The histone demethylase Fbxl11/Kdm2a plays an essential role in embryonic development by repressing cell-cycle regulators.

PubMed

Kawakami, Eri; Tokunaga, Akinori; Ozawa, Manabu; Sakamoto, Reiko; Yoshida, Nobuaki

2015-02-01

Methylation and de-methylation of histone lysine residues play pivotal roles in mammalian early development; these modifications influence chromatin architecture and regulate gene transcription. Fbxl11 (F-box and leucine-rich repeat 11)/Kdm2a is a histone demethylase that selectively removes mono- and di-methylation from histone H3K36. Previously, two other histone H3K36 demethylases (Jmjd5 or Fbxl10) were analyzed based on the phenotypes of the corresponding knockout (KO) mice; the results of those studies implicated H3K36 demethylases in cell proliferation, apoptosis, and senescence (Fukuda et al., 2011; Ishimura et al., 2012). To elucidate the physiological role of Fbxl11, we generated and examined Fbxl11 KO mice. Fbxl11 was expressed throughout the body during embryogenesis, and the Fbxl11 KO mice exhibited embryonic lethality at E10.5-12.5, accompanied with severe growth defects leading to reduced body size. Furthermore, knockout of Fbxl11 decreased cell proliferation and increased apoptosis. The lack of Fbxl11 resulted in downregulation of the Polycomb group protein (PcG) Ezh2, PcG mediated H2A ubiquitination and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Taken together, our findings suggest that Fbxl11 plays an essential role in embryonic development and homeostasis by regulating cell proliferation and survival. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases

PubMed Central

Lee, Sanghun; Xu, Siming; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

2016-01-01

Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity. PMID:27354553

Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases.

PubMed

Lee, Sanghun; Fu, Fuyou; Xu, Siming; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

2016-07-01

Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity. © 2016 American Society of Plant Biologists. All rights reserved.

Reader interactome of epigenetic histone marks in birds.

PubMed

Bluhm, Alina; Casas-Vila, Nuria; Scheibe, Marion; Butter, Falk

2016-02-01

Lysine methylation is part of the posttranscriptional histone code employed to recruit modification specific readers to chromatin. Unbiased, quantitative mass spectrometry approaches combined with peptide pull-downs have been used to study histone methylation-dependent binders in mammalian cells. Here, we extend the study to birds by investigating the interaction partners for H3K4me3, H3K9me3, H3K27me3 and H3K36me3 in chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) using label-free quantitative proteomics. In general, we find very strong overlap in interaction partners for the trimethyl marks in birds compared to mammals, underscoring the known conserved function of these modifications. In agreement with their epigenetic role, we find binding of PHF2 and members of the TFIID, SAGA, SET1 and NURF complex to the activation mark H3K4me3. Our data furthermore supports the existence of a LID complex in vertebrates recruited to the H3K4me3 mark. The repressive marks are bound by the HP1 proteins and the EED subunit of the PRC2 complex as well as by WIZ. Like reported in the previous mammalian screens, we found ZNF462, ZNF828 and POGZ enriched at H3K9me3. However, we noted some unexpected differences. N-PAC (also known as GLYR1), an H3K36me3 interactor in mammals, is reproducible not enriched at this modification in our screen in birds. This initial finding suggests that despite strong conservation of the histone tail sequence, a few species-specific differences in epigenetic readers may have evolved between birds and mammals. All MS data have been deposited in the ProteomeXchange with identifier PXD002282 (http://proteomecentral.proteomexchange.org/dataset/PXD002282). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation of strawberry anthocyanins using high-speed counter-current chromatography and the copigmentation with catechin or epicatechin by high pressure processing.

PubMed

Zou, Hui; Ma, Yan; Xu, Zhenzhen; Liao, Xiaojun; Chen, Ailiang; Yang, Shuming

2018-05-01

Three anthocyanins were isolated from strawberry extract by high-speed counter-current chromatography, using a biphasic mixture of tert-butyl methyl ether, n-butanol, acetonitrile, water and trifluoroacetic acid (2.5:2.0:2.5:5.0:1.0%). The anthocyanins were identified as pelargonidin-3-rutinoside, cyanidin-3-glucoside and pelargonidin-3-glucoside with purity of 95.6%, 96.2% and 99.3% respectively. Additionally, the copigmentation reaction rates between pelargonidin-3-glucoside and phenolic acids (catechin or epicatechin) at pH 1.5 and 3.6, pressure 0.1 and 500 MPa, and temperature 60 °C were calculated. The absorbance of pelargonidin-3-glucoside at pH 3.6, with high quantity of phenolic acids was significantly increased by high pressure. The complex of pelargonidin-3-glucoside/catechin has a binding energy of 78.64 kJ/mol at pH 3.6, and 39.13 kJ/mol at pH 1.5; pelargonidin-3-glucoside/epicatechin has a binding energy of 75.34 kJ/mol at pH 1.5 and 54.47 kJ/mol at pH 3.6. The hydrogen bond and van der Waals interaction were the main forces contributing to the structures of complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

Association between H3K4 methylation and cancer prognosis: A meta-analysis.

PubMed

Li, Simin; Shen, Luyan; Chen, Ke-Neng

2018-05-08

Histone H3 lysine 4 methylation (H3K4 methylation), including mono-methylation (H3K4me1), di-methylation (H3K4me2), or tri-methylation (H3K4me3), is one of the epigenetic modifications to histone proteins, which are related to the transcriptional activation of genes. H3K4 methylation has both tumor inhibiting and promoting effects, and the prognostic value of H3K4 methylation in cancer remains controversial. Therefore, we performed a systematic review and meta-analysis to examine the association between H3K4 methylation and cancer prognosis. A comprehensive search of PubMed, Web of Science, ScienceDirect, Embase, and Ovid databases was conducted to identify studies investigating the association between H3K4 methylation and prognosis of patients with malignant tumors. The data and characteristics of each study were extracted, and the hazard ratio (HR) at a 95% confidence interval (CI) was calculated to estimate the effect. A total of 1474 patients in 10 studies were enrolled in this meta-analysis. The pooled HR of 1.52 (95% CI 1.02-2.26) indicated that patients with a lower level of H3K4me2 expression were expected to have shorter overall survival, while the pooled HR of 0.45 (95% CI 0.27-0.74) indicated that patients with a lower level of H3K4me3 expression were expected to have longer overall survival. This meta-analysis indicates that increased H3K4me3 expression and decreased H3K4me2 expression might be predictive factors of poor prognosis in cancer. Further large cohort studies are needed to confirm these findings. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

PubMed Central

LeRoy, Gary; Bua, Dennis J; Garcia, Benjamin A; Gozani, Or; Richard, Stéphane

2010-01-01

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4me3) while heterochromatin contains the H3K9me3 mark. The H3K9me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP and SUV39H1 also fail to bind and to methylate H3K4me3 substrates. Accordingly, we provide in vivo evidence that H3K9me2-enriched histones are devoid of H3K4me2/3 and that histones depleted of H3K4me2/3 have elevated H3K9me2/3. The correlation between the loss of interaction of these KMTs with H3K4me3 and concomitant methylation impairment leads to the postulate that at least these four KMTs require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4me3 and H3K9me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners. PMID:21124070

[PHI regulates histone methylation and acetylation in Burkitt lymphoma Daudi cell line].

PubMed

Hong, Ling-Ling; Ma, Xu-Dong; Huang, Yi-Qun

2011-02-01

This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.

Links between DNA methylation and nucleosome occupancy in the human genome.

PubMed

Collings, Clayton K; Anderson, John N

2017-01-01

DNA methylation is an epigenetic modification that is enriched in heterochromatin but depleted at active promoters and enhancers. However, the debate on whether or not DNA methylation is a reliable indicator of high nucleosome occupancy has not been settled. For example, the methylation levels of DNA flanking CTCF sites are higher in linker DNA than in nucleosomal DNA, while other studies have shown that the nucleosome core is the preferred site of methylation. In this study, we make progress toward understanding these conflicting phenomena by implementing a bioinformatics approach that combines MNase-seq and NOMe-seq data and by comprehensively profiling DNA methylation and nucleosome occupancy throughout the human genome. The results demonstrated that increasing methylated CpG density is correlated with nucleosome occupancy in the total genome and within nearly all subgenomic regions. Features with elevated methylated CpG density such as exons, SINE-Alu sequences, H3K36-trimethylated peaks, and methylated CpG islands are among the highest nucleosome occupied elements in the genome, while some of the lowest occupancies are displayed by unmethylated CpG islands and unmethylated transcription factor binding sites. Additionally, outside of CpG islands, the density of CpGs within nucleosomes was shown to be important for the nucleosomal location of DNA methylation with low CpG frequencies favoring linker methylation and high CpG frequencies favoring core particle methylation. Prominent exceptions to the correlations between methylated CpG density and nucleosome occupancy include CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, distinguish the major silencing mechanisms of the human epigenome. Thus, the relationship between DNA methylation and nucleosome occupancy is influenced by the density of methylated CpG dinucleotides and by other epigenomic components in chromatin.

U.S. EPA, Pesticide Product Label, METHYL PARATHION TOXAPHENE 3-6, 04/11/1975

EPA Pesticide Factsheets

2011-04-14

... '. ttt'f ~),'fl)r .. , .. r~ll.k,I~ll}, ",Itlf'lq (Ir (irlrlk.lf)\\-lc .\\fTtH WUR!<., I I"" I~t .Iii 'N,'f'" l I"t""'" II,d ..... h~,t, .... S~I~:V"¥'t'r, ~; ,Ul~~ :.,tJ.i,-> tnd "'.,)t+'f. ...

DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

PubMed

Drouin, Simon; Laramée, Louise; Jacques, Pierre-Étienne; Forest, Audrey; Bergeron, Maxime; Robert, François

2010-10-28

Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alamdar, Ambreen; Xi, Guochen

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Sincemore » H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. - Highlights: • Epigenetic mechanisms of arsenic-induced male reproductive toxicity remain unclear. • Arsenic disturbs the expression of key steroidogenic genes in MLTC-1 cells. • Histone H3K9 di- and tri-methylation was suppressed in arsenic-exposed cells. • Arsenic activates 3β-HSD expression through repression of histone H3K9 methylation.« less

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

PubMed

Fuchs, Jörg; Jovtchev, Gabriele; Schubert, Ingo

2008-01-01

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

Histone H3.3 mutations drive paediatric glioblastoma through upregulation of MYCN

PubMed Central

Bjerke, Lynn; Mackay, Alan; Nandhabalan, Meera; Burford, Anna; Jury, Alexa; Popov, Sergey; Bax, Dorine A; Carvalho, Diana; Taylor, Kathryn R; Vinci, Maria; Bajrami, Ilirjana; McGonnell, Imelda M; Lord, Christopher J; Reis, Rui M; Hargrave, Darren; Ashworth, Alan; Workman, Paul; Jones, Chris

2013-01-01

Glioblastomas of children and young adults have a median survival of only 12-15months and are clinically and biologically distinct from histologically similar cancers in older adults1. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A2, occurring either at or close to key residues marked by methylation for regulation of transcription – K27 and G34. Here we show that the cerebral hemispheric-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem cell maintenance, cell fate decisions and self-renewal. Critically, H3F3A G34 mutations cause profound upregulation of MYCN, a potent oncogene which is causative of glioblastomas when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population by inhibiting kinases responsible for stabilisation of the protein. PMID:23539269

Genome-wide map of quantified epigenetic changes during in vitro chondrogenic differentiation of primary human mesenchymal stem cells.

PubMed

Herlofsen, Sarah R; Bryne, Jan Christian; Høiby, Torill; Wang, Li; Issner, Robbyn; Zhang, Xiaolan; Coyne, Michael J; Boyle, Patrick; Gu, Hongcang; Meza-Zepeda, Leonardo A; Collas, Philippe; Mikkelsen, Tarjei S; Brinchmann, Jan E

2013-02-15

For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.

Arsenic activates the expression of 3β-HSD in mouse Leydig cells through repression of histone H3K9 methylation.

PubMed

Alamdar, Ambreen; Xi, Guochen; Huang, Qingyu; Tian, Meiping; Eqani, Syed Ali Musstjab Akber Shah; Shen, Heqing

2017-07-01

Arsenic exposure has been associated with male reproductive dysfunction by disrupting steroidogenesis; however, the roles of epigenetic drivers, especially histone methylation in arsenic-induced steroidogenic toxicity remain not well documented. In this study, we investigated the role of histone H3 lysine 9 (H3K9) methylation in steroidogenesis disturbance in mouse Leydig cells (MLTC-1) due to arsenic exposure. Our results indicated that mRNA and protein expression levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) were both significantly up-regulated while the rest of key genes involved in steroidogenesis were down-regulated. Moreover, arsenic exposure significantly decreased the histone H3K9 di- and tri-methylation (H3K9me2/3) levels in MLTC-1 cells. Since H3K9 demethylation leads to gene activation, we further investigated whether the induction of 3β-HSD expression was ascribed to reduced H3K9 methylation. The results showed that H3K9me2/3 demethylase (JMJD2A) inhibitor, quercetin (Que) significantly attenuated the decrease of H3K9me2/3 and increase of 3β-HSD expression induced by arsenic. To further elucidate the mechanism for the activation of 3β-HSD, we determined the histone H3K9 methylation levels in Hsd3b gene promoter, which also showed significant decrease of H3K9me2/3 in the investigated region after arsenic exposure. Considering these results, we conclude that arsenic exposure induced 3β-HSD up-regulation by suppressing H3K9me2/3 status, which is suggested as a compensatory mechanism for steroidogenic disturbance in MLTC-1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

PubMed Central

Rizzardi, Lindsay F.; Dorn, Elizabeth S.; Strahl, Brian D.; Cook, Jeanette Gowen

2012-01-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication. PMID:22851644

DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

PubMed

Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

2012-10-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

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.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis.

PubMed

Martinelli, G; Mancini, M; De Benedittis, C; Rondoni, M; Papayannidis, C; Manfrini, M; Meggendorfer, M; Calogero, R; Guadagnuolo, V; Fontana, M C; Bavaro, L; Padella, A; Zago, E; Pagano, L; Zanotti, R; Scaffidi, L; Specchia, G; Albano, F; Merante, S; Elena, C; Savini, P; Gangemi, D; Tosi, P; Ciceri, F; Poletti, G; Riccioni, L; Morigi, F; Delledonne, M; Haferlach, T; Cavo, M; Valent, P; Soverini, S

2018-01-01

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

PubMed Central

Bell, Joshua S. K.; Kagey, Jacob D.; Barwick, Benjamin G.; Dwivedi, Bhakti; McCabe, Michael T.; Kowalski, Jeanne; Vertino, Paula M.

2016-01-01

ABSTRACT Aberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2′-deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumor-specific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome. PMID:27082926

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

G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis

PubMed Central

Tachibana, Makoto; Sugimoto, Kenji; Nozaki, Masami; Ueda, Jun; Ohta, Tsutomu; Ohki, Misao; Fukuda, Mikiko; Takeda, Naoki; Niida, Hiroyuki; Kato, Hiroyuki; Shinkai, Yoichi

2002-01-01

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes. PMID:12130538

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

CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell lines

PubMed Central

Guerra-Calderas, Lissania; González-Barrios, Rodrigo; Patiño, Carlos César; Alcaraz, Nicolás; Salgado-Albarrán, Marisol; de León, David Cantú; Hernández, Clementina Castro; Sánchez-Pérez, Yesennia; Maldonado-Martínez, Héctor Aquiles; De la Rosa-Velazquez, Inti A.; Vargas-Romero, Fernanda; Herrera, Luis A.; García-Carrancá, Alejandro; Soto-Reyes, Ernesto

2018-01-01

Histone demethylase KDM4A is involved in H3K9me3 and H3K36me3 demethylation, which are epigenetic modifications associated with gene silencing and RNA Polymerase II elongation, respectively. KDM4A is abnormally expressed in cancer, affecting the expression of multiple targets, such as the CHD5 gene. This enzyme localizes at the first intron of CHD5, and the dissociation of KDM4A increases gene expression. In vitro assays showed that KDM4A-mediated demethylation is enhanced in the presence of CTCF, suggesting that CTCF could increase its enzymatic activity in vivo, however the specific mechanism by which CTCF and KDM4A might be involved in the CHD5 gene repression is poorly understood. Here, we show that CTCF and KDM4A form a protein complex, which is recruited into the first intron of CHD5. This is related to a decrease in H3K36me3/2 histone marks and is associated with its transcriptional downregulation. Depletion of CTCF or KDM4A by siRNA, triggered the reactivation of CHD5 expression, suggesting that both proteins are involved in the negative regulation of this gene. Furthermore, the knockout of KDM4A restored the CHD5 expression and H3K36me3 and H3K36me2 histone marks. Such mechanism acts independently of CHD5 promoter DNA methylation. Our findings support a novel mechanism of epigenetic repression at the gene body that does not involve promoter silencing. PMID:29682202

Citrullination/Methylation Crosstalk on Histone H3 Regulates ER-Target Gene Transcription.

PubMed

Clancy, Kathleen W; Russell, Anna-Maria; Subramanian, Venkataraman; Nguyen, Hannah; Qian, Yuewei; Campbell, Robert M; Thompson, Paul R

2017-06-16

Posttranslational modifications of histone tails are a key contributor to epigenetic regulation. Histone H3 Arg26 and Lys27 are both modified by multiple enzymes, and their modifications have profound effects on gene expression. Citrullination of H3R26 by PAD2 and methylation of H3K27 by PRC2 have opposing downstream impacts on gene regulation; H3R26 citrullination activates gene expression, and H3K27 methylation represses gene expression. Both of these modifications are drivers of a variety of cancers, and their writer enzymes, PAD2 and EZH2, are the targets of drug therapies. After biochemical and cell-based analysis of these modifications, a negative crosstalk interaction is observed. Methylation of H3K27 slows citrullination of H3R26 30-fold, whereas citrullination of H3R26 slows methylation 30,000-fold. Examination of the mechanism of this crosstalk interaction uncovered a change in structure of the histone tail upon citrullination which prevents methylation by the PRC2 complex. This mechanism of crosstalk is reiterated in cell lines using knockdowns and inhibitors of both enzymes. Based our data, we propose a model in which, after H3 Cit26 formation, H3K27 demethylases are recruited to the chromatin to activate transcription. In total, our studies support the existence of crosstalk between citrullination of H3R26 and methylation of H3K27.

Comprehensive epigenetic landscape of rheumatoid arthritis fibroblast-like synoviocytes.

PubMed

Ai, Rizi; Laragione, Teresina; Hammaker, Deepa; Boyle, David L; Wildberg, Andre; Maeshima, Keisuke; Palescandolo, Emanuele; Krishna, Vinod; Pocalyko, David; Whitaker, John W; Bai, Yuchen; Nagpal, Sunil; Bachman, Kurtis E; Ainsworth, Richard I; Wang, Mengchi; Ding, Bo; Gulko, Percio S; Wang, Wei; Firestein, Gary S

2018-05-15

Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.

Arabidopsis COMPASS-Like Complexes Mediate Histone H3 Lysine-4 Trimethylation to Control Floral Transition and Plant Development

PubMed Central

Jiang, Danhua; Kong, Nicholas C.; Gu, Xiaofeng; Li, Zicong; He, Yuehui

2011-01-01

Histone H3 lysine-4 (H3K4) methylation is associated with transcribed genes in eukaryotes. In Drosophila and mammals, both di- and tri-methylation of H3K4 are associated with gene activation. In contrast to animals, in Arabidopsis H3K4 trimethylation, but not mono- or di-methylation of H3K4, has been implicated in transcriptional activation. H3K4 methylation is catalyzed by the H3K4 methyltransferase complexes known as COMPASS or COMPASS-like in yeast and mammals. Here, we report that Arabidopsis homologs of the COMPASS and COMPASS-like complex core components known as Ash2, RbBP5, and WDR5 in humans form a nuclear subcomplex during vegetative and reproductive development, which can associate with multiple putative H3K4 methyltransferases. Loss of function of ARABIDOPSIS Ash2 RELATIVE (ASH2R) causes a great decrease in genome-wide H3K4 trimethylation, but not in di- or mono-methylation. Knockdown of ASH2R or the RbBP5 homolog suppresses the expression of a crucial Arabidopsis floral repressor, FLOWERING LOCUS C (FLC), and FLC homologs resulting in accelerated floral transition. ASH2R binds to the chromatin of FLC and FLC homologs in vivo and is required for H3K4 trimethylation, but not for H3K4 dimethylation in these loci; overexpression of ASH2R causes elevated H3K4 trimethylation, but not H3K4 dimethylation, in its target genes FLC and FLC homologs, resulting in activation of these gene expression and consequent late flowering. These results strongly suggest that H3K4 trimethylation in FLC and its homologs can activate their expression, providing concrete evidence that H3K4 trimethylation accumulation can activate eukaryotic gene expression. Furthermore, our findings suggest that there are multiple COMPASS-like complexes in Arabidopsis and that these complexes deposit trimethyl but not di- or mono-methyl H3K4 in target genes to promote their expression, providing a molecular explanation for the observed coupling of H3K4 trimethylation (but not H3K4 dimethylation) with active gene expression in Arabidopsis. PMID:21423667

Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

PubMed Central

Jackel, Jamie N.; Storer, Jessica M.; Coursey, Tami

2016-01-01

ABSTRACT In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin. IMPORTANCE In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible. PMID:27279611

Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin.

PubMed

Jackel, Jamie N; Storer, Jessica M; Coursey, Tami; Bisaro, David M

2016-08-15

In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin. In plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The COMPASS Family of Histone H3K4 Methylases: Mechanisms of Regulation in Development and Disease Pathogenesis

PubMed Central

Shilatifard, Ali

2014-01-01

The Saccharomyces cerevisiae Set1/COMPASS was the first histone H3 lysine 4 (H3K4) methylase identified over ten years ago. Since then, it has been demonstrated that Set1/COMPASS and its enzymatic product, H3K4 methylation, is highly conserved across the evolutionary tree. Although there is only one COMPASS in yeast, human cells bear at least six COMPASS family members each capable of methylating H3K4 with non-redundant functions. In yeast, the monoubiquitination of histone H2B by Rad6/Bre1 is required for proper H3K4 and H3K79 trimethylations. This histone crosstalk and its machinery are also highly conserved from yeast to human. In this review, the process of histone H2B monoubiquitination-dependent and independent histone H3K4 methylation as a mark of active transcription, enhancer signatures, and developmentally poised genes will be discussed. The misregulation of histone H2B monoubiquitination and H3K4 methylation results in the pathogenesis of human diseases including cancer. Recent findings in this regard will also be examined. PMID:22663077

Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

PubMed Central

Selker, Eric U.

2017-01-01

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa. Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1–mediated histone deacetylation in heterochromatin spreading and gene silencing. PMID:29078403

P110β Inhibition Reduces Histone H3K4 Di-Methylation in Prostate Cancer.

PubMed

Pang, Jun; Yang, Yue-Wu; Huang, Yiling; Yang, Jun; Zhang, Hao; Chen, Ruibao; Dong, Liang; Huang, Yan; Wang, Dongying; Liu, Jihong; Li, Benyi

2017-02-01

Epigenetic alteration plays a major role in the development and progression of human cancers, including prostate cancer. Histones are the key factors in modulating gene accessibility to transcription factors and post-translational modification of the histone N-terminal tail including methylation is associated with either transcriptional activation (H3K4me2) or repression (H3K9me3). Furthermore, phosphoinositide 3-kinase (PI3 K) signaling and the androgen receptor (AR) are the key determinants in prostate cancer development and progression. We recently showed that prostate-targeted nano-micelles loaded with PI3 K/p110beta specific inhibitor TGX221 blocked prostate cancer growth in vitro and in vivo. Our objective of this study was to determine the role of PI3 K signaling in histone methylation in prostate cancer, with emphasis on histone H3K4 methylation. PI3 K non-specific inhibitor LY294002 and p110beta-specific inhibitor TGX221 were used to block PI3 K/p110beta signaling. The global levels of H3K4 and H3K9 methylation in prostate cancer cells and tissue specimens were evaluated by Western blot assay and immunohistochemical staining. A synthetic androgen R1881 was used to stimulate AR activity in prostate cancer cells. A castration-resistant prostate cancer (CRPC) specific human tissue microarray (TMA) was used to assess the global levels of H3K4me2 methylation by immunostaining approach. Our data revealed that H3K4me2 levels were significantly elevated after androgen stimulation. With RNA silencing and pharmacology approaches, we further defined that inhibition of PI3 K/p110beta activity through gene-specific knocking down and small chemical inhibitor TGX221 abolished androgen-stimulated H3K4me2 methylation. Consistently, prostate cancer-targeted delivery of TGX221 in vivo dramatically reduced the global levels of H3K4me2 as assessed by immunohistochemical staining on tissue section of mouse xenografts from CRPC cell lines 22RV1 and C4-2. Finally, immunostaining data revealed a strong H3K4me2 immunosignal in CRPC tissues compared to primary tumors and benign prostate tissues. Taken together, our results suggest that PI3 K/p110beta-dependent signaling is involved in androgen-stimulated H3K4me2 methylation in prostate cancer, which might be used as a novel biomarker for disease prognosis and targeted therapy. Prostate 77:299-308, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Redundant mechanisms to form silent chromatin at pericentromeric regions rely on BEND3 and DNA methylation.

PubMed

Saksouk, Nehmé; Barth, Teresa K; Ziegler-Birling, Celine; Olova, Nelly; Nowak, Agnieszka; Rey, Elodie; Mateos-Langerak, Julio; Urbach, Serge; Reik, Wolf; Torres-Padilla, Maria-Elena; Imhof, Axel; Déjardin, Jérome; Simboeck, Elisabeth

2014-11-20

Constitutive heterochromatin is typically defined by high levels of DNA methylation and H3 lysine 9 trimethylation (H3K9Me3), whereas facultative heterochromatin displays DNA hypomethylation and high H3 lysine 27 trimethylation (H3K27Me3). The two chromatin types generally do not coexist at the same loci, suggesting mutual exclusivity. During development or in cancer, pericentromeric regions can adopt either epigenetic state, but the switching mechanism is unknown. We used a quantitative locus purification method to characterize changes in pericentromeric chromatin-associated proteins in mouse embryonic stem cells deficient for either the methyltransferases required for DNA methylation or H3K9Me3. DNA methylation controls heterochromatin architecture and inhibits Polycomb recruitment. BEND3, a protein enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, allows Polycomb recruitment and H3K27Me3, resulting in a redundant pathway to generate repressive chromatin. This suggests that BEND3 is a key factor in mediating a switch from constitutive to facultative heterochromatin. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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

2013-02-15

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

ATXR5 and ATXR6 are novel H3K27 monomethyltransferases required for chromatin structure and gene silencing

PubMed Central

Jacob, Yannick; Feng, Suhua; LeBlanc, Chantal A.; Bernatavichute, Yana V.; Stroud, Hume; Cokus, Shawn; Johnson, Lianna M.; Pellegrini, Matteo; Jacobsen, Steven E.; Michaels, Scott D.

2009-01-01

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications including DNA methylation, histone H3 dimethylation at lysine 9 (H3K9me2), and monomethylation at lysine 27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified and mutations in these proteins lead to the reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6 exhibit H3K27 monomethyltransferase activity and double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Interestingly, H3K9me2 and DNA methylation are unaffected in the double mutant. These results indicate that ATXR5 and ATXR6 form a novel class of H3K27 methyltransferases and that H3K27me1 represents a new pathway required for transcriptional repression in Arabidopsis. PMID:19503079

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

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

Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

PubMed Central

Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

2014-01-01

Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660

Modes of Interaction of KMT2 Histone H3 Lysine 4 Methyltransferase/COMPASS Complexes with Chromatin

PubMed Central

Bochyńska, Agnieszka; Lüscher-Firzlaff, Juliane

2018-01-01

Regulation of gene expression is achieved by sequence-specific transcriptional regulators, which convey the information that is contained in the sequence of DNA into RNA polymerase activity. This is achieved by the recruitment of transcriptional co-factors. One of the consequences of co-factor recruitment is the control of specific properties of nucleosomes, the basic units of chromatin, and their protein components, the core histones. The main principles are to regulate the position and the characteristics of nucleosomes. The latter includes modulating the composition of core histones and their variants that are integrated into nucleosomes, and the post-translational modification of these histones referred to as histone marks. One of these marks is the methylation of lysine 4 of the core histone H3 (H3K4). While mono-methylation of H3K4 (H3K4me1) is located preferentially at active enhancers, tri-methylation (H3K4me3) is a mark found at open and potentially active promoters. Thus, H3K4 methylation is typically associated with gene transcription. The class 2 lysine methyltransferases (KMTs) are the main enzymes that methylate H3K4. KMT2 enzymes function in complexes that contain a necessary core complex composed of WDR5, RBBP5, ASH2L, and DPY30, the so-called WRAD complex. Here we discuss recent findings that try to elucidate the important question of how KMT2 complexes are recruited to specific sites on chromatin. This is embedded into short overviews of the biological functions of KMT2 complexes and the consequences of H3K4 methylation. PMID:29498679

Folate Polyglutamylation Is Involved in Chromatin Silencing by Maintaining Global DNA Methylation and Histone H3K9 Dimethylation in Arabidopsis[C][W

PubMed Central

Zhou, Hao-Ran; Zhang, Fang-Fang; Ma, Ze-Yang; Huang, Huan-Wei; Jiang, Ling; Cai, Tao; Zhu, Jian-Kang; Zhang, Chuyi; He, Xin-Jian

2013-01-01

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation. PMID:23881414

JMJD1A, H3K9me1, H3K9me2 and ADM expression as prognostic markers in oral and oropharyngeal squamous cell carcinoma.

PubMed

Maia, Lucas de Lima; Peterle, Gabriela Tonini; Dos Santos, Marcelo; Trivilin, Leonardo Oliveira; Mendes, Suzanny Oliveira; de Oliveira, Mayara Mota; Dos Santos, Joaquim Gasparini; Stur, Elaine; Agostini, Lidiane Pignaton; Couto, Cinthia Vidal Monteiro da Silva; Dalbó, Juliana; de Assis, Aricia Leone Evangelista Monteiro; Archanjo, Anderson Barros; Mercante, Ana Maria Da Cunha; Lopez, Rossana Veronica Mendoza; Nunes, Fábio Daumas; de Carvalho, Marcos Brasilino; Tajara, Eloiza Helena; Louro, Iúri Drumond; Álvares-da-Silva, Adriana Madeira

2018-01-01

Jumonji Domain-Containing 1A (JMJD1A) protein promotes demethylation of histones, especially at lysin-9 of di-methylated histone H3 (H3K9me2) or mono-methylated (H3K9me1). Increased levels of H3 histone methylation at lysin-9 (H3K9) is related to tumor suppressor gene silencing. JMJD1A gene target Adrenomeduline (ADM) has shown to promote cell growth and tumorigenesis. JMJD1A and ADM expression, as well as H3K9 methylation level have been related with development risk and prognosis of several tumor types. We aimed to evaluate JMJD1A, ADM, H3K9me1 and H3K9me2expression in paraffin-embedded tissue microarrays from 84 oral and oropharyngeal squamous cell carcinoma samples through immunohistochemistry analysis. Our results showed that nuclear JMJD1A expression was related to lymph node metastasis risk. In addition, JMJD1A cytoplasmic expression was an independent risk marker for advanced tumor stages. H3K9me1 cytoplasmic expression was associated with reduced disease-specific death risk. Furthermore, high H3K9me2 nuclear expression was associated with worse specific-disease and disease-free survival. Finally, high ADM cytoplasmic expression was an independent marker of lymph node metastasis risk. JMJD1A, H3K9me1/2 and ADM expression may be predictor markers of progression and prognosis in oral and oropharynx cancer patients, as well as putative therapeutic targets.

JMJD1A, H3K9me1, H3K9me2 and ADM expression as prognostic markers in oral and oropharyngeal squamous cell carcinoma

PubMed Central

Peterle, Gabriela Tonini; dos Santos, Marcelo; Trivilin, Leonardo Oliveira; Mendes, Suzanny Oliveira; de Oliveira, Mayara Mota; dos Santos, Joaquim Gasparini; Stur, Elaine; Agostini, Lidiane Pignaton; Couto, Cinthia Vidal Monteiro da Silva; Dalbó, Juliana; de Assis, Aricia Leone Evangelista Monteiro; Archanjo, Anderson Barros; Mercante, Ana Maria Da Cunha; Lopez, Rossana Veronica Mendoza; Nunes, Fábio Daumas; de Carvalho, Marcos Brasilino; Tajara, Eloiza Helena; Louro, Iúri Drumond; Álvares-da-Silva, Adriana Madeira

2018-01-01

Aims Jumonji Domain-Containing 1A (JMJD1A) protein promotes demethylation of histones, especially at lysin-9 of di-methylated histone H3 (H3K9me2) or mono-methylated (H3K9me1). Increased levels of H3 histone methylation at lysin-9 (H3K9) is related to tumor suppressor gene silencing. JMJD1A gene target Adrenomeduline (ADM) has shown to promote cell growth and tumorigenesis. JMJD1A and ADM expression, as well as H3K9 methylation level have been related with development risk and prognosis of several tumor types. Methods and results We aimed to evaluate JMJD1A, ADM, H3K9me1 and H3K9me2expression in paraffin-embedded tissue microarrays from 84 oral and oropharyngeal squamous cell carcinoma samples through immunohistochemistry analysis. Our results showed that nuclear JMJD1A expression was related to lymph node metastasis risk. In addition, JMJD1A cytoplasmic expression was an independent risk marker for advanced tumor stages. H3K9me1 cytoplasmic expression was associated with reduced disease-specific death risk. Furthermore, high H3K9me2 nuclear expression was associated with worse specific-disease and disease-free survival. Finally, high ADM cytoplasmic expression was an independent marker of lymph node metastasis risk. Conclusion JMJD1A, H3K9me1/2 and ADM expression may be predictor markers of progression and prognosis in oral and oropharynx cancer patients, as well as putative therapeutic targets. PMID:29590186

Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

PubMed

Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

2016-09-01

In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Tissue-Specific Expression of DNA Methyltransferases Involved in Early-Life Nutritional Stress of Chicken, Gallus gallus

PubMed Central

Kang, Seong W.; Madkour, Mahmoud; Kuenzel, Wayne J.

2017-01-01

DNA methylation was reported as a possible stress-adaptation mechanism involved in the transcriptional regulation of stress responsive genes. Limited data are available on effects of psychological stress and early-life nutritional stress on DNA methylation regulators [DNMTs: DNA (cytosine-5)-methyltransferase 1 (DNMT1), DNMT1 associated protein (DMAP1), DNMT 3 alpha (DNMT3A) and beta (DNMT3B)] in avian species. The objectives of this study were to: (1) investigate changes in expression of DNMT1, DMAP1, DNMT3A, and DNMT3B following acute (AS) or chronic immobilization stress (CS); (2) test immediate effect of early-life nutritional stress [food deprivation (FD) for 12 h (12hFD) or 36 h (36hFD) at the post-hatching period] on expression of DNA methylation regulators and glucocorticoid receptor (GR), and the long-term effect of early-life nutritional stress at 6 weeks of age. Expression of DNMTs and plasma corticosterone (CORT) concentration decreased by CS compared to AS (p < 0.05), indicating differential roles of DNA methylation regulators in the stress response. Plasma CORT at 12hFD and 36hFD birds increased compared to control birds (12hF and 36hF), but there were no significant differences in plasma CORT of 12hFD and 36hFD birds at 6 weeks of age compared to 6 week controls. DNMT1, DMAP1, and DNMT3B expression in the anterior pituitary increased by 12hFD, but decreased at 36hFD compared to their controls (P < 0.05). In liver, DNMT1, DNMT3A, and DNMT3B expression decreased by 12hFD, however, no significant changes occurred at 36hFD. Expression of DMAP1, DNMT3A, and DNMT3B in anterior pituitary and DMAP1 and DNMT3A expression in liver at 6 weeks of age were higher in 36hFD stressed birds compared to controls as well as 12hFD stressed birds. Hepatic GR expression decreased by 12hFD and increased by 36hFD (p < 0.05). Expression patterns of GR in the liver of FD stress-induced birds persisted until 6 weeks of age, suggesting the possible lifelong involvement of liver GR in early-life nutritional stress response of birds. Taken together, results suggest that DNA methylation regulator genes are tissue-specifically responsive to acute and chronic stress, and hepatic GR may play a critical role in regulating the early-life nutritional stress response of birds. In addition, the downregulation of DNMT1 and DMAP1 may be one of the adaptive mechanisms to chronic early-life nutritional stress via passive demethylation. PMID:29270191

Dynamics simulation on the flexibility and backbone motions of HP1 chromodomain bound to free and lysine 9-methylated histone H3 tails

NASA Astrophysics Data System (ADS)

Jiang, Yanke; Zou, Jianwei; Zeng, Min; Zhang, Na; Yu, Qingsen

Histone methylation has emerged as a central epigenetic modification with both activating and repressive roles in eukaryotic chromatin. Drosophila HP1 (heterochromatin-associated protein 1) is one of the chromodomain proteins that contain the essential aromatic residues as the recognition pocket for lysine methylated histone H3 tail. The aromatic cage indicates that the complex of chromodomain protein binding lysine methylated histone H3 tail can be seen as a typical host-guest system between protein and protein. About 10-ns molecular dynamics simulations have been carried out in this study to examine how the presence of mono-, trimethylated lysine 9 histone H3 tail (Me1K9, Me3K9 H3) influences the motions of HP1 protein receptor. The study shows that the conformation of HP1 protein free of H3 tail easily changes, whereas that of HP1 protein bound to methylated H3 tail does not. But the conformation of inserted Me1K9 H3 changes obviously as the Me1K recognition makes hydrogen-bonded interactions associated with the aromatic cage even more unstable than those in free HP1 protein. The conformational change of Me1K9 H3 is correlated with the motions of HP1 protein. As the recognition factor going from Me1K to Me3K produces a more favorable interaction for aromatic ring, hydrogen-bonded interactions associated with aromatic cage in Me3K9 H3-HP1 complex were observed to be much more stable than those in Me1K9 H3-HP1 complex and free HP1. Because of correlation, the flexibility of Me3K9 H3 decreases. The simulations indicate that both the MeK and the surrounding histone tail sequence are necessary features of recognition which significantly affect the flexibility and backbone motions of HP1 chromodomain. These findings confirm a regulatory mechanism of protein-protein interactions through a trimethylated post-translational modification.

Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection.

PubMed

Coursey, Tami; Milutinovic, Milica; Regedanz, Elizabeth; Brkljacic, Jelena; Bisaro, David M

2018-06-06

Histone post-translational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirus Cabbage leaf curl virus (CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 and 3 (EML1 and EML3), which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutant Arabidopsis plants exhibit contrasting hypersusceptible ( eml1 ) and tolerant ( eml3 ) responses to CaLCuV infection, and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Further, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting. IMPORTANCE Histone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter chromatin structure or activity. In this study, CaLCuV was used to characterize the activities of two Arabidopsis Agenet domain histone readers, EML1 and EML3. We show that eml1 mutants are hypersusceptible to CaLCuV, whereas eml3 plants are more tolerant of infection than wild type plants. We also demonstrate that EML1 and EML3 associate with histones and viral chromatin in planta , and that both proteins bind peptides containing H3K36, a PTM associated with active gene expression. Consistent with antiviral activity, EML1 suppresses CaLCuV gene expression and reduces Pol II access to viral chromatin. By linking EML1 and EML3 to pathogenesis, these studies have expanded our knowledge of histone reader proteins and uncovered an additional level of viral chromatin regulation. Copyright © 2018 American Society for Microbiology.

Environmental enrichment reverses histone methylation changes in the aged hippocampus and restores age-related memory deficits.

PubMed

Morse, Sarah J; Butler, Anderson A; Davis, Robin L; Soller, Ian J; Lubin, Farah D

2015-04-01

A decline in long-term memory (LTM) formation is a common feature of the normal aging process, which corresponds with abnormal expression of memory-related genes in the aged hippocampus. Epigenetic modulation of chromatin structure is required for proper transcriptional control of genes, such as the brain-derived neurotrophic factor (Bdnf) and Zif268 in the hippocampus during the consolidation of new memories. Recently, the view has emerged that aberrant transcriptional regulation of memory-related genes may be reflective of an altered epigenetic landscape within the aged hippocampus, resulting in memory deficits with aging. Here, we found that baseline resting levels for tri-methylation of histone H3 at lysine 4 (H3K4me3) and acetylation of histone H3 at lysine 9 and 14 (H3K9,K14ac) were altered in the aged hippocampus as compared to levels in the hippocampus of young adult rats. Interestingly, object learning failed to increase activity-dependent H3K4me3 and di-methylation of histone H3 at lysine 9 (H3K9me2) levels in the hippocampus of aged adults as compared to young adults. Treatment with the LSD-1 histone demethylase inhibitor, t-PCP, increased baseline resting H3K4me3 and H3K9,K14ac levels in the young adult hippocampus, while young adult rats exhibited similar memory deficits as observed in aged rats. After environmental enrichment (EE), we found that object learning induced increases in H3K4me3 levels around the Bdnf, but not the Zif268, gene region in the aged hippocampus and rescued memory deficits in aged adults. Collectively, these results suggest that histone lysine methylation levels are abnormally regulated in the aged hippocampus and identify histone lysine methylation as a transcriptional mechanism by which EE may serve to restore memory formation with aging.

Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity.

PubMed

Gursoy-Yuzugullu, Ozge; Carman, Chelsea; Serafim, Rodolfo Bortolozo; Myronakis, Marios; Valente, Valeria; Price, Brendan D

2017-04-11

Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.

Fumonisin FB1 treatment acts synergistically with methyl donor deficiency during rat pregnancy to produce alterations of H3- and H4-histone methylation patterns in fetuses.

PubMed

Pellanda, Hélène; Forges, Thierry; Bressenot, Aude; Chango, Abalo; Bronowicki, Jean-Pierre; Guéant, Jean-Louis; Namour, Fares

2012-06-01

Prenatal folate and methyl donor malnutrition lead to epigenetic alterations that could enhance susceptibility to disease. Methyl-deficient diet (MDD) and fumonisin FB1 are risk factors for neural tube defects and cancers. Evidence indicates that FB1 impairs folate metabolism. Folate receptors and four heterochromatin markers were investigated in rat fetuses liver derived from dams exposed to MDD and/or FB1 administered at a dose twice higher than the provisional maximum tolerable daily intake (PMTDI = 2 μg/kg/day). Even though folate receptors transcription seemed up-regulated by methyl depletion regardless of FB1 treatment, combined MDD/FB1 exposure might reverse this up-regulation since folate receptors transcripts were lower in the MDD/FB1 versus MDD group. Methyl depletion decreased H4K20me3. Combined MDD/FB1 decreased H4K20me3 even more and increased H3K9me3. The elevated H3K9me3 can be viewed as a defense mechanism inciting the cell to resist heterochromatin disorganization. H3R2me2 and H4K16Ac varied according to this mechanism even though statistical significance was not consistent. Considering that humans are exposed to FB1 levels above the PMTDI, this study is relevant because it suggests that low doses of FB1 interact with MDD thus contributing to disrupt the epigenetic landscape. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The profile of repeat-associated histone lysine methylation states in the mouse epigenome

PubMed Central

Martens, Joost H A; O'Sullivan, Roderick J; Braunschweig, Ulrich; Opravil, Susanne; Radolf, Martin; Steinlein, Peter; Jenuwein, Thomas

2005-01-01

Histone lysine methylation has been shown to index silenced chromatin regions at, for example, pericentric heterochromatin or of the inactive X chromosome. Here, we examined the distribution of repressive histone lysine methylation states over the entire family of DNA repeats in the mouse genome. Using chromatin immunoprecipitation in a cluster analysis representing repetitive elements, our data demonstrate the selective enrichment of distinct H3-K9, H3-K27 and H4-K20 methylation marks across tandem repeats (e.g. major and minor satellites), DNA transposons, retrotransposons, long interspersed nucleotide elements and short interspersed nucleotide elements. Tandem repeats, but not the other repetitive elements, give rise to double-stranded (ds) RNAs that are further elevated in embryonic stem (ES) cells lacking the H3-K9-specific Suv39h histone methyltransferases. Importantly, although H3-K9 tri- and H4-K20 trimethylation appear stable at the satellite repeats, many of the other repeat-associated repressive marks vary in chromatin of differentiated ES cells or of embryonic trophoblasts and fibroblasts. Our data define a profile of repressive histone lysine methylation states for the repetitive complement of four distinct mouse epigenomes and suggest tandem repeats and dsRNA as primary triggers for more stable chromatin imprints. PMID:15678104

Histone H3K4 methylation-dependent and -independent functions of Set1A/COMPASS in embryonic stem cell self-renewal and differentiation.

PubMed

Sze, Christie C; Cao, Kaixiang; Collings, Clayton K; Marshall, Stacy A; Rendleman, Emily J; Ozark, Patrick A; Chen, Fei Xavier; Morgan, Marc A; Wang, Lu; Shilatifard, Ali

2017-09-01

Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1A ΔSET ); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1A ΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1A ΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency. © 2017 Sze et al.; Published by Cold Spring Harbor Laboratory Press.

Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition

NASA Astrophysics Data System (ADS)

Fang, Jian; Cheng, Jingdong; Wang, Jiaolong; Zhang, Qiao; Liu, Mengjie; Gong, Rui; Wang, Ping; Zhang, Xiaodan; Feng, Yangyang; Lan, Wenxian; Gong, Zhou; Tang, Chun; Wong, Jiemin; Yang, Huirong; Cao, Chunyang; Xu, Yanhui

2016-04-01

UHRF1 is an important epigenetic regulator for maintenance DNA methylation. UHRF1 recognizes hemi-methylated DNA (hm-DNA) and trimethylation of histone H3K9 (H3K9me3), but the regulatory mechanism remains unknown. Here we show that UHRF1 adopts a closed conformation, in which a C-terminal region (Spacer) binds to the tandem Tudor domain (TTD) and inhibits H3K9me3 recognition, whereas the SET-and-RING-associated (SRA) domain binds to the plant homeodomain (PHD) and inhibits H3R2 recognition. Hm-DNA impairs the intramolecular interactions and promotes H3K9me3 recognition by TTD-PHD. The Spacer also facilitates UHRF1-DNMT1 interaction and enhances hm-DNA-binding affinity of the SRA. When TTD-PHD binds to H3K9me3, SRA-Spacer may exist in a dynamic equilibrium: either recognizes hm-DNA or recruits DNMT1 to chromatin. Our study reveals the mechanism for regulation of H3K9me3 and hm-DNA recognition by URHF1.

A histone methylation network regulates transgenerational epigenetic memory in C. elegans

PubMed Central

Greer, Eric L.; Beese-Sims, Sara E.; Brookes, Emily; Spadafora, Ruggero; Zhu, Yun; Rothbart, Scott B.; Aristizábal-Corrales, David; Chen, Shuzhen; Badeaux, Aimee I.; Jin, Qiuye; Wang, Wei; Strahl, Brian D.; Colaiácovo, Monica P.; Shi, Yang

2014-01-01

Summary How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans Histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here we identified multiple chromatin-modifying factors, including novel H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. Our findings uncover a network of chromatin regulators that control the trans-generational flow of epigenetic information, and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates trans-generational effects on fertility. PMID:24685137

Molecular responses of cells to 2-mercapto-1-methylimidazole gold nanoparticles (AuNPs)-mmi: investigations of histone methylation changes

NASA Astrophysics Data System (ADS)

Polverino, Arianna; Longo, Angela; Donizetti, Aldo; Drongitis, Denise; Frucci, Maria; Schiavo, Loredana; Carotenuto, Gianfranco; Nicolais, Luigi; Piscopo, Marina; Vitale, Emilia; Fucci, Laura

2014-07-01

While nanomedicine has an enormous potential to improve the precision of specific therapy, the ability to efficiently deliver these materials to regions of disease in vivo remains limited. In this study, we describe analyses of (AuNPs)-mmi cellular intake via fluorescence microscopy and its effects on H3K4 and H3K9 histone dimethylation. Specifically, we studied the level of H3K4 dimethylation in serving the role of an epigenetic marker of euchromatin, and of H3K9 dimethylation as a marker of transcriptional repression in four different cell lines. We analyzed histone di-methyl-H3K4 and di-methyl-H3K9 using either variable concentrations of nanoparticles or variable time points after cellular uptake. The observed methylation effects decreased consistently with decreasing (AuNPs)-mmi concentrations. Fluorescent microscopy and a binarization algorithm based on a thresholding process with RGB input images demonstrated the continued presence of (AuNPs)-mmi in cells at the lowest concentration used. Furthermore, our results show that the treated cell line used is able to rescue the untreated cell phenotype.

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication.

PubMed

Brustel, Julien; Kirstein, Nina; Izard, Fanny; Grimaud, Charlotte; Prorok, Paulina; Cayrou, Christelle; Schotta, Gunnar; Abdelsamie, Alhassan F; Déjardin, Jérôme; Méchali, Marcel; Baldacci, Giuseppe; Sardet, Claude; Cadoret, Jean-Charles; Schepers, Aloys; Julien, Eric

2017-09-15

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila , partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se , but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes. © 2017 The Authors.

FOXP3 Orchestrates H4K16 Acetylation and H3K4 Tri-Methylation for Activation of Multiple Genes through Recruiting MOF and Causing Displacement of PLU-1

PubMed Central

Katoh, Hiroto; Qin, Zhaohui S.; Liu, Runhua; Wang, Lizhong; Li, Weiquan; Li, Xiangzhi; Wu, Lipeng; Du, Zhanwen; Lyons, Robert; Liu, Chang-Gong; Liu, Xiuping; Dou, Yali; Zheng, Pan; Liu, Yang

2011-01-01

SUMMARY Both H4K16 acetylation and H3K4 tri-methylation are required for gene activation. However, it is still largely unclear how these modifications are orchestrated by transcriptional factors. Here we analyzed the mechanism of the transcriptional activation by FOXP3, an X-linked suppressor of autoimmune diseases and cancers. FOXP3 binds near transcriptional start sites of its target genes. By recruiting MOF and displacing histone H3K4 demethylase PLU-1, FOXP3 increases both H4K16 acetylation and H3K4 tri-methylation at the FOXP3-associated chromatins of multiple FOXP3-activated genes. RNAi-mediated silencing of MOF reduced both gene activation and tumor suppression by FOXP3, while both somatic mutations in clinical cancer samples and targeted mutation of FOXP3 in mouse prostate epithelial disrupted nuclear localization of MOF. Our data demonstrate a pull-push model in which a single transcription factor orchestrates two epigenetic alterations necessary for gene activation and provide a mechanism for somatic inactivation of the FOXP3 protein function in cancer cells. PMID:22152480

Vitamin C induces specific demethylation of H3K9me2 in mouse embryonic stem cells via Kdm3a/b.

