with increased oocyte competence, improved histone modification and a normal NSN-to-SN transition, harsher OH conditions induced a severe apoptosis with decreased oocyte competence, impaired histone modification and a pseudo (premature) NSN-to-SN transition. Observations on Fas/FasL expression and using the gld (generalized lymphoproliferative disorder) mice harboring FasL mutations indicated that OH triggered oocyte apoptosis with activation of the Fas signaling. It was concluded that OH stress caused oocyte apoptosis with activation of the Fas/FasL system and that oocyte competence was more closely correlated with histone modification than with chromatin configuration. PMID:27321442

  1. Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen-Induced Arthritis

    PubMed Central

    Wen, Cai-Yu-Zhu; Chen, Zhe; Wang, Yu; Huang, Ying; Hu, Yong-Hong; Tu, Sheng-Hao

    2016-01-01

    Background. Wutou decoction (WTD) has been wildly applied in the treatment of rheumatoid arthritis and experimental arthritis in rats for many years. Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis; however, the effects of WTD on epigenetic changes are unclear. This study is set to explore the effects of WTD on DNA methylation and histone modifications in rats with collagen-induced arthritis (CIA). Methods. The CIA model was established by the stimulation of collagen and adjuvant. The knee synovium was stained with hematoxylin and eosin. The DNA methyltransferase 1 (DNMT1) and methylated CpG binding domain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR. The global DNA histone H3-K4/H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected. Results. Our data demonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (P < 0.05). The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (P < 0.05). Moreover, H3 acetylation of PBMCs was overexpressed in WTD compared with CIA (P < 0.05). Conclusions. WTD may modulate DNA methylation and histone modifications, functioning as anti-inflammatory potential. PMID:27042192

  2. Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling

    PubMed Central

    Li, Bing; Pattenden, Samantha G.; Lee, Daeyoup; Gutiérrez, José; Chen, Jie; Seidel, Chris; Gerton, Jennifer; Workman, Jerry L.

    2005-01-01

    The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation. PMID:16344463

  3. Serine and SAM Responsive Complex SESAME Regulates Histone Modification Crosstalk by Sensing Cellular Metabolism.

    PubMed

    Li, Shanshan; Swanson, Selene K; Gogol, Madelaine; Florens, Laurence; Washburn, Michael P; Workman, Jerry L; Suganuma, Tamaki

    2015-11-05

    Pyruvate kinase M2 (PKM2) is a key enzyme for glycolysis and catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate, which supplies cellular energy. PKM2 also phosphorylates histone H3 threonine 11 (H3T11); however, it is largely unknown how PKM2 links cellular metabolism to chromatin regulation. Here, we show that the yeast PKM2 homolog, Pyk1, is a part of a novel protein complex named SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex), which contains serine metabolic enzymes, SAM (S-adenosylmethionine) synthetases, and an acetyl-CoA synthetase. SESAME interacts with the Set1 H3K4 methyltransferase complex, which requires SAM synthesized from SESAME, and recruits SESAME to target genes, resulting in phosphorylation of H3T11. SESAME regulates the crosstalk between H3K4 methylation and H3T11 phosphorylation by sensing glycolysis and glucose-derived serine metabolism. This leads to auto-regulation of PYK1 expression. Thus, our study provides insights into the mechanism of regulating gene expression, responding to cellular metabolism via chromatin modifications.

  4. Distinct histone modifications define initiation and repair of meiotic recombination in the mouse.

    PubMed

    Buard, Jérôme; Barthès, Pauline; Grey, Corinne; de Massy, Bernard

    2009-09-02

    Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11(-/-) mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination.

  5. Distinct histone modifications define initiation and repair of meiotic recombination in the mouse

    PubMed Central

    Buard, Jérôme; Barthès, Pauline; Grey, Corinne; de Massy, Bernard

    2009-01-01

    Little is known about the factors determining the location and activity of the rapidly evolving meiotic crossover hotspots that shape genome diversity. Here, we show that several histone modifications are enriched at the active mouse Psmb9 hotspot, and we distinguish those marks that precede from those that follow hotspot recombinational activity. H3K4Me3, H3K4Me2 and H3K9Ac are specifically enriched in the chromatids that carry an active initiation site, and in the absence of DNA double-strand breaks (DSBs) in Spo11−/− mice. We thus propose that these marks are part of the substrate for recombination initiation at the Psmb9 hotspot. In contrast, hyperacetylation of H4 is increased as a consequence of DSB formation, as shown by its dependency on Spo11 and by the enrichment detected on both recombining chromatids. In addition, the comparison with another hotspot, Hlx1, strongly suggests that H3K4Me3 and H4 hyperacetylation are common features of DSB formation and repair, respectively. Altogether, the chromatin signatures of the Psmb9 and Hlx1 hotspots provide a basis for understanding the distribution of meiotic recombination. PMID:19644444

  6. Potential role of adolescent alcohol exposure-induced amygdaloid histone modifications in anxiety and alcohol intake during adulthood.

    PubMed

    Pandey, Subhash C; Sakharkar, Amul J; Tang, Lei; Zhang, Huaibo

    2015-10-01

    Binge drinking is common during adolescence and can lead to the development of psychiatric disorders, including alcoholism in adulthood. Here, the role and persistent effects of histone modifications during adolescent intermittent ethanol (AIE) exposure in the development of anxiety and alcoholism in adulthood were investigated. Rats received intermittent ethanol exposure during post-natal days 28-41, and anxiety-like behaviors were measured after 1 and 24 h of the last AIE. The effects of AIE on anxiety-like and alcohol-drinking behaviors in adulthood were measured with or without treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Amygdaloid brain regions were collected to measure HDAC activity, global and gene-specific histone H3 acetylation, expression of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated (Arc) protein and dendritic spine density (DSD). Adolescent rats displayed anxiety-like behaviors after 24 h, but not 1 h, of last AIE with a concomitant increase in nuclear and cytosolic amygdaloid HDAC activity and HDAC2 and HDAC4 levels leading to deficits in histone (H3-K9) acetylation in the central (CeA) and medial (MeA), but not in basolateral nucleus of amygdala (BLA). Interestingly, some of AIE-induced epigenetic changes such as, increased nuclear HDAC activity, HDAC2 expression, and decreased global histone acetylation persisted in adulthood. In addition, AIE decreased BDNF exons I and IV and Arc promoter specific histone H3 acetylation that was associated with decreased BDNF, Arc expression and DSD in the CeA and MeA during adulthood. AIE also induced anxiety-like behaviors and enhanced ethanol intake in adulthood, which was attenuated by TSA treatment via normalization of deficits in histone H3 acetylation of BDNF and Arc genes. These novel results indicate that AIE induces long-lasting effects on histone modifications and deficits in synaptic events in the amygdala, which are

  7. Potential role of adolescent alcohol exposure-induced amygdaloid histone modifications in anxiety and alcohol intake during adulthood

    PubMed Central

    Pandey, Subhash C.; Sakharkar, Amul J.; Tang, Lei; Zhang, Huaibo

    2015-01-01

    Binge drinking is common during adolescence and can lead to the development of psychiatric disorders, including alcoholism in adulthood. Here, the role and persistent effects of histone modifications during adolescent intermittent ethanol (AIE) exposure in the development of anxiety and alcoholism in adulthood were investigated. Rats received intermittent ethanol exposure during post-natal days 28-41, and anxiety-like behaviors were measured after 1 and 24 hrs of the last AIE. The effects of AIE on anxiety-like and alcohol-drinking behaviors in adulthood were measured with or without treatment with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA). Amygdaloid brain regions were collected to measure HDAC activity, global and gene-specific histone H3 acetylation, expression of brain-derived neurotrophic factor (BDNF) and activity-regulated cytoskeleton-associated (Arc) protein and dendritic spine density (DSD). Adolescent rats displayed anxiety-like behaviors after 24 hrs, but not 1 hr, of last AIE with a concomitant increase in nuclear and cytosolic amygdaloid HDAC activity and HDAC2 and HDAC4 levels leading to deficits in histone (H3-K9) acetylation in the central (CeA) and medial (MeA), but not in basolateral nucleus of amygdala (BLA). Interestingly, some of AIE-induced epigenetic changes such as, increased nuclear HDAC activity, HDAC2 expression, decreased global histone acetylation persisted in adulthood. In addition, AIE decreased BDNF exon I, IV and Arc promoter specific histone H3 acetylation that was associated with decreased BDNF, Arc expression and DSD in the CeA and MeA during adulthood. AIE also induced anxiety-like behaviors and enhanced ethanol intake in adulthood, which was attenuated by TSA treatment via normalization of deficits in histone H3 acetylation of BDNF and Arc genes. These novel results indicate that AIE induces long-lasting effects on histone modifications and deficits in synaptic events in the amygdala, which are associated

  8. In silico modification of suberoylanilide hydroxamic acid (SAHA) as potential inhibitor for class II histone deacetylase (HDAC)

    PubMed Central

    2011-01-01

    Background The cervical cancer is the second most prevalent cancer for the woman in the world. It is caused by the oncogenic human papilloma virus (HPV). The inhibition activity of histone deacetylase (HDAC) is a potential strategy for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is widely known as a low toxicity HDAC inhibitor. This research presents in silico SAHA modification by utilizing triazole, in order to obtain a better inhibitor. We conducted docking of the SAHA inhibitor and 12 modified versions to six class II HDAC enzymes, and then proceeded with drug scanning of each one of them. Results The docking results show that the 12 modified inhibitors have much better binding affinity and inhibition potential than SAHA. Based on drug scan analysis, six of the modified inhibitors have robust pharmacological attributes, as revealed by drug likeness, drug score, oral bioavailability, and toxicity levels. Conclusions The binding affinity, free energy and drug scan screening of the best inhibitors have shown that 1c and 2c modified inhibitors are the best ones to inhibit class II HDAC. PMID:22373132

  9. SET1 and p300 Act Synergistically, through Coupled Histone Modifications, in Transcriptional Activation by p53

    PubMed Central

    Tang, Zhanyun; Chen, Wei-Yi; Shimada, Miho; Nguyen, Uyen T.T.; Kim, Jaehoon; Sun, Xiao-Jian; Sengoku, Toru; McGinty, Robert K.; Fernandez, Joseph P.; Muir, Tom W.; Roeder, Robert G.

