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

    PubMed Central

    Yan, Chunhong; Boyd, Douglas D.

    2006-01-01

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

  2. Regulation of histone H3K4 methylation in brain development and disease.

    PubMed

    Shen, Erica; Shulha, Hennady; Weng, Zhiping; Akbarian, Schahram

    2014-09-26

    The growing list of mutations implicated in monogenic disorders of the developing brain includes at least seven genes (ARX, CUL4B, KDM5A, KDM5C, KMT2A, KMT2C, KMT2D) with loss-of-function mutations affecting proper regulation of histone H3 lysine 4 methylation, a chromatin mark which on a genome-wide scale is broadly associated with active gene expression, with its mono-, di- and trimethylated forms differentially enriched at promoter and enhancer and other regulatory sequences. In addition to these rare genetic syndromes, dysregulated H3K4 methylation could also play a role in the pathophysiology of some cases diagnosed with autism or schizophrenia, two conditions which on a genome-wide scale are associated with H3K4 methylation changes at hundreds of loci in a subject-specific manner. Importantly, the reported alterations for some of the diseased brain specimens included a widespread broadening of H3K4 methylation profiles at gene promoters, a process that could be regulated by the UpSET(KMT2E/MLL5)-histone deacetylase complex. Furthermore, preclinical studies identified maternal immune activation, parental care and monoaminergic drugs as environmental determinants for brain-specific H3K4 methylation. These novel insights into the epigenetic risk architectures of neurodevelopmental disease will be highly relevant for efforts aimed at improved prevention and treatment of autism and psychosis spectrum disorders. PMID:25135975

  3. Histone H3K4 methylation regulates deactivation of the spindle assembly checkpoint through direct binding of Mad2.

    PubMed

    Schibler, Andria; Koutelou, Evangelia; Tomida, Junya; Wilson-Pham, Marenda; Wang, Li; Lu, Yue; Cabrera, Alexa Parra; Chosed, Renee J; Li, Wenqian; Li, Bing; Shi, Xiaobing; Wood, Richard D; Dent, Sharon Y R

    2016-05-15

    Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition. PMID:27198228

  4. Disrupted intricacy of histone H3K4 methylation in neurodevelopmental disorders

    PubMed Central

    Vallianatos, Christina N; Iwase, Shigeki

    2015-01-01

    MethylationofhistoneH3lysine4(H3K4me)isanintricatelyregulatedposttranslational modification, which is broadly associated with enhancers and promoters of actively transcribed genomic loci. Recent advances in next-generation sequencing have identified a number of H3K4me regulators mutated in neurodevelopmental disorders including intellectual disabilities, autism spectrum disorders, and schizophrenia. Here, we aim to summarize the molecular function of H3K4me-regulating enzymes in brain development and function. We describe four H3K4me methyltransferases (KMT2A, KMT2C, KMT2D, KMT2F), four demethylases (KDM1A, KDM5A, KDM5B, KDM5C), and two reader proteins (PHF21A, PHF8) mutated in neurodevelopmental disorders. Understanding the role of these chromatin regulators in the development and maintenance of neural connections will advance therapeutic opportunities for prevention and treatment of these lifelong neurodevelopmental disorders. PMID:26077434

  5. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription.

    PubMed

    Ramakrishnan, Saravanan; Pokhrel, Srijana; Palani, Sowmiya; Pflueger, Christian; Parnell, Timothy J; Cairns, Bradley R; Bhaskara, Srividya; Chandrasekharan, Mahesh B

    2016-01-01

    Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation-demethylation together control chromatin dynamics during various facets of transcriptional regulation. PMID:27325136

  6. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription

    PubMed Central

    Ramakrishnan, Saravanan; Pokhrel, Srijana; Palani, Sowmiya; Pflueger, Christian; Parnell, Timothy J.; Cairns, Bradley R.; Bhaskara, Srividya; Chandrasekharan, Mahesh B.

    2016-01-01

    Histone H3K4 methylation is connected to gene transcription from yeast to humans, but its mechanistic roles in transcription and chromatin dynamics remain poorly understood. We investigated the functions for Set1 and Jhd2, the sole H3K4 methyltransferase and H3K4 demethylase, respectively, in S. cerevisiae. Here, we show that Set1 and Jhd2 predominantly co-regulate genome-wide transcription. We find combined activities of Set1 and Jhd2 via H3K4 methylation contribute to positive or negative transcriptional regulation. Providing mechanistic insights, our data reveal that Set1 and Jhd2 together control nucleosomal turnover and occupancy during transcriptional co-regulation. Moreover, we find a genome-wide co-regulation of chromatin structure by Set1 and Jhd2 at different groups of transcriptionally active or inactive genes and at different regions within yeast genes. Overall, our study puts forth a model wherein combined actions of Set1 and Jhd2 via modulating H3K4 methylation−demethylation together control chromatin dynamics during various facets of transcriptional regulation. PMID:27325136

  7. Histone H3K4 methylation regulates hyphal growth, secondary metabolism and multiple stress responses in Fusarium graminearum.

    PubMed

    Liu, Ye; Liu, Na; Yin, Yanni; Chen, Yun; Jiang, Jinhua; Ma, Zhonghua

    2015-11-01

    Histone H3 lysine 4 methylation (H3K4me) is generally associated with actively transcribed genes in a variety of eukaryotes. The function of H3K4me in phytopathogenic fungi remains unclear. Here, we report that FgSet1 is predominantly responsible for mono-, di- and trimethylation of H3K4 in Fusarium graminearum. The FgSET1 deletion mutant (ΔFgSet1) was crippled in hyphal growth and virulence. H3K4me is required for the active transcription of genes involved in deoxynivalenol and aurofusarin biosyntheses. Unexpectedly, FgSet1 plays an important role in the response of F. graminearum to cell wall-damaging agents via negatively regulating phosphorylation of FgMgv1, a core kinase in the cell wall integrity pathway. In addition, ΔFgSet1 exhibited increased resistance to the transcription elongation inhibitor mycophenolic acid. Yeast two-hybrid assays showed that FgSet1 physically interacts with multiple proteins including FgBre2, FgSpp1 and FgSwd2. FgBre2 further interacts with FgSdc1. Western blotting analyses showed that FgBre2 and FgSdc1 are associated with H3K4me. Both proteins are also involved in regulating deoxynivalenol biosynthesis and in responses to mycophenolic acid and cell wall-damaging agents. Taken together, these data indicate that H3K4me plays critical roles not only in regulation of fungal growth and secondary metabolism but also in multiple stress responses in F. graminearum. PMID:26234386

  8. KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    PubMed Central

    2014-01-01

    Background Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function. Results Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes. Conclusions Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation. PMID:24495580

  9. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

    PubMed Central

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-01-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  10. Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development.

    PubMed

    Aoshima, Keisuke; Inoue, Erina; Sawa, Hirofumi; Okada, Yuki

    2015-07-01

    Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K-M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K-M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice. PMID:25925669

  11. Balancing of histone H3K4 methylation states by the Kdm5c/SMCX histone demethylase modulates promoter and enhancer function.

    PubMed

    Outchkourov, Nikolay S; Muiño, Jose M; Kaufmann, Kerstin; van Ijcken, Wilfred F J; Groot Koerkamp, Marian J; van Leenen, Dik; de Graaf, Petra; Holstege, Frank C P; Grosveld, Frank G; Timmers, H T Marc

    2013-04-25

    The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters. PMID:23545502

  12. H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation.

    PubMed

    Matsumura, Yoshihiro; Nakaki, Ryo; Inagaki, Takeshi; Yoshida, Ayano; Kano, Yuka; Kimura, Hiroshi; Tanaka, Toshiya; Tsutsumi, Shuichi; Nakao, Mitsuyoshi; Doi, Takefumi; Fukami, Kiyoko; Osborne, Timothy F; Kodama, Tatsuhiko; Aburatani, Hiroyuki; Sakai, Juro

    2015-11-19

    Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis. PMID:26590716

  13. Stage and Gene Specific Signatures Defined by Histones H3K4me2 and H3K27me3 Accompany Mammalian Retina Maturation In Vivo

    PubMed Central

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

    2012-01-01

    The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By in silico analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue in vivo. PMID:23056497

  14. Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation.

    PubMed

    Xiong, Yujing; Wang, Enyin; Huang, Yan; Guo, Xiaoyi; Yu, Yiping; Du, Qingyun; Ding, Xiaoyan; Sun, Yingpu

    2016-06-01

    Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs. PMID:27059868

  15. Oncofetal Epigenetic Bivalency in Breast Cancer Cells: H3K4 and H3K27 Tri-Methylation as a Biomarker for Phenotypic Plasticity.

    PubMed

    Messier, Terri L; Boyd, Joseph R; Gordon, Jonathan A R; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2016-11-01

    Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well-established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co-residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this "oncofetal epigenetic" signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment-resistant cancers. J. Cell. Physiol. 231: 2474-2481, 2016. © 2016 Wiley Periodicals, Inc. PMID:26916849

  16. Identification of cis regulatory features in the embryonic zebrafish genome through large-scale profiling of H3K4me1 and H3K4me3 binding sites

    PubMed Central

    Aday, Aaron W.; Zhu, Lihua Julie; Lakshmanan, Abirami; Wang, Jie; Lawson, Nathan D.

    2011-01-01

    An organism’s genome sequence serves as a blueprint for the proteins and regulatory RNAs essential for cellular function. The genome also harbors cis-acting non-coding sequences that control gene expression and are essential to coordinate regulatory programs during embryonic development. However, the genome sequence is largely identical between cell types within a multi-cellular organism indicating that factors such as DNA accessibility and chromatin structure play a crucial role in governing cell-specific gene expression. Recent studies have identified particular chromatin modifications that define functionally distinct cis regulatory elements. Among these are forms of histone 3 that are mono- or tri-methylated at lysine 4 (H3K4me1 or H3K4me3, respectively), which bind preferentially to promoter and enhancer elements in the mammalian genome. In this work, we investigated whether these modified histones could similarly identify cis regulatory elements within the zebrafish genome. By applying chromatin immunoprecipitation followed by deep sequencing, we find that H3K4me1 and H3K4me3 are enriched at transcriptional start sites in the genome of the developing zebrafish embryo and that this association correlates with gene expression. We further find that these modifications associate with distal non-coding conserved elements, including known active enhancers. Finally, we demonstrate that it is possible to utilize H3K4me1 and H3K4me3 binding profiles in combination with available expression data to computationally identify relevant cis regulatory sequences flanking syn-expressed genes in the developing embryo. Taken together, our results indicate that H3K4me1 and H3K4me3 generally mark cis regulatory elements within the zebrafish genome and indicate that further characterization of the zebrafish using this approach will prove valuable in defining transcriptional networks in this model system. PMID:21435340

  17. DNA methylation and not H3K4 trimethylation dictates the expression status of miR-152 gene which inhibits migration of breast cancer cells via DNMT1/CDH1 loop.

    PubMed

    Sengupta, Dipta; Deb, Moonmoon; Rath, Sandip Kumar; Kar, Swayamsiddha; Parbin, Sabnam; Pradhan, Nibedita; Patra, Samir Kumar

    2016-08-15

    MicroRNAs (miRNA) are small non-coding RNAs which targets most protein-coding transcripts (mRNA) and destroy them. Thus miRNA controls the abundance of those specific proteins and impact on developmental, physiological and pathological processes. Dysregulation of miRNA function thus may lead to various clinicopathological complications, including breast cancer. Silencing of miR-152 gene due to promoter DNA methylation alter the expression pattern of several other genes. E-cadherin (CDH1) forms the core of adherent junctions between surrounding epithelial cells, link with actin cytoskeleton and affects cell signaling. CDH1 gene is down regulated by promoter DNA methylation during cancer progression. In this investigation, we attempt to elucidate the correlation of miR-152 and CDH1 function, as it is well known that the loss of CDH1 function is one of the major reasons for cancer metastasis and aggressiveness of spreading. For the first time we have shown that loss of CDH1 expression is directly proportional to the loss of miR-152 function in breast cancer cells. mRNA and protein expression profile of DNMT1 implicate that miR-152 targets DNMT1 mRNA and inhibits its protein expression. Tracing the molecular marks on DNA and histone 3 for understanding the mechanism of gene regulation by ChIP analyses leads to a paradoxical result that shows DNA methylation adjacent to active histone marking (enrichment of H3K4me3) silence miR-152 gene. Further experiments revealed that DNMT1 plays crucial role for regulation of miR-152 gene. When DNMT1 protein function is blocked miR-152 expression prevails and destroys the mRNA of DNMT1; this molecular regulatory mechanism is creating a cyclic feedback loop, which is now focused as DNMT1/miR-152 switch for on/off of DNMT1 target genes. We discovered modulation of CDH1 gene expression by DNMT1/miR-152 switches. We have demonstrated further that DNMT1 down regulation mediated upregulation of CDH1 (hereafter, DNMT1/CDH1 loop) in

  18. Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4.

    PubMed

    Dhar, Shilpa S; Lee, Sung-Hun; Kan, Pu-Yeh; Voigt, Philipp; Ma, Li; Shi, Xiaobing; Reinberg, Danny; Lee, Min Gyu

    2012-12-15

    Mixed-lineage leukemia 4 (MLL4; also called MLL2 and ALR) enzymatically generates trimethylated histone H3 Lys 4 (H3K4me3), a hallmark of gene activation. However, how MLL4-deposited H3K4me3 interplays with other histone marks in epigenetic processes remains largely unknown. Here, we show that MLL4 plays an essential role in differentiating NT2/D1 stem cells by activating differentiation-specific genes. A tandem plant homeodomain (PHD(4-6)) of MLL4 recognizes unmethylated or asymmetrically dimethylated histone H4 Arg 3 (H4R3me0 or H4R3me2a) and is required for MLL4's nucleosomal methyltransferase activity and MLL4-mediated differentiation. Kabuki syndrome mutations in PHD(4-6) reduce PHD(4-6)'s binding ability and MLL4's catalytic activity. PHD(4-6)'s binding strength is inhibited by H4R3 symmetric dimethylation (H4R3me2s), a gene-repressive mark. The protein arginine methyltransferase 7 (PRMT7), but not PRMT5, represses MLL4 target genes by up-regulating H4R3me2s levels and antagonizes MLL4-mediated differentiation. Consistently, PRMT7 knockdown increases MLL4-catalyzed H3K4me3 levels. During differentiation, decreased H4R3me2s levels are associated with increased H3K4me3 levels at a cohort of genes, including many HOXA and HOXB genes. These findings indicate that the trans-tail inhibition of MLL4-generated H3K4me3 by PRMT7-regulated H4R3me2s may result from H4R3me2s's interference with PHD(4-6)'s binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation. PMID:23249737

  19. An High-Throughput In Vivo Screening System to Select H3K4-Specific Histone Demethylase Inhibitors

    PubMed Central

    Mannironi, Cecilia; Proietto, Marco; Bufalieri, Francesca; Cundari, Enrico; Alagia, Angela; Danovska, Svetlana; Rinaldi, Teresa; Famiglini, Valeria; Coluccia, Antonio; La Regina, Giuseppe; Silvestri, Romano; Negri, Rodolfo

    2014-01-01

    Background Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes. Methodology/Principal Findings In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity. Conclusions/Significance Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases. PMID:24489688

  20. Chromatin H3K27me3/H3K4me3 histone marks define gene sets in high-grade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells.

    PubMed

    Chapman-Rothe, N; Curry, E; Zeller, C; Liber, D; Stronach, E; Gabra, H; Ghaem-Maghami, S; Brown, R

    2013-09-19

    In embryonic stem (ES) cells, bivalent chromatin domains containing H3K4me3 and H3K27me3 marks silence developmental genes, while keeping them poised for activation following differentiation. We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high-grade ovarian serous tumour and examined their association with epigenetic silencing and malignant progression. This revealed novel silenced bivalent marked genes, not described previously for ES cells, which are significantly enriched for the PI3K (P<10(-7)) and TGF-β signalling pathways (P<10(-5)). We matched histone marked gene sets to gene expression sets of eight normal fallopian tubes and 499 high-grade serous malignant ovarian samples. This revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets in malignant tissue. We then correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumour 'stem cell-like' sustaining cells versus non-sustaining cells. This showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumour-sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared with the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer. However it also suggests that additional tumour specific bivalent marks, to those known in ES cells, are present in tumours and may potentially influence the subsequent development of drug resistance and tumour progression. PMID:23128397

  1. H3K4 demethylase activities repress proliferative and postmitotic aging

    PubMed Central

    Alvares, Stacy M; Mayberry, Gaea A; Joyner, Ebony Y; Lakowski, Bernard; Ahmed, Shawn

    2014-01-01

    Homeostasis of postmitotic and proliferating cells is maintained by pathways that repress stress. We found that the Caenorhabditis elegans histone 3 lysine 4 (H3K4) demethylases RBR-2 and SPR-5 promoted postmitotic longevity of stress-resistant daf-2 adults, altered pools of methylated H3K4, and promoted silencing of some daf-2 target genes. In addition, RBR-2 and SPR-5 were required for germ cell immortality at a high temperature. Transgenerational proliferative aging was enhanced for spr-5; rbr-2 double mutants, suggesting that these histone demethylases may function sequentially to promote germ cell immortality by targeting distinct H3K4 methyl marks. RBR-2 did not play a comparable role in the maintenance of quiescent germ cells in dauer larvae, implying that it represses stress that occurs as a consequence of germ cell proliferation, rather than stress that accumulates in nondividing cells. We propose that H3K4 demethylase activities promote the maintenance of chromatin states during stressful growth conditions, thereby repressing postmitotic aging of somatic cells as well as proliferative aging of germ cells. PMID:24134677

  2. The COMPASS Family of H3K4 Methylases in Drosophila ▿

    PubMed Central

    Mohan, Man; Herz, Hans-Martin; Smith, Edwin R.; Zhang, Ying; Jackson, Jessica; Washburn, Michael P.; Florens, Laurence; Eissenberg, Joel C.; Shilatifard, Ali

    2011-01-01

    Methylation of histone H3 lysine 4 (H3K4) in Saccharomyces cerevisiae is implemented by Set1/COMPASS, which was originally purified based on the similarity of yeast Set1 to human MLL1 and Drosophila melanogaster Trithorax (Trx). While humans have six COMPASS family members, Drosophila possesses a representative of the three subclasses within COMPASS-like complexes: dSet1 (human SET1A/SET1B), Trx (human MLL1/2), and Trr (human MLL3/4). Here, we report the biochemical purification and molecular characterization of the Drosophila COMPASS family. We observed a one-to-one similarity in subunit composition with their mammalian counterparts, with the exception of LPT (lost plant homeodomains [PHDs] of Trr), which copurifies with the Trr complex. LPT is a previously uncharacterized protein that is homologous to the multiple PHD fingers found in the N-terminal regions of mammalian MLL3/4 but not Drosophila Trr, indicating that Trr and LPT constitute a split gene of an MLL3/4 ancestor. Our study demonstrates that all three complexes in Drosophila are H3K4 methyltransferases; however, dSet1/COMPASS is the major monoubiquitination-dependent H3K4 di- and trimethylase in Drosophila. Taken together, this study provides a springboard for the functional dissection of the COMPASS family members and their role in the regulation of histone H3K4 methylation throughout development in Drosophila. PMID:21875999

  3. Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3.

    PubMed

    Kim, Sophia; Natesan, Senthil; Cornilescu, Gabriel; Carlson, Samuel; Tonelli, Marco; McClurg, Urszula L; Binda, Olivier; Robson, Craig N; Markley, John L; Balaz, Stefan; Glass, Karen C

    2016-08-26

    Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 μm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities. PMID:27281824

  4. Mechanism of Histone H3K4me3 Recognition by the Plant Homeodomain of Inhibitor of Growth 3*

    PubMed Central

    Kim, Sophia; Natesan, Senthil; Cornilescu, Gabriel; Carlson, Samuel; Tonelli, Marco; McClurg, Urszula L.; Binda, Olivier; Robson, Craig N.; Markley, John L.; Balaz, Stefan

    2016-01-01

    Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 μm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities. PMID:27281824

  5. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor suppressor genes

    PubMed Central

    Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

    2016-01-01

    Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs, and our experimental data from clinical samples, we discovered broad H3K4me3 (wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity together leading to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Broad H3K4me3 conserved across normal cells may represent pan-cancer tumor suppressors, such as P53 and PTEN, whereas cell-type-specific broad H3K4me3 may indicate cell-identity genes and cell-type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 in cancers is associated with repression of tumor suppressors. Together, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of novel tumor suppressors. PMID:26301496

  6. NAD+-SIRT1 control of H3K4 trimethylation through circadian deacetylation of MLL1

    PubMed Central

    Aguilar-Arnal, Lorena; Katada, Sayako; Orozco-Solis, Ricardo

    2015-01-01

    The circadian clock controls the transcription of hundred genes through specific chromatin remodeling events. The histone methyltransferase Mixed-Lineage Leukemia 1 (MLL1) coordinates recruitment of CLOCK–BMAL1 activator complexes to chromatin, an event associated to cyclic H3K4 tri-methylation at circadian promoters. Remarkably, in mouse liver circadian H3K4me3 is modulated by SIRT1, a NAD+ dependent deacetylase involved in clock control. We show that mammalian MLL1 is acetylated at two conserved residues, K1130 and K1133. Notably, MLL1 acetylation is cyclic, controlled by the clock and by SIRT1, and impacts the methyltransferase activity of MLL1. Moreover, H3K4 methylation at clock-controlled gene promoters is influenced by pharmacological or genetic inactivation of SIRT1. Finally, MLL1 acetylation and H3K4me3 levels at circadian gene promoters depend on NAD+ circadian levels. These findings reveal a previously unappreciated regulatory pathway between energy metabolism and histone methylation. PMID:25751424

  7. Control of transposon activity by a histone H3K4 demethylase in rice

    PubMed Central

    Cui, Xiekui; Jin, Ping; Cui, Xia; Gu, Lianfeng; Lu, Zhike; Xue, Yongming; Wei, Liya; Qi, Jianfei; Song, Xianwei; Luo, Ming; An, Gynheung; Cao, Xiaofeng

    2013-01-01

    Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many of which are retrotransposons, including 14% LTR- and ∼1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways. PMID:23319643

  8. CXXC finger protein 1 is critical for T-cell intrathymic development through regulating H3K4 trimethylation

    PubMed Central

    Cao, Wenqiang; Guo, Jing; Wen, Xiaofeng; Miao, Li; Lin, Feng; Xu, Guanxin; Ma, Ruoyu; Yin, Shengxia; Hui, Zhaoyuan; Chen, Tingting; Guo, Shixin; Chen, Wei; Huang, Yingying; Liu, Yizhi; Wang, Jianli; Wei, Lai; Wang, Lie

    2016-01-01

    T-cell development in the thymus is largely controlled by an epigenetic program, involving in both DNA methylation and histone modifications. Previous studies have identified Cxxc1 as a regulator of both cytosine methylation and histone 3 lysine 4 trimethylation (H3K4me3). However, it is unknown whether Cxxc1 plays a role in thymocyte development. Here we show that T-cell development in the thymus is severely impaired in Cxxc1-deficient mice. Furthermore, we identify genome-wide Cxxc1-binding sites and H3K4me3 modification sites in wild-type and Cxxc1-deficient thymocytes. Our results demonstrate that Cxxc1 directly controls the expression of key genes important for thymocyte survival such as RORγt and for T-cell receptor signalling including Zap70 and CD8, through maintaining the appropriate H3K4me3 on their promoters. Importantly, we show that RORγt, a direct target of Cxxc1, can rescue the survival defects in Cxxc1-deficient thymocytes. Our data strongly support a critical role of Cxxc1 in thymocyte development. PMID:27210293

  9. CXXC finger protein 1 is critical for T-cell intrathymic development through regulating H3K4 trimethylation.

    PubMed

    Cao, Wenqiang; Guo, Jing; Wen, Xiaofeng; Miao, Li; Lin, Feng; Xu, Guanxin; Ma, Ruoyu; Yin, Shengxia; Hui, Zhaoyuan; Chen, Tingting; Guo, Shixin; Chen, Wei; Huang, Yingying; Liu, Yizhi; Wang, Jianli; Wei, Lai; Wang, Lie

    2016-01-01

    T-cell development in the thymus is largely controlled by an epigenetic program, involving in both DNA methylation and histone modifications. Previous studies have identified Cxxc1 as a regulator of both cytosine methylation and histone 3 lysine 4 trimethylation (H3K4me3). However, it is unknown whether Cxxc1 plays a role in thymocyte development. Here we show that T-cell development in the thymus is severely impaired in Cxxc1-deficient mice. Furthermore, we identify genome-wide Cxxc1-binding sites and H3K4me3 modification sites in wild-type and Cxxc1-deficient thymocytes. Our results demonstrate that Cxxc1 directly controls the expression of key genes important for thymocyte survival such as RORγt and for T-cell receptor signalling including Zap70 and CD8, through maintaining the appropriate H3K4me3 on their promoters. Importantly, we show that RORγt, a direct target of Cxxc1, can rescue the survival defects in Cxxc1-deficient thymocytes. Our data strongly support a critical role of Cxxc1 in thymocyte development. PMID:27210293

  10. Histone H3K4 trimethylation: dynamic interplay with pre-mRNA splicing.

    PubMed

    Davie, James R; Xu, Wayne; Delcuve, Genevieve P

    2016-02-01

    Histone H3 lysine 4 trimethylation (H3K4me3) is often stated as a mark of transcriptionally active promoters. However, closer study of the positioning of H3K4me3 shows the mark locating primarily after the first exon at the 5' splice site and overlapping with a CpG island in mammalian cells. There are several enzyme complexes that are involved in the placement of the H3K4me3 mark, including multiple protein complexes containing SETD1A, SETD1B, and MLL1 enzymes (writers). CXXC1, which is associated with SETD1A and SETD1B, target these enzymes to unmethylated CpG islands. Lysine demethylases (KDM5 family members, erasers) demethylate H3K4me3. The H3K4me3 mark is recognized by several proteins (readers), including lysine acetyltransferase complexes, chromatin remodelers, and RNA bound proteins involved in pre-mRNA splicing. Interestingly, attenuation of H3K4me3 impacts pre-mRNA splicing, and inhibition of pre-mRNA splicing attenuates H3K4me3. PMID:26352678

  11. Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4

    PubMed Central

    Herz, Hans-Martin; Mohan, Man; Garruss, Alexander S.; Liang, Kaiwei; Takahashi, Yoh-hei; Mickey, Kristen; Voets, Olaf; Verrijzer, C. Peter; Shilatifard, Ali

    2012-01-01

    Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers. PMID:23166019

  12. Histone H3 K4 demethylation during activation and attenuation of GAL1 transcription in Saccharomyces cerevisiae.

    PubMed

    Ingvarsdottir, Kristin; Edwards, Chris; Lee, Min Gyu; Lee, Jung Shin; Schultz, David C; Shilatifard, Ali; Shiekhattar, Ramin; Berger, Shelley L

    2007-11-01

    In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes. PMID:17875926

  13. CG hypomethylation in Lsh−/− mouse embryonic fibroblasts is associated with de novo H3K4me1 formation and altered cellular plasticity

    PubMed Central

    Yu, Weishi; Briones, Victorino; Lister, Ryan; McIntosh, Carl; Han, Yixing; Lee, Eunice Y.; Ren, Jianke; Terashima, Minoru; Leighty, Robert M.; Ecker, Joseph R.; Muegge, Kathrin

    2014-01-01

    DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh−/− mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh−/− MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh−/− iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh−/− iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity. PMID:24711395

  14. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes.