PubMed

Ebata, Kevin T; Mesh, Kathryn; Liu, Shichong; Bilenky, Misha; Fekete, Alexander; Acker, Michael G; Hirst, Martin; Garcia, Benjamin A; Ramalho-Santos, Miguel

2017-01-01

Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in naïve ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 h and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes at specific loci. Lastly, we document Kdm3a/b are partially required for the upregulation of germline genes by vitamin C. These results reveal a specific role for vitamin C in histone demethylation in ES cells and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.

Repressive but not activating epigenetic modifications are aberrant on the inactive X chromosome in live cloned cattle.

PubMed

Geng-Sheng, Cao; Yu, Gao; Kun, Wang; Fang-Rong, Ding; Ning, Li

2009-08-01

X inactivation is the process of a chromosome-wide silencing of the majority of genes on the X chromosome during early mammalian development. This process may be aberrant in cloned animals. Here we show that repressive modifications, such as methylation of DNA, and the presence of methylated histones, H3K9me2 and H3K27me3, exhibit distinct aberrance on the inactive X chromosome in live clones. In contrast, H3K4me3, an active gene marker, is obviously missing from the inactive X chromosome in all cattle studied. This suggests that the disappearance of active histone modifications (H3K4me3) seems to be more important for X inactivation than deposition of marks associated with heterochromatin (DNA methylation, H3K27me3 and H3K9me2). It also implies that even apparently normal clones may have subtle abnormalities in repressive, but not activating epigenetic modifications on the inactive X when they survive to term. We also found that the histone H3 methylations were enriched and co-localized at q21-31 of the active X chromosome, which may be associated with an abundance of LINE1 repeat elements. © 2009 The Authors. Journal compilation © 2009 Japanese Society of Developmental Biologists.

The DNA methylation landscape of human melanoma.

PubMed

Jin, Seung-Gi; Xiong, Wenying; Wu, Xiwei; Yang, Lu; Pfeifer, Gerd P

2015-12-01

Using MIRA-seq, we have characterized the DNA methylome of metastatic melanoma and normal melanocytes. Individual tumors contained several thousand hypermethylated regions. We discovered 179 tumor-specific methylation peaks present in all (27/27) melanomas that may be effective disease biomarkers, and 3113 methylation peaks were seen in >40% of the tumors. We found that 150 of the approximately 1200 tumor-associated methylation peaks near transcription start sites (TSSs) were marked by H3K27me3 in melanocytes. DNA methylation in melanoma was specific for distinct H3K27me3 peaks rather than for broadly covered regions. However, numerous H3K27me3 peak-associated TSS regions remained devoid of DNA methylation in tumors. There was no relationship between BRAF mutations and the number of methylation peaks. Gene expression analysis showed upregulated immune response genes in melanomas presumably as a result of lymphocyte infiltration. Down-regulated genes were enriched for melanocyte differentiation factors; e.g., KIT, PAX3 and SOX10 became methylated and downregulated in melanoma. Copyright © 2015 Elsevier Inc. All rights reserved.

DOT1L and H3K79 Methylation in Transcription and Genomic Stability.

PubMed

Wood, Katherine; Tellier, Michael; Murphy, Shona

2018-02-27

The organization of eukaryotic genomes into chromatin provides challenges for the cell to accomplish basic cellular functions, such as transcription, DNA replication and repair of DNA damage. Accordingly, a range of proteins modify and/or read chromatin states to regulate access to chromosomal DNA. Yeast Dot1 and the mammalian homologue DOT1L are methyltransferases that can add up to three methyl groups to histone H3 lysine 79 (H3K79). H3K79 methylation is implicated in several processes, including transcription elongation by RNA polymerase II, the DNA damage response and cell cycle checkpoint activation. DOT1L is also an important drug target for treatment of mixed lineage leukemia (MLL)-rearranged leukemia where aberrant transcriptional activation is promoted by DOT1L mislocalisation. This review summarizes what is currently known about the role of Dot1/DOT1L and H3K79 methylation in transcription and genomic stability.

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

Functional analysis of a Wheat Homeodomain protein, TaR1, reveals that host chromatin remodelling influences the dynamics of the switch to necrotrophic growth in the phytopathogenic fungus Zymoseptoria tritici.

PubMed

Lee, Jack; Orosa, Beatriz; Millyard, Linda; Edwards, Martin; Kanyuka, Kostya; Gatehouse, Angharad; Rudd, Jason; Hammond-Kosack, Kim; Pain, Naomi; Sadanandom, Ari

2015-04-01

A distinguishing feature of Septoria leaf blotch disease in wheat is the long symptomless growth of the fungus amongst host cells followed by a rapid transition to necrotrophic growth resulting in disease lesions. Global reprogramming of host transcription marks this switch to necrotrophic growth. However no information exists on the components that bring about host transcriptional reprogramming. Gene-silencing, confocal-imaging and protein-protein interaction assays where employed to identify a plant homeodomain (PHD) protein, TaR1 in wheat that plays a critical role during the transition from symptomless to necrotrophic growth of Septoria. TaR1-silenced wheat show earlier symptom development upon Septoria infection but reduced fungal sporulation indicating that TaR1 is key for prolonging the symptomless phase and facilitating Septoria asexual reproduction. TaR1 is localized to the nucleus and binds to wheat Histone 3. Trimethylation of Histone 3 at lysine 4 (H3K4) and lysine 36 (H3K36) are found on open chromatin with actively transcribed genes, whereas methylation of H3K27 and H3K9 are associated with repressed loci. TaR1 specifically recognizes dimethylated and trimethylated H3K4 peptides suggesting that it regulates transcriptional activation at open chromatin. We conclude that TaR1 is an important component for the pathogen life cycle in wheat that promotes successful colonization by Septoria. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance

PubMed Central

Zhu, Kunshou; Deng, Yujie; Weng, Guoxing; Hu, Dan; Huang, Cheng; Matsumoto, Keitaro; Nagayasu, Takeshi; Koji, Takehiko; Zheng, Xiongwei; Jiang, Wenhui; Lin, Gen; Cai, Yibin; Weng, Guibin; Chen, Xiaohui

2018-01-01

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA. PMID:29616099

Dynamic regulation of six histone H3 lysine (K) methyltransferases in response to prolonged anoxia exposure in a freshwater turtle.

PubMed

Wijenayake, Sanoji; Hawkins, Liam J; Storey, Kenneth B

2018-04-05

The importance of histone lysine methylation is well established in health, disease, early development, aging, and cancer. However, the potential role of histone H3 methylation in regulating gene expression in response to extended periods of oxygen deprivation (anoxia) in a natural, anoxia-tolerant model system is underexplored. Red-eared sliders (Trachemys scripta elegans) can tolerate and survive three months of absolute anoxia and recover without incurring detrimental cellular damage, mainly by reducing the overall metabolic rate by 90% when compared to normoxia. Stringent regulation of gene expression is a vital aspect of metabolic rate depression in red-eared sliders, and as such we examined the anoxia-responsive regulation of histone lysine methylation in the liver during 5 h and 20 h anoxia exposure. Interestingly, this is the first study to illustrate the existence of histone lysine methyltransferases (HKMTs) and corresponding histone H3 lysine methylation levels in the liver of anoxia-tolerant red-eared sliders. In brief, H3K4me1, a histone mark associated with active transcription, and two corresponding histone lysine methyltransferases that modify H3K4me1 site, significantly increased in response to anoxia. On the contrary, H3K27me1, another transcriptionally active histone mark, significantly decreased during 20 h anoxia, and a transcriptionally repressive histone mark, H3K9me3, and the corresponding KMTs, similarly increased during 20 h anoxia. Overall, the results suggest a dynamic regulation of histone H3 lysine methylation in the liver of red-eared sliders that could theoretically aid in the selective upregulation of genes that are necessary for anoxia survival, while globally suppressing others to conserve energy. Copyright © 2018 Elsevier B.V. All rights reserved.

Specific histone modification responds to arsenic-induced oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Lu

To explore whether specific histone modifications are associated with arsenic-induced oxidative damage, we recruited 138 arsenic-exposed and arsenicosis subjects from Jiaole Village, Xinren County of Guizhou province, China where the residents were exposed to arsenic from indoor coal burning. 77 villagers from Shang Batian Village that were not exposed to high arsenic coal served as the control group. The concentrations of urine and hair arsenic in the arsenic-exposure group were 2.4-fold and 2.1-fold (all P < 0.001) higher, respectively, than those of the control group. Global histone modifications in human peripheral lymphocytes (PBLCs) were examined by ELISA. The results showedmore » that altered global levels of H3K18ac, H3K9me2, and H3K36me3 correlated with both urinary and hair-arsenic levels of the subjects. Notably, H3K36me3 and H3K18ac modifications were associated with urinary 8-OHdG (H3K36me3: β = 0.16; P = 0.042, H3K18ac: β = − 0.24; P = 0.001). We also found that the modifications of H3K18ac and H3K36me3 were enriched in the promoters of oxidative stress response (OSR) genes in human embryonic kidney (HEK) cells and HaCaT cells, providing evidence that H3K18ac and H3K36me3 modifications mediate transcriptional regulation of OSR genes in response to NaAsO{sub 2} treatment. Particularly, we found that reduced H3K18ac modification correlated with suppressed expression of OSR genes in HEK cells with long term arsenic treatment and in PBLCs of all the subjects. Taken together, we reveal a critical role for specific histone modification in response to arsenic-induced oxidative damage. - Highlights: • H3K18ac, H3K9me2 and H3K36me3 were associated with arsenic exposed levels. • H3K18ac and H3K36me3 were correlated with oxidative damage induced by arsenic. • H3K18ac and H3K36me3 might involve in transcriptional regulation of OSR genes. • Dysregulation of H3K18ac and H3K36me3 might be biomarkers of arsenic toxicity.« less

Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines.

PubMed

Riffo-Campos, Ángela L; Gimeno-Valiente, Francisco; Rodríguez, Fernanda M; Cervantes, Andrés; López-Rodas, Gerardo; Franco, Luis; Castillo, Josefa

2018-04-17

Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

Metabolites from the endophytic fungus Sporormiella minimoides isolated from Hintonia latiflora

USDA-ARS?s Scientific Manuscript database

An extract of the solid cultures of Sporormiella minimoides (Sporormiaceae) isolated as an endophytic fungus from Hintonia latiflora (Rubiaceae), yielded three polyketides, 3,6-dimethoxy-8-methyl-1H,6H-benzo[de]isochromene-1,9-dione, 3-hydroxy-1,6,10-trimethoxy-8-methyl-1H,3H-benzo[de]isochromen-9-o...

Dimerization of a Viral SET Protein Endows its Function

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Wei; M Zhou

Histone modifications are regarded as the most indispensible phenomena in epigenetics. Of these modifications, lysine methylation is of the greatest complexity and importance as site- and state-specific lysine methylation exerts a plethora of effects on chromatin structure and gene transcription. Notably, paramecium bursaria chlorella viruses encode a conserved SET domain methyltransferase, termed vSET, that functions to suppress host transcription by methylating histone H3 at lysine 27 (H3K27), a mark for eukaryotic gene silencing. Unlike mammalian lysine methyltransferases (KMTs), vSET functions only as a dimer, but the underlying mechanism has remained elusive. In this study, we demonstrate that dimeric vSET operatesmore » with negative cooperativity between the two active sites and engages in H3K27 methylation one site at a time. New atomic structures of vSET in the free form and a ternary complex with S-adenosyl homocysteine and a histone H3 peptide and biochemical analyses reveal the molecular origin for the negative cooperativity and explain the substrate specificity of H3K27 methyltransferases. Our study suggests a 'walking' mechanism, by which vSET acts all by itself to globally methylate host H3K27, which is accomplished by the mammalian EZH2 KMT only in the context of the Polycomb repressive complex.« less

Intricate Effects of α-Amino and Lysine Modifications on Arginine Methylation of the N-Terminal Tail of Histone H4.

PubMed

Fulton, Melody D; Zhang, Jing; He, Maomao; Ho, Meng-Chiao; Zheng, Y George

2017-07-18

Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

Chromatin signaling to kinetochores: Trans-regulation of Dam1 methylation by histone H2B ubiquitination

PubMed Central

Latham, John A.; Chosed, Renée J.; Wang, Shanzhi; Dent, Sharon Y.R.

2011-01-01

Summary Histone H3K4 trimethylation by the Set1/MLL family of proteins provides a hallmark for transcriptional activity from yeast to humans. In S. cerevisiae, H3K4 methylation is mediated by the Set1-containing COMPASS complex and is regulated in trans by prior ubiquitination of histone H2BK123. All of the events that regulate H2BK123ub and H3K4me are thought to occur at gene promoters. Here we report that this pathway is indispensable for methylation of the only other known substrate of Set1, K233 in Dam1, at kinetochores. Deletion of RAD6, BRE1, or Paf1 complex members abolishes Dam1 methylation, as does mutation of H2BK123. Our results demonstrate that Set1-mediated methylation is regulated by a general pathway regardless of substrate that is composed of transcriptional regulatory factors functioning independently of transcription. Moreover, our data identify a node of regulatory cross-talk in trans between a histone modification and modification on a non-histone protein, demonstrating that changing chromatin states can signal functional changes in other essential cellular proteins and machineries. PMID:21884933

Neurospora Importin α Is Required for Normal Heterochromatic Formation and DNA Methylation

PubMed Central

Klocko, Andrew D.; Rountree, Michael R.; Grisafi, Paula L.; Hays, Shan M.; Adhvaryu, Keyur K.; Selker, Eric U.

2015-01-01

Heterochromatin and associated gene silencing processes play roles in development, genome defense, and chromosome function. In many species, constitutive heterochromatin is decorated with histone H3 tri-methylated at lysine 9 (H3K9me3) and cytosine methylation. In Neurospora crassa, a five-protein complex, DCDC, catalyzes H3K9 methylation, which then directs DNA methylation. Here, we identify and characterize a gene important for DCDC function, dim-3 (defective in methylation-3), which encodes the nuclear import chaperone NUP-6 (Importin α). The critical mutation in dim-3 results in a substitution in an ARM repeat of NUP-6 and causes a substantial loss of H3K9me3 and DNA methylation. Surprisingly, nuclear transport of all known proteins involved in histone and DNA methylation, as well as a canonical transport substrate, appear normal in dim-3 strains. Interactions between DCDC members also appear normal, but the nup-6dim-3 allele causes the DCDC members DIM-5 and DIM-7 to mislocalize from heterochromatin and NUP-6dim-3 itself is mislocalized from the nuclear envelope, at least in conidia. GCN-5, a member of the SAGA histone acetyltransferase complex, also shows altered localization in dim-3, raising the possibility that NUP-6 is necessary to localize multiple chromatin complexes following nucleocytoplasmic transport. PMID:25793375

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 suggest that it may play the same role in meiosis.

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 suggest that it may play the same role in meiosis. PMID:27362481

Hyperglycemia induces a dynamic cooperativity of histone methylase and demethylase enzymes associated with gene-activating epigenetic marks that coexist on the lysine tail.

PubMed

Brasacchio, Daniella; Okabe, Jun; Tikellis, Christos; Balcerczyk, Aneta; George, Prince; Baker, Emma K; Calkin, Anna C; Brownlee, Michael; Cooper, Mark E; El-Osta, Assam

2009-05-01

Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as "hyperglycemic memory." We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. Models of transient hyperglycemia were used to link NFkappaB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFkappaB-p65 chromatin. The sustained upregulation of the NFkappaB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. These studies indicate that the active transcriptional state of the NFkappaB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

PubMed Central

Brasacchio, Daniella; Okabe, Jun; Tikellis, Christos; Balcerczyk, Aneta; George, Prince; Baker, Emma K.; Calkin, Anna C.; Brownlee, Michael; Cooper, Mark E.; El-Osta, Assam

2009-01-01

OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes. PMID:19208907

Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1.

PubMed

Leung, Danny; Du, Tingting; Wagner, Ulrich; Xie, Wei; Lee, Ah Young; Goyal, Preeti; Li, Yujing; Szulwach, Keith E; Jin, Peng; Lorincz, Matthew C; Ren, Bing

2014-05-06

During mammalian development, DNA methylation patterns need to be reset in primordial germ cells (PGCs) and preimplantation embryos. However, many LTR retrotransposons and imprinted genes are impervious to such global epigenetic reprogramming via hitherto undefined mechanisms. Here, we report that a subset of such genomic regions are resistant to widespread erasure of DNA methylation in mouse embryonic stem cells (mESCs) lacking the de novo DNA methyltransferases (Dnmts) Dnmt3a and Dnmt3b. Intriguingly, these loci are enriched for H3K9me3 in mESCs, implicating this mark in DNA methylation homeostasis. Indeed, deletion of the H3K9 methyltransferase SET domain bifurcated 1 (Setdb1) results in reduced H3K9me3 and DNA methylation levels at specific loci, concomitant with increased 5-hydroxymethylation (5hmC) and ten-eleven translocation 1 binding. Taken together, these data reveal that Setdb1 promotes the persistence of DNA methylation in mESCs, likely reflecting one mechanism by which DNA methylation is maintained at LTR retrotransposons and imprinted genes during developmental stages when DNA methylation is reprogrammed.

Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis.

PubMed

Coward, William R; Brand, Oliver J; Pasini, Alice; Jenkins, Gisli; Knox, Alan J; Pang, Linhua

2018-04-01

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-β1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism

NASA Astrophysics Data System (ADS)

Torres, Idelisse Ortiz; Kuchenbecker, Kristopher M.; Nnadi, Chimno I.; Fletterick, Robert J.; Kelly, Mark J. S.; Fujimori, Danica Galonić

2015-02-01

The retinoblastoma binding protein KDM5A removes methyl marks from lysine 4 of histone H3 (H3K4). Misregulation of KDM5A contributes to the pathogenesis of lung and gastric cancers. In addition to its catalytic jumonji C domain, KDM5A contains three PHD reader domains, commonly recognized as chromatin recruitment modules. It is unknown whether any of these domains in KDM5A have functions beyond recruitment and whether they regulate the catalytic activity of the demethylase. Here using biochemical and nuclear magnetic resonance (NMR)-based structural studies, we show that the PHD1 preferentially recognizes unmethylated H3K4 histone tail, product of KDM5A-mediated demethylation of tri-methylated H3K4 (H3K4me3). Binding of unmodified H3 peptide to the PHD1 stimulates catalytic domain-mediated removal of methyl marks from H3K4me3 peptide and nucleosome substrates. This positive-feedback mechanism—enabled by the functional coupling between a reader and a catalytic domain in KDM5A—suggests a model for the spread of demethylation on chromatin.

Degron Protease Blockade Sensor to Image Epigenetic Histone Protein Methylation in Cells and Living Animals

PubMed Central

2015-01-01

Lysine methylation of histone H3 and H4 has been identified as a promising therapeutic target in treating various cellular diseases. The availability of an in vivo assay that enables rapid screening and preclinical evaluation of drugs that potentially target this cellular process will significantly expedite the pace of drug development. This study is the first to report the development of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)3]-[H3-K9]-[cODC]) that can detect and monitor the methylation status of a specific histone lysine methylation mark (H3-K9) in live animals. The sensitivity of this sensor was assessed in various cell lines, in response to down-regulation of methyltransferase EHMT2 by specific siRNA, and in nude mice with lysine replacement mutants. In vivo imaging in response to a combination of methyltransferase inhibitors BIX01294 and Chaetocin in mice reveals the potential of this sensor for preclinical drug evaluation. This biosensor thus has demonstrated its utility in the detection of H3-K9 methylations in vivo and potential value in preclinical drug development. PMID:25489787

Methylation of DNA Ligase 1 by G9a/GLP Recruits UHRF1 to Replicating DNA and Regulates DNA Methylation.

PubMed

Ferry, Laure; Fournier, Alexandra; Tsusaka, Takeshi; Adelmant, Guillaume; Shimazu, Tadahiro; Matano, Shohei; Kirsh, Olivier; Amouroux, Rachel; Dohmae, Naoshi; Suzuki, Takehiro; Filion, Guillaume J; Deng, Wen; de Dieuleveult, Maud; Fritsch, Lauriane; Kudithipudi, Srikanth; Jeltsch, Albert; Leonhardt, Heinrich; Hajkova, Petra; Marto, Jarrod A; Arita, Kyohei; Shinkai, Yoichi; Defossez, Pierre-Antoine

2017-08-17

DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance. Copyright © 2017 Elsevier Inc. All rights reserved.

Histone H3 Lysine 36 Trimethylation Is Established over the Xist Promoter by Antisense Tsix Transcription and Contributes to Repressing Xist Expression

PubMed Central

Ohhata, Tatsuya; Matsumoto, Mika; Leeb, Martin; Shibata, Shinwa; Sakai, Satoshi; Kitagawa, Kyoko; Niida, Hiroyuki

2015-01-01

One of the two X chromosomes in female mammals is inactivated by the noncoding Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix, which represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 leads to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited by Tsix cotranscriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression of Xist. Moreover, depletion of the H3K36 methylase Setd2 leads to upregulation of Xist, suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over the Xist promoter, indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter. PMID:26370508

The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

PubMed Central

Zou, Tie; Yao, Annie Y.; Cooper, Marcus P.; Boyartchuk, Victor; Wang, Yong-Xu

2012-01-01

Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes. PMID:22719268

Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

PubMed Central

Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

2012-01-01

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359

H3K9me2/3 Binding of the MBT Domain Protein LIN-61 Is Essential for Caenorhabditis elegans Vulva Development

PubMed Central

Koester-Eiserfunke, Nora; Fischle, Wolfgang

2011-01-01

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3 binding proteins, LIN-61 and HPL-2, as well as the H3K9MT MET-2 in distinct developmental pathways. PMID:21437264

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

PubMed Central

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

2016-01-01

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 terms of gene expression and behavior. PMID:27078100

Thermal Decomposition of Potential Ester Biofuels. Part I: Methyl Acetate and Methyl Butanoate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Porterfield, Jessica P.; Bross, David H.; Ruscic, Branko

2017-06-09

Two methyl esters have been examined as models for the pyrolysis of biofuels. Dilute samples (0.06 - 0.13%) of methyl acetate (CH 3COOCH 3) and methyl butanoate (CH 3CH 2CH 2COOCH 3) were entrained in (He, Ar) carrier gas and decomposed in a set of flash-pyrolysis micro-reactors. The pyrolysis products resulting from the methyl esters were detected and identified by vacuum ultraviolet photoionization mass spectrometry. Complementary product identification was provided by matrix infrared absorption spectroscopy. Pyrolysis pressures in the pulsed micro-reactor were roughly 20 Torr and residence times through the reactors were approximately 25 - 150 µs. Reactor temperatures ofmore » 300 – 1600 K were explored. Decomposition of CH 3COOCH 3 commences at 1000 K and the initial products are (CH 2=C=O and CH 3OH). As the micro-reactor is heated to 1300 K, a mixture of (CH 2=C=O and CH 3OH, CH 3, CH 2=O, H, CO, CO 2) appears. The thermal cracking of CH 3CH 2CH 2COOCH 3 begins at 800 K with the formation of (CH 3CH 2CH=C=O, CH 3OH). By 1300 K, the pyrolysis of methyl butanoate yields a complex mixture of (CH 3CH 2CH=C=O, CH 3OH, CH 3, CH 2=O, CO, CO 2, CH 3CH=CH 2, CH 2CHCH 2, CH 2=C=CH 2, HCCCH 2, CH 2=C=C=O, CH 2=CH 2, HCΞCH, CH 2=C=O). Based on the results from the thermal cracking of methyl acetate and methyl butanoate, we predict several important decomposition channels for the pyrolysis of fatty acid methyl esters, R CH 2-COOCH 3. The lowest energy fragmentation will be a 4-center elimination of methanol to form the ketene, RCH=C=O. At higher temperatures, concerted fragmentation to radicals will ensue to produce a mixture of species: (RCH 2 + CO 2 + CH 3) and (RCH 2 + CO + CH 2=O + H). Thermal cracking of the β C-C bond of the methyl ester will generate the radicals (R and H) as well as CH 2=C=O + CH 2=O. The thermochemistry of methyl acetate and its fragmentation products have been obtained via the Active Thermochemical Tables (ATcT) approach, resulting in Δ fH 298(CH 3COOCH 3) = -98.7 ± 0.2 kcal mol -1, Δ fH 298(CH 3CO 2) = -45.7 ± 0.3 kcal mol -1, and Δ fH 298(COOCH 3) = -38.3 ± 0.4 kcal mol -1.« less

Distinct molecular profile of diffuse cerebellar gliomas.

PubMed

Nomura, Masashi; Mukasa, Akitake; Nagae, Genta; Yamamoto, Shogo; Tatsuno, Kenji; Ueda, Hiroki; Fukuda, Shiro; Umeda, Takayoshi; Suzuki, Tomonari; Otani, Ryohei; Kobayashi, Keiichi; Maruyama, Takashi; Tanaka, Shota; Takayanagi, Shunsaku; Nejo, Takahide; Takahashi, Satoshi; Ichimura, Koichi; Nakamura, Taishi; Muragaki, Yoshihiro; Narita, Yoshitaka; Nagane, Motoo; Ueki, Keisuke; Nishikawa, Ryo; Shibahara, Junji; Aburatani, Hiroyuki; Saito, Nobuhito

2017-12-01

Recent studies have demonstrated that tumor-driving alterations are often different among gliomas that originated from different brain regions and have underscored the importance of analyzing molecular characteristics of gliomas stratified by brain region. Therefore, to elucidate molecular characteristics of diffuse cerebellar gliomas (DCGs), 27 adult, mostly glioblastoma cases were analyzed. Comprehensive analysis using whole-exome sequencing, RNA sequencing, and Infinium methylation array (n = 17) demonstrated their distinct molecular profile compared to gliomas in other brain regions. Frequent mutations in chromatin-modifier genes were identified including, noticeably, a truncating mutation in SETD2 (n = 4), which resulted in loss of H3K36 trimethylation and was mutually exclusive with H3F3A K27M mutation (n = 3), suggesting that epigenetic dysregulation may lead to DCG tumorigenesis. Alterations that cause loss of p53 function including TP53 mutation (n = 9), PPM1D mutation (n = 2), and a novel type of PPM1D fusion (n = 1), were also frequent. On the other hand, mutations and copy number changes commonly observed in cerebral gliomas were infrequent. DNA methylation profile analysis demonstrated that all DCGs except for those with H3F3A mutations were categorized in the "RTK I (PDGFRA)" group, and those DCGs had a gene expression signature that was highly associated with PDGFRA. Furthermore, compared with the data of 315 gliomas derived from different brain regions, promoter methylation of transcription factors genes associated with glial development showed a characteristic pattern presumably reflecting their tumor origin. Notably, SOX10, a key transcription factor associated with oligodendroglial differentiation and PDGFRA regulation, was up-regulated in both DCG and H3 K27M-mutant diffuse midline glioma, suggesting their developmental and biological commonality. In contrast, SOX10 was silenced by promoter methylation in most cerebral gliomas. These findings may suggest potential tailored targeted therapy for gliomas according to their brain region, in addition to providing molecular clues to identify the region-related cellular origin of DCGs.

Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases

PubMed Central

2015-01-01

JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of “methylation state” and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the “methylation state” and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators. PMID:25335116

Chemical constituents from Hericium erinaceus and their ability to stimulate NGF-mediated neurite outgrowth on PC12 cells.

PubMed

Zhang, Cheng-Chen; Yin, Xia; Cao, Chen-Yu; Wei, Jing; Zhang, Qiang; Gao, Jin-Ming

2015-11-15

One new meroterpenoid, named hericenone K (11), along with 10 known compounds (1-10), ergosterol peroxide (1), cerevisterol (2), 3β,5α,9α-trihydroxy-ergosta-7,22-dien-6-one (3), inoterpene A (4), astradoric acid C (5), betulin (6), oleanolic acid (7), ursolic acid (8), hemisceramide (9), and 3,4-dihydro-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl)-9(7H)-oxo-2H-furo[3,4-h]benzopyran (10), was isolated from the fruiting bodies of the mushroom Hericium erinaceus. Their structures were characterized on the basis of spectroscopic methods, as well as through comparison with previously reported data. Compounds 3-6, 8, and 9 were isolated from Hericium species for the first time. Compounds 10 and 11 was suggested to be racemic by the CD spectrum data and specific rotations, which ware resolved by chiral HPLC into respective enantiomers. Compounds 1-3, (±)-10, (-)-10 and (+)-10 in the presence of NGF (20 ng/mL) exerted a significant increase in neurite-bearing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

PubMed

Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

2017-10-16

Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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 response to LPS. Conclusions These data suggest that prenatal ethanol modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction of POMC gene expression. Gestational choline supplementation prevents the adverse effects of ethanol on these neurons. PMID:23413810

KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation

PubMed Central

McDowell-Sargent, Maria; Uppal, Timsy; Purushothaman, Pravinkumar

2017-01-01

Kaposi’s sarcoma associated herpesvirus (KSHV) persists in a highly-ordered chromatin structure inside latently infected cells with the majority of the viral genome having repressive marks. However, upon reactivation the viral chromatin landscape changes into ‘open’ chromatin through the involvement of lysine demethylases and methyltransferases. Besides methylation of lysine residues of histone H3, arginine methylation of histone H4 plays an important role in controlling the compactness of the chromatin. Symmetric methylation of histone H4 at arginine 3 (H4R3me2s) negatively affects the methylation of histone H3 at lysine 4 (H3K4me3), an active epigenetic mark deposited on the viral chromatin during reactivation. We identified a novel binding partner to KSHV viral DNA processivity factor, ORF59-a protein arginine methyl transferase 5 (PRMT5). PRMT5 is an arginine methyltransferase that dimethylates arginine 3 (R3) of histone H4 in a symmetric manner, one hallmark of condensed chromatin. Our ChIP-seq data of symmetrically methylated H4 arginine 3 showed a significant decrease in H4R3me2s on the viral genome of reactivated cells as compared to the latent cells. Reduction in arginine methylation correlated with the binding of ORF59 on the viral chromatin and disruption of PRMT5 from its adapter protein, COPR5 (cooperator of PRMT5). Binding of PRMT5 through COPR5 is important for symmetric methylation of H4R3 and the expression of ORF59 competitively reduces the association of PRMT5 with COPR5, leading to a reduction in PRMT5 mediated arginine methylation. This ultimately resulted in a reduced level of symmetrically methylated H4R3 and increased levels of H3K4me3 marks, contributing to the formation of an open chromatin for transcription and DNA replication. Depletion of PRMT5 levels led to a decrease in symmetric methylation and increase in viral gene transcription confirming the role of PRMT5 in viral reactivation. In conclusion, ORF59 modulates histone-modifying enzymes to alter the chromatin structure during lytic reactivation. PMID:28678843

KSHV encoded ORF59 modulates histone arginine methylation of the viral genome to promote viral reactivation.

PubMed

Strahan, Roxanne C; McDowell-Sargent, Maria; Uppal, Timsy; Purushothaman, Pravinkumar; Verma, Subhash C

2017-07-01

Kaposi's sarcoma associated herpesvirus (KSHV) persists in a highly-ordered chromatin structure inside latently infected cells with the majority of the viral genome having repressive marks. However, upon reactivation the viral chromatin landscape changes into 'open' chromatin through the involvement of lysine demethylases and methyltransferases. Besides methylation of lysine residues of histone H3, arginine methylation of histone H4 plays an important role in controlling the compactness of the chromatin. Symmetric methylation of histone H4 at arginine 3 (H4R3me2s) negatively affects the methylation of histone H3 at lysine 4 (H3K4me3), an active epigenetic mark deposited on the viral chromatin during reactivation. We identified a novel binding partner to KSHV viral DNA processivity factor, ORF59-a protein arginine methyl transferase 5 (PRMT5). PRMT5 is an arginine methyltransferase that dimethylates arginine 3 (R3) of histone H4 in a symmetric manner, one hallmark of condensed chromatin. Our ChIP-seq data of symmetrically methylated H4 arginine 3 showed a significant decrease in H4R3me2s on the viral genome of reactivated cells as compared to the latent cells. Reduction in arginine methylation correlated with the binding of ORF59 on the viral chromatin and disruption of PRMT5 from its adapter protein, COPR5 (cooperator of PRMT5). Binding of PRMT5 through COPR5 is important for symmetric methylation of H4R3 and the expression of ORF59 competitively reduces the association of PRMT5 with COPR5, leading to a reduction in PRMT5 mediated arginine methylation. This ultimately resulted in a reduced level of symmetrically methylated H4R3 and increased levels of H3K4me3 marks, contributing to the formation of an open chromatin for transcription and DNA replication. Depletion of PRMT5 levels led to a decrease in symmetric methylation and increase in viral gene transcription confirming the role of PRMT5 in viral reactivation. In conclusion, ORF59 modulates histone-modifying enzymes to alter the chromatin structure during lytic reactivation.

The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns.

PubMed

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

2016-01-01

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

The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

PubMed Central

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

2016-01-01

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

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection

PubMed Central

Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

2017-01-01

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were “DNA methylation-sensitive” genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A. The other half were “DNA methylation-resistant” genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site. PMID:28903418

Histone modification alteration coordinated with acquisition of promoter DNA methylation during Epstein-Barr virus infection.

PubMed

Funata, Sayaka; Matsusaka, Keisuke; Yamanaka, Ryota; Yamamoto, Shogo; Okabe, Atsushi; Fukuyo, Masaki; Aburatani, Hiroyuki; Fukayama, Masashi; Kaneda, Atsushi

2017-08-15

Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer. Epstein-Barr virus positive gastric cancer is the most frequently hypermethylated tumor among human malignancies. Herein, we performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration during EBV infection into gastric cancer cell line MKN7. Among 7,775 genes with increased DNA methylation in promoter regions, roughly half were "DNA methylation-sensitive" genes, which acquired DNA methylation in the whole promoter regions and thus were repressed. These included anti-oncogenic genes, e.g. CDKN2A . The other half were "DNA methylation-resistant" genes, where DNA methylation is acquired in the surrounding of promoter regions, but unmethylated status is protected in the vicinity of transcription start site. These genes thereby retained gene expression, and included DNA repair genes. Histone modification was altered dynamically and coordinately with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks, H3K4me3 and H3K27ac. Apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes. Genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Our data show that orchestrated epigenetic alterations are important in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at transcription start site.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer

PubMed Central

Hon, Gary C.; Hawkins, R. David; Caballero, Otavia L.; Lo, Christine; Lister, Ryan; Pelizzola, Mattia; Valsesia, Armand; Ye, Zhen; Kuan, Samantha; Edsall, Lee E.; Camargo, Anamaria Aranha; Stevenson, Brian J.; Ecker, Joseph R.; Bafna, Vineet; Strausberg, Robert L.; Simpson, Andrew J.; Ren, Bing

2012-01-01

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells. PMID:22156296

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

PubMed Central

Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

2016-01-01

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

Investigating the effects of in utero benzene exposure on epigenetic modifications in maternal and fetal CD-1 mice.

PubMed

Philbrook, Nicola A; Winn, Louise M

2015-11-15

Exposure to the ubiquitous environmental pollutant benzene is positively correlated with leukemia in adults and may be associated with childhood leukemia following in utero exposure. While numerous studies implicate oxidative stress and DNA damage as playing a role in benzene-mediated carcinogenicity, emerging evidence suggests that alterations in epigenetic regulations may be involved. The present study aimed to determine whether DNA methylation and/or various histone modifications were altered following in utero benzene exposure in CD-1 mice. Global DNA methylation and promoter-specific methylation of the tumor suppressor gene, p15, were assessed. Additionally, levels of acetylated histones H3, H4, and H3K56, as well as methylated histones H3K9 and H3K27 were assessed by Western blotting. A significant decrease in global DNA methylation of maternal bone marrow was observed following benzene exposure; however no effect on global DNA methylation was detected in fetal livers. Additionally, no effect of benzene exposure was observed on p15 promoter methylation or any measured histone modifications in both maternal bone marrow and fetal livers. These results suggest that the methodology used in the present study did not reveal alterations in DNA methylation and histone modifications following in utero exposure to benzene; however further experimentation investigating these modifications at the whole genome/epigenome level, as well as at later stages of benzene-induced carcinogenesis, are warranted. Copyright © 2015 Elsevier Inc. All rights reserved.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

PubMed Central

McCabe, Michael T.; Graves, Alan P.; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S.; Jiang, Yong; Smitheman, Kimberly N.; Ott, Heidi M.; Pappalardi, Melissa B.; Allen, Kimberly E.; Chen, Stephanie B.; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M.; Gilbert, Seth A.; Thrall, Sara H.; Tummino, Peter J.; Kruger, Ryan G.; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L.

2012-01-01

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 kcat/Km ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 kcat/Km ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 kcat/Km ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2. PMID:22323599

Associations between Blood and Urine Arsenic Concentrations and Global Levels of Post-Translational Histone Modifications in Bangladeshi Men and Women

PubMed Central

Howe, Caitlin G.; Liu, Xinhua; Hall, Megan N.; Slavkovich, Vesna; Ilievski, Vesna; Parvez, Faruque; Siddique, Abu B.; Shahriar, Hasan; Uddin, Mohammad N.; Islam, Tariqul; Graziano, Joseph H.; Costa, Max; Gamble, Mary V.

2016-01-01

Background: Exposure to inorganic arsenic is associated with numerous adverse health outcomes, with susceptibility differing by sex. Although evidence from in vitro studies suggests that arsenic alters post-translational histone modifications (PTHMs), evidence in humans is limited. Objectives: The objectives were to determine: a) if arsenic exposure is associated with global (percent) levels of PTHMs H3K36me2, H3K36me3, and H3K79me2 in a sex-dependent manner, and b) if %PTHMs are stable when arsenic exposure is reduced. Methods: We examined associations between arsenic, measured in blood and urine, and %PTHMs in peripheral blood mononuclear cells from 317 participants enrolled in the Bangladesh Folic Acid and Creatine Trial (FACT). We also examined the stability of %PTHMs after the use of arsenic-removal water filters (n = 60). Results: Associations between natural log–transformed (ln) urinary arsenic, adjusted for creatinine (uAsCr), and %H3K36me2 differed significantly between men and women (p = 0.01). ln(uAsCr) was positively associated with %H3K36me2 in men [β = 0.12; 95% confidence interval (CI): 0.01, 0.23, p = 0.03] but was negatively associated with %H3K36me2 in women (β = –0.05; 95% CI: –0.12, 0.02, p = 0.19). The patterns of associations with blood arsenic were similar. On average, water filter use was also associated with reductions in %H3K36me2 (p < 0.01), but this did not differ significantly by sex. Arsenic was not significantly associated with %H3K36me3 or %H3K79me2 in men or women. Conclusions: Arsenic exposure was associated with %H3K36me2 in a sex-specific manner but was not associated with %H3K36me3 or %H3K79me2. Additional studies are needed to assess changes in %H3K36me2 after arsenic removal. Citation: Howe CG, Liu X, Hall MN, Slavkovich V, Ilievski V, Parvez F, Siddique AB, Shahriar H, Uddin MN, Islam T, Graziano JH, Costa M, Gamble MV. 2016. Associations between blood and urine arsenic concentrations and global levels of post-translational histone modifications in Bangladeshi men and women. Environ Health Perspect 124:1234–1240; http://dx.doi.org/10.1289/ehp.1510412 PMID:26967670

DOE Office of Scientific and Technical Information (OSTI.GOV)

Philbrook, Nicola A.; Winn, Louise M., E-mail: winnl@queensu.ca; School of Environmental Studies, Queen's University, Kingston, ON K7L3N6

Exposure to the ubiquitous environmental pollutant benzene is positively correlated with leukemia in adults and may be associated with childhood leukemia following in utero exposure. While numerous studies implicate oxidative stress and DNA damage as playing a role in benzene-mediated carcinogenicity, emerging evidence suggests that alterations in epigenetic regulations may be involved. The present study aimed to determine whether DNA methylation and/or various histone modifications were altered following in utero benzene exposure in CD-1 mice. Global DNA methylation and promoter-specific methylation of the tumor suppressor gene, p15, were assessed. Additionally, levels of acetylated histones H3, H4, and H3K56, as wellmore » as methylated histones H3K9 and H3K27 were assessed by Western blotting. A significant decrease in global DNA methylation of maternal bone marrow was observed following benzene exposure; however no effect on global DNA methylation was detected in fetal livers. Additionally, no effect of benzene exposure was observed on p15 promoter methylation or any measured histone modifications in both maternal bone marrow and fetal livers. These results suggest that the methodology used in the present study did not reveal alterations in DNA methylation and histone modifications following in utero exposure to benzene; however further experimentation investigating these modifications at the whole genome/epigenome level, as well as at later stages of benzene-induced carcinogenesis, are warranted. - Highlights: • Benzene exposure in pregnant mice decreased global DNA methylation in maternal bone marrow. • Benzene exposure in pregnant mice had no effect on global DNA methylation in fetal livers. • No effect of benzene exposure was observed on p15 promoter methylation. • No effect of benzene on measured histone modifications in both maternal bone marrow and fetal livers was observed.« less

Rate Constant and RRKM Product Study for the Reaction Between CH3 and C2H3 at T = 298K

NASA Technical Reports Server (NTRS)

Thorn, R. Peyton, Jr.; Payne, Walter A., Jr.; Chillier, Xavier D. F.; Stief, Louis J.; Nesbitt, Fred L.; Tardy, D. C.

2000-01-01

The total rate constant k1 has been determined at P = 1 Torr nominal pressure (He) and at T = 298 K for the vinyl-methyl cross-radical reaction CH3 + C2H3 yields products. The measurements were performed in a discharge flow system coupled with collision-free sampling to a mass spectrometer operated at low electron energies. Vinyl and methyl radicals were generated by the reactions of F with C2H4 and CH4, respectively. The kinetic studies were performed by monitoring the decay of C2H3 with methyl in excess, 6 < |CH3|(sub 0)/|C2H3|(sub 0) < 21. The overall rate coefficient was determined to be k1(298 K) = (1.02 +/- 0.53)x10(exp -10) cubic cm/molecule/s with the quoted uncertainty representing total errors. Numerical modeling was required to correct for secondary vinyl consumption by reactions such as C2H3 + H and C2H3 + C2H3. The present result for k1 at T = 298 K is compared to two previous studies at high pressure (100-300 Torr He) and to a very recent study at low pressure (0.9-3.7 Torr He). Comparison is also made with the rate constant for the similar reaction CH3 + C2H5 and with a value for k1 estimated by the geometric mean rule employing values for k(CH3 + CH3) and k(C2H3 + C2H3). Qualitative product studies at T = 298 K and 200 K indicated formation of C3H6, C2H2, and C2H5 as products of the combination-stabilization, disproportionation, and combination-decomposition channels, respectively, of the CH3 + C2H3 reaction. We also observed the secondary C4H8 product of the subsequent reaction of C3H5 with excess CH3; this observation provides convincing evidence for the combination-decomposition channel yielding C3H5 + H. RRKM calculations with helium as the deactivator support the present and very recent experimental observations that allylic C-H bond rupture is an important path in the combination reaction. The pressure and temperature dependencies of the branching fractions are also predicted.

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).

PubMed

McCabe, Michael T; Graves, Alan P; Ganji, Gopinath; Diaz, Elsie; Halsey, Wendy S; Jiang, Yong; Smitheman, Kimberly N; Ott, Heidi M; Pappalardi, Melissa B; Allen, Kimberly E; Chen, Stephanie B; Della Pietra, Anthony; Dul, Edward; Hughes, Ashley M; Gilbert, Seth A; Thrall, Sara H; Tummino, Peter J; Kruger, Ryan G; Brandt, Martin; Schwartz, Benjamin; Creasy, Caretha L

2012-02-21

Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.

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

PubMed Central

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

2015-01-01

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. PMID:25538301

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

PubMed

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

2015-06-02

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

Histone H3K9 Demethylase JMJD2B Activates Adipogenesis by Regulating H3K9 Methylation on PPARγ and C/EBPα during Adipogenesis

PubMed Central

Jang, Min-Kyung; Kim, Ji-Hyun; Jung, Myeong Ho

2017-01-01

Previous studies have shown that tri- or di-methylation of histone H3 at lysine 9 (H3K9me3/me2) on the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) contribute to the repression of PPARγ and C/EBPα and inhibition of adipogenesis in 3T3-L1 preadipocytes. The balance of histone methylation is regulated by histone methyltransferases and demethylases. However, it is poorly understood which demethylases are responsible for removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα. JMJD2B is a H3K9me3/me2 demethylase that was previously shown to activate adipogenesis by promoting mitotic clonal expansion. Nevertheless, it remains unclear whether JMJD2B plays a role in the regulation of adipogenesis by removing H3K9me3/me2 on the promoter of PPARγ and C/EBPα and subsequently activating PPARγ and C/EBPα expression. Here, we showed that JMJD2B decreased H3K9me3/me2 on the promoter of PPARγ and C/EBPα, which in turn stimulated the expression of PPARγ and C/EBPα. JMJD2B knockdown using siRNA in 3T3-L1 preadipocytes repressed the expression of PPARγ and C/EBPα, resulting in inhibition of adipogenesis. This was accompanied by increased enrichment of H3K9me3/me2 on the promoter of PPARγ and C/EBPα. In contrast, overexpression of JMJD2B increased the expression of PPARγ and C/EBPα, which was accompanied by decreased enrichment of H3K9me3/me2 on the promoter and activated adipogenesis. Together, these results indicate that JMJD2B regulates PPARγ and C/EBPα during adipogenesis. PMID:28060835

HDAC inhibitors induce global changes in histone lysine and arginine methylation and alter expression of lysine demethylases.

PubMed

Lillico, Ryan; Sobral, Marina Gomez; Stesco, Nicholas; Lakowski, Ted M

2016-02-05

Histone deacetylase (HDAC) inhibitors are cancer treatments that inhibit the removal of the epigenetic modification acetyllysine on histones, resulting in altered gene expression. Such changes in expression may influence other histone epigenetic modifications. We describe a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify lysine acetylation and methylation and arginine methylation on histones extracted from cultured cells treated with HDAC inhibitors. The HDAC inhibitors vorinostat, mocetinostat and entinostat induced 400-600% hyperacetylation in HEK 293 and K562 cells. All HDAC inhibitors decreased histone methylarginines in HEK 293 cells but entinostat produced dose dependent reductions in asymmetric dimethylarginine, not observed in K562 cells. Vorinostat produced increases in histone lysine methylation and decreased expression of some lysine demethylases (KDM), measured by quantitative PCR. Entinostat had variable effects on lysine methylation and decreased expression of some KDM while increasing expression of others. Mocetinostat produced dose dependent increases in histone lysine methylation by LC-MS/MS. This was corroborated with a multiplex colorimetric assay showing increases in histone H3 lysine 4, 9, 27, 36 and 79 methylation. Increases in lysine methylation were correlated with dose dependent decreases in the expression of seven KDM. Mocetinostat functions as an HDAC inhibitor and a de facto KDM inhibitor. Copyright © 2015 Elsevier B.V. All rights reserved.

H3 K79 dimethylation marks developmental activation of the beta-globin gene but is reduced upon LCR-mediated high-level transcription.

PubMed

Sawado, Tomoyuki; Halow, Jessica; Im, Hogune; Ragoczy, Tobias; Bresnick, Emery H; Bender, M A; Groudine, Mark

2008-07-15

Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired beta-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Delta locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the beta-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and DeltaLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the DeltaLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with beta-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level beta-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

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

PubMed

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

2013-07-01

Prenatal exposure to ethanol (EtOH) 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, because 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 EtOH on these neurons. 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 immunosorbent assay. Prenatal EtOH treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10), and increased the repressive marks (H3K9me2, G9a, Setdb1), 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 EtOH-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 response to LPS. These data suggest that prenatal EtOH modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction in POMC gene expression. Gestational choline supplementation prevents the adverse effects of EtOH on these neurons. Copyright © 2013 by the Research Society on Alcoholism.

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

PubMed

Diaz, Elsie; Machutta, Carl A; Chen, Stephanie; Jiang, Yong; Nixon, Christopher; Hofmann, Glenn; Key, Danielle; Sweitzer, Sharon; Patel, Mehul; Wu, Zining; Creasy, Caretha L; Kruger, Ryan G; LaFrance, Louis; Verma, Sharad K; Pappalardi, Melissa B; Le, Baochau; Van Aller, Glenn S; McCabe, Michael T; Tummino, Peter J; Pope, Andrew J; Thrall, Sara H; Schwartz, Benjamin; Brandt, Martin

2012-12-01

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

High expression of trimethylated histone H3 at lysine 27 predicts better prognosis in non-small cell lung cancer.

PubMed

Chen, Xiaohui; Song, Ning; Matsumoto, Keitaro; Nanashima, Atsushi; Nagayasu, Takeshi; Hayashi, Tomayoshi; Ying, Mingang; Endo, Daisuke; Wu, Zhiren; Koji, Takehiko

2013-11-01

Epigenetic parameters such as DNA methylation and histone modifications play pivotal roles in carcinogenesis. Global histone modification patterns have been implicated as possible predictors of cancer recurrence and prognoses in a great variety of tumor entities. Our study was designed to evaluate the association among trimethylated histone H3 at lysine 27 (H3K27me3), clinicopathological variables and outcome in early-stage non-small cell lung cancer (NSCLC). The expression of H3K27me3 and its methyl-transferase, enhancer of zeste homolog 2 (EZH2) together with proliferating cell nuclear antigen (PCNA) were evaluated by immunohistochemistry in normal lung tissue (n=5) and resected NSCLC patients (n=42). In addition, the specificity of antibody for H3K27me3 was tested by western blot analysis. The optimal cut-off point of H3K27me3 expression for prognosis was determined by the X-tile program. The prognostic significance was determined by means of Kaplan-Meier survival estimates and log-rank tests. As a result, enhanced trimethylation of H3K27me3 was correlated with longer overall survival (OS) and better prognosis (P<0.05). Moreover, both univariate and multivariate analyses indicated that H3K27me3 level was a significant and independent predictor of better survival (hazard ratio, 0.187; 95% confidence interval, 0.066-0.531, P=0.002). Furthermore, H3K27me3 expression was positively correlated with DNA methylation level at CCGG sites while reversely related to EZH2 expression (P<0.05). In conclusion, H3K27me3 level defines unrecognized subgroups of NSCLC patients with distinct epigenetic phenotype and clinical outcome, and can probably be used as a novel predictor for better prognosis in NSCLC patients.