    2014-01-01

    SUMMARY The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53. PMID:23870121

  10. In silico modification of suberoylanilide hydroxamic acid (SAHA) as potential inhibitor for class II histone deacetylase (HDAC).

    PubMed

    Tambunan, Usman S F; Bramantya, N; Parikesit, Arli A

    2011-01-01

    The cervical cancer is the second most prevalent cancer for the woman in the world. It is caused by the oncogenic human papilloma virus (HPV). The inhibition activity of histone deacetylase (HDAC) is a potential strategy for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is widely known as a low toxicity HDAC inhibitor. This research presents in silico SAHA modification by utilizing triazole, in order to obtain a better inhibitor. We conducted docking of the SAHA inhibitor and 12 modified versions to six class II HDAC enzymes, and then proceeded with drug scanning of each one of them. The docking results show that the 12 modified inhibitors have much better binding affinity and inhibition potential than SAHA. Based on drug scan analysis, six of the modified inhibitors have robust pharmacological attributes, as revealed by drug likeness, drug score, oral bioavailability, and toxicity levels. The binding affinity, free energy and drug scan screening of the best inhibitors have shown that 1c and 2c modified inhibitors are the best ones to inhibit class II HDAC.

  11. 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

  12. Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process

    PubMed Central

    Xu, Jidi; Xu, Haidan; Liu, Yuanlong; Wang, Xia; Xu, Qiang; Deng, Xiuxin

    2015-01-01

    In eukaryotes, histone acetylation and methylation have been known to be involved in regulating diverse developmental processes and plant defense. These histone modification events are controlled by a series of histone modification gene families. To date, there is no study regarding genome-wide characterization of histone modification related genes in citrus species. Based on the two recent sequenced sweet orange genome databases, a total of 136 CsHMs (Citrus sinensis histone modification genes), including 47 CsHMTs (histone methyltransferase genes), 23 CsHDMs (histone demethylase genes), 50 CsHATs (histone acetyltransferase genes), and 16 CsHDACs (histone deacetylase genes) were identified. These genes were categorized to 11 gene families. A comprehensive analysis of these 11 gene families was performed with chromosome locations, phylogenetic comparison, gene structures, and conserved domain compositions of proteins. In order to gain an insight into the potential roles of these genes in citrus fruit development, 42 CsHMs with high mRNA abundance in fruit tissues were selected to further analyze their expression profiles at six stages of fruit development. Interestingly, a numbers of genes were expressed highly in flesh of ripening fruit and some of them showed the increasing expression levels along with the fruit development. Furthermore, we analyzed the expression patterns of all 136 CsHMs response to the infection of blue mold (Penicillium digitatum), which is the most devastating pathogen in citrus post-harvest process. The results indicated that 20 of them showed the strong alterations of their expression levels during the fruit-pathogen infection. In conclusion, this study presents a comprehensive analysis of the histone modification gene families in sweet orange and further elucidates their behaviors during the fruit development and the blue mold infection responses. PMID:26300904

  13. Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process.

    PubMed

    Xu, Jidi; Xu, Haidan; Liu, Yuanlong; Wang, Xia; Xu, Qiang; Deng, Xiuxin

    2015-01-01

    In eukaryotes, histone acetylation and methylation have been known to be involved in regulating diverse developmental processes and plant defense. These histone modification events are controlled by a series of histone modification gene families. To date, there is no study regarding genome-wide characterization of histone modification related genes in citrus species. Based on the two recent sequenced sweet orange genome databases, a total of 136 CsHMs (Citrus sinensis histone modification genes), including 47 CsHMTs (histone methyltransferase genes), 23 CsHDMs (histone demethylase genes), 50 CsHATs (histone acetyltransferase genes), and 16 CsHDACs (histone deacetylase genes) were identified. These genes were categorized to 11 gene families. A comprehensive analysis of these 11 gene families was performed with chromosome locations, phylogenetic comparison, gene structures, and conserved domain compositions of proteins. In order to gain an insight into the potential roles of these genes in citrus fruit development, 42 CsHMs with high mRNA abundance in fruit tissues were selected to further analyze their expression profiles at six stages of fruit development. Interestingly, a numbers of genes were expressed highly in flesh of ripening fruit and some of them showed the increasing expression levels along with the fruit development. Furthermore, we analyzed the expression patterns of all 136 CsHMs response to the infection of blue mold (Penicillium digitatum), which is the most devastating pathogen in citrus post-harvest process. The results indicated that 20 of them showed the strong alterations of their expression levels during the fruit-pathogen infection. In conclusion, this study presents a comprehensive analysis of the histone modification gene families in sweet orange and further elucidates their behaviors during the fruit development and the blue mold infection responses.

  14. Pathway analysis of whole exome sequence data provides further support for the involvement of histone modification in the aetiology of schizophrenia.

    PubMed

    Curtis, David

    2016-10-01

    Weighted burden pathway analysis was applied to whole exome sequence data for 2045 schizophrenic patients and 2045 controls. Overall, there was a statistically significant excess of pathways with more rare, functional variants in cases than in controls. Among the highest ranked were pathways relating to histone modification, as well as neuron differentiation and membrane and vesicle function. This bolsters the evidence from previous studies that histone modification pathways may be important in the aetiology of schizophrenia.

  15. A computational model for histone mark propagation reproduces the distribution of heterochromatin in different human cell types.

    PubMed

    Schwämmle, Veit; Jensen, Ole Nørregaard

    2013-01-01

    Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.

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

    PubMed

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

    2017-01-01

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

  17. Epigenetic Interactions between Alcohol and Cannabinergic Effects: Focus on Histone Modification and DNA Methylation

    PubMed Central

    Parira, Tiyash; Laverde, Alejandra; Agudelo, Marisela

    2017-01-01

    Epigenetic studies have led to a more profound understanding of the mechanisms involved in chronic conditions. In the case of alcohol addiction, according to the National Institute on Alcohol Abuse and Alcoholism, 16 million adults suffer from Alcohol Use Disorders (AUDs). Even though therapeutic interventions like behavioral therapy and medications to prevent relapse are currently available, no robust cure exists, which stems from the lack of understanding the mechanisms of action of alcohol and the lack of development of precision medicine approaches to treat AUDs. Another common group of addictive substance, cannabinoids, have been studied extensively to reveal they work through cannabinoid receptors. Therapeutic applications have been found for the cannabinoids and a deeper understanding of the endocannabinoid system has been gained over the years. Recent reports of cannabinergic mechanisms in AUDs has opened an exciting realm of research that seeks to elucidate the molecular mechanisms of alcohol-induced end organ diseases and hopefully provide insight into new therapeutic strategies for the treatment of AUDs. To date, several epigenetic mechanisms have been associated with alcohol and cannabinoids independently. Therefore, the scope of this review is to compile the most recent literature regarding alcohol and cannabinoids in terms of a possible epigenetic connection between the endocannabinoid system and alcohol effects. First, we will provide an overview of epigenetics, followed by an overview of alcohol and epigenetic mechanisms with an emphasis on histone modifications and DNA methylations. Then, we will provide an overview of cannabinoids and epigenetic mechanisms. Lastly, we will discuss evidence of interactions between alcohol and cannabinergic pathways and possible insights into the novel epigenetic mechanisms underlying alcohol-cannabinergic pathway activity. Finalizing the review will be a discussion of future directions and therapeutic applications

  18. Developmentally arrested Austrofundulus limnaeus embryos have changes in post-translational modifications of histone H3.

    PubMed

    Toni, Lee S; Padilla, Pamela A

    2016-02-01

    Although vertebrate embryogenesis is typically a continuous and dynamic process, some embryos have evolved mechanisms to developmentally arrest. The embryos of Austrofundulus limnaeus, a killifish that resides in ephemeral ponds, routinely enter diapause II (DII), a reversible developmental arrest promoted by endogenous cues rather than environmental stress. DII, which starts at 24-26 days post-fertilization and can persist for months, is characterized by a significant decline in heart rate and an arrest of development and differentiation. Thus, A. limnaeus is a unique model to study epigenetic features associated with embryonic arrest. To investigate chromosome structures associated with mitosis or gene expression, we examined the post-translational modifications of histone H3 (phosphorylation of serine 10, mono-, di- and tri-methylation of lysine 4 or 27) in preDII, DII and postDII embryos. As seen by microscopy analysis, DII embryos have a significant decrease in the H3S10P marker for mitotic nuclei and an inner nuclear membrane localization of the H3K27me2 marker associated with silencing of gene expression. ELISA experiments reveal that the levels of methylation at H3K4 and H3K27 are significantly different between preDII, DII and postDII embryos, indicating that there are molecular differences between embryos of different chronological age and stage of development. Furthermore, in DII embryos relative to preDII embryos, there are differences in the level of H3K27me3 and H3K4me3, which may reflect critical chromatin remodeling that occurs prior to arrest of embryogenesis. This work helps lay a foundation for chromatin analysis of vertebrate embryo diapause, an intriguing yet greatly understudied phenomenon.

  19. Differences in histone modifications between slow- and fast-twitch muscle of adult rats and following overload, denervation, or valproic acid administration.

    PubMed

    Kawano, Fuminori; Nimura, Keisuke; Ishino, Saki; Nakai, Naoya; Nakata, Ken; Ohira, Yoshinobu

    2015-11-15

    Numerous studies have reported alterations in skeletal muscle properties and phenotypes in response to various stimuli such as exercise, unloading, and gene mutation. However, a shift in muscle fiber phenotype from fast twitch to slow twitch is not completely induced by stimuli. This limitation is hypothesized to result from the epigenetic differences between muscle types. The main purpose of the present study was to identify the differences in histone modification for the plantaris (fast) and soleus (slow) muscles of adult rats. Genome-wide analysis by chromatin immunoprecipitation followed by DNA sequencing revealed that trimethylation at lysine 4 and acetylation of histone 3, which occurs at transcriptionally active gene loci, was less prevalent in the genes specific to the slow-twitch soleus muscle. Conversely, gene loci specific to the fast-twitch plantaris muscle were associated with the aforementioned histone modifications. We also found that upregulation of slow genes in the plantaris muscle, which are related to enhanced muscular activity, is not associated with activating histone modifications. Furthermore, silencing of muscle activity by denervation caused the displacement of acetylated histone and RNA polymerase II (Pol II) in 5' ends of genes in plantaris, but minor effects were observed in soleus. Increased recruitment of Pol II induced by forced acetylation of histone was also suppressed in valproic acid-treated soleus. Our present data indicate that the slow-twitch soleus muscle has a unique set of histone modifications, which may relate to the preservation of the genetic backbone against physiological stimuli.