    PubMed

    Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

    2015-10-01

    Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs and our experimental data from clinical samples, we discovered broad peaks for trimethylation of histone H3 at lysine 4 (H3K4me3; wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity, which together lead to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Genes with broad H3K4me3 peaks conserved across normal cells may represent pan-cancer tumor suppressors, such as TP53 and PTEN, whereas genes with cell type-specific broad H3K4me3 peaks may represent cell identity genes and cell type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 peaks in cancers is associated with repression of tumor suppressors. Thus, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of new tumor suppressors. PMID:26301496

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

    SciTech Connect

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

    2011-09-28

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

  16. Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain.

    PubMed

    Dincer, A; Gavin, D P; Xu, K; Zhang, B; Dudley, J T; Schadt, E E; Akbarian, S

    2015-01-01

    Regulators of the histone H3-trimethyl lysine-4 (H3K4me3) mark are significantly associated with the genetic risk architecture of common neurodevelopmental disease, including schizophrenia and autism. Typical H3K4me3 is primarily localized in the form of sharp peaks, extending in neuronal chromatin on average only across 500-1500 base pairs mostly in close proximity to annotated transcription start sites. Here, through integrative computational analysis of epigenomic and transcriptomic data based on next-generation sequencing, we investigated H3K4me3 landscapes of sorted neuronal and non-neuronal nuclei in human postmortem, non-human primate and mouse prefrontal cortex (PFC), and blood. To explore whether H3K4me3 peak signals could also extend across much broader domains, we examined broadest domain cell-type-specific H3K4me3 peaks in an unbiased manner with an innovative approach on 41+12 ChIP-seq and RNA-seq data sets. In PFC neurons, broadest H3K4me3 distribution ranged from 3.9 to 12 kb, with extremely broad peaks (~10 kb or broader) related to synaptic function and GABAergic signaling (DLX1, ELFN1, GAD1, IGSF9B and LINC00966). Broadest neuronal peaks showed distinct motif signatures and were centrally positioned in prefrontal gene-regulatory Bayesian networks and sensitive to defective neurodevelopment. Approximately 120 of the broadest H3K4me3 peaks in human PFC neurons, including many genes related to glutamatergic and dopaminergic signaling, were fully conserved in chimpanzee, macaque and mouse cortical neurons. Exploration of spread and breadth of lysine methylation markings could provide novel insights into epigenetic mechanism involved in neuropsychiatric disease and neuronal genome evolution. PMID:26575220

  17. The H3K4me3/2 histone demethylase RBR-2 controls axon guidance by repressing the actin-remodeling gene wsp-1.

    PubMed

    Mariani, Luca; Lussi, Yvonne C; Vandamme, Julien; Riveiro, Alba; Salcini, Anna Elisabetta

    2016-03-01

    The dynamic regulation of histone modifications is important for modulating transcriptional programs during development. Aberrant H3K4 methylation is associated with neurological disorders, but how the levels and the recognition of this modification affect specific neuronal processes is unclear. Here, we show that RBR-2, the sole homolog of the KDM5 family of H3K4me3/2 demethylases in Caenorhabditis elegans, ensures correct axon guidance by controlling the expression of the actin regulator wsp-1. Loss of rbr-2 results in increased levels of H3K4me3 at the transcriptional start site of wsp-1, with concomitant higher wsp-1 expression responsible for defective axon guidance. In agreement, overexpression of WSP-1 mimics rbr-2 loss, and its depletion restores normal axon guidance in rbr-2 mutants. NURF-1, an H3K4me3-binding protein and member of the chromatin-remodeling complex NURF, is required for promoting aberrant wsp-1 transcription in rbr-2 mutants and its ablation restores wild-type expression of wsp-1 and axon guidance. Thus, our results establish a precise role for epigenetic regulation in neuronal development by demonstrating a functional link between RBR-2 activity, H3K4me3 levels, the NURF complex and the expression of WSP-1. PMID:26811384

  18. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner

    PubMed Central

    Cano-Rodriguez, David; Gjaltema, Rutger A F.; Jilderda, Laura J; Jellema, Pytrick; Dokter-Fokkens, Jelleke; Ruiters, Marcel H J.; Rots, Marianne G

    2016-01-01

    Histone modifications reflect gene activity, but the relationship between cause and consequence of transcriptional control is heavily debated. Recent developments in rewriting local histone codes of endogenous genes elucidated instructiveness of certain marks in regulating gene expression. Maintenance of such repressive epigenome editing is controversial, while stable reactivation is still largely unexplored. Here we demonstrate sustained gene re-expression using two types of engineered DNA-binding domains fused to a H3K4 methyltransferase. Local induction of H3K4me3 is sufficient to allow re-expression of silenced target genes in various cell types. Maintenance of the re-expression is achieved, but strongly depends on the chromatin microenvironment (that is, DNA methylation status). We further identify H3K79me to be essential in allowing stable gene re-expression, confirming its role in epigenetic crosstalk for stable reactivation. Our approach uncovers potent epigenetic modifications to be directly written onto genomic loci to stably activate any given gene. PMID:27506838

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. Physical and functional interactions between the histone H3K4 demethylase KDM5A and the nucleosome remodeling and deacetylase (NuRD) complex.

    PubMed

    Nishibuchi, Gohei; Shibata, Yukimasa; Hayakawa, Tomohiro; Hayakawa, Noriyo; Ohtani, Yasuko; Sinmyozu, Kaori; Tagami, Hideaki; Nakayama, Jun-ichi

    2014-10-17

    Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here, we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing histone deacetylase complex and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two histone deacetylase complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the Caenorhabditis elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes. PMID:25190814

  2. Physical and Functional Interactions between the Histone H3K4 Demethylase KDM5A and the Nucleosome Remodeling and Deacetylase (NuRD) Complex*

    PubMed Central

    Nishibuchi, Gohei; Shibata, Yukimasa; Hayakawa, Tomohiro; Hayakawa, Noriyo; Ohtani, Yasuko; Sinmyozu, Kaori; Tagami, Hideaki; Nakayama, Jun-ichi

    2014-01-01

    Histone H3K4 methylation has been linked to transcriptional activation. KDM5A (also known as RBP2 or JARID1A), a member of the KDM5 protein family, is an H3K4 demethylase, previously implicated in the regulation of transcription and differentiation. Here, we show that KDM5A is physically and functionally associated with two histone deacetylase complexes. Immunoaffinity purification of KDM5A confirmed a previously described association with the SIN3B-containing histone deacetylase complex and revealed an association with the nucleosome remodeling and deacetylase (NuRD) complex. Sucrose density gradient and sequential immunoprecipitation analyses further confirmed the stable association of KDM5A with these two histone deacetylase complexes. KDM5A depletion led to changes in the expression of hundreds of genes, two-thirds of which were also controlled by CHD4, the NuRD catalytic subunit. Gene ontology analysis confirmed that the genes commonly regulated by both KDM5A and CHD4 were categorized as developmentally regulated genes. ChIP analyses suggested that CHD4 modulates H3K4 methylation levels at the promoter and coding regions of target genes. We further demonstrated that the Caenorhabditis elegans homologues of KDM5 and CHD4 function in the same pathway during vulva development. These results suggest that KDM5A and the NuRD complex cooperatively function to control developmentally regulated genes. PMID:25190814

  3. SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes.

    PubMed

    Kim, Jung-Hyun; Baddoo, Melody C; Park, Eun Young; Stone, Joshua K; Park, Hyeonsoo; Butler, Thomas W; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M; Tan, Ming; Flemington, Erik K; Lim, Ssang-Taek; Ahn, Eun-Young Erin

    2016-03-17

    Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

  4. LSD1-Mediated Demethylation of H3K4me2 Is Required for the Transition from Late Progenitor to Differentiated Mouse Rod Photoreceptor.

    PubMed

    Popova, Evgenya Y; Pinzon-Guzman, Carolina; Salzberg, Anna C; Zhang, Samuel Shao-Min; Barnstable, Colin J

    2016-09-01

    Epigenetic modifiers can work in concert with transcription factors to control the transition of cells from proliferating progenitors into quiescent terminally differentiated cells. This transition involves changes in histone methylation and one of the key regulators of this is the H3K4me2/1 histone demethylase LSD1. Here, we show that the highest expression of LSD1 occurs in postmitotic retinal cells during the peak period of rod photoreceptor differentiation. Pharmacological inhibition of LSD1 in retinal explants cultured from PN1 to PN8 had three major effects. It prevented the normal decrease in expression of genes associated with progenitor function, it blocked rod photoreceptor development, and it increased expression of genes associated with other retinal cell types. The maintained expression of progenitor genes was associated with a maintained level of H3K4me2 over the gene and its promoter. Among the genes whose expression was maintained was Hes1, a repressor known to block rod photoreceptor development. The inhibition of rod photoreceptor gene expression occurred in spite of the normal expression of transcription factors CRX and NRL, and the normal accumulation of H3K4me2 marks over the promoter and gene body. We suggest that LSD1 acts in concert with a series of nuclear receptors to modify chromatin structure and repress progenitor genes as well as to inhibit ectopic patterns of gene expression in the differentiating postmitotic retinal cells. PMID:26298666

  5. Mutations in Mll2, an H3K4 Methyltransferase, Result in Insulin Resistance and Impaired Glucose Tolerance in Mice

    PubMed Central

    Schröter, David; Matthews, Helen C.; Bogani, Debora; Moir, Lee; Long, Anna; Church, Christopher; Hugill, Alison; Anstee, Quentin M.; Goldin, Rob; Thursz, Mark; Hollfelder, Florian; Cox, Roger D.

    2013-01-01

    We employed a random mutagenesis approach to identify novel monogenic determinants of type 2 diabetes. Here we show that haplo-insufficiency of the histone methyltransferase myeloid-lineage leukemia (Mll2/Wbp7) gene causes type 2 diabetes in the mouse. We have shown that mice heterozygous for two separate mutations in the SET domain of Mll2 or heterozygous Mll2 knockout mice were hyperglycaemic, hyperinsulinaemic and developed non-alcoholic fatty liver disease. Consistent with previous Mll2 knockout studies, mice homozygous for either ENU mutation (or compound heterozygotes) died during embryonic development at 9.5–14.5 days post coitum. Heterozygous deletion of Mll2 induced in the adult mouse results in a normal phenotype suggesting that changes in chromatin methylation during development result in the adult phenotype. Mll2 has been shown to regulate a small subset of genes, a number of which Neurod1, Enpp1, Slc27a2, and Plcxd1 are downregulated in adult mutant mice. Our results demonstrate that histone H3K4 methyltransferase Mll2 is a component of the genetic regulation necessary for glucose homeostasis, resulting in a specific disease pattern linking chromatin modification with causes and progression of type 2 diabetes, providing a basis for its further understanding at the molecular level. PMID:23826075

  6. The Role of H3K4me3 in Transcriptional Regulation Is Altered in Huntington’s Disease

    PubMed Central

    Labadorf, Adam; Roussos, Panos; Chen, Jiang-Fan; Myers, Richard H.; Akbarian, Schahram; Weng, Zhiping

    2015-01-01

    Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion of CAG repeats in the Huntingtin (HTT) gene. Previous studies have shown mutant HTT can alter expression of genes associated with dysregulated epigenetic modifications. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls, and its association with gene expression measured by RNA-sequencing. We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down-regulated in HD. Although H3K4me3 signals are expected to be associated with mRNA levels, we found an unexpected discordance between altered H3K4me3 peaks and mRNA levels. Gene ontology (GO) term enrichment analysis of the genes with differential H3K4me3 peaks, revealed statistically significantly enriched GO terms only in the genes with down-regulated signals in HD. The most frequently implicated biological process terms are organ morphogenesis and positive regulation of gene expression. More than 9,000 H3K4me3 peaks were located not near any recognized transcription start sites and approximately 36% of these “distal” peaks co-localized to known enhancer sites. Six transcription factors and chromatin remodelers are differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, PRC2 repressive state was significantly depleted in HD-enriched peaks, suggesting the epigenetic role of PRC2 inhibition associated with up-regulated H3K4me3 in Huntington’s disease. In summary, our study provides new insights into transcriptional dysregulation of Huntington’s disease by analyzing the differentiation of H3K4me3 enrichment. PMID:26636336

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

    PubMed Central

    Shen, Yuan; Conde e Silva, Natalia; Audonnet, Laure; Servet, Caroline; Wei, Wei; Zhou, Dao-Xiu

    2014-01-01

    Histone H3 lysine 4 trimethylation (H3K4me3) has been shown to be involved in stress-responsive gene expression and gene priming in plants. However, the role of H3K4me3 resetting in the processes is not clear. In this work we studied the expression and function of Arabidopsis H3K4 demethylase gene JMJ15. We show that the expression of JMJ15 was relatively low and was limited to a number of tissues during vegetative growth but was higher in young floral organs. Over-expression of the gene in gain-of-function mutants reduced the plant height with accumulation of lignin in stems, while the loss-of-function mutation did not produce any visible phenotype. The gain-of-function mutants showed enhanced salt tolerance, whereas the loss-of-function mutant was more sensitive to salt compared to the wild type. Transcriptomic analysis revealed that over-expression of JMJ15 down-regulated many genes which are preferentially marked by H3K4me3 and H3K4me2. Many of the down-regulated genes encode transcription regulators involved in stress responses. The data suggest that increased JMJ15 levels may regulate the gene expression program that enhances stress tolerance. PMID:25009544

  8. Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1 Pre-mRNA Splicing.

    PubMed

    Khan, Dilshad H; Gonzalez, Carolina; Tailor, Nikesh; Hamedani, Mohammad K; Leygue, Etienne; Davie, James R

    2016-10-01

    Pre-mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre-mRNA by splicing factors, histone deacetylases (HDACs) and K-acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA-MB-231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA-MB-231 cells. We show that H3K4me3 steady-state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA-MB-231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre-mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre-mRNA. J. Cell. Physiol. 231: 2196-2204, 2016. © 2016 Wiley Periodicals, Inc. PMID:26864447

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

    PubMed Central

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

    2016-01-01

    Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis. PMID:27080563

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

    PubMed

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

    2016-01-01

    Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis. PMID:27080563

  11. Mouse MORC3 is a GHKL ATPase that localizes to H3K4me3 marked chromatin.

    PubMed

    Li, Sisi; Yen, Linda; Pastor, William A; Johnston, Jonathan B; Du, Jiamu; Shew, Colin J; Liu, Wanlu; Ho, Jamie; Stender, Bryan; Clark, Amander T; Burlingame, Alma L; Daxinger, Lucia; Patel, Dinshaw J; Jacobsen, Steven E

    2016-08-30

    Microrchidia (MORC) proteins are GHKL (gyrase, heat-shock protein 90, histidine kinase, MutL) ATPases that function in gene regulation in multiple organisms. Animal MORCs also contain CW-type zinc finger domains, which are known to bind to modified histones. We solved the crystal structure of the murine MORC3 ATPase-CW domain bound to the nucleotide analog AMPPNP (phosphoaminophosphonic acid-adenylate ester) and in complex with a trimethylated histone H3 lysine 4 (H3K4) peptide (H3K4me3). We observed that the MORC3 N-terminal ATPase domain forms a dimer when bound to AMPPNP. We used native mass spectrometry to show that dimerization is ATP-dependent, and that dimer formation is enhanced in the presence of nonhydrolyzable ATP analogs. The CW domain uses an aromatic cage to bind trimethylated Lys4 and forms extensive hydrogen bonds with the H3 tail. We found that MORC3 localizes to promoters marked by H3K4me3 throughout the genome, consistent with its binding to H3K4me3 in vitro. Our work sheds light on aspects of the molecular dynamics and function of MORC3. PMID:27528681

  12. Computational inference of H3K4me3 and H3K27ac domain length.

    PubMed

    Zubek, Julian; Stitzel, Michael L; Ucar, Duygu; Plewczynski, Dariusz M

    2016-01-01

    Background. Recent epigenomic studies have shown that the length of a DNA region covered by an epigenetic mark is not just a byproduct of the assaying technologies and has functional implications for that locus. For example, expanded regions of DNA sequences that are marked by enhancer-specific histone modifications, such as acetylation of histone H3 lysine 27 (H3K27ac) domains coincide with cell-specific enhancers, known as super or stretch enhancers. Similarly, promoters of genes critical for cell-specific functions are marked by expanded H3K4me3 domains in the cognate cell type, and these can span DNA regions from 4-5kb up to 40-50kb in length. These expanded H3K4me3 domains are known as buffer domains or super promoters. Methods. To ask what correlates with-and potentially regulates-the length of loci marked with these two important histone marks, H3K4me3 and H3K27ac, we built Random Forest regression models. With these models, we computationally identified genomic and epigenomic patterns that are predictive for the length of these marks in seven ENCODE cell lines. Results. We found that certain epigenetic marks and transcription factors explain the variability of the length of H3K4me3 and H3K27ac marks across different cell types, which implies that the lengths of these two epigenetic marks are tightly regulated in a given cell type. Our source code for the regression models and data can be found at our GitHub page: https://github.com/zubekj/broad_peaks. Discussion. Our Random Forest based regression models enabled us to estimate the individual contribution of different epigenetic marks and protein binding patterns to the length of H3K4me3 and H3K27ac deposition patterns, therefore potentially revealing genomic signatures at cell specific regulatory elements. PMID:26989607

  13. Computational inference of H3K4me3 and H3K27ac domain length

    PubMed Central

    Zubek, Julian; Stitzel, Michael L.

    2016-01-01

    Background. Recent epigenomic studies have shown that the length of a DNA region covered by an epigenetic mark is not just a byproduct of the assaying technologies and has functional implications for that locus. For example, expanded regions of DNA sequences that are marked by enhancer-specific histone modifications, such as acetylation of histone H3 lysine 27 (H3K27ac) domains coincide with cell-specific enhancers, known as super or stretch enhancers. Similarly, promoters of genes critical for cell-specific functions are marked by expanded H3K4me3 domains in the cognate cell type, and these can span DNA regions from 4–5kb up to 40–50kb in length. These expanded H3K4me3 domains are known as buffer domains or super promoters. Methods. To ask what correlates with—and potentially regulates—the length of loci marked with these two important histone marks, H3K4me3 and H3K27ac, we built Random Forest regression models. With these models, we computationally identified genomic and epigenomic patterns that are predictive for the length of these marks in seven ENCODE cell lines. Results. We found that certain epigenetic marks and transcription factors explain the variability of the length of H3K4me3 and H3K27ac marks across different cell types, which implies that the lengths of these two epigenetic marks are tightly regulated in a given cell type. Our source code for the regression models and data can be found at our GitHub page: https://github.com/zubekj/broad_peaks. Discussion. Our Random Forest based regression models enabled us to estimate the individual contribution of different epigenetic marks and protein binding patterns to the length of H3K4me3 and H3K27ac deposition patterns, therefore potentially revealing genomic signatures at cell specific regulatory elements. PMID:26989607

  14. PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

    PubMed Central

    Chung, Ho-Ryun; Xu, Chao; Fuchs, Alisa; Mund, Andreas; Lange, Martin; Staege, Hannah; Schubert, Tobias; Bian, Chuanbing; Dunkel, Ilona; Eberharter, Anton; Regnard, Catherine; Klinker, Henrike; Meierhofer, David; Cozzuto, Luca; Winterpacht, Andreas; Di Croce, Luciano; Min, Jinrong; Will, Hans; Kinkley, Sarah

    2016-01-01

    PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes. DOI: http://dx.doi.org/10.7554/eLife.10607.001 PMID:27223324

  15. Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive.

    PubMed

    Gatchalian, Jovylyn; Gallardo, Carmen Mora; Shinsky, Stephen A; Ospina, Ruben Rosas; Liendo, Andrea Mansilla; Krajewski, Krzysztof; Klein, Brianna J; Andrews, Forest H; Strahl, Brian D; M van Wely, Karel H; Kutateladze, Tatiana G

    2016-07-27

    Histone post-translational modifications, and specific combinations they create, mediate a wide range of nuclear events. However, the mechanistic bases for recognition of these combinations have not been elucidated. Here, we characterize crosstalk between H3T3 and H3T6 phosphorylation, occurring in mitosis, and H3K4me3, a mark associated with active transcription. We detail the molecular mechanisms by which H3T3ph/K4me3/T6ph switches mediate activities of H3K4me3-binding proteins, including those containing plant homeodomain (PHD) and double Tudor reader domains. Our results derived from nuclear magnetic resonance chemical shift perturbation analysis, orthogonal binding assays and cell fluorescence microscopy studies reveal a strong anti-correlation between histone H3T3/T6 phosphorylation and retention of PHD finger proteins in chromatin during mitosis. Together, our findings uncover the mechanistic rules of chromatin engagement for H3K4me3-specific readers during cell division. PMID:27016734

  16. Locus co-occupancy, nucleosome positioning, and H3K4me1 regulate the functionality of FOXA2-, HNF4A-, and PDX1-bound loci in islets and liver

    PubMed Central

    Hoffman, Brad G.; Robertson, Gordon; Zavaglia, Bogard; Beach, Mike; Cullum, Rebecca; Lee, Sam; Soukhatcheva, Galina; Li, Leping; Wederell, Elizabeth D.; Thiessen, Nina; Bilenky, Mikhail; Cezard, Timothee; Tam, Angela; Kamoh, Baljit; Birol, Inanc; Dai, Derek; Zhao, YongJun; Hirst, Martin; Verchere, C. Bruce; Helgason, Cheryl D.; Marra, Marco A.; Jones, Steven J.M.; Hoodless, Pamela A.