DNA topoisomerase 1α promotes transcriptional silencing of transposable elements through DNA methylation and histone lysine 9 dimethylation in Arabidopsis.

PubMed

Dinh, Thanh Theresa; Gao, Lei; Liu, Xigang; Li, Dongming; Li, Shengben; Zhao, Yuanyuan; O'Leary, Michael; Le, Brandon; Schmitz, Robert J; Manavella, Pablo A; Manavella, Pablo; Li, Shaofang; Weigel, Detlef; Pontes, Olga; Ecker, Joseph R; Chen, Xuemei

2014-07-01

RNA-directed DNA methylation (RdDM) and histone H3 lysine 9 dimethylation (H3K9me2) are related transcriptional silencing mechanisms that target transposable elements (TEs) and repeats to maintain genome stability in plants. RdDM is mediated by small and long noncoding RNAs produced by the plant-specific RNA polymerases Pol IV and Pol V, respectively. Through a chemical genetics screen with a luciferase-based DNA methylation reporter, LUCL, we found that camptothecin, a compound with anti-cancer properties that targets DNA topoisomerase 1α (TOP1α) was able to de-repress LUCL by reducing its DNA methylation and H3K9me2 levels. Further studies with Arabidopsis top1α mutants showed that TOP1α silences endogenous RdDM loci by facilitating the production of Pol V-dependent long non-coding RNAs, AGONAUTE4 recruitment and H3K9me2 deposition at TEs and repeats. This study assigned a new role in epigenetic silencing to an enzyme that affects DNA topology.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

E Rajakumara; Z Wang; H Ma

2011-12-31

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajakumara, Eerappa; Wang, Zhentian; Ma, Honghui

2011-08-29

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

Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3-HP1-Me(3)K20H4 pathway.

PubMed

Bongiorni, Silvia; Pasqualini, Barbara; Taranta, Monia; Singh, Prim B; Prantera, Giorgio

2007-03-15

Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain 'lagging' chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set.

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

PubMed Central

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

2016-01-01

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer. PMID:27528607

Molecular structure and conformational composition of methyl chloroacetate: An electron-diffraction and ab initio molecular orbital investigation

NASA Astrophysics Data System (ADS)

Aarset, Kirsten; Boldermo, Kjell Gunnar; Hagen, Kolbjørn

2010-08-01

The molecular structure and conformational composition of methyl chloroacetate, H 2ClC sbnd C( dbnd O) sbnd O sbnd CH 3, have been determined by gas-phase electron-diffraction (GED), using results from ab initio molecular orbital calculations (HF, MP2 and MP3/6-311+G(d,p)) to obtain constraints on some of the structural parameters. The molecules exist in the gas-phase at 25 °C as a mixture of two stable conformers: syn with C sbnd Cl eclipsing C dbnd O and gauche with C sbnd H approximately eclipsing C dbnd O. In both of these conformers O sbnd CH 3 is also eclipsing C dbnd O. The experimentally observed conformational composition at 25 °C was 36(8)% syn and 64(8)% gauche (parenthesised values are 2 σ), corresponding to a free energy difference between conformers of ΔGexp° = 1.4(9) kJ/mol. The corresponding theoretical values obtained for Δ G° are 1.1 kJ/mol (HF), 2.3 kJ/mol (MP2), and 2.4 kJ/mol (MP3). The results for the principal distances ( rh1) and angles ( ∠h1) for the major gauche conformer obtained from the combined GED/ ab initio study (2 σ uncertainties) are r(CO sbnd CCl) = 1.502(9) Å, r(C sbnd H) = 1.084(6) Å (average value), r(C sbnd Cl) = 1.782(4) Å, r(C dbnd O) = 1.213(4) Å, r(CO sbnd O) = 1.346(4) Å, r(CH 3sbnd O) = 1.468(10) Å, ∠C sbnd C sbnd Cl = 110.0(6)°, ∠C sbnd C dbnd O = 124.7(6)°, ∠C sbnd C sbnd O = 108.3(10)°, ∠C sbnd O sbnd C = 115.9(8)°, ϕ(Cl sbnd C sbnd C dbnd O) = 111(2)°, ϕ(C sbnd O sbnd C dbnd O) = 3(3)°.

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

PubMed Central

Song, Ning; Liu, Jie; An, Shucai; Nishino, Tomoya; Hishikawa, Yoshitaka; Koji, Takehiko

2011-01-01

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis. PMID:21927517

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis.

PubMed

Song, Ning; Liu, Jie; An, Shucai; Nishino, Tomoya; Hishikawa, Yoshitaka; Koji, Takehiko

2011-08-27

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.

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

PubMed

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

2013-07-15

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

The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2

PubMed Central

Du, Yipeng; Cao, Lin-lin; Li, Meiting; Shen, Changchun; Hou, Tianyun; Zhao, Ying; Wang, Haiying; Deng, Dajun; Wang, Lina; He, Qihua; Zhu, Wei-Guo

2015-01-01

Cyclooxygenase-2 (COX-2) is overexpressed in a variety of human epithelial cancers, including lung cancer, and is highly associated with a poor prognosis and a low survival rate. Understanding how COX-2 is regulated in response to carcinogens will offer insight into designing anti-cancer strategies and preventing cancer development. Here, we analyzed COX-2 expression in several human lung cancer cell lines and found that COX-2 expression was absent in the H719 and H460 cell lines by a DNA methylation-independent mechanism. The re-expression of COX-2 was observed after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment in both cell lines. Further investigation found that H3K36 dimethylation was significantly reduced near the COX-2 promoter because histone demethylase 2A (KDM2A) was recruited to the COX-2 promoter after TPA treatment. In addition, the transcription factor c-Fos was found to be required to recruit KDM2A to the COX-2 promoter for reactivation of COX-2 in response to TPA treatment in both the H719 and H460 cell lines. Together, our data reveal a novel mechanism by which the carcinogen TPA activates COX-2 expression by regulating H3K36 dimethylation near the COX-2 promoter. PMID:26430963

Chromatin remodeling in somatic cells injected into mature pig oocytes.

PubMed

Bui, Hong-Thuy; Van Thuan, Nguyen; Wakayama, Teruhiko; Miyano, Takashi

2006-06-01

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle.

PubMed

Pesavento, James J; Yang, Hongbo; Kelleher, Neil L; Mizzen, Craig A

2008-01-01

Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G(2), M, and G(1) phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

PubMed

Ikegami, Kohta; Iwatani, Misa; Suzuki, Masako; Tachibana, Makoto; Shinkai, Yoichi; Tanaka, Satoshi; Greally, John M; Yagi, Shintaro; Hattori, Naka; Shiota, Kunio

2007-01-01

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

PubMed Central

Zoabi, Muhammad; Nadar-Ponniah, Prathamesh T.; Khoury-Haddad, Hanan; Usaj, Marko; Budowski-Tal, Inbal; Haran, Tali; Henn, Arnon; Mandel-Gutfreund, Yael; Ayoub, Nabieh

2014-01-01

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin. PMID:25378304

Enhanced antioxidant activity of gold nanoparticle embedded 3,6-dihydroxyflavone: a combinational study

NASA Astrophysics Data System (ADS)

Medhe, Sharad; Bansal, Prachi; Srivastava, Man Mohan

2014-02-01

The antioxidative effect of selected dietary compounds (3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine) was determined in single and combination using DPPH (2,2-diphenyl-l-picrylhydrazyl), OH (hydroxyl), H2O2 (hydrogen peroxide) and NO (nitric oxide) radical scavenging assays. Radical scavenging effect of the dietary phytochemicals individually are found to be in the order: ascorbic acid (standard) > lutein > 3,6-dihydroxyflavone > selenium methyl selenocysteine, at concentration 100 μg/ml, confirmed by all the four bioassays (p < 0.05). Among the various combinations studied, the triplet combination of 3,6-dihydroxyflavone, lutein and selenium methyl selenocysteine (1:1:1), exhibited enhancement in the target activity at same concentration level. Synthesized gold nanoparticle embedded 3,6-dihydroxyflavone further enhanced the target antioxidant activity. The combinational study including gold nanoparticle embedded 3,6-dihydroxyflavone with other native dietary nutrients showed remarkable increase in antioxidant activity at the same concentration level. The present in vitro study on combinational and nanotech enforcement of dietary phytochemicals shows the utility in the architecture of nanoparticle embedded phytoproducts having a wide range of applications in medical science.

Thermal Decomposition of Methyl Acetate (CH_3COOCH_3) in a Flash-Pyrolysis Micro-Reactor

NASA Astrophysics Data System (ADS)

Porterfield, Jessica P.; Bross, David H.; Ruscic, Branko; Thorpe, James H.; Nguyen, Thanh Lam; Baraban, Joshua H.; Stanton, John F.; Daily, John W.; Ellison, Barney

2017-06-01

The thermal decomposition of methyl acetate (CH_3COOCH_3) has been studied in a set of flash pyrolysis micro-reactors. Samples were diluted to (0.06 - 0.13%) in carrier gases (He, Ar) and subjected to temperatures of 300 - 1600 K at roughly 20 Torr. After residence times of approximately 25 - 150 μseconds, the unimolecular pyrolysis products were detected by vacuum ultraviolet photoionization mass spectrometry at 10.487 eV (118.2 nm). Complementary product identification was provided by matrix isolation infrared spectroscopy. Decomposition began at 1000 K with the observation of (CH_2=C=O, CH_3OH), products of a four centered rearrangement with a Δ_{rxn}H_{298} = 39.1 ± 0.2 kcal mol^{-1}. As the micro-reactor was heated to 1300 K, a mixture of (CH_2=C=O, CH_3OH, CH_3, CH_2=O, H, CO, CO_2) appeared. A new novel pathway is calculated in which both methyl groups leave behind CO_2 simultaneously, Δ_{rxn}H_{298} = 74.5 ± 0.4 kcal mol^{-1}. This pathway is in contrast to step-wise loss of methyl radical, which can go in two ways: Δ_{rxn}H_{298} (CH_3COOCH_3 → CH_3 + COOCH_3) = 95.4 ± 0.4 kcal mol^{-1}, Δ_{rxn}H_{298} (CH_3COOCH_3 → CH_3COO + CH_3) = 88.0 ± 0.3 kcal mol^{-1}.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

PubMed Central

2012-01-01

Background Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis. Results We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly. Conclusion There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets. PMID:23253144

1H NMR spectrum of the native human insulin monomer. Evidence for conformational differences between the monomer and aggregated forms.

PubMed

Roy, M; Lee, R W; Brange, J; Dunn, M F

1990-04-05

The effects of high dilution on the 1H Fourier transform NMR spectrum of native human insulin at pH* 8.0 and 9.3 have been examined at 500 MHz resolution. The dependence of the spectrum on concentration and comparison with the spectrum of a biologically highly potent monomeric insulin mutant (SerB9----Asp) establish that at 36 microM (pH* 9.3) or 18 microM (pH* 8) and no added buffer or salts, human insulin is monomeric. Under these conditions of dilution, ionic strength, and pH*, human insulin and the SerB9----Asp mutant exhibit nearly identical 1H NMR spectra. At higher concentrations (i.e. greater than 36 microM to 0.91 mM), native human insulin dimerizes, and this aggregation causes a change in insulin conformation. Although there are many changes in the spectrum, the TyrB26 ring H3,5 proton signals located at 6.63 ppm and the methyl signal located at 0.105 ppm (characteristics of monomeric insulin) are particularly distinct signatures of the conformation change that accompanies dimerization. Magnetization transfer experiments show that the 0.105 ppm methyl signal shifts downfield to a new position at 0.45 ppm. We conclude that the 0.105 ppm methyl signal is due to a conformation in which a Leu methyl group is centered over and in van der Waals contact with the ring of an aromatic side chain. Dimerization causes a conformation change that alters this interaction, thereby causing the downfield shift. Nuclear Overhauser studies indicate that the methyl group involved is located within a cluster of aromatic side chains and that the closest ring-methyl group interaction is with the ring of PheB24.

Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus.

PubMed

Hamada, Yoshimasa; Bando, Tetsuya; Nakamura, Taro; Ishimaru, Yoshiyasu; Mito, Taro; Noji, Sumihare; Tomioka, Kenji; Ohuchi, Hideyo

2015-09-01

Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression. © 2015. Published by The Company of Biologists Ltd.

Epigenetic-Imprinting Changes Caused by Neonatal Fasting Stress Protect From Future Fasting Stress.

PubMed

Jiang, Y; Denbow, C; Meiri, N; Denbow, D M

2016-01-01

Unfavourable nutritional conditions during the neonatal critical period can cause both acute metabolic disorders and severe metabolic syndromes in later life. These phenomena have been tightly related to the epigenetic modification controlling the balance between satiety and hunger in the hypothalamus. In the present study, we investigated epigenetic modification associated with both the fasting stress effects and the short-term resilience to fasting stress in the hypothalamic paraventricular nucleus (PVN) of chicks. Fasting for 24 h at 3 days of age (D) (i.e. D3) significantly increased global methylation at lysine 27 of histone 3 (H3K27) and its specific histone methyltransferase (HMT) expression level in the PVN. Because global methylation could not fully reveal the changes at specific genes, the regulation of the gene for brain-derived neurotrophic factor (Bdnf), which was recently also found to have an anorexigenic effect, was evaluated as a potential target. Chromatin immunoprecipitation assay analysis revealed that tri- (me3) and di-methylated (me2) H3K27 exhibited an instant (on D4 only) and latent increase (on both D11 and D41), respectively, at the putative promoter of Bdnf after 24 h of fasting on D3. This indicated that fasting could regulate energy-expenditure-related genes via modifying methylation at H3K27, which we suspected might be a protective mechanism for keeping the inner environment homeostatic. To test this hypothesis, a short-term repetitive fasting stress was applied to chickens, which were fasted for 24 h either on D10 only or on both D3 and D10. It was found that pre-existing fasting on D3 could induce a short-term fasting resilience, which rescued the reduction of Bdnf expression from future fasting on D10. We call this phenomenon the ‘molecular memory’, which was mainly conducted by HMTs and H3K27me2/me3 in the PVN. In conclusion, chicks respond to fasting with dynamic methylation at H3K27 in the PVN during the neonatal critical period. This allows the PVN to form a ‘molecular memory’, which keeps the individual inner environment homeostatic and resilient to future fasting over the short term.

Clr4 specificity and catalytic activity beyond H3K9 methylation.

PubMed

Kusevic, Denis; Kudithipudi, Srikanth; Iglesias, Nahid; Moazed, Danesh; Jeltsch, Albert

2017-04-01

In fission yeast, the catalytic activity of the protein lysine methyltransferase (PKMT) Clr4, the sole homolog of the mammalian SUV39H1 and SUV39H2 enzymes, majorly contributes to the formation of heterochromatin. The enzyme introduces histone 3 lysine 9 (H3K9) di- and tri-methylation, a central heterochromatic histone modification, and later it was also found to methylate the Mlo3 protein, which has a role in heterochromatin formation as well. Herein, we have investigated the substrate specificity of Clr4 using custom made mutational scanning peptide arrays. Our data show, that Clr4 recognizes an RK core motif, showing high preference for R8. In addition, it exhibits specific contacts at the S10, T11, G12 and G13 positions of the H3 peptide recognizing an R-K-SKRT-TCS-G sequence. Based on the specificity profile and in vitro methyltransferase assay targeted searches, 11 putative methylation sites in S. pombe proteins were identified from reported Clr4 interacting proteins including Mlo3. Peptide methylation was observed on Mlo3 and 7 novel target sites with strongest methylation signals on Spbc28F2.11 (HMG box-containing protein) at lysine 292 and Hrp3 (Chromodomain ATP-dep DNA helicase) at lysine 89. These data suggest that Clr4 has additional methylation substrates and it will be important to study the biological function of these novel methylation events. Furthermore, the specificity profile of Clr4 has been used to develop a quantitative method to compare and cluster specificity profiles of PKMTs. It shows that the specificity profile of Clr4 is most similar to that of the SUV39H2 enzyme, one of its human homologs. This approach will be helpful in the comparison of the recognition profiles of other families of PKMTs as well. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Specific Modifications of Histone Tails, but Not DNA Methylation, Mirror the Temporal Variation of Mammalian Recombination Hotspots

PubMed Central

Zeng, Jia; Yi, Soojin V.

2014-01-01

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called “recombination hotspot paradox”) remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy “bivalent” chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. PMID:25326136

Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

PubMed

Zeng, Jia; Yi, Soojin V

2014-10-16

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Histone H3 Lysine 9 Methyltransferase DIM-5 Modifies Chromatin at frequency and Represses Light-Activated Gene Expression

PubMed Central

Ruesch, Catherine E.; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S.; Zaman, Riasat; Joska, Tammy M.; Belden, William J.

2014-01-01

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1bd. These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. PMID:25429045

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

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

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

NMDA receptor- and ERK-dependent histone methylation changes in the lateral amygdala bidirectionally regulate fear memory formation.

PubMed

Gupta-Agarwal, Swati; Jarome, Timothy J; Fernandez, Jordan; Lubin, Farah D

2014-07-01

It is well established that fear memory formation requires de novo gene transcription in the amygdala. We provide evidence that epigenetic mechanisms in the form of histone lysine methylation in the lateral amygdala (LA) are regulated by NMDA receptor (NMDAR) signaling and involved in gene transcription changes necessary for fear memory consolidation. Here we found increases in histone H3 lysine 9 dimethylation (H3K9me2) levels in the LA at 1 h following auditory fear conditioning, which continued to be temporally regulated up to 25 h following behavioral training. Additionally, we demonstrate that inhibiting the H3K9me2 histone lysine methyltransferase G9a (H/KMTs-G9a) in the LA impaired fear memory, while blocking the H3K9me2 histone lysine demethylase LSD1 (H/KDM-LSD1) enhanced fear memory, suggesting that H3K9me2 in the LA can bidirectionally regulate fear memory formation. Furthermore, we show that NMDAR activity differentially regulated the recruitment of H/KMT-G9a, H/KDM-LSD1, and subsequent H3K9me2 levels at a target gene promoter. This was largely regulated by GluN2B- but not GluN2A-containing NMDARs via ERK activation. Moreover, fear memory deficits associated with NMDAR or ERK blockade were successfully rescued through pharmacologically inhibiting LSD1, suggesting that enhancements of H3K9me2 levels within the LA can rescue fear memory impairments that result from hypofunctioning NMDARs or loss of ERK signaling. Together, the present study suggests that histone lysine methylation regulation in the LA via NMDAR-ERK-dependent signaling is involved in fear memory formation. © 2014 Gupta-Agarwal et al.; Published by Cold Spring Harbor Laboratory Press.

Epigenomic diversification within the genus Lupinus

PubMed Central

Bewick, Adam J.; Hasterok, Robert; Schmitz, Robert J.; Naganowska, Barbara

2017-01-01

Deciphering the various chemical modifications of both DNA and the histone compound of chromatin not only leads to a better understanding of the genome-wide organisation of epigenetic landmarks and their impact on gene expression but may also provide some insights into the evolutionary processes. Although both histone modifications and DNA methylation have been widely investigated in various plant genomes, here we present the first study for the genus Lupinus. Lupins, which are members of grain legumes (pulses), are beneficial for food security, nutrition, health and the environment. In order to gain a better understanding of the epigenetic organisation of genomes in lupins we applied the immunostaining of methylated histone H3 and DNA methylation as well as whole-genome bisulfite sequencing. We revealed variations in the patterns of chromatin modifications at the chromosomal level among three crop lupins, i.e. L. angustifolius (2n = 40), L. albus (2n = 50) and L. luteus (2n = 52), and the legume model plant Medicago truncatula (2n = 16). Different chromosomal patterns were found depending on the specific modification, e.g. H3K4me2 was localised in the terminal parts of L. angustifolius and M. truncatula chromosomes, which is in agreement with the results that have been obtained for other species. Interestingly, in L. albus and L. luteus this modification was limited to one arm in the case of all of the chromosomes in the complement. Additionally, H3K9me2 was detected in all of the analysed species except L. luteus. DNA methylation sequencing (CG, CHG and CHH contexts) of aforementioned crop but also wild lupins such as L. cosentinii (2n = 32), L. digitatus (2n = 36), L. micranthus (2n = 52) and L. pilosus (2n = 42) supported the range of interspecific diversity. The examples of epigenetic modifications illustrate the diversity of lupin genomes and could be helpful for elucidating further epigenetic changes in the evolution of the lupin genome. PMID:28640886

Epigenomic diversification within the genus Lupinus.

PubMed

Susek, Karolina; Braszewska-Zalewska, Agnieszka; Bewick, Adam J; Hasterok, Robert; Schmitz, Robert J; Naganowska, Barbara

2017-01-01

Deciphering the various chemical modifications of both DNA and the histone compound of chromatin not only leads to a better understanding of the genome-wide organisation of epigenetic landmarks and their impact on gene expression but may also provide some insights into the evolutionary processes. Although both histone modifications and DNA methylation have been widely investigated in various plant genomes, here we present the first study for the genus Lupinus. Lupins, which are members of grain legumes (pulses), are beneficial for food security, nutrition, health and the environment. In order to gain a better understanding of the epigenetic organisation of genomes in lupins we applied the immunostaining of methylated histone H3 and DNA methylation as well as whole-genome bisulfite sequencing. We revealed variations in the patterns of chromatin modifications at the chromosomal level among three crop lupins, i.e. L. angustifolius (2n = 40), L. albus (2n = 50) and L. luteus (2n = 52), and the legume model plant Medicago truncatula (2n = 16). Different chromosomal patterns were found depending on the specific modification, e.g. H3K4me2 was localised in the terminal parts of L. angustifolius and M. truncatula chromosomes, which is in agreement with the results that have been obtained for other species. Interestingly, in L. albus and L. luteus this modification was limited to one arm in the case of all of the chromosomes in the complement. Additionally, H3K9me2 was detected in all of the analysed species except L. luteus. DNA methylation sequencing (CG, CHG and CHH contexts) of aforementioned crop but also wild lupins such as L. cosentinii (2n = 32), L. digitatus (2n = 36), L. micranthus (2n = 52) and L. pilosus (2n = 42) supported the range of interspecific diversity. The examples of epigenetic modifications illustrate the diversity of lupin genomes and could be helpful for elucidating further epigenetic changes in the evolution of the lupin genome.

Histone Methylation and Epigenetic Silencing in Breast Cancer

DTIC Science & Technology

2011-02-01

Chen, S., Bohrer, L.R., Nair-Rai, A., Pan , Y., Gan, L., Zhou, X., Bagchi, A., Simon, J.A. and Huang, H. (2010). Cyclin-dependent kinases regulate...EZH2 suppresses methylation of lysine 27 in histone H3. Science 310(5746): 306-310. Chen, S., Bohrer, L.R., Rai, A.N., Pan , Y., Gan, L., Zhou, X...recent study finds that ri-methyl-H3-K27 levels are decreased in breast, ovarian and pan - reatic cancer samples [65], which could reflect a shift in

Kinetics of the Reaction of CH3O2 Radicals with OH Studied over the 292-526 K Temperature Range.

PubMed

Yan, Chao; Kocevska, Stefani; Krasnoperov, Lev N

2016-08-11

Reaction of methyl peroxy radicals with hydroxyl radicals, CH3O2 + OH → CH3O + HO2 (1a) and CH3O2 + OH → CH2OO + H2O (1b) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 292-526 K temperature range and pressure 1 bar (bath gas He). Hydroxyl radicals were generated in the reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N2O at 193.3 nm, with H2O. Methyl peroxy radicals were generated in the reaction of methyl radicals, CH3, produced in the photolysis of acetone at 193.3 nm, and subsequent reaction of CH3 with O2. Temporal profiles of OH were monitored via transient absorption of light from a DC discharge H2O/Ar low-pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light was determined by accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The overall rate constant of the reaction is k1a+1b = (8.4 ± 1.7) × 10(-11)(T/298 K)(-0.81) cm(3) molecule(-1) s(-1) (292-526 K). The branching ratio of channel 1b at 298 K is less than 5%.

De novo mutations in histone modifying genes in congenital heart disease

PubMed Central

Zaidi, Samir; Choi, Murim; Wakimoto, Hiroko; Ma, Lijiang; Jiang, Jianming; Overton, John D.; Romano-Adesman, Angela; Bjornson, Robert D.; Breitbart, Roger E.; Brown, Kerry K.; Carriero, Nicholas J.; Cheung, Yee Him; Deanfield, John; DePalma, Steve; Fakhro, Khalid A.; Glessner, Joseph; Hakonarson, Hakon; Italia, Michael; Kaltman, Jonathan R.; Kaski, Juan; Kim, Richard; Kline, Jennie K.; Lee, Teresa; Leipzig, Jeremy; Lopez, Alexander; Mane, Shrikant M.; Mitchell, Laura E.; Newburger, Jane W.; Parfenov, Michael; Pe'er, Itsik; Porter, George; Roberts, Amy; Sachidanandam, Ravi; Sanders, Stephan J.; Seiden, Howard S.; State, Mathew W.; Subramanian, Sailakshmi; Tikhonova, Irina R.; Wang, Wei; Warburton, Dorothy; White, Peter S.; Williams, Ismee A.; Zhao, Hongyu; Seidman, Jonathan G.; Brueckner, Martina; Chung, Wendy K.; Gelb, Bruce D.; Goldmuntz, Elizabeth; Seidman, Christine E.; Lifton, Richard P.

2013-01-01

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. By analysis of exome sequencing of parent-offspring trios, we compared the incidence of de novo mutations in 362 severe CHD cases and 264 controls. CHD cases showed a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging mutations. Similar odds ratios were seen across major classes of severe CHD. We found a marked excess of de novo mutations in genes involved in production, removal or reading of H3K4 methylation (H3K4me), or ubiquitination of H2BK120, which is required for H3K4 methylation2–4. There were also two de novo mutations in SMAD2; SMAD2 signaling in the embryonic left-right organizer induces demethylation of H3K27me5. H3K4me and H3K27me mark `poised' promoters and enhancers that regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundred genes that collectively contribute to ~10% of severe CHD. PMID:23665959

A novel assessment of the role of the methyl radical and water formation channel in the CH3OH + H reaction.

PubMed

Sanches-Neto, Flávio O; Coutinho, Nayara D; Carvalho-Silva, Valter H

2017-09-20

A number of experimental and theoretical papers accounted almost exclusively for two channels in the reaction of atomic hydrogen with methanol: H-abstraction from the methyl (R1) and hydroxyl (R2) functional groups. Recently, several astrochemical studies claimed the importance of another channel for this reaction, which is crucial for kinetic simulations related to the abundance of molecular constituents in planetary atmospheres: methyl radical and water formation (R3 channel). Here, motivated by the lack of and uncertainties about the experimental and theoretical kinetic rate constants for the third channel, we developed first-principles Car-Parrinello molecular dynamics thermalized at two significant temperatures - 300 and 2500 K. Furthermore, the kinetic rate constant of all three channels was calculated using a high-level deformed-transition state theory (d-TST) at a benchmark electronic structure level. d-TST is shown to be suitable for describing the overall rate constant for the CH 3 OH + H reaction (an archetype of the moderate tunnelling regime) with the precision required for practical applications. Considering the experimental ratios at 1000 K, k R1 /k R2 ≈ 0.84 and k R1 /k R3 ≈ 15-40, we provided a better estimate when compared with previous theoretical work: 7.47 and 637, respectively. The combination of these procedures explicitly demonstrates the role of the third channel in a significant range of temperatures and indicates its importance considering the thermodynamic control to estimate methyl radical and water formation. We expect that these results can help to shed new light on the fundamental kinetic rate equations for the CH 3 OH + H reaction.

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation

PubMed Central

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-01-01

Background Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Methods Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine–phosphate–guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Results Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine–phosphate–guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Conclusions Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation–specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. PMID:24997139

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC

PubMed Central

Wolstenholme, Jennifer T.; Mahmood, Tariq; Harris, Guy M.; Abbas, Shahroze; Miles, Michael F.

2017-01-01

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol’s actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood. PMID:29018328

Intermittent Ethanol during Adolescence Leads to Lasting Behavioral Changes in Adulthood and Alters Gene Expression and Histone Methylation in the PFC.

PubMed

Wolstenholme, Jennifer T; Mahmood, Tariq; Harris, Guy M; Abbas, Shahroze; Miles, Michael F

2017-01-01

Adolescents primarily consume alcohol in binges, which can be particularly harmful to the developing frontal cortex and increase risk for an adult alcohol use disorder. We conducted a study investigating immediate and long lasting changes to the prefrontal cortex (PFC) transcriptome to determine the molecular mechanisms underlying adult ethanol behavioral sensitivity following binge ethanol in adolescence. DBA/2J mice were orally dosed with 4 g/kg ethanol intermittently from day 29 to 42. Adolescent mice were tested for anxiety-like behavior and ethanol sensitivity using the loss of righting reflex task. As adults, mice were tested for cognitive changes using the novel object recognition task, ethanol-induced anxiolysis and ethanol sensitivity. Adolescent binge ethanol altered ethanol sensitivity in young mice and led to lasting memory deficits in the object recognition test and greater ethanol sensitivity in adulthood. Using genomic profiling of transcripts in the PFC, we found that binge ethanol reduced myelin-related gene expression and altered chromatin modifying genes involved in histone demethylation at H3K9 and H3K36. We hypothesize that ethanol's actions on histone methylation may be a switch for future transcriptional changes that underlie the behavioral changes lasting into adulthood.

Influence of Threonine Metabolism on S-adenosyl-methionine and Histone Methylation

PubMed Central

Shyh-Chang, Ng; Locasale, Jason W.; Lyssiotis, Costas A.; Zheng, Yuxiang; Teo, Ren Yi; Ratanasirintrawoot, Sutheera; Zhang, Jin; Onder, Tamer; Unternaehrer, Juli J.; Zhu, Hao; Asara, John M.; Daley, George Q.; Cantley, Lewis C.

2013-01-01

Threonine is the only amino acid critically required for the pluripotency of mouse embryonic stem cells (mESCs) but the detailed mechanism remains unclear. We found that threonine (Thr) and S-adenosyl-methionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation. Isotope labeling of mESCs revealed that Thr provides a substantial fraction of both the cellular glycine (Gly) and the acetyl-coenzyme A (CoA) needed for SAM synthesis. Depletion of Thr from the culture medium or threonine dehydrogenase (Tdh) from mESCs decreased accumulation of SAM and decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth, and increased differentiation. Thus abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. PMID:23118012

dSet1 Is the Main H3K4 Di- and Tri-Methyltransferase Throughout Drosophila Development

PubMed Central

Hallson, Graham; Hollebakken, Robert E.; Li, Taosui; Syrzycka, Monika; Kim, Inho; Cotsworth, Shawn; Fitzpatrick, Kathleen A.; Sinclair, Donald A. R.; Honda, Barry M.

2012-01-01

In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a “global” or general H3K4 di- and trimethyl HMT in Drosophila. Levels of H3K4 di- and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di- and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans. PMID:22048023

Discovery of sodium R-(+)-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyrate (elagolix), a potent and orally available nonpeptide antagonist of the human gonadotropin-releasing hormone receptor.

PubMed

Chen, Chen; Wu, Dongpei; Guo, Zhiqiang; Xie, Qiu; Reinhart, Greg J; Madan, Ajay; Wen, Jenny; Chen, Takung; Huang, Charles Q; Chen, Mi; Chen, Yongsheng; Tucci, Fabio C; Rowbottom, Martin; Pontillo, Joseph; Zhu, Yun-Fei; Wade, Warren; Saunders, John; Bozigian, Haig; Struthers, R Scott

2008-12-11

The discovery of novel uracil phenylethylamines bearing a butyric acid as potent human gonadotropin-releasing hormone receptor (hGnRH-R) antagonists is described. A major focus of this optimization was to improve the CYP3A4 inhibition liability of these uracils while maintaining their GnRH-R potency. R-4-{2-[5-(2-fluoro-3-methoxyphenyl)-3-(2-fluoro-6-[trifluoromethyl]benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-pyrimidin-1-yl]-1-phenylethylamino}butyric acid sodium salt, 10b (elagolix), was identified as a potent and selective hGnRH-R antagonist. Oral administration of 10b suppressed luteinizing hormone in castrated macaques. These efforts led to the identification of 10b as a clinical compound for the treatment of endometriosis.

The histone H3 lysine 9 methyltransferase DIM-5 modifies chromatin at frequency and represses light-activated gene expression.

PubMed

Ruesch, Catherine E; Ramakrishnan, Mukund; Park, Jinhee; Li, Na; Chong, Hin S; Zaman, Riasat; Joska, Tammy M; Belden, William J

2014-11-25

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output. Copyright © 2015 Ruesch et al.

Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory.

PubMed

Webb, William M; Sanchez, Richard G; Perez, Gabriella; Butler, Anderson A; Hauser, Rebecca M; Rich, Megan C; O'Bierne, Aidan L; Jarome, Timothy J; Lubin, Farah D

2017-07-01

Epigenetic mechanisms such as DNA methylation and histone methylation are critical regulators of gene transcription changes during memory consolidation. However, it is unknown how these epigenetic modifications coordinate control of gene expression following reactivation of a previously consolidated memory. Here, we found that retrieval of a recent contextual fear conditioned memory increased global levels of H3 lysine 4-trimethylation (H3K4me3) and DNA 5-hydroxymethylation (5hmC) in area CA1 of the dorsal hippocampus. Further experiments revealed increased levels of H3K4me3 and DNA 5hmC within a CpG-enriched coding region of the Npas4, but not c-fos, gene. Intriguingly, retrieval of a 30-day old memory increased H3K4me3 and DNA 5hmC levels at a CpG-enriched coding region of c-fos, but not Npas4, in the anterior cingulate cortex, suggesting that while these two epigenetic mechanisms co-occur following the retrieval of a recent or remote memory, their gene targets differ depending on the brain region. Additionally, we found that in vivo siRNA-mediated knockdown of the H3K4me3 methyltransferase Mll1 in CA1 abolished retrieval-induced increases in DNA 5hmC levels at the Npas4 gene, suggesting that H3K4me3 couples to DNA 5hmC mechanisms. Consistent with this, loss of Mll1 prevented retrieval-induced increases in Npas4 mRNA levels in CA1 and impaired fear memory. Collectively, these findings suggest an important link between histone methylation and DNA hydroxymethylation mechanisms in the epigenetic control of de novo gene transcription triggered by memory retrieval. Copyright © 2017 Elsevier Inc. All rights reserved.

N-(4-bromophenethyl) Caffeamide Inhibits Melanogenesis by Regulating AKT/Glycogen Synthase Kinase 3 Beta/Microphthalmia-associated Transcription Factor and Tyrosinase-related Protein 1/Tyrosinase.

PubMed

Kuo, Yueh-Hsiung; Chen, Chien-Chia; Lin, Ping; You, Ya-Jhen; Chiang, Hsiu-Mei

2015-01-01

Skin color is primarily produced by melanin, which is a crucial pigment that protects the skin from UV-induced damage and prevents carcinogenesis. However, accumulated melanin in the skin may cause hyperpigmentation and related disorders. Melanin synthesis comprises consecutive oxidative reactions, and tyrosinase is the enzyme that catalyzes the rate-limiting process of melanogenesis. In this study, tyrosinase-related protein 1 (TRP-1) and TRP-2 contributed to melanin formation. N-(4-bromophenethyl) caffeamide ((E)-N-(4-bromophenethyl)-3-(3,4-dihydroxyphenyl)acrylamide; K36H), a caffeic acid phenyl amide derivative, inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and tyrosinase activity in B16F0 cells. In addition, K36H reduced the protein expression of the phospho-cAMP response element binding protein (p-CREB), microphthalmia-associated transcription factor (MITF), tyrosinase, and TRP-1. Moreover, K36H promoted AKT and glycogen synthase kinase 3 beta (GSK3β) phosphorylation, thereby inhibiting MITF transcription activity. Thus, K36H attenuated α-MSH-induced cAMP pathways, contributing to hypopigmentation. The results of a safety assay revealed that K36H did not exhibit cytotoxicity or irritate the skin or eyes. According to these results, K36H may have the potential to be used as a whitening agent in the cosmetic and pharmaceutical industries.

The molecular basis for stability of heterochromatin-mediated silencing in mammals.

PubMed

Hiragami-Hamada, Kyoko; Xie, Sheila Q; Saveliev, Alexander; Uribe-Lewis, Santiago; Pombo, Ana; Festenstein, Richard

2009-11-04

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo.

Biosynthesis of methanopterin

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, R.H.

1990-06-05

The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. (methylene{sup 2}H)-6-(hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from (methyl-{sup 2}H{sub 3})methionine. Cells grown in the presence of (methylene-{sup 2}H)-6-(hydroxymethyl)pterin, (ethyl-{sup 2}H{sub 4})-6-(1 (RS)-hydroxyethyl)pterin, (methyl-{sup 2}H{sub 3})-6-(hydroxymethyl)-7-methylpterin, (ethyl-{sup 2}H{sub 4}, methyl-{sup 2}H{submore » 3})-6-(1 (RS)-hydroxyethyl)-7-methylpterin, and (1-ethyl-{sup 3}H)-6-(1 (RS)-hydroxyethyl)-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic (methylene-{sup 3}H)-7,8-H{sub 2}-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H{sub 2}-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.« less

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

PubMed

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

2015-07-01

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. © 2015 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Histone Modification Is Correlated With Reverse Left Ventricular Remodeling in Nonischemic Dilated Cardiomyopathy.

PubMed

Ito, Emiko; Miyagawa, Shigeru; Fukushima, Satsuki; Yoshikawa, Yasushi; Saito, Shunsuke; Saito, Tetsuya; Harada, Akima; Takeda, Maki; Kashiyama, Noriyuki; Nakamura, Yuki; Shiozaki, Motoko; Toda, Koichi; Sawa, Yoshiki

2017-11-01

Although implantation of a left ventricular assist device (LVAD) induces reverse remodeling of the left ventricle in end-stage nonischemic dilated cardiomyopathy (DCM), the underlying mechanism is not fully understood. It has been shown that epigenetic modification, such as methylation or acetylation of the histone, is one of the most important upstream signals in cardiac failure. This study hypothesized that histone profiles may be modified by LVAD implantation for end-stage nonischemic DCM, in association with reverse left ventricular remodeling. Hemodynamic changes associated with histone modification profiles in the left ventricle were comprehensively assessed in 14 patients with a diagnosis of end-stage nonischemic DCM. These patients underwent LVAD implantation and subsequent cardiac transplantation in our institution (Osaka University Hospital, Osaka, Japan). Samples of normal left ventricle from 3 different people were used as a control. After LVAD support for 2.5 ± 1.2 years, the study cohort showed a significant reverse remodeling of left ventricular function associated with histopathologic changes in the left ventricle, such as reduction of myocyte size. Although the left ventricle of the cohort histologically expressed less 3 histone methylation-related molecules (eg, H3 lysine 4 trimethylation [H3K4me3], H3 lysine 9 dimethylation [H3K9me2], and H3 lysine 9 trimethylation [H3K9me3]) compared with normal left ventricle, LVAD support reversed expression of these molecules, associated with up-regulation of H3 lysine 9 [H3K9] methyltransferase and suppressor of variegation 3-9 homologue 1 [SUV39H1] and with down-regulation of H3K9 demethylase and jumonji domains [JMJDs] in the LVAD-supported left ventricle. Moreover, expression of atrial natriuretic peptide and brain natriuretic peptide (ANP and BNP) was negatively correlated with that of H3K9me2 and H3K9me3. The epigenetic state of cardiac myocytes (eg, as histone methylation) was substantially modulated in end-stage nonischemic DCM. LVAD support partially reversed the epigenetic state and its upstream signals, in association with pathologic and functional reverse remodeling. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Synthesis of polyhydroxysterols (III): synthesis and structural elucidation of 24-methylenecholest-4-en-3beta,6 alpha-diol.

PubMed

Cui, Jian Guo; Lin, Cui Wu; Zeng, Long Mei; Su, Jing Yu

2002-12-01

Using stigmasterol as the starting material, 24-methylenecholest-4-en-3beta,6 alpha-diol (2) was synthesized in eight steps in 13% overall yield. The introduction of the sterol side-chain was carried out using (3-methyl-2-oxobutyl)-triphenylarsonium bromide (11) and K(2)CO(3) in a solid-liquid phase-transfer Wittig reaction. Construction of the steroidal nucleus was finished by oxidation of 24-methylenecholest-5-en-3beta-ol (9) with pyridinium chlorochromate (PCC) in dichloromethane at ambient temperature and by reduction of 24-methylenecholest-4-en-3,6-dione (10) with NaBH(4) in the presence of CeCl(3).7H(2)O.

[Dot1 and Set2 Histone Methylases Control the Spontaneous and UV-Induced Mutagenesis Levels in the Saccharomyces cerevisiae Yeasts].

PubMed

Kozhina, T N; Evstiukhina, T A; Peshekhonov, V T; Chernenkov, A Yu; Korolev, V G

2016-03-01

In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of hi- stone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-in- duced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the sponta- neous mutagenesis rate in both single and d ouble mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the ho- mologous-recombination-based and the postreplicative DNA repair.

Comparative analysis of Histone modifications and DNA methylation at OsBZ8 locus under salinity stress in IR64 and Nonabokra rice varieties.

PubMed

Paul, Amit; Dasgupta, Pratiti; Roy, Dipan; Chaudhuri, Shubho

2017-09-01

Rice being an important cereal crop is highly sensitive to salinity stress causing growth retardation and loss in productivity. However, certain rice genotypes like Nonabokra and Pokkali show a high level of tolerance towards salinity stress compared to IR64 variety. This differential response of tolerant varieties towards salinity stress may be a cumulative effect of genetic and epigenetic factors. In this study, we have compared the salinity-induced changes in chromatin modifications at the OsBZ8 locus in salt-tolerant Nonabokra and salt-sensitive IR64 rice varieties. Expression analysis indicates that the OsBZ8 gene is highly induced in Nonabokra plants even in the absence of salt stress, whereas in IR64, the expression significantly increases only during salt stress. Sequence analysis and nucleosomal arrangement within the region -2000 to +1000 of OsBZ8 gene show no difference between the two rice varieties. However, there was a considerable difference in histone modifications and DNA methylation at the locus between these varieties. In Nonabokra, the upstream region was hyperacetylated at H3K9 and H3K27, and this acetylation did not change during salt stress. However, in IR64, histone acetylation was observed only during salt stress. Moreover, the upstream region of OsBZ8 gene has highly dynamic nucleosome arrangement in Nonabokra, compared to IR64. Furthermore, loss of DNA methylation was observed at OsBZ8 locus in Nonabokra control plants along with low H3K27me3 and high H3K4me3. Control IR64 plants show high DNA methylation and enriched H3K27me3. Collectively these results indicate a significant difference in chromatin modifications between the rice varieties that regulates differential expression of OsBZ8 gene during salt stress.

Dynamics of H3K27me3 methylation and demethylation in plant development

PubMed Central

Gan, Eng-Seng; Xu, Yifeng; Ito, Toshiro

2015-01-01

Epigenetic regulation controls multiple aspects of the plant development. The N-terminal tail of histone can be differently modified to regulate various chromatin activities. One of them, the trimethylation of histone H3 lysine 27 (H3K27me3) confers a repressive chromatin state with gene silencing. H3K27me3 is dynamically deposited and removed throughout development. While components of the H3K27me3 writer, Polycomb repressive complex 2 (PRC2), have been reported for almost 2 decades, it is only recently that JUMONJI (JMJ) proteins are reported as H3K27me3 demethylases, affirming the dynamic nature of histone modifications. This review highlights recent progress in plant epigenetic research, focusing on the H3K27me3 demethylases. PMID:26313233

Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes.

PubMed

Dindar, Gülcin; Anger, Andreas M; Mehlhorn, Christine; Hake, Sandra B; Janzen, Christian J

2014-11-12

DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono- and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes.

Role of annexin gene and its regulation during zebrafish caudal fin regeneration.

PubMed

Saxena, Sandeep; Purushothaman, Sruthi; Meghah, Vuppalapaty; Bhatti, Bhawna; Poruri, Akhila; Meena Lakshmi, Mula G; Sarath Babu, Nukala; Narasimha Murthy, Ch Lakshmi; Mandal, Komal K; Kumar, Arvind; Idris, Mohammed M

2016-05-01

The molecular mechanism of epimorphic regeneration is elusive due to its complexity and limitation in mammals. Epigenetic regulatory mechanisms play a crucial role in development and regeneration. This investigation attempted to reveal the role of epigenetic regulatory mechanisms, such as histone H3 and H4 lysine acetylation and methylation during zebrafish caudal fin regeneration. It was intriguing to observe that H3K9,14 acetylation, H4K20 trimethylation, H3K4 trimethylation and H3K9 dimethylation along with their respective regulatory genes, such as GCN5, SETd8b, SETD7/9, and SUV39h1, were differentially regulated in the regenerating fin at various time points of post-amputation. Annexin genes have been associated with regeneration; this study reveals the significant up-regulation of ANXA2a and ANXA2b transcripts and their protein products during the regeneration process. Chromatin immunoprecipitation and PCR analysis of the regulatory regions of the ANXA2a and ANXA2b genes demonstrated the ability to repress two histone methylations, H3K27me3 and H4K20me3, in transcriptional regulation during regeneration. It is hypothesized that this novel insight into the diverse epigenetic mechanisms that play a critical role during the regeneration process may help to strategize the translational efforts, in addition to identifying the molecules involved in vertebrate regeneration. © 2016 by the Wound Healing Society.

Reversible conversion of immortal human cells from telomerase-positive to telomerase-negative cells.

PubMed

Kumakura, Shin-ichi; Tsutsui, Takeo W; Yagisawa, Junko; Barrett, J Carl; Tsutsui, Takeki

2005-04-01

Immortal cell lines and tumors maintain their telomeres via the telomerase pathway or via a telomerase-independent pathway, referred to as alternative lengthening of telomeres (ALT). Here, we show the reversible conversion of the human papillomavirus type 16 E6-induced immortal human fibroblasts E6 Cl 6 from telomerase-positive (Tel(+)) to telomerase-negative (Tel(-)) cells. Tel(+) cells converted spontaneously to Tel(-) cells that reverted to Tel(+) cells following treatment with trichostatin A (TSA) and/or 5-aza-2'-deoxycytidine (5-AZC), which induced the reversion from complete to partial methylation of the CpG islands of the human telomerase reverse transcriptase (hTERT) promoter in Tel(-) E6 Cl 6 cells. Tel(-) E6 Cl 6 cells lacked the phenotypes characteristic of ALT cell lines such as very long and heterogenous telomeres and ALT-associated promyelocytic leukemia nuclear bodies (APB) but grew for >240 population doublings (PD) after they became telomerase negative. The ratios of histone H3 (H3) lysine (K) 9 methylation to each of H3-K4 methylation, H3-K9 acetylation, and H3-K14 acetylation of the chromatin containing the hTERT promoter in Tel(-) E6 Cl 6 cells and ALT cell lines were greater than those in Tel(+) cells and decreased following treatment with TSA and/or 5-AZC, inversely corresponding to telomerase activity. Our findings suggest the possibility that human tumors may be able to reversibly interconvert their telomere maintenance phenotypes by chromatin structure-mediated regulation of hTERT expression.

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome

PubMed Central

Girardot, Michael; Hirasawa, Ryutaro; Kacem, Salim; Fritsch, Lauriane; Pontis, Julien; Kota, Satya K.; Filipponi, Doria; Fabbrizio, Eric; Sardet, Claude; Lohmann, Felix; Kadam, Shilpa; Ait-Si-Ali, Slimane; Feil, Robert

2014-01-01

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus. PMID:24097435

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

PubMed

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

2014-12-01

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

Small-Molecule Modulators of Methyl-Lysine Binding for the CBX7 Chromodomain

DOE PAGES

Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N.; ...

2015-02-05

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes tri-methylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. Here in this study, we report discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show thatmore » a lead compound MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. Ultimately, these small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.« less

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

PubMed

Chater-Diehl, Eric J; Laufer, Benjamin I; 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.

PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation

PubMed Central

Yang, Yan; Li, Wencheng; Hoque, Mainul; Hou, Liming; Shen, Steven; Tian, Bin; Dynlacht, Brian D.

2016-01-01

The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs. PMID:26765774

The molecular basis for stability of heterochromatin-mediated silencing in mammals

PubMed Central

2009-01-01

The archetypal epigenetic phenomenon of position effect variegation (PEV) in Drosophila occurs when a gene is brought abnormally close to heterochromatin, resulting in stochastic silencing of the affected gene in a proportion of cells that would normally express it. PEV has been instrumental in unraveling epigenetic mechanisms. Using an in vivo mammalian model for PEV we have extensively investigated the molecular basis for heterochromatin-mediated gene silencing. Here we distinguish 'epigenetic effects' from other cellular differences by studying ex vivo cells that are identical, apart from the expression of the variegating gene which is silenced in a proportion of the cells. By separating cells according to transgene expression we show here that silencing appears to be associated with histone H3 lysine 9 trimethylation (H3K9me3), DNA methylation and the localization of the silenced gene to a specific nuclear compartment enriched in these modifications. In contrast, histone H3 acetylation (H3Ac) and lysine 4 di or tri methylation (H3K4me2/3) are the predominant modifications associated with expression where we see the gene in a euchromatic compartment. Interestingly, DNA methylation and inaccessibility, rather than H3K9me3, correlated most strongly with resistance to de-repression by cellular activation. These results have important implications for understanding the contribution of specific factors involved in the establishment and maintenance of gene silencing and activation in vivo. PMID:19889207

The combination of dimethoxycurcumin with DNA methylation inhibitor enhances gene re-expression of promoter-methylated genes and antagonizes their cytotoxic effect

PubMed Central

Hassan, Hazem E.; Keita, Jean-Arnaud; Narayan, Lawrence; Brady, Sean M.; Frederick, Richard; Carlson, Samuel; C. Glass, Karen; Natesan, Senthil; Buttolph, Thomm; Fandy, Tamer E.