  20. POTAMOS mass spectrometry calculator: computer aided mass spectrometry to the post-translational modifications of proteins. A focus on histones.

    PubMed

    Vlachopanos, A; Soupsana, E; Politou, A S; Papamokos, G V

    2014-12-01

    Mass spectrometry is a widely used technique for protein identification and it has also become the method of choice in order to detect and characterize the post-translational modifications (PTMs) of proteins. Many software tools have been developed to deal with this complication. In this paper we introduce a new, free and user friendly online software tool, named POTAMOS Mass Spectrometry Calculator, which was developed in the open source application framework Ruby on Rails. It can provide calculated mass spectrometry data in a time saving manner, independently of instrumentation. In this web application we have focused on a well known protein family of histones whose PTMs are believed to play a crucial role in gene regulation, as suggested by the so called "histone code" hypothesis. The PTMs implemented in this software are: methylations of arginines and lysines, acetylations of lysines and phosphorylations of serines and threonines. The application is able to calculate the kind, the number and the combinations of the possible PTMs corresponding to a given peptide sequence and a given mass along with the full set of the unique primary structures produced by the possible distributions along the amino acid sequence. It can also calculate the masses and charges of a fragmented histone variant, which carries predefined modifications already implemented. Additional functionality is provided by the calculation of the masses of fragments produced upon protein cleavage by the proteolytic enzymes that are most widely used in proteomics studies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. The progeny of Arabidopsis thaliana plants exposed to salt exhibit changes in DNA methylation, histone modifications and gene expression.

    PubMed

    Bilichak, Andriy; Ilnystkyy, Yaroslav; Hollunder, Jens; Kovalchuk, Igor

    2012-01-01

    Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5' and 3' ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants.

  2. Quantitatively profiling genome-wide patterns of histone modifications in Arabidopsis thaliana using ChIP-seq.

    PubMed

    Luo, Chongyuan; Lam, Eric

    2014-01-01

    Genome-wide quantitative profiling of chromatin modifications is a critical experimental approach to study epigenetic and transcriptional control mechanisms. Since first being reported in 2007, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) has soon became a popular method-of-choice for profiling chromatin modifications and transcription factor-binding sites in eukaryote genomes. ChIP-seq has the advantage over the earlier ChIP-chip approach in multiple aspects including the lower amount of input DNA required, an expanded dynamic range and compatibility with sample multiplexing. Here we describe a detailed protocol for profiling histone modification in the Arabidopsis thaliana genome with ChIP-seq using the SOLiD™ 2.0 high-throughput sequencing platform. As read length and sequencing depth are two critical factors determining data quality and cost, we have developed bioinformatics approach to evaluate the effect of read length and sequencing depth on the alignment accuracy and the generated chromatin profile, respectively. Our analyses suggest that 2-3 million high quality sequencing tags with a read length of 35 nucleotides would be sufficient to profile the majority of histone modifications in this popular model plant species.

  3. 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.

  4. Calibrating ChIP-seq with nucleosomal internal standards to measure histone modification density genome-wide

    PubMed Central

    Grzybowski, Adrian T.; Chen, Zhonglei; Ruthenburg, Alexander J.

    2015-01-01

    SUMMARY Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison amongst experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons and reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls, as well as providing a critical examination of untested assumptions inherent to conventional ChIP. PMID:26004229

  5. Metabolically Derived Lysine Acylations and Neighboring Modifications Tune the Binding of the BET Bromodomains to Histone H4.

    PubMed

    Olp, Michael D; Zhu, Nan; Smith, Brian C

    2017-10-05

    Recent proteomic studies discovered histone lysines are modified by acylations beyond acetylation. These acylations derive from acyl-CoA metabolites, potentially linking metabolism to transcription. Bromodomains bind lysine acylation on histones and other nuclear proteins to influence transcription. However, the extent bromodomains bind non-acetyl acylations is largely unknown. Also unclear are the effects of neighboring post-translational modifications, especially within heavily modified histone tails. Using peptide arrays, binding assays, sucrose gradients, and computational methods, we quantified 10 distinct acylations for binding to the bromodomain and extraterminal domain (BET) family. Four of these acylations (hydroxyisobutyrylation, malonylation, glutarylation, and homocitrullination) had never been tested for bromodomain binding. We found N-terminal BET bromodomains bound acetylated and propionylated peptides, consistent with previous studies. Interestingly, all other acylations inhibited binding of the BET bromodomains to peptides and nucleosomes. To understand how context tunes bromodomain binding, effects of neighboring methylation, phosphorylation, and acylation within histone H4 tails were determined. Serine 1 phosphorylation inhibited binding of the BRD4 N-terminal bromodomain to polyacetylated H4 tails by >5-fold, whereas methylation had no effect. Furthermore, binding of BRDT and BRD4 N-terminal bromodomains to H4K5acetyl was enhanced 1.4-9.5-fold by any neighboring acylation of lysine 8, indicating a secondary H4K8acyl binding site that is more permissive of non-acetyl acylations than previously appreciated. In contrast, C-terminal BET bromodomains exhibited 9.9-13.5-fold weaker binding for polyacylated than for monoacylated H4 tails, indicating the C-terminal bromodomains do not cooperatively bind multiple acylations. These results suggest acyl-CoA levels tune or block recruitment of the BET bromodomains to histones, linking metabolism to

  6. Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism

    PubMed Central

    Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

    2016-01-01

    Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression. PMID:27598153

  7. Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism.

    PubMed

    Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

    2016-09-03

    Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression.

  8. Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations.

    PubMed

    Savickienė, Jūratė; Matuzevičius, Dalius; Baronaitė, Sandra; Treigytė, Gražina; Krasovskaja, Natalija; Zaikova, Ilona; Navakauskas, Dalius; Utkus, Algirdas; Navakauskienė, Rūta

    2017-04-05

    Human amniotic fluid (AF)-derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus-affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5-Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF-MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF-MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 9999: 1-12, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

    PubMed

    Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

    2017-02-28

    Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies.

  10. Structure of the human histone chaperone FACT Spt16 N-terminal domain.

    PubMed

    Marcianò, G; Huang, D T

    2016-02-01

    The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  11. Genome-wide analysis of histone modifications by ChIP-chip to identify silenced genes in gastric cancer.

    PubMed

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

    2015-05-01

    The present study aimed to identify novel histone modification markers in gastric cancer (GC) by chromatin immunoprecipitation microarray (ChIP-chip) analysis and to determine whether these markers were able to discriminate between normal and GC cells. We also tested for correlations with DNA methylation. We probed a human CpG island microarray with DNA from a GC cell line (MKN45) by chromatin immunoprecipitation (ChIP). ChIP-reverse-transcriptase quantitative polymerase chain reaction PCR (RT-qPCR) was used to validate the microarray results. Additionally, mRNA expression levels and the DNA methylation of potential target genes were evaluated by RT-qPCR and methylation-specific PCR (MSP). The moults showed that 134 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over acetylation and 46 genes exhibited the highest signal-to-noise ratio of H3-K9 trimethylation over H3-K4 trimethylation in MKN45 cells. The ChIP-qPCR results agreed with those obtained from the ChIP-chip analysis. Aberrant DNA methylation status and mRNA expression levels were also identified for selected genes (PSD, SMARCC1 and Vps37A) in the GC cell lines. The results suggest that CpG island microarray coupled with ChIP (ChIP-chip) can identify novel targets of gene silencing in GC. Additionally, ChIP-chip is the best approach for assessing the genome-wide status of epigenetic regulation, which may allow for a broader genomic understanding compared to the knowledge that has been accumulated from single-gene studies.

  12. Liganded thyroid hormone receptor induces nucleosome removal and histone modifications to activate transcription during larval intestinal cell death and adult stem cell development.

    PubMed

    Matsuura, Kazuo; Fujimoto, Kenta; Fu, Liezhen; Shi, Yun-Bo

    2012-02-01

    Thyroid hormone (T(3)) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T(3) signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T(3)-dependent Xenopus metamorphosis as a model to study how T(3) regulates transcription during vertebrate development. T(3) exerts its metamorphic effects through T(3) receptors (TR). TR recruits, in a T(3)-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T(3) target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo.

  13. Alteration in inflammatory/apoptotic pathway and histone modifications by nordihydroguaiaretic acid prevents acute pancreatitis in swiss albino mice.

    PubMed

    Mahajan, Ujwal Mukund; Gupta, Chanchal; Wagh, Preshit Ravindra; Karpe, Pinakin Arun; Tikoo, Kulbhushan

    2011-11-01

    Reactive oxygen radicals, pro-inflammatory mediators and cytokines have been implicated in caerulein induced acute pancreatitis. Nordihydroguaiaretic acid (NDGA), a plant lignin, has marked anti-inflammatory properties. The present study aimed to investigate the possible protective effect of NDGA against caerulein induced pancreatitis. Acute pancreatitis was induced by intraperitoneal administration of eight doses of caerulein in male swiss albino mice. NDGA was administered after 9 h of acute pancreatitis induction. Pancreatic damage and the protective effect of NDGA were assessed by oxidative stress parameters and histopathology of pancreas. The mRNA expression of heat shock proteins (DNAJ C15 and HSPD1) was examined by real-time RT-PCR analysis. Expression of HSP 27, NF-κB, TNF-α, p-p38, Bcl-2, p-PP2A, procaspase-3, caspase-3 and histone modifications were examined by western blotting. NDGA attenuated the oxidative stress, led to increased plasma α-amylase and decreased IGF-1 in AP mice. It modulated the mRNA and protein levels of heat shock proteins and reduced the expression of NF-κB, TNF-α and p-p38. It increased the number of TUNEL positive apoptotic cells in the pancreas of AP mice. In addition, NDGA prevented the changes in modifications of histone H3 in acute pancreatitis. To best of our knowledge, this is the first report which suggests that NDGA prevents the progression of acute pancreatitis by involving alteration of histone H3 modifications and modulating the expression of genes involved in inflammatory/apoptotic cascade, which may be responsible for decreased necrosis and increased apoptosis in this model of acute pancreatitis.