    2010-01-01

    The liver and pancreas share a common origin and coexpress several transcription factors. To gain insight into the transcriptional networks regulating the function of these tissues, we globally identify binding sites for FOXA2 in adult mouse islets and liver, PDX1 in islets, and HNF4A in liver. Because most eukaryotic transcription factors bind thousands of loci, many of which are thought to be inactive, methods that can discriminate functionally active binding events are essential for the interpretation of genome-wide transcription factor binding data. To develop such a method, we also generated genome-wide H3K4me1 and H3K4me3 localization data in these tissues. By analyzing our binding and histone methylation data in combination with comprehensive gene expression data, we show that H3K4me1 enrichment profiles discriminate transcription factor occupied loci into three classes: those that are functionally active, those that are poised for activation, and those that reflect pioneer-like transcription factor activity. Furthermore, we demonstrate that the regulated presence of H3K4me1-marked nucleosomes at transcription factor occupied promoters and enhancers controls their activity, implicating both tissue-specific transcription factor binding and nucleosome remodeling complex recruitment in determining tissue-specific gene expression. Finally, we apply these approaches to generate novel insights into how FOXA2, PDX1, and HNF4A cooperate to drive islet- and liver-specific gene expression. PMID:20551221

  17. Impairment of Preimplantation Porcine Embryo Development by Histone Demethylase KDM5B Knockdown Through Disturbance of Bivalent H3K4me3-H3K27me3 Modifications1

    PubMed Central

    Huang, Jiaojiao; Zhang, Hongyong; Wang, Xianlong; Dobbs, Kyle B.; Yao, Jing; Qin, Guosong; Whitworth, Kristin; Walters, Eric M.; Prather, Randall S.; Zhao, Jianguo

    2015-01-01

    ABSTRACT KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications. PMID:25609834

  18. ZMYND8 Reads the Dual Histone Mark H3K4me1-H3K14ac to Antagonize the Expression of Metastasis-Linked Genes.

    PubMed

    Li, Na; Li, Yuanyuan; Lv, Jie; Zheng, Xiangdong; Wen, Hong; Shen, Hongjie; Zhu, Guangjing; Chen, Tsai-Yu; Dhar, Shilpa S; Kan, Pu-Yeh; Wang, Zhibin; Shiekhattar, Ramin; Shi, Xiaobing; Lan, Fei; Chen, Kaifu; Li, Wei; Li, Haitao; Lee, Min Gyu

    2016-08-01

    Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating functions of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a metastasis-suppressive epigenetic mechanism in which ZMYND8's PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes. PMID:27477906

  19. Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae)

    PubMed Central

    Serrano, Érica Alves; Araya-Jaime, Cristian; Suárez-Villota, Elkin Y.; Oliveira, Claudio; Foresti, Fausto

    2016-01-01

    Abstract Characidium gomesi Travasso, 1956 specimens from the Pardo River have up to four heterochromatic supernumerary chromosomes, derived from the sex chromosomes. To access the meiotic behavior and distribution of an active chromatin marker, males and females of Characidium gomesi with two or three B chromosomes were analyzed. Mitotic chromosomes were characterized using C-banding and FISH with B chromosome probes. Meiocytes were subjected to immunofluorescence-FISH assay using anti-SYCP3, anti-H3K4m, and B chromosomes probes. Molecular homology of supernumeraries was confirmed by FISH and by its bivalent conformation in individuals with two of these chromosomes. In individuals with three Bs, these elements formed a bivalent and a univalent. Supernumerary and sex chromosomes exhibited H3K4m signals during pachytene contrasting with their heterochromatic and asynaptic nature, which suggest a more structural role than functional of this histone modification. The implications of this result are discussed in light of the homology, meiotic nuclear organization, and meiotic silencing of unsynapsed chomatin. PMID:27551347

  20. Meiotic behavior and H3K4m distribution in B chromosomes of Characidium gomesi (Characiformes, Crenuchidae).

    PubMed

    Serrano, Érica Alves; Araya-Jaime, Cristian; Suárez-Villota, Elkin Y; Oliveira, Claudio; Foresti, Fausto

    2016-01-01

    Characidium gomesi Travasso, 1956 specimens from the Pardo River have up to four heterochromatic supernumerary chromosomes, derived from the sex chromosomes. To access the meiotic behavior and distribution of an active chromatin marker, males and females of Characidium gomesi with two or three B chromosomes were analyzed. Mitotic chromosomes were characterized using C-banding and FISH with B chromosome probes. Meiocytes were subjected to immunofluorescence-FISH assay using anti-SYCP3, anti-H3K4m, and B chromosomes probes. Molecular homology of supernumeraries was confirmed by FISH and by its bivalent conformation in individuals with two of these chromosomes. In individuals with three Bs, these elements formed a bivalent and a univalent. Supernumerary and sex chromosomes exhibited H3K4m signals during pachytene contrasting with their heterochromatic and asynaptic nature, which suggest a more structural role than functional of this histone modification. The implications of this result are discussed in light of the homology, meiotic nuclear organization, and meiotic silencing of unsynapsed chomatin. PMID:27551347

  1. Triptolide Induces Cell Apoptosis by Targeting H3K4me3 and Downstream Effector Proteins in KM3 Multiple Myeloma Cells.

    PubMed

    Wen, Lu; Chen, Yan; Zeng, Ling L; Zhao, Fei; Yi, Sha; Yang, Li J; Zhang, Ben P; Zhao, Jie; Zhao, Zi C; Zhang, Chun

    2015-01-01

    As the principal active ingredient in the Chinese herb Tripterygium wilfordii Hook.F (TwHF), triptolide has been shown to have very strong antitumor properties. The trimethylation of lysine 4 on histone H3 (H3K4me3) has been proposed to promote gene expression, and the accumulation of H3K4me3 at the transcriptional start sites of oncogenes is involved in carcinogenesis. To identify the association between the reduction of H3K4me3 and the apoptosis of MM cells induced by triptolide, we investigated the global patterns of H3K4me3 occupancy in the MM cell genome. Combined analyses using ChIP-on-chip and western blotting showed that H3K4me3 were highly enriched on the gene promoters of c-Myc and VEGFA and were associated with the up-regulation of both genes. Treatment of KM3 cells with triptolide and siRNA targeting ASH2L reduced the expression of c-Myc and VEGFA. These results suggest that triptolide can down-regulate c-Myc and VEGFA expression by blocking the accumulation of H3K4me3 on their promoters,and thus play an important role in anti-MM mechanism. PMID:26420049

  2. The MLL3/MLL4 Branches of the COMPASS Family Function as Major Histone H3K4 Monomethylases at Enhancers

    PubMed Central

    Hu, Deqing; Gao, Xin; Morgan, Marc A.; Herz, Hans-Martin; Smith, Edwin R.

    2013-01-01

    Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (complex of proteins associated with Set1) family from Saccharomyces cerevisiae to humans, and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which is enriched at transcription start sites in all eukaryotes. Our recent studies of Drosophila melanogaster demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 (GeneID 58508) and MLL4 (GeneID 8085), are most closely related to Drosophila Trr. Here, we use chromatin immunoprecipitation-sequencing (ChIP-seq) of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and find that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, we demonstrate that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, we find a redundant role between Mll3 (GeneID 231051) and Mll4 (GeneID 381022) in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3 and MLL4 function in the regulation of enhancer activity and that mutations of MLL3 and MLL4 that are found in cancers could exert their properties through malfunction of these Trr/MLL3/MLL4-specific (Trrific) enhancers. PMID:24081332

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

    PubMed Central

    Kooistra, Susanne M.; Malatesta, Martina; Morales Torres, Cristina; Rekling, Jens C.; Johansen, Jens V.; Abarrategui, Iratxe; Helin, Kristian

    2013-01-01

    Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development, and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb target genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants, supporting a functional interplay between Polycomb proteins and Jarid1b. To understand how Jarid1b regulates mouse development, we performed a genome-wide analysis of histone modifications, which demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 during early embryogenesis in Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators like Pax6 and Otx2 in Jarid1b knockout brains. Taken together, these results suggest that Jarid1b regulates mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications. PMID:23637629

  4. The H3K4me3 Histone Demethylase Fbxl10 Is a Regulator of Chemokine Expression, Cellular Morphology, and the Metabolome of Fibroblasts

    PubMed Central

    Janzer, Andreas; Stamm, Katrin; Becker, Astrid; Zimmer, Andreas; Buettner, Reinhard; Kirfel, Jutta

    2012-01-01

    Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts. PMID:22825849

  5. Pro Isomerization in MLL1 PHD3-Bromo Cassette Connects H3K4me Readout to CyP33 and HDAC-Mediated Repression

    SciTech Connect

    Wang, Zhanxin; Song, Jikui; Milne, Thomas A.; Wang, Gang G.; Li, Haitao; Allis, C. David; Patel, Dinshaw J.

    2010-09-13

    The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.

  6. Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity.

    PubMed

    Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

    2016-01-01

    Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. PMID:27087825

  7. Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity

    PubMed Central

    Lu, Jun; Zhang, Xiaoli; Shen, Tingting; Ma, Chao; Wu, Jun; Kong, Hualei; Tian, Jing; Shao, Zhifeng; Zhao, Xiaodong; Xu, Ling

    2016-01-01

    Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P < 0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. PMID:27087825

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem Cells Is Associated With the Activating H3K4me3 Histone Mark.

    PubMed

    Zaidi, Sayyed K; Boyd, Joseph R; Grandy, Rodrigo A; Medina, Ricardo; Lian, Jane B; Stein, Gary S; Stein, Janet L

    2016-09-01

    Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation, and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run-on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome-wide chromatin immunoprecipitation-deep sequencing and bioinformatics approaches, we discovered that, RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M, and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down-regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007-2013, 2016. © 2016 Wiley Periodicals, Inc. PMID:26755341

  11. H3K4me3 Stimulates the V(D)J RAG Complex for Both Nicking and Hairpinning in Trans in Addition to Tethering in Cis: Implications for Translocations

    PubMed Central

    Shimazaki, Noriko; Tsai, Albert G.; Lieber, Michael R.

    2010-01-01

    SUMMARY The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. However, in the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 has not been clear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2 to 5-fold) and hairpinning (3 to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans). This indicates that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (kcat) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T-cell acute lymphoblastic lymphomas. PMID:19524534

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

    PubMed Central

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

    2014-01-01

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

  13. reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4(+) memory T cells.

    PubMed

    Kinkley, Sarah; Helmuth, Johannes; Polansky, Julia K; Dunkel, Ilona; Gasparoni, Gilles; Fröhler, Sebastian; Chen, Wei; Walter, Jörn; Hamann, Alf; Chung, Ho-Ryun

    2016-01-01

    The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analysis tool, normR that allows for the sequential enrichment and detection of co-localizing DNA-associated proteins in an unbiased and genome-wide manner. We illustrate the utility of the reChIP-seq method and normR by identifying H3K4me3 or H3K27me3 bivalently modified nucleosomes in primary human CD4(+) memory T cells. We unravel widespread bivalency at hypomethylated CpG-islands coinciding with inactive promoters of developmental regulators. reChIP-seq additionally uncovered heterogeneous bivalency in the population, which was undetectable by intersecting H3K4me3 and H3K27me3 ChIP-seq tracks. Finally, we provide evidence that bivalency is established and stabilized by an interplay between the genome and epigenome. Our reChIP-seq approach augments conventional ChIP-seq and is broadly applicable to unravel combinatorial modes of action. PMID:27530917

  14. reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4+ memory T cells

    PubMed Central

    Kinkley, Sarah; Helmuth, Johannes; Polansky, Julia K.; Dunkel, Ilona; Gasparoni, Gilles; Fröhler, Sebastian; Chen, Wei; Walter, Jörn; Hamann, Alf; Chung, Ho-Ryun

    2016-01-01

    The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analysis tool, normR that allows for the sequential enrichment and detection of co-localizing DNA-associated proteins in an unbiased and genome-wide manner. We illustrate the utility of the reChIP-seq method and normR by identifying H3K4me3 or H3K27me3 bivalently modified nucleosomes in primary human CD4+ memory T cells. We unravel widespread bivalency at hypomethylated CpG-islands coinciding with inactive promoters of developmental regulators. reChIP-seq additionally uncovered heterogeneous bivalency in the population, which was undetectable by intersecting H3K4me3 and H3K27me3 ChIP-seq tracks. Finally, we provide evidence that bivalency is established and stabilized by an interplay between the genome and epigenome. Our reChIP-seq approach augments conventional ChIP-seq and is broadly applicable to unravel combinatorial modes of action. PMID:27530917

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

    PubMed Central

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

    2015-01-01

    DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3. PMID:25712104

  16. A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos

    PubMed Central

    Akkers, Robert C.; van Heeringen, Simon J.; Jacobi, Ulrike G.; Janssen-Megens, Eva M.; Françoijs, Kees-Jan; Stunnenberg, Hendrik G.; Veenstra, Gert Jan C.

    2009-01-01

    SUMMARY Epigenetic mechanisms set apart the active and inactive regions in the genome of multicellular organisms to produce distinct cell fates during embryogenesis. Here we report on the epigenetic and transcriptome genome-wide maps of gastrula-stage Xenopus tropicalis embryos using massive parallel sequencing of cDNA (RNA-seq) and DNA obtained by chromatin immunoprecipitation (ChIP-seq) of histone H3 K4 and K27 trimethylation and RNA Polymerase II (RNAPII). These maps identify promoters and transcribed regions. Strikingly, genomic regions featuring opposing histone modifications are mostly transcribed, reflecting spatially regulated expression rather than bivalency as determined by expression profile analyses, sequential ChIP, and ChIP-seq on dissected embryos. Spatial differences in H3K27me3 deposition are predictive of localized gene expression. Moreover, the appearance of H3K4me3 coincides with zygotic gene activation, whereas H3K27me3 is predominantly deposited upon subsequent spatial restriction or repression of transcriptional regulators. These results reveal a hierarchy in the spatial control of zygotic gene activation. PMID:19758566

  17. High-Resolution Mapping of Epigenetic Modifications of the Rice Genome Uncovers Interplay between DNA Methylation, Histone Methylation, and Gene Expression[W

    PubMed Central

    Li, Xueyong; Wang, Xiangfeng; He, Kun; Ma, Yeqin; Su, Ning; He, Hang; Stolc, Viktor; Tongprasit, Waraporn; Jin, Weiwei; Jiang, Jiming; Terzaghi, William; Li, Songgang; Deng, Xing Wang

    2008-01-01

    We present high-resolution maps of DNA methylation and H3K4 di- and trimethylation of two entire chromosomes and two fully sequenced centromeres in rice (Oryza sativa) shoots and cultured cells. This analysis reveals combinatorial interactions between these epigenetic modifications and chromatin structure and gene expression. Cytologically densely stained heterochromatin had less H3K4me2 and H3K4me3 and more methylated DNA than the less densely stained euchromatin, whereas centromeres had a unique epigenetic composition. Most transposable elements had highly methylated DNA but no H3K4 methylation, whereas more than half of protein-coding genes had both methylated DNA and di- and/or trimethylated H3K4. Methylation of DNA but not H3K4 was correlated with suppressed transcription. By contrast, when both DNA and H3K4 were methylated, transcription was only slightly reduced. Transcriptional activity was positively correlated with the ratio of H3K4me3/H3K4me2: genes with predominantly H3K4me3 were actively transcribed, whereas genes with predominantly H3K4me2 were transcribed at moderate levels. More protein-coding genes contained all three modifications, and more transposons contained DNA methylation in shoots than cultured cells. Differential epigenetic modifications correlated to tissue-specific expression between shoots and cultured cells. Collectively, this study provides insights into the rice epigenomes and their effect on gene expression and plant development. PMID:18263775

  18. CpG islands under selective pressure are enriched with H3K4me3, H3K27ac and H3K36me3 histone modifications

    PubMed Central

    2013-01-01

    Background Histone modification is an epigenetic mechanism that influences gene regulation in eukaryotes. In particular, histone modifications in CpG islands (CGIs) are associated with different chromatin states and with transcription activity. Changes in gene expression play a crucial role in adaptation and evolution. Results In this paper, we have studied, using a computational biology approach, the relationship between histone modifications in CGIs and selective pressure in Homo sapiens. We considered three histone modifications: histone H3 lysine 4 trimethylation (H3K4me3), histone H3 lysine 27 acetylation (H3K27ac) and histone H3 lysine 36 trimethylation (H3K36me3), and we used the publicly available genomic-scale histone modification data of thirteen human cell lines. To define regions under selective pressure, we used three distinct signatures that mark selective events from different evolutionary periods. We found that CGIs under selective pressure showed significant enrichments for histone modifications. Conclusion Our result suggests that, CGIs that have undergone selective events are characterized by epigenetic signatures, in particular, histone modifications that are distinct from CGIs with no evidence of selection. PMID:23837650

  19. Histone H3 lysine 4 methylation revisited

    PubMed Central

    Kusch, Thomas

    2012-01-01

    Since its discovery more than a decade ago, H3K4 methylation has become synonymous with transcription. We only now have begun to realize that the distinct states of H3K4 methylation have unique distributions and specialized roles in other chromatin-related processes. Here, I discuss recent findings addressing their regulation and functions. PMID:23117820

  20. Pygo2 functions as a prognostic factor for glioma due to its up-regulation of H3K4me3 and promotion of MLL1/MLL2 complex recruitment

    PubMed Central

    Zhou, Cefan; Zhang, Yi; Dai, Jun; Zhou, Mengzhou; Liu, Miao; Wang, Yefu; Chen, Xing-Zhen; Tang, Jingfeng

    2016-01-01

    Pygo2 has been discovered as an important Wnt signaling component contributing to the activation of Wnt-target gene transcription. In the present study, we discovered that Pygo2 mRNA and protein levels were up-regulated in the majority of (152/209) human brain glioma tissues and five glioma cell lines, and significantly correlated with the age, the WHO tumor classification and poor patient survival. The histone methyltransferase complex components (WDR5, Ash2, and menin, but not CXCC1 or NCOA6) were down-regulated at the promoter loci of Wnt target genes after Pygo2 knockdown, and this was accompanied by the down-regulation of Wnt/β-catenin pathway activity. Further, we demonstrated that the involvement of Pygo2 in the activation of the Wnt pathway in human glioma progression is through up-regulation of the H3K4me3 (but not H3K4me2) by promoting the recruitment of the histone methyltransferase MLL1/MLL2 complex to Wnt target gene promoters. Thus, our study provided evidence that Pygo2 functions as a novel prognostic marker and represents a potential therapeutic target. PMID:26902498

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

    PubMed Central

    Gacek-Matthews, Agnieszka; Sasaki, Takahiko; Wittstein, Kathrin; Gruber, Clemens; Strauss, Joseph

    2016-01-01

    Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes—usually physically linked in co-regulated clusters—are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production. PMID:27548260

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

    PubMed

    Gacek-Matthews, Agnieszka; Berger, Harald; Sasaki, Takahiko; Wittstein, Kathrin; Gruber, Clemens; Lewis, Zachary A; Strauss, Joseph

    2016-08-01

    Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production. PMID:27548260

  3. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

    PubMed

    Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

    2015-01-01

    Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. PMID:25848745

  4. Dnmt2-dependent methylomes lack defined DNA methylation patterns

    PubMed Central

    Raddatz, Günter; Guzzardo, Paloma M.; Olova, Nelly; Fantappié, Marcelo Rosado; Rampp, Markus; Schaefer, Matthias; Reik, Wolf; Hannon, Gregory J.; Lyko, Frank

    2013-01-01

    Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms. PMID:23641003

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

    PubMed Central

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

    2015-01-01

    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. PMID:26584620

  6. 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. PMID:26584620

  7. Defining the cutoff value of MGMT gene promoter methylation and its predictive capacity in glioblastoma.

    PubMed

    Brigliadori, Giovanni; Foca, Flavia; Dall'Agata, Monia; Rengucci, Claudia; Melegari, Elisabetta; Cerasoli, Serenella; Amadori, Dino; Calistri, Daniele; Faedi, Marina

    2016-06-01

    Despite advances in the treatment of glioblastoma (GBM), median survival is 12-15 months. O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation status is acknowledged as a predictive marker for temozolomide (TMZ) treatment. When MGMT promoter values fall into a "methylated" range, a better response to chemotherapy is expected. However, a cutoff that discriminates between "methylated" and "unmethylated" status has yet to be defined. We aimed to identify the best cutoff value and to find out whether variability in methylation profiles influences the predictive capacity of MGMT promoter methylation. Data from 105 GBM patients treated between 2008 and 2013 were analyzed. MGMT promoter methylation status was determined by analyzing 10 CpG islands by pyrosequencing. Patients were treated with radiotherapy followed by TMZ. MGMT promoter methylation status was classified into unmethylated 0-9 %, methylated 10-29 % and methylated 30-100 %. Statistical analysis showed that an assumed methylation cutoff of 9 % led to an overestimation of responders. All patients in the 10-29 % methylation group relapsed before the 18-month evaluation. Patients with a methylation status ≥30 % showed a median overall survival of 25.2 months compared to 15.2 months in all other patients, confirming this value as the best methylation cutoff. Despite wide variability among individual profiles, single CpG island analysis did not reveal any correlation between single CpG island methylation values and relapse or death. Specific CpG island methylation status did not influence the predictive value of MGMT. The predictive role of MGMT promoter methylation was maintained only with a cutoff value ≥30 %. PMID:27029617

  8. Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases.

    PubMed

    Huang, Po-Hsien; Chen, Chun-Han; Chou, Chih-Chien; Sargeant, Aaron M; Kulp, Samuel K; Teng, Che-Ming; Byrd, John C; Chen, Ching-Shih

    2011-01-01

    This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications. PMID:20959362

  9. Reduced DNA methylation patterning and transcriptional connectivity define human skin aging.

    PubMed

    Bormann, Felix; Rodríguez-Paredes, Manuel; Hagemann, Sabine; Manchanda, Himanshu; Kristof, Boris; Gutekunst, Julian; Raddatz, Günter; Haas, Rainer; Terstegen, Lara; Wenck, Horst; Kaderali, Lars; Winnefeld, Marc; Lyko, Frank

    2016-06-01

    Epigenetic changes represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Age-related changes in DNA methylation at the genome scale have been termed 'epigenetic drift', but the defining features of this phenomenon remain to be established. Human epidermis represents an excellent model for understanding age-related epigenetic changes because of its substantial cell-type homogeneity and its well-known age-related phenotype. We have now generated and analyzed the currently largest set of human epidermis methylomes (N = 108) using array-based profiling of 450 000 methylation marks in various age groups. Data analysis confirmed that age-related methylation differences are locally restricted and characterized by relatively small effect sizes. Nevertheless, methylation data could be used to predict the chronological age of sample donors with high accuracy. We also identified discontinuous methylation changes as a novel feature of the aging methylome. Finally, our analysis uncovered an age-related erosion of DNA methylation patterns that is characterized by a reduced dynamic range and increased heterogeneity of global methylation patterns. These changes in methylation variability were accompanied by a reduced connectivity of transcriptional networks. Our findings thus define the loss of epigenetic regulatory fidelity as a key feature of the aging epigenome. PMID:27004597

  10. On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-1 (MLL1) core complex.

    PubMed

    Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E; Cosgrove, Michael S

    2009-09-01

    Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked approximately 600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. PMID:19556245

  11. On the Mechanism of Multiple Lysine Methylation by the Human Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex*♦

    PubMed Central

    Patel, Anamika; Dharmarajan, Venkatasubramanian; Vought, Valarie E.; Cosgrove, Michael S.

    2009-01-01

    Transcription in eukaryotic genomes depends on enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation. The mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of H3K4 methyltransferases and is frequently rearranged in acute leukemias. Despite sequence comparisons that predict that SET1 family enzymes should only monomethylate their substrates, mono-, di-, and trimethylation of H3K4 has been attributed to SET1 family complexes in vivo and in vitro. To better understand this paradox, we have biochemically reconstituted and characterized a five-component 200-kDa MLL1 core complex containing human MLL1, WDR5, RbBP5, Ash2L, and DPY-30. We demonstrate that the isolated MLL1 SET domain is a slow monomethyltransferase and that tyrosine 3942 of MLL1 prevents di- and trimethylation of H3K4. In contrast, a complex containing the MLL1 SET domain, WDR5, RbBP5, Ash2L, and DPY-30, displays a marked ∼600-fold increase in enzymatic activity but only to the dimethyl form of H3K4. Single turnover kinetic experiments reveal that the reaction leading to H3K4 dimethylation involves the transient accumulation of a monomethylated species, suggesting that the MLL1 core complex uses a non-processive mechanism to catalyze multiple lysine methylation. We have also discovered that the non-SET domain components of the MLL1 core complex possess a previously unrecognized methyltransferase activity that catalyzes H3K4 dimethylation within the MLL1 core complex. Our results suggest that the mechanism of multiple lysine methylation by the MLL1 core complex involves the sequential addition of two methyl groups at two distinct active sites within the complex. PMID:19556245

  12. Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.