2016-01-01

ABSTRACT Curcumin and its analogs exhibited antileukemic activity either as single agent or in combination therapy. Dimethoxycurcumin (DMC) is a more metabolically stable curcumin analog that was shown to induce the expression of promoter-methylated genes without reversing DNA methylation. Accordingly, co-treatment with DMC and DNA methyltransferase (DNMT) inhibitors could hypothetically enhance the re-expression of promoter-methylated tumor suppressor genes. In this study, we investigated the cytotoxic effects and epigenetic changes associated with the combination of DMC and the DNMT inhibitor decitabine (DAC) in primary leukemia samples and cell lines. The combination demonstrated antagonistic cytotoxic effects and was minimally cytotoxic to primary leukemia cells. The combination did not affect the metabolic stability of DMC. Although the combination enhanced the downregulation of nuclear DNMT proteins, the hypomethylating activity of the combination was not increased significantly compared to DAC alone. On the other hand, the combination significantly increased H3K27 acetylation (H3K27Ac) compared to the single agents near the promoter region of promoter-methylated genes. Furthermore, sequential chromatin immunoprecipitation (ChIP) and DNA pyrosequencing of the chromatin-enriched H3K27Ac did not show any significant decrease in DNA methylation compared to other regions. Consequently, the enhanced induction of promoter-methylated genes by the combination compared to DAC alone is mediated by a mechanism that involves increased histone acetylation and not through potentiation of the DNA hypomethylating activity of DAC. Collectively, our results provide the mechanistic basis for further characterization of this combination in leukemia animal models and early phase clinical trials. PMID:27588609

Laboratory study of methyl isocyanate ices under astrophysical conditions

NASA Astrophysics Data System (ADS)

Maté, B.; Molpeceres, G.; Timón, V.; Tanarro, I.; Escribano, R.; Guillemin, J. C.; Cernicharo, J.; Herrero, V. J.

2017-10-01

Methyl isocyanate has been recently detected in comet 67P/Churyumov-Gerasimenko (67P/CG) and in the interstellar medium. New physicochemical studies on this species are now necessary as tools for subsequent studies in astrophysics. In this work, infrared spectra of solid CH3NCO have been obtained at temperatures of relevance for astronomical environments. The spectra are dominated by a strong, characteristic multiplet feature at 2350-2250 cm-1, which can be attributed to the asymmetric stretching of the NCO group. A phase transition from amorphous to crystalline methyl isocyanate is observed at ˜90 K. The band strengths for the absorptions of CH3NCO in ice at 20 K have been measured. Deuterated methyl isocyanate is used to help with the spectral assignment. No X-ray structure has been reported for crystalline CH3NCO. Here we advance a tentative theoretical structure, based on density functional theory (DFT) calculations, derived taking the crystal of isocyanic acid as a starting point. A harmonic theoretical spectrum is then calculated for the proposed structure and compared with the experimental data. A mixed ice of H2O and CH3NCO was formed by simultaneous deposition of water and methyl isocyanate at 20 K. The absence of new spectral features indicates that methyl isocyanate and water do not react appreciably at 20 K, but form a stable mixture. The high CH3NCO/H2O ratio reported for comet 67P/CG, and the characteristic structure of the 2350-2250 cm-1 band, makes it a very good candidate for future astronomical searches.

Comprehensive physicochemical studies of a new hybrid material: 2-Amino-4-methyl-3-nitropyridinium hydrogen oxalate

NASA Astrophysics Data System (ADS)

Bryndal, I.; Kucharska, E.; Wandas, M.; Lorenc, J.; Hermanowicz, K.; Mączka, M.; Lis, T.; Marchewka, M.; Hanuza, J.

2014-01-01

A new organic-organic salt, 2-amino-4-methyl-3-nitropyridinium hydrogen oxalate (AMNPO), and its deuterium analogue have been synthesized and characterized by means of FT-IR, FT-Raman, DSC and single crystal X-ray studies. The DSC measurements and temperature dependence of the IR and Raman spectra in the range 4-295 K show that it undergoes a reversible phase transition at ∼240 K. At room temperature it crystallizes in noncentrosymmetric space group P21. The unit-cell is built of the 2-amino-4-methyl-3-nitropyridinium cations and oxalate monoanions which are connected via the Nsbnd H⋯O and Osbnd H⋯O hydrogen bonds. The geometrical and hydrogen bond parameters are similar for non-deuterated (at 120 and 293 K) and deuterated compounds (at 90 K). The phase transition is probably a consequence of order-disorder transition inside of hydrogen network. The 6-311G(2d,2p) basis set with B3LYP functional have been used to discuss the structure and vibrational spectra of the studied compound.

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

PubMed Central

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

2014-01-01

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

Methylation-based classification of benign and malignant peripheral nerve sheath tumors.

PubMed

Röhrich, Manuel; Koelsche, Christian; Schrimpf, Daniel; Capper, David; Sahm, Felix; Kratz, Annekathrin; Reuss, Jana; Hovestadt, Volker; Jones, David T W; Bewerunge-Hudler, Melanie; Becker, Albert; Weis, Joachim; Mawrin, Christian; Mittelbronn, Michel; Perry, Arie; Mautner, Victor-Felix; Mechtersheimer, Gunhild; Hartmann, Christian; Okuducu, Ali Fuat; Arp, Mirko; Seiz-Rosenhagen, Marcel; Hänggi, Daniel; Heim, Stefanie; Paulus, Werner; Schittenhelm, Jens; Ahmadi, Rezvan; Herold-Mende, Christel; Unterberg, Andreas; Pfister, Stefan M; von Deimling, Andreas; Reuss, David E

2016-06-01

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.

The role of DNA methylation in directing the functional organization of the cancer epigenome.

PubMed

Lay, Fides D; Liu, Yaping; Kelly, Theresa K; Witt, Heather; Farnham, Peggy J; Jones, Peter A; Berman, Benjamin P

2015-04-01

The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes. © 2015 Lay et al.; Published by Cold Spring Harbor Laboratory Press.

Arabidopsis thaliana telomeres exhibit euchromatic features

PubMed Central

Vaquero-Sedas, María I.; Gámez-Arjona, Francisco M.; Vega-Palas, Miguel A.

2011-01-01

Telomere function is influenced by chromatin structure and organization, which usually involves epigenetic modifications. We describe here the chromatin structure of Arabidopsis thaliana telomeres. Based on the study of six different epigenetic marks we show that Arabidopsis telomeres exhibit euchromatic features. In contrast, subtelomeric regions and telomeric sequences present at interstitial chromosomal loci are heterochromatic. Histone methyltransferases and the chromatin remodeling protein DDM1 control subtelomeric heterochromatin formation. Whereas histone methyltransferases are required for histone H3K92Me and non-CpG DNA methylation, DDM1 directs CpG methylation but not H3K92Me or non-CpG methylation. These results argue that both kinds of proteins participate in different pathways to reinforce subtelomeric heterochromatin formation. PMID:21071395

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

PubMed

Noh, Kyung-Min; Wang, Haibo; Kim, Hyunjae R; Wenderski, Wendy; Fang, Fang; Li, Charles H; Dewell, Scott; Hughes, Stephen H; Melnick, Ari M; Patel, Dinshaw J; Li, Haitao; Allis, C David

2015-07-02

Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Lymphocytes From Patients With Type 1 Diabetes Display a Distinct Profile of Chromatin Histone H3 Lysine 9 Dimethylation

PubMed Central

Miao, Feng; Smith, David D.; Zhang, Lingxiao; Min, Andrew; Feng, Wei; Natarajan, Rama

2008-01-01

OBJECTIVE—The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications. RESEARCH DESIGN AND METHODS—We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools. RESULTS—A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-β, nuclear factor-κB, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes. CONCLUSIONS—The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes–related biological pathways and also a new understanding of the pathology of type 1 diabetes. PMID:18776137

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

PubMed

Upadhyay, Anup K; Judge, Russell A; Li, Leiming; Pithawalla, Ron; Simanis, Justin; Bodelle, Pierre M; Marin, Violeta L; Henry, Rodger F; Petros, Andrew M; Sun, Chaohong

2018-06-01

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain. Copyright © 2018 Elsevier Ltd. All rights reserved.

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

PubMed

Sneeringer, Christopher J; Scott, Margaret Porter; Kuntz, Kevin W; Knutson, Sarah K; Pollock, Roy M; Richon, Victoria M; Copeland, Robert A

2010-12-07

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

Binding selectivity of vitamin K3 based chemosensors towards nickel(II) and copper(II) metal ions

NASA Astrophysics Data System (ADS)

Patil, Amit; Lande, Dipali N.; Nalkar, Archana; Gejji, Shridhar P.; Chakrovorty, Debamitra; Gonnade, Rajesh; Moniz, Tânia; Rangel, Maria; Pereira, Eulália; Salunke-Gawali, Sunita

2017-09-01

The vitamin K3 derivatives 2-methyl-3-[(pyridin-2-ylmethyl)-amino]-1,4-naphthoquinone (M-1), 2-methyl-3-[(pyridin-2-ylethyl)-amino]-1,4-naphthoquinone (M-2), 2-methyl-3-((2-(thiophen-2-yl)methyl)amino)naphthalene-1,4-dione (M-3) and 2-methyl-3-((2-(thiophen-2-yl)ethyl)amino)naphthalene-1,4-dione (M-4) have been synthesized, characterized and studied for their chemosensor abilities towards transition metal ions. Crystal structures of M-1 to M-4 revealed a variety of Nsbnd H⋯O, Csbnd H⋯O, Csbnd H⋯π and π⋯π interactions. Minor variations in such interactions by chemical stimuli such as metal ions, results in change in color that can be visualized by naked eyes. It has been shown that electronic structure and 1H NMR, vibrational as well as electronic spectra from the density functional theory agree well with the experiments. The metal ion binding in ethanol, ethanol-water and in mild base triethylamine brings forth recognizing ability of M-1 toward Ni2+ whereas M-2 exhibits large sensing ability for Cu2+ ion. Interestingly M-1 display varying metal ion binding specificity in different solvents with the association constant in ethanol being 11,786 M-1 for Ni2+ compared to 9462 M-1 for the Cu2+. A reversal in preferential binding of M-2 with the respective association constants being 4190 M-1 and 6370 M-1 is discernible.

New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

PubMed Central

Nic-Can, Geovanny I.; López-Torres, Adolfo; Barredo-Pool, Felipe; Wrobel, Kazimierz; Loyola-Vargas, Víctor M.; Rojas-Herrera, Rafael; De-la-Peña, Clelia

2013-01-01

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed. PMID:23977240

Lack of the COMPASS Component Ccl1 Reduces H3K4 Trimethylation Levels and Affects Transcription of Secondary Metabolite Genes in Two Plant-Pathogenic Fusarium Species.

PubMed

Studt, Lena; Janevska, Slavica; Arndt, Birgit; Boedi, Stefan; Sulyok, Michael; Humpf, Hans-Ulrich; Tudzynski, Bettina; Strauss, Joseph

2016-01-01

In the two fungal pathogens Fusarium fujikuroi and Fusarium graminearum , secondary metabolites (SMs) are fitness and virulence factors and there is compelling evidence that the coordination of SM gene expression is under epigenetic control. Here, we characterized Ccl1, a subunit of the COMPASS complex responsible for methylating lysine 4 of histone H3 (H3K4me). We show that Ccl1 is not essential for viability but a regulator of genome-wide trimethylation of H3K4 (H3K4me3). Although, recent work in Fusarium and Aspergillus spp. detected only sporadic H3K4 methylation at the majority of the SM gene clusters, we show here that SM profiles in CCL1 deletion mutants are strongly deviating from the wild type. Cross-complementation experiments indicate high functional conservation of Ccl1 as phenotypes of the respective △ ccl1 were rescued in both fungi. Strikingly, biosynthesis of the species-specific virulence factors gibberellic acid and deoxynivalenol produced by F. fujikuroi and F. graminearum , respectively, was reduced in axenic cultures but virulence was not attenuated in these mutants, a phenotype which goes in line with restored virulence factor production levels in planta. This suggests that yet unknown plant-derived signals are able to compensate for Ccl1 function during pathogenesis.

Architecture of epigenetic reprogramming following Twist1-mediated epithelial-mesenchymal transition

PubMed Central

2013-01-01

Background Epithelial-mesenchymal transition (EMT) is known to impart metastasis and stemness characteristics in breast cancer. To characterize the epigenetic reprogramming following Twist1-induced EMT, we characterized the epigenetic and transcriptome landscapes using whole-genome transcriptome analysis by RNA-seq, DNA methylation by digital restriction enzyme analysis of methylation (DREAM) and histone modifications by CHIP-seq of H3K4me3 and H3K27me3 in immortalized human mammary epithelial cells relative to cells induced to undergo EMT by Twist1. Results EMT is accompanied by focal hypermethylation and widespread global DNA hypomethylation, predominantly within transcriptionally repressed gene bodies. At the chromatin level, the number of gene promoters marked by H3K4me3 increases by more than one fifth; H3K27me3 undergoes dynamic genomic redistribution characterized by loss at half of gene promoters and overall reduction of peak size by almost half. This is paralleled by increased phosphorylation of EZH2 at serine 21. Among genes with highly altered mRNA expression, 23.1% switch between H3K4me3 and H3K27me3 marks, and those point to the master EMT targets and regulators CDH1, PDGFRα and ESRP1. Strikingly, Twist1 increases the number of bivalent genes by more than two fold. Inhibition of the H3K27 methyltransferases EZH2 and EZH1, which form part of the Polycomb repressive complex 2 (PRC2), blocks EMT and stemness properties. Conclusions Our findings demonstrate that the EMT program requires epigenetic remodeling by the Polycomb and Trithorax complexes leading to increased cellular plasticity. This suggests that inhibiting epigenetic remodeling and thus decrease plasticity will prevent EMT, and the associated breast cancer metastasis. PMID:24367927

Contrasting molecular pathology of colorectal carcinoma in Egyptian and Western patients

PubMed Central

Soliman, A S; Bondy, M L; El-Badawy, S A; Mokhtar, N; Eissa, S; Bayoumy, S; Seifeldin, I A; Houlihan, P S; Lukish, J R; Watanabe, T; Chan, A On On; Zhu, D; Amos, C I; Levin, B; Hamilton, S R

2001-01-01

Colorectal carcinoma is uncommon in Egypt, but a high proportion of cases occurs before age 40 years and in the rectum. We compared the molecular pathology of 59 representative Egyptian patients aged 10–72 to Western patients with sporadic, young-onset, or hereditary non-polyposis colorectal cancer syndrome (HNPCC)-associated carcinoma and found significant differences. Most Egyptian cancers were rectal (51%) and poorly differentiated (58%). High levels of microsatellite instability (MSI-H) were frequent (37%) and attributable in some cases (36%) to methylation of the promoter of the hMLH1 mismatch repair gene, but no MSI-H cancer had loss of hMSH2 mismatch repair gene product of the type seen with germline hMSH2 mutation in HNPCC. K-ras mutation was uncommon (11%). In subset analyses, high frequencies of MSI-H in rectal carcinomas (36%) and p53 gene product overexpression in MSI-H cancers (50%) were found. MSI-H and K-ras mutation in Egyptians under age 40 were unusual (17% and 0%, respectively), and schistosomiasis was associated with MSI and K-ras mutation. Cluster analysis identified 2 groups: predominantly young men with poorly differentiated mucinous and signet-ring cell colorectal carcinoma lacking K-ras mutation; older patients who had well- or moderately differentiated adenocarcinoma often with MSI-H, K-ras mutation and schistosomiasis. Our findings show that the molecular pathology of colorectal cancer in older as well as younger Egyptians has unique differences from Western patients, and schistosomiasis influences the molecular pathogenesis of some tumours. © 2001 Cancer Research Campaignhttp://www.bjcancer.com PMID:11592777

Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation.

PubMed

Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

2014-12-01

Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chronic exposure to a low concentration of bisphenol A during follicle culture affects the epigenetic status of germinal vesicles and metaphase II oocytes.

PubMed

Trapphoff, Tom; Heiligentag, Martyna; El Hajj, Nady; Haaf, Thomas; Eichenlaub-Ritter, Ursula

2013-12-01

To determine whether exposure to low concentrations of the endocrine disrupting chemical bisphenol A (BPA) during follicle culture and oocyte growth alters the methylation status of differentially methylated regions (DMRs) of imprinted genes and histone posttranslational modification patterns in mammalian oocytes. Comparative and control study. Experimental laboratory. C57/Bl6JxCBA/Ca mice. Exposure of oocytes to 3 nM or 300 nM BPA during follicle culture from preantral to antral stage. Methylation status of DMRs of maternally imprinted (Snrpn, Igf2r, and Mest) and paternally imprinted gene(s) (H19) in mouse germinal vesicle oocytes; trimethylation of histone H3K9, acetylation of histone H4K12, and distance between centromeres of sister chromatids in metaphase II oocytes. Exposure to 3 nM BPA was associated with slightly accelerated follicle development, statistically significant increases in allele methylation errors in DMRs of maternally imprinted genes, and statistically significant decreases in histone H3K9 trimethylation and interkinetochore distance. The disturbances in oocyte genomic imprinting and modification of posttranslational histone and centromere architecture provide the first link between low BPA exposures and induction of epigenetic changes that may contribute to chromosome congression failures and meiotic errors, and to altered gene expression that might affect health of the offspring. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Higher chylomicron remnants and LDL particle numbers associate with CD36 SNPs and DNA methylation sites that reduce CD36.

PubMed

Love-Gregory, Latisha; Kraja, Aldi T; Allum, Fiona; Aslibekyan, Stella; Hedman, Åsa K; Duan, Yanan; Borecki, Ingrid B; Arnett, Donna K; McCarthy, Mark I; Deloukas, Panos; Ordovas, Jose M; Hopkins, Paul N; Grundberg, Elin; Abumrad, Nada A

2016-12-01

Cluster of differentiation 36 (CD36) variants influence fasting lipids and risk of metabolic syndrome, but their impact on postprandial lipids, an independent risk factor for cardiovascular disease, is unclear. We determined the effects of SNPs within a ∼410 kb region encompassing CD36 and its proximal and distal promoters on chylomicron (CM) remnants and LDL particles at fasting and at 3.5 and 6 h following a high-fat meal (Genetics of Lipid Lowering Drugs and Diet Network study, n = 1,117). Five promoter variants associated with CMs, four with delayed TG clearance and five with LDL particle number. To assess mechanisms underlying the associations, we queried expression quantitative trait loci, DNA methylation, and ChIP-seq datasets for adipose and heart tissues that function in postprandial lipid clearance. Several SNPs that associated with higher serum lipids correlated with lower adipose and heart CD36 mRNA and aligned to active motifs for PPARγ, a major CD36 regulator. The SNPs also associated with DNA methylation sites that related to reduced CD36 mRNA and higher serum lipids, but mixed-model analyses indicated that the SNPs and methylation independently influence CD36 mRNA. The findings support contributions of CD36 SNPs that reduce adipose and heart CD36 RNA expression to inter-individual variability of postprandial lipid metabolism and document changes in CD36 DNA methylation that influence both CD36 expression and lipids. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human

PubMed Central

Takahashi, Yoh-hei; Westfield, Gerwin H.; Oleskie, Austin N.; Trievel, Raymond C.; Shilatifard, Ali; Skiniotis, Georgios

2011-01-01

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies. PMID:22158900

Structural analysis of the core COMPASS family of histone H3K4 methylases from yeast to human.

PubMed

Takahashi, Yoh-hei; Westfield, Gerwin H; Oleskie, Austin N; Trievel, Raymond C; Shilatifard, Ali; Skiniotis, Georgios

2011-12-20

Histone H3 lysine 4 (H3K4) methylation is catalyzed by the highly evolutionarily conserved multiprotein complex known as Set1/COMPASS or MLL/COMPASS-like complexes from yeast to human, respectively. Here we have reconstituted fully functional yeast Set1/COMPASS and human MLL/COMPASS-like complex in vitro and have identified the minimum subunit composition required for histone H3K4 methylation. These subunits include the methyltransferase C-terminal SET domain of Set1/MLL, Cps60/Ash2L, Cps50/RbBP5, Cps30/WDR5, and Cps25/Dpy30, which are all common components of the COMPASS family from yeast to human. Three-dimensional (3D) cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and two-dimensional (2D) EM analysis of the individual subcomplexes reveal a Y-shaped architecture with Cps50 and Cps30 localizing on the top two adjacent lobes and Cps60-Cps25 forming the base at the bottom. EM analysis of the human complex reveals a striking similarity to its yeast counterpart, suggesting a common subunit organization. The SET domain of Set1 is located at the juncture of Cps50, Cps30, and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation. This structural arrangement suggested that COMPASS family members function as exo-methylases, which we have confirmed by in vitro and in vivo studies.

EG-17SUV420-MEDIATED HETEROCHROMATIN CHANGES IN PEDIATRIC BRAIN CANCERS

PubMed Central

Van Meter, Timothy E.; Terry, Jocelyn; Rockwell, Nathan; Goggin, Sarah; Nethala, Priya; Khan, Asadullah

2014-01-01

Silencing mechanisms play a role in genomic stability by maintaining condensed, non-active regions of the genome. SUV420 enzymes contain a SET domain conferring methyltransferase activity toward histones. The Histone H4 lysine 20 trimethylation (H4K20me3) mark maintained by SUV420H2 is associated with heterochromatin formation and gene silencing, whereas the dimethylated mark (H4K20me2) is associated with DNA repair. In studies of epigenetic factors in large patient cohorts with ependymoma, it was found that SUV420H2 expression was lost or diminished in patients with reciprocal increases in prognostic markers such as hTERT. To better understand the normal function of Suv4-20H1/H2 enzyme in neural progenitors, and pathological changes in cancers, a variety of differentiation paradigms were used. The NT2D1 neurally restricted cell line, and BGO1V and H9 human embryonic stem cells (ESCs), and differentiated progeny, were used alongside tumors to better understand enzyme targets and functional outcomes (e.g.,lineage, differentiation, regional chromatin modifications). Lineage stages were verified with stage-specific markers by immunofluorescence and qPCR. Suv4-20 H1 and H2 were present in ESCs and neural progenitors and decreased thereafter. RNAi knockdown of SUV420 enzymes led to decreased H4K20 methylation in cancer cells. DNA methylation microarrays and ChIP-PCR suggest 1) that SUV420 is not regulated by DNA methylation in ependymomas; 2) that active chromatin marks such as H3K4 dimethylation are enriched near the transcriptional start site in the SUV420H2 gene, and 3) that hTERT is hyper-methylated at specific CpG islands and histones in a tumor sub-group-specific manner. This data supports the hypothesis that Suv4-20H2 is highly active in progenitor cells and functionally lost in some brain cancers. These studies begin to elucidate coincident mechanisms of gene silencing active in neural progenitors that may be altered in a subset of pediatric brain cancers.

KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp

PubMed Central

2012-01-01

Background The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors. Results To gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2. Conclusions Agave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants. PMID:23126409

Thermal stability of spent coffee ground polysaccharides: galactomannans and arabinogalactans.

PubMed

Simões, Joana; Maricato, Elia; Nunes, Fernando M; Domingues, M Rosário; Coimbra, Manuel A

2014-01-30

In order to better understand the thermal stability of spent coffee grounds (SCG) galactomannans and arabinogalactans and the reactions that can occur upon roasting, long term isothermal exposures, up to 3h, were performed at 160, 180, 200, 220, and 240 °C. The resultant products were analysed according to the sugars and linkage composition and also by electrospray mass spectrometry. Galactomannans did not loss mass at T ≤ 200 °C during exposures up to 3h whereas the arabinogalactans showed that thermal stability only for T ≤ 180 °C. This was in accordance with the estimated activation energies of their thermal decomposition of 138 kJ/mol and 94 kJ/mol, respectively. The roasting of galactomannans promoted the formation of new glycosidic linkages, with occurrence of 2-, 6-, 2,3-, 2,6-, 3,6-, 2,3,6-, 3,4,6-linked mannose residues, 3,4,6-linked galactose residues, and terminally-linked glucose residues, observed by methylation analysis. Depolymerisation and formation of anhydrohexose residues at the reducing end and mannose-glucose isomerisation were also observed. The roasting of galactomannans at 200 °C promoted their solubility in water upon alkali extraction and neutralisation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Arsenic silences hepatic PDK4 expression through activation of histone H3K9 methylatransferase G9a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xi; Wu, Jianguo; Choiniere, Jonathan

It is well established that increased liver cancer incidence is strongly associated with epigenetic silencing of tumor suppressor genes; the latter is contributed by the environmental exposure to arsenic. Pyruvate dehydrogenase kinase 4 (PDK4) is a mitochondrial protein that regulates the TCA cycle. However, the epigenetic mechanisms mediated by arsenic to control PDK4 expression remain elusive. In the present study, we showed that histone methyltransferase G9a- and Suv39H-mediated histone H3 lysine 9 (H3K9) methylations contributed to PDK4 silencing in hepatic cells. The PDK4 expression was induced by G9a inhibitor BRD4770 (BRD) and Suv39H inhibitor Chaetocin (CHA). In contrast, arsenic exposuremore » decreased PDK4 expression by inducing G9a and increasing H3K9 di- and tri-methylations levels (H3K9me2/3). In addition, arsenic exposure antagonizes the effect of BRD by enhancing the enrichment of H3K9me2/3 in the PKD4 promoter. Moreover, knockdown of G9a using siRNA induced PDK4 expression in HCC cells. Furthermore, arsenic decreased hepatic PDK4 expression as well as diminished the induction of PDK4 by BRD in mouse liver and hepatocytes. Overall, the results suggest that arsenic causes aberrant repressive histone modification to silence PDK4 in both HCC cells and in mouse liver. - Graphical abstract: Schematic showing arsenic-mediated epigenetic pathway that inhibits PDK4 expression. (A) BRD induces PDK4 expression by decreasing G9a protein and histone H3K9me2 and H3K9me3 levels as well as diminishing their recruitment to the PDK4 promoter. (B) Arsenic counteracts the effect of BRD by increasing histone H3K9me2 and H3K9me3 levels as well as enhancing their enrichment to the PDK4 promoter. Display Omitted - Highlights: • Histone methyltrasferase G9a inhibitor BRD induces PDK4 expression. • Arsenic decreases PDK4 expression and increases H3K9me2 and me3 levels. • Arsenic enhances H3K9me2/me3 enrichment in the PDK4 promoter. • Arsenic antagonizes the activation of PDK4 by BRD.« less

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

PubMed

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

2017-06-01

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

Cluster size resolving analysis of CH3F-(ortho-H2)n in solid para-hydrogen using FTIR absorption spectroscopy at 3 μm region.

PubMed

Miyamoto, Yuki; Momose, Takamasa; Kanamori, Hideto

2012-11-21

Infrared absorption spectra of methyl fluoride with ortho-hydrogen (ortho-H(2)) clusters in a solid para-hydrogen (para-H(2)) crystal at 3.6 K were studied in the C-H stretching fundamental region (~3000 cm(-1)) using an FTIR spectrometer. As shown previously, the ν(3) C-F stretching fundamental band of CH(3)F-(ortho-H(2))(n) (n = 0, 1, 2, ...) clusters at 1040 cm(-1) shows a series of n discrete absorption lines, which correspond to different-sized clusters. We observed three unresolved broad peaks in the C-H stretching region and applied this cluster model to them assuming the same intensity distribution function as the ν(3) band. A fitting analysis successfully gave us the linewidth and lineshift of the components in each vibrational band. It was found that the separately determined linewidth, matrix shift of the band origin, and cluster shift are dependent on the vibrational mode. From the transition intensities of the monomer component derived from the fitting analysis, we discuss the mixing ratio of the vibrational modes due to Fermi resonance.

Regulation of tumour related genes by dynamic epigenetic alteration at enhancer regions in gastric epithelial cells infected by Epstein-Barr virus.

PubMed

Okabe, Atsushi; Funata, Sayaka; Matsusaka, Keisuke; Namba, Hiroe; Fukuyo, Masaki; Rahmutulla, Bahityar; Oshima, Motohiko; Iwama, Atsushi; Fukayama, Masashi; Kaneda, Atsushi

2017-08-11

Epstein-Barr virus (EBV) infection is associated with tumours such as Burkitt lymphoma, nasopharyngeal carcinoma, and gastric cancer. We previously showed that EBV(+) gastric cancer presents an extremely high-methylation epigenotype and this aberrant DNA methylation causes silencing of multiple tumour suppressor genes. However, the mechanisms that drive EBV infection-mediated tumorigenesis, including other epigenomic alteration, remain unclear. We analysed epigenetic alterations induced by EBV infection especially at enhancer regions, to elucidate their contribution to tumorigenesis. We performed ChIP sequencing on H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K9me3 in gastric epithelial cells infected or not with EBV. We showed that repressive marks were redistributed after EBV infection, resulting in aberrant enhancer activation and repression. Enhancer dysfunction led to the activation of pathways related to cancer hallmarks (e.g., resisting cell death, disrupting cellular energetics, inducing invasion, evading growth suppressors, sustaining proliferative signalling, angiogenesis, and tumour-promoting inflammation) and inactivation of tumour suppressive pathways. Deregulation of cancer-related genes in EBV-infected gastric epithelial cells was also observed in clinical EBV(+) gastric cancer specimens. Our analysis showed that epigenetic alteration associated with EBV-infection may contribute to tumorigenesis through enhancer activation and repression.

Improved Therapeutic Effect against Leukemia by a Combination of the Histone Methyltransferase Inhibitor Chaetocin and the Histone Deacetylase Inhibitor Trichostatin A

PubMed Central

Tran, Huong Thi Thanh; Kim, Hee Nam; Lee, Il-Kwon; Nguyen-Pham, Thanh-Nhan; Ahn, Jae-Sook; Kim, Yeo-Kyeoung; Lee, Je-Jung; Park, Kyeong-Soo; Kook, Hoon

2013-01-01

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia. PMID:23400519

Immunohistochemical analysis of H3K27me3 demonstrates global reduction in group-A childhood posterior fossa ependymoma and is a powerful predictor of outcome.

PubMed

Panwalkar, Pooja; Clark, Jonathan; Ramaswamy, Vijay; Hawes, Debra; Yang, Fusheng; Dunham, Christopher; Yip, Stephen; Hukin, Juliette; Sun, Yilun; Schipper, Matthew J; Chavez, Lukas; Margol, Ashley; Pekmezci, Melike; Chung, Chan; Banda, Adam; Bayliss, Jill M; Curry, Sarah J; Santi, Mariarita; Rodriguez, Fausto J; Snuderl, Matija; Karajannis, Matthias A; Saratsis, Amanda M; Horbinski, Craig M; Carret, Anne-Sophie; Wilson, Beverly; Johnston, Donna; Lafay-Cousin, Lucie; Zelcer, Shayna; Eisenstat, David; Silva, Marianna; Scheinemann, Katrin; Jabado, Nada; McNeely, P Daniel; Kool, Marcel; Pfister, Stefan M; Taylor, Michael D; Hawkins, Cynthia; Korshunov, Andrey; Judkins, Alexander R; Venneti, Sriram

2017-11-01

Posterior fossa ependymomas (EPN_PF) in children comprise two morphologically identical, but biologically distinct tumor entities. Group-A (EPN_PFA) tumors have a poor prognosis and require intensive therapy. In contrast, group-B tumors (EPN_PFB) exhibit excellent prognosis and the current consensus opinion recommends future clinical trials to test the possibility of treatment de-escalation in these patients. Therefore, distinguishing these two tumor subtypes is critical. EPN_PFA and EPN_PFB can be distinguished based on DNA methylation signatures, but these assays are not routinely available. We have previously shown that a subset of poorly prognostic childhood EPN_PF exhibits global reduction in H3K27me3. Therefore, we set out to determine whether a simple immunohistochemical assay for H3K27me3 could be used to segregate EPN_PFA from EPN_PFB tumors. We assembled a cohort of 230 childhood ependymomas and H3K27me3 immunohistochemistry was assessed as positive or negative in a blinded manner. H3K27me3 staining results were compared with DNA methylation-based subgroup information available in 112 samples [EPN_PFA (n = 72) and EPN_PFB tumors (n = 40)]. H3K27me3 staining was globally reduced in EPN_PFA tumors and immunohistochemistry showed 99% sensitivity and 100% specificity in segregating EPN_PFA from EPN_PFB tumors. Moreover, H3K27me3 immunostaining was sufficient to delineate patients with worse prognosis in two independent, non-overlapping cohorts (n = 133 and n = 97). In conclusion, immunohistochemical evaluation of H3K27me3 global reduction is an economic, easily available and readily adaptable method for defining high-risk EPN_PFA from low-risk posterior fossa EPN_PFB tumors to inform prognosis and to enable the design of future clinical trials.

β-glucuronidase inhibitory studies on coumarin derivatives.

PubMed

Khan, Khalid Mohammed; Fakhri, Muhammad Imran; Shaikh, Nimra Naveed; Saad, Syed Muhammad; Hussain, Shafqat; Perveen, Shahnaz; Choudhary, Muhammad Iqbal

2014-01-01

Twenty-three (23) derivatives of coumarin (5-27) were synthesized and screened for their in vitro β- glucuronidase (E. coli) inhibitory activities. Only three compounds, 7,8-dihydroxy-4-methyl-2H-chromen-2-one (9) (IC50 = 52.39 ± 1.85 µM), 3-chloro-6-hexyl-7-hydroxy-4-methyl-2H-chromen-2-one (18) (IC50 = 60.50 ± 0.87 µM), and 3,6- dichloro-7-hydroxy-4-methyl-2H-chromen-2-one (15) (IC50 = 380.26 ± 0.92 µM) displayed activities against β- glucuronidase as compared to standard D-saccharic acid 1,4-lactone (IC50 = 45.75 ± 2.16 µM). The results indicated that the activity of the synthetic coumarins depends upon the substituents present on the coumarin skeleton.

Deuterium and oxygen-18 isotope effects on nucleophilic displacement by water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.

1981-01-01

Kinetic deuterium isotope effects (k/sub H/sub 2/O//k/sub D/sub 2/O/) have been measured for nucleophilic attack by water at primary alkyl carbon (S-methylthiophenium ion (MeTh+), methyl trifluoromethane-sulfonate (MeOTf), methyl and ethyl perchlorate (MeOClO/sub 3/, EtOClO/sub 3/)) in aprotic solvents (acetonitrile (MeCN) and tetrahydrothiophene-1, 1-dioxide (TMSO/sub 2/; solfolane)) and L/sub 2/O. In L/sub 2/O solvent k/sub H/sub 2/O//k/sub D/sub 2/O/ is significantly greater than unity while it is reduced to near unity in aprotic solvents. The oxygen-18 isotope effect has also been observed to be 1.002 +- 0.004 for the reaction of MeTh/sup +/ with dilute H/sub 2/O in TMSO/sub 2/ atmore » 35/sup 0/C. For the reaction of MeTh/sup +/ and MeOClO/sub 3/ in aprotic solvents the chemical reaction kinetics are discussed.« less

Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones as protoporphyrinogen oxidase inhibitors.

PubMed

Zuo, Yang; Yang, Sheng-Gang; Luo, Yan-Ping; Tan, Ying; Hao, Ge-Fei; Wu, Qiong-You; Xi, Zhen; Yang, Guang-Fu

2013-06-01

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a-z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K(i) values ranging from 0.06 to 17.79 μM. Compound 2e, ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K(i) value of 0.06 μM against mtPPO, comparable to (K(i)=0.03 μM) sulfentrazone. Further green house assays showed that compound 2f (K(i)=0.24 μM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 gai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 gai/ha, whereas they are susceptible to sulfentrazone even at 75 gai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ischemic Preconditioning Confers Epigenetic Repression of Mtor and Induction of Autophagy Through G9a-Dependent H3K9 Dimethylation.

PubMed

Gidlöf, Olof; Johnstone, Andrea L; Bader, Kerstin; Khomtchouk, Bohdan B; O'Reilly, Jiaqi J; Celik, Selvi; Van Booven, Derek J; Wahlestedt, Claes; Metzler, Bernhard; Erlinge, David

2016-12-22

Ischemic preconditioning (IPC) protects the heart from prolonged ischemic insult and reperfusion injury through a poorly understood mechanism. Post-translational modifications of histone residues can confer rapid and drastic switches in gene expression in response to various stimuli, including ischemia. The aim of this study was to investigate the effect of histone methylation in the response to cardiac ischemic preconditioning. We used cardiac biopsies from mice subjected to IPC to quantify global levels of 3 of the most well-studied histone methylation marks (H3K9me2, H3K27me3, and H3K4me3) with Western blot and found that H3K9me2 levels were significantly increased in the area at risk compared to remote myocardium. In order to assess which genes were affected by the increase in H3K9me2 levels, we performed ChIP-Seq and transcriptome profiling using microarray. Two hundred thirty-seven genes were both transcriptionally repressed and enriched in H3K9me2 in the area at risk of IPC mice. Of these, Mtor (Mechanistic target of rapamycin) was chosen for mechanistic studies. Knockdown of the major H3K9 methyltransferase G9a resulted in a significant decrease in H3K9me2 levels across Mtor, increased Mtor expression, as well as decreased autophagic activity in response to rapamycin and serum starvation. IPC confers an increase of H3K9me2 levels throughout the Mtor gene-a master regulator of cellular metabolism and a key player in the cardioprotective effect of IPC-leading to transcriptional repression via the methyltransferase G9a. The results of this study indicate that G9a has an important role in regulating cardiac autophagy and the cardioprotective effect of IPC. © 2016 The Authors and University of Miami Miller School of Medicine. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation.

PubMed

Trindade, Lucas Vinícius; Desagiacomo, Carla; Polizeli, Maria de Lourdes Teixeira de Moraes; Damasio, André Ricardo de Lima; Lima, Aline Margarete Furuyama; Gomes, Eleni; Bonilla-Rodriguez, Gustavo Orlando

2016-01-01

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca 2+ , and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. E a ( d ) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and Δ G ( d ) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus . Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.

Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic Fungus Rhizomucor pusillus A13.36 Obtained by Submerged Cultivation

PubMed Central

Trindade, Lucas Vinícius; Desagiacomo, Carla; Damasio, André Ricardo de Lima; Lima, Aline Margarete Furuyama; Gomes, Eleni

2016-01-01

This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5–47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. E a(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG (d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot. PMID:28025649

Solubility Report of 1-Methyl-3,5-Dinitro-1H-1,2,4-Triazole (MDNT) and 2-Methyl-4,5-Dinitro-2H-1,2,3-Triazole 1-Oxide (MDNTO) for Co-Crystallization Screen

DTIC Science & Technology

2015-11-01

New Co-Crystals,” Crystal Growth & Design, Vol. 9, No. 3, pp. 1531-1537, 2009. 2 Landenberger, K., Matzger, A., “ Cocrystal Engineering of a...Y., Li, H., Zhou, Y., Zhou, J., Geo, T., Zhang, H., Jiang, G., “Toward Low- Sensitive and High-Energetic Cocrystal 1: Evaluation of the Power and the...Safety of Observed Energetic Cocrystals ,” CrystEngComm, 15, 4003-4014, 2013. 4 Levinthal, M.L., “Propellant Made With Cocrystals of

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

PubMed

Rodríguez-Miguel, Cristina; Moral, Raquel; Escrich, Raquel; Vela, Elena; Solanas, Montserrat; Escrich, Eduard

2015-01-01

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Doubly end-on azido bridged mixed-valence cobalt trinuclear complex: Spectral study, VTM, inhibitory effect and antimycobacterial activity on human carcinoma and tuberculosis cells

NASA Astrophysics Data System (ADS)

Datta, Amitabha; Das, Kuheli; Sen, Chandana; Karan, Nirmal Kumar; Huang, Jui-Hsien; Lin, Chia-Her; Garribba, Eugenio; Sinha, Chittaranjan; Askun, Tulin; Celikboyun, Pinar; Mane, Sandeep B.

2015-09-01

Doubly end-on azido-bridged mixed-valence trinuclear cobalt complex, [Co3(L)2(N3)6(CH3OH)2] (1) is afforded by employing a potential monoanionic tetradentate-N2O2 Schiff base precursor (2-[{[2-(dimethylamino)ethyl]imino}methyl]-6-methoxyphenol; HL). Single crystal X-ray structure reveals that in 1, the adjacent CoII and CoIII ions are linked by double end-on azido bridges and thus the full molecule is generated by the site symmetry of a crystallographic twofold rotation axis. Complex 1 is subjected on different spectral analysis such as IR, UV-vis, emission and EPR spectroscopy. On variable temperature magnetic study, we observe that during cooling, the χMT values decrease smoothly until 15 K and then reaches to the value 1.56 cm3 K mol-1 at 2 K. Complex 1 inhibits the cell growth on human lung carcinoma (A549 cells), human colorectal (COLO 205 and HT-29 cells), and human heptacellular (PLC5 cells) carcinoma cells. Complex 1 exhibits anti-mycobacterial activity and considerable efficacy on Mycobacterium tuberculosis H37Rv ATCC 27294 and H37Ra ATCC 25177 strains.

Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata.

PubMed

Valledor, Luis; Pascual, Jesús; Meijón, Mónica; Escandón, Mónica; Cañal, María Jesús

2015-01-01

Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species.

Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata

PubMed Central

Meijón, Mónica; Escandón, Mónica; Cañal, María Jesús

2015-01-01

Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species. PMID:25965766

Epigenetic regulation of normal human mammary cell type-specific miRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

Enthalpies of melting of LnSF compounds (Ln = La, Ce, Pr, Nd, Sm)

NASA Astrophysics Data System (ADS)

Andreev, P. O.; Mikhalkina, O. G.; Andreev, O. V.; Elyshev, A. V.

2015-05-01

The melting temperatures and enthalpies of such congruently melting compounds as LaSF ( T m = 1713 ± 7 K, Δ H = 45.7 ± 4.6 kJ/mol), CeSF ( T m = 1683 ± 7 K, Δ H = 40.7 ± 4.1 kJ/mol), PrSF ( T m = 1661 ± 7 K, Δ H = 39.7 ± 4.0 kJ/mol), NdSF ( T m = 1654 ± 7 K, Δ H = 40.2 ± 4.0 kJ/mol), and SmSF ( T m = 1587 ± 7 K, Δ H = 36.1 ± 3.6 kJ/mol) are determined via synchronous thermal analysis. The tetrad effect is evident in the change of the melting temperatures and enthalpies of LnSF compounds (Ln = La, Ce, Pr, Nd, Sm) depending on r(Ln3+).

Synthesis, DNA interaction, and cytotoxic activity of a novel proflavine-dithiazolidinone pharmacophore.

PubMed

Janovec, Ladislav; Sabolova, Danica; Kozurkova, Maria; Paulíkova, Helena; Kristian, Pavol; Ungvarský, Jan; Moravcíkova, Erika; Bajdichova, Maria; Podhradský, Dusan; Imrich, Jan

2007-01-01

Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K = 6.2-2.2 x 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2',2' '-[(Acridin-3,6-diyl)diimino]-3',3' '-dipropyl-1,3-dithiazolidin-4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 microM and 12.9 microM over the course of 72 h.

Synthesis and characterization of n-alkylamino derivatives of vitamin K3: Molecular structure of 2-propylamino-3-methyl-1,4-naphthoquinone and antibacterial activities

NASA Astrophysics Data System (ADS)

Chadar, Dattatray; Camilles, Maria; Patil, Rishikesh; Khan, Ayesha; Weyhermüller, Thomas; Salunke-Gawali, Sunita

2015-04-01

We would like to introduce eight analogues of n-alkylamino derivatives of vitamin K3 (2-methyl-1,4-naphthoquinone) viz, 2-(n-alkylamino)-3-methyl-1,4-naphthoquinone (where n-alkyl is methyl; LM-1, ethyl; LM-2, propyl; LM-3, butyl; LM-4, pentyl; LM-5, hexyl; LM-6, heptyl; LM-7, octyl; LM-8). All the above analogues have been successfully synthesized from vitamin K3 and characterized using different analytical techniques. Furthermore, in order to understand the mechanistic aspects of formation of LM-1 to LM-8 compounds, we could propose the mechanism. The FT-IR analysis of LM-1 to LM-8 indicate the presence of characteristic band of Nsbnd H group ∼3287-3364 cm-1, the variation was attributed to extensive intramolecular hydrogen bonding interaction. The molecular structure of LM-3 compound has been confirmed by single crystal X-ray diffraction analysis. LM-3 compound crystallises in triclinic space group P1. There were four independent molecules in asymmetric unit cell and their molecular interactions observed via Nsbnd H⋯O, Csbnd H⋯O and π-π stacking of quinonoid rings. Pharmacological potential of all compounds has been evaluated in terms of their antibacterial activities against Pseudomonas aeruginosa and Staphylococcus aureus. All the compounds were active against both the strains while LM-2 was found to be more effective with a minimum inhibition concentration of 0.3125 μg/mL and 0.156 μg/mL respectively.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Rapid Conversion of Mutant IDH1 from Driver to Passenger in a Model of Human Gliomagenesis

PubMed Central

Johannessen, Tor-Christian Aase; Mukherjee, Joydeep; Viswanath, Pavithra; Ohba, Shigeo; Ronen, Sabrina M.; Bjerkvig, Rolf; Pieper, Russell O.

2016-01-01

Missense mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. While mutant IDH1 expression is thought to drive the gliomagenesis process, the extent to which it remains a viable therapeutic target remains unknown. To address this question we exposed immortalized (p53/pRb-deficient), untransformed human astrocytes to the mutant IDH1 inhibitor AGI-5198 prior to, concomitant with, or at intervals after, introduction of transforming mutant IDH1, then measured effects on 2-HG levels, histone methylation (H3K4me3, H3K9me2, H3K9me3 or H3K27me3) and growth in soft-agar. Addition of AGI-5198 prior to, or concomitant with, introduction of mutant IDH1 blocked all mutant IDH1-driven changes including cellular transformation. Addition at time intervals as short as 4 days following introduction of mutant IDH1 also suppressed 2-HG levels, but had minimal effects on histone methylation, and lost the ability to suppress clonogenicity in a time-dependent manner. Furthermore, in two different models of mutant IDH1-driven gliomagenesis, AGI-5198 exposures that abolished production of 2-HG also failed to decrease histone methylation, adherent cell growth, or anchorage-independent growth in soft-agar over a prolonged period. These studies show although mutant IDH1 expression drives gliomagenesis, mutant IDH1 itself rapidly converts from driver to passenger. Implications Agents that target mutant IDH may be effective for a narrow time and may require further optimization or additional therapeutics in glioma. PMID:27430238

Epigenome profiling and editing of neocortical progenitor cells during development.

PubMed

Albert, Mareike; Kalebic, Nereo; Florio, Marta; Lakshmanaperumal, Naharajan; Haffner, Christiane; Brandl, Holger; Henry, Ian; Huttner, Wieland B

2017-09-01

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo , which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development. © 2017 The Authors.

MeCP2 deficiency in Rett syndrome causes epigenetic aberrations at the PWS/AS imprinting center that affects UBE3A expression.

PubMed

Makedonski, Kirill; Abuhatzira, Liron; Kaufman, Yotam; Razin, Aharon; Shemer, Ruth

2005-04-15

Rett syndrome (RS) is a severe and progressive neurodevelopmental disorder caused by heterozygous mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene. MeCP2 is a nuclear protein that binds specifically to methylated DNA and functions as a general transcription repressor in the context of chromatin remodeling complexes. RS shares clinical features with those of Angelman syndrome (AS), an imprinting neurodevelopmental disorder. In AS patients, the maternally expressed copy of UBE3A that codes for the ubiquitin protein ligase 3A (E6-AP) is repressed. The similar phenotype of these two syndromes led us to hypothesize that part of the RS phenotype is due to MeCP2-associated silencing of UBE3A. Indeed, UBE3A mRNA and protein are shown here to be significantly reduced in human and mouse MECP2 deficient brains. This reduced UBE3A level was associated with biallelic production of the UBE3A antisense RNA. In addition, MeCP2 deficiency resulted in elevated histone H3 acetylation and H3(K4) methylation and reduced H3(K9) methylation at the PWS/AS imprinting center, with no effect on DNA methylation or SNRPN expression. We conclude, therefore, that MeCP2 deficiency causes epigenetic aberrations at the PWS imprinting center. These changes in histone modifications result in loss of imprinting of the UBE3A antisense gene in the brain, increase in UBE3A antisense RNA level and, consequently reduction in UBE3A production.

Cleavage of sp3 C-O bonds via oxidative addition of C-H bonds.

PubMed

Choi, Jongwook; Choliy, Yuriy; Zhang, Xiawei; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

2009-11-04

(PCP)Ir (PCP = kappa(3)-C(6)H(3)-2,6-[CH(2)P(t-Bu)(2)](2)) is found to undergo oxidative addition of the methyl-oxygen bond of electron-poor methyl aryl ethers, including methoxy-3,5-bis(trifluoromethyl)benzene and methoxypentafluorobenzene, to give the corresponding aryloxide complexes (PCP)Ir(CH(3))(OAr). Although the net reaction is insertion of the Ir center into the C-O bond, density functional theory (DFT) calculations and a significant kinetic isotope effect [k(CH(3))(OAr)/k(CD(3))(OAr) = 4.3(3)] strongly argue against a simple insertion mechanism and in favor of a pathway involving C-H addition and alpha-migration of the OAr group to give a methylene complex followed by hydride-to-methylene migration to give the observed product. Ethoxy aryl ethers, including ethoxybenzene, also undergo C-O bond cleavage by (PCP)Ir, but the net reaction in this case is 1,2-elimination of ArO-H to give (PCP)Ir(H)(OAr) and ethylene. DFT calculations point to a low-barrier pathway for this reaction that proceeds through C-H addition of the ethoxy methyl group followed by beta-aryl oxide elimination and loss of ethylene. Thus, both of these distinct C-O cleavage reactions proceed via initial addition of a C(sp(3))-H bond, despite the fact that such bonds are typically considered inert and are much stronger than C-O bonds.

H3.1 K36M mutation in a congenital-onset soft tissue neoplasm.

PubMed

Kernohan, Kristin D; Grynspan, David; Ramphal, Raveena; Bareke, Eric; Wang, You Chang; Nizalik, Elizabeth; Ragoussis, Jiannis; Jabado, Nada; Boycott, Kym M; Majewski, Jacek; Sawyer, Sarah L

2017-12-01

We describe a patient who presented with a congenital soft tissue lesion initially diagnosed as infantile fibromatosis at 15 days of age. Unusually, the mass demonstrated malignant progression leading to death at 20 months of age. Biological progression to malignancy is not known to occur in fibromatosis, and fibrosarcoma is not known to progress from a benign lesion. Whole-exome sequencing of the tumor identified a driver mutation in histone H3.1 at lysine (K)36. Our findings support the link between oncohistones and infantile soft tissue tumors and provide additional evidence for the oncogenic effects of p.K36M in H3 variants. © 2017 Wiley Periodicals, Inc.

Scopadulciol, an inhibitor of gastric H+, K(+)-ATPase from Scoparia dulcis, and its structure-activity relationships.