  14. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    DOE PAGES

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; ...

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less

  15. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility

    SciTech Connect

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S.; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A.; Cheng, Yong; Jain, Deepti; Visel, Axel; Pennacchio, Len A.; Weiss, Mitchell J.; Blobel, Gerd A.; Hardison, Ross C.

    2015-04-23

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

  16. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility.

    PubMed

    Dogan, Nergiz; Wu, Weisheng; Morrissey, Christapher S; Chen, Kuan-Bei; Stonestrom, Aaron; Long, Maria; Keller, Cheryl A; Cheng, Yong; Jain, Deepti; Visel, Axel; Pennacchio, Len A; Weiss, Mitchell J; Blobel, Gerd A; Hardison, Ross C

    2015-01-01

    Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

  17. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    PubMed Central

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  18. Modelling complex features from histone modification signatures using genetic algorithm for the prediction of enhancer region.

    PubMed

    Lee, Nung Kion; Fong, Pui Kwan; Abdullah, Mohd Tajuddin

    2014-01-01

    Using Genetic Algorithm, this paper presents a modelling method to generate novel logical-based features from DNA sequences enriched with H3K4mel histone signatures. Current histone signature is mostly represented using k-mers content features incapable of representing all the possible complex interactions of various DNA segments. The main contributions are, among others: (a) demonstrating that there are complex interactions among sequence segments in the histone regions; (b) developing a parse tree representation of the logical complex features. The proposed novel feature is compared to the k-mers content features using datasets from the mouse (mm9) genome. Evaluation results show that the new feature improves the prediction performance as shown by f-measure for all datasets tested. Also, it is discovered that tree-based features generated from a single chromosome can be generalized to predict histone marks in other chromosomes not used in the training. These findings have a great impact on feature design considerations for histone signatures as well as other classifier design features.

  19. Inhibitors of histone demethylation and histone deacetylation cooperate in regulating gene expression and inhibiting growth in human breast cancer cells

    PubMed Central

    Vasilatos, Shauna N.; Boric, Lamia; Shaw, Patrick G.; Davidson, Nancy E.

    2013-01-01

    Abnormal activities of histone lysine demethylases (KDMs) and lysine deacetylases (HDACs) are associated with aberrant gene expression in breast cancer development. However, the precise molecular mechanisms underlying the crosstalk between KDMs and HDACs in chromatin remodeling and regulation of gene transcription are still elusive. In this study, we showed that treatment of human breast cancer cells with inhibitors targeting the zinc cofactor dependent class I/II HDAC, but not NAD+ dependent class III HDAC, led to significant increase of H3K4me2 which is a specific substrate of histone lysine-specific demethylase 1 (LSD1) and a key chromatin mark promoting transcriptional activation. We also demonstrated that inhibition of LSD1 activity by a pharmacological inhibitor, pargyline, or siRNA resulted in increased acetylation of H3K9 (AcH3K9). However, siRNA knockdown of LSD2, a homolog of LSD1, failed to alter the level of AcH3K9, suggesting that LSD2 activity may not be functionally connected with HDAC activity. Combined treatment with LSD1 and HDAC inhibitors resulted in enhanced levels of H3K4me2 and AcH3K9, and exhibited synergistic growth inhibition of breast cancer cells. Finally, microarray screening identified a unique subset of genes whose expression was significantly changed by combination treatment with inhibitors of LSD1 and HDAC. Our study suggests that LSD1 intimately interacts with histone deacetylases in human breast cancer cells. Inhibition of histone demethylation and deacetylation exhibits cooperation and synergy in regulating gene expression and growth inhibition, and may represent a promising and novel approach for epigenetic therapy of breast cancer. PMID:21452019

  20. 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

  1. Prenatal Exposure to a Maternal High-Fat Diet Affects Histone Modification of Cardiometabolic Genes in Newborn Rats.

    PubMed

    Upadhyaya, Bijaya; Larsen, Tricia; Barwari, Shivon; Louwagie, Eli J; Baack, Michelle L; Dey, Moul

    2017-04-20

    Infants born to women with diabetes or obesity are exposed to excess circulating fuels during fetal heart development and are at higher risk of cardiac diseases. We have previously shown that late-gestation diabetes, especially in conjunction with a maternal high-fat (HF) diet, impairs cardiac functions in rat-offspring. This study investigated changes in genome-wide histone modifications in newborn hearts from rat-pups exposed to maternal diabetes and HF-diet. Chromatin-immunoprecipitation-sequencing revealed a differential peak distribution on gene promoters in exposed pups with respect to acetylation of lysines 9 and 14 and to trimethylation of lysines 4 and 27 in histone H3 (all, false discovery rate, FDR < 0.1). In the HF-diet exposed offspring, 54% of the annotated genes showed the gene-activating mark trimethylated lysine 4. Many of these genes (1) are associated with the "metabolic process" in general and particularly with "positive regulation of cholesterol biosynthesis" (FDR = 0.03); (2) overlap with 455 quantitative trait loci for blood pressure, body weight, serum cholesterol (all, FDR < 0.1); and (3) are linked to cardiac disease susceptibility/progression, based on disease ontology analyses and scientific literature. These results indicate that maternal HF-diet changes the cardiac histone signature in offspring suggesting a fuel-mediated epigenetic reprogramming of cardiac tissue in utero.

  2. Rapid Histone-Catalyzed DNA Lesion Excision and Accompanying Protein Modification in Nucleosomes and Nucleosome Core Particles.

    PubMed

    Weng, Liwei; Greenberg, Marc M

    2015-09-02

    C5'-Hydrogen atoms are frequently abstracted during DNA oxidation. The oxidized abasic lesion 5'-(2-phosphoryl-1,4-dioxobutane) (DOB) is an electrophilic product of the C5'-radical. DOB is a potent irreversible inhibitor of DNA polymerase β, and forms interstrand cross-links in free DNA. We examined the reactivity of DOB within nucleosomes and nucleosome core particles (NCPs), the monomeric component of chromatin. Depending upon the position at which DOB is generated within a NCP, it is excised from nucleosomal DNA at a rate 275-1500-fold faster than that in free DNA. The half-life of DOB (7.0-16.8 min) in NCPs is shorter than any other abasic lesion. DOB's lifetime in NCPs is also significantly shorter than the estimated lifetime of an abasic site within a cell, suggesting that the observed chemistry would occur intracellularly. Histones also catalyze DOB excision when the lesion is present in the DNA linker region of a nucleosome. Schiff-base formation between DOB and histone proteins is detected in nucleosomes and NCPs, resulting in pyrrolone formation at the lysine residues. The lysines modified by DOB are often post-translationally modified. Consequently, the histone modifications described herein could affect the regulation of gene expression and may provide a chemical basis for the cytotoxicity of the DNA damaging agents that produce this lesion.

  3. The functional interactome landscape of the human histone deacetylase family

    PubMed Central

    Joshi, Preeti; Greco, Todd M; Guise, Amanda J; Luo, Yang; Yu, Fang; Nesvizhskii, Alexey I; Cristea, Ileana M

    2013-01-01

    Histone deacetylases (HDACs) are a diverse family of essential transcriptional regulatory enzymes, that function through the spatial and temporal recruitment of protein complexes. As the composition and regulation of HDAC complexes are only partially characterized, we built the first global protein interaction network for all 11 human HDACs in T cells. Integrating fluorescence microscopy, immunoaffinity purifications, quantitative mass spectrometry, and bioinformatics, we identified over 200 unreported interactions for both well-characterized and lesser-studied HDACs, a subset of which were validated by orthogonal approaches. We establish HDAC11 as a member of the survival of motor neuron complex and pinpoint a functional role in mRNA splicing. We designed a complementary label-free and metabolic-labeling mass spectrometry-based proteomics strategy for profiling interaction stability among different HDAC classes, revealing that HDAC1 interactions within chromatin-remodeling complexes are largely stable, while transcription factors preferentially exist in rapid equilibrium. Overall, this study represents a valuable resource for investigating HDAC functions in health and disease, encompassing emerging themes of HDAC regulation in cell cycle and RNA processing and a deeper functional understanding of HDAC complex stability. PMID:23752268

  4. Caspase-mediated specific cleavage of human histone deacetylase 4.

    PubMed

    Liu, Fang; Dowling, Melissa; Yang, Xiang-Jiao; Kao, Gary D

    2004-08-13

    Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation.

  5. Experience Affects Critical Period Plasticity in the Visual Cortex through an Epigenetic Regulation of Histone Post-Translational Modifications.