    PubMed

    Patel, Anamika; Vought, Valarie E; Swatkoski, Stephen; Viggiano, Susan; Howard, Benny; Dharmarajan, Venkatasubramanian; Monteith, Kelsey E; Kupakuwana, Gillian; Namitz, Kevin E; Shinsky, Stephen A; Cotter, Robert J; Cosgrove, Michael S

    2014-01-10

    The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex. PMID:24235145

  13. Automethylation Activities within the Mixed Lineage Leukemia-1 (MLL1) Core Complex Reveal Evidence Supporting a “Two-active Site” Model for Multiple Histone H3 Lysine 4 Methylation*

    PubMed Central

    Patel, Anamika; Vought, Valarie E.; Swatkoski, Stephen; Viggiano, Susan; Howard, Benny; Dharmarajan, Venkatasubramanian; Monteith, Kelsey E.; Kupakuwana, Gillian; Namitz, Kevin E.; Shinsky, Stephen A.; Cotter, Robert J.; Cosgrove, Michael S.

    2014-01-01

    The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a “two-active site” model for multiple H3K4 methylation by the MLL1 core complex. PMID:24235145

  14. Effects of nickel, chromate, and arsenite on histone 3 lysine methylation

    SciTech Connect

    Zhou Xue; Li Qin; Arita, Adriana; Sun Hong; Costa, Max

    2009-04-01

    Occupational exposure to nickel (Ni), chromium (Cr), and arsenic (As) containing compounds has been associated with lung cancer and other adverse health effects. Their carcinogenic properties may be attributable in part, to activation and/or repression of gene expression induced by changes in the DNA methylation status and histone tail post-translational modifications. Here we show that individual treatment with nickel, chromate, and arsenite all affect the gene activating mark H3K4 methylation. We found that nickel (1 mM), chromate (10 {mu}M), and arsenite (1 {mu}M) significantly increase tri-methyl H3K4 after 24 h exposure in human lung carcinoma A549 cells. Seven days of exposure to lower levels of nickel (50 and 100 {mu}M), chromate (0.5 and 1 {mu}M) or arsenite (0.1, 0.5 and 1 {mu}M) also increased tri-methylated H3K4 in A549 cells. This mark still remained elevated and inherited through cell division 7 days following removal of 1 {mu}M arsenite. We also demonstrate by dual staining immunofluorescence microscopy that both H3K4 tri-methyl and H3K9 di-methyl marks increase globally after 24 h exposure to each metal treatment in A549 cells. However, the tri-methyl H3K4 and di-methyl H3K9 marks localize in different regions in the nucleus of the cell. Thus, our study provides further evidence that a mechanism(s) of carcinogenicity of nickel, chromate, and arsenite metal compounds may involve alterations of various histone tail modifications that may in turn affect the expression of genes that may cause transformation.

  15. Methamphetamine-Associated Memory is Regulated by a Writer and an Eraser of Permissive Histone Methylation

    PubMed Central

    Griggs, Erica M.; Mikaelsson, Mikael A.; Takács, Irma F.; Young, Erica J.; Rumbaugh, Gavin; Miller, Courtney A.

    2013-01-01

    Background Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder by triggering craving. The nucleus accumbens (NAc) is essential to these drug-associated memories, but underlying mechanisms are poorly understood. Posttranslational chromatin modifications, such as histone methylation, modulate gene transcription, thus we investigated the role of the associated epigenetic modifiers in METH-associated memory. Methods Conditioned place preference was used to assess the epigenetic landscape in the NAc supporting METH-associated memory (n=79). The impact of histone methylation (H3K4me2/3) on the formation and expression of METH-associated memory was determined by focal, intra NAc knockdown (KD) of a writer, the methyltransferase MLL1 (n=26), and an eraser, the histone demethylase KDM5C (n=38), of H3K4me2/3. Results A survey of chromatin modifications in the NAc of animals forming a METH-associated memory revealed the global induction of several modifications associated with active transcription. This correlated with a pattern of gene activation, as revealed by microarray analysis, including upregulation of Oxtr and Fos, whose promoters also had increased H3K4me3. KD of Mll1 reduced H3K4me3, Fos and Oxtr levels and disrupted METH-associated memory. KD of Kdm5c resulted in hypermethylation of H3K4 and prevented the expression of METH-associated memory. Conclusions The development and expression of METH-associated memory are supported by regulation of H3K4me2/3 levels by MLL1 and KDM5C, respectively, in the NAc. These data indicate that permissive histone methylation, and the associated epigenetic writers and erasers, represent potential targets for the treatment of substance abuse relapse, a psychiatric condition perpetuated by unwanted associative memories. PMID:24183790

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

    PubMed Central

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

    2016-01-01

    KMT2D, which encodes a histone H3K4 methyltransferase, has been implicated in human congenital heart disease in the context of Kabuki syndrome. However, its role in heart development is not understood. Here, we demonstrate a requirement for KMT2D in cardiac precursors and cardiomyocytes during cardiogenesis in mice. Gene expression analysis revealed downregulation of ion transport and cell cycle genes, leading to altered calcium handling and cell cycle defects. We further determined that myocardial Kmt2d deletion led to decreased H3K4me1 and H3K4me2 at enhancers and promoters. Finally, we identified KMT2D-bound regions in cardiomyocytes, of which a subset was associated with decreased gene expression and decreased H3K4me2 in mutant hearts. This subset included genes related to ion transport, hypoxia-reoxygenation and cell cycle regulation, suggesting that KMT2D is important for these processes. Our findings indicate that KMT2D is essential for regulating cardiac gene expression during heart development primarily via H3K4 di-methylation. PMID:26932671

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

    PubMed

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

    2016-03-01

    KMT2D, which encodes a histone H3K4 methyltransferase, has been implicated in human congenital heart disease in the context of Kabuki syndrome. However, its role in heart development is not understood. Here, we demonstrate a requirement for KMT2D in cardiac precursors and cardiomyocytes during cardiogenesis in mice. Gene expression analysis revealed downregulation of ion transport and cell cycle genes, leading to altered calcium handling and cell cycle defects. We further determined that myocardial Kmt2d deletion led to decreased H3K4me1 and H3K4me2 at enhancers and promoters. Finally, we identified KMT2D-bound regions in cardiomyocytes, of which a subset was associated with decreased gene expression and decreased H3K4me2 in mutant hearts. This subset included genes related to ion transport, hypoxia-reoxygenation and cell cycle regulation, suggesting that KMT2D is important for these processes. Our findings indicate that KMT2D is essential for regulating cardiac gene expression during heart development primarily via H3K4 di-methylation. PMID:26932671

  18. Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

    SciTech Connect

    Islam, Kabirul; Chen, Yuling; Wu, Hong; Bothwell, Ian R.; Blum, Gil J.; Zeng, Hong; Dong, Aiping; Zheng, Weihong; Min, Jinrong; Deng, Haiteng; Luo, Minkui

    2013-11-18

    Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

  19. Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns

    PubMed Central

    Edwards, John R.; O'Donnell, Anne H.; Rollins, Robert A.; Peckham, Heather E.; Lee, Clarence; Milekic, Maria H.; Chanrion, Benjamin; Fu, Yutao; Su, Tao; Hibshoosh, Hanina; Gingrich, Jay A.; Haghighi, Fatemeh; Nutter, Robert; Bestor, Timothy H.

    2010-01-01

    Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di- or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity. PMID:20488932

  20. Methylation of the PTEN promoter defines low-grade gliomas and secondary glioblastoma

    PubMed Central

    Wiencke, John K.; Zheng, Shichun; Jelluma, Nanette; Tihan, Tarik; Vandenberg, Scott; Tamgüney, Tanja; Baumber, Rachel; Parsons, Ramon; Lamborn, Kathleen R.; Berger, Mitchel S.; Wrensch, Margaret R.; Haas-Kogan, Daphne Adele; Stokoe, David

    2007-01-01

    Glioblastoma multiforme (GBM) can present as either de novo or secondary tumors arising from previously diagnosed low-grade gliomas. Although these tumor types are phenotypically indistinguishable, de novo and secondary GBMs are associated with distinct genetic characteristics. PTEN mutations, which result in activation of the phosphoinositide 3-kinase (PI3K) signal transduction pathway, are frequent in de novo but not in secondary GBMs or their antecedent low-grade tumors. Results we present here show that grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas commonly display methylation of the PTEN promoter, a finding that is absent in nontumor brain specimens and rare in de novo GBMs. Methylation of the PTEN promoter correlates with protein kinase B (PKB/Akt) phosphorylation, reflecting functional activation of the PI3K pathway. Our results also demonstrate frequent methylation of the PTEN promoter in grade III astrocytomas and secondary GBMs, consistent with the hypothesis that these tumors arise from lower grade precursors. PTEN methylation is rare in de novo GBMs and is mutually exclusive with PTEN mutations. We conclude that methylation of the PTEN promoter may represent an alternate mechanism by which PI3K signaling is increased in grade II and III gliomas as well as secondary GBMs, a finding that offers new therapeutic approaches in these patients. PMID:17504928

  1. Methylation of the PTEN promoter defines low-grade gliomas and secondary glioblastoma.

    PubMed

    Wiencke, John K; Zheng, Shichun; Jelluma, Nanette; Tihan, Tarik; Vandenberg, Scott; Tamgüney, Tanja; Baumber, Rachel; Parsons, Ramon; Lamborn, Kathleen R; Berger, Mitchel S; Wrensch, Margaret R; Haas-Kogan, Daphne Adele; Stokoe, David

    2007-07-01

    Glioblastoma multiforme (GBM) can present as either de novo or secondary tumors arising from previously diagnosed low-grade gliomas. Although these tumor types are phenotypically indistinguishable, de novo and secondary GBMs are associated with distinct genetic characteristics. PTEN mutations, which result in activation of the phosphoinositide 3-kinase (PI3K) signal transduction pathway, are frequent in de novo but not in secondary GBMs or their antecedent low-grade tumors. Results we present here show that grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas commonly display methylation of the PTEN promoter, a finding that is absent in nontumor brain specimens and rare in de novo GBMs. Methylation of the PTEN promoter correlates with protein kinase B (PKB/Akt) phosphorylation, reflecting functional activation of the PI3K pathway. Our results also demonstrate frequent methylation of the PTEN promoter in grade III astrocytomas and secondary GBMs, consistent with the hypothesis that these tumors arise from lower grade precursors. PTEN methylation is rare in de novo GBMs and is mutually exclusive with PTEN mutations. We conclude that methylation of the PTEN promoter may represent an alternate mechanism by which PI3K signaling is increased in grade II and III gliomas as well as secondary GBMs, a finding that offers new therapeutic approaches in these patients. PMID:17504928

  2. Regulation of DNA methylation using different tensions of double strands constructed in a defined DNA nanostructure.

    PubMed

    Endo, Masayuki; Katsuda, Yousuke; Hidaka, Kumi; Sugiyama, Hiroshi

    2010-02-10

    A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure. PMID:20078043

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

    PubMed

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

    2014-10-01

    Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation. PMID:25141862

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

    PubMed Central

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

    2014-01-01

    Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation. PMID:25141862

  5. Methylation profiling defines an extensive field defect in histologically normal prostate tissues associated with prostate cancer.

    PubMed

    Yang, Bing; Bhusari, Sachin; Kueck, Jessica; Weeratunga, Pushpa; Wagner, Jennifer; Leverson, Glen; Huang, Wei; Jarrard, David F

    2013-04-01

    Prostate cancer (PCa) is typically found as a multifocal disease suggesting the potential for molecular defects within the morphologically normal tissue. The frequency and spatial extent of DNA methylation changes encompassing a potential field defect are unknown. A comparison of non-tumor-associated (NTA) prostate to histologically indistinguishable tumor-associated (TA) prostate tissues detected a distinct profile of DNA methylation alterations (0.2%) using genome-wide DNA arrays based on the Encyclopedia of DNA Elements 18 sequence that tile both gene-rich and poor regions. Hypomethylation (87%) occurred more frequently than hypermethylation (13%). Several of the most significantly altered loci (CAV1, EVX1, MCF2L, and FGF1) were then used as probes to map the extent of these DNA methylation changes in normal tissues from prostates containing cancer. In TA tissues, the extent of methylation was similar both adjacent (2 mm) and at a distance (>1 cm) from tumor foci. These loci were also able to distinguish NTA from TA tissues in a validation set of patient samples. These mapping studies indicate that a spatially widespread epigenetic defect occurs in the peripheral prostate tissues of men who have PCa that may be useful in the detection of this disease. PMID:23555185

  6. Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

    PubMed Central

    Gaykalova, Daria A.; Vatapalli, Rajita; Wei, Yingying; Tsai, Hua-Ling; Wang, Hao; Zhang, Chi; Hennessey, Patrick T.; Guo, Theresa; Tan, Marietta; Li, Ryan; Ahn, Julie; Khan, Zubair; Westra, William H.; Bishop, Justin A.; Zaboli, David; Koch, Wayne M.; Khan, Tanbir; Ochs, Michael F.; Califano, Joseph A.

    2015-01-01

    Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC. PMID:26544568

  7. Histone Methylation in Nickel-Smelting Industrial Workers

    PubMed Central

    Ma, Li; Bai, Yana; Pu, Hongquan; Gou, Faxiang; Dai, Min; Wang, Hui; He, Jie; Zheng, Tongzhang; Cheng, Ning

    2015-01-01

    Background Nickel is an essential trace metal naturally found in the environment. It is also common in occupational settings, where it associates with various levels of both occupational and nonoccupational exposure In vitro studies have shown that nickel exposure can lead to intracellular accumulation of Ni2+, which has been associated with global decreases in DNA methylation, increases in chromatin condensation, reductions in H3K9me2, and elevated levels of H3K4me3. Histone modifications play an important role in modulating chromatin structure and gene expression. For example, tri-methylation of histone H3k4 has been found to be associated with transcriptional activation, and tri-methylation of H3k27 has been found to be associated with transcriptional repression. Aberrant histone modifications have been found to be associated with various human diseases, including cancer. The purpose of this work was to identify biomarkers for populations with occupational nickel exposure and to examine the relationship between histone methylation and nickel exposure. This may provide a scientific indicator of early health impairment and facilitate exploration of the molecular mechanism underlying cancer pathogenesis. Methods One hundred and forty subjects with occupational exposure to Ni and 140 referents were recruited. H3K4 and H3K27 trimethylation levels were measured in subjects’ blood cells. Results H3K4me3 levels were found to be higher in nickel smelting workers (47.24±20.85) than in office workers (22.65±8.81; P = 0.000), while the opposite was found for levels of H3K27me3(nickel smelting workers, 13.88± 4.23; office workers, 20.67± 5.96; P = 0.000). H3K4me3 was positively (r = 0.267, P = 0.001) and H3K27 was negatively (r = -0.684, P = 0.000) associated with age and length of service in smelting workers. Conclusion This study indicated that occupational exposure to Ni is associated with alterations in levels of histone modification. PMID:26474320

  8. Histone Lysine Methylation in TGF-β1 Mediated p21 Gene Expression in Rat Mesangial Cells

    PubMed Central

    Guo, Qiaoyan; Li, Xiaoxia; Han, Hongbo; Li, Chaoyuan; Liu, Shujun; Gao, Wenhui

    2016-01-01

    Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases. PMID:27247942

  9. Epigenomic Analysis of Sézary Syndrome Defines Patterns of Aberrant DNA Methylation and Identifies Diagnostic Markers.

    PubMed

    van Doorn, Remco; Slieker, Roderick C; Boonk, Stéphanie E; Zoutman, Willem H; Goeman, Jelle J; Bagot, Martine; Michel, Laurence; Tensen, Cornelis P; Willemze, Rein; Heijmans, Bas T; Vermeer, Maarten H

    2016-09-01

    Sézary syndrome (Sz) is a malignancy of skin-homing CD4(+) memory T cells that is clinically characterized by erythroderma, lymphadenopathy, and blood involvement. Distinction of Sz from erythroderma secondary to inflammatory skin diseases (erythrodermic inflammatory dermatosis [EID]) is often challenging. Recent studies identified recurrent mutations in epigenetic enzymes involved in DNA modification in Sz. Here we defined the DNA methylomes of purified CD4(+) T cells from patients with Sz, EID, and healthy control subjects. Sz showed extensive global DNA methylation alterations, with 7.8% of 473,921 interrogated autosomal CpG sites showing hypomethylation and 3.2% hypermethylation. Promoter CpG islands were markedly enriched for hypermethylation. The 126 genes with recurrent promoter hypermethylation in Sz included multiple candidate tumor suppressors that showed transcriptional repression, implicating aberrant methylation in the pathogenesis of Sz. Validation in an independent sample set showed promoter hypermethylation of CMTM2, C2orf40, G0S2, HSPB6, PROM1, and PAM in 94-100% of Sz samples but not in EID samples. Notably, promoter hypermethylation of a single gene, the chemokine-like factor CMTM2, was sufficient to accurately distinguish Sz from EID in all cases. This study shows that Sz is characterized by widespread yet distinct DNA methylation alterations, which can be used clinically as epigenetic diagnostic markers. PMID:27113428

  10. Deletions of a differentially methylated CpG island at SNRPN define a putative imprinting control region

    SciTech Connect

    Sutcliffe, J.S.,; Nakao, M.; Beaudet, A.L.

    1994-09-01

    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies, respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy, or other mutations. Four transcripts designated PAR-5, PAR-7, PAR-1 and PAR-4 were isolated and localized to a region within 300 kb telomeric to the gene encoding small nuclear ribonucleoprotein-associated polypeptide N (SNRPN). Analysis of the transcripts in cultured fibroblasts and lymphoblasts from deletion patients demonstrated that SNRPN, PAR-5 and PAR-1 are expressed exclusively from the paternal chromosome, defining an imprinted domain that spans at least 200 kb. All three imprinted transcripts were absent in cells from three PWS patients (one pair of sibs and one sporadic case) with small deletions that involve a differentially methylated CpG island containing a previously undescribed 5{prime} untranslated exon ({alpha}) of SNRPN. Methylation of the CpG island is specific for the maternal chromosome consistent with paternal expression of the imprinted domain. One deletion, which is benign when maternally transmitted, extends upstream <30 kb from the CpG island, and is associated with altered methylation centromeric to SNRPN, and loss of transcription telomeric to SNRPN, implying the presence of an imprinting control region around the CpG island containing exon {alpha}.

  11. An NF-Y-Dependent Switch of Positive and Negative Histone Methyl Marks on CCAAT Promoters

    PubMed Central

    Dolfini, Diletta; Fossati, Andrea; Ceribelli, Michele; Mantovani, Roberto

    2008-01-01

    Background Histone tails have a plethora of different post-translational modifications, which are located differently in “open” and “closed” parts of genomes. H3K4me3/H3K79me2 and H4K20me3 are among the histone marks associated with the early establishment of active and inactive chromatin, respectively. One of the most widespread promoter elements is the CCAAT box, bound by the NF-Y trimer. Two of NF-Y subunits have an H2A-H2B-like structure. Principal findings We established the causal relationship between NF-Y binding and positioning of methyl marks, by ChIP analysis of mouse and human cells infected with a dominant negative NF-YA: a parallel decrease in NF-Y binding, H3K4me3, H3K79me2 and transcription was observed in promoters that are dependent upon NF-Y. On the contrary, changes in the levels of H3K9-14ac were more subtle. Components of the H3K4 methylating MLL complex are not recruited in the absence of NF-Y. As for repressed promoters, NF-Y removal leads to a decrease in the H4K20me3 mark and deposition of H3K4me3. Conclusions Two relevant findings are reported: (i) NF-Y gains access to its genomic locations independently from the presence of methyl histone marks, either positive or negative; (ii) NF-Y binding has profound positive or negative consequences on the deposition of histone methyl marks. Therefore NF-Y is a fundamental switch at the heart of decision between gene activation and repression in CCAAT regulated genes. PMID:18446193

  12. The Challenge of Research and Extension to Define and Implement Alternatives to Methyl Bromide

    PubMed Central

    Noling, J. W.; Becker, J. O.

    1994-01-01

    Over the past 30 years, methyl bromide (MBr), a broad spectrum fumigant, has been used extensively for soilborne disease and pest control in the production of many fruit, vegetable, turf, and nursery crops. Recently, agricultural emissions of MBr were implicated as a potentially significant contributor to stratospheric ozone depletion. As a precautionary measure for global ozone protection, the U.S. Environmental Protection Agency has enforced federal legislation which mandates a complete phase-out of MBr use within the United States by 1 January 2001. Thus, new cost effective, environmentally compatible strategies for control of nematodes and other soilborne pests and pathogens must be developed and tested in a relatively short time to avoid significant losses in crop productivity. The extent to which certain agricultural industries that are now heavily reliant on MBr are affected will depend on the development of sustainable, integrated tactics to pest control, such as combinations of cultural, chemical, and biological tactics. New muhidisciplinary research and extension programs must be developed to address and overcome major constraints and incompatibilities that have prevented such tactics from being widely adopted. PMID:19279928

  13. Disruption of histone methylation in developing sperm impairs offspring health transgenerationally.

    PubMed

    Siklenka, Keith; Erkek, Serap; Godmann, Maren; Lambrot, Romain; McGraw, Serge; Lafleur, Christine; Cohen, Tamara; Xia, Jianguo; Suderman, Matthew; Hallett, Michael; Trasler, Jacquetta; Peters, Antoine H F M; Kimmins, Sarah

    2015-11-01

    A father's lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions. PMID:26449473

  14. A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes

    PubMed Central

    Varier, Radhika A; Outchkourov, Nikolay S; de Graaf, Petra; van Schaik, Frederik M A; Ensing, Henk Jan L; Wang, Fangwei; Higgins, Jonathan M G; Kops, Geert J P L; Timmers, HTh Marc

    2010-01-01

    Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho–methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle. PMID:20953165

  15. Abnormal Histone Methylation is Responsible for Increased VEGF165a Secretion from Airway Smooth Muscle Cells in Asthma

    PubMed Central

    Clifford, Rachel L.; John, Alison E.; Brightling, Christopher E.; Knox, Alan J.

    2012-01-01

    Vascular Endothelial Growth Factor (VEGF), a key angiogenic molecule, is aberrantly expressed in several diseases including asthma where it contributes to bronchial vascular remodelling and chronic inflammation. Asthmatic human airway smooth muscle (HASM) cells hypersecrete VEGF but the mechanism is unclear. Here we defined the mechanism in HASM cells from non-asthmatic (NA) and asthmatic (A) patients. We found that asthmatic cells lacked a repression complex at the VEGF promoter which was present in non-asthmatic cells. Recruitment of G9A, trimethylation of histone H3 at lysine 9 (H3K9me3) and a resultant decrease in RNA polymerase II (RNA pol II) at the VEGF promoter was critical to repression of VEGF secretion in non-asthmatic cells. At the asthmatic promoter H3K9me3 was absent due to failed recruitment of G9a; RNA pol II binding, in association with TAF1, was increased, H3K4me3 was present and Sp1 binding was exaggerated and sustained. In contrast DNA methylation and histone acetylation were similar in A and NA cells. This is the first study to show that airway cells in asthma have altered epigenetic regulation of remodelling gene(s). Histone methylation at genes such as VEGF may be an important new therapeutic target. PMID:22689881

  16. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

    PubMed

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E; Oltz, Eugene M; Jarvis, James N; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

  17. Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy

    PubMed Central

    Miller-Delaney, Suzanne F.C.; Bryan, Kenneth; Das, Sudipto; McKiernan, Ross C.; Bray, Isabella M.; Reynolds, James P.; Gwinn, Ryder; Stallings, Raymond L.

    2015-01-01

    Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain. PMID

  18. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome

    PubMed Central

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E.; Oltz, Eugene M.; Jarvis, James N.; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F.; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

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

    PubMed

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

    2014-01-01

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

  20. DNA methylation on N6-adenine in C. elegans

    PubMed Central

    Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei; Sendinc, Erdem; Liu, Jianzhao; Aristizábal-Corrales, David; Hsu, Chih-Hung; Aravind, L.; He, Chuan; Shi, Yang

    2015-01-01

    Summary In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. PMID:25936839

  1. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    SciTech Connect

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

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

    PubMed Central

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

    2015-01-01

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

  3. A Mouse Model of X-linked Intellectual Disability Associated with Impaired Removal of Histone Methylation.

    PubMed

    Iwase, Shigeki; Brookes, Emily; Agarwal, Saurabh; Badeaux, Aimee I; Ito, Hikaru; Vallianatos, Christina N; Tomassy, Giulio Srubek; Kasza, Tomas; Lin, Grace; Thompson, Andrew; Gu, Lei; Kwan, Kenneth Y; Chen, Chinfei; Sartor, Maureen A; Egan, Brian; Xu, Jun; Shi, Yang

    2016-02-01

    Mutations in a number of chromatin modifiers are associated with human neurological disorders. KDM5C, a histone H3 lysine 4 di- and tri-methyl (H3K4me2/3)-specific demethylase, is frequently mutated in X-linked intellectual disability (XLID) patients. Here, we report that disruption of the mouse Kdm5c gene recapitulates adaptive and cognitive abnormalities observed in XLID, including impaired social behavior, memory deficits, and aggression. Kdm5c-knockout brains exhibit abnormal dendritic arborization, spine anomalies, and altered transcriptomes. In neurons, Kdm5c is recruited to promoters that harbor CpG islands decorated with high levels of H3K4me3, where it fine-tunes H3K4me3 levels. Kdm5c predominantly represses these genes, which include members of key pathways that regulate the development and function of neuronal circuitries. In summary, our mouse behavioral data strongly suggest that KDM5C mutations are causal to XLID. Furthermore, our findings suggest that loss of KDM5C function may impact gene expression in multiple regulatory pathways relevant to the clinical phenotypes. PMID:26804915

  4. Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression.