PubMed

Hayashi, T; Asano, S; Mizutani, M; Takeguchi, N; Kojima, T; Okamura, K; Morita, N

1991-01-01

A new tetracyclic diterpenoid, scopadulciol [3], together with 6-methoxybenzoxazolinone, glutinol, and acacetin, was isolated from the 70% EtOH extract of Scoparia dulcis collected in Taiwan. Its structure was elucidated to be 6 beta-benzoyl-12-methyl-13-oxo-9(12)a,9(12)b-dihomo-18-podocarpanol on the basis of spectral data. It mildly inhibited hog gastric H+, K(+)-ATPase. Examination of the inhibitory activities of derivatives of scopadulcic acid B [2], including 3, revealed that methylation of the carboxyl group and introduction of an acetyl group or oxime at C-13 or C-18 markedly enhanced the inhibitory activity, while debenzoylation reduced the activity. Among the 30 compounds tested, compound 12, a methyl ester of scopadulcic acid B [2], showed the most potent activity.

Histone demethylase JMJD3 regulates CD11a expression through changes in histone H3K27 tri-methylation levels in CD4+ T cells of patients with systemic lupus erythematosus.

PubMed

Yin, Heng; Wu, Haijing; Zhao, Ming; Zhang, Qing; Long, Hai; Fu, Siqi; Lu, Qianjin

2017-07-25

Aberrant CD11a overexpression in CD4+ T cells induces T cell auto-reactivity, which is an important factor for systemic lupus erythematosus (SLE) pathogenesis. Although many studies have focused on CD11a epigenetic regulation, little is known about histone methylation. JMJD3, as a histone demethylase, is capable of specifically removing the trimethyl group from the H3K27 lysine residue, triggering target gene activation. Here, we examined the expression and function of JMJD3 in CD4+ T cells from SLE patients. Significantly decreased H3K27me3 levels and increased JMJD3 binding were detected within the ITGAL (CD11a) promoter locus in SLE CD4+ T cells compared with those in healthy CD4+ T cells. Moreover, overexpressing JMJD3 through the transfection of pcDNA3.1-JMJD3 into healthy donor CD4+ T cells increased JMJD3 enrichment and decreased H3K27me3 enrichment within the ITGAL (CD11a) promoter and up-regulated CD11a expression, leading to T and B cell hyperactivity. Inhibition of JMJD3 via JMJD3-siRNA in SLE CD4+ T cells showed the opposite effects. These results demonstrated that histone demethylase JMJD3 regulates CD11a expression in lupus T cells by affecting the H3K27me3 levels in the ITGAL (CD11a) promoter region, and JMJD3 might thereby serve as a potential therapeutic target for SLE.

Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men.

PubMed

Hammoud, Saher Sue; Nix, David A; Hammoud, Ahmad O; Gibson, Mark; Cairns, Bradley R; Carrell, Douglas T

2011-09-01

The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome. Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing. Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men. This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.

Distinct modes of DNA accessibility in plant chromatin.

PubMed

Shu, Huan; Wildhaber, Thomas; Siretskiy, Alexey; Gruissem, Wilhelm; Hennig, Lars

2012-01-01

The accessibility of DNA to regulatory proteins is a major property of the chromatin environment that favours or hinders transcription. Recent studies in flies reported that H3K9me2-marked heterochromatin is accessible while H3K27me3-marked chromatin forms extensive domains of low accessibility. Here we show that plants regulate DNA accessibility differently. H3K9me2-marked heterochromatin is the least accessible in the Arabidopsis thaliana genome, and H3K27me3-marked chromatin also has low accessibility. We see that very long genes without H3K9me2 or H3K27me3 are often inaccessible and generated significantly lower amounts of antisense transcripts than other genes, suggesting that reduced accessibility is associated with reduced recognition of alternative promoters. Low accessibility of H3K9me2-marked heterochromatin and long genes depend on cytosine methylation, explaining why chromatin accessibility differs between plants and flies. Together, we conclude that restriction of DNA accessibility is a local property of chromatin and not necessarily a consequence of microscopically visible compaction.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy

PubMed Central

Mack, S. C.; Witt, H.; Piro, R. M.; Gu, L.; Zuyderduyn, S.; Stütz, A. M.; Wang, X.; Gallo, M.; Garzia, L.; Zayne, K.; Zhang, X.; Ramaswamy, V.; Jäger, N.; Jones, D. T. W.; Sill, M.; Pugh, T. J.; Ryzhova, M.; Wani, K. M.; Shih, D. J. H.; Head, R.; Remke, M.; Bailey, S. D.; Zichner, T.; Faria, C. C.; Barszczyk, M.; Stark, S.; Seker-Cin, H.; Hutter, S.; Johann, P.; Bender, S.; Hovestadt, V.; Tzaridis, T.; Dubuc, A. M.; Northcott, P. A.; Peacock, J.; Bertrand, K. C.; Agnihotri, S.; Cavalli, F. M. G.; Clarke, I.; Nethery-Brokx, K.; Creasy, C. L.; Verma, S. K.; Koster, J.; Wu, X.; Yao, Y.; Milde, T.; Sin-Chan, P.; Zuccaro, J.; Lau, L.; Pereira, S.; Castelo-Branco, P.; Hirst, M.; Marra, M. A.; Roberts, S. S.; Fults, D.; Massimi, L.; Cho, Y. J.; Van Meter, T.; Grajkowska, W.; Lach, B.; Kulozik, A. E.; von Deimling, A.; Witt, O.; Scherer, S. W.; Fan, X.; Muraszko, K. M.; Kool, M.; Pomeroy, S. L.; Gupta, N.; Phillips, J.; Huang, A.; Tabori, U.; Hawkins, C.; Malkin, D.; Kongkham, P. N.; Weiss, W. A.; Jabado, N.; Rutka, J. T.; Bouffet, E.; Korbel, J. O.; Lupien, M.; Aldape, K. D.; Bader, G. D.; Eils, R.; Lichter, P.; Dirks, P. B.; Pfister, S. M.; Korshunov, A.; Taylor, M. D.

2014-01-01

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland. PMID:24553142

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

PubMed

Mack, S C; Witt, H; Piro, R M; Gu, L; Zuyderduyn, S; Stütz, A M; Wang, X; Gallo, M; Garzia, L; Zayne, K; Zhang, X; Ramaswamy, V; Jäger, N; Jones, D T W; Sill, M; Pugh, T J; Ryzhova, M; Wani, K M; Shih, D J H; Head, R; Remke, M; Bailey, S D; Zichner, T; Faria, C C; Barszczyk, M; Stark, S; Seker-Cin, H; Hutter, S; Johann, P; Bender, S; Hovestadt, V; Tzaridis, T; Dubuc, A M; Northcott, P A; Peacock, J; Bertrand, K C; Agnihotri, S; Cavalli, F M G; Clarke, I; Nethery-Brokx, K; Creasy, C L; Verma, S K; Koster, J; Wu, X; Yao, Y; Milde, T; Sin-Chan, P; Zuccaro, J; Lau, L; Pereira, S; Castelo-Branco, P; Hirst, M; Marra, M A; Roberts, S S; Fults, D; Massimi, L; Cho, Y J; Van Meter, T; Grajkowska, W; Lach, B; Kulozik, A E; von Deimling, A; Witt, O; Scherer, S W; Fan, X; Muraszko, K M; Kool, M; Pomeroy, S L; Gupta, N; Phillips, J; Huang, A; Tabori, U; Hawkins, C; Malkin, D; Kongkham, P N; Weiss, W A; Jabado, N; Rutka, J T; Bouffet, E; Korbel, J O; Lupien, M; Aldape, K D; Bader, G D; Eils, R; Lichter, P; Dirks, P B; Pfister, S M; Korshunov, A; Taylor, M D

2014-02-27

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Metsulfuron-methyl sorption/desorption behavior on volcanic ash-derived soils. effect of phosphate and pH.

PubMed

Cáceres, Lizethly; Fuentes, Roxana; Escudey, Mauricio; Fuentes, Edwar; Báez, María E

2010-06-09

Metsulfuron-methyl sorption/desorption behavior was studied through batch sorption experiments in three typical volcanic ash-derived soils belonging to Andisol and Ultisol orders. Their distinctive physical and chemical properties are acidic pH and variable surface charge. Organic matter content and mineral composition affected in different ways sorption of metsulfuron-methyl (K(OC) ranging from 113 to 646 mL g(-1)): organic matter and iron and aluminum oxides mainly through hydrophilic rather than hydrophobic interactions in Andisols, and Kaolinite group minerals, as major constituents of Ultisols, and iron and aluminum oxides only through hydrophilic interactions. The Freundlich model described metsulfuron-methyl behavior in all cases (R(2) > 0.992). K(f) values (3.1-14.4 microg(1-1/n) mL(1/n) g(-1)) were higher than those reported for different class of soils including some with variable charge. Hysteresis was more significant in Ultisols. A strong influence of pH and phosphate was established for both kinds of soil, intensive soil fertilization and liming being the most probable scenario for leaching of metsulfuron-methyl, particularly in Ultisols.

Beta-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent delta opioid antagonists.

PubMed

Tóth, Géza; Ioja, Eniko; Tömböly, Csaba; Ballet, Steven; Tourwé, Dirk; Péter, Antal; Martinek, Tamás; Chung, Nga N; Schiller, Peter W; Benyhe, Sándor; Borsodi, Anna

2007-01-25

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2',6'-dimethyltyrosine) and a new unnatural amino acid, beta-MeCha (beta-methyl-cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar delta antagonist activity and mu agonist or antagonist properties depending on the configuration of the beta-MeCha residue. The most promising analog, H-Dmt-Tic-(2S,3S)-beta-MeCha-Phe-OH was a very selective delta antagonist both in the mouse vas deferens (MVD) assay (Ke = 0.241 +/- 0.05 nM) and in radioligand binding assay (K i delta = 0.48 +/- 0.05 nM, K i mu/K i delta = 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-beta-MeCha-Phe-OH and the corresponding peptide amide turned out to be mixed partial mu agonist/delta antagonists in the guinea pig ileum and MVD assays. Our results constitute further examples of the influence of Dmt and beta-methyl substitution as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.

Distinct epigenomic landscapes of pluripotent and lineage-committed human cells.

PubMed

Hawkins, R David; Hon, Gary C; Lee, Leonard K; Ngo, Queminh; Lister, Ryan; Pelizzola, Mattia; Edsall, Lee E; Kuan, Samantha; Luu, Ying; Klugman, Sarit; Antosiewicz-Bourget, Jessica; Ye, Zhen; Espinoza, Celso; Agarwahl, Saurabh; Shen, Li; Ruotti, Victor; Wang, Wei; Stewart, Ron; Thomson, James A; Ecker, Joseph R; Ren, Bing

2010-05-07

Human embryonic stem cells (hESCs) share an identical genome with lineage-committed cells, yet possess the remarkable properties of self-renewal and pluripotency. The diverse cellular properties in different cells have been attributed to their distinct epigenomes, but how much epigenomes differ remains unclear. Here, we report that epigenomic landscapes in hESCs and lineage-committed cells are drastically different. By comparing the chromatin-modification profiles and DNA methylomes in hESCs and primary fibroblasts, we find that nearly one-third of the genome differs in chromatin structure. Most changes arise from dramatic redistributions of repressive H3K9me3 and H3K27me3 marks, which form blocks that significantly expand in fibroblasts. A large number of potential regulatory sequences also exhibit a high degree of dynamics in chromatin modifications and DNA methylation. Additionally, we observe novel, context-dependent relationships between DNA methylation and chromatin modifications. Our results provide new insights into epigenetic mechanisms underlying properties of pluripotency and cell fate commitment.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N.

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes tri-methylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. Here in this study, we report discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show thatmore » a lead compound MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. Ultimately, these small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.« less

Concepts of epigenetics in prostate cancer development.

PubMed

Cooper, C S; Foster, C S

2009-01-27

Substantial evidence now supports the view that epigenetic changes have a role in the development of human prostate cancer. Analyses of the patterns of epigenetic alteration are providing important insights into the origin of this disease and have identified specific alterations that may serve as useful diagnostic and prognostic biomarkers. Examination of cancer methylation patterns supports a stem cell origin of prostate cancer. It is well established that methylation of GSTpi is a marker of prostate cancer, and global patterns of histone marking appear to be linked to cancer prognosis with levels of acetylated histones H3K9, H3K18, and H4K12, and of dimethylated H4R3 and H3K4, dividing low-grade prostate cancer (Gleason 6 or less) into two prognostically separate groups. Elevated levels of several components of the polycomb group protein complex, EZH2, BMI1, and RING1, can also act as biomarkers of poor clinical outcome. Many components of the epigenetic machinery, including histone deacetylase (whose expression level is linked to the TMPRSS2:ERG translocation) and the histone methylase EZH2, are potential therapeutic targets. The recent discovery of the role of small RNAs in governing the epigenetic status of individual genes offers exciting new possibilities in therapeutics and chemoprevention.

Evaluation of Different Blood Collection Tubes and Blood Storage Conditions for the Preservation and Stability of Cell-Free Circulating DNA for the Analysis of the Methylated mSEPT9 Colorectal Cancer Screening Marker.

PubMed

Distler, Jurgen; Tetzner, Reimo; Weiss, Gunter; König, Thomas; Schlegel, Anne; Bagrowski, Michal

2016-01-01

For the subsequent analysis of the methylated mSEPT9 colorectal cancer screening marker in plasma, different blood collection tubes and blood storage conditions were investigated. The study demonstrated that methylated Septin 9 ( m SEPT9) can be consistently detected in plasma samples derived from whole blood samples collected with S-Monovette® K3E and BD Vacutainer ® K2EDTA tubes stored at 2-8 °C for a maximum of 24 h and for samples collected in S-Monovette CPDA tubes stored at 18-25 °C for up to 48 h.

The Progeny of Arabidopsis thaliana Plants Exposed to Salt Exhibit Changes in DNA Methylation, Histone Modifications and Gene Expression

PubMed Central

Bilichak, Andriy; Ilnystkyy, Yaroslav; Hollunder, Jens; Kovalchuk, Igor

2012-01-01

Plants are able to acclimate to new growth conditions on a relatively short time-scale. Recently, we showed that the progeny of plants exposed to various abiotic stresses exhibited changes in genome stability, methylation patterns and stress tolerance. Here, we performed a more detailed analysis of methylation patterns in the progeny of Arabidopsis thaliana (Arabidopsis) plants exposed to 25 and 75 mM sodium chloride. We found that the majority of gene promoters exhibiting changes in methylation were hypermethylated, and this group was overrepresented by regulators of the chromatin structure. The analysis of DNA methylation at gene bodies showed that hypermethylation in the progeny of stressed plants was primarily due to changes in the 5′ and 3′ ends as well as in exons rather than introns. All but one hypermethylated gene tested had lower gene expression. The analysis of histone modifications in the promoters and coding sequences showed that hypermethylation and lower gene expression correlated with the enrichment of H3K9me2 and depletion of H3K9ac histones. Thus, our work demonstrated a high degree of correlation between changes in DNA methylation, histone modifications and gene expression in the progeny of salt-stressed plants. PMID:22291972

S-adenosylmethionine decreases the peak blood alcohol levels 3 h after an acute bolus of ethanol by inducing alcohol metabolizing enzymes in the liver.

PubMed

Bardag-Gorce, Fawzia; Oliva, Joan; Wong, Wesley; Fong, Stephanie; Li, Jun; French, Barbara A; French, Samuel W

2010-12-01

An alcohol bolus causes the blood alcohol level (BAL) to peak at 1-2 h post ingestion. The ethanol elimination rate is regulated by alcohol metabolizing enzymes, primarily alcohol dehydrogenase (ADH1), acetaldehyde dehydrogenase (ALDH), and cytochrome P450 (CYP2E1). Recently, S-adenosylmethionine (SAMe) was found to reduce acute BALs 3 h after an alcohol bolus. The question, then, was: what is the mechanism involved in this reduction of BAL by feeding SAMe? To answer this question, we investigated the changes in ethanol metabolizing enzymes and the epigenetic changes that regulate the expression of these enzymes during acute binge drinking and chronic drinking. Rats were fed a bolus of ethanol with or without SAMe, and were sacrificed at 3 h or 12 h after the bolus. RT-PCR and Western blot analyses showed that SAMe significantly induced ADH1 levels in the 3 h liver samples. However, SAMe did not affect the changes in ADH1 protein levels 12 h post bolus. Since SAMe is a methyl donor, it was postulated that the ADH1 gene expression up regulation at 3 h was due to a histone modification induced by methylation from methyl transferases. Dimethylated histone 3 lysine 4 (H3K4me2), a modification responsible for gene expression activation, was found to be significantly increased by SAMe at 3 h post bolus. These results correlated with the low BAL found at 3 h post bolus, and support the concept that SAMe increased the gene expression to increase the elimination rate of ethanol in binge drinking by increasing H3K4me2. Copyright © 2010 Elsevier Inc. All rights reserved.

Developmental bisphenol A (BPA) exposure leads to sex-specific modification of hepatic gene expression and epigenome at birth that may exacerbate high-fat diet-induced hepatic steatosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Strakovsky, Rita S.; Wang, Huan; Engeseth, Nicki J.

Developmental bisphenol A (BPA) exposure increases adulthood hepatic steatosis with reduced mitochondrial function. To investigate the potential epigenetic mechanisms behind developmental BPA-induced hepatic steatosis, pregnant Sprague–Dawley rats were dosed with vehicle (oil) or BPA (100 μg/kg/day) from gestational day 6 until postnatal day (PND) 21. After weaning, offspring were either challenged with a high-fat (HF; 45% fat) or remained on a control (C) diet until PND110. From PND60 to 90, both BPA and HF diet increased the fat/lean ratio in males only, and the combination of BPA and HF diet appeared to cause the highest ratio. On PND110, Oil-HF, BPA-C,more » and BPA-HF males had higher hepatic lipid accumulation than Oil-C, with microvesicular steatosis being marked in the BPA-HF group. Furthermore, on PND1, BPA increased and modified hepatic triglyceride (TG) and free fatty acid (FFA) compositions in males only. In PND1 males, BPA increased hepatic expression of FFA uptake gene Fat/Cd36, and decreased the expression of TG synthesis- and β-oxidation-related genes (Dgat, Agpat6, Cebpα, Cebpβ, Pck1, Acox1, Cpt1a, Cybb). BPA altered DNA methylation and histone marks (H3Ac, H4Ac, H3Me2K4, H3Me3K36), and decreased the binding of several transcription factors (Pol II, C/EBPβ, SREBP1) within the male Cpt1a gene, the key β-oxidation enzyme. In PND1 females, BPA only increased the expression of genes involved in FFA uptake and TG synthesis (Lpl, Fasn, and Dgat). These data suggest that developmental BPA exposure alters and reprograms hepatic β-oxidation capacity in males, potentially through the epigenetic regulation of genes, and further alters the response to a HF diet. - Highlights: • Developmental BPA exposure exacerbates HF-diet induced steatosis in adult males. • Gestational BPA exposure increases hepatic lipid accumulation in neonatal males. • BPA decreases Cpt1a and other hepatic β-oxidation genes in neonatal males. • BPA alters neonatal male Cpt1a DNA methylation, histones, and transcription factors.« less

Densely ionizing radiation affects DNA methylation of selective LINE-1 elements1

PubMed Central

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

2016-01-01

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

DNA methylation pathways and their crosstalk with histone methylation

PubMed Central

Du, Jiamu; Johnson, Lianna M.; Jacobsen, Steven E.; Patel, Dinshaw J.

2015-01-01

Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk. PMID:26296162

Degradation of cyanidin-3-rutinoside and formation of protocatechuic acid methyl ester in methanol solution by gamma irradiation.

PubMed

Lee, Seung Sik; Kim, Tae Hoon; Lee, Eun Mi; Lee, Min Hee; Lee, Ha Yeong; Chung, Byung Yeoup

2014-08-01

Anthocyanins are naturally occurring phenolic compounds having broad biological activities including anti-mutagenesis and anti-carcinogenesis. We studied the effects and the degradation mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside (cya-3-rut), by using gamma ray. Cya-3-rut in methanol (1mg/ml) was exposed to gamma-rays from 1 to 10kGy. We found that the reddish colour of cya-3-rut in methanol disappeared gradually in a dose-dependent manner and effectively disappeared (>97%) at 10kGy of gamma ray. Concomitantly, a new phenolic compound was generated and identified as a protocatechuic acid methyl ester by liquid chromatography, (1)H, and (13)C NMR. The formation of protocatechuic acid methyl ester increased with increasing irradiation and the amount of protocatechuic acid methyl ester formed by decomposition of cya-3-rut (20μg) at 10kGy of gamma ray was 1.95μg. In addition, the radical-scavenging activities were not affected by gamma irradiation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Glutathione-S-transferase pi 1(GSTP1) gene silencing in prostate cancer cells is reversed by the histone deacetylase inhibitor depsipeptide.

PubMed

Hauptstock, Vera; Kuriakose, Sapuna; Schmidt, Doris; Düster, Robert; Müller, Stefan C; von Ruecker, Alexander; Ellinger, Jörg

2011-09-09

Gene silencing by epigenetic mechanisms is frequent in prostate cancer (PCA). The link between DNA hypermethylation and histone modifications is not completely understood. We chose the GSTP1 gene which is silenced by hypermethylation to analyze the effect of the histone deacetylase inhibitor depsipeptide on DNA methylation and histone modifications at the GSTP1 promoter site. Prostate cell lines (PC-3, LNCaP, and BPH-1) were treated with depsipeptide; apoptosis (FACS analysis), GSTP1 mRNA levels (quantitative real-time PCR), DNA hypermethylation (methylation-specific PCR), and histone modifications (chromatin immunoprecipitation) were studied. Depsipeptide induced apoptosis in PCA cells, but not a cell cycle arrest. Depispeptide reversed DNA hypermethylation and repressive histone modifications (reduction of H3K9me2/3 and H3K27me2/3; increase of H3K18Ac), thereby inducing GSTP1 mRNA re-expression. Successful therapy requires both, DNA demethylation and activating histone modifications, to induce complete gene expression of epigenetically silenced genes and depsipeptide fulfils both criteria. Copyright © 2011 Elsevier Inc. All rights reserved.

Protonation-dissociation reactions of imazamethabenz-methyl and imazamethabenz-Acid in relation to their soil sorption and abiotic degradation.

PubMed

Pintado, Sara; Montoya, Mercedes Ruiz; Mellado, José Miguel Rodríguez

2009-12-09

This paper present ultraviolet-visible absorption spectra of imazamethabenz-methyl (IMBM) (mixture of the isomers methyl 6-[(RS)-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-m-toluate, m-imazamethabenz, and methyl 2-[(RS)-4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-p-toluate, p-imazamethabenz) and the corresponding carboxylic acid, imazamethabenz-acid (IMBA). The spectral characteristics are determined as functions of the pH. The appreciable absorbance in the visible (or near-ultraviolet) region of the spectra indicates that the natural photolytic degradation is possible. From variations of the maximum absorbances of the bands, the pK values of 3.4 +/- 0.2 and 9.4 +/- 0.2 were obtained for protonation of the imidazol (=N-) nitrogen and dissociation of the NH imidazol nitrogen of IMBM, respectively. For IMBA, the dissociation pK of the carboxylic group is very close to that of the imidazol (=N-) nitrogen, both being close to 3.3. The dissociation pK of the NH imidazol nitrogen for IMBA is 9.6 +/- 0.2. The role of the acid-base reactions on the sorption on soils of these herbicides is discussed.

Berberine acts as a putative epigenetic modulator by affecting the histone code.

PubMed

Wang, Zhixiang; Liu, Yuan; Xue, Yong; Hu, Haiyan; Ye, Jieyu; Li, Xiaodong; Lu, Zhigang; Meng, Fanyi; Liang, Shuang

2016-10-01

Berberine, an isoquinoline plant alkaloid, exhibits a wide range of biochemical and pharmacological effects. However, the precise mechanism of these bioactivities remains poorly understood. In this study, we found significant similarity between berberine and two epigenetic modulators (CG-1521 and TSA). Reverse-docking using berberine as a ligand identified lysine-N-methyltransferase as a putative target of berberine. These findings suggested the potential role of berberine in epigenetic modulation. The results of PCR array analysis of epigenetic chromatin modification enzymes supported our hypothesis. Furthermore, the analysis showed that enzymes involved in histone acetylation and methylation were predominantly affected by treatment with berberine. Up-regulation of histone acetyltransferase CREBBP and EP300, histone deacetylase SIRT3, histone demethylase KDM6A as well as histone methyltransferase SETD7, and down-regulation of histone acetyltransferase HDAC8, histone methyltransferase WHSC1I, WHSC1II and SMYD3, in addition to 38 genes from histone clusters 1-3 were observed in berberine-treated cells using real-time PCR. In parallel, western blotting analyses revealed that the expression of H3K4me3, H3K27me3 and H3K36me3 proteins decreased with berberine treatment. These results were further confirmed in acute myelocytic leukemia (AML) cell lines HL-60/ADR and KG1-α. Taken together, this study suggests that berberine might modulate the expression of epigenetic regulators important for many downstream pathways, resulting in the variation of its bioactivities. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Epigenetic Changes and Suppression of the Nuclear Factor of Activated T Cell 1 (NFATC1) Promoter in Human Lymphomas with Defects in Immunoreceptor Signaling

PubMed Central

Akimzhanov, Askar; Krenacs, Laszlo; Schlegel, Timm; Klein-Hessling, Stefan; Bagdi, Enikö; Stelkovics, Eva; Kondo, Eisaku; Chuvpilo, Sergei; Wilke, Philipp; Avots, Andris; Gattenlöhner, Stefan; Müller-Hermelink, Hans-Konrad; Palmetshofer, Alois; Serfling, Edgar

2008-01-01

The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin’s lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a “window of hypomethylation,” which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing. PMID:18156209

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

PubMed

Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

1990-07-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

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

PubMed

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

2012-12-01

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.

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

Aminoacid N-substituted 1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclododecane Zn2+, Cd2+ and Cu2+ complexes. A preparative, potentiometric titration and NMR spectroscopic study.

PubMed

Plush, Sally E; Lincoln, Stephen F; Wainwright, Kevin P

2004-05-07

The pK(a)s and Zn2+, Cd2+ and Cu2+ complexation constants (K) for 1,4,7-tris[(2''S)-acetamido-2''-(methyl-3''-phenylpropionate)]-1,4,7-triazacyclononane, 1, 1,4,7-tris[(2''S)-acetamido-2''-(1''-carboxy-3''-phenylpropane)]-1,4,7-triazacyclononane, H(3)2, 1,4,7-tris[(2''S)-acetamido-2''-(methyl-3''-(1H-3-indolyl)propionate)]-1,4,7-triazacyclononane, 3, and 1,4,7,10-tetrakis[(2''S)-acetamido-2''-(methyl-3''-phenylpropionate)]-1,4,7,10-tetraazacyclododecane, 4, 1,4,7,10-tetrakis[(2''S)-acetamido-2''-(1''-carboxy-3''-phenylpropane)]-1,4,7,10-tetraazacyclododecane, H(4)5, in 20 : 80 v/v water-methanol solution are reported. The pK(a)s within the potentiometric detection range for H(3)1(3+) = 8.69 and 3.59, for H(6)2(3+) = 9.06, 6.13, 4.93 and 4.52, H(3)3(3+) = 8.79 and 3.67, H(4)4(4+) = 8.50, 5.62 and 3.77 and for H(8)5(4+) = 9.89, 7.06, 5.53, 5.46, 4.44 and 4.26 where each tertiary amine nitrogen is protonated. The complexes of 1: [Zn(1)]2+(9.00), [Cd(1)]2+ (6.49), [Cd(H1)]3+ (4.54) and [Cu(1)]2+ (10.01) are characterized by the log(K/dm3 mol(-1)) values shown in parentheses. Analogous complexes are formed by 3 and 4: [Zn(3)]2+ (10.19), [Cd(3)]2+ (8.54), [Cu(3)]2+ (10.77), [Zn(4)]2+ (11.41) [Cd(4)]2+ (9.16), [Cd(H4)]3+ (6.16) and [Cu(4)]2+ (11.71). The tricarboxylic acid H(3)2 generates a greater variety of complexes as exemplified by: [Zn(2)-] (10.68) [Zn(H2)] (6.60) [Zn(H(2)2)+] (5.15), [Cd(2)](-) (4.99), [Cd(H2)] (4.64), [Cd(H2(2))]+ (3.99), [Cd(H(3)2)]2+ (3.55), [Cu(2)](-) (12.55) [Cu(H2)] (7.66), [Cu(H(2)2)]+ (5.54) and [Cu(2)2](4-) (3.23). The complexes of H(4)5 were insufficiently soluble to study in this way. The 1H and 13C NMR spectra of the ligands are consistent with formation of a predominant Zn2+ and Cd2+ Delta or Lambda diastereomer. The preparations of the new pendant arm macrocycles H(3)2, 3, 4 and H(4)5 are reported.

Intra- and intermolecular H-bond mediated tautomerization and dimerization of 3-methyl-1,2-cyclopentanedione: Infrared spectroscopy in argon matrix and CCl 4 solution

NASA Astrophysics Data System (ADS)

Samanta, Amit K.; Pandey, Prasenjit; Bandyopadhyay, Biman; Mukhopadhyay, Anamika; Chakraborty, Tapas

2011-05-01

Mid-infrared spectra of 3-methyl-1,2-cyclopentanedione (3-MeCPD) have been recorded by isolating the molecule in a cold argon matrix (8 K) and also in CCl 4 solution at room temperature. The spectral features reveal that in both media, the molecule exists exclusively in an enol tautomeric form, which is stabilized by an intramolecular O sbnd H⋯O hydrogen bond. NBO analysis shows that the preferred conformer is further stabilized because of hyperconjugation interaction between the methyl and vinyl group of the enol tautomer. In CCl 4 solution, the molecule undergoes extensive self association and generates a doubly hydrogen bonded centrosymmetric dimer. The dimerization constant ( K d) is estimated to have a value of ˜9 L mol -1 at room temperature (25 °C) and the thermodynamic parameters, Δ H°, Δ S° and Δ G°, of dimerization are estimated by measuring K d at several temperatures within the range 22-60 °C. The same dimer is also produced when the matrix is annealed at a higher temperature. In addition, a non-centrosymmetric singly hydrogen bonded dimer is also identified in the argon matrix. A comparison between the spectral features of the two dimers indicates that the dimerization effect on doubly H-bonded case is influenced by cooperative interaction between the two H-bonds.

Impact of dietary fibers [methyl cellulose, chitosan, and pectin] on digestion of lipids under simulated gastrointestinal conditions.

PubMed

Espinal-Ruiz, Mauricio; Parada-Alfonso, Fabián; Restrepo-Sánchez, Luz-Patricia; Narváez-Cuenca, Carlos-Eduardo; McClements, David Julian

2014-12-01

A simulated in vitro digestion model was used to elucidate the impact of dietary fibers on the digestion rate of emulsified lipids. The influence of polysaccharide type (chitosan (cationic), methyl cellulose (non-ionic), and pectin (anionic)) and initial concentration (0.4 to 3.6% (w/w)) was examined. 2% (w/w) corn oil-in-water emulsions stabilized by 0.2% (w/w) Tween-80 were prepared, mixed with polysaccharide, and then subjected to an in vitro digestion model (37 °C): initial (pH 7.0); oral (pH 6.8; 10 min); gastric (pH 2.5; 120 min); and, intestinal (pH 7.0; 120 min) phases. The impact of polysaccharides on lipid digestion, ζ-potential, particle size, viscosity, and stability was determined. The rate and extent of lipid digestion decreased with increasing pectin, methyl cellulose, and chitosan concentrations. The free fatty acids released after 120 min of lipase digestion were 46, 63, and 81% (w/w) for methyl cellulose, pectin, and chitosan, respectively (3.6% (w/w) initial polysaccharide), indicating that methyl cellulose had the highest capacity to inhibit lipid digestion, followed by pectin, and then chitosan. The impact of the polysaccharides on lipid digestion was attributed to their ability to induce droplet flocculation, and/or due to their interactions with molecular species involved in lipid hydrolysis, such as bile salts, fatty acids, and calcium. These results have important implications for understanding the influence of dietary fibers on lipid digestion. The control of lipid digestibility within the gastrointestinal tract might be important for the development of reduced-calorie emulsion-based functional food products.

H3K27me3 and H3K4me3 chromatin environment at super-induced dehydration stress memory genes of Arabidopsis thaliana.

PubMed

Liu, Ning; Fromm, Michael; Avramova, Zoya

2014-03-01

Pre-exposure to a stress may alter the plant's cellular, biochemical, and/or transcriptional responses during future encounters as a 'memory' from the previous stress. Genes increasing transcription in response to a first dehydration stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced 'transcription memory' genes in Arabidopsis thaliana. The chromatin environment (histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3) studied at five dehydration stress memory genes revealed existence of distinct memory-response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities during the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress-responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function independently and are not mutually exclusive at the dehydration stress-responding memory genes.

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

2011-02-04

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.

Construction of combustion models for rapeseed methyl ester bio-diesel fuel for internal combustion engine applications.

PubMed

Golovitchev, Valeri I; Yang, Junfeng

2009-01-01

Bio-diesel fuels are non-petroleum-based diesel fuels consisting of long chain alkyl esters produced by the transesterification of vegetable oils, that are intended for use (neat or blended with conventional fuels) in unmodified diesel engines. There have been few reports of studies proposing theoretical models for bio-diesel combustion simulations. In this study, we developed combustion models based on ones developed previously. We compiled the liquid fuel properties, and the existing detailed mechanism of methyl butanoate ester (MB, C(5)H(10)O(2)) oxidation was supplemented by sub-mechanisms for two proposed fuel constituent components, C(7)H(16) and C(7)H(8)O (and then, by mp2d, C(4)H(6)O(2) and propyne, C(3)H(4)) to represent the combustion model for rapeseed methyl ester described by the chemical formula, C(19)H(34)O(2) (or C(19)H(36)O(2)). The main fuel vapor thermal properties were taken as those of methyl palmitate C(19)H(36)O(2) in the NASA polynomial form of the Burcat database. The special global reaction was introduced to "crack" the main fuel into its constituent components. This general reaction included 309 species and 1472 reactions, including soot and NO(x) formation processes. The detailed combustion mechanism was validated using shock-tube ignition-delay data under diesel engine conditions. For constant volume and diesel engine (Volvo D12C) combustion modeling, this mechanism could be reduced to 88 species participating in 363 reactions.

Contrast between the mechanisms for dissociative electron attachment to CH3SCN and CH3NCS.

PubMed

Miller, Thomas M; Viggiano, Albert A; Shuman, Nicholas S

2018-05-14

The kinetics of thermal electron attachment to methyl thiocyanate (CH 3 SCN), methyl isothiocyanate (CH 3 NCS), and ethyl thiocyanate (C 2 H 5 SCN) were measured using flowing afterglow-Langmuir probe apparatuses at temperatures between 300 and 1000 K. CH 3 SCN and C 2 H 5 SCN undergo inefficient dissociative attachment to yield primarily SCN - at 300 K (k = 2 × 10 -10 cm 3 s -1 ), with increasing efficiency as temperature increases. The increase is well described by activation energies of 0.17 eV (CH 3 SCN) and 0.14 eV (C 2 H 5 SCN). CN - product is formed at <1% branching at 300 K, increasing to ∼30% branching at 1000 K. Attachment to CH 3 NCS yields exclusively SCN - ionic product but at a rate at 300 K that is below our detection threshold (k < 10 -12 cm 3 s -1 ). The rate coefficient increases rapidly with increasing temperature (k = 6 × 10 -11 cm 3 s -1 at 600 K), in a manner well described by an activation energy of 0.51 eV. Calculations at the B3LYP/def2-TZVPPD level suggest that attachment to CH 3 SCN proceeds through a dissociative state of CH 3 SCN - , while attachment to CH 3 NCS initially forms a weakly bound transient anion CH 3 NCS -* that isomerizes over an energetic barrier to yield SCN - . Kinetic modeling of the two systems is performed in an attempt to identify a kinetic signature differentiating the two mechanisms. The kinetic modeling reproduces the CH 3 NCS data only if dissociation through the transient anion is considered.

Contrast between the mechanisms for dissociative electron attachment to CH3SCN and CH3NCS

NASA Astrophysics Data System (ADS)

Miller, Thomas M.; Viggiano, Albert A.; Shuman, Nicholas S.

2018-05-01

The kinetics of thermal electron attachment to methyl thiocyanate (CH3SCN), methyl isothiocyanate (CH3NCS), and ethyl thiocyanate (C2H5SCN) were measured using flowing afterglow-Langmuir probe apparatuses at temperatures between 300 and 1000 K. CH3SCN and C2H5SCN undergo inefficient dissociative attachment to yield primarily SCN- at 300 K (k = 2 × 10-10 cm3 s-1), with increasing efficiency as temperature increases. The increase is well described by activation energies of 0.17 eV (CH3SCN) and 0.14 eV (C2H5SCN). CN- product is formed at <1% branching at 300 K, increasing to ˜30% branching at 1000 K. Attachment to CH3NCS yields exclusively SCN- ionic product but at a rate at 300 K that is below our detection threshold (k < 10-12 cm3 s-1). The rate coefficient increases rapidly with increasing temperature (k = 6 × 10-11 cm3 s-1 at 600 K), in a manner well described by an activation energy of 0.51 eV. Calculations at the B3LYP/def2-TZVPPD level suggest that attachment to CH3SCN proceeds through a dissociative state of CH3SCN-, while attachment to CH3NCS initially forms a weakly bound transient anion CH3NCS-* that isomerizes over an energetic barrier to yield SCN-. Kinetic modeling of the two systems is performed in an attempt to identify a kinetic signature differentiating the two mechanisms. The kinetic modeling reproduces the CH3NCS data only if dissociation through the transient anion is considered.

Analysis of Biological Features Associated with Meiotic Recombination Hot and Cold Spots in Saccharomyces cerevisiae

PubMed Central

Hansen, Loren; Kim, Nak-Kyeong; Mariño-Ramírez, Leonardo; Landsman, David

2011-01-01

Meiotic recombination is not distributed uniformly throughout the genome. There are regions of high and low recombination rates called hot and cold spots, respectively. The recombination rate parallels the frequency of DNA double-strand breaks (DSBs) that initiate meiotic recombination. The aim is to identify biological features associated with DSB frequency. We constructed vectors representing various chromatin and sequence-based features for 1179 DSB hot spots and 1028 DSB cold spots. Using a feature selection approach, we have identified five features that distinguish hot from cold spots in Saccharomyces cerevisiae with high accuracy, namely the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. Previous studies have associated H3K4me3, H3K36me3, and GC content with areas of mitotic recombination. H3K14ac and H3K79me3 are novel predictions and thus represent good candidates for further experimental study. We also show nucleosome occupancy maps produced using next generation sequencing exhibit a bias at DSB hot spots and this bias is strong enough to obscure biologically relevant information. A computational approach using feature selection can productively be used to identify promising biological associations. H3K14ac and H3K79me3 are novel predictions of chromatin marks associated with meiotic DSBs. Next generation sequencing can exhibit a bias that is strong enough to lead to incorrect conclusions. Care must be taken when interpreting high throughput sequencing data where systematic biases have been documented. PMID:22242140

An examination of the thermodynamics of fusion, vaporization, and sublimation of several parabens by correlation gas chromatography.

PubMed

Umnahanant, Patamaporn; Chickos, James

2011-05-01

The vaporization, fusion, and sublimation enthalpies of methyl, ethyl, propyl, and butyl paraben are reported and compared with literature values. The vaporization enthalpies were measured by correlation gas chromatography and the fusion enthalpies by differential scanning calorimetry. Adjusted to T = 298.15 K, these enthalpies were combined to yield the sublimation enthalpy. The results compare favorably to some of the literature values but do not support the reversal in magnitude of both the vaporization and sublimation enthalpy previously reported for propyl and butyl paraben. The following fusion and vaporization enthalpies were measured for methyl through to butyl paraben, respectively: ΔH(fus) (T(fus) ) 26.3 ± 0.1 (398.6 K), 26.5 ± 0.1 (388.5 K), 27.3 ± 0.1 (368.8), and 25.9 ± 0.3 (340.7 K) kJ·mol(-1); ΔH(vap) (298.15 K) 79.5 ± 0.5, 84.0 ± 0.5, 89.7 ± 0.6, and 95.8 ± 0.6 kJ·mol(-1). The results are believed to be accurate to ± 4 kJ·mol(-1). Copyright © 2011 Wiley-Liss, Inc.

ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwase, Shigeki; Xiang, Bin; Ghosh, Sharmistha

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD{sub ATRX}), whose function has remained elusive. Here we identify ADD{sub ATRX} as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD{sub ATRX} bound to H3{sub 1-15}K9me3 peptide reveals an atypical composite H3K9me3-binding pocket,more » which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.« less

ATRX ADD Domain Links an Atypical Histone Methylation Recognition Mechanism to Human Mental-Retardation Syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Iwase; B Xiang; S Ghosh

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD{sub ATRX}), whose function has remained elusive. Here we identify ADD{sub ATRX} as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD{sub ATRX} bound to H3{sub 1-15}K9me3 peptide reveals an atypical composite H3K9me3-binding pocket,more » which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.« less

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

PubMed Central

Kleb, Brittany; Estécio, Marcos R.H.; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M.; Tahir, Salahaldin; Marquez, Victor E.; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-01-01

ABSTRACT Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR−SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR+ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR− and AR+ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR−SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR−SCPC cell lines. We conclude that the epigenome of AR− is distinct from that of AR+ castration-resistant prostate carcinomas, and that the AR− phenotype can be reversed with epigenetic drugs. PMID:26890396

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

PubMed

Kleb, Brittany; Estécio, Marcos R H; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M; Tahir, Salahaldin; Marquez, Victor E; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-03-03

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

The Dynamics and Regulatory Mechanism of Pronuclear H3k9me2 Asymmetry in Mouse Zygotes

PubMed Central

Ma, Xue-Shan; Chao, Shi-Bin; Huang, Xian-Ju; Lin, Fei; Qin, Ling; Wang, Xu-Guang; Meng, Tie-Gang; Zhu, Cheng-Cheng; Schatten, Heide; Liu, Hong-Lin; Sun, Qing-Yuan

2015-01-01

H3K9 methylation is an important histone modification that is correlated with gene transcription repression. The asymmetric H3K9 dimethylation (H3K9me2) pattern between paternal and maternal genomes is generated soon after fertilization. In the present study, we carefully determined the dynamics of H3K9me2 changes in mouse zygotes, and investigated the regulatory mechanisms. The results indicated that histone methyltransferase G9a, but not GLP, was involved in the regulation of asymmetric H3K9me2, and G9a was the methyltransferase that induced the appearance of H3K9me2 in the male pronucleus of the zygote treated with cycloheximide. We found that there were two distinct mechanisms that regulate H3K9me2 in the male pronucleus. Before 8 h of in vitro fertilization (IVF), a mechanism exists that inhibits the association of G9a with the H3K9 sites. After 10 h of IVF the inhibition of G9a activity depends on yet unknown novel protein(s) synthesis. The two mechanisms of transfer take place between 8–10 h of IVF, and the novel protein failed to inhibit G9a activity in time, resulting in the appearance of a low level de novo H3K9me2 in the male pronucleus. PMID:26639638

36 h fasting of young men influences adipose tissue DNA methylation of LEP and ADIPOQ in a birth weight-dependent manner.

PubMed

Hjort, Line; Jørgensen, Sine W; Gillberg, Linn; Hall, Elin; Brøns, Charlotte; Frystyk, Jan; Vaag, Allan A; Ling, Charlotte

2017-01-01

Subjects born with low birth weight (LBW) display a more energy-conserving response to fasting compared with normal birth weight (NBW) subjects. However, the molecular mechanisms explaining these metabolic differences remain unknown. Environmental influences may dynamically affect epigenetic marks, also in postnatal life. Here, we aimed to study the effects of short-term fasting on leptin ( LEP ) and adiponectin ( ADIPOQ ) DNA methylation and gene expression in subcutaneous adipose tissue (SAT) from subjects with LBW and NBW. Twenty-one young LBW men and 18 matched NBW controls were studied during 36 h fasting. Eight subjects from each group completed a control study (overnight fast). We analyzed SAT LEP and ADIPOQ methylation (Epityper MassARRAY), gene expression (q-PCR), and adipokine plasma levels. After overnight fast (control study), LEP and ADIPOQ DNA methylation levels were higher in LBW compared to those in NBW subjects ( p  ≤ 0.03) and increased with 36 h fasting in NBW subjects only ( p  ≤ 0.06). Both LEP and ADIPOQ methylation levels were positively associated with total body fat percentage ( p  ≤ 0.05). Plasma leptin levels were higher in LBW versus NBW subjects after overnight fasting ( p  = 0.04) and decreased more than threefold in both groups after 36 h fasting ( p  ≤ 0.0001). This is the first study to demonstrate that fasting induces changes in DNA methylation. This was shown in LEP and ADIPOQ promoters in SAT among NBW but not LBW subjects. The altered epigenetic flexibility in LBW subjects might contribute to their differential response to fasting, adipokine levels, and increased risk of metabolic disease.

Trans-suppression of host CDH3 and LOXL4 genes during Cryptosporidium parvum infection involves nuclear delivery of parasite Cdg7_FLc_1000 RNA.

PubMed

Ming, Zhenping; Gong, Ai-Yu; Wang, Yang; Zhang, Xin-Tian; Li, Min; Li, Yao; Pang, Jing; Dong, Stephanie; Strauss-Soukup, Juliane K; Chen, Xian-Ming

2018-05-01

Intestinal infection by Cryptosporidium parvum causes significant alterations in the gene expression profile in host epithelial cells. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of human intestinal cryptosporidiosis, we report here that trans-suppression of the cadherin 3 (CDH3) and lysyl oxidase like 4 (LOXL4) genes in human intestinal epithelial cells following C. parvum infection involves host delivery of the Cdg7_FLc_1000 RNA, a C. parvum RNA that has been previously demonstrated to be delivered into the nuclei of infected host cells. Downregulation of CDH3 and LOXL4 genes was detected in host epithelial cells following C. parvum infection or in cells expressing the parasite Cdg7_FLc_1000 RNA. Knockdown of Cdg7_FLc_1000 attenuated the trans-suppression of CDH3 and LOXL4 genes in host cells induced by infection. Interestingly, Cdg7_FLc_1000 was detected to be recruited to the promoter regions of both CDH3 and LOXL4 gene loci in host cells following C. parvum infection. Host delivery of Cdg7_FLc_1000 promoted the PH domain zinc finger protein 1 (PRDM1)-mediated H3K9 methylation associated with trans-suppression in the CDH3 gene locus, but not the LOXL4 gene. Therefore, our data suggest that host delivery of Cdg7_FLc_1000 causes CDH3 trans-suppression in human intestinal epithelial cells following C. parvum infection through PRDM1-mediated H3K9 methylation in the CDH3 gene locus, whereas Cdg7_FLc_1000 induces trans-suppression of the host LOXL4 gene through H3K9/H3K27 methylation-independent mechanisms. Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

Viral genome methylation as an epigenetic defense against geminiviruses.

PubMed

Raja, Priya; Sanville, Bradley C; Buchmann, R Cody; Bisaro, David M

2008-09-01

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

Ring-like distribution of constitutive heterochromatin in bovine senescent cells.

PubMed

Pichugin, Andrey; Beaujean, Nathalie; Vignon, Xavier; Vassetzky, Yegor

2011-01-01

Cells that reach "Hayflick limit" of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli. Constitutive heterochromatin in bovine senescent cells is organized in ring-like structures.

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells*

PubMed Central

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

2016-01-01

How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di- and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di- or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2. PMID:27226594

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

PubMed

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

2016-07-15

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

Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

PubMed

Tsutsumi, T; Tokumura, A; Kitazawa, S

1998-02-05

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between the internalization rates of methyl-PAF in HL-60 and K562 cells was correlated with their different susceptibilities to the cytotoxic effect of methyl-PAF, we suggest that the capacities for uptake of methyl-PAF and its accumulation in intracellular membranes are critical factor for its induction of apoptosis. (c) 1998 Elsevier Science B.V.

PHF1 Tudor and N-terminal domains synergistically target partially unwrapped nucleosomes to increase DNA accessibility

PubMed Central

Gibson, Matthew D.; Gatchalian, Jovylyn; Slater, Andrew; Kutateladze, Tatiana G.

2017-01-01

Abstract The Tudor domain of human PHF1 recognizes trimethylated lysine 36 on histone H3 (H3K36me3). PHF1 relies on this interaction to regulate PRC2 methyltransferase activity, localize to DNA double strand breaks and mediate nucleosome accessibility. Here, we investigate the impact of the PHF1 N-terminal domain (NTD) on the Tudor domain interaction with the nucleosome. We show that the NTD is partially ordered when it is natively attached to the Tudor domain. Through a combination of FRET and single molecule studies, we find that the increase of DNA accessibility within the H3K36me3-containing nucleosome, instigated by the Tudor binding to H3K36me3, is dramatically enhanced by the NTD. We demonstrate that this nearly order of magnitude increase is due to preferential binding of PHF1 to partially unwrapped nucleosomes, and that PHF1 alters DNA–protein binding within the nucleosome by decreasing dissociation rates. These results highlight the potency of a PTM-binding protein to regulate DNA accessibility and underscores the role of the novel mechanism by which nucleosomes control DNA–protein binding through increasing protein dissociation rates. PMID:28082396

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

PubMed

Gahurova, Lenka; Tomizawa, Shin-Ichi; Smallwood, Sébastien A; Stewart-Morgan, Kathleen R; Saadeh, Heba; Kim, Jeesun; Andrews, Simon R; Chen, Taiping; Kelsey, Gavin

2017-01-01

Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

SETDB1 modulates PRC2 activity at developmental genes independently of H3K9 trimethylation in mouse ES cells

PubMed Central

Fei, Qi; Yang, Xiaoqin; Jiang, Hua; Wang, Qian; Yu, Yanyan; Yu, Yiling; Yi, Wei; Zhou, Shaolian; Chen, Taiping; Lu, Chris; Atadja, Peter; Liu, Xiaole Shirley; Li, En; Zhang, Yong; Shou, Jianyong

2015-01-01

SETDB1, a histone methyltransferase responsible for methylation of histone H3 lysine 9 (H3K9), is involved in maintenance of embryonic stem (ES) cells and early embryonic development of the mouse. However, how SETDB1 regulates gene expression during development is largely unknown. Here, we characterized genome-wide SETDB1 binding and H3K9 trimethylation (H3K9me3) profiles in mouse ES cells and uncovered two distinct classes of SETDB1 binding sites, termed solo and ensemble peaks. The solo peaks were devoid of H3K9me3 and enriched near developmental regulators while the ensemble peaks were associated with H3K9me3. A subset of the SETDB1 solo peaks, particularly those near neural development–related genes, was found to be associated with Polycomb Repressive Complex 2 (PRC2) as well as PRC2-interacting proteins JARID2 and MTF2. Genetic deletion of Setdb1 reduced EZH2 binding as well as histone 3 lysine 27 (H3K27) trimethylation level at SETDB1 solo peaks and facilitated neural differentiation. Furthermore, we found that H3K27me3 inhibits SETDB1 methyltransferase activity. The currently identified reciprocal action between SETDB1 and PRC2 reveals a novel mechanism underlying ES cell pluripotency and differentiation regulation. PMID:26160163

Aberrant Epigenetic Alterations of Glutathione-S-Transferase P1 in Age-Related Nuclear Cataract.