    PubMed

    Baroncelli, Laura; Scali, Manuela; Sansevero, Gabriele; Olimpico, Francesco; Manno, Ilaria; Costa, Mario; Sale, Alessandro

    2016-03-23

    During an early phase of enhanced sensitivity called the critical period (CP), monocular deprivation causes a shift in the response of visual cortex binocular neurons in favor of the nondeprived eye, a process named ocular dominance (OD) plasticity. While the time course of the CP for OD plasticity can be modulated by genetic/pharmacological interventions targeting GABAergic inhibition, whether an increased sensory-motor experience can affect this major plastic phenomenon is not known. We report that exposure to environmental enrichment (EE) accelerated the closure of the CP for OD plasticity in the rat visual cortex. Histone H3 acetylation was developmentally regulated in primary visual cortex, with enhanced levels being detectable early in enriched pups, and chromatin immunoprecipitation revealed an increase at the level of the BDNF P3 promoter. Administration of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) to animals reared in a standard cage mimicked the increase in H3 acetylation observed in the visual cortex and resulted in an accelerated decay of OD plasticity. Finally, exposure to EE in adulthood upregulated H3 acetylation and was paralleled by a reopening of the CP. These findings demonstrate a critical involvement of the epigenetic machinery as a mediator of visual cortex developmental plasticity and of the impact of EE on OD plasticity. While it is known that an epigenetic remodeling of chromatin structure controls developmental plasticity in the visual cortex, three main questions have remained open. Which is the physiological time course of histone modifications? Is it possible, by manipulating the chromatin epigenetic state, to modulate plasticity levels during the critical period? How can we regulate histone acetylation in the adult brain in a noninvasive manner? We show that the early exposure of rat pups to enriching environmental conditions accelerates the critical period for plasticity in the primary visual cortex

  6. Structure of the human histone chaperone FACT Spt16 N-terminal domain

    SciTech Connect

    Marcianò, G.; Huang, D. T.

    2016-01-22

    The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

  7. Large scale analysis of co-existing post-translational modifications in histone tails reveals global fine structure of cross-talk.

    PubMed

    Schwämmle, Veit; Aspalter, Claudia-Maria; Sidoli, Simone; Jensen, Ole N

    2014-07-01

    Mass spectrometry (MS) is a powerful analytical method for the identification and quantification of co-existing post-translational modifications in histone proteins. One of the most important challenges in current chromatin biology is to characterize the relationships between co-existing histone marks, the order and hierarchy of their deposition, and their distinct biological functions. We developed the database CrossTalkDB to organize observed and reported co-existing histone marks as revealed by MS experiments of histone proteins and their derived peptides. Statistical assessment revealed sample-specific patterns for the co-frequency of histone post-translational modifications. We implemented a new method to identify positive and negative interplay between pairs of methylation and acetylation marks in proteins. Many of the detected features were conserved between different cell types or exist across species, thereby revealing general rules for cross-talk between histone marks. The observed features are in accordance with previously reported examples of cross-talk. We observed novel types of interplay among acetylated residues, revealing positive cross-talk between nearby acetylated sites but negative cross-talk for distant ones, and for discrete methylation states at Lys-9, Lys-27, and Lys-36 of histone H3, suggesting a more differentiated functional role of methylation beyond the general expectation of enhanced activity at higher methylation states.

  8. SUMO modification of a heterochromatin histone demethylase JMJD2A enables viral gene transactivation and viral replication

    PubMed Central

    Yang, Wan-Shan; Campbell, Mel

    2017-01-01

    Small ubiquitin-like modifier (SUMO) modification of chromatin has profound effects on transcription regulation. By using Kaposi’s sarcoma associated herpesvirus (KSHV) as a model, we recently demonstrated that epigenetic modification of viral chromatin by SUMO-2/3 is involved in regulating gene expression and viral reactivation. However, how this modification orchestrates transcription reprogramming through targeting histone modifying enzymes remains largely unknown. Here we show that JMJD2A, the first identified Jumonji C domain-containing histone demethylase, is the histone demethylase responsible for SUMO-2/3 enrichment on the KSHV genome during viral reactivation. Using in vitro and in vivo SUMOylation assays, we found that JMJD2A is SUMOylated on lysine 471 by KSHV K-bZIP, a viral SUMO-2/3-specific E3 ligase, in a SUMO-interacting motif (SIM)-dependent manner. SUMOylation is required for stabilizing chromatin association and gene transactivation by JMJD2A. These finding suggest that SUMO-2/3 modification plays an essential role in the epigenetic regulatory function of JMJD2A. Consistently, hierarchical clustering analysis of RNA-seq data showed that a SUMO-deficient mutant of JMJD2A was more closely related to JMJD2A knockdown than to wild-type. Our previous report demonstrated that JMJD2A coated and maintained the “ready to activate” status of the viral genome. Consistent with our previous report, a SUMO-deficient mutant of JMJD2A reduced viral gene expression and virion production. Importantly, JMJD2A has been implicated as an oncogene in various cancers by regulating proliferation. We therefore further analyzed the role of SUMO modification of JMJD2A in regulating cell proliferation. Interestingly, the SUMO-deficient mutant of JMJD2A failed to rescue the proliferation defect of JMJD2A knockdown cells. Emerging specific inhibitors of JMJD2A have been generated for evaluation in cancer studies. Our results revealed that SUMO conjugation mediates an

  9. Changes in histone modification and DNA methylation of the StAR and Cyp19a1 promoter regions in granulosa cells undergoing luteinization during ovulation in rats.

    PubMed

    Lee, Lifa; Asada, Hiromi; Kizuka, Fumie; Tamura, Isao; Maekawa, Ryo; Taketani, Toshiaki; Sato, Shun; Yamagata, Yoshiaki; Tamura, Hiroshi; Sugino, Norihiro

    2013-01-01

    The ovulatory LH surge induces rapid up-regulation of steroidogenic acute regulatory (StAR) protein and rapid down-regulation of aromatase (Cyp19a1) in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic mechanisms including histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h)CG injection. StAR mRNA levels rapidly increased after hCG injection, reached a peak at 4 h, and then remained higher compared with 0 h until 12 h. Cyp19a1 mRNA levels gradually decreased after hCG injection and reached their lowest level at 12 h. A chromatin immunoprecipitation assay revealed that levels of histone-H4 acetylation (Ac-H4) and trimethylation of histone-H3 lysine-4 (H3K4me3) increased whereas H3K9me3 and H3K27me3 decreased in the StAR promoter after hCG injection. On the other hand, the levels of Ac-H3 and -H4 and H3K4me3 decreased, and H3K27me3 increased in the Cyp19a1 promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the StAR promoter and increased in the Cyp19a1 promoter after hCG injection. A chromatin immunoprecipitation assay also showed that binding activities of CAATT/enhancer-binding protein β to the StAR promoter increased and binding activities of phosphorylated-cAMP response element binding protein to the Cyp19a1 promoter decreased after hCG injection. These results provide in vivo evidence that histone modifications are involved in the rapid changes of StAR and Cyp19a1 gene expression by altering chromatin structure of the promoters in GCs undergoing luteinization during ovulation.

  10. Bioinformatic dissecting of TP53 regulation pathway underlying butyrate-induced histone modification in epigenetic regulation

    USDA-ARS?s Scientific Manuscript database

    Butyrate affects cell proliferation, differentiation and motility. Butyrate inhibits histone deacetylase (HDAC) activities and induces cell cycle arrest and apoptosis. TP53 is one of the most active upstream regulators discovered by IPA in our RNA sequencing data set. The TP53 signaling pathway pl...

  11. Transcriptomic sequencing reveals a set of unique genes activated by butyrate-induced histone modification

    USDA-ARS?s Scientific Manuscript database

    Butyrate is a nutritional element with strong epigenetic regulatory activity as an inhibitor of histone deacetylases (HDACs). Based on the analysis of differentially expressed genes induced by butyrate in the bovine epithelial cell using deep RNA-sequencing technology (RNA-seq), a set of unique gen...

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

    PubMed Central

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

    2014-01-01

    Trimethylation of lysine 36 of histone H3 (H3K36me3) is found to be associated with various transcription events. In Arabidopsis, the H3K36me3 level peaks in the first half of coding regions, which is in contrast to the 3′-end enrichment in animals. The MRG15 family proteins function as ‘reader’ proteins by binding to H3K36me3 to control alternative splicing or prevent spurious intragenic transcription in animals. Here, we demonstrate that two closely related Arabidopsis homologues (MRG1 and MRG2) are localised to the euchromatin and redundantly ensure the increased transcriptional levels of two flowering time genes with opposing functions, FLOWERING LOCUS C and FLOWERING LOCUS T (FT). MRG2 directly binds to the FT locus and elevates the expression in an H3K36me3-dependent manner. MRG1/2 binds to H3K36me3 with their chromodomain and interact with the histone H4-specific acetyltransferases (HAM1 and HAM2) to achieve a high expression level through active histone acetylation at the promoter and 5′ regions of target loci. Together, this study presents a mechanistic link between H3K36me3 and histone H4 acetylation. Our data also indicate that the biological functions of MRG1/2 have diversified from their animal homologues during evolution, yet they still maintain their conserved H3K36me3-binding molecular function. PMID:25183522

  13. DNA methylation, histone acetylation and methylation of epigenetic modifications as a therapeutic approach for cancers.

    PubMed

    Yen, Ching-Yu; Huang, Hurng-Wern; Shu, Chih-Wen; Hou, Ming-Feng; Yuan, Shyng-Shiou F; Wang, Hui-Ru; Chang, Yung-Ting; Farooqi, Ammad Ahmad; Tang, Jen-Yang; Chang, Hsueh-Wei

    2016-04-10

    Epigenetic modifications play important roles in regulating carcinogenesis, and specific epigenetic modifications have emerged as potential tumor markers. Herein, we summarize several types of epigenetic modifications, explore the role played by epigenetic modifications in gene regulation, and describe the patterns of epigenetic modifications in cancers. Since epigenetic modifications have been reported to regulate the Warburg effect in cancers, the roles of epigenetic modifications in sugar metabolism are discussed. In addition, oxidative stress may play an important role in carcinogenesis, and the role of oxidative stress and epigenetic modification in carcinogenesis is addressed. We also discuss the role of epigenetic modifications as therapeutic targets. Finally, the synergistic effects of the combined treatment of epigenetic regulator and anticancer drugs for cancer therapy are described. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Promoter methylation and histone modifications affect the expression of the exogenous DsRed gene in transgenic goats.