    PubMed

    Mersman, Douglas P; Du, Hai-Ning; Fingerman, Ian M; South, Paul F; Briggs, Scott D

    2009-04-15

    The identification of histone methyltransferases and demethylases has uncovered a dynamic methylation system needed to modulate appropriate levels of gene expression. Gene expression levels of various histone demethylases, such as the JARID1 family, show distinct patterns of embryonic and adult expression and respond to different environmental cues, suggesting that histone demethylase protein levels must be tightly regulated for proper development. In our study, we show that the protein level of the yeast histone H3 Lys 4 (H3 K4) demethylase Jhd2/Kdm5 is modulated through polyubiquitination by the E3 ubiquitin ligase Not4 and turnover by the proteasome. We determine that polyubiquitin-mediated degradation of Jhd2 controls in vivo H3 K4 trimethylation and gene expression levels. Finally, we show that human NOT4 can polyubiquitinate human JARID1C/SMCX, a homolog of Jhd2, suggesting that this is likely a conserved mechanism. We propose that Not4 is an E3 ubiquitin ligase that monitors and controls a precise amount of Jhd2 protein so that the proper balance between histone demethylase and histone methyltransferase activities occur in the cell, ensuring appropriate levels of H3 K4 trimethylation and gene expression. PMID:19346402

  5. A mouse model of X-linked intellectual disability associated with impaired removal of histone methylation

    PubMed Central

    Iwase, Shigeki; Brookes, Emily; Agarwal, Saurabh; Badeaux, Aimee I; Ito, Hikaru; Vallianatos, Christina N; Tomassy, Giulio Srubek; Kasza, Tomas; Lin, Grace; Thompson, Andrew; Gu, Lei; Kwan, Kenneth Y.; Chen, Chinfei; Sartor, Maureen A.; Egan, Brian; Xu, Jun; Shi, Yang

    2015-01-01

    Mutations in a number of chromatin modifiers are associated with human neurological disorders. KDM5C, a histone H3 lysine 4 di- and tri-methyl (H3K4me2/3)-specific demethylase, is frequently mutated in X-linked intellectual disability (XLID) patients. Here, we report that disruption of the mouse Kdm5c gene recapitulates adaptive and cognitive abnormalities observed in XLID, including impaired social behavior and memory, and aggression. Kdm5c-knockout brains exhibit impaired dendritic arborization, spine abnormalities, and altered transcriptomes. In neurons, Kdm5c is recruited to promoters that harbor CpG islands decorated with high levels of H3K4me3, where it fine-tunes H3K4me3 levels. Kdm5c predominantly represses these genes, which include members of key pathways that regulate the development and function of neuronal circuitries. In summary, our mouse behavioral data strongly suggests that KDM5C mutations are causal to XLID. Furthermore, our findings suggest that loss of KDM5C function may impact gene expression in multiple regulatory pathways relevant to the clinical phenotypes. PMID:26804915

  6. Loss of CclA, required for histone 3 lysine 4 methylation, decreases growth but increases secondary metabolite production in Aspergillus fumigatus

    PubMed Central

    Lee, Seul; Dagenais, Taylor R.T.; Andes, David R.; Kontoyiannis, Dimitrios P.

    2013-01-01

    Secondary metabolite (SM) production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested. PMID:23638376

  7. Methylation Profiling Defines an Extensive Field Defect in Histologically Normal Prostate Tissues Associated with Prostate Cancer12

    PubMed Central

    Yang, Bing; Bhusari, Sachin; Kueck, Jessica; Weeratunga, Pushpa; Wagner, Jennifer; Leverson, Glen; Huang, Wei; Jarrard, David F

    2013-01-01

    Prostate cancer (PCa) is typically found as a multifocal disease suggesting the potential for molecular defects within the morphologically normal tissue. The frequency and spatial extent of DNA methylation changes encompassing a potential field defect are unknown. A comparison of non-tumor-associated (NTA) prostate to histologically indistinguishable tumor-associated (TA) prostate tissues detected a distinct profile of DNA methylation alterations (0.2%) using genome-wide DNA arrays based on the Encyclopedia of DNA Elements 18 sequence that tile both gene-rich and poor regions. Hypomethylation (87%) occurred more frequently than hypermethylation (13%). Several of the most significantly altered loci (CAV1, EVX1, MCF2L, and FGF1) were then used as probes to map the extent of these DNA methylation changes in normal tissues from prostates containing cancer. In TA tissues, the extent of methylation was similar both adjacent (2 mm) and at a distance (>1 cm) from tumor foci. These loci were also able to distinguish NTA from TA tissues in a validation set of patient samples. These mapping studies indicate that a spatially widespread epigenetic defect occurs in the peripheral prostate tissues of men who have PCa that may be useful in the detection of this disease. PMID:23555185

  8. The Essential WD Repeat Protein Swd2 Has Dual Functions in RNA Polymerase II Transcription Termination and Lysine 4 Methylation of Histone H3

    PubMed Central

    Cheng, Hailing; He, Xiaoyuan; Moore, Claire

    2004-01-01

    Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3′ end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled. PMID:15024081

  9. Broad Shifts in Gene Expression during Early Postnatal Life Are Associated with Shifts in Histone Methylation Patterns

    PubMed Central

    Lui, Julian C.; Chen, Weiping; Cheung, Crystal S. F.; Baron, Jeffrey

    2014-01-01

    During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration

  10. Differential DNA methylation and expression of inflammatory and zinc transporter genes defines subgroups of osteoarthritic hip patients

    PubMed Central

    Rushton, Michael D; Young, David A; Loughlin, John; Reynard, Louise N

    2015-01-01

    Objectives We have previously shown that the cartilage DNA methylome delineates two clusters of osteoarthritic (OA) hip patients, characterised by differential methylation of inflammatory genes, while others have demonstrated a link between zinc homeostasis and inflammation in OA. We aimed to investigate these effects at the methylation and gene expression level. Methods We used our previously generated methylation data while quantitative PCR was used to measure gene expression using RNA from the hip cartilage of members of both clusters and from control individuals without hip OA. Results One of the OA clusters is characterised by the promoter hypomethylation and increased expression of inflammation-associated genes including IL1A and TNF. Furthermore, we show that the increase in expression of these genes is accompanied by increased expression of several zinc transporter genes. In addition, the zinc responsive transcription factor MTF1 is also upregulated, which is accompanied by an increase in the expression of its targets the metalloproteinases MMP13 and ADAMTS5. Conclusions We have identified a subgroup of OA hip patients that are epigenetically and transcriptiomically characterised by a cartilage inflammatory phenotype with concurrent differential regulation of zinc regulators. The identification of subgroups enhances stratified phenotyping of OA patients and has important implications for future therapeutic applications. PMID:25854584

  11. Histone lysine methylation exhibits a distinct distribution during spermatogenesis in pigs.

    PubMed

    An, Junhui; Qin, Jinzhou; Wan, Yi; Zhang, Yaqing; Hu, Yuan; Zhang, Chunfang; Zeng, Wenxian

    2015-12-01

    Spermatogenesis is a continual process throughout the adult life of a male, which is governed by unique transcriptional regulation and massive alterations of chromatin. Histone modification was one of the underlying epigenetic mechanisms during spermatogenesis. It has been shown that methylation of histone lysine exhibits a distinct distribution in mice during spermatogenesis and some histone lysine methylation is essential for male fertility. However, the dynamic change of methylated histone in porcine testis tissue was largely unknown. Here, we studied the dynamic modulation of three types of methylation (monomethylation, dimethylation, and trimethylation) of H3K4, H3K27, and H4K20 during spermatogenesis in pigs. The results showed that H3K4me2/3, H3K27me3, and H4K20me1/2/3 were extensively localized in adult pig testis. Interestingly, we found that undifferentiated spermatogonia contained strongly H4K20me2 and H4K20me3, but little H4K20me1, whereas the differentiated spermatogonia possessed H4K20me1 and H4K20me2 and little H4K20me3. The findings of this study help for the understanding of epigenetic modifications during spermatogenesis in pigs and provide information for further studies. PMID:26409824

  12. DNA Methylation and Somatic Mutations Converge on the Cell Cycle and Define Similar Evolutionary Histories in Brain Tumors.

    PubMed

    Mazor, Tali; Pankov, Aleksandr; Johnson, Brett E; Hong, Chibo; Hamilton, Emily G; Bell, Robert J A; Smirnov, Ivan V; Reis, Gerald F; Phillips, Joanna J; Barnes, Michael J; Idbaih, Ahmed; Alentorn, Agusti; Kloezeman, Jenneke J; Lamfers, Martine L M; Bollen, Andrew W; Taylor, Barry S; Molinaro, Annette M; Olshen, Adam B; Chang, Susan M; Song, Jun S; Costello, Joseph F

    2015-09-14

    The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution. PMID:26373278

  13. Embryonic transcription is controlled by maternally defined chromatin state

    PubMed Central

    Hontelez, Saartje; van Kruijsbergen, Ila; Georgiou, Georgios; van Heeringen, Simon J.; Bogdanovic, Ozren; Lister, Ryan; Veenstra, Gert Jan C.

    2015-01-01

    Histone-modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3- and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300 recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. PMID:26679111

  14. Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation.

    PubMed

    Chen, Xu; Xie, Weibin; Gu, Peng; Cai, Qingqing; Wang, Bo; Xie, Yun; Dong, Wen; He, Wang; Zhong, Guangzheng; Lin, Tianxin; Huang, Jian

    2015-01-01

    WD repeat domain 5 (WDR5) plays an important role in various biological functions through the epigenetic regulation of gene transcription; however, its role in bladder cancer remains largely unknown. Our study investigated the role of WDR5 in bladder cancer and demonstrated that WDR5 was upregulated in bladder cancer tissues, and elevated WDR5 protein levels positively correlated with advanced tumor stage and poor survival. Through gain or loss of function, we demonstrated that WDR5 promoted proliferation, self-renewal and chemoresistance to cisplatin in bladder cancer cells in vitro, and tumor growth in vivo. Mechanistically, WDR5 regulated various functions in bladder cancer by mediating the transcription of cyclin B1, cyclin E1, cyclin E2, UHMK1, MCL1, BIRC3 and Nanog by histone H3 lysine 4 trimethylation. Therefore, we have discovered that WDR5 plays an important role in bladder cancer suggesting that WDR5 is a potential biomarker and a promising target in the treatment of bladder cancer. PMID:25656485

  15. Alternation of histone and DNA methylation in human atherosclerotic carotid plaques.

    PubMed

    Greißel, A; Culmes, M; Napieralski, R; Wagner, E; Gebhard, H; Schmitt, M; Zimmermann, A; Eckstein, H-H; Zernecke, A; Pelisek, J

    2015-08-01

    Little is known about epigenetics and its possible role in atherosclerosis. We here analysed histone and DNA methylation and the expression of corresponding methyltransferases in early and advanced human atherosclerotic carotid lesions in comparison to healthy carotid arteries. Western Blotting was performed on carotid plaques from our biobank with early (n=60) or advanced (n=60) stages of atherosclerosis and healthy carotid arteries (n=12) to analyse di-methylation patterns of histone H3 at positions K4, K9 and K27. In atherosclerotic lesions, di-methylation of H3K4 was unaltered and that of H3K9 and H3K27 significantly decreased compared to control arteries. Immunohistochemistry revealed an increased appearance of di-methylated H3K4 in smooth muscle cells (SMCs), a decreased expression of di-methylated H3K9 in SMCs and inflammatory cells, and reduced di-methylated H3K27 in inflammatory cells in advanced versus early atherosclerosis. Expression of corresponding histone methyltransferases MLL2 and G9a was increased in advanced versus early atherosclerosis. Genomic DNA hypomethylation, as determined by PCR for methylated LINE1 and SAT-alpha, was observed in early and advanced plaques compared to control arteries and in cell-free serum of patients with high-grade carotid stenosis compared to healthy volunteers. In contrast, no differences in DNA methylation were observed in blood cells. Expression of DNA-methyltransferase DNMT1 was reduced in atherosclerotic plaques versus controls, DNMT3A was undetectable, and DNMT3B not altered. DNA-demethylase TET1 was increased in atherosclerosisc plaques. The extent of histone and DNA methylation and expression of some corresponding methyltransferases are significantly altered in atherosclerosis, suggesting a possible contribution of epigenetics in disease development. PMID:25993995

  16. Well-defined inorganic/organic nanocomposite by nano silica core-poly(methyl methacrylate/butylacrylate/trifluoroethyl methacrylate) shell.

    PubMed

    Chang, Gang; He, Ling; Zheng, Wei; Pan, Aizhao; Liu, Jing; Li, Yingjun; Cao, Ruijun

    2013-04-15

    The novel inorganic/organic core-shell SiO2/P(MMA/BA/3FMA) nanocomposite for coating application is synthesized in this paper by seed emulsion polymerization, in which the inorganic phase is composed of nano-SiO2 modified by vinyl-trimethoxysilane (VMS) or γ-methacryloxy propyl trimethoxylsilane (MPMS), and the organic phase is made of terpolymer by 2,2,2-trifluoroethyl methacrylate (3FMA), methyl methacrylate (MMA), and n-butyl acrylate (BA). The chemical structure of SiO2/P(MMA/BA/3FMA) is characterized by FTIR. The effect of surfactant polyvinylpyrrolidone (PVP), sodium dodecyl sulfate (SDS)/octyl phenyl polyoxyethylene ether (TX-10), sodium dodecyl benzene sulfonate (SDBS)/TX-10 and sodium hexametaphosphate (SHMP) on the grafting ratio (GR) of VMS and MPMS, the dispersion of nano-SiO2 particles and the film properties of SiO2/P(MMA/BA/3FMA) are investigated by TGA, DLS, TEM, SEM, and XPS. The morphology variation and the particle size distributions of SiO2/P(MMA/BA/3FMA) with the content of surfactant and P(MMA/BA/3FMA) are characterized. It is found that MPMS is more effective than VMS in improving GR and the dispersion of nano-SiO2 particles. The surfactants are favor of gaining the higher GR in the multilayer grafted nano-SiO2, especially SDS/TX-10 for 17.6% GR. The morphology of SiO2/P(MMA/BA/3FMA) is controlled by the amount of SDS/TX-10 and P(MMA/BA/3FMA) as the core-shell particles, the stacked pomegranate seed with multicore and the multicore-single shell structure when w(MMA)/w(BA)/w(3FMA)=1.3/1/1. Among the different surfactants, SDBS/TX-10 and PVP could give the monodispersing nano-SiO2 in the terpolymer matrix of the films, but SDS/TX-10 and SDBS/TX-10 could perform the fluorine-rich surface. PMID:23403111

  17. 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

  18. Synthesis of Well-Defined Miktoarm Star Copolymer composed of Poly(3-hexylthiophene) and Poly(methyl methacrylate) via combining anionic polymerization and click reaction

    NASA Astrophysics Data System (ADS)

    Park, Jicheol; Moon, Hong Chul; Kim, Jin Kon

    2013-03-01

    We synthesized well-defined miktoarm star copolymer composed of regioregular poly(3-hexylthiophene) and poly(methyl methacrylate) ((P3HT)2- b-PMMA) by combining anionic polymerization and click reaction. First, we synthesized PMMA terminated with 1,3,5-tris(bromomethy)lbenzene (PMMA-(br)2) by anionic polymerization. Then, the bromide end groups transformed to azide group (PMMA-(N3)2) . For the synthesis (P3HT)2- b-PMMA, click reaction between ethynyl-capped P3HT and PMMA-(N3)2 was performed. The optical property and thin film morphology of (P3HT)2- b-PMMA were investigated by using UV-Vis spectra and atomic force microscopy, respectively.

  19. A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects.

    PubMed

    McClay, Joseph L; Aberg, Karolina A; Clark, Shaunna L; Nerella, Srilaxmi; Kumar, Gaurav; Xie, Lin Y; Hudson, Alexandra D; Harada, Aki; Hultman, Christina M; Magnusson, Patrik K E; Sullivan, Patrick F; Van Den Oord, Edwin J C G

    2014-03-01

    The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25-92 years (mean = 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 × 10(-8)). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10(-30)). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays. PMID:24135035

  20. Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes.

    PubMed

    Ali, Muzaffar; Daze, Kevin D; Strongin, Daniel E; Rothbart, Scott B; Rincon-Arano, Hector; Allen, Hillary F; Li, Janessa; Strahl, Brian D; Hof, Fraser; Kutateladze, Tatiana G

    2015-09-18

    Plant homeodomain (PHD) finger-containing proteins are implicated in fundamental biological processes, including transcriptional activation and repression, DNA damage repair, cell differentiation, and survival. The PHD finger functions as an epigenetic reader that binds to posttranslationally modified or unmodified histone H3 tails, recruiting catalytic writers and erasers and other components of the epigenetic machinery to chromatin. Despite the critical role of the histone-PHD interaction in normal and pathological processes, selective inhibitors of this association have not been well developed. Here we demonstrate that macrocyclic calixarenes can disrupt binding of PHD fingers to methylated lysine 4 of histone H3 in vitro and in vivo. The inhibitory activity relies on differences in binding affinities of the PHD fingers for H3K4me and the methylation state of the histone ligand, whereas the composition of the aromatic H3K4me-binding site of the PHD fingers appears to have no effect. Our approach provides a novel tool for studying the biological roles of methyllysine readers in epigenetic signaling. PMID:26229108

  1. Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation

    PubMed Central

    Ueno, Kazuko; Iwagawa, Toshiro; Kuribayashi, Hiroshi; Baba, Yukihiro; Nakauchi, Hiromitsu; Murakami, Akira; Nagasaki, Masao; Suzuki, Yutaka; Watanabe, Sumiko

    2016-01-01

    To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms. PMID:27377164

  2. Transition of differential histone H3 methylation in photoreceptors and other retinal cells during retinal differentiation.

    PubMed

    Ueno, Kazuko; Iwagawa, Toshiro; Kuribayashi, Hiroshi; Baba, Yukihiro; Nakauchi, Hiromitsu; Murakami, Akira; Nagasaki, Masao; Suzuki, Yutaka; Watanabe, Sumiko

    2016-01-01

    To analyze cell lineage-specific transitions in global transcriptional and epigenetic changes during retinogenesis, we purified retinal cells from normal mice during postnatal development into two fractions, namely, photoreceptors and other retinal cells, based on Cd73 expression, and performed RNA sequencing and ChIP sequencing of H3K27me3 and H3K4me3. Genes expressed in the photoreceptor lineage were marked with H3K4me3 in the Cd73-positive cell fraction; however, the level of H3K27me3 was very low in both Cd73-positive and -negative populations. H3K27me3 may be involved in spatio-temporal onset of a subset of bipolar-related genes. Subsets of genes expressed in amacrine and retinal ganglion cells, which are early-born retinal cell types, were suggested to be maintained in a silent state by H3K27me3 during late-stage retinogenesis. In the outer nuclear layer, upregulation of Rho and rod-related genes were observed in Ezh2-ablated retina, suggesting a role for H3K27me3 in the maintenance of proper expression levels. Taken together, our data on the transition of lineage-specific molecular signatures during development suggest that histone methylation is involved in retinal differentiation and maintenance through cell lineage-specific mechanisms. PMID:27377164

  3. Genome-wide analysis of histone methylation reveals chromatin state-based complex regulation of differential gene transcription and function of CD8 memory T cells

    PubMed Central

    Araki, Yasuto; Wang, Zhibin; Zang, Chongzhi; Wood, William H.; Schones, Dustin; Cui, Kairong; Roh, Tae-Young; Lhotsky, Brad; Wersto, Robert P.; Peng, Weiqun; Becker, Kevin G.; Zhao, Keji; Weng, Nan-ping

    2009-01-01

    Summary Memory lymphocytes are characterized by their ability to exhibit a rapid response to the recall antigen, in which differential transcription plays a significant role, yet the underlying mechanism is not understood. We report here a genome-wide analysis of histone methylation on two histone H3 lysine residues (H3K4me3 and H3K27me3) and gene expression profiles in naïve and memory CD8 T cells. We found that a general correlation exists between the levels of gene expression and the levels of H3K4me3 (positive correlation) and H3K27me3 (negative correlation) across the gene body. These correlations display four distinct modes: repressive, active, poised, and bivalent, reflecting different functions of these genes. Furthermore, a permissive chromatin state of each gene is established by a combination of different histone modifications. Our findings reveal a complex regulation by histone methylation in differential gene expression and suggest that histone methylation may be responsible for memory CD8 T cell function. PMID:19523850

  4. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  5. Chromatin states of developmentally-regulated genes revealed by DNA and histone methylation patterns in zebrafish embryos.

    PubMed

    Lindeman, Leif C; Winata, Cecilia L; Aanes, Hvard; Mathavan, Sinnakaruppan; Alestrom, Peter; Collas, Philippe

    2010-01-01

    Embryo development proceeds from a cascade of gene activation and repression events controlled by epigenetic modifications of DNA and histones. Little is known about epigenetic states in the developing zebrafish, despite its importance as a model organism. We report here DNA methylation and histone modification profiles of promoters of developmentally-regulated genes (pou5f1, sox2, sox3, klf4, nnr, otx1b, nes, vasa), as well as tert and bactin2, in zebrafish embryos at the mid-late blastula transition, shortly after embryonic genome activation. We identify four classes of promoters based on the following profiles: (i) those enriched in marks of active genes (H3K9ac, H4ac, H3K4me3) without transcriptionally repressing H3K9me3 or H3K27me3; (ii) those enriched in H3K9ac, H4ac and H3K27me3, without H3K9me3; one such gene was klf4, shown by in situ hybridization to be mosaically expressed, likely accounting for the detection of both activating and repressive marks on its promoter; (iii) those enriched in H3K4me3 and H3K27me3 without acetylation; and (iv) those enriched in all histone modifications examined. Culture of embryo-derived cells under differentiation conditions leads to H3K9 and H4 deacetylation and H3K9 and H3K27 trimethylation on genes that are inactivated, yielding an epigenetic profile similar to those of fibroblasts or muscle. All promoters however retain H3K4me3, indicating an uncoupling of H3K4me3 occupancy and gene expression. All non-CpG island developmentally-regulated promoters are DNA unmethylated in embryos, but hypermethylated in fibroblasts. Our results suggest that differentially expressed embryonic genes are regulated by various patterns of histone modifications on unmethylated DNA, which create a developmentally permissive chromatin state. PMID:20336603

  6. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements.

    PubMed

    Prior, Sara; Miousse, Isabelle R; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R; Allen, Antiño R; Raber, Jacob; Tackett, Alan J; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2016-10-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2'-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5'-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

  7. Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger

    SciTech Connect

    Wang, Gang G.; Song, Jikui; Wang, Zhanxin; Dormann, Holger L.; Casadio, Fabio; Li, Haitao; Luo, Jun-Li; Patel, Dinshaw J.; Allis, C. David

    2009-07-21

    Histone H3 lysine4 methylation (H3K4me) has been proposed as a critical component in regulating gene expression, epigenetic states, and cellular identities. The biological meaning of H3K4me is interpreted by conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states. The dysregulation of PHD fingers has been implicated in several human diseases, including cancers and immune or neurological disorders. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the carboxy-terminal PHD finger of PHF23 or JARID1A (also known as KDM5A or RBBP2), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukaemias, generated potent oncoproteins that arrested haematopoietic differentiation and induced acute myeloid leukaemia in murine models. In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukaemogenesis. Mutations in PHD fingers that abrogated H3K4me3 binding also abolished leukaemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1 and Pbx1), and enforced their active gene transcription in murine haematopoietic stem/progenitor cells. Mechanistically, NUP98-PHD fusions act as 'chromatin boundary factors', dominating over polycomb-mediated gene silencing to 'lock' developmentally critical loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukaemia stem cells. Collectively, our studies represent, to our knowledge, the first report that deregulation of the PHD finger, an 'effector' of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during mammalian development.