PubMed

Chen, Jia; Zhou, Jing; Wu, Jian; Zhang, Guowei; Kang, Lihua; Ben, Jindong; Wang, Yong; Qin, Bai; Guan, Huaijin

2017-03-01

Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients. The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay. GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1. Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

AURKA induces EMT by regulating histone modification through Wnt/β-catenin and PI3K/Akt signaling pathway in gastric cancer

PubMed Central

Liu, Xi; Li, Zhaoxia; Song, Yue; Wang, Rui; Han, Lei; Wang, Qixue; Jiang, Kui; Kang, Chunsheng; Zhang, Qingyu

2016-01-01

Gastric cancer, a highly invasive and aggressive malignancy, is the third leading cause of death from cancer worldwide. Genetic association studies have successfully revealed several important genes consistently associated with gastric cancer to date. However, these robust gastric cancer-associated genes do not fully elucidate the mechanisms underlying the development and progression of the disease. In the present study, we performed an alternative approach, a gene expression-based genome-wide association study (eGWAS) across 13 independent microarray experiments (including 251 gastric cancer cases and 428 controls), to identify top candidates (p<0.00001). Additionally, we conducted gene ontology analysis, pathway analysis and network analysis and identified aurora kinase A (AURKA) as our candidate. We observed that MLN8237, which is a specific inhibitor of AURKA, decreased the β-catenin and the phosphorylation of Akt1 and GSK-3β, as well as blocked the Akt and Wnt signaling pathways. Furthermore, MLN8237 arrested the cells in the G2/M phase. The activity of Wnt and Akt signaling pathways affected the level of histone methylation significantly, and we supposed that MLN8237 affected the level of histone methylation through these two signaling pathways. Additionally, the treatment of MLN8237 influenced the level of H3K4 me1/2/3 and H3K27 me1/2/3. Chip data on cell lines suggested that MLN8237 increases the level of H3K27 me3 on the promoter of Twist and inhibits EMT (epithelial-mesenchymal transition). In summary, AURKA is a potential therapeutic target in gastric cancer and induces EMT through histone methylation. PMID:27121204

Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

PubMed

Geens, M; Seriola, A; Barbé, L; Santalo, J; Veiga, A; Dée, K; Van Haute, L; Sermon, K; Spits, C

2016-04-01

Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture take-over of chromosome abnormalities and was independent of the parental origin of the X chromosome. Therefore, we suggest that a culture advantage conferred by alleles on the X chromosome or by XCI-related mechanisms may be at the basis of this phenomenon. Finally, differentiated populations inherited the aberrant XCI patterns from the undifferentiated cells they were derived from. All hPSC lines in this study were cultured in highly similar conditions. Our results may therefore be specific for these conditions and alternative culture conditions might lead to different findings. Our findings are only a first step towards elucidating the molecular events leading to the phenomena we observed. Our results highlight the significant extent of aberrant XCI in female hPSC. The fact that these aberrations are inherited by the differentiated progeny may have a significant impact on downstream research and clinical uses of hPSC. In order to achieve the full potential of hPSC, more insight into the XCI status and its stability in hPSC and its effect on the properties of the differentiated progeny is needed. Not applicable. Our research is supported by grants from the Research Foundation - Flanders (FWO-Vlaanderen, grant 1502512N), Generalitat de Catalunya (2014SGR-005214) and the Methusalem grant of the Research Council of the Vrije Universiteit Brussel, on name of K.S. L.V.H. is funded by EMBO (ALTF 701-2013). The authors declare no potential conflict of interest. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.

PubMed

Porter, Louise F; Galli, Giorgio G; Williamson, Sally; Selley, Julian; Knight, David; Elcioglu, Nursel; Aydin, Ali; Elcioglu, Mustafa; Venselaar, Hanka; Lund, Anders H; Bonshek, Richard; Black, Graeme C; Manson, Forbes D

2015-12-01

Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Monoclinic modification of 1,1,3,3,5,5-hexa­methyl-cyclo-1,3,5-tris­tannathiane

PubMed Central

Singh, Nanhai; Kumar, Abhinav; Molloy, Kieran C.; Kociok-Köhn, G.

2008-01-01

The asymmetric unit of the title compound, [Sn3(CH3)6S3], contains two molecules with twist-boat conformations. There are intermolecular S⋯H (2.929 Å), S⋯S (3.433 Å), S⋯C (3.465 Å) and C⋯H (2.898 Å) inter­actions in addition to prominent intermolecular Sn⋯S inter­actions of 3.692 and 3.769 Å. PMID:21200475

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

PubMed Central

Fernández, Agustín F.; Bayón, Gustavo F.; Urdinguio, Rocío G.; Toraño, Estela G.; García, María G.; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M.; Riancho, José A.; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A.

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. PMID:25271306

Privileged Communication Embryonic Development Following Somatic Cell Nuclear Transfer Impeded by Persisting Histone Methylation

PubMed Central

Matoba, Shogo; Liu, Yuting; Lu, Falong; Iwabuchi, Kumiko A.; Shen, Li; Inoue, Azusa; Zhang, Yi

2014-01-01

SUMMARY Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major epigenetic barrier for efficient reprogramming by SCNT. Comparative transcriptome analysis identified reprogramming resistant regions (RRRs) that are expressed normally at 2-cell mouse embryos generated by IVF but not SCNT. RRRs are enriched for H3K9me3 in donor somatic cells, and its removal by ectopic expression of the H3K9me3 demethylase Kdm4d not only reactivates the majority of RRRs, but also greatly improves SCNT efficiency. Furthermore, use of donor somatic nuclei depleted of H3K9 methyltransferases markedly improves SCNT efficiency. Our study thus identifies H3K9me3 as a critical epigenetic barrier in SCNT-mediated reprogramming and provides a promising approach for improving mammalian cloning efficiency. PMID:25417163

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1.

PubMed

Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Altelaar, A F Maarten; van Leeuwen, Fred

2016-12-06

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features.

Direct screening for chromatin status on DNA barcodes in yeast delineates the regulome of H3K79 methylation by Dot1

PubMed Central

Vlaming, Hanneke; Molenaar, Thom M; van Welsem, Tibor; Poramba-Liyanage, Deepani W; Smith, Desiree E; Velds, Arno; Hoekman, Liesbeth; Korthout, Tessy; Hendriks, Sjoerd; Maarten Altelaar, AF; van Leeuwen, Fred

2016-01-01

Given the frequent misregulation of chromatin in cancer, it is important to understand the cellular mechanisms that regulate chromatin structure. However, systematic screening for epigenetic regulators is challenging and often relies on laborious assays or indirect reporter read-outs. Here we describe a strategy, Epi-ID, to directly assess chromatin status in thousands of mutants. In Epi-ID, chromatin status on DNA barcodes is interrogated by chromatin immunoprecipitation followed by deep sequencing, allowing for quantitative comparison of many mutants in parallel. Screening of a barcoded yeast knock-out collection for regulators of histone H3K79 methylation by Dot1 identified all known regulators as well as novel players and processes. These include histone deposition, homologous recombination, and adenosine kinase, which influences the methionine cycle. Gcn5, the acetyltransferase within the SAGA complex, was found to regulate histone methylation and H2B ubiquitination. The concept of Epi-ID is widely applicable and can be readily applied to other chromatin features. DOI: http://dx.doi.org/10.7554/eLife.18919.001 PMID:27922451

Specific labelling of serotonin 5-HT(1B) receptors in rat frontal cortex with the novel, phenylpiperazine derivative, [3H]GR125,743. A pharmacological characterization.

PubMed

Millan, M J; Newman-Tancredi, A; Lochon, S; Touzard, M; Aubry, S; Audinot, V

2002-04-01

Although several tritiated agonists have been used for radiolabelling serotonin (5-hydroxytryptamine, 5-HT)(1B) receptors in rats, data with a selective, radiolabelled antagonist have not been presented. Inasmuch as [3H]GR125,743 specifically labels cloned, human and native guinea pig 5-HT(1B) receptors and has been employed for characterization of cerebral 5-HT(1B) receptor in the latter species [Eur. J. Pharmacol. 327 (1997) 247.], the present study evaluated its utility for characterization of native, cerebral 5-HT(1B) sites in the rat. In homogenates of frontal cortex, [3H]GR125,743 (0.8 nM) showed rapid association (t(1/2)=3.4 min), >90% specific binding and high affinity (K(d)=0.6 nM) for a homogeneous population of receptors with a density (B(max)) of 160 fmol/mg protein. In competition binding studies, affinities were determined for 15 chemically diverse 5-HT(1B) agonists, including 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694,247; pK(i), 10.4), 5-carboxamidotryptamine (5-CT; 9.7), 3-[3-(2-dimethylamino-ethyl)-1H-indol-6-yl]-N-(4-methoxybenzyl)acrylamide (GR46,611; 9.6), 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU24,969; 9.5), dihydroergotamine (DHE; 8.6), 5-H-pyrrolo[3,2-b]pyridin-5-one,1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl (CP93,129; 8.4), anpirtoline (7.9), sumatriptan (7.4), 1-[2-(3-fluorophenyl)ethyl]-4-[3-[5-(1,2,4-triazol-4-yl)-1H-indol-3-yl]propyl]piperazine (L775,606; 6.4) and (minus sign)-1(S)-[2-[4-(4-methoxyphenyl)piperazin-1-yl]ethyl]-N-methyl-3,4-dihydro-1H-2-benzopyran-6-carboxamide (PNU109,291; <5.0). Similarly, affinities were established for 13 chemically diverse antagonists, including N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR125,743; pK(i), 9.1), (-)cyanopindolol (9.0), (-)-tertatolol (8.2), N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiozol-3-yl)biphenyl-4-carboxamide (GR127,935; 8.2), N-[3-(1,4-benzodioxan-5-yl)piperidin-4-yl]N-(indan-2yl)amine (S18127; 7.9), metergoline (7.8), (-)-pindolol (7.6), 1'-methyl-5-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5H-spiro[furo[2,3-f]indole-3,4'-piperidine] (SB224,289; 7.5) and ketanserin (<5.0). These rank orders of affinity correspond to the binding profile of 5-HT(1B) rather than 5-HT(1D) receptors. The low affinities of L775,066 and PNU109,291 versus L694,247 should be noted, as well as the low affinity of ketanserin as compared to SB224,289. Finally, in line with species differences, the affinities of several ligands including CP93,129, RU24,969, (-)-pindolol and (-)-propanolol in rat 5-HT(1B) sites were markedly different to guinea pig 5-HT(1B) sites labelled with [3H]GR125,743. In conclusion, [3H]GR125,743 is an appropriate tool for the radiolabelling of native, rat 5-HT(1B) receptors and permitted determination of the affinities of an extensive series of ligands at these sites.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

A shock tube and theory study of the dissociation of acetone and subsequent recombination of methyl radicals.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, A.; Kiefer, J. H.; Klippenstein, S. J.

The dissociation of acetone: CH{sub 3}C{double_bond}OCH{sub 3} {yields} CH{sub 3}C{double_bond}O + CH{sub 3}, quickly followed by CH{sub 3}CO {yields} CH{sub 3} + CO, has been examined with Laser-Schlieren measurements in incident shock waves over 32-717 Torr and 1429-1936 K using 5% acetone dilute in krypton. A few very low pressure experiments ({approx}10 Torr) were used in a marginal effort to resolve the extremely fast vibrational relaxation of this molecule. This effort was partly motivated as a test for molecular, 'roaming methyl' reactions, and also as a source of methyl radicals to test the application of a recent high-temperature mechanism formore » ethane decomposition [J.H. Kiefer, S. Santhanam, N.K. Srinivasan, R.S. Tranter, S.J. Klippenstein, M.A. Oehlschlaeger, Proc. Combust. Inst. 30 (2005) 1129-1135] on the reverse methyl combination. The gradient profiles show strong initial positive gradients and following negative values fully consistent with methyl radical formation and its following recombination. Thus C-C fission is certainly a large part of the process and molecular channels cannot be responsible for more than 30% of the dissociation. Rates obtained for the C-C fission show strong falloff well fit by variable reaction coordinate transition state theory when combined with a master equation. The calculated barrier is 82.8 kcal/mol, the fitted <{Delta}E>{sub down} = 400 (T/298) cm{sup -1}, similar to what was found in a recent study of C-C fission in acetaldehyde, and the extrapolated k{sub {infinity}} = 10{sup 25.86} T{sup -2.72} exp(?87.7 (kcal/mol)/RT), which agrees with the literature rate for CH{sub 3} + CH{sub 3}CO. Large negative (exothermic) gradients appearing late from methyl combination are accurately fit in both time of onset and magnitude by the earlier ethane dissociation mechanism. The measured dissociation rates are in close accord with one earlier shock-tube study [K. Sato, Y. Hidaka, Combust. Flame 122 (2000) 291-311], but show much less falloff than the high pressure experiments of Ernst et al. [J. Ernst, K. Spindler, H.Gg. Wagner, Ber. Bunsenges. Phys. Chem. 80 (1976) 645-650].« less

Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis

PubMed Central

Wen, Luan; Fu, Liezhen; Guo, Xiaogang; Chen, Yonglong; Shi, Yun-Bo

2015-01-01

Histone methylations have been implicated to play important roles in diverse cellular processes. Of particular interest is the methylation of histone H3K79, which is catalyzed by an evolutionarily conserved methyltransferase, disruptor of telomeric silencing (Dot1)-like (Dot1L). To investigate the role of Dot1L during vertebrate development, we have generated a Dot1L-specific transcription activator-like effector nuclease (TALEN) nuclease to knockdown endogenous Dot1L in Xenopus tropicalis, a diploid species highly related to the well-known developmental model Xenopus laevis, a pseudotetraploid amphibian. We show that the TALEN was extremely efficient in mutating Dot1L when expressed in fertilized eggs, creating essentially Dot1L knockout embryos with little H3K79 methylation. Importantly, we observed that Dot1L knockdown had no apparent effect on embryogenesis because normally feeding tadpoles were formed, consistent with the lack of maternal Dot1L expression. On the other hand, Dot1L knockdown severely retarded the growth of the tadpoles and led to tadpole lethality prior to metamorphosis. These findings suggest that Dot1L and H3K79 methylation play an important role for tadpole growth and development prior to metamorphosis into a frog. Our findings further reveal interesting similarities and differences between Xenopus and mouse development and suggest the existence of 2 separate phases of vertebrate development with distinct requirements for epigenetic modifications.—Wen, L., Fu, L., Guo, X., Chen, Y., Shi, Y.-B. Histone methyltransferase Dot1L plays a role in postembryonic development in Xenopus tropicalis. PMID:25366346

Histone H3 Lysine 36 Methyltransferase Whsc1 Promotes the Association of Runx2 and p300 in the Activation of Bone-Related Genes

PubMed Central

Lee, Yu Fei; Nimura, Keisuke; Lo, Wan Ning; Saga, Kotaro; Kaneda, Yasufumi

2014-01-01

The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1 −/− embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression. PMID:25188294

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prior, Sara; Miousse, Isabelle R.

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 promotermore » 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. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.« less

Oligoamine analogues in combination with 2-difluoromethylornithine synergistically induce re-expression of aberrantly silenced tumour-suppressor genes

PubMed Central

Wu, Yu; Steinbergs, Nora; Murray-Stewart, Tracy; Marton, Laurence J.; Casero, Robert A.

2011-01-01

Epigenetic gene silencing is an important mechanism in the initiation and progression of cancer. Abnormal DNA CpG island hypermethylation and histone modifications are involved in aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) was the first enzyme identified to specifically demethylate H3K4 (Lys4 of histone H3). Methylated H3K4 is an important mark associated with transcriptional activation. The flavin adenine dinucleotide-binding amine oxidase domain of LSD1 is homologous with two polyamine oxidases, SMO (spermine oxidase) and APAO (N1-acetylpolyamine oxidase). We have demonstrated previously that long-chain polyamine analogues, the oligoamines, are inhibitors of LSD1. In the present paper we report the synergistic effects of specific oligoamines in combination with DFMO (2-difluoromethylornithine), an inhibitor of ornithine decarboxylase, in human colorectal cancer cells. DFMO treatment depletes natural polyamines and increases the uptake of exogenous polyamines. The combination of oligoamines and DFMO results in a synergistic re-expression of aberrantly silenced tumour-suppressor genes, including SFRP2 (secreted frizzled-related protein 2), which encodes a Wnt signalling pathway antagonist and plays an anti-tumorigenic role in colorectal cancer. The treatment-induced re-expression of SFRP2 is associated with increased H3K4me2 (di-methyl H3K4) in the gene promoter. The combination of LSD1-inhibiting oligoamines and DFMO represents a novel approach to epigenetic therapy of cancer. PMID:22132744

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition

PubMed Central

Singhal, N. K.; Huang, H.; Li, S.; Clements, R.; Gadd, J.; Daniels, A.; Kooijman, E. E.; Bannerman, P.; Burns, T.; Guo, F.; Pleasure, D.; Freeman, E.; Shriver, L.

2017-01-01

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L−/−) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L−/− mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination. PMID:27709268

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

PubMed

Singhal, N K; Huang, H; Li, S; Clements, R; Gadd, J; Daniels, A; Kooijman, E E; Bannerman, P; Burns, T; Guo, F; Pleasure, D; Freeman, E; Shriver, L; McDonough, J

2017-01-01

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L -/- ) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L -/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

PubMed

Grandy, Rodrigo A; Whitfield, Troy W; Wu, Hai; Fitzgerald, Mark P; VanOudenhove, Jennifer J; Zaidi, Sayyed K; Montecino, Martin A; Lian, Jane B; van Wijnen, André J; Stein, Janet L; Stein, Gary S

2016-02-15

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation

PubMed Central

Grandy, Rodrigo A.; Whitfield, Troy W.; Wu, Hai; Fitzgerald, Mark P.; VanOudenhove, Jennifer J.; Zaidi, Sayyed K.; Montecino, Martin A.; Lian, Jane B.; van Wijnen, André J.; Stein, Janet L.

2015-01-01

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established. By mapping genomic enrichment of H3K4me3/H3K27me3 in pure populations of hESCs in G2, mitotic, and G1 phases of the cell cycle, we found striking variations in the levels of H3K4me3 through the G2-M-G1 transition. Analysis of a representative set of bivalent genes revealed that chromatin modifiers involved in H3K4 methylation/demethylation are recruited to bivalent gene promoters in a cell cycle-dependent fashion. Interestingly, bivalent genes enriched with H3K4me3 exclusively during mitosis undergo the strongest upregulation after induction of differentiation. Furthermore, the histone modification signature of genes that remain bivalent in differentiated cells resolves into a cell cycle-independent pattern after lineage commitment. These results establish a new dimension of chromatin regulation important in the maintenance of pluripotency. PMID:26644406

Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barnash, Kimberly D.; The, Juliana; Norris-Drouin, Jacqueline L.

The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein–protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to the embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EED’s recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2) peptide tomore » a smaller, more potent peptidomimetic ligand (K d = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EED’s reader function can lead to allosteric inhibition of PRC2 catalytic activity.« less

Histone Methylation Restrains the Expression of Subtype-Specific Genes during Terminal Neuronal Differentiation in Caenorhabditis elegans

PubMed Central

Chiang, Victor; Chalfie, Martin

2013-01-01

Although epigenetic control of stem cell fate choice is well established, little is known about epigenetic regulation of terminal neuronal differentiation. We found that some differences among the subtypes of Caenorhabditis elegans VC neurons, particularly the expression of the transcription factor gene unc-4, require histone modification, most likely H3K9 methylation. An EGF signal from the vulva alleviated the epigenetic repression of unc-4 in vulval VC neurons but not the more distant nonvulval VC cells, which kept unc-4 silenced. Loss of the H3K9 methyltransferase MET-2 or H3K9me2/3 binding proteins HPL-2 and LIN-61 or a novel chromodomain protein CEC-3 caused ectopic unc-4 expression in all VC neurons. Downstream of the EGF signaling in vulval VC neurons, the transcription factor LIN-11 and histone demethylases removed the suppressive histone marks and derepressed unc-4. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit. Thus, epigenetic mechanisms help establish subtype-specific gene expression, which are needed for optimal activity of a neural circuit. PMID:24348272

Developmental bisphenol A (BPA) exposure leads to sex-specific modification of hepatic gene expression and epigenome at birth that may exacerbate high-fat diet-induced hepatic steatosis.

PubMed

Strakovsky, Rita S; Wang, Huan; Engeseth, Nicki J; Flaws, Jodi A; Helferich, William G; Pan, Yuan-Xiang; Lezmi, Stéphane

2015-04-15

Developmental bisphenol A (BPA) exposure increases adulthood hepatic steatosis with reduced mitochondrial function. To investigate the potential epigenetic mechanisms behind developmental BPA-induced hepatic steatosis, pregnant Sprague-Dawley rats were dosed with vehicle (oil) or BPA (100μg/kg/day) from gestational day 6 until postnatal day (PND) 21. After weaning, offspring were either challenged with a high-fat (HF; 45% fat) or remained on a control (C) diet until PND110. From PND60 to 90, both BPA and HF diet increased the fat/lean ratio in males only, and the combination of BPA and HF diet appeared to cause the highest ratio. On PND110, Oil-HF, BPA-C, and BPA-HF males had higher hepatic lipid accumulation than Oil-C, with microvesicular steatosis being marked in the BPA-HF group. Furthermore, on PND1, BPA increased and modified hepatic triglyceride (TG) and free fatty acid (FFA) compositions in males only. In PND1 males, BPA increased hepatic expression of FFA uptake gene Fat/Cd36, and decreased the expression of TG synthesis- and β-oxidation-related genes (Dgat, Agpat6, Cebpα, Cebpβ, Pck1, Acox1, Cpt1a, Cybb). BPA altered DNA methylation and histone marks (H3Ac, H4Ac, H3Me2K4, H3Me3K36), and decreased the binding of several transcription factors (Pol II, C/EBPβ, SREBP1) within the male Cpt1a gene, the key β-oxidation enzyme. In PND1 females, BPA only increased the expression of genes involved in FFA uptake and TG synthesis (Lpl, Fasn, and Dgat). These data suggest that developmental BPA exposure alters and reprograms hepatic β-oxidation capacity in males, potentially through the epigenetic regulation of genes, and further alters the response to a HF diet. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas.

PubMed

Pajtler, Kristian W; Wen, Ji; Sill, Martin; Lin, Tong; Orisme, Wilda; Tang, Bo; Hübner, Jens-Martin; Ramaswamy, Vijay; Jia, Sujuan; Dalton, James D; Haupfear, Kelly; Rogers, Hazel A; Punchihewa, Chandanamali; Lee, Ryan; Easton, John; Wu, Gang; Ritzmann, Timothy A; Chapman, Rebecca; Chavez, Lukas; Boop, Fredrick A; Klimo, Paul; Sabin, Noah D; Ogg, Robert; Mack, Stephen C; Freibaum, Brian D; Kim, Hong Joo; Witt, Hendrik; Jones, David T W; Vo, Baohan; Gajjar, Amar; Pounds, Stan; Onar-Thomas, Arzu; Roussel, Martine F; Zhang, Jinghui; Taylor, J Paul; Merchant, Thomas E; Grundy, Richard; Tatevossian, Ruth G; Taylor, Michael D; Pfister, Stefan M; Korshunov, Andrey; Kool, Marcel; Ellison, David W

2018-06-16

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

The catalytic potential of cosmic dust: implications for prebiotic chemistry in the solar nebula and other protoplanetary systems.

PubMed

Hill, Hugh G M; Nuth, Joseph A

2003-01-01

The synthesis of important prebiotic molecules is fundamentally reliant on basic starting ingredients: water, organic species [e.g., methane (CH(4))], and reduced nitrogen compounds [e.g., ammonia (NH(3)), methyl cyanide (CH(3)CN) etc.]. However, modern studies conclude that the primordial Earth's atmosphere was too rich in CO, CO(2), and water to permit efficient synthesis of such reduced molecules as envisioned by the classic Miller-Urey experiment. Other proposed sources of terrestrial nitrogen reduction, like those within submarine vent systems, also seem to be inadequate sources of chemically reduced C-H-O-N compounds. Here, we demonstrate that nebular dust analogs have impressive catalytic properties for synthesizing prebiotic molecules. Using a catalyst analogous to nebular iron silicate condensate, at temperatures ranging from 500K to 900K, we catalyzed both the Fischer-Tropsch conversion of CO and H(2) to methane and water, and the corresponding Haber-Bosch synthesis of ammonia from N(2) and H(2). Remarkably, when CO, N(2), and H(2) were allowed to react simultaneously, these syntheses also yielded nitrogen-containing organics such as methyl amine (CH(3)NH(2)), acetonitrile (CH(3)CN), and N-methyl methylene imine (H(3)CNCH(2)). A fundamental consequence of this work for astrobiology is the potential for a natural chemical pathway to produce complex chemical building blocks of life throughout our own Solar System and beyond.

BORIS up-regulates OCT4 via histone methylation to promote cancer stem cell-like properties in human liver cancer cells.

PubMed

Liu, Qiuying; Chen, Kefei; Liu, Zhongjian; Huang, Yuan; Zhao, Rongce; Wei, Ling; Yu, Xiaoqin; He, Jingyang; Liu, Jun; Qi, Jianguo; Qin, Yang; Li, Bo

2017-09-10

Accumulating evidence has revealed the importance of cancer stem cells (CSCs) in chemoresistance and recurrence. BORIS, a testes-specific CTCF paralog, has been shown to be associated with stemness traits of embryonic cancer cells and epithelial CSCs. We previously reported that BORIS is correlated with the expression of the CSC marker CD90 in hepatocellular carcinoma (HCC). These results encourage us to wonder whether BORIS exerts functions on CSC-like traits of human liver cancer cells. Here, we report that BORIS was enriched in HCC tissues. Exogenous overexpression of BORIS promoted CSC-like properties, including self-renewal, chemoresistance, migration and invasion in Huh7 and HCCLM3 cells. Conversely, BORIS knockdown suppressed CSC-like properties in SMMC-7721 and HepG2 cells and inhibited tumorigenicity in SMMC-7721 cells. Moreover, BORIS alteration did not affect the DNA methylation status of the minimal promoter and exon 1 region of OCT4. However, BORIS overexpression enhanced the amount of BORIS bound on the OCT4 promoter and increased H3K4me2, while reducing H3K27me3; BORIS depletion decreased BORIS and H3K4me2 on the OCT4 promoter, while increasing H3K27me3. These results revealed that BORIS is associated with the CSC-like traits of human liver cancer cells through the epigenetic regulation of OCT4. Copyright © 2017 Elsevier B.V. All rights reserved.

Chronic Δ⁸-THC Exposure Differently Affects Histone Modifications in the Adolescent and Adult Rat Brain.

PubMed

Prini, Pamela; Penna, Federica; Sciuccati, Emanuele; Alberio, Tiziana; Rubino, Tiziana

2017-10-04

Adolescence represents a vulnerable period for the psychiatric consequences of delta9-tetrahydrocannabinol (Δ⁸-THC) exposure, however, the molecular underpinnings of this vulnerability remain to be established. Histone modifications are emerging as important epigenetic mechanisms involved in the etiopathogenesis of psychiatric diseases, thus, we investigated the impact of chronic Δ⁸-THC exposure on histone modifications in different brain areas of female rats. We checked histone modifications associated to both transcriptional repression (H3K9 di- and tri-methylation, H3K27 tri-methylation) and activation (H3K9 and H3K14 acetylation) after adolescent and adult chronic Δ⁸-THC exposure in the hippocampus, nucleus accumbens, and amygdala. Chronic exposure to increasing doses of Δ⁸-THC for 11 days affected histone modifications in a region- and age-specific manner. The primary effect in the adolescent brain was represented by changes leading to transcriptional repression, whereas the one observed after adult treatment led to transcriptional activation. Moreover, only in the adolescent brain, the primary effect was followed by a homeostatic response to counterbalance the Δ⁸-THC-induced repressive effect, except in the amygdala. The presence of a more complex response in the adolescent brain may be part of the mechanisms that make the adolescent brain vulnerable to Δ⁸-THC adverse effects.

A flash photolysis resonance fluorescence investigation of the reaction OH + CH3CCl3 yields H2O + CH2CCl3. [in troposphere

NASA Technical Reports Server (NTRS)

Kurylo, M. J.; Anderson, P. C.; Klais, O.

1979-01-01

The absolute rate constant for the reaction OH + CH3CCl3 yields H2O + CH2CCl3 was determined by the flash photolysis resonance fluorescence method from 253 to 363K. The use of the Arrhenius equation with atmospheric observational data on methyl chloroform nearly doubles the predicted tropospheric OH reaction sink strength for the removal of atmospheric gases whose lifetimes are controlled by OH. The increased use of methyl chloroform instead of the restricted trichloroethylene focused attention to its role in stratospheric ozone depletion, producing modeling analyses to determine the amount of released methyl chloroform which reaches the stratosphere. Since the primary atmospheric loss of CH3CCl3 is considered by reaction with OH radicals, these data are used to compute an average tropospheric OH concentration and the strength of the 'global tropospheric OH reaction sink'.

H3K27 methylation and H3S28 phosphorylation-dependent transcriptional regulation by INHAT subunit SET/TAF-Iβ.

PubMed

Kim, Ji-Young; Kim, Kee-Beom; Son, Hye-Ju; Chae, Yun-Cheol; Oh, Si-Taek; Kim, Dong-Wook; Pak, Jhang Ho; Seo, Sang-Beom

2012-09-21

Significant progress has been made in understanding the relationship between histone modifications and 'reader' molecules and their effects on transcriptional regulation. A previously identified INHAT complex subunit, SET/TAF-Iβ, binds to histones and inhibits histone acetylation. To investigate the binding specificities of SET/TAF-Iβ to various histone modifications, we employed modified histone tail peptide array analyses. SET/TAF-Iβ strongly recognized PRC2-mediated H3K27me1/2/3; however, the bindings were completely disrupted by H3S28 phosphorylation. We have demonstrated that SET/TAF-Iβ is sequentially recruited to the target gene promoter ATF3 after the PRC2 complex via H3K27me recognition and may offer additive effects in the repression of the target gene. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synthesis and conformational studies of carrabiose and its 4'-sulphate and 2,4'-disulphate.

PubMed

Parra, E; Caro, H N; Jiménez-Barbero, J; Martín-Lomas, M; Bernabé, M

1990-12-15

Methyl alpha-carrabioside (13), and its 4-sulphate (19) and 2,4-disulphate (20) have been synthesised via glycosylation of methyl 3,6-anhydro-2-O-benzyl-alpha-D-galactopyranoside with 2,3,6-tri-O-acetyl-4-O-benzyl-beta-D-galactopyranosyl bromide and subsequent partial or complete debenzylation, sulphation, and deprotection of the resulting disaccharide derivatives. Conformational studies have been carried out on 13, 19, and 20 on the basis of 1D and 2D 1H-n.m.r. spectroscopy and molecular mechanics calculations.

X-ray structural studies and physicochemical characterization of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl- 3,4-dihydro-2(1H)-pyrimidinone polymorphs.

PubMed

Miyamae, A; Kitamura, S; Tada, T; Koda, S; Yasuda, T

1991-10-01

The polymorphism of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-di hydro- 2(1 H)-pyrimidinone (FK664; 1) was characterized by using X-ray powder diffractometry, differential scanning calorimetry (DSC), and IR spectroscopy. Structures of two polymorphs (Forms A and B) were determined by X-ray crystallographic analysis. Form A crystallized in the monoclinic space group P2(1)/c, with a = 13.504(2), b = 6.733(1), c = 24.910(8) A, beta = 96.55(4) degrees, z = 4, and dcal = 1.203 g/cm3, while Form B crystallized in the same space group, with a = 8.067(2), b = 15.128(4), c = 18.657(4) A, beta = 102.34(3) degrees, z = 4, and dcal = 1.216 g/cm3. The conformational features of 1 were very similar between the two polymorphs. Compound 1, in both crystal forms, took an energetically reasonable conformation in three rigid planes, such as 2-pyrimidone, trimethylphenyl, and dimethoxyphenyl rings, but the molecules were packed in different ways between the two polymorphs. In the Form B crystal, a short contact was possible, to form pi-pi interactions between two dimethoxyphenyl groups related with the inversion center in the crystal lattice; this interaction seems to contribute to stabilizing the crystal structure of Form B. Both Forms A and B showed only one endothermic peak due to fusion at 115 and 140 degrees C, respectively, on the DSC thermograms; therefore, it is suggested that there are no transition points between the two polymorphs. The heats of fusion obtained from the DSC thermograms were 33.2(2) kJ/mol for Form A and 36.8(1) kJ/mol for Form B.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Landscape of Modified Histones in Drosophila Heterochromatic Genes and Euchromatin-Heterochromatin Transition Zones

PubMed Central

Yasuhara, Jiro C; Wakimoto, Barbara T

2008-01-01

Constitutive heterochromatin is enriched in repetitive sequences and histone H3-methylated-at-lysine 9. Both components contribute to heterochromatin's ability to silence euchromatic genes. However, heterochromatin also harbors hundreds of expressed genes in organisms such as Drosophila. Recent studies have provided a detailed picture of sequence organization of D. melanogaster heterochromatin, but how histone modifications are associated with heterochromatic sequences at high resolution has not been described. Here, distributions of modified histones in the vicinity of heterochromatic genes of normal embryos and embryos homozygous for a chromosome rearrangement were characterized using chromatin immunoprecipitation and genome tiling arrays. We found that H3-di-methylated-at-lysine 9 (H3K9me2) was depleted at the 5′ ends but enriched throughout transcribed regions of heterochromatic genes. The profile was distinct from that of euchromatic genes and suggests that heterochromatic genes are integrated into, rather than insulated from, the H3K9me2-enriched domain. Moreover, the profile was only subtly affected by a Su(var)3–9 null mutation, implicating a histone methyltransferase other than SU(VAR)3–9 as responsible for most H3K9me2 associated with heterochromatic genes in embryos. On a chromosomal scale, we observed a sharp transition to the H3K9me2 domain, which coincided with increased retrotransposon density in the euchromatin-heterochromatin (eu-het) transition zones on the long chromosome arms. Thus, a certain density of retrotransposons, rather than specific boundary elements, may demarcate Drosophila pericentric heterochromatin. We also demonstrate that a chromosome rearrangement that created a new eu-het junction altered H3K9me2 distribution and induced new euchromatic sites of enrichment as far as several megabases away from the breakpoint. Taken together, the findings argue against simple classification of H3K9me as the definitive signature of silenced genes, and clarify roles of histone modifications and repetitive DNAs in heterochromatin. The results are also relevant for understanding the effects of chromosome aberrations and the megabase scale over which epigenetic position effects can operate in multicellular organisms. PMID:18208336

MLL4 Is Required to Maintain Broad H3K4me3 Peaks and Super-Enhancers at Tumor Suppressor Genes.

PubMed

Dhar, Shilpa S; Zhao, Dongyu; Lin, Tao; Gu, Bingnan; Pal, Khusboo; Wu, Sarah J; Alam, Hunain; Lv, Jie; Yun, Kyuson; Gopalakrishnan, Vidya; Flores, Elsa R; Northcott, Paul A; Rajaram, Veena; Li, Wei; Shilatifard, Ali; Sillitoe, Roy V; Chen, Kaifu; Lee, Min Gyu

2018-06-07

Super-enhancers are large clusters of enhancers that activate gene expression. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines active tumor suppressor genes. However, how these epigenomic signatures are regulated for tumor suppression is little understood. Here we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (a COMPASS-like enzyme, also known as KMT2D) in mice spontaneously induces medulloblastoma. Mll4 loss upregulates oncogenic Ras and Notch pathways while downregulating neuronal gene expression programs. MLL4 enhances DNMT3A-catalyzed DNA methylation and SIRT1/BCL6-mediated H4K16 deacetylation, which antagonize expression of Ras activators and Notch pathway components, respectively. Notably, Mll4 loss downregulates tumor suppressor genes (e.g., Dnmt3a and Bcl6) by diminishing broad H3K4me3 and super-enhancers and also causes widespread impairment of these epigenomic signatures during medulloblastoma genesis. These findings suggest an anti-tumor role for super-enhancers and provide a unique tumor-suppressive mechanism in which MLL4 is necessary to maintain broad H3K4me3 and super-enhancers at tumor suppressor genes. Copyright © 2018 Elsevier Inc. All rights reserved.

Detection of Turner syndrome using X-chromosome inactivation specific differentially methylated CpG sites: A pilot study.

PubMed

Zhang, Qiang; Guo, Xiaohong; Tian, Tian; Wang, Teng; Li, Qiaoli; Wang, Lei; Liu, Yun; Xing, Qinghe; He, Lin; Zhao, Xinzhi

2017-05-01

Early diagnosis of Turner syndrome (TS) may improve preventive measures and treatment. X-chromosome inactivation specific differentially methylated CpG sites (XIDMSs) that are high methylated in inactive X chromosomes (Xi) and unmethylated in active X chromosomes (Xa) may be potential makers for TS detection. The candidate XIDMSs were screened from 9 male and 12 female DNA samples with normal karyotypes using the Illumina 450k array and validated by bisulfite sequencing PCR and pyrosequencing assay. X chromosome dosage was calculated according to the methylation level of multiple XIDMSs. Overall, 108 candidate XIDMSs were screened by the 450k array. Validations indicated that XIDMSs gathered and formed the X-chromosome inactivation specific differentially methylated regions (XIDMRs). Using 3 XIDMRs at SAT1, UXT and UTP14A loci, 36 TS, 22 normal female and 6 male samples were analyzed. Methylation levels of the 20 XIDMSs in the XIDMRs could distinguish between TS and normal female DNA samples, the X chromosome dosage was consistent with karyotyping data. Analyzing samples of 2 triple X syndrome and 3 Klinefelter syndrome patients suggested that this method could be used to detect X chromosome aneuploids other than TS. XIDMSs are widely spread along the X chromosome and might be effective markers for detection of TS and other X chromosome aneuploids. Copyright © 2017 Elsevier B.V. All rights reserved.

Methyl 3-O-α-l-fucopyranosyl β-d-glucopyran­oside tetra­hydrate

PubMed Central

Eriksson, Lars; Widmalm, Göran

2012-01-01

The title compound, C13H24O10·4H2O, is the methyl glycoside of a disaccharide structural element present in the backbone of the capsular polysaccharide from Klebsiella K1, which contains only three sugars and a substituent in the polysaccharide repeating unit. The conformation of the title disaccharide is described by the glycosidic torsion angles ϕH = 51.1 (1)° and ψH = 25.8 (1)°. In the crystal, a number of O—H⋯O hydrogen bonds link the methyl glycoside and water mol­ecules, forming a three-dimensional network. One water mol­ecule is disordered over two positions with occupancies of 0.748 (4) and 0.252 (4). PMID:23284493

Epigenetic-Mediated Downregulation of μ-Protocadherin in Colorectal Tumours

PubMed Central

Mateusz, Bujko; Paulina, Kober; Małgorzata, Statkiewicz; Michal, Mikula; Marcin, Ligaj; Lech, Zwierzchowski; Jerzy, Ostrowski; Aleksander, Siedlecki Janusz

2015-01-01

Carcinogenesis involves altered cellular interaction and tissue morphology that partly arise from aberrant expression of cadherins. Mucin-like protocadherin is implicated in intercellular adhesion and its expression was found decreased in colorectal cancer (CRC). This study has compared MUPCDH (CDHR5) expression in three key types of colorectal tissue samples, for normal mucosa, adenoma, and carcinoma. A gradual decrease of mRNA levels and protein expression was observed in progressive stages of colorectal carcinogenesis which are consistent with reports of increasing MUPCDH 5′ promoter region DNA methylation. High MUPCDH methylation was also observed in HCT116 and SW480 CRC cell lines that revealed low gene expression levels compared to COLO205 and HT29 cell lines which lack DNA methylation at the MUPCDH locus. Furthermore, HCT116 and SW480 showed lower levels of RNA polymerase II and histone H3 lysine 4 trimethylation (H3K4me3) as well as higher levels of H3K27 trimethylation at the MUPCDH promoter. MUPCDH expression was however restored in HCT116 and SW480 cells in the presence of 5-Aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Results indicate that μ-protocadherin downregulation occurs during early stages of tumourigenesis and progression into the adenoma-carcinoma sequence. Epigenetic mechanisms are involved in this silencing. PMID:25972897

A repertoire of pyridinium-phenyl-methyl cross-talk through a cascade of intramolecular electrostatic interactions.

PubMed

Acharya, P; Plashkevych, O; Morita, C; Yamada, S; Chattopadhyaya, J

2003-02-21

Direct intramolecular cation-pi interaction between phenyl and pyridinium moieties in 1a(+) has been experimentally evidenced through pH-dependent (1)H NMR titration. The basicity of the pyridinyl group (pK(a) 2.9) in 1a can be measured both from the pH-dependent chemical shifts of the pyridinyl protons as well as from the protons of the neighboring phenyl and methyl groups as a result of electrostatic interaction between the phenyl and the pyridinium ion in 1a(+) at the ground state. The net result of this nearest neighbor electrostatic interaction is that the pyridinium moiety in 1a becomes more basic (pK(a) 2.92) compared to that in the standard 2a (pK(a) 2.56) as a consequence of edge-to-face cation (pyridinium)-pi (phenyl) interaction, giving a free energy of stabilization (DeltaDeltaG(o)pKa) of -2.1 kJ mol(-1). The fact that the pH-dependent downfield shifts of the phenyl and methyl protons give the pK(a) of the pyridine moiety of 1a also suggests that the nearest neighbor cation (pyridinium)-pi (phenyl) interaction also steers the CH (methyl)-pi (phenyl) interaction in tandem. This means that the whole pyridine-phenyl-methyl system in 1a(+) is electronically coupled at the ground state, cross-modulating the physicochemical property of the next neighbor by using the electrostatics as the engine, and the origin of this electrostatics is a far away point in the molecule-the pyridinyl-nitrogen. The relative chemical shift changes and the pK(a) differences show that the cation (pyridinium)-pi (phenyl) interaction is indeed more stable (DeltaDeltaG(o)pKa = -2.1 kJ mol(-1)) than that of the CH (methyl)-pi (phenyl) interaction (DeltaDeltaG(o)pKa = -0.8 kJ mol(-1)). Since the pK(a) of the pyridine moiety in 1a is also obtained through the pH-dependent shifts of both phenyl and methyl protons, it suggests that the net electrostatic mediated charge transfer from the phenyl to the pyridinium and its effect on the CH (methyl)-pi (phenyl) interaction corresponds to DeltaG(o)pKa of the pyridinium ion (approximately 17.5 kJ mol(-1)), which means that the aromatic characters of the phenyl and the pyridinium rings in 1a(+) have been cross-modulated owing to the edge-to-face interaction proportional to this DeltaG(o)pKa change.

Histone methylation mediates plasticity of human FOXP3(+) regulatory T cells by modulating signature gene expressions.

PubMed

He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-03-01

CD4(+) FOXP3(+) regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4(+) CD25(high) CD127(low/-) Treg cells convert to two subpopulations with distinctive FOXP3(+) and FOXP3(-) phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. © 2013 John Wiley & Sons Ltd.

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence1

PubMed Central

Liu, Jian; Ji, Yingbin; Zhou, Jun; Xing, Da

2016-01-01

PI3K and its product PI3P are both involved in plant development and stress responses. In this study, the down-regulation of PI3K activity accelerated leaf senescence induced by methyl jasmonate (MeJA) and suppressed the activation of vacuolar H+-ATPase (V-ATPase). Yeast two-hybrid analyses indicated that PI3K bound to the V-ATPase B subunit (VHA-B). Analysis of bimolecular fluorescence complementation in tobacco guard cells showed that PI3K interacted with VHA-B2 in the tonoplasts. Through the use of pharmacological and genetic tools, we found that PI3K and V-ATPase promoted vacuolar acidification and stomatal closure during leaf senescence. Vacuolar acidification was suppressed by the PIKfyve inhibitor in 35S:AtVPS34-YFP Arabidopsis during MeJA-induced leaf senescence, but the decrease was lower than that in YFP-labeled Arabidopsis. These results suggest that PI3K promotes V-ATPase activation and consequently induces vacuolar acidification and stomatal closure, thereby delaying MeJA-induced leaf senescence. PMID:26739232

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

PubMed

Fernández, Agustín F; Bayón, Gustavo F; Urdinguio, Rocío G; Toraño, Estela G; García, María G; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M; Riancho, José A; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A; Fraga, Mario F

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. © 2015 Fernández et al.; Published by Cold Spring Harbor Laboratory Press.

Effects of BPA on global DNA methylation and global histone 3 lysine modifications in SH-SY5Y cells: An epigenetic mechanism linking the regulation of chromatin modifiying genes.

PubMed

Senyildiz, Mine; Karaman, Ecem Fatma; Bas, Serap Sancar; Pirincci, Pelin Arda; Ozden, Sibel

2017-10-01

Bisphenol A (BPA), an estrogenic endocrine disruptor, is widely used in the production of polycarbonate plastic and epoxy resins, resulting in high risk on human health. In present study we aimed to investigate the effects of BPA on global and gene specific DNA methylation, global histone modifications and regulation of chromatin modifiying enzymes in human neuroblastoma cells (SH-SY5Y). Cells were treated with BPA at 0.1, 1 and 10μM concentrations for 48 and 96h. IC 50 value of BPA was determined as 183 and 129μM in SH-SY5Y cells after 24h by MTT and NRU tests, respectively. We observed significant alterations on the 5-mC% levels (1.3 fold) and 5-hmC% levels (1.67 fold) after 10μM of BPA for 96h. Significant decrease was identified in H3K9me3 and H3K9ac after 10μM of BPA for 96h while decrease was observed in H3K4me3 at 10μM of BPA for 48h. Alterations were observed in chromatin modifiying genes including G9a, EZH2, SETD8, SETD1A, HAT1, SIRT1, DNMT1, RIZ1 and Suv39h1 after 96h of BPA exposure. Taken together, this study suggests that BPA might modulate the epigenetic regulators which would be key molecular events in the toxicity of endocrine disrupting chemicals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

PubMed Central

Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

2013-01-01

Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

PubMed

Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

2018-01-15

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

Regulation of transcriptional silencing and chromodomain protein localization at centromeric heterochromatin by histone H3 tyrosine 41 phosphorylation in fission yeast.

PubMed

Ren, Bingbing; Tan, Hwei Ling; Nguyen, Thi Thuy Trang; Sayed, Ahmed Mahmoud Mohammed; Li, Ying; Mok, Yu-Keung; Yang, Henry; Chen, Ee Sin

2018-01-09

Heterochromatin silencing is critical for genomic integrity and cell survival. It is orchestrated by chromodomain (CD)-containing proteins that bind to methylated histone H3 lysine 9 (H3K9me), a hallmark of heterochromatin. Here, we show that phosphorylation of tyrosine 41 (H3Y41p)-a novel histone H3 modification-participates in the regulation of heterochromatin in fission yeast. We show that a loss-of-function mutant of H3Y41 can suppress heterochromatin de-silencing in the centromere and subtelomere repeat regions, suggesting a de-silencing role for H3Y41p on heterochromatin. Furthermore, we show both in vitro and in vivo that H3Y41p differentially regulates two CD-containing proteins without the change in the level of H3K9 methylation: it promotes the binding of Chp1 to histone H3 and the exclusion of Swi6. H3Y41p is preferentially enriched on centromeric heterochromatin during M- to early S phase, which coincides with the localization switch of Swi6/Chp1. The loss-of-function H3Y41 mutant could suppress the hypersensitivity of the RNAi mutants towards hydroxyurea (HU), which arrests replication in S phase. Overall, we describe H3Y41p as a novel histone modification that differentially regulates heterochromatin silencing in fission yeast via the binding of CD-containing proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Oral acetate supplementation attenuates N-methyl D-aspartate receptor hypofunction-induced behavioral phenotypes accompanied by restoration of acetyl-histone homeostasis.

PubMed

Singh, Seema; Choudhury, Arnab; Gusain, Priya; Parvez, Suhel; Palit, Gautam; Shukla, Shubha; Ganguly, Surajit

2016-04-01

Aberrations in cellular acetate-utilization processes leading to global histone hypoacetylation have been implicated in the etiology of neuropsychiatric disorders like schizophrenia. Here, we investigated the role of acetate supplementation in the form of glyceryl triacetate (GTA) for the ability to restore the N-methyl D-aspartate (NMDA) receptor-induced histone hypoacetylation and to ameliorate associated behavioral phenotypes in mice. Taking cues from the studies in SH-SY5Y cells, we monitored acetylation status of specific lysine residues of histones H3 and H4 (H3K9 and H4K8) to determine the impact of oral GTA supplementation in vivo. Mice treated chronically with MK-801 (10 days; 0.15 mg/kg daily) induced hypoacetylation of H3K9 and H4K8 in the hippocampus. Daily oral supplementation of GTA (2.9 g/kg) was able to prevent this MK801-induced hypoacetylation significantly. Though MK-801-stimulated decreases in acetyl-H3K9 and acetyl-H4K8 were found to be associated with ERK1/2 activation, GTA seemed to act independent of this pathway. Simultaneously, GTA administration was able to attenuate the chronic MK-801-induced cognitive behavior phenotypes in elevated plus maze and novel object recognition tests. Not only MK-801, GTA also demonstrated protective effects against behavioral phenotypes generated by another NMDA receptor antagonist, ketamine. Acute (single injection) ketamine-mediated hyperactivity phenotype and chronic (10 days treatment) ketamine-induced phenotype of exaggerated immobility in forced swim test were ameliorated by GTA. The signature behavioral phenotypes induced by acute and chronic regimen of NMDA receptor antagonists seemed to be attenuated by GTA. This study thus provides a therapeutic paradigm of using dietary acetate supplement in psychiatric disorders.

Characterization of muscarinic receptors on intact human neuroblastoma cells: coupling to phosphoinositide hydrolysis and phosphorylation by phorbol esters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serra, M.; Watson, M.; Roeske, W.R.