    PubMed

    Nuo, M T; Yuan, J L; Yang, W L; Gao, X Y; He, N; Liang, H; Cang, M; Liu, D J

    2016-08-29

    Transgene silencing, which is common in transgenic plants and animals, limits the generation and application of genetically modified organisms, and is associated with the exogenous gene copy number, the methylation status of its promoters, and histone modification abnormalities. Here, we analyzed the expression of the exogenous gene DsRed and the methylation status of its cytomegalovirus (CMV) promoter in six healthy transgenic cashmere goats and transgenic nuclear donor cells. The CMV promoter exhibited high methylation levels (74.4-88.2%) in four of the goats, a moderate methylation level (58.7%) in one, and a low methylation level (21.2%) in one, while the methylation level of the transgenic nuclear donor cells was comparatively low (14.3%). DsRed expression was negatively correlated with promoter methylation status. Transgenic cashmere goats carried one to three copies of the CMV promoter fragment and one to six copies of the DsRed fragment, but copy number showed no obvious correlation with DsRed expression. After treatment with the methylation inhibitor 5-azacytidine, DsRed expression in transgenic goat cells significantly increased and CMV promoter methylation significantly decreased; this indicated an inverse correlation between promoter methylation status and DsRed expression. After treatment with the histone deacetylase inhibitor trichostatin A, DsRed expression increased, indicating that an abnormal histone modification in transgenic goats is also involved in exogenous gene silencing. These findings indicate the potential of trichostatin A and 5-azacytidine to rescue the biological activity of silenced exogenous transgenes in adult-derived transgenic cells under culture conditions.

  15. Stability of single copy transgene expression in CHOK1 cells is affected by histone modifications but not by DNA methylation.

    PubMed

    Spencer, Shawal; Gugliotta, Agustina; Koenitzer, Jennifer; Hauser, Hansjörg; Wirth, Dagmar

    2015-02-10

    Intraclonal heterogeneity of genetically modified mammalian cells has been observed as a phenomenon that has a strong impact on overall transgene expression levels and that limits the predictability of transgene expression in genetically modified cells, thereby hampering single cell based screening approaches. The underlying mechanism(s) leading to this variance are poorly understood. To study the dynamics and mechanisms of heterogeneity of early stage silencing we analyzed the expression in more than 100 independent clones of CHOK1 cells that harbour genetically stable integrates of single copy reporter cassettes driven by EF1α and CMV promoters. Single cell analysis showed intraclonal variability with heterogeneity in expression in genetically uniform populations. DNA methylation is a well known mechanism responsible for silencing of gene expression. Interestingly, loss of expression was not associated with DNA methylation of the CMV promoter. However, in most of the clonal populations expression could be increased by inhibitors of the histone deacetylases (HDACi) suggesting that heterogeneity of transgene expression is crucially governed by histone modifications. Further, to determine if the epigenetic status of transgene expression is governed by the chromosomal integration locus we targeted heterologous expression cassettes into two chromosomal sites using recombinase mediated cassette exchange (RMCE). The expression status of a particular clone was faithfully re-established when the same promoter used. In this way the problem of early stage cell clone instability can be bypassed. However, upon introduction of an unrelated promoter methylation-independent silencing was observed. Together, these results suggest that histone modifications are the relevant mechanisms by which epigenetic modulation of transgene expression cassettes is governed in the early phase of clone generation.

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

    PubMed Central

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

    2012-01-01

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

  17. Histone deacetylase inhibitor-induced cell death in bladder cancer is associated with chromatin modification and modifying protein expression: A proteomic approach

    PubMed Central

    LI, QINGDI QUENTIN; HAO, JIAN-JIANG; ZHANG, ZHENG; HSU, IAWEN; LIU, YI; TAO, ZHEN; LEWI, KEIDREN; METWALLI, ADAM R.; AGARWAL, PIYUSH K.

    2016-01-01

    The Cancer Genome Atlas (TCGA) project recently identified the importance of mutations in chromatin remodeling genes in human carcinomas. These findings imply that epigenetic modulators might have a therapeutic role in urothelial cancers. To exploit histone deacetylases (HDACs) as targets for cancer therapy, we investigated the HDAC inhibitors (HDACIs) romidepsin, trichostatin A, and vorinostat as potential chemotherapeutic agents for bladder cancer. We demonstrate that the three HDACIs suppressed cell growth and induced cell death in the bladder cancer cell line 5637. To identify potential mechanisms associated with the anti-proliferative and cytotoxic effects of the HDACIs, we used quantitative proteomics to determine the proteins potentially involved in these processes. Our proteome studies identified a total of 6003 unique proteins. Of these, 2472 proteins were upregulated and 2049 proteins were downregulated in response to HDACI exposure compared to the untreated controls (P<0.05). Bioinformatic analysis further revealed that those differentially expressed proteins were involved in multiple biological functions and enzyme-regulated pathways, including cell cycle progression, apoptosis, autophagy, free radical generation and DNA damage repair. HDACIs also altered the acetylation status of histones and non-histone proteins, as well as the levels of chromatin modification proteins, suggesting that HDACIs exert multiple cytotoxic actions in bladder cancer cells by inhibiting HDAC activity or altering the structure of chromatin. We conclude that HDACIs are effective in the inhibition of cell proliferation and the induction of apoptosis in the 5637 bladder cancer cells through multiple cell death-associated pathways. These observations support the notion that HDACIs provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration. PMID:27082124

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

    PubMed

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

    2013-05-31

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

  19. Macro Histone Variants Are Critical for the Differentiation of Human Pluripotent Cells*

    PubMed Central

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

    2013-01-01

    We have previously shown that macro histone variants (macroH2A) are expressed at low levels in stem cells and are up-regulated during differentiation. Here we show that the knockdown of macro histone variants impaired the in vitro and in vivo differentiation of human pluripotent cells, likely through defects in the silencing of pluripotency-related genes. ChIP experiments showed that during differentiation macro histone variants are recruited to the regulatory regions of pluripotency and developmental genes marked with H3K27me3 contributing to the silencing of these genes. PMID:23595991

  20. Maternal Betaine Supplementation throughout Gestation and Lactation Modifies Hepatic Cholesterol Metabolic Genes in Weaning Piglets via AMPK/LXR-Mediated Pathway and Histone Modification

    PubMed Central

    Cai, Demin; Yuan, Mengjie; Liu, Haoyu; Pan, Shifeng; Ma, Wenqiang; Hong, Jian; Zhao, Ruqian

    2016-01-01

    Betaine serves as an animal and human nutrient which has been heavily investigated in glucose and lipid metabolic regulation, yet the underlying mechanisms are still elusive. In this study, feeding sows with betaine-supplemented diets during pregnancy and lactation increased cholesterol content and low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI) gene expression, but decreasing bile acids content and cholesterol-7a-hydroxylase (CYP7a1) expression in the liver of weaning piglets. This was associated with the significantly elevated serum betaine and methionine levels and hepatic S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) content. Concurrently, the hepatic nuclear transcription factor liver X receptor LXR was downregulated along with activated signal protein AMP-activated protein kinase (AMPK). Moreover, a chromatin immunoprecipitation assay showed lower LXR binding on CYP7a1 gene promoter and more enriched activation histone marker H3K4me3 on LDLR and SR-BI promoters. These results suggest that gestational and lactational betaine supplementation modulates hepatic gene expression involved in cholesterol metabolism via an AMPK/LXR pathway and histone modification in the weaning offspring. PMID:27763549

  1. Maternal Betaine Supplementation throughout Gestation and Lactation Modifies Hepatic Cholesterol Metabolic Genes in Weaning Piglets via AMPK/LXR-Mediated Pathway and Histone Modification.

    PubMed

    Cai, Demin; Yuan, Mengjie; Liu, Haoyu; Pan, Shifeng; Ma, Wenqiang; Hong, Jian; Zhao, Ruqian

    2016-10-18

    Betaine serves as an animal and human nutrient which has been heavily investigated in glucose and lipid metabolic regulation, yet the underlying mechanisms are still elusive. In this study, feeding sows with betaine-supplemented diets during pregnancy and lactation increased cholesterol content and low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI) gene expression, but decreasing bile acids content and cholesterol-7a-hydroxylase (CYP7a1) expression in the liver of weaning piglets. This was associated with the significantly elevated serum betaine and methionine levels and hepatic S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) content. Concurrently, the hepatic nuclear transcription factor liver X receptor LXR was downregulated along with activated signal protein AMP-activated protein kinase (AMPK). Moreover, a chromatin immunoprecipitation assay showed lower LXR binding on CYP7a1 gene promoter and more enriched activation histone marker H3K4me3 on LDLR and SR-BI promoters. These results suggest that gestational and lactational betaine supplementation modulates hepatic gene expression involved in cholesterol metabolism via an AMPK/LXR pathway and histone modification in the weaning offspring.

  2. 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

  3. Chemotherapy-induced cognitive impairment is associated with decreases in cell proliferation and histone modifications

    PubMed Central

    2011-01-01

    Background In this study, we examined the effects of cyclophosphamide, methothrexate, and 5-Fluorouracil (CMF) drug combination on various aspects of learning and memory. We also examined the effects of CMF on cell proliferation and chromatin remodeling as possible underlying mechanisms to explain chemotherapy-associated cognitive dysfunction. Twenty-four adult female Wistar rats were included in the study and had minimitter implantation for continuous activity monitoring two weeks before the chemotherapy regimen was started. Once baseline activity data were collected, rats were randomly assigned to receive either CMF or saline injections given intraperitoneally. Treatments were given once a week for a total of 4 weeks. Two weeks after the last injection, rats were tested in the water maze for spatial learning and memory ability as well as discrimination learning. Bromodeoxyuridine (BrdU) injection was given at 100 mg/Kg intraperitoneally 4 hours prior to euthanasia to determine hippocampal cell proliferation while histone acetylation and histone deacetylase activity was measured to determine CMF effects on chromatin remodeling. Results Our data showed learning and memory impairment following CMF administration independent of the drug effects on physical activity. In addition, CMF-treated rats showed decreased hippocampal cell proliferation, associated with increased histone acetylation and decreased histone deacetylase activity. Conclusions These results suggest the negative consequences of chemotherapy on brain function and that anti-cancer drugs can adversely affect the self-renewal potential of neural progenitor cells and also chromatin remodeling in the hippocampus. The significance of our findings lie on the possible usefulness of animal models in addressing the clinical phenomenon of 'chemobrain.' PMID:22152030

  4. 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.

  5. 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.

  6. Oncogenic RAS alters the global and gene-specific histone modification pattern during epithelial-mesenchymal transition in colorectal carcinoma cells.