  8. Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger

    PubMed Central

    Wang, Gang G.; Song, Jikui; Wang, Zhanxin; Dormann, Holger L.; Casadio, Fabio; Li, Haitao; Luo, Jun-Li; Patel, Dinshaw J.; Allis, C. David

    2009-01-01

    Histone H3 Lys4 methylation (H3K4me) was proposed as a critical component in regulating the gene expression, epigenetic states, and cellular identities1. The biological meaning of H3K4me is interpreted via conserved modules including plant homeodomain (PHD) fingers that recognize varied H3K4me states1,2. The dysregulation of PHD finger has been implicated in a variety of human diseases including cancers and immune or neurological disorders3. Here we report that fusing an H3K4-trimethylation (H3K4me3)-binding PHD finger, such as the C-terminal PHD finger of JARID1A or PHF23 (JARID1APHD3, PHF23PHD), to a common fusion partner nucleoporin-98 (NUP98) as identified in human leukemias4,5, generated potent oncoproteins that arrested hematopoietic differentiation and induced acute myeloid leukemia (AML). In these processes, a PHD finger that specifically recognizes H3K4me3/2 marks was essential for leukemogenesis. Mutations in PHD fingers that abrogated H3K4me3-binding also abolished leukemic transformation. NUP98-PHD fusion prevented the differentiation-associated removal of H3K4me3 at many loci encoding lineage-specific transcription factors (Hox(s), Gata3, Meis1, Eya1, Pbx1), and enforced their active gene transcription. Mechanistically, NUP98-PHD fusions act as ‘chromatin boundary factors’, dominating over polycomb-mediated gene silencing to ‘lock’ developmentally crucial loci into an active chromatin state (H3K4me3 with induced histone acetylation), a state that defined leukemia stem cells. Collectively, our studies represent the first report wherein the deregulation of PHD finger, ‘effector’ of specific histone modification, perturbs the epigenetic dynamics on developmentally critical loci, catastrophizes cellular fate decision-making, and even causes oncogenesis during development. PMID:19430464

  9. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing

    PubMed Central

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L.

    2016-01-01

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from −938 to −337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34+ selected hematopoietic stem and progenitor cells. PMID:27570841

  10. SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    PubMed Central

    Li, Shaofang; Liu, Lin; Li, Shengben; Gao, Lei; Zhao, Yuanyuan; Kim, Yun Ju; Chen, Xuemei

    2016-01-01

    Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3–9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3–9 family that has previously been associated with silencing through H3K9 methylation. PMID:26400170

  11. SUVH1, a Su(var)3-9 family member, promotes the expression of genes targeted by DNA methylation.

    PubMed

    Li, Shaofang; Liu, Lin; Li, Shengben; Gao, Lei; Zhao, Yuanyuan; Kim, Yun Ju; Chen, Xuemei

    2016-01-29

    Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. Here, an anti-silencing factor was identified using a forward genetic screen on a reporter line that harbors a LUCIFERASE (LUC) gene driven by a promoter that undergoes DNA methylation. SUVH1, a Su(var)3-9 homolog, was identified as a factor promoting the expression of the LUC gene. Treatment with a cytosine methylation inhibitor completely suppressed the LUC expression defects of suvh1, indicating that SUVH1 is dispensable for LUC expression in the absence of DNA methylation. SUVH1 also promotes the expression of several endogenous genes with promoter DNA methylation. However, the suvh1 mutation did not alter DNA methylation levels at the LUC transgene or on a genome-wide scale; thus, SUVH1 functions downstream of DNA methylation. Histone H3 lysine 4 (H3K4) trimethylation was reduced in suvh1; in contrast, H3K9 methylation levels remained unchanged. This work has uncovered a novel, anti-silencing function for a member of the Su(var)3-9 family that has previously been associated with silencing through H3K9 methylation. PMID:26400170

  12. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer.

    PubMed

    Kim, Tae Woo; Lee, Seon-Jin; Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-26

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells. PMID:26675260

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

    PubMed Central

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

    2014-01-01

    We evaluated whether epigenetic changes contributed to improve ethanol tolerance in mutant populations of Saccharomyces cerevisiae (S. cerevisiae). Two ethanol-tolerant variants of S. cerevisiae were used to evaluate the genetic stability in the process of stress-free passage cultures. We found that acquired ethanol tolerance was lost and transcription level of some genes (HSP104, PRO1, TPS1, and SOD1) closely related to ethanol tolerance decreased significantly after the 10th passage in ethanol-free medium. Tri-methylation of lysine 4 on histone H3 (H3K4) enhanced at the promoter of HSP104, PRO1, TPS1 and SOD1 in ethanol-tolerant variants of S. cerevisiae was also diminished after tenth passage in stress-free cultures. The ethanol tolerance was reacquired when exogenous SOD1 transferred in some tolerance-lost strains. This showed that H3K4 methylation is involved in phenotypic variation with regard to ethanol tolerance with respect to classic breeding methods used in yeast. PMID:24779776

  14. Epigenetic modification of TLR4 promotes activation of NF-κB by regulating methyl-CpG-binding domain protein 2 and Sp1 in gastric cancer

    PubMed Central

    Oh, Byung Moo; Lee, Heesoo; Uhm, Tae Gi; Min, Jeong-Ki; Park, Young-Jun; Yoon, Suk Ran; Kim, Bo-Yeon; Kim, Jong Wan; Choe, Yong-Kyung; Lee, Hee Gu

    2016-01-01

    Toll-like receptor 4 (TLR4) is important in promoting the immune response in various cancers. Recently, TLR4 is highly expressed in a stage-dependent manner in gastric cancer, but the regulatory mechanism of TLR4 expression has been not elucidated it. Here, we investigated the mechanism underlying regulation of TLR4 expression through promoter methylation and histone modification between transcriptional regulation and silencing of the TLR4 gene in gastric cancer cells. Chromatin immunoprecipitation was carried out to screen for factors related to TLR4 methylation such as MeCP2, HDAC1, and Sp1 on the TLR4 promoter. Moreover, DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC) induced demethylation of the TLR4 promoter and increased H3K4 trimethylation and Sp1 binding to reactivate silenced TLR4. In contrast, although the silence of TLR4 activated H3K9 trimethylation and MeCP2 complex, combined treatment with TLR4 agonist and 5-aza-dC upregulated H3K4 trimethylation and activated with transcription factors as Sp1 and NF-κB. This study demonstrates that recruitment of the MeCP2/HDAC1 repressor complex increases the low levels of TLR4 expression through epigenetic modification of DNA and histones on the TLR4 promoter, but Sp1 activates TLR4 high expression by hypomethylation and NF-κB signaling in gastric cancer cells. PMID:26675260

  15. Coupling of histone methylation and RNA processing by the nuclear mRNA cap-binding complex.

    PubMed

    Li, Zicong; Jiang, Danhua; Fu, Xing; Luo, Xiao; Liu, Renyi; He, Yuehui

    2016-01-01

    In eukaryotes, genes are transcribed into pre-mRNAs that are subsequently processed into mature mRNAs by adding a 5'-cap and a 3'-polyA tail and splicing introns. Pre-mRNA processing involves their binding proteins and processing factors, whereas gene transcription often involves chromatin modifiers. It has been unclear how the factors involved in chromatin modifications and RNA processing function in concert to control mRNA production. Here, we show that in Arabidopsis thaliana, the evolutionarily conserved nuclear mRNA cap-binding complex (CBC) forms multi-protein complexes with a conserved histone 3 lysine 4 (H3K4) methyltransferase complex called COMPASS-like and a histone 3 lysine 36 (H3K36) methyltransferase to integrate active histone methylations with co-transcriptional mRNA processing and cap preservation, leading to a high level of mature mRNA production. We further show that CBC is required for H3K4 and H3K36 trimethylation, and the histone methyltransferases are required for CBC-mediated mRNA cap preservation and efficient pre-mRNA splicing at their target loci, suggesting that these factors are functionally interdependent. Our study reveals novel roles for histone methyltransferases in RNA-processing-related events and provides mechanistic insights into how the 'downstream' RNA CBC controls eukaryotic gene transcription. PMID:27249350

  16. Disclosing the crosstalk among DNA methylation, transcription factors, and histone marks in human pluripotent cells through discovery of DNA methylation motifs

    PubMed Central

    Luu, Phuc-Loi; Schöler, Hans R.; Araúzo-Bravo, Marcos J.

    2013-01-01

    Gene expression regulation is gated by promoter methylation states modulating transcription factor binding. The known DNA methylation/unmethylation mechanisms are sequence unspecific, but different cells with the same genome have different methylomes. Thus, additional processes bringing specificity to the methylation/unmethylation mechanisms are required. Searching for such processes, we demonstrated that CpG methylation states are influenced by the sequence context surrounding the CpGs. We used such a property to develop a CpG methylation motif discovery algorithm. The newly discovered motifs reveal “methylation/unmethylation factors” that could recruit the “methylation/unmethylation machinery” to the loci specified by the motifs. Our methylation motif discovery algorithm provides a synergistic approach to the differently methylated region algorithms. Since our algorithm searches for commonly methylated regions inside the same sample, it requires only a single sample to operate. The motifs that were found discriminate between hypomethylated and hypermethylated regions. The hypomethylation-associated motifs have a high CG content, their targets appear in conserved regions near transcription start sites, they tend to co-occur within transcription factor binding sites, they are involved in breaking the H3K4me3/H3K27me3 bivalent balance, and they transit the enhancers from repressive H3K27me3 to active H3K27ac during ES cell differentiation. The new methylation motifs characterize the pluripotent state shared between ES and iPS cells. Additionally, we found a collection of motifs associated with the somatic memory inherited by the iPS from the initial fibroblast cells, thus revealing the existence of epigenetic somatic memory on a fine methylation scale. PMID:24149073

  17. Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

    PubMed Central

    Lepikhov, Konstantin; Zakhartchenko, Valeri; Hao, Ru; Yang, Feikun; Wrenzycki, Christine; Niemann, Heiner; Wolf, Eckhard; Walter, Joern

    2008-01-01

    Background In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit. Results When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer. Conclusion Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species. PMID:19014417

  18. DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination

    PubMed Central

    Zamudio, Natasha; Barau, Joan; Teissandier, Aurélie; Walter, Marius; Borsos, Maté; Servant, Nicolas; Bourc'his, Déborah

    2015-01-01

    DNA methylation is essential for protecting the mammalian germline against transposons. When DNA methylation-based transposon control is defective, meiotic chromosome pairing is consistently impaired during spermatogenesis: How and why meiosis is vulnerable to transposon activity is unknown. Using two DNA methylation-deficient backgrounds, the Dnmt3L and Miwi2 mutant mice, we reveal that DNA methylation is largely dispensable for silencing transposons before meiosis onset. After this, it becomes crucial to back up to a developmentally programmed H3K9me2 loss. Massive retrotransposition does not occur following transposon derepression, but the meiotic chromatin landscape is profoundly affected. Indeed, H3K4me3 marks gained over transcriptionally active transposons correlate with formation of SPO11-dependent double-strand breaks and recruitment of the DMC1 repair enzyme in Dnmt3L−/− meiotic cells, whereas these features are normally exclusive to meiotic recombination hot spots. Here, we demonstrate that DNA methylation restrains transposons from adopting chromatin characteristics amenable to meiotic recombination, which we propose prevents the occurrence of erratic chromosomal events. PMID:26109049

  19. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure

    PubMed Central

    Chater-Diehl, Eric J.; Castellani, Christina A.; Alberry, Bonnie L.; Singh, Shiva M.

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse’s lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as “Free radical scavenging”. We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was “Peroxisome biogenesis”; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD. PMID:27136348

  20. Activin/Nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark

    PubMed Central

    Bertero, Alessandro; Madrigal, Pedro; Galli, Antonella; Hubner, Nina C.; Moreno, Inmaculada; Burks, Deborah; Brown, Stephanie; Pedersen, Roger A.; Gaffney, Daniel; Mendjan, Sasha; Pauklin, Siim

    2015-01-01

    Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin–SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs. PMID:25805847

  1. Activin/nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark.

    PubMed

    Bertero, Alessandro; Madrigal, Pedro; Galli, Antonella; Hubner, Nina C; Moreno, Inmaculada; Burks, Deborah; Brown, Stephanie; Pedersen, Roger A; Gaffney, Daniel; Mendjan, Sasha; Pauklin, Siim; Vallier, Ludovic

    2015-04-01

    Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin-SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs. PMID:25805847

  2. Human-specific histone methylation signatures at transcription start sites in prefrontal neurons.

    PubMed

    Shulha, Hennady P; Crisci, Jessica L; Reshetov, Denis; Tushir, Jogender S; Cheung, Iris; Bharadwaj, Rahul; Chou, Hsin-Jung; Houston, Isaac B; Peter, Cyril J; Mitchell, Amanda C; Yao, Wei-Dong; Myers, Richard H; Chen, Jiang-Fan; Preuss, Todd M; Rogaev, Evgeny I; Jensen, Jeffrey D; Weng, Zhiping; Akbarian, Schahram

    2012-01-01

    Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5-1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans. PMID:23185133

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

    SciTech Connect

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

    2010-07-19

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

  4. Biological function and regulation of histone and non-histone lysine methylation in response to DNA damage.

    PubMed

    Chen, Yongcan; Zhu, Wei-Guo

    2016-07-01

    DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources. Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways. Post-translational modifications of histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP-ribosyl)ation, acetylation, and methylation are all involved in the spatial-temporal regulation of DDR, among which phosphorylation and ubiquitylation are well studied. Studies in the past decade also revealed extensive roles of lysine methylation in response to DNA damage. Lysine methylation is finely regulated by plenty of lysine methyltransferases, lysine demethylases, and can be recognized by proteins with chromodomain, plant homeodomain, Tudor domain, malignant brain tumor domain, or proline-tryptophan-tryptophan-proline domain. In this review, we outline the dynamics and regulation of histone lysine methylation at canonical (H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20) and non-canonical sites after DNA damage, and discuss their context-specific functions in DDR protein recruitment or extraction, chromatin environment establishment, and transcriptional regulation. We also present the emerging advances of lysine methylation in non-histone proteins during DDR. PMID:27217472

  5. 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

  6. Effects of Wutou Decoction on DNA Methylation and Histone Modifications in Rats with Collagen-Induced Arthritis.

    PubMed

    Liu, Ya-Fei; Wen, Cai-Yu-Zhu; Chen, Zhe; Wang, Yu; Huang, Ying; Hu, Yong-Hong; Tu, Sheng-Hao

    2016-01-01

    Background. Wutou decoction (WTD) has been wildly applied in the treatment of rheumatoid arthritis and experimental arthritis in rats for many years. Epigenetic deregulation is associated with the aetiology of rheumatoid arthritis; however, the effects of WTD on epigenetic changes are unclear. This study is set to explore the effects of WTD on DNA methylation and histone modifications in rats with collagen-induced arthritis (CIA). Methods. The CIA model was established by the stimulation of collagen and adjuvant. The knee synovium was stained with hematoxylin and eosin. The DNA methyltransferase 1 (DNMT1) and methylated CpG binding domain 2 (MBD2) expression of peripheral blood mononuclear cells (PBMCs) were determined by Real-Time PCR. The global DNA histone H3-K4/H3-K27 methylation and total histones H3 and H4 acetylation of PBMCs were detected. Results. Our data demonstrated that the DNMT1 mRNA expression was significantly lowered in group WTD compared to that in group CIA (P < 0.05). The DNA methylation level was significantly reduced in group WTD compared to that in group CIA (P < 0.05). Moreover, H3 acetylation of PBMCs was overexpressed in WTD compared with CIA (P < 0.05). Conclusions. WTD may modulate DNA methylation and histone modifications, functioning as anti-inflammatory potential. PMID:27042192

  7. Comparative methylome analysis in solid tumors reveals aberrant methylation at chromosome 6p in nasopharyngeal carcinoma

    PubMed Central

    Dai, Wei; Cheung, Arthur Kwok Leung; Ko, Josephine Mun Yee; Cheng, Yue; Zheng, Hong; Ngan, Roger Kai Cheong; Ng, Wai Tong; Lee, Anne Wing Mui; Yau, Chun Chung; Lee, Victor Ho Fu; Lung, Maria Li

    2015-01-01

    Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10−9), but was less obvious in other types of solid tumors except for prostate and Epstein–Barr virus (EBV)-positive gastric cancer (FDR<10−3). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection. PMID:25924914

  8. Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation and POMC gene expression in β-endorphin-producing POMC neurons of the hypothalamus

    PubMed Central

    Bekdash, Rola A.; Zhang, Changqing; Sarkar, Dipak K.

    2013-01-01

    Background Prenatal exposure to ethanol reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, since these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of ethanol on these neurons. Methods Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes, and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunoabsorbent assay. Results Prenatal ethanol treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10) increased the repressive marks (H3K9me2, G9a, Setdb1) and DNA methylating enzyme (Dnmt1) and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and β-EP, and elevated corticosterone response to LPS. Gestational choline normalized the ethanol-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1 and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, β-EP production and the corticosterone

  9. Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species.

    PubMed

    Braszewska-Zalewska, Agnieszka; Dziurlikowska, Alina; Maluszynska, Jolanta

    2012-01-01

    Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific. PMID:22195975

  10. HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis1[W][OA

    PubMed Central

    Liu, Xuncheng; Yu, Chun-Wei; Duan, Jun; Luo, Ming; Wang, Koching; Tian, Gang; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status. PMID:21994348

  11. Silver nanoparticle-induced hemoglobin decrease involves alteration of histone 3 methylation status.

    PubMed

    Qian, Yi; Zhang, Jie; Hu, Qinglin; Xu, Ming; Chen, Yue; Hu, Guoqing; Zhao, Meirong; Liu, Sijin

    2015-11-01

    Silver nanoparticles (nanosilver, AgNPs) have been shown to induce toxicity in vitro and in vivo; however, the molecular bases underlying the detrimental effects have not been thoroughly understood. Although there are numerous studies on its genotoxicity, only a few studies have investigated the epigenetic changes, even less on the changes of histone modifications by AgNPs. In the current study, we probed the AgNP-induced alterations to histone methylation that could be responsible for globin reduction in erythroid cells. AgNP treatment caused a significant reduction of global methylation level for histone 3 (H3) in erythroid MEL cells at sublethal concentrations, devoid of oxidative stress. The ChIP-PCR analyses demonstrated that methylation of H3 at lysine (Lys) 4 (H3K4) and Lys 79 (H3K79) on the β-globin locus was greatly reduced. The reduction in methylation could be attributed to decreased histone methyltransferase DOT-1L and MLL levels as well as the direct binding between AgNPs to H3/H4 that provide steric hindrance to prevent methylation as predicted by the all-atom molecular dynamics simulations. This direct interaction was further proved by AgNP-mediated pull-down assay and immunoprecipitation assay. These changes, together with decreased RNA polymerase II activity and chromatin binding at this locus, resulted in decreased hemoglobin production. By contrast, Ag ion-treated cells showed no alterations in histone methylation level. Taken together, these results showed a novel finding in which AgNPs could alter the methylation status of histone. Our study therefore opens a new avenue to study the biological effects of AgNPs at sublethal concentrations from the perspective of epigenetic mechanisms. PMID:26295435

  12. Nucleosome competition reveals processive acetylation by the SAGA HAT module.

    PubMed

    Ringel, Alison E; Cieniewicz, Anne M; Taverna, Sean D; Wolberger, Cynthia

    2015-10-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications. PMID:26401015

  13. Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus

    PubMed Central

    Wilson, Gareth A.; Rakyan, Vardhman K.; Teschendorff, Andrew E.; Akan, Pelin; Stupka, Elia; Down, Thomas A.; Prokopenko, Inga; Morison, Ian M.; Mill, Jonathan; Pidsley, Ruth; Deloukas, Panos; Frayling, Timothy M.; Hattersley, Andrew T.; McCarthy, Mark I.; Beck, Stephan; Hitman, Graham A.

    2010-01-01

    Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases. PMID:21124985

  14. Modulation of histone methylation and MLH1 gene silencing by hexavalent chromium

    SciTech Connect

    Sun Hong; Zhou Xue; Chen Haobin; Li Qin; Costa, Max

    2009-06-15

    Hexavalent chromium [Cr(VI)] is a mutagen and carcinogen, and occupational exposure can lead to lung cancers and other adverse health effects. Genetic changes resulting from DNA damage have been proposed as an important mechanism that mediates chromate's carcinogenicity. Here we show that chromate exposure of human lung A549 cells increased global levels of di- and tri-methylated histone H3 lysine 9 (H3K9) and lysine 4 (H3K4) but decreased the levels of tri-methylated histone H3 lysine 27 (H3K27) and di-methylated histone H3 arginine 2 (H3R2). Most interestingly, H3K9 dimethylation was enriched in the human MLH1 gene promoter following chromate exposure and this was correlated with decreased MLH1 mRNA expression. Chromate exposure increased the protein as well as mRNA levels of G9a a histone methyltransferase that specifically methylates H3K9. This Cr(VI)-induced increase in G9a may account for the global elevation of H3K9 dimethylation. Furthermore, supplementation with ascorbate, the primary reductant of Cr(VI) and also an essential cofactor for the histone demethylase activity, partially reversed the H3K9 dimethylation induced by chromate. Thus our studies suggest that Cr(VI) may target histone methyltransferases and demethylases, which in turn affect both global and gene promoter specific histone methylation, leading to the silencing of specific tumor suppressor genes such as MLH1.

  15. Polycomb repressive complex 2 epigenomic signature defines age-associated hypermethylation and gene expression changes

    PubMed Central

    Dozmorov, Mikhail G

    2015-01-01

    Although age-associated gene expression and methylation changes have been reported throughout the literature, the unifying epigenomic principles of aging remain poorly understood. Recent explosion in availability and resolution of functional/regulatory genome annotation data (epigenomic data), such as that provided by the ENCODE and Roadmap Epigenomics projects, provides an opportunity for the identification of epigenomic mechanisms potentially altered by age-associated differentially methylated regions (aDMRs) and regulatory signatures in the promoters of age-associated genes (aGENs). In this study we found that aDMRs and aGENs identified in multiple independent studies share a common Polycomb Repressive Complex 2 signature marked by EZH2, SUZ12, CTCF binding sites, repressive H3K27me3, and activating H3K4me1 histone modification marks, and a “poised promoter” chromatin state. This signature is depleted in RNA Polymerase II-associated transcription factor binding sites, activating H3K79me2, H3K36me3, H3K27ac marks, and an “active promoter” chromatin state. The PRC2 signature was shown to be generally stable across cell types. When considering the directionality of methylation changes, we found the PRC2 signature to be associated with aDMRs hypermethylated with age, while hypomethylated aDMRs were associated with enhancers. In contrast, aGENs were associated with the PRC2 signature independently of the directionality of gene expression changes. In this study we demonstrate that the PRC2 signature is the common epigenomic context of genomic regions associated with hypermethylation and gene expression changes in aging. PMID:25880792

  16. Substrate-Induced Transcriptional Activation of the MoCel7C Cellulase Gene Is Associated with Methylation of Histone H3 at Lysine 4 in the Rice Blast Fungus Magnaporthe oryzae

    PubMed Central

    Vu, Ba Van; Pham, Kieu Thi Minh

    2013-01-01

    The mechanisms involved in substrate-dependent regulation of a Magnaporthe oryzae gene encoding a cellulase which we designate MoCel7C (MGG_14954) were investigated. The levels of MoCel7C transcript were dramatically increased more than 1,000-fold, 16 to 24 h after transfer to a medium containing 2% carboxymethylcellulose (CMC), while levels were very low or undetectable in conventional rich medium. Green fluorescent protein reporter assays showed that the MoCel7C promoter was activated by cello-oligosaccharides larger than a pentamer. CMC-induced activation of the MoCel7C promoter was suppressed by glucose and cellobiose. Chromatin immunoprecipitation assays revealed that histone H3 methylation on lysine 4 (H3K4) at the MoCel7C locus was associated with activation of the gene by CMC. Consistently, CMC-induced MoCel7C gene activation was drastically diminished in a knockout (KO) mutant of the MoSET1 gene, which encodes a histone lysine methyltransferase that catalyzes H3K4 methylation in M. oryzae. Interestingly, however, MoCel7C transcript levels under noninducing conditions were significantly increased in the MoSET1 KO mutant, suggesting that MoSET1 directly or indirectly plays a role in both activation and suppression of the MoCel7C gene in response to environmental signals. In addition, gene expression and silencing vectors using the MoCel7C promoter were constructed. PMID:23995923

  17. Defining "Development".

    PubMed

    Pradeu, Thomas; Laplane, Lucie; Prévot, Karine; Hoquet, Thierry; Reynaud, Valentine; Fusco, Giuseppe; Minelli, Alessandro; Orgogozo, Virginie; Vervoort, Michel

    2016-01-01

    Is it possible, and in the first place is it even desirable, to define what "development" means and to determine the scope of the field called "developmental biology"? Though these questions appeared crucial for the founders of "developmental biology" in the 1950s, there seems to be no consensus today about the need to address them. Here, in a combined biological, philosophical, and historical approach, we ask whether it is possible and useful to define biological development, and, if such a definition is indeed possible and useful, which definition(s) can be considered as the most satisfactory. PMID:26969977

  18. Defining Infertility

    MedlinePlus

    ... of the American Society for Reproductive Medicine Defining infertility What is infertility? Infertility is “the inability to conceive after 12 months ... to conceive after 6 months is generally considered infertility. How common is it? Infertility affects 10%-15% ...