Cloned human neuroblastoma cells (SH-SY5Y) were grown. High affinity binding of (/sup 3/H)(-)quinuclidinyl benzilate ((/sup 3/H)(-)QNB) and its quaternary derivative (/sup 3/H)(-)methyl QNB to muscarinic receptors (MR) on intact SH-SY5Y cells was studied. A 30 min rinse time gave a ratio of specific/total binding of 90% for both ligands. Association rates of (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB were determined. Both ligands reached steady state by 60 min at 37/sup 0/C. Rates of dissociation for both radioligands were biphasic, although (/sup 3/H)(-)methyl QNB was faster. Saturation studies yielded K/sub d/ (dissociation constant) values of 16 and 260 pM and B/submore » max/ (maximal MR density) values of 172 and 134 fmoles/mg prot for (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB, respectively. Activation of protein kinase C by phorbol esters produced increased phosphorylation of cellular proteins. Pretreatment with 100 nM of 4..beta..-phorbol 12..beta..-myristate 13..cap alpha..-acetate (PMA) induced a decrease in agonist affinity for MR, suggesting a PMA-promoted phosphorylation of the MR protein. Phosphoinositide (PhI) turnover was measured by MR agonist-induced accumulation of inositol-1-phosphate in the presence of Li/sup + +/ (10 mM). Only carbachol and acetylcholine elicited potent responses with oxotremorine (16%) pilocarpine (17%) and McN-A-343 (8%) appearing to be weak partial agonist of low efficacy.« less

CpG Island Methylator Phenotype and Prognosis of Colorectal Cancer in Northeast China

PubMed Central

Li, Xia; Hu, Fulan; Yao, Xiaoping; Zhang, Zuoming; Wang, Fan; Sun, Guizhi; Cui, Bin-Bin; Dong, Xinshu; Zhao, Yashuang

2014-01-01

Purpose. To investigate the association between CpG island methylator phenotype (CIMP) and the overall survival of sporadic colorectal cancer (CRC) in Northeast China. Methods. 282 sporadic CRC patients were recruited in this study. We selected MLH1, MGMT, p16, APC, MINT1, MINT31, and RUNX3 as the CIMP panel markers. The promoter methylation was assessed by methylation sensitive high resolution melting (MS-HRM). Proportional hazards-regression models were fitted with computing hazard ratios (HR) and the corresponding 95% confidence intervals (95% CI). Results. 12.77% (36/282) of patients were CIMP-0, 74.1% (209/282) of patients were CIMP-L, and 13.12% (37/282) of patients were CIMP-H. The five-year survival of the 282 CRC patients was 58%. There was significant association between APC gene promoter methylation and CRC overall survival (HR = 1.61; 95% CI: 1.05–2.46; P = 0.03). CIMP-H was significantly associated with worse prognosis compared to CIMP-0 (HR = 3.06; 95% CI: 1.19–7.89; P = 0.02) and CIMP-L (HR = 1.97; 95% CI: 1.11–3.48; P = 0.02), respectively. While comparing with the combine of CIMP-L and CIMP-0 (CIMP-L/0), CIMP-H also presented a worse prognosis (HR = 2.31; 95% CI: 1.02–5.24; P = 0.04). Conclusion. CIMP-H may be a predictor of a poor prognosis of CRC in Northeast China patients. PMID:25243122

CpG island methylator phenotype and prognosis of colorectal cancer in Northeast China.

PubMed

Li, Xia; Hu, Fulan; Wang, Yibaina; Yao, Xiaoping; Zhang, Zuoming; Wang, Fan; Sun, Guizhi; Cui, Bin-Bin; Dong, Xinshu; Zhao, Yashuang

2014-01-01

To investigate the association between CpG island methylator phenotype (CIMP) and the overall survival of sporadic colorectal cancer (CRC) in Northeast China. 282 sporadic CRC patients were recruited in this study. We selected MLH1, MGMT, p16, APC, MINT1, MINT31, and RUNX3 as the CIMP panel markers. The promoter methylation was assessed by methylation sensitive high resolution melting (MS-HRM). Proportional hazards-regression models were fitted with computing hazard ratios (HR) and the corresponding 95% confidence intervals (95% CI). 12.77% (36/282) of patients were CIMP-0, 74.1% (209/282) of patients were CIMP-L, and 13.12% (37/282) of patients were CIMP-H. The five-year survival of the 282 CRC patients was 58%. There was significant association between APC gene promoter methylation and CRC overall survival (HR = 1.61; 95% CI: 1.05-2.46; P = 0.03). CIMP-H was significantly associated with worse prognosis compared to CIMP-0 (HR = 3.06; 95% CI: 1.19-7.89; P = 0.02) and CIMP-L (HR = 1.97; 95% CI: 1.11-3.48; P = 0.02), respectively. While comparing with the combine of CIMP-L and CIMP-0 (CIMP-L/0), CIMP-H also presented a worse prognosis (HR = 2.31; 95% CI: 1.02-5.24; P = 0.04). CIMP-H may be a predictor of a poor prognosis of CRC in Northeast China patients.

Electron impact dissociation of amorphous cis-methyl acetate ice analogs

NASA Astrophysics Data System (ADS)

Sivaraman, B.; Mukherjee, R.; Subramanian, K. P.; Banerjee, S. B.

2014-08-01

We have carried out, for the first time, electron irradiation on methyl acetate (CH3COOCH3) ices grown, under astrochemical condition, at 85 K and the chemical changes were probed by FTIR spectroscopy in the mid-IR region (4000-500 cm-1). Carbon dioxide (CO2), carbon monoxide (CO) molecules were found to be the major products indicating a competing dissociation pathway along with the by-products ethane (C2H6) and dimethyl ether (CH3OCH3), respectively. CH3OH (methyl alcohol) formation showed the possibility of acetate to alcohol conversion by keV electron irradiation. In addition irradiation induced aggregation of CH3COOCH3 molecules is also proposed.

Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

PubMed Central

Małecki, Jędrzej; Nilges, Benedikt S.; Moen, Anders; Leidel, Sebastian A.

2017-01-01

Abstract Many cellular proteins are methylated on lysine residues and this has been most intensively studied for histone proteins. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. Several recently discovered KMTs belong to the so-called seven-β-strand (7BS) class of MTases and we have here investigated an uncharacterized human 7BS MTase currently annotated as part of the endothelin converting enzyme 2, but which should be considered a separate enzyme. Combining in vitro enzymology and analyzes of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we show that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site and we demonstrate the importance of this motif for catalytic activity. PMID:28520920

12,12′-[2,2′-Oxybis(ethane-2,1-di­yl)bis­(­oxy)]bis­[(R p)-4-bromo­[2.2]paracyclo­phane

PubMed Central

Hong, Bing; Ma, Yudao; Duan, Wenzeng; He, Fuyan; Zhao, Lei

2011-01-01

The title compound, C36H36Br2O3, was synthesized from (R p)-4-bromo-12-hy­droxy[2.2]paracyclo­phane and oxydiethane-2,1-diyl bis­(4-methyl­benzene­sulfonate). The crystal packing exhibits a short O⋯Br inter­action [Br⋯O = 3.185 (3) Å] and a weak inter­molecular C—H⋯O contact. PMID:21754216

A new half-condensed Schiff base compound: highly selective and sensitive pH-responsive fluorescent sensor.

PubMed

Saha, Uday Chand; Dhara, Koushik; Chattopadhyay, Basab; Mandal, Sushil Kumar; Mondal, Swastik; Sen, Supriti; Mukherjee, Monika; van Smaalen, Sander; Chattopadhyay, Pabitra

2011-09-02

A new probe, 3-[(3-benzyloxypyridin-2-ylimino)methyl]-2-hydroxy-5-methylbenzaldehyde (1-H) behaves as a highly selective fluorescent pH sensor in a Britton-Robinson buffer at 25 °C. The pH titrations show a 250-fold increase in fluorescence intensity within the pH range of 4.2 to 8.3 with a pK(a) value of 6.63 which is valuable for studying many of the biological organelles.

Histone H4 methyltransferase Suv420h2 maintains fidelity of osteoblast differentiation

PubMed Central

Farzaneh, Khani; Thaler, Roman; Paradise, Christopher R.; Deyle, David R.; Julio, Marianne Kruijthof-de; Galindo, Mario; Gordon, Jonathan A.; Stein, Gary S.; Dudakovic, Amel; van Wijnen, Andre J.

2017-01-01

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones (‘histone code’), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. PMID:27862226

Intricate Conformational Tunneling in Carbonic Acid Monomethyl Ester.

PubMed

Linden, Michael M; Wagner, J Philipp; Bernhardt, Bastian; Bartlett, Marcus A; Allen, Wesley D; Schreiner, Peter R

2018-04-05

Disentangling internal and external effects is a key requirement for understanding conformational tunneling processes. Here we report the s- trans/ s- cis tunneling rotamerization of carbonic acid monomethyl ester (1) under matrix isolation conditions and make comparisons to its parent carbonic acid (3). The observed tunneling rate of 1 is temperature-independent in the 3-20 K range and accelerates when using argon instead of neon as the matrix material. The methyl group increases the effective half life (τ eff ) of the energetically disfavored s- trans-conformer from 3-5 h for 3 to 11-13 h for 1. Methyl group deuteration slows the rotamerization further (τ eff ≈ 35 h). CCSD(T)/cc-pVQZ//MP2/aug-cc-pVTZ computations of the tunneling probability suggest that the rate should be almost unaffected by methyl substitution or its deuteration. Thus the observed relative rates are puzzling, and they disagree with previous explanations involving fast vibrational relaxation after the tunneling event facilitated by the alkyl rotor.

Dipotassium tetra-aqua-bis-[3,5-bis-(dicyano-methyl-ene)cyclo-pentane-1,2,4-trionato(1-)-κN]cobaltate(II).

PubMed

Chagas, Luciano Honorato; Janczak, Jan; Machado, Flavia C; de Oliveira, Luiz Fernando C; Diniz, Renata

2010-11-27

The title structure, K(2)[Co(C(11)N(4)O(3))(2)(H(2)O)(4)], is isotypic with K(2)[Fe(C(11)N(4)O(3))(2)(H(2)O)(4)]. The Co(II) atom is in a distorted octa-hedral CoN(2)O(4) geometry, forming a dianionic mononuclear entity. Each dianionic unit is associated with two potassium cations and inter-acts with adjacent units through O-H⋯N and O-H⋯O hydrogen bonds.

The use of a selective saturation pulse to suppress t1 noise in two-dimensional 1H fast magic angle spinning solid-state NMR spectroscopy

NASA Astrophysics Data System (ADS)

Robertson, Aiden J.; Pandey, Manoj Kumar; Marsh, Andrew; Nishiyama, Yusuke; Brown, Steven P.

2015-11-01

A selective saturation pulse at fast magic angle spinning (MAS) frequencies (60+ kHz) suppresses t1 noise in the indirect dimension of two-dimensional 1H MAS NMR spectra. The method is applied to a synthetic nucleoside with an intense methyl 1H signal due to triisopropylsilyl (TIPS) protecting groups. Enhanced performance in terms of suppressing the methyl signal while minimising the loss of signal intensity of nearby resonances of interest relies on reducing spin diffusion - this is quantified by comparing two-dimensional 1H NOESY-like spin diffusion spectra recorded at 30-70 kHz MAS. For a saturation pulse centred at the methyl resonance, the effect of changing the nutation frequency at different MAS frequencies as well as the effect of changing the pulse duration is investigated. By applying a pulse of duration 30 ms and nutation frequency 725 Hz at 70 kHz MAS, a good compromise of significant suppression of the methyl resonance combined with the signal intensity of resonances greater than 5 ppm away from the methyl resonance being largely unaffected is achieved. The effectiveness of using a selective saturation pulse is demonstrated for both homonuclear 1H-1H double quantum (DQ)/single quantum (SQ) MAS and 14N-1H heteronuclear multiple quantum coherence (HMQC) two-dimensional solid-state NMR experiments.

A687V EZH2 is a driver of histone H3 lysine 27 (H3K27) hypertrimethylation.

PubMed

Ott, Heidi M; Graves, Alan P; Pappalardi, Melissa B; Huddleston, Michael; Halsey, Wendy S; Hughes, Ashley M; Groy, Arthur; Dul, Edward; Jiang, Yong; Bai, Yuchen; Annan, Roland; Verma, Sharad K; Knight, Steven D; Kruger, Ryan G; Dhanak, Dashyant; Schwartz, Benjamin; Tummino, Peter J; Creasy, Caretha L; McCabe, Michael T

2014-12-01

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. ©2014 American Association for Cancer Research.

Reactivity of 3-hydroxy-3-methyl-2-butanone: Photolysis and OH reaction kinetics

NASA Astrophysics Data System (ADS)

Bouzidi, H.; Laversin, H.; Tomas, A.; Coddeville, P.; Fittschen, C.; El Dib, G.; Roth, E.; Chakir, A.

2014-12-01

Hydroxycarbonyl compounds are important secondary reaction products in the oxidation of Volatile Organic Compounds (VOCs) in the atmosphere. The atmospheric fate of these oxygenated VOCs is however poorly understood, especially the relevance of the photolytic pathway. In this work, a combined investigation of the photolysis and temperature-dependent OH radical reaction of 3-hydroxy-3-methyl-2-butanone (3H3M2B) is presented. A photolysis lifetime of about 4-5 days was estimated with a global quantum yield of 0.10. The OH reaction rate coefficient follows the Arrhenius trend (298-356 K) and could be modelled through the following expression: k3H3M2B(T) = (5.12 ± 0.07) × 10-12 exp(-563 ± 119/T) in cm3 molecule-1 s-1. A 3H3M2B atmospheric lifetime of 15 days towards the OH radical was evaluated. Our results showed that the photolysis pathway is the major degradation channel for 3H3M2B. Photolysis products were identified and quantified in the present work with a carbon balance of around 80% enabling a reaction mechanism to be proposed. The present work underlines the need for further studies on the atmospheric chemistry of oxygenated VOCs.

Electronic absorption spectroscopy of matrix-isolated polycyclic aromatic hydrocarbon cations. II. The phenanthrene cation (C14H10+) and its 1-methyl derivative

NASA Technical Reports Server (NTRS)

Salama, F.; Joblin, C.; Allamandola, L. J.

1994-01-01

The ultraviolet, visible, and near infrared absorption spectra of phenanthrene (C14H10), 1-methylphenanthrene [(CH3)C14H9], and their radical ions [C14H10+; (CH3)C14H9+], formed by vacuum-ultraviolet irradiation, were measured in neon matrices at 4.2 K. The associated vibronic band systems and their spectroscopic assignments are discussed. The oscillator strengths were calculated for the phenanthrene ion and found lower than the theoretical predictions. This study presents the first spectroscopic data for phenanthrene and its methyl derivative trapped in a neon matrix where the perturbation of the isolated species by its environment is minimum; a condition crucial to astrophysical applications.

Inhibition of SET Domain–Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis

PubMed Central

Sasaki, Kensuke; Nakashima, Ayumu; Irifuku, Taisuke; Yamada, Kyoko; Kokoroishi, Keiko; Ueno, Toshinori; Doi, Toshiki; Hida, Eisuke; Arihiro, Koji; Kohno, Nobuoki

2016-01-01

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain–containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent. PMID:26045091

In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level

NASA Astrophysics Data System (ADS)

Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M.; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

2017-10-01

ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

In silico probing and biological evaluation of SETDB1/ESET-targeted novel compounds that reduce tri-methylated histone H3K9 (H3K9me3) level.

PubMed

Park, Insun; Hwang, Yu Jin; Kim, TaeHun; Viswanath, Ambily Nath Indu; Londhe, Ashwini M; Jung, Seo Yun; Sim, Kyoung Mi; Min, Sun-Joon; Lee, Ji Eun; Seong, Jihye; Kim, Yun Kyung; No, Kyoung Tai; Ryu, Hoon; Pae, Ae Nim

2017-10-01

ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.

Human papillomavirus dysregulates the cellular apparatus controlling the methylation status of H3K27 in different human cancers to consistently alter gene expression regardless of tissue of origin

PubMed Central

Zhang, Ali; Barrett, John W.; Nichols, Anthony C.; Torchia, Joe; Mymryk, Joe S.

2017-01-01

High-risk human papillomaviruses (HPV) cause cancer at multiple distinct anatomical locations. Regardless of the tissue of origin, most HPV positive (HPV+) cancers show highly upregulated expression of the p16 product of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene. Paradoxically, HPV+ tumor cells require continuous expression of this tumor suppressor for survival. Thus, restoration of normal p16 regulation has potential therapeutic value against HPV induced cancers. Normally, p16 transcription is tightly controlled at the epigenetic level via polycomb repressive complex-mediated tri-methylation of histone 3 lysine 27 (H3K27me3). Although a mechanism by which HPV induces p16 has been proposed based on tissue culture models, it has not been extensively validated in human tumors. In this study, we used data from over 800 human cervical and head and neck tumors from The Cancer Genome Atlas (TCGA) to test this model. We determined the impact of HPV status on expression from the CDKN2A locus, the adjacent CDKN2B locus, and transcript levels of key epigenetic regulators of these loci. As expected, HPV+ tumors from both anatomical sites exhibited high levels of p16. Furthermore, HPV+ tumors expressed higher levels of KDM6A, which demethylates H3K27me3. CpG methylation of the CDKN2A locus was also consistently altered in HPV+ tumors. This data validates previous tissue culture studies and identifies remarkable similarities between the effects of HPV on gene expression and DNA methylation in both cervical and oral tumors in large human cohorts. Furthermore, these results support a model whereby HPV-mediated dysregulation of CDKN2A transcription requires KDM6A, a potentially druggable target. PMID:29069809

Self-assembly of Metallamacrocycles Employing a New Benzil-based Organometallic Bisplatinum(II) Acceptor.

PubMed

Roy, Bijan; Shanmugaraju, Sankarasekaran; Saha, Rupak; Mukherjee, Partha Sarathi

2015-01-01

A benzil-based semi-rigid dinuclear-organometallic acceptor 4,4'-bis[trans-Pt(PEt(3))(2)(NO(3))(ethynyl)]benzil (bisPt-NO(3)) containing a Pt-ethynyl functionality was synthesized in good yield and characterized by multinuclear NMR ((1)H, (31)P, and (13)C), electrospray ionization mass spectrometry (ESI-MS), and single-crystal X-ray diffraction analysis of the iodide analogue bisPt-I. The stoichiometric (1:1) combination of the acceptor bisPt-NO(3) separately with four different ditopic donors (L(1)-L(4); L(1) = 9-ethyl-3,6-di(1H-imidazol-1-yl)-9H-carbazole, L(2) = 1,4-bis((1H-imidazol-1-yl)methyl)benzene, L(3) = 1,3-bis((1H-imidazol-1-yl)methyl)benzene and L(4) = 9,10-bis((1H-imidazol-1-yl) methyl)anthracene) yielded four [2 + 2] self-assembled metallacycles M(1)-M(4) in quantitative yields, respectively. All these newly synthesized assemblies were characterized by various spectroscopic techniques (NMR, IR, ESI-MS) and their sizes/shapes were predicted through geometry optimization employing the PM6 semi-empirical method. The benzil moiety was introduced in the backbone of the acceptor bisPt-NO(3) due to the interesting structural feature of long carbonyl C-C bond (∼1.54 Å), which enabled us to probe the role of conformational flexibility on size and shapes of the resulting coordination ensembles.

8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors

PubMed Central

2016-01-01

We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay. PMID:26741168

A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation.

PubMed

Duncan, Christopher G; Barwick, Benjamin G; Jin, Genglin; Rago, Carlo; Kapoor-Vazirani, Priya; Powell, Doris R; Chi, Jen-Tsan; Bigner, Darell D; Vertino, Paula M; Yan, Hai

2012-12-01

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

Synthesis, biological evaluation and docking analysis of 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones as potential cyclooxygenase-2 (COX-2) inhibitors.

PubMed

Grover, Jagdeep; Kumar, Vivek; Sobhia, M Elizabeth; Jachak, Sanjay M

2014-10-01

As a part of our continued efforts to discover new COX inhibitors, a series of 3-methyl-1-phenylchromeno[4,3-c]pyrazol-4(1H)-ones were synthesized and evaluated for in vitro COX inhibitory potential. Within this series, seven compounds (3a-d, 3h, 3k and 3q) were identified as potential and selective COX-2 inhibitors (COX-2 IC50's in 1.79-4.35μM range; COX-2 selectivity index (SI)=6.8-16.7 range). Compound 3b emerged as most potent (COX-2 IC50=1.79μM; COX-1 IC50 >30μM) and selective COX-2 inhibitor (SI >16.7). Further, compound 3b displayed superior anti-inflammatory activity (59.86% inhibition of edema at 5h) in comparison to celecoxib (51.44% inhibition of edema at 5h) in carrageenan-induced rat paw edema assay. Structure-activity relationship studies suggested that N-phenyl ring substituted with p-CF3 substituent (3b, 3k and 3q) leads to more selective inhibition of COX-2. To corroborate obtained experimental biological data, molecular docking study was carried out which revealed that compound 3b showed stronger binding interaction with COX-2 as compared to COX-1. Copyright © 2014 Elsevier Ltd. All rights reserved.

Histone modifier gene mutations in peripheral T-cell lymphoma not otherwise specified.

PubMed

Ji, Meng-Meng; Huang, Yao-Hui; Huang, Jin-Yan; Wang, Zhao-Fu; Fu, Di; Liu, Han; Liu, Feng; Leboeuf, Christophe; Wang, Li; Ye, Jing; Lu, Yi-Ming; Janin, Anne; Cheng, Shu; Zhao, Wei-Li

2018-04-01

Due to heterogeneous morphological and immunophenotypic features, approximately 50% of peripheral T-cell lymphomas are unclassifiable and categorized as peripheral T-cell lymphomas, not otherwise specified. These conditions have an aggressive course and poor clinical outcome. Identification of actionable biomarkers is urgently needed to develop better therapeutic strategies. Epigenetic alterations play a crucial role in tumor progression. Histone modifications, particularly methylation and acetylation, are generally involved in chromatin state regulation. Here we screened the core set of genes related to histone methylation ( KMT2D , SETD2 , KMT2A , KDM6A ) and acetylation ( EP300 , CREBBP ) and identified 59 somatic mutations in 45 of 125 (36.0%) patients with peripheral T-cell lymphomas, not otherwise specified. Histone modifier gene mutations were associated with inferior progression-free survival time of the patients, irrespective of chemotherapy regimens, but an increased response to the histone deacetylase inhibitor chidamide. In vitro , chidamide significantly inhibited the growth of EP300-mutated T-lymphoma cells and KMT2D-mutated T-lymphoma cells when combined with the hypomethylating agent decitabine. Mechanistically, decitabine acted synergistically with chidamide to enhance the interaction of KMT2D with transcription factor PU.1, regulated H3K4me-associated signaling pathways, and sensitized T-lymphoma cells to chidamide. In a xenograft KMT2D-mutated T-lymphoma model, dual treatment with chidamide and decitabine significantly retarded tumor growth and induced cell apoptosis through modulation of the KMT2D/H3K4me axis. Our work thus contributes to the understanding of aberrant histone modification in peripheral T-cell lymphomas, not otherwise specified and the stratification of a biological subset that can benefit from epigenetic treatment. Copyright© 2018 Ferrata Storti Foundation.

Antidepressant-Like Effects of Acupuncture-Insights From DNA Methylation and Histone Modifications of Brain-Derived Neurotrophic Factor.

PubMed

Jiang, Huili; Zhang, Xuhui; Lu, Jun; Meng, Hong; Sun, Yang; Yang, Xinjing; Zhao, Bingcong; Bao, Tuya

2018-01-01

Sensitive and stable biomarkers that facilitate depression detection and monitor the antidepressant efficiency are currently unavailable. Thus, the objective is to investigate the potential of DNA methylation and histone modifications of brain-derived neurotrophic factor (BDNF) in monitoring severity and antidepressive effects of acupuncture. The depression rat model was imitated by social isolation and chronic unpredicted mild stress (CUMS). The expression of serum BDNF was detected by enzyme-linked immunosorbent assay (ELISA), the hippocampal BDNF, acetylation levels in histone H3 lysine 9 (acH3K9), and HDAC2 by Western blot, the hippocampal mRNA of BDNF by RT-polymerase chain reaction (PCR). The DNA methylation patterns of the promoter I of BDNF was detected by MS-PCR. We investigated that the expression of BDNF in serum and hippocampus were significantly downregulated compared with controls. The same trend was found in mRNA of BDNF. Notably, acupuncture reversed the downregulation of BDNF in serum and hippocampus and mRNA of BDNF compared with model group. Acupuncture reversed the CUMS-induced downregulation of hippocampal acH3K9. On the contrary, the CUMS-induced upregulation of hippocampal HDAC2 in model group was significantly reversed by acupuncture. Collectively, the antidepressant effect of acupuncture might be mediated by regulating the DNA methylation and histone modifications of BDNF, which may represent novel biomaker for detection of depression and monitoring severity and antidepressive effects.

DNA adenine methylation modulates pathogenicity of Klebsiella pneumoniae genotype K1.

PubMed

Fang, Chi-Tai; Yi, Wen-Ching; Shun, Chia-Tung; Tsai, Shih-Feng

2017-08-01

Klebsiella pneumoniae genotype K1 is a highly virulent pathogen that causes liver abscess and metastatic endophthalmitis/meningitis. Whether its pathogenicity is controlled by DNA adenine methylase (Dam), an epigenetic regulator of bacterial virulence gene expression, is yet unknown. We aimed to study the role of DNA adenine methylation in the pathogenicity of K. pneumoniae genotype K1. We identified the dam gene in the prototype tissue-invasive strain (NTUH-K2044) of K. pneumoniae genotype K1, using the strain's complete genome sequence in GenBank. We constructed a dam - mutant and compared it with the wild type, in terms of in vitro serum resistance and in vivo BALB/cByl mice inoculation. Loss of Dam activity in the mutant was verified by MboI restriction digestion of the genomic DNA and a 1000-fold increase in spontaneous mutation rate. The dam mutant lost at least 68% of serum resistance when compared with the wild type (survival ratio at 1 hour: 2.6 ± 0.4 vs. 8.2 ± 1.9; at 2 hours: 3.9 ± 1.6 vs. 17.4 ± 3.6; p values < 0.05). Likewise, virulence to mice decreased by 40-fold in an intraperitoneal injection model [lethal dose, 50% (LD 50 ): 2 × 10 3 colony-forming units (CFUs) vs. 5 × 10 1 CFUs] and by sixfold in a gastric ingestion model (LD 50 : 3 × 10 4 CFUs vs. 5 × 10 3 CFUs). Attenuation of the dam mutant was not attributable to its growth rate, which was similar to that of the wild type. Our results support the view that DNA adenine methylation plays an important role in modulating the pathogenicity of K. pneumoniae genotype K1. The incomplete attenuation indicates the existence of other regulatory factors. Copyright © 2015. Published by Elsevier B.V.

FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis

PubMed Central

Kato, Yasuko; Alavattam, Kris G.; Sin, Ho-Su; Meetei, Amom Ruhikanta; Pang, Qishen; Andreassen, Paul R.; Namekawa, Satoshi H.

2015-01-01

Fanconi anemia (FA) is a recessive X-linked and autosomal genetic disease associated with bone marrow failure and increased cancer, as well as severe germline defects such as hypogonadism and germ cell depletion. Although deficiencies in FA factors are commonly associated with germ cell defects, it remains unknown whether the FA pathway is involved in unique epigenetic events in germ cells. In this study, we generated Fancb mutant mice, the first mouse model of X-linked FA, and identified a novel function of the FA pathway in epigenetic regulation during mammalian gametogenesis. Fancb mutant mice were infertile and exhibited primordial germ cell (PGC) defects during embryogenesis. Further, Fancb mutation resulted in the reduction of undifferentiated spermatogonia in spermatogenesis, suggesting that FANCB regulates the maintenance of undifferentiated spermatogonia. Additionally, based on functional studies, we dissected the pathway in which FANCB functions during meiosis. The localization of FANCB on sex chromosomes is dependent on MDC1, a binding partner of H2AX phosphorylated at serine 139 (γH2AX), which initiates chromosome-wide silencing. Also, FANCB is required for FANCD2 localization during meiosis, suggesting that the role of FANCB in the activation of the FA pathway is common to both meiosis and somatic DNA damage responses. H3K9me2, a silent epigenetic mark, was decreased on sex chromosomes, whereas H3K9me3 was increased on sex chromosomes in Fancb mutant spermatocytes. Taken together, these results indicate that FANCB functions at critical stages of germ cell development and reveal a novel function of the FA pathway in the regulation of H3K9 methylation in the germline. PMID:26123487

Kinetic effects of sulfur oxidation on catalytic nitrile hydration: nitrile hydratase insights from bioinspired ruthenium(II) complexes.

PubMed

Kumar, Davinder; Nguyen, Tho N; Grapperhaus, Craig A

2014-12-01

Kinetic investigations inspired by the metalloenzyme nitrile hydratase were performed on a series of ruthenium(II) complexes to determine the effect of sulfur oxidation on catalytic nitrile hydration. The rate of benzonitrile hydration was quantified as a function of catalyst, nitrile, and water concentrations. Precatalysts L(n)RuPPh3 (n = 1-3; L(1) = 4,7-bis(2'-methyl-2'-mercapto-propyl)-1-thia-4,7-diazacyclononane; L(2) = 4-(2'-methyl-2'-sulfinatopropyl)-7-(2'-methyl-2'-mercapto-propyl)-1-thia-4,7-diazacyclononane; L(3) = 4-(2'-methyl-2'-sulfinatopropyl)-7-(2'-methyl-2'-sulfenato-propyl)-1-thia-4,7-diazacyclononane) were activated by substitution of triphenylphosphine with substrate in hot dimethylformamide solution. Rate measurements are consistent with a dynamic equilibrium between inactive aqua (L(n)Ru-OH2) and active nitrile (L(n)Ru-NCR) derivatives with K = 21 ± 1, 9 ± 0.9, and 23 ± 3 for L(1) to L(3), respectively. Subsequent hydration of the L(n)Ru-NCR intermediate yields the amide product with measured hydration rate constants (k's) of 0.37 ± 0.01, 0.82 ± 0.07, and 1.59 ± 0.12 M(-1) h(-1) for L(1) to L(3), respectively. Temperature dependent studies reveal that sulfur oxidation lowers the enthalpic barrier by 27 kJ/mol, but increases the entropic barrier by 65 J/(mol K). Density functional theory (DFT) calculations (B3LYP/LanL2DZ (Ru); 6-31G(d) (all other atoms)) support a nitrile bound catalytic cycle with lowering of the reaction barrier as a consequence of sulfur oxidation through enhanced nitrile binding and attack of the water nucleophile through a highly organized transition state.

Gas-phase kinetics study of reaction of OH radical with CH3NHNH2 by second-order multireference perturbation theory.

PubMed

Sun, Hongyan; Zhang, Peng; Law, Chung K

2012-05-31

The gas-phase kinetics of H-abstraction reactions of monomethylhydrazine (MMH) by OH radical was investigated by second-order multireference perturbation theory and two-transition-state kinetic model. It was found that the abstractions of the central and terminal amine H atoms by the OH radical proceed through the formation of two hydrogen bonded preactivated complexes with energies of 6.16 and 5.90 kcal mol(-1) lower than that of the reactants, whereas the abstraction of methyl H atom is direct. Due to the multireference characters of the transition states, the geometries and ro-vibrational frequencies of the reactant, transition states, reactant complexes, and product complexes were optimized by the multireference CASPT2/aug-cc-pVTZ method, and the energies of the stationary points of the potential energy surface were refined at the QCISD(T)/CBS level via extrapolation of the QCISD(T)/cc-pVTZ and QCISD(T)/cc-pVQZ energies. It was found that the abstraction reactions of the central and two terminal amine H atoms of MMH have the submerged energy barriers with energies of 2.95, 2.12, and 1.24 kcal mol(-1) lower than that that of the reactants respectively, and the abstraction of methyl H atom has a real energy barrier of 3.09 kcal mol(-1). Furthermore, four MMH radical-H(2)O complexes were found to connect with product channels and the corresponding transition states. Consequently, the rate coefficients of MMH + OH for the H-abstraction of the amine H atoms were determined on the basis of a two-transition-state model, with the total energy E and angular momentum J conserved between the two transition-state regions. In units of cm(3) molecule(-1) s(-1), the rate coefficient was found to be k(1) = 3.37 × 10(-16)T(1.295) exp(1126.17/T) for the abstraction of the central amine H to form the CH(3)N(•)NH(2) radical, k(2) = 2.34 × 10(-17)T(1.907) exp(1052.26/T) for the abstraction of the terminal amine H to form the trans-CH(3)NHN(•)H radical, k(3) = 7.41 × 10(-20)T(2.428) exp(1343.20/T) for the abstraction of the terminal amine H to form the cis-CH(3)NHN(•)H radical, and k(4) = 9.13 × 10(-21)T(2.964) exp(-114.09/T) for the abstraction of the methyl H atom to form the C(•)H(2)NHNH(2) radical, respectively. Assuming that the rate coefficients are additive, the total rate coefficient of these theoretical predictions quantitatively agrees with the measured rate constant at temperatures of 200-650 K, with no adjustable parameters.

Furosemide- and bumetanide-sensitive ion transport and volume control in primary astrocyte cultures from rat brain.

PubMed

Kimelberg, H K; Frangakis, M V

1985-12-30

K+ and Cl- transport using 42K+ and 36Cl- was studied in primary astrocyte cultures prepared from neonatal rat brains. A component of 42K+ uptake was sensitive to both furosemide and bumetanide with maximum inhibition being obtained at 1 and 0.01 mM concentrations of the inhibitors, respectively. Furosemide and bumetanide also markedly inhibited uptake of 36Cl-. 42K+ uptake in the presence of ouabain was also sensitive to the omission of medium Na+ and Cl-. These results suggest the existence of a K+ + Na+ + Cl- cotransport system in astrocyte cultures which in many cells has been shown to be involved in volume regulation. We studied volume changes using uptake of [14C]3-O- methyl-D-glucose ([14C]3-OMG), and also ion transport, in attached cells in response to exposure to hyper- or hypotonic medium. Exposure to medium made hypertonic with mannitol resulted in shrinkage of the [14C]3-OMG space of the cells, but did not affect 36Cl- content, expressed as nmol/mg protein. Exposure to hypotonic medium led to a marked increase in the [14C]3-OMG space, rapidly followed by a decrease towards control values. After the cells were then exposed to isotonic medium there was an immediate decrease followed by a slower increase in the [14C]3-OMG space. The increase in the [14C]3-OMG space was partially inhibited by 1 mM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

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

PubMed

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

2017-05-01

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

Changes in DNA methylation over the growing season differ between North Carolina farmworkers and non-farmworkers.

PubMed

Howard, Timothy D; Hsu, Fang-Chi; Chen, Haiying; Quandt, Sara A; Talton, Jennifer W; Summers, Phillip; Arcury, Thomas A

2016-10-01

The occupational risk to farmworkers, particularly chronic exposure to pesticides, is an acknowledged environmental and work-related health problem. Epigenetics has recently been shown to contribute to a number of complex diseases and traits, including measures of cognitive function and preclinical neurodegenerative disease. We sought to determine whether changes in DNA methylation existed between farmworker and non-farmworker populations and to identify the genes most likely involved in those changes. Eighty-three farmworkers and 60 non-farmworkers were selected from PACE4, a community-based, participatory research project comparing occupational exposures between immigrant Latino farmworker and non-farmworker manual workers. Measurements of DNA methylation were performed with the Infinium HumanMethylation450 BeadChip, at the beginning and end of the 2012 growing season. Bonferroni adjustment was used to identify significant findings (p = 1.03 × 10(-7), based on 485,000 tested methylation sites), although less stringent criteria (i.e., p ≤ 1 × 10(-6)) were used to identify sites of interest. Expression quantitative trait locus (eQTL) databases were used to help identify the most likely functional genes for each associated methylation site. Methylation at 36 CpG sites, located in or near 72 genes, differed between the two groups (p ≤ 1 × 10(-6)). The difference between the two groups was generally due to an increase in methylation in the farmworkers and a slight decrease in methylation in the non-farmworkers. Enrichment was observed in several biological pathways, including those involved in the immune response, as well as growth hormone signaling, role of BRCA1 in DNA damage response, p70S6K signaling, and PI3K signaling in B lymphocytes. We identified considerable changes in DNA methylation at 36 CpG sites over the growing season that differed between farmworkers and non-farmworkers. Dominant pathways included immune-related (HLA) processes, as well as a number of diverse biological systems. Further studies are necessary to determine which exposures or behaviors are responsible for the observed changes, and whether these changes eventually lead to disease-related phenotypes in this population.

Auxin-induced ethylene triggers abscisic acid biosynthesis and growth inhibition.

PubMed

Hansen, H; Grossmann, K

2000-11-01

The growth-inhibiting effects of indole-3-acetic acid (IAA) at high concentration and the synthetic auxins 7-chloro-3-methyl-8-quinolinecarboxylic acid (quinmerac), 2-methoxy-3,6-dichlorobenzoic acid (dicamba), 4-amino-3,6, 6-trichloropicolinic acid (picloram), and naphthalene acetic acid, were investigated in cleavers (Galium aparine). When plants were root treated with 0.5 mM IAA, shoot epinasty and inhibition of root and shoot growth developed during 24 h. Concomitantly, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, and ACC and ethylene production were transiently stimulated in the shoot tissue within 2 h, followed by increases in immunoreactive (+)-abscisic acid (ABA) and its precursor xanthoxal (xanthoxin) after 5 h. After 24 h of treatment, levels of xanthoxal and ABA were elevated up to 2- and 24-fold, relative to control, respectively. In plants treated with IAA, 7-chloro-3-methyl-8-quinolinecarboxylic acid, naphthalene acetic acid, 2-methoxy-3,6-dichlorobenzoic acid, and 4-amino-3,6,6-trichloropicolinic acid, levels of ethylene, ACC, and ABA increased in close correlation with inhibition of shoot growth. Aminoethoxyvinyl-glycine and cobalt ions, which inhibit ethylene synthesis, decreased ABA accumulation and growth inhibition, whereas the ethylene-releasing ethephon promoted ABA levels and growth inhibition. In accordance, tomato mutants defective in ethylene perception (never ripe) did not produce the xanthoxal and ABA increases and growth inhibition induced by auxins in wild-type plants. This suggests that auxin-stimulated ethylene triggers ABA accumulation and the consequent growth inhibition. Reduced catabolism most probably did not contribute to ABA increase, as indicated by immunoanalyses of ABA degradation and conjugation products in shoot tissue and by pulse experiments with [(3)H]-ABA in cell suspensions of G. aparine. In contrast, studies using inhibitors of ABA biosynthesis (fluridone, naproxen, and tungstate), ABA-deficient tomato mutants (notabilis, flacca, and sitiens), and quantification of xanthophylls indicate that ABA biosynthesis is influenced, probably through stimulated cleavage of xanthophylls to xanthoxal in shoot tissue.

Polymethylated [Fe(η6-arene)2]2+ dications: methyl-group rearrangements and application of the EINS mechanism.

PubMed

Štíbr, Bohumil; Bakardjiev, Mario; Hájková, Zuzana; Holub, Josef; Padělková, Zdenka; Růžička, Aleš; Kennedy, John D

2011-06-14

Reactions between the methylated arenes ArMe(n) [where ArMe(n) = C(6)Me(n)H((6-n)), and n = 1-6] and FeCl(2) in heptane at 90 °C in the presence of anhydrous AlCl(3) give, for the arenes with n = 1-5, extensive isomerisations and disproportionations involving the methyl groups on the arene rings, and the formation of mixtures of [Fe(ArMe(n))(2)](2+) dications that defy separation into pure species. GC-MS studies of AlCl(3)/mesitylene and AlCl(3)/durene reactions in the absence of FeCl(2) (90 °C, 2 h) allow quantitative assessments of the rearrangements, and the EINS mechanism (electrophile-induced nucleophilic substitution) is applied to rationalise the phenomena. By contrast, ArMe(n) / FeCl(2) /AlCl(3) reactions in heptane for 24-36 h at room-temperature proceed with no rearrangements, allowing the synthesis of the complete series of pure [Fe(ArMen)](2+) cations in yields of 48-71%. The pure compounds are characterised by (1)H NMR spectroscopy and electrospray-ionization mass-spectrometry (ESI-MS), and the structures of [Fe(m-xylene)(2)][PF(6)](2) and [Fe(durene)(2)][PF(6)](2) are established by single-crystal X-ray diffraction analyses.

Global analysis of H3K27me3 as an epigenetic marker in prostate cancer progression.

PubMed

Ngollo, Marjolaine; Lebert, Andre; Daures, Marine; Judes, Gaelle; Rifai, Khaldoun; Dubois, Lucas; Kemeny, Jean-Louis; Penault-Llorca, Frederique; Bignon, Yves-Jean; Guy, Laurent; Bernard-Gallon, Dominique

2017-04-12

H3K27me3 histone marks shape the inhibition of gene transcription. In prostate cancer, the deregulation of H3K27me3 marks might play a role in prostate tumor progression. We investigated genome-wide H3K27me3 histone methylation profile using chromatin immunoprecipitation (ChIP) and 2X400K promoter microarrays to identify differentially-enriched regions in biopsy samples from prostate cancer patients. H3K27me3 marks were assessed in 34 prostate tumors: 11 with Gleason score > 7 (GS > 7), 10 with Gleason score ≤ 7 (GS ≤ 7), and 13 morphologically normal prostate samples. Here, H3K27me3 profiling identified an average of 386 enriched-genes on promoter regions in healthy control group versus 545 genes in GS ≤ 7 and 748 genes in GS > 7 group. We then ran a factorial discriminant analysis (FDA) and compared the enriched genes in prostate-tumor biopsies and normal biopsies using ANOVA to identify significantly differentially-enriched genes. The analysis identified ALG5, EXOSC8, CBX1, GRID2, GRIN3B, ING3, MYO1D, NPHP3-AS1, MSH6, FBXO11, SND1, SPATS2, TENM4 and TRA2A genes. These genes are possibly associated with prostate cancer. Notably, the H3K27me3 histone mark emerged as a novel regulatory mechanism in poor-prognosis prostate cancer. Our findings point to epigenetic mark H3K27me3 as an important event in prostate carcinogenesis and progression. The results reported here provide new molecular insights into the pathogenesis of prostate cancer.

Influence of the Reaction Temperature on the Nature of the Active and Deactivating Species During Methanol-to-Olefins Conversion over H-SAPO-34

PubMed Central

2017-01-01

The selectivity toward lower olefins during the methanol-to-olefins conversion over H-SAPO-34 at reaction temperatures between 573 and 773 K has been studied with a combination of operando UV–vis diffuse reflectance spectroscopy and online gas chromatography. It was found that the selectivity toward propylene increases in the temperature range of 573–623 K, while it decreases in the temperature range of 623–773 K. The high degree of incorporation of olefins, mainly propylene, into the hydrocarbon pool affects the product selectivity at lower reaction temperatures. The nature and dynamics of the active and deactivating hydrocarbon species with increasing reaction temperature were revealed by a non-negative matrix factorization of the time-resolved operando UV–vis diffuse reflectance spectra. The active hydrocarbon pool species consist of mainly highly methylated benzene carbocations at temperatures between 573 and 598 K, of both highly methylated benzene carbocations and methylated naphthalene carbocations at 623 K, and of only methylated naphthalene carbocations at temperatures between 673 and 773 K. The operando spectroscopy results suggest that the nature of the active species also influences the olefin selectivity. In fact, monoenylic and highly methylated benzene carbocations are more selective to the formation of propylene, whereas the formation of the group of low methylated benzene carbocations and methylated naphthalene carbocations at higher reaction temperatures (i.e., 673 and 773 K) favors the formation of ethylene. At reaction temperatures between 573 and 623 K, catalyst deactivation is caused by the gradual filling of the micropores with methylated naphthalene carbocations, while between 623 and 773 K the formation of neutral poly aromatics and phenanthrene/anthracene carbocations are mainly responsible for catalyst deactivation, their respective contribution increasing with increasing reaction temperature. Methanol pulse experiments at different temperatures demonstrate the dynamics between methylated benzene and methylated naphthalene carbocations. It was found that methylated naphthalene carbocations species are deactivating and block the micropores at low reaction temperatures, while acting as the active species at higher reaction temperatures, although they give rise to the formation of extended hydrocarbon deposits. PMID:28824823

EZH2 mutations and promoter hypermethylation in childhood acute lymphoblastic leukemia.

PubMed

Schäfer, Vivien; Ernst, Jana; Rinke, Jenny; Winkelmann, Nils; Beck, James F; Hochhaus, Andreas; Gruhn, Bernd; Ernst, Thomas

2016-07-01

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and young adults. The polycomb repressive complex 2 (PRC2) has been identified as one of the most frequently mutated epigenetic protein complexes in hematologic cancers. PRC2 acts as an epigenetic repressor through histone H3 lysine 27 trimethylation (H3K27me3), catalyzed by the histone methyltransferase enhancer of zeste homolog 2 protein (EZH2). To study the prevalence and clinical impact of PRC2 aberrations in an unselected childhood ALL cohort (n = 152), we performed PRC2 mutational screenings by Sanger sequencing and promoter methylation analyses by quantitative pyrosequencing for the three PRC2 core component genes EZH2, suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). Targeted deep next-generation sequencing of 30 frequently mutated genes in leukemia was performed to search for cooperating mutations in patients harboring PRC2 aberrations. Finally, the functional consequence of EZH2 promoter hypermethylation on H3K27me3 was studied by Western blot analyses of primary cells. Loss-of-function EZH2 mutations were detected in 2/152 (1.3 %) patients with common-ALL and early T-cell precursor (ETP)-ALL, respectively. In one patient, targeted deep sequencing identified cooperating mutations in ASXL1 and TET2. EZH2 promoter hypermethylation was found in one patient with ETP-ALL which led to reduced H3K27me3. In comparison with healthy children, the EZH2 promoter was significantly higher methylated in T-ALL patients. No mutations or promoter methylation changes were identified for SUZ12 or EED genes, respectively. Although PRC2 aberrations seem to be rare in childhood ALL, our findings indicate that EZH2 aberrations might contribute to the disease in specific cases. Hereby, EZH2 promoter hypermethylation might have functionally similar consequences as loss-of-function mutations.

Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia.

PubMed

Adal, Ayelign M; Sarker, Lukman S; Lemke, Ashley D; Mahmoud, Soheil S

2017-04-01

A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17 µM and 2.01 s -1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k cat /K m = 0.56 µM -1 s -1 ) than NPP (k cat /K m = 0.044 µM -1 s -1 ). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8-72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.

Induction of Maltose Release by Light in the Endosymbiont Chlorella variabilis of Paramecium bursaria.

PubMed

Shibata, Aika; Takahashi, Fumio; Kasahara, Masahiro; Imamura, Nobutaka

2016-11-01

The endosymbiotic green algae of Paramecium bursaria are known to release a photosynthate to the host cells. The endosymbiont Chlorella variabilis F36-ZK isolated in Japan releases maltose under acidic conditions, and such release requires both light and low pH. However, whether photosynthate release is due to light sensing by photoreceptors or is merely a consequence of active photosynthesis is unclear. Herein, we studied the effect of light on maltose release from C. variabilis F36-ZK; we measured maltose release using a combination of 1-phenyl-3-methyl-5-pyrazolone derivative and 14 C-tracer methods. Blue (450nm) or red (around 600nm) light was most effective to stimulate maltose release. This suggests that the photosynthetic pathway probably participates in maltose release, because the effective wavelength corresponds to the absorption spectrum of chlorophyll. Furthermore, maltose release was slightly affected by addition of a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but was abolished by another inhibitor of photosynthesis, 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone, suggesting that electron flow through photosystem I may be more involved in maltose release. Interestingly, starving F36-ZK cells cultured under prolonged dark conditions did not release maltose but retained their photosynthetic capacity. Our results thus show that maltose release is regulated by light and cellular conditions in endosymbiotic Chlorella. Copyright © 2016. Published by Elsevier GmbH.

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

C-Methylated Flavonoid Glycosides from Pentarhizidium orientale Rhizomes and Their Inhibitory Effects on the H1N1 Influenza Virus.

PubMed

Huh, Jungmoo; Ha, Thi Kim Quy; Kang, Kyo Bin; Kim, Ki Hyun; Oh, Won Keun; Kim, Jinwoong; Sung, Sang Hyun

2017-10-27

Thirteen C-methylated flavonoid glycosides (1-13), along with 15 previously known flavonoids (14-28), were isolated from rhizomes of Pentarhizidium orientale. Among these compounds, matteuorienates D-K (1-8) were obtained as analogues of matteuorienates A-C (14-16), which contain a characteristic 3-hydroxy-3-methylglutaryl (HMG) moiety. The structures of 1-13 were characterized by spectroscopic analysis and chemical derivatization. The isolates were evaluated for their antiviral activities against influenza virus (H1N1), with compounds 21, 22, 23, 25, and 26 showing inhibitory effects (IC 50 of 23.9-30.3 μM) against neuraminidases.

Chromodomains direct integration of retrotransposons to heterochromatin

PubMed Central

Gao, Xiang; Hou, Yi; Ebina, Hirotaka; Levin, Henry L.; Voytas, Daniel F.

2008-01-01

The enrichment of mobile genetic elements in heterochromatin may be due, in part, to targeted integration. The chromoviruses are Ty3/gypsy retrotransposons with chromodomains at their integrase C termini. Chromodomains are logical determinants for targeting to heterochromatin, because the chromodomain of heterochromatin protein 1 (HP1) typically recognizes histone H3 K9 methylation, an epigenetic mark characteristic of heterochromatin. We describe three groups of chromoviruses based on amino acid sequence relationships of their integrase C termini. Genome sequence analysis indicates that representative chromoviruses from each group are enriched in gene-poor regions of the genome relative to other retrotransposons, and when fused to fluorescent marker proteins, the chromodomains target proteins to specific subnuclear foci coincident with heterochromatin. The chromodomain of the fungal element, MAGGY, interacts with histone H3 dimethyl- and trimethyl-K9, and when the MAGGY chromodomain is fused to integrase of the Schizosaccharomyces pombe Tf1 retrotransposon, new Tf1 insertions are directed to sites of H3 K9 methylation. Repetitive sequences such as transposable elements trigger the RNAi pathway resulting in their epigenetic modification. Our results suggest a dynamic interplay between retrotransposons and heterochromatin, wherein mobile elements recognize heterochromatin at the time of integration and then perpetuate the heterochromatic mark by triggering epigenetic modification. PMID:18256242

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

PubMed Central

Majumder, Syamantak; Thieme, Karina; Batchu, Sri N.; Alghamdi, Tamadher A.; Bowskill, Bridgit B.; Kabir, M. Golam; Liu, Youan; Advani, Suzanne L.; White, Kathryn E.; Geldenhuys, Laurette; Tennankore, Karthik K.; Poyah, Penelope; Siddiqi, Ferhan S.