    PubMed

    Peláez, Ignacio Mazón; Kalogeropoulou, Margarita; Ferraro, Angelo; Voulgari, Angeliki; Pankotai, Tibor; Boros, Imre; Pintzas, Alexander

    2010-06-01

    The presence of different forms of histone covalent modifications, such as phosphorylation, acetylation and methylation in localized promoter regions are markers for chromatin packing and transcription. Activation of RAS signalling pathways through oncogenic RAS mutations is a hallmark of colorectal cancer. Overexpression of Harvey-Ras oncogene induces epithelial-mesenchymal transition (EMT) in Caco-2 cells. We focused on the role of epigenetic modifications of histone H3 and its dependence on RAS signal transduction pathways and oncogenic transformation. Using cell lines stably overexpressing oncogenic Harvey-RAS with EMT phenotype, we studied the acquired changes in the H3 histone modification patterns. Two genes show inverse protein expression patterns after Ha-RAS overexpression: Cyclin D1, a cell cycle-related gene, and the EMT marker-gene E-cadherin. We report that these two genes demonstrate matching inverse histone repression patterns on their promoter, while histone markers associated with an active state of genes were affected by the RAS-activated signalling pathway MEK-ERK-MSK1. Furthermore, we show that though the level of methyltransferases enzymes was increased, the status of H3 three-methylation at lysine 27 (H3K27me(3)), associated with gene repression on the promoter of Cyclin D1, was lower. Together, these results suggest that histone covalent modifications can be affected by oncogenic RAS pathways to regulate the expression of target genes like Cyclin D1 or E-cadherin and that the dynamic balance of opposing histone-modifying enzymes is critical for the regulation of cell proliferation.

  7. Quick fluorescent in situ hybridization protocol for Xist RNA combined with immunofluorescence of histone modification in X-chromosome inactivation.

    PubMed

    Yue, Minghui; Charles Richard, John Lalith; Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

    2014-11-26

    Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

  8. A histone modification identifies a DNA element controlling slo BK channel gene expression in muscle

    PubMed Central

    Li, Xiaolei; Ghezzi, Alfredo; Krishnan, Harish R.; Pohl, Jascha B.; Bohm, Arun Y.; Atkinson, Nigel S.

    2016-01-01

    The slo gene encodes BK type Ca2+-activated K+ channels. In Drosophila, expression of slo is induced by organic solvent sedation (benzyl alcohol and ethanol) and this increase in neural slo expression contributes to the production of functional behavioral tolerance (inducible resistance) to these drugs. Within the slo promoter region, we observed that benzyl alcohol sedation produces a localized spike of histone acetylation over a 65 n conserved DNA element called 55b. Changes in histone acetylation are commonly the consequence of transcription factor activity and previously, a localized histone acetylation spike was used to successfully map a DNA element involved in benzyl alcohol-induced slo expression. To determine whether the 55b element was also involved in benzyl alcohol-induced neural expression of slo we deleted it from the endogenous slo gene by homologous recombination. Flies lacking the 55b element were normal with respect to basal and benzyl alcohol-induced neural slo expression, the capacity to acquire and maintain functional tolerance, their threshold for electrically-induced seizures and most slo-related behaviors. Removal of the 55b element did however increase the level of basal expression from the muscle/tracheal cell-specific slo core promoter and produced a slight increase in overall locomotor activity. We conclude that the 55b element is involved in control of slo expression from the muscle and tracheal-cell promoter but is not involved in the production of functional benzyl alcohol tolerance. PMID:25967280

  9. 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.

  10. 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

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

    PubMed

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

    2010-02-01

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

  12. Jarid2 (Jumonji, AT rich interactive domain 2) regulates NOTCH1 expression via histone modification in the developing heart.

    PubMed

    Mysliwiec, Matthew R; Carlson, Clayton D; Tietjen, Josh; Hung, Holly; Ansari, Aseem Z; Lee, Youngsook

    2012-01-06

    Jarid2/Jumonji, the founding member of the Jmj factor family, critically regulates various developmental processes, including cardiovascular development. The Jmj family was identified as histone demethylases, indicating epigenetic regulation by Jmj proteins. Deletion of Jarid2 in mice resulted in cardiac malformation and increased endocardial Notch1 expression during development. Although Jarid2 has been shown to occupy the Notch1 locus in the developing heart, the precise molecular role of Jarid2 remains unknown. Here we show that deletion of Jarid2 results in reduced methylation of lysine 9 on histone H3 (H3K9) at the Notch1 genomic locus in embryonic hearts. Interestingly, SETDB1, a histone H3K9 methyltransferase, was identified as a putative cofactor of Jarid2 by yeast two-hybrid screening, and the physical interaction between Jarid2 and SETDB1 was confirmed by coimmunoprecipitation experiments. Concurrently, accumulation of SETDB1 at the site of Jarid2 occupancy was significantly reduced in Jarid2 knock out (KO) hearts. Employing genome-wide approaches, putative Jarid2 target genes regulated by SETDB1 via H3K9 methylation were identified in the developing heart by ChIP-chip. These targets are involved in biological processes that, when dysregulated, could manifest in the phenotypic defects observed in Jarid2 KO mice. Our data demonstrate that Jarid2 functions as a transcriptional repressor of target genes, including Notch1, through a novel process involving the modification of H3K9 methylation via specific interaction with SETDB1 during heart development. Therefore, our study provides new mechanistic insights into epigenetic regulation by Jarid2, which will enhance our understanding of the molecular basis of other organ development and biological processes.

  13. Release and activity of histone in diseases

    PubMed Central

    Chen, R; Kang, R; Fan, X-G; Tang, D

    2014-01-01

    Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Anti-histone treatment (e.g., neutralizing antibodies, activated protein C, recombinant thrombomodulin, and heparin) protect mice against lethal endotoxemia, sepsis, ischemia/reperfusion injury, trauma, pancreatitis, peritonitis, stroke, coagulation, and thrombosis. In addition, elevated serum histone and nucleosome levels have been implicated in multiple pathophysiological processes and progression of diseases including autoimmune diseases, inflammatory diseases, and cancer. Therefore, extracellular histones could serve as biomarkers and novel therapeutic targets in human diseases. PMID:25118930

  14. 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.

  15. Histone H2B monoubiquitination: roles to play in human malignancy.

    PubMed

    Cole, Alexander J; Clifton-Bligh, Roderick; Marsh, Deborah J

    2015-02-01

    Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20-RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy. © 2015 Society for Endocrinology.

  16. Histone Modification Associated with Initiation of DNA Replication | Center for Cancer Research

    Cancer.gov

    Before cells are able to divide, they must first duplicate their chromosomes accurately. DNA replication and packaging of DNA into chromosomes by histone proteins need to be coordinated by the cell to ensure proper transmission of genetic and epigenetic information to the next generation. Mammalian DNA replication begins at specific chromosomal sites, called replication origins, which are located throughout the genome. The replication origins are tightly regulated to start replication only once per cell division so that genomic stability is maintained and cancer development is prevented.

  17. The many roles of the conserved eukaryotic Paf1 complex in regulating transcription, histone modifications, and disease states

    PubMed Central

    Tomson, Brett N.; Arndt, Karen M.

    2012-01-01

    The Paf1 complex was originally identified over fifteen years ago in budding yeast through its physical association with RNA polymerase II. The Paf1 complex is now known to be conserved throughout eukaryotes and is well studied for promoting RNA polymerase II transcription elongation and transcription-coupled histone modifications. Through these critical regulatory functions, the Paf1 complex participates in numerous cellular processes such as gene expression and silencing, RNA maturation, DNA repair, cell cycle progression and prevention of disease states in higher eukaryotes. In this review, we describe the historic and current research involving the eukaryotic Paf1 complex to explain the cellular roles that underlie its conservation and functional importance. PMID:22982193

  18. 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

  19. Neutrophil histone modification by peptidylarginine deiminase 4 is critical for deep vein thrombosis in mice.

    PubMed

    Martinod, Kimberly; Demers, Melanie; Fuchs, Tobias A; Wong, Siu Ling; Brill, Alexander; Gallant, Maureen; Hu, Jing; Wang, Yanming; Wagner, Denisa D

    2013-05-21

    Deep vein thrombosis and pulmonary embolism are major health problems associated with high mortality. Recently, DNA-based neutrophil extracellular traps (NETs) resulting from the release of decondensed chromatin, were found to be part of the thrombus scaffold and to promote coagulation. However, the significance of nuclear decondensation and NET generation in thrombosis is largely unknown. To address this, we adopted a stenosis model of deep vein thrombosis and analyzed venous thrombi in peptidylarginine deiminase 4 (PAD4)-deficient mice that cannot citrullinate histones, a process required for chromatin decondensation and NET formation. Intriguingly, less than 10% of PAD4(-/-) mice produced a thrombus 48 h after inferior vena cava stenosis whereas 90% of wild-type mice did. Neutrophils were abundantly present in thrombi formed in both groups, whereas extracellular citrullinated histones were seen only in thrombi from wild-type mice. Bone marrow chimera experiments indicated that PAD4 in hematopoietic cells was the source of the prothrombotic effect in deep vein thrombosis. Thrombosis could be rescued by infusion of wild-type neutrophils, suggesting that neutrophil PAD4 was important and sufficient. Endothelial activation and platelet aggregation were normal in PAD4(-/-) mice, as was hemostatic potential determined by bleeding time and platelet plug formation after venous injury. Our results show that PAD4-mediated chromatin decondensation in the neutrophil is crucial for pathological venous thrombosis and present neutrophil activation and PAD4 as potential drug targets for deep vein thrombosis.