  19. Defining Risk.

    ERIC Educational Resources Information Center

    Tholkes, Ben F.

    1998-01-01

    Defines camping risks and lists types and examples: (1) objective risk beyond control; (2) calculated risk based on personal choice; (3) perceived risk; and (4) reckless risk. Describes campers to watch ("immortals" and abdicators), and several "treatments" of risk: avoidance, safety procedures and well-trained staff, adequate insurance, and a…

  20. Stress-induced gene expression and behavior are controlled by DNA methylation and methyl donor availability in the dentate gyrus.

    PubMed

    Saunderson, Emily A; Spiers, Helen; Mifsud, Karen R; Gutierrez-Mecinas, Maria; Trollope, Alexandra F; Shaikh, Abeera; Mill, Jonathan; Reul, Johannes M H M

    2016-04-26

    Stressful events evoke long-term changes in behavioral responses; however, the underlying mechanisms in the brain are not well understood. Previous work has shown that epigenetic changes and immediate-early gene (IEG) induction in stress-activated dentate gyrus (DG) granule neurons play a crucial role in these behavioral responses. Here, we show that an acute stressful challenge [i.e., forced swimming (FS)] results in DNA demethylation at specific CpG (5'-cytosine-phosphate-guanine-3') sites close to the c-Fos (FBJ murine osteosarcoma viral oncogene homolog) transcriptional start site and within the gene promoter region of Egr-1 (early growth response protein 1) specifically in the DG. Administration of the (endogenous) methyl donor S-adenosyl methionine (SAM) did not affect CpG methylation and IEG gene expression at baseline. However, administration of SAM before the FS challenge resulted in an enhanced CpG methylation at the IEG loci and suppression of IEG induction specifically in the DG and an impaired behavioral immobility response 24 h later. The stressor also specifically increased the expression of the de novo DNA methyltransferase Dnmt3a [DNA (cytosine-5-)-methyltransferase 3 alpha] in this hippocampus region. Moreover, stress resulted in an increased association of Dnmt3a enzyme with the affected CpG loci within the IEG genes. No effects of SAM were observed on stress-evoked histone modifications, including H3S10p-K14ac (histone H3, phosphorylated serine 10 and acetylated lysine-14), H3K4me3 (histone H3, trimethylated lysine-4), H3K9me3 (histone H3, trimethylated lysine-9), and H3K27me3 (histone H3, trimethylated lysine-27). We conclude that the DNA methylation status of IEGs plays a crucial role in FS-induced IEG induction in DG granule neurons and associated behavioral responses. In addition, the concentration of available methyl donor, possibly in conjunction with Dnmt3a, is critical for the responsiveness of dentate neurons to environmental stimuli in

  1. Defining cure.

    PubMed

    Hilton, Paul; Robinson, Dudley

    2011-06-01

    This paper is a summary of the presentations made as Proposal 2-"Defining cure" to the 2nd Annual meeting of the ICI-Research Society, in Bristol, 16th June 2010. It reviews definitions of 'cure' and 'outcome', and considers the impact that varying definition may have on prevalence studies and cure rates. The difference between subjective and objective outcomes is considered, and the significance that these different outcomes may have for different stakeholders (e.g. clinicians, patients, carers, industry etc.) is discussed. The development of patient reported outcome measures and patient defined goals is reviewed, and consideration given to the use of composite end-points. A series of proposals are made by authors and discussants as to how currently validated outcomes should be applied, and where our future research activity in this area might be directed. PMID:21661023

  2. DNA Methylation and Colorectal Cancer

    PubMed Central

    Ashktorab, Hassan; Brim, Hassan

    2014-01-01

    Colorectal cancer (CRC) is one of the major cancers in the world and second death-causing cancer in the US. CRC development involves genetic and epigenetic alterations. Changes in DNA methylation status are believed to be involved at different stages of CRC. Promoter silencing via DNA methylation and hypomethylation of oncogenes alter genes’ expression, and can be used as a tool for the early detection of colonic lesions. DNA methylation use as diagnostic and prognostic marker has been described for many cancers including CRC. CpG Islands Methylator Phenotype (CIMP) is one of the underlying CRC mechanisms. This review aims to define methylation signatures in CRC. The analysis of DNA methylation profile in combination with the pathological diagnosis would be useful in predicting CRC tumors’ evolution and their prognostic behavior. PMID:25580099

  3. Methyl Iodide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methyl iodide (MeI, iodomethane, CH3I) was reported as a potential alternative to the stratospheric ozone-depleting fumigant methyl bromide (MeBr) in the mid-1990s (Sims et al., 1995; Ohr et al., 1996). It has since received significant research attention to determine its environmental fate and tran...

  4. Defining chaos

    SciTech Connect

    Hunt, Brian R.; Ott, Edward

    2015-09-15

    In this paper, we propose, discuss, and illustrate a computationally feasible definition of chaos which can be applied very generally to situations that are commonly encountered, including attractors, repellers, and non-periodically forced systems. This definition is based on an entropy-like quantity, which we call “expansion entropy,” and we define chaos as occurring when this quantity is positive. We relate and compare expansion entropy to the well-known concept of topological entropy to which it is equivalent under appropriate conditions. We also present example illustrations, discuss computational implementations, and point out issues arising from attempts at giving definitions of chaos that are not entropy-based.

  5. Methyl chloroform

    SciTech Connect

    Wray, T.K.

    1994-04-01

    Methyl chloroform is identified as a Class 1 ozone-depleting substance under Title VI of the CAA Amendments. On Nov. 30, 1993, EPA ordered the phaseout of Class 1 ozone-depleting substances -- chlorofluorocarbons (CFCs), halons, carbon tetrachloride and methyl chloroform -- by Jan. 1, 1996. Methyl chloroform and other Class 1 substances may be used after the dead-line if sources can be found through recycling or existing inventories. Methyl chloroform is listed as a hazardous air pollutant under CAA. It also is a SARA Title III, Sec. 313 compound with a reportable quantity of 1,000 pounds. OSHA and the American Conference of Government Industrial Hygienists have set 350 ppm as the time-weighted average airborne exposure level for methyl chloroform. NIOSH lists its immediately dangerous to life or health'' concentration as 1,000 parts per million. DOT identifies the substance as a hazardous material, Class 6.1 (poison).

  6. A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer.

    PubMed

    Kumegawa, Kohei; Maruyama, Reo; Yamamoto, Eiichiro; Ashida, Masami; Kitajima, Hiroshi; Tsuyada, Akihiro; Niinuma, Takeshi; Kai, Masahiro; Yamano, Hiro-O; Sugai, Tamotsu; Tokino, Takashi; Shinomura, Yasuhisa; Imai, Kohzoh; Suzuki, Hiromu

    2016-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA. PMID:27215978

  7. A genomic screen for long noncoding RNA genes epigenetically silenced by aberrant DNA methylation in colorectal cancer

    PubMed Central

    Kumegawa, Kohei; Maruyama, Reo; Yamamoto, Eiichiro; Ashida, Masami; Kitajima, Hiroshi; Tsuyada, Akihiro; Niinuma, Takeshi; Kai, Masahiro; Yamano, Hiro-o; Sugai, Tamotsu; Tokino, Takashi; Shinomura, Yasuhisa; Imai, Kohzoh; Suzuki, Hiromu

    2016-01-01

    Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA. PMID:27215978

  8. Protection of CpG islands against de novo DNA methylation during oogenesis is associated with the recognition site of E2f1 and E2f2

    PubMed Central

    2014-01-01

    Background Epigenetic reprogramming during early mammalian embryonic and germ cell development is a genome-wide process. CpG islands (CGIs), central to the regulation of mammalian gene expression, are exceptional in terms of whether, when and how they are affected by epigenetic reprogramming. Results We investigated the DNA sequences of CGIs in the context of genome-wide data on DNA methylation and transcription during oogenesis and early embryogenesis to identify signals associated with methylation establishment and protection from de novo methylation in oocytes and associated with post-fertilisation methylation maintenance. We find no evidence for a characteristic DNA sequence motif in oocyte-methylated CGIs. Neither do we find evidence for a general role of regular CpG spacing in methylation establishment at CGIs in oocytes. In contrast, the resistance of most CGIs to de novo methylation during oogenesis is associated with the motif CGCGC, the recognition site of E2f1 and E2f2, transcription factors highly expressed specifically in oocytes. This association is independent of prominent known hypomethylation-associated factors: CGI promoter activity, H3K4me3, Cfp1 binding or R-loop formation potential. Conclusions Our results support a DNA sequence-independent and transcription-driven model of de novo CGI methylation during oogenesis. In contrast, our results for CGIs that remain unmethylated are consistent with a model of protection from methylation involving sequence recognition by DNA-binding proteins, E2f1 and E2f2 being probable candidates. PMID:25478011

  9. DNA Methylation in the Exon 1 Region and Complex Regulation of Twist1 Expression in Gastric Cancer Cells

    PubMed Central

    Sakamoto, Ayuna; Akiyama, Yoshimitsu; Shimada, Shu; Zhu, Wei-Guo; Yuasa, Yasuhito; Tanaka, Shinji

    2015-01-01

    Twist1 overexpression is frequently observed in various cancers including gastric cancer (GC). Although DNA methylation of the Twist1 gene has been reported in cancer cells, the mechanisms underlying transcriptional activation remain uncertain. In this study, we first examined epigenetic alterations of the Twist1 using Twist1 transcription-positive and -negative cell lines that are derived from our established diffuse-type GC mouse model. Treatment with a DNA demethylation agent 5-aza-dC re-activated Twist1 expression in Twist1 expression-negative GC cells. According to methylation-specific PCR and bisulfite sequencing analysis, methylation at the CpG-rich region within Twist1 coding exon 1, rather than its promoter region, was tightly linked to transcriptional silencing of the Twist1 expression in mouse GC cells. Chromatin immunoprecipitation assays revealed that active histone mark H3K4me3 was enriched in Twist1 expression-positive cells, and inactive histone mark H3K9me3 was enriched in Twist1 expression-negative cells. The expression levels of Suv39h1 and Suv39h2, histone methyltransferases for H3K9me3, were inversely correlated with Twist1 expression, and knockdown of Suv39h1 or Suv39h2 induced Twist1 expression. Moreover, Sp1 transcription factor bound to the exon 1 CpG-rich region in Twist1 expression-positive cell lines, and Twist1 expression was diminished by mithramycin, which that interferes with Sp1 binding to CpG-rich regulatory sequences. Our studies suggested that the Twist1 transcription in GC cells might be regulated through potential cooperation of DNA methylation, histone modification in complex with Sp1 binding to CpG-rich regions within the exon 1 region. PMID:26695186

  10. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  11. Structural Basis for Lower Lysine Methylation State-Specific Readout by MBT Repeats of L3MBTL1 and an Engineered PHD Finger

    SciTech Connect

    Li, Haitao; Fischle, Wolfgang; Wang, Wooikoon; Duncan, Elizabeth M.; Liang, Lena; Murakami-Ishibe, Satoko; Allis, C. David; Patel, Dinshaw J.

    2008-09-17

    Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the 'cavity insertion' (L3MBTL1) and 'surface groove' (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.

  12. Diversity of two forms of DNA methylation in the brain

    PubMed Central

    Chen, Yuanyuan; Damayanti, Nur P.; Irudayaraj, Joseph; Dunn, Kenneth; Zhou, Feng C.

    2014-01-01

    DNA methylation 5-methylcytosine (5mC) predicts a compacting chromatin inaccessible to transcription. The discovery of 5-hydroxymethylcytosine (5hmC), which is derived from 5mC, adds a new dimension to the mechanism and role of DNA methylation in epigenetics. Genomic evidence indicates that the 5hmC is located in the alternate regions to 5mC. However, the nature of 5hmC, as compared with classical 5mC remains unclear. Observing the mouse brain through embryonic development to the adult, first, we found that 5hmC is not merely an intermediate metabolite of demethylation, but is long lasting, chromatically distinct, and dynamically changing during neurodevelopment. Second, we found that 5hmC distinctly differs from 5mC in its chromatin affiliation during neural stem cell (NSC) development. Thirdly, we found both 5mC and 5hmC to be uniquely polarized and dynamic through the NSC development. 5mC was found to progressively polarize with MBD1 and MeCP2, and recruits H3K9me3 and H3K27me3; while 5hmC progressively co-localizes with MBD3 and recruits H3K4me2. Critical differential binding of 5mC with MBD1, and 5hmC with MBD3 was validated by Resonance Energy Transfer technique FLIM-FRET. This transition and polarization coincides with neuroprogenitor differentiation. Finally, at the time of synaptogenesis, 5mC gradually accumulates in the heterochromatin while 5hmC accumulates in the euchromatin, which is consistent with the co-localization of 5hmC with PolII, which mediates RNA transcription. Our data indicate that 5mC and 5hmC are diverse in their functional interactions with chromatin. This diversity is likely to contribute to the versatile epigenetic control of transcription mediating brain development and functional maintenance of adult brain. PMID:24653733

  13. TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

    PubMed

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R; Kantarjian, Hagop M; Liang, Shoudan; Estecio, Marcos R; Godley, Lucy A; Issa, Jean-Pierre J

    2015-07-15

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites. PMID:25972343

  14. Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma.

    PubMed

    Asmar, Fazila; Punj, Vasu; Christensen, Jesper; Pedersen, Marianne T; Pedersen, Anja; Nielsen, Anders B; Hother, Christoffer; Ralfkiaer, Ulrik; Brown, Peter; Ralfkiaer, Elisabeth; Helin, Kristian; Grønbæk, Kirsten

    2013-12-01

    The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10(-30)). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells. PMID:23831920

  15. Methyl chloride

    Integrated Risk Information System (IRIS)

    Methyl chloride ; CASRN 74 - 87 - 3 ( 07 / 17 / 2001 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for

  16. Methyl acrylate

    Integrated Risk Information System (IRIS)

    Methyl acrylate ; CASRN 96 - 33 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  17. Methyl isocyanate

    Integrated Risk Information System (IRIS)

    Methyl isocyanate ; CASRN 624 - 83 - 9 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  18. Methyl iodide

    Integrated Risk Information System (IRIS)

    Methyl iodide ; CASRN 74 - 88 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effe

  19. Methyl parathion

    Integrated Risk Information System (IRIS)

    Methyl parathion ; CASRN 298 - 00 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  20. Methyl chlorocarbonate

    Integrated Risk Information System (IRIS)

    Methyl chlorocarbonate ; CASRN 79 - 22 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  1. Methyl methacrylate

    Integrated Risk Information System (IRIS)

    Methyl methacrylate ; CASRN 80 - 62 - 6 ( 03 / 02 / 98 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments f

  2. Monocyte enhancers are highly altered in systemic lupus erythematosus

    PubMed Central

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

    2015-01-01

    Objective: Histone modifications set transcriptional competency and can perpetuate pathologic expression patterns. We defined systemic lupus erythematosus (SLE)-specific changes in H3K4me3 and K3K27me3, histone marks of gene activation and repression, respectively. Methods: We used ChIP-seq to define histone modifications in monocytes from SLE patients and controls. Results: Both promoters and enhancers exhibited significant changes in histone methylation in SLE. Regions with differential H3K4me3 in SLE were significantly enriched in potential interferon-related transcription factor binding sites and pioneer transcription factor sites. Conclusion: Enhancer activation defines the character of the cell and our data support extensive disease effects in monocytes, a particularly plastic lineage. Type I interferons not only drive altered gene expression but may also alter the character of the cell through chromatin modifications. PMID:26442457

  3. Altered DNA methylation in PAH deficient phenylketonuria.

    PubMed

    Dobrowolski, Steven F; Lyons-Weiler, James; Spridik, Kayla; Biery, Amy; Breck, Jane; Vockley, Jerry; Yatsenko, Svetlana; Sultana, Tamanna

    2015-01-01

    While phenylalanine (PHE) is the toxic insult in phenylketonuria (PKU), mechanisms underlying PHE toxicity remain ill-defined. Altered DNA methylation in response to toxic exposures is well-recognized. DNA methylation patterns were assessed in blood and brain from PKU patients to determine if PHE toxicity impacts methylation. Methylome assessment, utilizing methylated DNA immunoprecipitation and paired-end sequencing, was performed in DNA obtained from brain tissue of classical PKU patients, leukocytes from poorly controlled PKU patients, leukocytes from well controlled PKU patients, and appropriate control tissues. In PKU brain tissue, expression analysis determined the impact of methylation on gene function. Differential methylation was observed in brain tissue of PKU patients and expression studies identified downstream impact on gene expression. Altered patterns of methylation were observed in leukocytes of well controlled and poorly controlled patients with more extensive methylation in patients with high PHE exposure. Differential methylation of noncoding RNA genes was extensive in patients with high PHE exposure but minimal in well controlled patients. Methylome repatterning leading to altered gene expression was present in brain tissue of PKU patients, suggesting a role in neuropathology. Aberrant methylation is observed in leukocytes of PKU patients and is influenced by PHE exposure. DNA methylation may provide a biomarker relating to historic PHE exposure. PMID:25990862

  4. From the laboratory to the field: assaying histone methylation at FLOWERING LOCUS C in naturally growing Arabidopsis halleri.

    PubMed

    Nishio, Haruki; Buzas, Diana Mihaela; Nagano, Atsushi J; Suzuki, Yutaka; Sugano, Sumio; Ito, Motomi; Morinaga, Shin-Ichi; Kudoh, Hiroshi

    2016-07-20

    Gene regulatory mechanisms are often defined in studies performed in the laboratory but are seldom validated for natural habitat conditions, i.e., in natura. Vernalization, the promotion of flowering by winter cold, is a prominent naturally occurring phenomenon, so far best characterized using artificial warm and cold treatments. The floral inhibitor FLOWERING LOCUS C (FLC) gene of Arabidopsis thaliana has been identified as the central regulator of vernalization. FLC shows an idiosyncratic pattern of histone modification at different stages of cold exposure, believed to regulate transcriptional responses of FLC. Chromatin modifications, including H3K4me3 and H3K27me3, are routinely quantified using chromatin immunoprecipitation (ChIP), standardized for laboratory samples. In this report, we modified a ChIP protocol to make it suitable for analysis of field samples. We first validated candidate normalization control genes at two stages of cold exposure in the laboratory and two seasons in the field, also taking into account nucleosome density. We further describe experimental conditions for performing sampling and sample preservation in the field and demonstrate that these conditions give robust results, comparable with those from laboratory samples. The ChIP protocol incorporating these modifications, "Field ChIP", was used to initiate in natura chromatin analysis of AhgFLC, an FLC orthologue in A. halleri, of which a natural population is already under investigation. Here, we report results on levels of H3K4me3 and H3K27me3 at three representative regions of AhgFLC in controlled cold and field samples, before and during cold exposure. We directly compared the results in the field with those from laboratory samples. These data revealed largely similar trends in histone modification dynamics between laboratory and field samples at AhgFLC, but also identified some possible differences. The Field ChIP method described here will facilitate comprehensive chromatin

  5. Methyl gallate.

    PubMed

    Bebout, Deborah; Pagola, Silvina

    2009-01-01

    THE CRYSTAL STRUCTURE OF THE TITLE COMPOUND (SYSTEMATIC NAME: methyl 3,4,5-trihydroxy-benzoate), C(8)H(8)O(5), is composed of essentially planar mol-ecules [maximum departures from the mean carbon and oxygen skeleton plane of 0.0348 (10) Å]. The H atoms of the three hydroxyl groups, which function as hydrogen-bond donors and acceptors simultaneously, are oriented in the same direction around the aromatic ring. In addition to two intra-molecular hydrogen bonds, each mol-ecule is hydrogen bonded to six others, creating a three-dimensional hydrogen-bonded network. PMID:21581923

  6. Methyl gallate

    PubMed Central

    Bebout, Deborah; Pagola, Silvina

    2009-01-01

    The crystal structure of the title compound (systematic name: methyl 3,4,5-trihydroxy­benzoate), C8H8O5, is composed of essentially planar mol­ecules [maximum departures from the mean carbon and oxygen skeleton plane of 0.0348 (10) Å]. The H atoms of the three hydroxyl groups, which function as hydrogen-bond donors and acceptors simultaneously, are oriented in the same direction around the aromatic ring. In addition to two intra­molecular hydrogen bonds, each mol­ecule is hydrogen bonded to six others, creating a three-dimensional hydrogen-bonded network. PMID:21581923

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

    PubMed

    Pedersen, Marianne Terndrup; Kooistra, Susanne Marije; Radzisheuskaya, Aliaksandra; Laugesen, Anne; Johansen, Jens Vilstrup; Hayward, Daniel Geoffrey; Nilsson, Jakob; Agger, Karl; Helin, Kristian

    2016-07-15

    Chromatin-associated proteins are essential for the specification and maintenance of cell identity. They exert these functions through modulating and maintaining transcriptional patterns. To elucidate the functions of the Jmjd2 family of H3K9/H3K36 histone demethylases, we generated conditional Jmjd2a/Kdm4a, Jmjd2b/Kdm4b and Jmjd2c/Kdm4c/Gasc1 single, double and triple knockout mouse embryonic stem cells (ESCs). We report that while individual Jmjd2 family members are dispensable for ESC maintenance and embryogenesis, combined deficiency for specifically Jmjd2a and Jmjd2c leads to early embryonic lethality and impaired ESC self-renewal, with spontaneous differentiation towards primitive endoderm under permissive culture conditions. We further show that Jmjd2a and Jmjd2c both localize to H3K4me3-positive promoters, where they have widespread and redundant roles in preventing accumulation of H3K9me3 and H3K36me3. Jmjd2 catalytic activity is required for ESC maintenance, and increased H3K9me3 levels in knockout ESCs compromise the expression of several Jmjd2a/c targets, including genes that are important for ESC self-renewal. Thus, continual removal of H3K9 promoter methylation by Jmjd2 demethylases represents a novel mechanism ensuring transcriptional competence and stability of the pluripotent cell identity. PMID:27266524

  8. Wp specific methylation of highly proliferated LCLs

    SciTech Connect

    Park, Jung-Hoon; Jeon, Jae-Pil; Shim, Sung-Mi; Nam, Hye-Young; Kim, Joon-Woo; Han, Bok-Ghee; Lee, Suman . E-mail: suman@cha.ac.kr

    2007-06-29

    The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes.

  9. A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex*

    PubMed Central

    Patel, Anamika; Vought, Valarie E.; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S.

    2011-01-01

    Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex. PMID:21106533

  10. A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex.

    PubMed

    Patel, Anamika; Vought, Valarie E; Dharmarajan, Venkatasubramanian; Cosgrove, Michael S

    2011-02-01

    Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex. PMID:21106533

  11. Neuronal Kmt2a/Mll1 histone methyltransferase is essential for prefrontal synaptic plasticity and working memory.

    PubMed

    Jakovcevski, Mira; Ruan, Hongyu; Shen, Erica Y; Dincer, Aslihan; Javidfar, Behnam; Ma, Qi; Peter, Cyril J; Cheung, Iris; Mitchell, Amanda C; Jiang, Yan; Lin, Cong L; Pothula, Venu; Stewart, A Francis; Ernst, Patricia; Yao, Wei-Dong; Akbarian, Schahram

    2015-04-01

    Neuronal histone H3-lysine 4 methylation landscapes are defined by sharp peaks at gene promoters and other cis-regulatory sequences, but molecular and cellular phenotypes after neuron-specific deletion of H3K4 methyl-regulators remain largely unexplored. We report that neuronal ablation of the H3K4-specific methyltransferase, Kmt2a/Mixed-lineage leukemia 1 (Mll1), in mouse postnatal forebrain and adult prefrontal cortex (PFC) is associated with increased anxiety and robust cognitive deficits without locomotor dysfunction. In contrast, only mild behavioral phenotypes were observed after ablation of the Mll1 ortholog Kmt2b/Mll2 in PFC. Impaired working memory after Kmt2a/Mll1 ablation in PFC neurons was associated with loss of training-induced transient waves of Arc immediate early gene expression critical for synaptic plasticity. Medial prefrontal layer V pyramidal neurons, a major output relay of the cortex, demonstrated severely impaired synaptic facilitation and temporal summation, two forms of short-term plasticity essential for working memory. Chromatin immunoprecipitation followed by deep sequencing in Mll1-deficient cortical neurons revealed downregulated expression and loss of the transcriptional mark, trimethyl-H3K4, at <50 loci, including the homeodomain transcription factor Meis2. Small RNA-mediated Meis2 knockdown in PFC was associated with working memory defects similar to those elicited by Mll1 deletion. Therefore, mature prefrontal neurons critically depend on maintenance of Mll1-regulated H3K4 methylation at a subset of genes with an essential role in cognition and emotion. PMID:25834037

  12. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors

    PubMed Central

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  13. Physical Interactions and Functional Coordination between the Core Subunits of Set1/Mll Complexes and the Reprogramming Factors.