  20. DNMT1 and DNMT3B modulate distinct polycomb-mediated histone modifications in colon cancer.

    PubMed

    Jin, Bilian; Yao, Bing; Li, Jian-Liang; Fields, C Robert; Delmas, Amber L; Liu, Chen; Robertson, Keith D

    2009-09-15

    DNA methylation patterns are established and maintained by three DNA methyltransferases (DNMT): DNMT1, DNMT3A, and DNMT3B. Although essential for development, methylation patterns are frequently disrupted in cancer and contribute directly to carcinogenesis. Recent studies linking polycomb group repression complexes (PRC1 and PRC2) to the DNMTs have begun to shed light on how methylation is targeted. We identified previously a panel of genes regulated by DNMT3B. Here, we compare these with known polycomb group targets to show that approximately 47% of DNMT3B regulated genes are also bound by PRC1 or PRC2. We chose 44 genes coregulated by DNMT3B and PRC1/PRC2 to test whether these criteria would accurately identify novel targets of epigenetic silencing in colon cancer. Using reverse transcription-PCR, bisulfite genomic sequencing, and pyrosequencing, we show that the majority of these genes are frequently silenced in colorectal cancer cell lines and primary tumors. Some of these, including HAND1, HMX2, and SIX3, repressed cell growth. Finally, we analyzed the histone code, DNMT1, DNMT3B, and PRC2 binding by chromatin immunoprecipitation at epigenetically silenced genes to reveal a novel link between DNMT3B and the mark mediated by PRC1. Taken together, these studies suggest that patterns of epigenetic modifiers and the histone code influence the propensity of a gene to become hypermethylated in cancer and that DNMT3B plays an important role in regulating PRC1 function.

  1. Discovering Cooperative Relationships of Chromatin Modifications in Human T Cells Based on a Proposed Closeness Measure

    PubMed Central

    Liu, Hongbo; Wu, Xueting; Zhu, Jiang; Su, Jianzhong; Wang, Fang; Cui, Ying; Zhang, Yan

    2010-01-01

    Background Eukaryotic transcription is accompanied by combinatorial chromatin modifications that serve as functional epigenetic markers. Composition of chromatin modifications specifies histone codes that regulate the associated gene. Discovering novel chromatin regulatory relationships are of general interest. Methodology/Principal Findings Based on the premise that the interaction of chromatin modifications is hypothesized to influence CpG methylation, we present a closeness measure to characterize the regulatory interactions of epigenomic features. The closeness measure is applied to genome-wide CpG methylation and histone modification datasets in human CD4+T cells to select a subset of potential features. To uncover epigenomic and genomic patterns, CpG loci are clustered into nine modules associated with distinct chromatin and genomic signatures based on terms of biological function. We then performed Bayesian network inference to uncover inherent regulatory relationships from the feature selected closeness measure profile and all nine module-specific profiles respectively. The global and module-specific network exhibits topological proximity and modularity. We found that the regulatory patterns of chromatin modifications differ significantly across modules and that distinct patterns are related to specific transcriptional levels and biological function. DNA methylation and genomic features are found to have little regulatory function. The regulatory relationships were partly validated by literature reviews. We also used partial correlation analysis in other cells to verify novel regulatory relationships. Conclusions/Significance The interactions among chromatin modifications and genomic elements characterized by a closeness measure help elucidate cooperative patterns of chromatin modification in transcriptional regulation and help decipher complex histone codes. PMID:21151929

  2. Histone Modifiers in Cancer

    PubMed Central

    Cohen, Idan; Poręba, Elżbieta; Kamieniarz, Kinga; Schneider, Robert

    2011-01-01

    Covalent modifications of histones can regulate all DNA-dependent processes. In the last few years, it has become more and more evident that histone modifications are key players in the regulation of chromatin states and dynamics as well as in gene expression. Therefore, histone modifications and the enzymatic machineries that set them are crucial regulators that can control cellular proliferation, differentiation, plasticity, and malignancy processes. This review discusses the biology and biochemistry of covalent histone posttranslational modifications (PTMs) and evaluates the dual role of their modifiers in cancer: as oncogenes that can initiate and amplify tumorigenesis or as tumor suppressors. PMID:21941619

  3. Unraveling Site-Specific and Combinatorial Histone Modifications Using High-Resolution Mass Spectrometry in Histone Deacetylase Mutants of Fission Yeast.

    PubMed

    Abshiru, Nebiyu; Rajan, Roshan Elizabeth; Verreault, Alain; Thibault, Pierre

    2016-07-01

    Histone deacetylases (HDACs) catalyze the removal of acetylation marks from lysine residues on histone and nonhistone substrates. Their activity is generally associated with essential cellular processes such as transcriptional repression and heterochromatin formation. Interestingly, abnormal activity of HDACs has been reported in various types of cancers, which makes them a promising therapeutic target for cancer treatment. In the current study, we aim to understand the mechanisms underlying the function of HDACs using an in-depth quantitative analysis of changes in histone acetylation levels in Schizosaccharomyces pombe (S. pombe) lacking major HDAC activities. We employed a targeted quantitative mass spectrometry approach to profile changes of acetylation and methylation at multiple lysine residues on the N-terminal tail of histones H3 and H4. Our analyses identified a number of histone acetylation sites that are significantly affected by S. pombe HDAC mutations. We discovered that mutation of the Class I HDAC known as Clr6 causes a major increase in the abundance of triacetylated H4 molecules at K5, K8, and K12. A clr6-1 hypomorphic mutation also increased the abundance of multiple acetyl-lysines in histone H3. In addition, our study uncovered a few crosstalks between histone acetylation and methylation upon deletion of HDACs Hos2 and Clr3. We anticipate that the results from this study will greatly improve our current understanding of the mechanisms involved in HDAC-mediated gene regulation and heterochromatin assembly.

  4. Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere

    PubMed Central

    1994-01-01

    Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2- terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin. PMID:7962047

  5. Electrospray ionization mass spectrometry for natural and radiation-induced modifications in histone proteins

    SciTech Connect

    Edmonds, C.G.; Fuciarelli, A.F.; Thrall, B.D.; Springer, D.L.

    1992-05-01

    Chick erythrocyle histone H2B was irradiated in the presence of thymine, the principle cross-linking base recognized in earlier studies, and the products were examined directly by electrospray ionization mass spectrometry (ESI-MS). Following exposure to 5 Gy of ionizing radiation the relative abundance of two unique species were increased by nearly 50% in irradiated samples over background response at the same m/z. The first corresponds to a mass increment increase similar to the expected value for thymine-H2B adduct formation (126.1 Da measured, 125.1 Da calculated). The mass increment increase for the second component (140.7 Da) was less easily explained. Additional dose-yield data are needed to confirm the significance of these changes.

  6. Development of primer sets that can verify the enrichment of histone modifications, and their application to examining vernalization-mediated chromatin changes in Brassica rapa L.

    PubMed

    Kawanabe, Takahiro; Osabe, Kenji; Itabashi, Etsuko; Okazaki, Keiichi; Dennis, Elizabeth S; Fujimoto, Ryo

    2016-07-20

    Epigenetic regulation is crucial for the development of plants and for adaptation to a changing environment. Recently, genome-wide profiles of histone modifications have been determined by a combination of chromatin immunoprecipitation (ChIP) and genomic tiling arrays (ChIP on chip) or ChIP and high-throughput sequencing (ChIP-seq) in species including Arabidopsis thaliana, rice and maize. Validation of ChIP analysis by PCR or qPCR using positive and negative regions of histone modification is necessary. In contrast, information about histone modifications is limited in Chinese cabbage, Brassica rapa. The aim of this study was to develop positive and negative control primer sets for H3K4me3 (trimethylation of the 4(th) lysine of H3), H3K9me2, H3K27me3 and H3K36me3 in B. rapa. The expression and histone modification of four FLC paralogs in B. rapa, before and after vernalization, were examined using the method developed here. After vernalization, expression of all four BrFLC genes was reduced, and accumulation of H3K27me3 was observed in three of them. As with A. thaliana, the vernalization response and stability of FLC repression correlated with the accumulation of H3K27me3. These results suggest that the epigenetic state during vernalization is important for high bolting resistance in B. rapa. The positive and negative control primer sets developed here revealed positive and negative histone modifications in B. rapa that can be used as a control for future studies.

  7. GATA2 regulates GATA1 expression through LSD1-mediated histone modification

    PubMed Central

    Guo, Yidi; Fu, Xueqi; Huo, Bo; Wang, Yongsen; Sun, Jing; Meng, Lingyuan; Hao, Tian; Zhao, Zhizhuang Joe; Hu, Xin

    2016-01-01

    The dynamic and reversed expression of GATA1 and GATA2 are essential for proper erythroid differentiation. Our previous work demonstrates that LSD1, a histone H3K4 demethylase, represses GATA2 expression at late stage of erythroid differentiation. K562 and MEL cells were used and cultured in Roswell Park Memorial Institute-1640 medium (RPMI) and Dulbecco’s modified Eagle’s medium (DMEM), respectively. Western blot assay was used to examine the GATA1, GATA2, TAL1, HDAC1, HDAC2, CoREST and β-actin protein. The immunoprecipitation assay and GST pull-down assay were employed to detect the precipitated protein complexes and investigate the interaction between the proteins. The small interfering RNA (siRNA) and nonspecific control siRNA were synthesized to silence the target genes. Double fluorescence immunostaining was used to observe the association of LSD1 with GATA2 in K562 cells. The results indicated that knockdown of LSD1 in K562 cell causes increased H3K4 di-methylation at GATA1 locus and activates GATA1 expression, demonstrating that LSD1 represses GATA1 expression through LSD1-mediated histone demethylation. Upon induced erythroid differentiation of K562 cells, the interaction between GATA2 and LSD1 is decreased, consistent with a de-repression of GATA1 expression. Meanwhile, the interaction between TAL1 and LSD1 is increased, which forms a complex that efficiently suppresses GATA2 expression. In conclusion, these observations reveal an elegant mechanism to modulate GATA1 and GATA2 expression during erythroid differentiation. While LSD1 mainly forms complex with GATA2 to repress GATA1 expression in hematopoietic progenitor cells, it mostly forms complex with TAL1 to repress GATA2 expression in differentiated cells. PMID:27347333

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

    SciTech Connect

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

    2011-12-10

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

  9. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    SciTech Connect

    Osada, Tomoharu; Rydén, Anna-Margareta; Masutani, Mitsuko

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  10. Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family

    PubMed Central

    Filippakopoulos, Panagis; Picaud, Sarah; Mangos, Maria; Keates