    PubMed

    Yang, Zhenhua; Augustin, Jonathan; Hu, Jing; Jiang, Hao

    2015-01-01

    Differentiated cells can be reprogrammed to the pluripotent state by overexpression of defined factors, and this process is profoundly influenced by epigenetic mechanisms including dynamic histone modifications. Changes in H3K4 methylation have been shown to be the predominant activating response in the early stage of cellular reprogramming. Mechanisms underlying such epigenetic priming, however, are not well understood. Here we show that the expression of the reprogramming factors (Yamanaka factors, Oct4, Sox2, Klf4 and Myc), especially Myc, directly promotes the expression of certain core subunits of the Set1/Mll family of H3K4 methyltransferase complexes. A dynamic recruitment of the Set1/Mll complexes largely, though not sufficiently in its own, explains the dynamics of the H3K4 methylation during cellular reprogramming. We then demonstrate that the core subunits of the Set1/Mll complexes physically interact with mainly Sox2 and Myc among the Yamanaka factors. We further show that Sox2 directly binds the Ash2l subunit in the Set1/Mll complexes and this binding is mediated by the HMG domain of Sox2. Functionally, we show that the Set1/Mll complex core subunits are required for efficient cellular reprogramming. We also show that Dpy30, one of the core subunits in the complexes, is required for the efficient target binding of the reprogramming factors. Interestingly, such requirement is not necessarily dependent on locus-specific H3K4 methylation. Our work provides a better understanding of how the reprogramming factors physically interact and functionally coordinate with a key group of epigenetic modulators to mediate transitions of the chromatin state involved in cellular reprogramming. PMID:26691508

  14. Viroid-induced DNA methylation in plants.

    PubMed

    Dalakouras, Athanasios; Dadami, Elena; Wassenegger, Michael

    2013-12-01

    In eukaryotes, DNA methylation refers to the addition of a methyl group to the fifth atom in the six-atom ring of cytosine residues. At least in plants, DNA regions that become de novo methylated can be defined by homologous RNA molecules in a process termed RNA-directed DNA methylation (RdDM). RdDM was first discovered in viroid-infected plants. Viroids are pathogenic circular, non-coding, single-stranded RNA molecules. Members of the Pospiviroidae family replicate in the nucleus through double-stranded RNA intermediates, attracting the host RNA silencing machinery. The recruitment of this machinery results in the production of viroid-derived small RNAs (vd-sRNAs) that mediate RNA degradation and DNA methylation of cognate sequences. Here, we provide an overview of the cumulative data on the field of viroid-induced RdDM and discuss three possible scenarios concerning the mechanistic details of its establishment. PMID:25436756

  15. Sunflower oil methyl ester as diesel fuel

    SciTech Connect

    Hassett, D.J.; Hasan, R.A.

    1982-01-01

    Methyl ester formation represents one approach to overcome the problems associated with the relatively high viscosity of sunflower oil when used as a diesel fuel replacement. Sunflower oil methyl ester is being prepared at the University of North Dakota Engieering Experiment Station. Physical and chemical properties of this material at varying levels of refinement and purity will be used to define fuel properties. Engine testing is being carried out to determine if the fouling characteristics of methyl ester are significantly less than those of sunflower oil. 1 figure, 1 table.

  16. Evidence for Regulation of ECM3 Expression by Methylation of Histone H3 Lysine 4 and Intergenic Transcription in Saccharomyces cerevisiae

    PubMed Central

    Raupach, Elizabeth A.; Martens, Joseph A.; Arndt, Karen M.

    2016-01-01

    Transcription of nonprotein-coding DNA is widespread in eukaryotes and plays important regulatory roles for many genes, including genes that are misregulated in cancer cells. Its pervasiveness presents the potential for a wealth of diverse regulatory roles for noncoding transcription. We previously showed that the act of transcribing noncoding DNA (ncDNA) across the promoter of the protein-coding SER3 gene in Saccharomyces cerevisiae positions nucleosomes over the upstream activating sequences, leading to strong repression of SER3 transcription. To explore the possibility of other regulatory roles for ncDNA transcription, we selected six candidate S. cerevisiae genes that express ncRNAs over their promoters and analyzed the regulation of one of these genes, ECM3, in detail. Because noncoding transcription can lead to changes in the local chromatin landscape that impinge on the expression of nearby coding genes, we surveyed the effects of various chromatin regulators on the expression of ECM3. These analyses identified roles for the Paf1 complex in positively regulating ECM3 transcription through methylation of histone H3 at lysine 4 (K4) and for Paf1 in controlling the pattern of intergenic transcription at this locus. By deleting a putative promoter for the noncoding transcription unit that lies upstream of ECM3, we provide evidence for a positive correlation between intergenic transcription and ECM3 expression. Our results are consistent with a model in which cotranscriptional methylation of histone H3 K4, mediated by the Paf1 complex and noncoding transcription, leads to activation of ECM3 transcription. PMID:27449519

  17. Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells

    SciTech Connect

    Vrba, Lukas; Jensen, Taylor J.; Garbe, James C.; Heimark, Ronald L.; Cress, Anne E.; Dickinson, Sally; Stampfer, Martha R.; Futscher, Bernard W.

    2009-12-23

    BACKGROUND: The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood. METHODOLOGY/ PRINCIPAL FINDINGS: Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141-negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-2'-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control. CONCLUSIONS/ SIGNIFICANCE: We report that DNA methylation plays a role in the normal cell type-specific expression of miR-200c and miR-141 and this role appears evolutionarily conserved, since similar results were obtained in mouse. Aberrant DNA methylation of the

  18. Gene methylation in gastric cancer.

    PubMed

    Qu, Yiping; Dang, Siwen; Hou, Peng

    2013-09-23

    Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide. Over 70% of new cases and deaths occur in developing countries. In the early years of the molecular biology revolution, cancer research mainly focuses on genetic alterations, including gastric cancer. Epigenetic mechanisms are essential for normal development and maintenance of tissue-specific gene expression patterns in mammals. Disruption of epigenetic processes can lead to altered gene function and malignant cellular transformation. Recent advancements in the rapidly evolving field of cancer epigenetics have shown extensive reprogramming of every component of the epigenetic machinery in cancer, including DNA methylation, histone modifications, nucleosome positioning, noncoding RNAs, and microRNAs. Aberrant DNA methylation in the promoter regions of gene, which leads to inactivation of tumor suppressor and other cancer-related genes in cancer cells, is the most well-defined epigenetic hallmark in gastric cancer. The advantages of gene methylation as a target for detection and diagnosis of cancer in biopsy specimens and non-invasive body fluids such as serum and gastric washes have led to many studies of application in gastric cancer. This review focuses on the most common and important phenomenon of epigenetics, DNA methylation, in gastric cancer and illustrates the impact epigenetics has had on this field. PMID:23669186

  19. DNA methylation in endometriosis (Review)

    PubMed Central

    KOUKOURA, OURANIA; SIFAKIS, STAVROS; SPANDIDOS, DEMETRIOS A.

    2016-01-01

    Endometriosis is defined by the presence and growth of functional endometrial tissue, outside the uterine cavity, primarily in the ovaries, pelvic peritoneum and rectovaginal septum. Although it is a benign disease, it presents with malignant characteristics, such as invasion to surrounding tissues, metastasis to distant locations and recurrence following treatment. Accumulating evidence suggests that various epigenetic aberrations may play an essential role in the pathogenesis of endometriosis. Aberrant DNA methylation represents a possible mechanism repsonsible for this disease, linking gene expression alterations observed in endometriosis with hormonal and environmental factors. Several lines of evidence indicate that endometriosis may partially be due to selective epigenetic deregulations influenced by extrinsic factors. Previous studies have shed light into the epigenetic component of endometriosis, reporting variations in the epigenetic patterns of genes known to be involved in the aberrant hormonal, immunologic and inflammatory status of endometriosis. Although recent studies, utilizing advanced molecular techniques, have allowed us to further elucidate the possible association of DNA methylation with altered gene expression, whether these molecular changes represent the cause or merely the consequence of the disease is a question which remains to be answered. This review provides an overview of the current literature on the role of DNA methylation in the pathophysiology and malignant evolution of endometriosis. We also provide insight into the mechanisms through which DNA methylation-modifying agents may be the next step in the research of the pharmaceutical treatment of endometriosis. PMID:26934855

  20. DNA methylation in endometriosis (Review).

    PubMed

    Koukoura, Ourania; Sifakis, Stavros; Spandidos, Demetrios A

    2016-04-01

    Endometriosis is defined by the presence and growth of functional endometrial tissue, outside the uterine cavity, primarily in the ovaries, pelvic peritoneum and rectovaginal septum. Although it is a benign disease, it presents with malignant characteristics, such as invasion to surrounding tissues, metastasis to distant locations and recurrence following treatment. Accumulating evidence suggests that various epigenetic aberrations may play an essential role in the pathogenesis of endometriosis. Aberrant DNA methylation represents a possible mechanism repsonsible for this disease, linking gene expression alterations observed in endometriosis with hormonal and environmental factors. Several lines of evidence indicate that endometriosis may partially be due to selective epigenetic deregulations influenced by extrinsic factors. Previous studies have shed light into the epigenetic component of endometriosis, reporting variations in the epigenetic patterns of genes known to be involved in the aberrant hormonal, immunologic and inflammatory status of endometriosis. Although recent studies, utilizing advanced molecular techniques, have allowed us to further elucidate the possible association of DNA methylation with altered gene expression, whether these molecular changes represent the cause or merely the consequence of the disease is a question which remains to be answered. This review provides an overview of the current literature on the role of DNA methylation in the pathophysiology and malignant evolution of endometriosis. We also provide insight into the mechanisms through which DNA methylation-modifying agents may be the next step in the research of the pharmaceutical treatment of endometriosis. PMID:26934855

  1. Methyl salicylate overdose

    MedlinePlus

    Methyl salicylate (oil of wintergreen) is a chemical that smells like wintergreen. It is used in many over- ... muscle ache creams. It is related to aspirin. Methyl salicylate overdose occurs when someone swallows a dangerous amount ...

  2. Techniques in protein methylation.

    PubMed

    Lee, Jaeho; Cheng, Donghang; Bedford, Mark T

    2004-01-01

    Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated. PMID:15173617

  3. Competitive Binding of a Benzimidazole to the Histone-Binding Pocket of the Pygo PHD Finger

    PubMed Central

    2014-01-01

    The Pygo-BCL9 complex is a chromatin reader, facilitating β-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo’s PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein–protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD–HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo–BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis. PMID:25323450

  4. 40 CFR 180.451 - Tribenuron methyl; tolerances for residues.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... residues of the herbicide tribenuron methyl and its metabolites and degradates in or on the commodities in.... Tolerances with regional registration, as defined in § 180.1(l) are established for residues of the...

  5. 40 CFR 180.451 - Tribenuron methyl; tolerances for residues.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... residues of the herbicide tribenuron methyl and its metabolites and degradates in or on the commodities in.... Tolerances with regional registration, as defined in § 180.1(l) are established for residues of the...

  6. 40 CFR 180.451 - Tribenuron methyl; tolerances for residues.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... residues of the herbicide tribenuron methyl and its metabolites and degradates in or on the commodities in.... Tolerances with regional registration, as defined in § 180.1(l) are established for residues of the...

  7. Defining Overweight and Obesity

    MedlinePlus

    ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs Defining Adult Overweight and ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs File Formats Help: How ...

  8. Interpolation and Definability

    NASA Astrophysics Data System (ADS)

    Gabbay, Dov M.; Maksimova, Larisa L.

    This chapter is on interpolation and definability. This notion is not only central in pure logic, but has significant meaning and applicability in all areas where logic itself is applied, especially in computer science, artificial intelligence, logic programming, philosophy of science and natural language. The notion may sometimes appear to the reader as too technical/mathematical but it does also have a general meaning in terms of expressibility and definability.

  9. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    PubMed

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  10. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  11. Cytoplasmic protein methylation is essential for neural crest migration

    PubMed Central

    Vermillion, Katie L.; Lidberg, Kevin A.

    2014-01-01

    As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration. PMID:24379414

  12. Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2.

    PubMed

    Vaidya, Himani; Rumph, Candie; Katula, Karen S

    2016-01-01

    WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a

  13. Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2

    PubMed Central

    Vaidya, Himani; Rumph, Candie; Katula, Karen S.

    2016-01-01

    WNT5A is a secreted ligand involved in Wnt pathway signaling and has a role in cell movement and differentiation. Altered WNT5A expression is associated with various cancers, although in most studies the focus has been on only one of the known WNT5A isoforms. In this study, we analyzed expression from two of the major WNT5A promoters, termed promoter A and promoter B, in normal human osteoblasts, SaOS-2 and U2OS osteosarcoma cell lines, and osteosarcoma tumor tissue. We found that both promoters A and B are active in normal osteoblasts with nearly 11-fold more promoter B than A transcripts. Promoter B but not promoter A transcripts are decreased or nearly undetectable in the SaOS-2 and U2OS cell lines and osteosarcoma tumor tissues. Transient transfection of promoter A and promoter B reporter constructs confirmed that SaOS-2 cells have the necessary factors to transcribe both promoters. Bisulfite sequencing analysis revealed that three CpG enriched regions upstream of the promoter B exon 1βare highly methylated in both SaOS-2 and U2OS cells. The CpG island sub-region R6 located in promoter B exon 1β was approximately 51% methylated in SaOS-2 and 25% methylated in U2OS. Region 3 was approximately 28% methylated in normal osteoblasts, whereas the others were unmethylated. Promoter B was re-activated by treatment of SaOS-2 cells with 1 μM 5-azacytidine, which was associated with only a small insignificant change in methylation of sub-region R6. ChIP analysis of U2OS and SaOS-2 cells indicated that the promoter B region is less enriched in the active histone mark H3K4me3, in comparison to promoter A and that there is increased enrichment of the repressive mark H3K27me3 in association with the promoter B genomic region in the cell line SaOS-2. These findings show that epigenetic inactivation of the WNT5A promoter B involves both DNA methylation and histone modifications and suggest that differential expression of the WNT5A alternative promoters A and B is a

  14. Defining Airflow Obstruction

    PubMed Central

    Eschenbacher, William L.

    2016-01-01

    Airflow obstruction has been defined using spirometric test results when the forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio is below a fixed cutoff (<70%) or lower limits of normal (LLN) from reference equations that are based on values from a normal population. However, similar to other positive or abnormal diagnostic test results that are used to identify the presence of disease, perhaps airflow obstruction should be defined based on the values of FEV1/FVC for a population of individuals with known disease such as chronic obstructive pulmonary disease (COPD). Unfortunately, we do not know such a distribution of values of FEV1/FVC for patients with COPD since there is no gold standard for this syndrome or condition. Yet, we have used this physiologic definition of airflow obstruction based on a normal population to identify patients with COPD. In addition, we have defined airflow obstruction as either being present or absent. Instead, we should use a different approach to define airflow obstruction based on the probability or likelihood that the airflow obstruction is present which in turn would give us the probability or likelihood of a disease state such as COPD. PMID:27239557

  15. Defining Equality in Education

    ERIC Educational Resources Information Center

    Benson, Ronald E.

    1977-01-01

    Defines equality of education in three areas: 1) by the degree of integration of school systems; 2) by a comparison of material resources and assets in education; and 3) by the effects of schooling as measured by the mean scores of groups on standardized tests. Available from: College of Education, 107 Quadrangle, Iowa State University, Ames, Iowa…

  16. Defining Supports Geometry

    ERIC Educational Resources Information Center

    Stephan, Michelle L.; McManus, George E.; Dickey, Ashley L.; Arb, Maxwell S.

    2012-01-01

    The process of developing definitions is underemphasized in most mathematics instruction. Investing time in constructing meaning is well worth the return in terms of the knowledge it imparts. In this article, the authors present a third approach to "defining," called "constructive." It involves modifying students' previous understanding of a term…

  17. On Defining Mass

    ERIC Educational Resources Information Center

    Hecht, Eugene

    2011-01-01

    Though central to any pedagogical development of physics, the concept of mass is still not well understood. Properly defining mass has proven to be far more daunting than contemporary textbooks would have us believe. And yet today the origin of mass is one of the most aggressively pursued areas of research in all of physics. Much of the excitement…

  18. Defining Effective Teaching

    ERIC Educational Resources Information Center

    Layne, L.

    2012-01-01

    The author looks at the meaning of specific terminology commonly used in student surveys: "effective teaching." The research seeks to determine if there is a difference in how "effective teaching" is defined by those taking student surveys and those interpreting the results. To investigate this difference, a sample group of professors and students…

  19. Defining structural limit zones

    NASA Technical Reports Server (NTRS)

    Merchant, D. H.

    1978-01-01

    Method for defining limit loads uses probability distribution of largest load occurring during given time intervals. Method is compatible with both deterministic and probabilistic structural design criteria. It also rationally accounts for fact that longer structure is exposed to random loading environment, greater is possibility that it will experience extreme load.

  20. Defining Faculty Work.

    ERIC Educational Resources Information Center

    Gray, Peter J.; Diamond, Robert M.

    1994-01-01

    A process of planned change is proposed for redefining college faculty work. Legitimate faculty work is defined in broad terms, and information sources and methods for collecting information to support redefinition are identified. The final step in the redefinition process is the development of new mission statements for the institution and its…

  1. Defined by Limitations

    ERIC Educational Resources Information Center

    Arriola, Sonya; Murphy, Katy

    2010-01-01

    Undocumented students are a population defined by limitations. Their lack of legal residency and any supporting paperwork (e.g., Social Security number, government issued identification) renders them essentially invisible to the American and state governments. They cannot legally work. In many states, they cannot legally drive. After the age of…

  2. [Accidental methyl alcohol poisoning].

    PubMed

    Xiao, J H

    1990-05-01

    An accidental poisoning due to drinking methyl alcohol in Chaoyang county is reported, analysing the accident. The poison came from the "retail white spirit" which was contaminated with methyl alcohol. Twenty-nine persons drank the wine, fourteen of them died, two of them became blind. After drinking this "retail white spirit" the drinkers showed symptoms of vertigo, headache, weakness, vomiting, night sweat, dyspnea and blurring of vision etc. within 6-120 hours. On examining the remaining spirit, we found the content of methyl alcohol to be between 16.6 and 40.69 g/100 ml. Some of the patients' urine and blood also contained methyl alcohol. We reckoned that each one of the twenty patients had taken more than 27 g of methyl alcohol and each of the ten dead drank more than 40 ml of the alcohol. PMID:2253526

  3. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of si

  4. Liver histone H3 methylation and acetylation may associate with type 2 diabetes development.

    PubMed

    Tu, Peipei; Li, Xiaodan; Ma, Baicheng; Duan, Huikun; Zhang, Yaofang; Wu, Ri; Ni, Zaizhong; Jiang, Pingzhe; Wang, Haisong; Li, Miao; Zhu, Jianhong; Li, Minggang

    2015-03-01

    Type 2 diabetes (T2D) is a complicated systemic disease, and the exact pathogenetic molecular mechanism is unclear. Distinct histone modifications regulate gene expression in certain diseases, but little is known about histone epigenetics in diabetes. In the current study, C57BL/6 J mice were used to build T2D model, then treated with exendin-4 (10 μg/kg). Histone H3K9 and H3K23 acetylation, H3K4 monomethylation and H3K9 dimethylation were explored by Western blotting of liver histone extracts. Real-time polymerase chain reaction (PCR) was used to examine expression levels of diabetes-related genes, while chromatin immunoprecipitation (ChIP) was applied to analyze H3 and H3K9 acetylation, H3K4 monomethylation, and H3K9 dimethylation in the promoter of facilitated glucose transporter member 2 (Glut2) gene. The results showed that liver's total H3K4 monomethylation and H3K9 dimethylation was increased in diabetic mice, which was abrogated with the treatment of exendin-4. In contrast, H3K9 and H3K23 acetylation were reduced in diabetic mice, while exendin-4 only alleviated the reduction of H3K9 acetylation. Our data indicated that the progression of type 2 diabetes mellitus (T2D) is associated with global liver histone H3K9 and H3K23 acetylation, H3K4 monomethylation, and H3K9 dimethylation. Exploiting exact histone modify enzyme inhibitors, which may represent a novel strategy to prevent T2D. PMID:25666660

  5. Defining Dynamic Route Structure

    NASA Technical Reports Server (NTRS)

    Zelinski, Shannon; Jastrzebski, Michael

    2011-01-01

    This poster describes a method for defining route structure from flight tracks. Dynamically generated route structures could be useful in guiding dynamic airspace configuration and helping controllers retain situational awareness under dynamically changing traffic conditions. Individual merge and diverge intersections between pairs of flights are identified, clustered, and grouped into nodes of a route structure network. Links are placed between nodes to represent major traffic flows. A parametric analysis determined the algorithm input parameters producing route structures of current day flight plans that are closest to todays airway structure. These parameters are then used to define and analyze the dynamic route structure over the course of a day for current day flight paths. Route structures are also compared between current day flight paths and more user preferred paths such as great circle and weather avoidance routing.

  6. Defining the paramedic process.

    PubMed

    Carter, Holly; Thompson, James

    2015-01-01

    The use of a 'process of care' is well established in several health professions, most evidently within the field of nursing. Now ingrained within methods of care delivery, it offers a logical approach to problem solving and ensures an appropriate delivery of interventions that are specifically suited to the individual patient. Paramedicine is a rapidly advancing profession despite a wide acknowledgement of limited research provisions. This frequently results in the borrowing of evidence from other disciplines. While this has often been useful, there are many concerns relating to the acceptable limit of evidence transcription between professions. To date, there is no formally recognised 'process of care'-defining activity within the pre-hospital arena. With much current focus on the professional classification of paramedic work, it is considered timely to formally define a formula that underpins other professional roles such as nursing. It is hypothesised that defined processes of care, particularly the nursing process, may have features that would readily translate to pre-hospital practice. The literature analysed was obtained through systematic searches of a range of databases, including Ovid MEDLINE, Cumulative Index to Nursing and Allied Health. The results demonstrated that the defined process of care provides nursing with more than just a structure for practice, but also has implications for education, clinical governance and professional standing. The current nursing process does not directly articulate to the complex and often unstructured role of the paramedic; however, it has many principles that offer value to the paramedic in their practice. Expanding the nursing process model to include the stages of Dispatch Considerations, Scene Assessment, First Impressions, Patient History, Physical Examination, Clinical Decision-Making, Interventions, Re-evaluation, Transport Decisions, Handover and Reflection would provide an appropriate model for pre

  7. Defining functional dyspepsia.

    PubMed

    Mearin, Fermín; Calleja, José Luis

    2011-12-01

    Dyspepsia and functional dyspepsia represent a highly significant public health issue. A good definition of dyspepsia is key for helping us to better approach symptoms, decision making, and therapy indications.During the last few years many attempts were made at establishing a definition of dyspepsia. Results were little successful on most occasions, and clear discrepancies arose on whether symptoms should be associated with digestion, which types of symptoms were to be included, which anatomic location should symptoms have, etc.The Rome III Committee defined dyspepsia as "a symptom or set of symptoms that most physicians consider to originate from the gastroduodenal area", including the following: postprandial heaviness, early satiety, and epigastric pain or burning. Two new entities were defined: a) food-induced dyspeptic symptoms (postprandial distress syndrome); and b) epigastric pain (epigastric pain syndrome). These and other definitions have shown both strengths and weaknesses. At times they have been much too complex, at times much too simple; furthermore, they have commonly erred on the side of being inaccurate and impractical. On the other hand, some (the most recent ones) are difficult to translate into the Spanish language. In a meeting of gastroenterologists with a special interest in digestive functional disorders, the various aspects of dyspepsia definition were discussed and put to the vote, and the following conclusions were arrived at: dyspepsia is defined as a set of symptoms, either related or unrelated to food ingestion, localized on the upper half of the abdomen. They include: a) epigastric discomfort (as a category of severity) or pain; b) postprandial heaviness; and c) early satiety. Associated complaints include: nausea, belching, bloating, and epigastric burn (heartburn). All these must be scored according to severity and frequency. Furthermore, psychological factors may be involved in the origin of functional dyspepsia. On the other hand

  8. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

  9. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

  10. 40 CFR 721.6920 - Butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... substituted methyl styrene, methyl methacrylate, and substituted silane. 721.6920 Section 721.6920 Protection... acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted silane. (a... butyl acrylate, polymer with substituted methyl styrene, methyl methacrylate, and substituted...