2017-01-01

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain–containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways. PMID:29227285

The use of a selective saturation pulse to suppress t1 noise in two-dimensional (1)H fast magic angle spinning solid-state NMR spectroscopy.

PubMed

Robertson, Aiden J; Pandey, Manoj Kumar; Marsh, Andrew; Nishiyama, Yusuke; Brown, Steven P

2015-11-01

A selective saturation pulse at fast magic angle spinning (MAS) frequencies (60+kHz) suppresses t1 noise in the indirect dimension of two-dimensional (1)H MAS NMR spectra. The method is applied to a synthetic nucleoside with an intense methyl (1)H signal due to triisopropylsilyl (TIPS) protecting groups. Enhanced performance in terms of suppressing the methyl signal while minimising the loss of signal intensity of nearby resonances of interest relies on reducing spin diffusion--this is quantified by comparing two-dimensional (1)H NOESY-like spin diffusion spectra recorded at 30-70 kHz MAS. For a saturation pulse centred at the methyl resonance, the effect of changing the nutation frequency at different MAS frequencies as well as the effect of changing the pulse duration is investigated. By applying a pulse of duration 30 ms and nutation frequency 725 Hz at 70 kHz MAS, a good compromise of significant suppression of the methyl resonance combined with the signal intensity of resonances greater than 5 ppm away from the methyl resonance being largely unaffected is achieved. The effectiveness of using a selective saturation pulse is demonstrated for both homonuclear (1)H-(1)H double quantum (DQ)/single quantum (SQ) MAS and (14)N-(1)H heteronuclear multiple quantum coherence (HMQC) two-dimensional solid-state NMR experiments. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

PubMed Central

Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas

2013-01-01

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

In situ tetrazole templated chair-like decanuclear azido-cobalt(II) SMM containing both tetra- and octa-hedral Co(II) ions.

PubMed

Zhang, Yuan-Zhu; Gao, Song; Sato, Osamu

2015-01-14

An azido-bridged chair-like decanuclear cluster: [Co(II)10(bzp)8(Metz)2(N3)18]·4MeOH·3H2O (1, bzp = 2-benzoylpyridine and HMetz = 5-methyl-1H-tetrazole) was prepared with in situ tetrazolate anions as templates in a sealed system. 1 containing both octahedral and tetrahedral Co(II) ions exhibited slow relaxation of magnetization with an effective barrier of 26 K under an applied dc field of 1 kOe.

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwiatkowska, Aleksandra, E-mail: A.Kwiatkows@gmail.com; Zebrowski, Jacek; Oklejewicz, Bernadetta

2014-05-02

Highlights: • A decrease in proliferation rate during long-term cultivation of Arabidopsis cells. • Age-dependent increase in senescence-associated gene expression in Arabidopsis cells. • Age-related increase in DNA methylation, H3K9me2, and H3K27me3 in Arabidopsis cells. • High potential of photosynthetic efficiency of long-term cultured Arabidopsis cells. - Abstract: Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic andmore » physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.« less

Tautomerism and isomerism in some antitrichinellosis active benzimidazoles: Morphological study in polarized light, quantum chemical computations

NASA Astrophysics Data System (ADS)

Anichina, Kameliya; Mavrova, Anelia; Yancheva, Denitsa; Tsenov, Jordan; Dimitrov, Rasho

2017-12-01

The morphology of the crystal structure of some antitrichinellosis active benzimidazole derivatives including (1H-benzimidazol-2-ylthio)acetic acids, [1,3]thiazolo[3,2-a]benzimidazol-3(2H)-ones, 1H-benzimidazol-2-ylthioacetylpiperazines and starting 2-mercapto benzimidazoles, was studied by the use of Polarized Light Microscopy (PLM). Characterization of the crystal phase was complimented by Differential scanning calorimetry analysis (DSC) and spectroscopic data. DFT computations were performed in order to investigate the prototropic tautomerism and the geometry of the molecule of the synthesized compounds. One distinct type of crystal structure for each one of 5 or 6-methyl-(1H-benzimidazol-2-ylthio)acetic acid 6 was observed by PLM - dendritic and needle-shaped formations. Compound 14, containing a methyl substituent in the benzimidazole ring crystallized also into two phases; while for the unsubstituted compound 13 a separation of phases does not take place. The influence of the both solvents - chloroform and ethanol on the phase separation and the formation of the crystalline structure of compound 14 was investigated. The morphological study showed that the cyclization of 6 in the presence of acetic anhydride in pyridine medium led to a mixture of 6-methyl-[1,3]tiazolo[3,2-a]benzimidazol-3(2H)-one (10a) and 7-methyl-[1,3]thiazolo[3,2-a]-benzimidazole-3(2H)-one (10b), which crystallized in the form of fibrils and spherulites respectively. It was found that a difference in the crystal structures of substituted and unsubstituted benzimidazol-2-thiones, respectively benzimidazol-2-thiol derivatives exists, which may be due not only to the thiol-thione tautomerism but to the prototropic properties of the hydrogen atom in first position of the ring. The calculation results indicated that the thione form is more stable than the thiol tautomer by 51-55 kJ mol-1. But at the same time ΔG for the two thiol tautomers is below 0.5 kJ mol-1. In solid phase the 5(6)-substituted-1H-benzimidazol-2-thiols crystallized in two different crystal structures while the unsubstituted 1H-benzimidazol-2-thiol possess one type of crystal structure.

H3K27me3 dynamics dictate evolving uterine states in pregnancy and parturition

PubMed Central

Nancy, Patrice; Siewiera, Johan; Tagliani, Elisa; Osokine, Ivan; Manandhar, Priyanka; Clementi, Caterina

2017-01-01

Uncovering the causes of pregnancy complications such as preterm labor requires greater insight into how the uterus remains in a noncontractile state until term and then surmounts this state to enter labor. Here, we show that dynamic generation and erasure of the repressive histone modification tri-methyl histone H3 lysine 27 (H3K27me3) in decidual stromal cells dictate both elements of pregnancy success in mice. In early gestation, H3K27me3-induced transcriptional silencing of select gene targets ensured uterine quiescence by preventing the decidua from expressing parturition-inducing hormone receptors, manifesting type 1 immunity, and most unexpectedly, generating myofibroblasts and associated wound-healing responses. In late gestation, genome-wide H3K27 demethylation allowed for target gene upregulation, decidual activation, and labor entry. Pharmacological inhibition of H3K27 demethylation in late gestation not only prevented term parturition, but also inhibited delivery while maintaining pup viability in a noninflammatory model of preterm parturition. Immunofluorescence analysis of human specimens suggested that similar regulatory events might occur in the human decidua. Together, these results reveal the centrality of regulated gene silencing in the uterine adaptation to pregnancy and suggest new areas in the study and treatment of pregnancy disorders. PMID:29202469

Epigenetic Regulation of Werner Syndrome Gene in Age-Related Cataract

PubMed Central

Guan, Huaijin

2015-01-01

Purpose. To examine the promoter methylation and histone modification of WRN (Werner syndrome gene), a DNA repair gene, and their relationship with the gene expression in age-related cataract (ARC) lens. Methods. We collected the lenses after cataract surgery from 117ARC patients and 39 age-matched non-ARC. WRN expression, DNA methylation and histone modification around the CpG island were assessed. The methylation status of Human-lens-epithelium cell (HLEB-3) was chemically altered to observe the relationship between methylation and expression of WRN. Results. The WRN expression was significantly decreased in the ARC anterior lens capsules comparing with the control. The CpG island of WRN promoter in the ARC anterior lens capsules displayed hypermethylation comparing with the controls. The WRN promoter was almost fully methylated in the cortex of ARC and control lens. Acetylated H3 was lower while methylated H3-K9 was higher in ARC anterior lens capsules than that of the controls. The expression of WRN in HLEB-3 increased after demethylation of the cells. Conclusions. A hypermethylation in WRN promoter and altered histone modification in anterior lens capsules might contribute to the ARC mechanism. The data suggest an association of altered DNA repair capability in lens with ARC pathogenesis. PMID:26509079

Hydrolysis of tert-butyl formate: Kinetics, products, and implications for the environmental impact of methyl tert-butyl ether

USGS Publications Warehouse

Church, Clinton D.; Pankow, James F.; Tratnyek, Paul G.

1999-01-01

Asessing the environmental fate of methyl tert-butyl ether (MTBE) has become a subject of renewed interest because of the large quantities of this compound that are being used as an oxygenated additive in gasoline. Various studies on the fate of MTBE have shown that it can be degraded to tert-butyl formate (TBF), particularly in the atmosphere. Although it is generally recognized that TBF is subject to hydrolysis, the kinetics and products of this reaction under environmentally relevant conditions have not been described previously. In this study, we determined the kinetics of TBF hydrolysis as a function of pH and temperature. Over the pH range of 5 to 7, the neutral hydrolysis pathway predominates, with kN = (1.0 ± 0.2) × 10−6/s. Outside this range, strong pH effects were observed because of acidic and basic hydrolyses, from which we determined that kA = (2.7 ± 0.5) × 10−3/(M·s) and kB = 1.7 ± 0.3/(M·s). Buffered and unbuffered systems gave the same hydrolysis rates for a given pH, indicating that buffer catalysis was not significant under the conditions tested. The activation energies corresponding to kN, kA, and kBwere determined to be 78 ± 5, 59 ± 4, and 88 ±11 kJ/mol, respectively. In all experiments, tert-butyl alcohol was found at concentrations corresponding to stoichiometric formation from TBF. Based on our kinetics data, the expected half-life for hydrolysis of TBF at pH = 2 and 4°C (as per some standard preservation protocols for water sampling) is 6 h. At neutral pH and 22°C, the estimated half-life is 5 d, and at pH = 11 and 22°C, the value is only 8 min.

Epigenetic silencing of the DNA mismatch repair gene, MLH1, induced by hypoxic stress in a pathway dependent on the histone demethylase, LSD1

PubMed Central

Lu, Yuhong; Wajapeyee, Narendra; Turker, Mitchell S.; Glazer, Peter M.

2014-01-01

SUMMARY Silencing of the MLH1 gene is frequently seen in sporadic cancers. We report that hypoxia causes decreased H3K4 methylation at the MLH1 promoter via the H3K4 demethylases, LSD1 and PLU-1, and promotes long-term silencing of the promoter in a pathway that requires LSD1. Knockdown of LSD1 or its co-repressor, CoREST, also prevents the re-silencing (and cytosine DNA methylation) of the endogenous MLH1 promoter in RKO colon cancer cells following transient reactivation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC). The results demonstrate that hypoxia is a critical driving force for silencing of MLH1 through chromatin modification and indicate that the LSD1/CoREST complex is essential for MLH1 silencing. PMID:25043185

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

Repression of Middle Sporulation Genes in Saccharomyces cerevisiae by the Sum1-Rfm1-Hst1 Complex Is Maintained by Set1 and H3K4 Methylation

PubMed Central

Jaiswal, Deepika; Jezek, Meagan; Quijote, Jeremiah; Lum, Joanna; Choi, Grace; Kulkarni, Rushmie; Park, DoHwan; Green, Erin M.

2017-01-01

The conserved yeast histone methyltransferase Set1 targets H3 lysine 4 (H3K4) for mono, di, and trimethylation and is linked to active transcription due to the euchromatic distribution of these methyl marks and the recruitment of Set1 during transcription. However, loss of Set1 results in increased expression of multiple classes of genes, including genes adjacent to telomeres and middle sporulation genes, which are repressed under normal growth conditions because they function in meiotic progression and spore formation. The mechanisms underlying Set1-mediated gene repression are varied, and still unclear in some cases, although repression has been linked to both direct and indirect action of Set1, associated with noncoding transcription, and is often dependent on the H3K4me2 mark. We show that Set1, and particularly the H3K4me2 mark, are implicated in repression of a subset of middle sporulation genes during vegetative growth. In the absence of Set1, there is loss of the DNA-binding transcriptional regulator Sum1 and the associated histone deacetylase Hst1 from chromatin in a locus-specific manner. This is linked to increased H4K5ac at these loci and aberrant middle gene expression. These data indicate that, in addition to DNA sequence, histone modification status also contributes to proper localization of Sum1. Our results also show that the role for Set1 in middle gene expression control diverges as cells receive signals to undergo meiosis. Overall, this work dissects an unexplored role for Set1 in gene-specific repression, and provides important insights into a new mechanism associated with the control of gene expression linked to meiotic differentiation. PMID:29066473

Design, synthesis and anticonvulsant activity of some new 6,8-halo-substituted-2h-[1,2,4]triazino[5,6-b]indole-3(5h)-one/-thione and 6,8-halo-substituted 5-methyl-2h-[1,2,4]triazino[5,6-b]indol-3(5h)-one/-thione

PubMed Central

Kumar, Rajeev; Singh, Tejendra; Singh, Hariram; Jain, Sandeep; Roy, R. K.

2014-01-01

A new series of 6,8-halo-substituted-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-one/-thione and 6,8-halo-substituted 5-methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one/-thione (5a-5l) were designed and synthesized keeping in view of the structural requirement of pharmacophore. The above compounds were characterized by thin layer chromatography and spectral analysis. Anticonvulsant activity of the synthesized compounds was evaluated by the maximal electroshock (MES) test. Neurotoxicity and CNS depressant effects were evaluated by the rotarod motor impairment and Porsolt’s force swim tests, respectively. A computational study was carried out, for calculation of pharmacophore pattern, prediction of pharmacokinetic properties and toxicity properties. The above study revealed that the compounds 8-chloro-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5e), 6,8-dibromo-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5i) and 6,8-dibromo-5-methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5k) possess excellent anticonvulsant activity in the series with little CNS depressant effect and no neurotoxicity as compared to standard drugs phenytoin and carbamazepine. PMID:26417257

On the Structure of Some Pentaerythritol-Related Molecular Crystals with Pseudotetrahedral Molecular Structure. Ordered Structure of 2,2-Bis(bromomethyl)-1,3-propanediol and Orientationally Disordered Structure of 2-Chloromethyl-2-methyl-1,3-dichloropropane; 35Cl NQR

NASA Astrophysics Data System (ADS)

Dou, Shi-qi; Fuess, Hartmut; Strauß, Roman; Weiss, Alarich

1996-06-01

C(CH2Cl)3(CH3), melting point Tm = 291.3 K, shows in the DTA/DSC experiment a phase tran-sition from an orientationally disordered plastic phase I into an ordered phase II at TI ⃗ II = 235.9 K. From 77 K up to TII ⃗ I the 35Cl NQR spectrum is a triplet with (ν in MHz, T=77 K) ν1 = 34.213, ν2 = 34.183, ν3 = 33.786. The 35Cl NQR fade-out temperature Tf = 247 K coincides with TII ⃗ I found from the DSC experiment. The plastic phase of 2-chloromethyl-2-methyl-l,3-propanediol is cubic bcc, Im3m, Z = 2 and the lattice constant increases linearly 755 pm at 235 K to 768 pm at 283 K. In the heating cycle we found (ΔH in kJ/mol): ΔHII ⃗ I = 12.0, ΔSm/R = 1.0. The compound belongs to the group of plastic molecules formed by ellipsoid like distorted tetrahedra and is derived by substitution from methane. The crystal structure of 2,2-bis(bromomethyl)-1,3-propanediol was determined. DTA-DSC show that an orientationally disordered plastic phase does not exist. At room temperature C(CH2Br)2(CH2OH)2 crystallizes monoclinic, space group Cc, Z = 4, a = 628.2 pm, 6 = 2015.5 pm, c = 659.6 pm, β = 94.27°. The molecules interact by intermolecular hydrogen bonds between the OH-groups and by van der Waals forces Br…Br.

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation.

PubMed

Petell, Christopher J; Alabdi, Lama; He, Ming; San Miguel, Phillip; Rose, Richard; Gowher, Humaira

2016-09-19

Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation

PubMed Central

Petell, Christopher J.; Alabdi, Lama; He, Ming; San Miguel, Phillip; Rose, Richard; Gowher, Humaira

2016-01-01

Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites. PMID:27179026

Crystal structure of K0.75[Fe(II) 3.75Fe(III) 1.25(HPO3)6]·0.5H2O, an open-framework iron phosphite with mixed-valent Fe(II)/Fe(III) ions.

PubMed

Larrea, Edurne S; Mesa, José Luis; Legarra, Estibaliz; Aguayo, Andrés Tomás; Arriortua, Maria Isabel

2016-01-01

Single crystals of the title compound, potassium hexa-phosphito-penta-ferrate(II,III) hemihydrate, K0.75[Fe(II) 3.75Fe(III) 1.25(HPO3)6]·0.5H2O, were grown under mild hydro-thermal conditions. The crystal structure is isotypic with Li1.43[Fe(II) 4.43Fe(III) 0.57(HPO3)6]·1.5H2O and (NH4)2[Fe(II) 5(HPO3)6] and exhibits a [Fe(II) 3.75Fe(III) 1.25(HPO3)6](0.75-) open framework with disordered K(+) (occupancy 3/4) as counter-cations. The anionic framework is based on (001) sheets of two [FeO6] octa-hedra (one with point group symmetry 3.. and one with point group symmetry .2.) linked along [001] through [HPO3](2-) oxoanions. Each sheet is constructed from 12-membered rings of edge-sharing [FeO6] octa-hedra, giving rise to channels with a radius of ca 3.1 Å where the K(+) cations and likewise disordered water mol-ecules (occupancy 1/4) are located. O⋯O contacts between the water mol-ecule and framework O atoms of 2.864 (5) Å indicate hydrogen-bonding inter-actions of medium strength. The infrared spectrum of the compound shows vibrational bands typical for phosphite and water groups. The Mössbauer spectrum is in accordance with the presence of Fe(II) and Fe(III) ions.

Conformation of methylated amino acids: structure of 3,4-dimethoxy-alpha-methyl-DL-phenylalanine sesquihydrate.

PubMed

Srikrishnan, T; Ravichandran, V; Chacko, K K

1988-05-15

C12H17NO4.1.5H2O, Mr = 266.3, triclinic, P1, a = 5.872 (1), b = 11.437 (2), c = 20.434 (1) A, alpha = 95.74 (1), beta = 96.91 (1), gamma = 89.18 (1) degrees, V = 1355.5 A3, Z = 4, Dm = 1.29, D chi = 1.305 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 8.3 cm-1, F(000) = 572, T = 294 K, R = 0.038 for 4006 reflections, I greater than or equal to 3 sigma(I). Both the molecules A and B in the asymmetric unit exist as zwitterions. With respect to the D enantiomer, the torsion angles psi 1 and psi 2 are +47.2 and -134.4 degrees in molecule A and +33.3 and -147.5 degrees in molecule B respectively. The torsion angles of the alpha-methyl group, NH3+ and COO- groups with respect to Cv are in molecules A and B respectively +67.2, +66.8, -174.3, -175.6, and -59.2 and -59.5 degrees. The hydrogen-bonding environment of water OW1 is trigonal nonplanar; OW2 is trigonal planar and OW3 is tetrahedral. The crystal structure is stabilized by a number of hydrogen bonds involving the amino and carboxylate groups of both molecules A and B and the water molecules.

SET DOMAIN GROUP701 encodes a H3K4-methytransferase and regulates multiple key processes of rice plant development.

PubMed

Liu, Kunpeng; Yu, Yu; Dong, Aiwu; Shen, Wen-Hui

2017-07-01

Chromatin-based epigenetic information plays an important role in developmental gene regulation, in response to environment, and in natural variation of gene expression levels. Histone H3 lysine 4 di/trimethylation (H3K4me2/3) is abundant in euchromatin and is generally associated with transcriptional activation. Strikingly, however, enzymes catalyzing H3K4me2/3 remain poorly characterized in crops so far. Here, we investigated the function of the rice SET DOMAIN GROUP 701 (SDG701) gene by molecular and biochemical characterization of the gene product, and by studying effects of its loss or gain of function on plant growth and development. We demonstrated that SDG701 encodes a methytransferase specifically catalyzing H3K4 methylation. Overexpression and knockdown experiments showed that SDG701 is crucial for proper sporophytic plant development as well as for gametophytic transmission that directly impacts rice grain production. In-depth analysis of plant flowering time revealed that SDG701 promotes rice flowering under either long-day or short-day photoperiods. Consistently, the SDG701 protein was found to bind chromatin to promote H3K4me3 and to enhance expression of the rice Hd3a and RFT1 florigens. Collectively, our results establish SDG701 as a major rice H3K4-specific methyltransferase and provide important insights into function of H3K4me3 deposition in transcription activation of florigens in promoting plant flowering. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

PubMed

Selvi B, Ruthrotha; Pradhan, Suman Kalyan; Shandilya, Jayasha; Das, Chandrima; Sailaja, Badi Sri; Shankar G, Naga; Gadad, Shrikanth S; Reddy, Ashok; Dasgupta, Dipak; Kundu, Tapas K

2009-02-27

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Synthesis and in vitro antiproliferative activity of 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives against human cancer cell lines.

PubMed

Mallesha, Lingappa; Mohana, Kikkeri N; Veeresh, Bantal; Alvala, Ravi; Mallika, Alvala

2012-01-01

A series of new 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives 6a-j were synthesized by a nucleophilic substitution reaction of 2-methyl-3-(2-piperazin-1-ylethyl)-pyrido[1,2-a]pyrimidin-4-one with various sulfonyl chlorides. The compounds were characterized by different spectral studies. All the compounds were evaluated for their antiproliferative effect using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method against four human cancer cell lines (K562, Colo-205, MDA-MB 231, IMR-32) for the time period of 24 h. Among the series, compounds 6d, 6e and 6i showed good activity on all cell lines except K562, whereas the other compounds in the series exhibited moderate activity. Compound 6d could be a potential anticancer agent and therefore deserves further research.

Biosorption potential of synthetic dyes by heat-inactivated and live Lentinus edodes CCB-42 immobilized in loofa sponges.

PubMed

Gimenez, Gabriela Gregolin; Ruiz, Suelen Pereira; Caetano, Wilker; Peralta, Rosane Marina; Matioli, Graciette

2014-12-01

Lentinus edodes CCB-42 was immobilized in loofa sponges and applied to the biosorption of the synthetic dyes congo red, bordeaux red and methyl violet. Live immobilized microorganisms achieved average decolorations of congo red, bordeaux red and methyl violet of 97.8, 99.7 and 90.6 %, respectively. The loofa sponge was the support and the coadjuvant promoting dye adsorption. The biosorption conditions were optimized for each dye, yielding 30 °C, pH 5.0 and a 12 h reaction time for congo red; 25 °C, pH 3.0 and 36 h for bordeaux red; and 25 °C, pH 8.0 and 24 h for methyl violet. Operational stability was evaluated over five consecutive cycles, with both bordeaux red and congo red exhibiting decolorations above 90 %, while the decoloration of methyl violet decreased after the third cycle. In the sixth month of storage, congo red, bordeaux red and methyl violet had decolorations of 93.1, 79.4 and 73.8 %, respectively. Biosorption process best fit the pseudo-second-order kinetic and Freundlich isotherm models. Maximum biosorption capacity of heat-treated L. edodes immobilized in loofa sponge was determined as 143.678, 500.00 and 381.679 mg/g for congo red, bordeaux red and methyl violet, respectively. Treatment with immobilized L. edodes reduced the phytotoxicity of the medium containing dyes. FT-Raman experiments suggested the occurrence of interactions between loofa sponge fibers, L. edodes and dye. L. edodes CCB-42 immobilized in loofa sponges represents a promising new mode of treatment of industrial effluents.

21 CFR 172.800 - Acesulfame potassium.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Acesulfame potassium. 172.800 Section 172.800 Food... Acesulfame potassium. Acesulfame potassium (CAS Reg. No. 55589-62-3), also known as acesulfame K, may be... following conditions: (a) Acesulfame potassium is the potassium salt of 6-methyl-1,2,3-oxathiazine-4(3H)-one...

The histone methyltransferase Smyd2 is a negative regulator of macrophage activation by suppressing interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) production.

PubMed

Xu, Guiliang; Liu, Guilin; Xiong, Sidong; Liu, Haiyan; Chen, Xi; Zheng, Biao

2015-02-27

SET and MYND domain-containing 2 (Smyd2), a histone 3 lysine 4- and histone 3 lysine 36 (H3K36)-specific methyltransferase, plays critical roles in cardiac development and tumorigenesis. However, the role of Smyd2 in immunity and inflammation remains poorly understood. In this study, we report that Smyd2 is a novel negative regulator for macrophage activation and M1 polarization. Elevated Smyd2 expression suppresses the production of proinflammatory cytokines, including IL-6 and TNF, and inhibits the expression of important cell surface molecules, including major MHC-II and costimulatory molecules. Furthermore, macrophages with high Smyd2 expression inhibit Th-17 cell differentiation but promote regulatory T cell differentiation as a result of increased TGF-β production and decreased IL-6 secretion. In macrophages, Smyd2 specifically facilitates H3K36 dimethylation at Tnf and Il6 promoters to suppress their transcription and inhibits NF-κB and ERK signaling. Therefore, our data demonstrate that epigenetic modification by Smyd2-mediated H3K36 dimethylation at Tnf and Il6 promoters plays an important role in the regulation of macrophage activation during inflammation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Histone methylation mediates plasticity of human FOXP3+ regulatory T cells by modulating signature gene expressions

PubMed Central

He, Haiqi; Ni, Bing; Tian, Yi; Tian, Zhiqiang; Chen, Yanke; Liu, Zhengwen; Yang, Xiaomei; Lv, Yi; Zhang, Yong

2014-01-01

CD4+ FOXP3+ regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4+ CD25high CD127low/− Treg cells convert to two subpopulations with distinctive FOXP3+ and FOXP3− phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events. PMID:24152290

Isotopic orientational order in acetyl salicylic acid

NASA Astrophysics Data System (ADS)

Schiebel, P.; Prandl, W.; Papoular, R.; Paulus, W.; Detken, A.; Haeberlen, U.; Zimmermann, H.

2000-03-01

Isotopically mixed methyl groups CD xH 3- x with zero averaged deuteron/hydrogen scattering length 0=< a>= xaD+(3- x) aH are expected to be invisible in a neutron diffraction experiment. We find, indeed, in the scattering length density of aspirin-CD xH 3- x, reconstructed by maximum-entropy methods, at room temperature only three very week minima. At 10 K, however, one positive and two negative extrema are visible: unique evidence for orientational isotopic order. From a combination of 1-d-Fourier and algebraic methods we deconvolute < a> and derive the orientational distribution function f( φ) which has three equivalent maxima/minima at 300 K and loses this 3 φ periodicity at 10 K. f( φ) is the basis for the determination of the hindrance potential with cos( φ) as the leading term.

The histone H3 variant H3.3 regulates gene body DNA methylation in Arabidopsis thaliana.

PubMed

Wollmann, Heike; Stroud, Hume; Yelagandula, Ramesh; Tarutani, Yoshiaki; Jiang, Danhua; Jing, Li; Jamge, Bhagyshree; Takeuchi, Hidenori; Holec, Sarah; Nie, Xin; Kakutani, Tetsuji; Jacobsen, Steven E; Berger, Frédéric

2017-05-18

Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. We propose that H3.3 prevents recruitment of H1, inhibiting H1's promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.

Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

PubMed Central

Luo, Wei; Karpf, Adam R.; Deeb, Kristin K.; Muindi, Josephia R.; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.

2010-01-01

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. PMID:20587525

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

PubMed Central

Pham, Kieu Thi Minh; Inoue, Yoshihiro; Vu, Ba Van; Nguyen, Hanh Hieu; Nakayashiki, Toru; Ikeda, Ken-ichi; Nakayashiki, Hitoshi

2015-01-01

Here we report the genetic analyses of histone lysine methyltransferase (KMT) genes in the phytopathogenic fungus Magnaporthe oryzae. Eight putative M. oryzae KMT genes were targeted for gene disruption by homologous recombination. Phenotypic assays revealed that the eight KMTs were involved in various infection processes at varying degrees. Moset1 disruptants (Δmoset1) impaired in histone H3 lysine 4 methylation (H3K4me) showed the most severe defects in infection-related morphogenesis, including conidiation and appressorium formation. Consequently, Δmoset1 lost pathogenicity on wheat host plants, thus indicating that H3K4me is an important epigenetic mark for infection-related gene expression in M. oryzae. Interestingly, appressorium formation was greatly restored in the Δmoset1 mutants by exogenous addition of cAMP or of the cutin monomer, 16-hydroxypalmitic acid. The Δmoset1 mutants were still infectious on the super-susceptible barley cultivar Nigrate. These results suggested that MoSET1 plays roles in various aspects of infection, including signal perception and overcoming host-specific resistance. However, since Δmoset1 was also impaired in vegetative growth, the impact of MoSET1 on gene regulation was not infection specific. ChIP-seq analysis of H3K4 di- and tri-methylation (H3K4me2/me3) and MoSET1 protein during infection-related morphogenesis, together with RNA-seq analysis of the Δmoset1 mutant, led to the following conclusions: 1) Approximately 5% of M. oryzae genes showed significant changes in H3K4-me2 or -me3 abundance during infection-related morphogenesis. 2) In general, H3K4-me2 and -me3 abundance was positively associated with active transcription. 3) Lack of MoSET1 methyltransferase, however, resulted in up-regulation of a significant portion of the M. oryzae genes in the vegetative mycelia (1,491 genes), and during infection-related morphogenesis (1,385 genes), indicating that MoSET1 has a role in gene repression either directly or more likely indirectly. 4) Among the 4,077 differentially expressed genes (DEGs) between mycelia and germination tubes, 1,201 and 882 genes were up- and down-regulated, respectively, in a Moset1-dependent manner. 5) The Moset1-dependent DEGs were enriched in several gene categories such as signal transduction, transport, RNA processing, and translation. PMID:26230995

Avoiding microRNA Function Through Alternative Polyadenylation in Prostate Cancer

DTIC Science & Technology

2012-04-01

H3K36 (REF. 22). Similarly to NSD1, Drosophila melanogaster MES-4 catalyses global mono- and dimeth- ylation of H3K36 in vivo, but SET2, the fly...analysed both the positions of nucleosomes and their modification status within the genomes of humans, C. elegans, D.  melanogaster and mice79–81. In...dosage compensation by enhancing recruitment of the MSL complex in Drosophila melanogaster . Mol. Cell. Biol. 28, 3401–3409 (2008). 73. Alekseyenko, A. A

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Jamy C.

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.« less

Euchromatin islands in large heterochromatin domains are enriched for CTCF binding and differentially DNA-methylated regions

PubMed Central

2012-01-01

Background The organization of higher order chromatin is an emerging epigenetic mechanism for understanding development and disease. We and others have previously observed dynamic changes during differentiation and oncogenesis in large heterochromatin domains such as Large Organized Chromatin K (lysine) modifications (LOCKs), of histone H3 lysine-9 dimethylation (H3K9me2) or other repressive histone posttranslational modifications. The microstructure of these regions has not previously been explored. Results We analyzed the genome-wide distribution of H3K9me2 in two human pluripotent stem cell lines and three differentiated cells lines. We identified > 2,500 small regions with very low H3K9me2 signals in the body of LOCKs, which were termed as euchromatin islands (EIs). EIs are 6.5-fold enriched for DNase I Hypersensitive Sites and 8-fold enriched for the binding of CTCF, the major organizer of higher-order chromatin. Furthermore, EIs are 2–6 fold enriched for differentially DNA-methylated regions associated with tissue types (T-DMRs), reprogramming (R-DMRs) and cancer (C-DMRs). Gene ontology (GO) analysis suggests that EI-associated genes are functionally related to organ system development, cell adhesion and cell differentiation. Conclusions We identify the existence of EIs as a finer layer of epigenomic architecture within large heterochromatin domains. Their enrichment for CTCF sites and DNAse hypersensitive sites, as well as association with DMRs, suggest that EIs play an important role in normal epigenomic architecture and its disruption in disease. PMID:23102236

Broad chromosomal domains of histone modification patterns in C. elegans

PubMed Central

Liu, Tao; Rechtsteiner, Andreas; Egelhofer, Thea A.; Vielle, Anne; Latorre, Isabel; Cheung, Ming-Sin; Ercan, Sevinc; Ikegami, Kohta; Jensen, Morten; Kolasinska-Zwierz, Paulina; Rosenbaum, Heidi; Shin, Hyunjin; Taing, Scott; Takasaki, Teruaki; Iniguez, A. Leonardo; Desai, Arshad; Dernburg, Abby F.; Kimura, Hiroshi; Lieb, Jason D.; Ahringer, Julie; Strome, Susan; Liu, X. Shirley

2011-01-01

Chromatin immunoprecipitation identifies specific interactions between genomic DNA and proteins, advancing our understanding of gene-level and chromosome-level regulation. Based on chromatin immunoprecipitation experiments using validated antibodies, we define the genome-wide distributions of 19 histone modifications, one histone variant, and eight chromatin-associated proteins in Caenorhabditis elegans embryos and L3 larvae. Cluster analysis identified five groups of chromatin marks with shared features: Two groups correlate with gene repression, two with gene activation, and one with the X chromosome. The X chromosome displays numerous unique properties, including enrichment of monomethylated H4K20 and H3K27, which correlate with the different repressive mechanisms that operate in somatic tissues and germ cells, respectively. The data also revealed striking differences in chromatin composition between the autosomes and between chromosome arms and centers. Chromosomes I and III are globally enriched for marks of active genes, consistent with containing more highly expressed genes, compared to chromosomes II, IV, and especially V. Consistent with the absence of cytological heterochromatin and the holocentric nature of C. elegans chromosomes, markers of heterochromatin such as H3K9 methylation are not concentrated at a single region on each chromosome. Instead, H3K9 methylation is enriched on chromosome arms, coincident with zones of elevated meiotic recombination. Active genes in chromosome arms and centers have very similar histone mark distributions, suggesting that active domains in the arms are interspersed with heterochromatin-like structure. These data, which confirm and extend previous studies, allow for in-depth analysis of the organization and deployment of the C. elegans genome during development. PMID:21177964

DOT1L regulates dystrophin expression and is critical for cardiac function

PubMed Central

Nguyen, Anh T.; Xiao, Bin; Neppl, Ronald L.; Kallin, Eric M.; Li, Juan; Chen, Taiping; Wang, Da-Zhi; Xiao, Xiao; Zhang, Yi

2011-01-01

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin–glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease. PMID:21289070

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

PubMed Central

Schwämmle, Veit; Sidoli, Simone; Ruminowicz, Chrystian; Wu, Xudong; Lee, Chung-Fan; Helin, Kristian; Jensen, Ole N.

2016-01-01

Histones are abundant chromatin constituents carrying numerous post-translational modifications (PTMs). Such PTMs mediate a variety of biological functions, including recruitment of enzymatic readers, writers and erasers that modulate DNA replication, transcription and repair. Individual histone molecules contain multiple coexisting PTMs, some of which exhibit crosstalk, i.e. coordinated or mutually exclusive activities. Here, we present an integrated experimental and computational systems level molecular characterization of histone PTMs and PTM crosstalk. Using wild type and engineered mouse embryonic stem cells (mESCs) knocked out in components of the Polycomb Repressive Complex 2 (PRC2, Suz12−/−), PRC1 (Ring1A/B−/−) and (Dnmt1/3a/3b−/−) we performed comprehensive PTM analysis of histone H3 tails (50 aa) by utilizing quantitative middle-down proteome analysis by tandem mass spectrometry. We characterized combinatorial PTM features across the four mESC lines and then applied statistical data analysis to predict crosstalk between histone H3 PTMs. We detected an overrepresentation of positive crosstalk (codependent marks) between adjacent mono-methylated and acetylated marks, and negative crosstalk (mutually exclusive marks) among most of the seven characterized di- and tri-methylated lysine residues in the H3 tails. We report novel features of PTM interplay involving hitherto poorly characterized arginine methylation and lysine methylation sites, including H3R2me, H3R8me and H3K37me. Integration of the H3 data with RNAseq data by coabundance clustering analysis of histone PTMs and histone modifying enzymes revealed correlations between PTM and enzyme levels. We conclude that middle-down proteomics is a powerful tool to determine conserved or dynamic interdependencies between histone marks, which paves the way for detailed investigations of the histone code. Histone H3 PTM data is publicly available in the CrossTalkDB repository at http://crosstalkdb.bmb.sdu.dk. PMID:27302890

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

PubMed

Huang, Jiaojiao; Zhang, Hongyong; Yao, Jing; Qin, Guosong; Wang, Feng; Wang, Xianlong; Luo, Ailing; Zheng, Qiantao; Cao, Chunwei; Zhao, Jianguo

2016-01-01

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression. © 2016 Society for Reproduction and Fertility.

Lack of detectable DNA alkylation for bromhexine in man.

PubMed

Farmer, P B; Parry, A; Franke, H; Schmid, J

1988-09-01

It is known that in vitro incubation of the expectorant drug bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammonium hydrochloride) with nitrite yields methylcyclohexyl nitrosamine (NMCA). NMCA is capable of methylating DNA when administered to rats. In vivo tests with bromhexine have also demonstrated that the drug methylates DNA when it is orally administered in the presence of sodium nitrite, presumably due to the intragastric formation of NMCA. In this study the potential of bromhexine to methylate nucleic acids in man, under physiological conditions, has been investigated. 20 volunteers were orally administered on each of three successive days 48 mg of bromhexine hydrochloride, labelled with three deuterium atoms in the N-methyl group. Urine was collected before treatment and subsequent to the last dose, and analysed by GC-MS for d0- and d3-7-methylguanine. 7-Methylguanine is naturally occurring in urine owing to the turnover of t-RNA of which it is a minor constituent. It is also a repair product from nucleic acids methylated by carcinogens, which is known to be excreted unmetabolised largely within 24 h of the methylation process. Unlabelled 7-methylguanine was present at levels of 7.36 +/- 2.43 mg/d in control urine and 6.12 +/- 2.36 mg/d in treated urine, in accord with previously published values. The excretion of isotopically labelled 7-methylguanine averaged 0.43 +/- 0.077% of the unlabelled concentration for control urines and 0.44 +/- 0.066% for treated urines, i.e. no d3-7-methylguanine could be detected following the drug treatment. The observed signals were largely accounted for by the naturally occurring isotopes 13C and 15N.(ABSTRACT TRUNCATED AT 250 WORDS)

Fungal cell-wall lytic enzymes, antifungal metabolite(s) production, and characterization from Streptomyces exfoliatus MT9 for controlling fruit-rotting fungi.

PubMed

Choudhary, Bharti; Nagpure, Anand; Gupta, Rajinder K

2014-12-01

An antifungal actinomycete strain MT9 was isolated from Loktak Lake, Manipur, India and its cultural characteristics, fatty acid methyl ester, 16S rRNA gene analysis suggests that strain MT9 is identical to Streptomyces exfoliatus. Strain MT9 displayed strong and broad-spectrum antagonism towards several fruit-rotting fungi by mycelial growth suppression. Crude fungal cell-wall lytic enzymes, i.e., chitinase, β-1,3-glucanase, and protease produced by S. exfoliatus MT9 were optimally active at pH 8.0 and 50 °C, pH 5.0 and 60 °C, pH 9.0 and 70 °C, respectively. All three mycolytic enzymes had good stability over a wide pH range of 5.0-10.0, with protease being more thermostable than both chitinase and β-1,3-glucanase. Interestingly zymogram analysis revealed that S. exfoliatus MT9 secretes six distinct chitinase isoenzymes with approximate molecular weights of 9.42, 13.93, 27.87, 36.43, 54.95, 103.27 kDa, six active protease isoenzymes with apparent molecular weights of 12.45, 30.20, 37.45, 46.32, 52.46, 131.46 kDa, and an active band of 119.39 kDa as β-1,3-glucanase enzyme. Extracellular fluid and its organic solvent extracts also exhibited inhibitory activity to various fruit-rotting fungi. The MIC value of n-butanol extract was 2-25 µg/ml against tested fruit-rotting fungi. Antifungal secondary metabolite(s) was found to be polyene in nature. To the best of our knowledge, this is the first report on extracellular production of fungal cell-wall lytic enzymes and antifungal metabolites by bioactive S. exfoliatus MT9 under submerged fermentation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probes for narcotic receptor mediated phenomena. 42. Synthesis and in vitro pharmacological characterization of the N-methyl and N-phenethyl analogues of the racemic ortho-c and para-c oxide-bridged phenylmorphans

PubMed Central

Kim, Jin-Hee; Deschamps, Jeffrey R.; Rothman, Richard B.; Dersch, Christina M.; Folk, John E.; Cheng, Kejun; Jacobson, Arthur E.; Rice, Kenner C.

2011-01-01

A new synthesis of N-methyl and N-phenethyl substituted ortho-c and para-c oxide-bridged phenylmorphans, using N-benzyl- rather than N-methyl-substituted intermediates, was used and the pharmacological properties of these compounds were determined. The N-phenethyl substituted ortho-c oxide-bridged phenylmorphan (rac-(3R,6aS,11aS)-2-phenethyl-2,3,4,5,6,11a-hexahydro-1H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol (12)) was found to have the highest μ-opioid receptor affinity (Ki = 1.1 nM) of all of the a- through f-oxide-bridged phenylmorphans. Functional data ([35S]GTP-γ-S) showed that the racemate 12 was more than three times more potent than naloxone as an μ-opioid antagonist. PMID:21570305

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

PubMed

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

2009-06-11

Histone H3 lysine 4 methylation (H3K4me) has been proposed as a critical component in regulating gene expression, epigenetic states, and cellular identities1. 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.

Development of a pair of differential H/D isotope-coded derivatization reagents d(0)/d(3)-4-(1-methyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenlamine and its application for determination of aldehydes in selected aquatic products by liquid chromatography-tandem mass spectrometry.

PubMed

Sun, Zhiwei; Wang, Xiaoxiang; Cai, Yiping; Fu, Junqing; You, Jinmao

2014-03-01

A new pair of derivatization reagents, d0-4-(1-methyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenlamine (d0-MPIA) and d3-4-(1-methyl-1H-phenanthro[9,10-d]imidazol-2-yl)phenlamine (d3-MPIA) have been designed and synthesized. It was successfully used to label aliphatic aldehydes and the aldehyde derivatives were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The new isotope-coded reagents could easily label aldehydes under acidic conditions in the presence of NaCNBH3. The target derivatives exhibited intense [M+H](+) and regular product ions with electrospray ionization source in positive mode. The d0/d3-MPIA-aldehydes were monitored by the transitions of [M+H](+)→m/z 322 and [M+H](+)→m/z 165, and the obtained detection limits were in the range of 0.18-15.9 pg/mL at signal to noise ratio of 3. The global isotope internal standard technology was employed for quantification analysis with d3-MPIA-aldehyde as internal standard for corresponding d0-MPIA-aldehyde. Excellent linear responses for relative quantification were observed in the range of 1/10-10/1 with coefficients >0.998. The developed method has been applied to the quantification of aliphatic aldehydes in selected aquatic products with RSD<3.6% and recoveries >85.2%. © 2013 Elsevier B.V. All rights reserved.

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor.

PubMed

Romagnoli, Romeo; Baraldi, Pier Giovanni; Pavani, Maria Giovanna; Tabrizi, Mojgan Aghazadeh; Moorman, Allan R; Di Virgilio, Francesco; Cattabriga, Elena; Pancaldi, Cecilia; Gessi, Stefania; Borea, Pier Andrea

2004-11-15

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.

[{(Mo)Mo5O21(H2O)3(SO4)}12(VO)30(H2O)20]36-: a molecular quantum spin icosidodecahedron.

PubMed

Botar, Bogdan; Kögerler, Paul; Hill, Craig L

2005-07-07

Self-assembly of aqueous solutions of molybdate and vanadate under reducing, mildly acidic conditions results in a polyoxomolybdate-based {Mo72V30} cluster compound Na8K16(VO)(H2O)5[K10 subset{(Mo)Mo5O21(H2O)3(SO4)}12(VO)30(H2O)20].150H2O, 1, a quantum spin-based Keplerate structure.

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

PubMed Central

Nakagawa, Takeya; Kajitani, Takuya; Togo, Shinji; Masuko, Norio; Ohdan, Hideki; Hishikawa, Yoshitaka; Koji, Takehiko; Matsuyama, Toshifumi; Ikura, Tsuyoshi; Muramatsu, Masami; Ito, Takashi

2008-01-01

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation. PMID:18172164

Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

PubMed

Khani, Farzaneh; Thaler, Roman; Paradise, Christopher R; Deyle, David R; Kruijthof-de Julio, Marianne; Galindo, Mario; Gordon, Jonathan A; Stein, Gary S; Dudakovic, Amel; van Wijnen, Andre J

2017-05-01

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Theoretical study of the regioselectivity of the interaction of 3-methyl-4-pyrimidone and 1-methyl-2-pyrimidone with Lewis acids.

PubMed

Kasende, Okuma Emile; Muya, Jules Tshishimbi; Broeckaert, Lies; Maes, Guido; Geerlings, Paul

2012-08-23

A density functional theory (DFT) study is performed to determine the stability of the complexes formed between either the N or O site of 3-methyl-4-pyrimidone and 1-methyl-2-pyrimidone molecules and different ligands. The studied ligands are boron and alkali Lewis acids, namely, B(CH(3))(3), HB(CH(3))(2), H(2)B(CH(3)), BH(3), H(2)BF, HBF(2), BF(3), Li(+), Na(+), and K(+). The acids are divided into two groups according to their hardness. The reactivity predictions, according to the molecular electrostatic potential (MEP) map and the natural bond orbital (NBO) analysis, are in agreement with the calculated relative stabilities. Our findings reveal a strong regioselectivity with borane and its derivatives preferring the nitrogen site in both pyrimidone isomers, while a preference for oxygen is observed for the alkali acids in the 3-methyl-4-pyrimidone molecule. The complexation of 1-methyl-2-pyrimidone with these hard alkali acids does not show any discrimination between the two sites due to the presence of a continuous delocalized density region between the nitrogen and the oxygen atoms. The preference of boron Lewis acids toward the N site is due to the stronger B-N bond as compared to the B-O bond. The influence of fluorine or methyl substitution on the boron atom is discussed through natural orbital analysis (NBO) concentrating on the overlap of the boron empty p-orbital with the F lone pairs and methyl hyperconjugation, respectively. The electrophilicity of the boron acids gives a good overall picture of the interaction capabilities with the Lewis base.

Chemical behavior of methylpyranomalvidin-3-O-glucoside in aqueous solution studied by NMR and UV-visible spectroscopy.

PubMed

Oliveira, Joana; Petrov, Vesselin; Parola, A Jorge; Pina, Fernando; Azevedo, Joana; Teixeira, Natércia; Brás, Natércia F; Fernandes, Pedro A; Mateus, Nuno; Ramos, Maria João; de Freitas, Victor

2011-02-17

In the present work, the proton-transfer reactions of the methylpyranomalvidin-3-O-glucoside pigment in water with different pH values was studied by NMR and UV-visible spectroscopies. The results showed four equilibrium forms: the methylpyranomalvidin-3-O-glucoside cation, the neutral quinoidal base, the respective anionic quinoidal base, and a dianionic base unprotonated at the methyl group. According to the NMR data, it seems that for methylpyranomalvidin-3-O-glucoside besides the acid-base equilibrium between the pyranoflavylium cation and the neutral quinoidal base, a new species is formed at pD 4.88-6.10. This is corroborated by the appearance of a new set of signals in the NMR spectrum that may be assigned to the formation of hemiketal/cis-chalcone species to a small extent. The two ionization constants (pK(a1) and pK(a2)) obtained by both methods (NMR and UV-visible) for methylpyranomalvidin-3-O-glucoside are in agreement (pK(a1) = 5.17 ± 0.03; pK(a2) = 8.85 ± 0.08; and pK(a1) = 4.57 ± 0.07; pK(a2) = 8.23 ± 0.04 obtained by NMR and UV-visible spectroscopies, respectively). Moreover, the fully dianionic unprotonated form (at the methyl group) of the methylpyranomalvidin-3-O-glucoside is converted slowly into a new structure that displays a yellow color at basic pH. On the basis of the results obtained through LC-MS and NMR, the proposed structure was found to correspond to the flavonol syringetin-3-glucoside.

Repressive histone methylation regulates cardiac myocyte cell cycle exit.

PubMed

El-Nachef, Danny; Oyama, Kyohei; Wu, Yun-Yu; Freeman, Miles; Zhang, Yiqiang; Robb MacLellan, W

2018-05-22

Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo. Copyright © 2017. Published by Elsevier Ltd.

QM/MM Investigation of Substrate and Product Specificities of Suv4-20h2: How Does This Enzyme Generate Dimethylated H4K20 from Monomethylated Substrate?

PubMed

Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong

2017-06-13

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.

Human mass balance, metabolite profile and identification of metabolic enzymes of [¹⁴C]ASP015K, a novel oral janus kinase inhibitor.

PubMed

Oda, Kazuo; Cao, Ying J; Sawamoto, Taiji; Nakada, Naoyuki; Fisniku, Ogert; Nagasaka, Yasuhisa; Sohda, Kin-Ya

2015-01-01

1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.

Design and syntheses of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione and N-(benzothiazol-5-yl)isoindoline-1,3-dione as potent protoporphyrinogen oxidase inhibitors.

PubMed

Jiang, Li-Li; Zuo, Yang; Wang, Zhi-Fang; Tan, Yin; Wu, Qiong-You; Xi, Zhen; Yang, Guang-Fu

2011-06-08

Discovery of protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors has been one of the hottest research areas in the field of herbicide development for many years. As a continuation of our research work on the development of new PPO-inhibiting herbicides, a series of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-diones (1a-p) and N-(benzothiazol-5-yl)isoindoline-1,3-diones (2a-h) were designed and synthesized according to the ring-closing strategy of two ortho-substituents. The bioassay results indicated that some newly synthesized compounds exhibited higher PPO inhibition activity than the control of sulfentrazone. Compound 1a, S-(5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl) O-methyl carbonothioate, was identified as the most potent inhibitor with k(i) value of 0.08 μM, about 9 times higher than that of sulfentrazone (k(i) = 0.72 μM). Further green house assay showed that compound 1b, methyl 2-((5-(1,3-dioxo-4,5,6,7-tetrahydro-1H-isoindol-2(3H)-yl)-6-fluorobenzothiazol-2-yl)thio)acetate, exhibited herbicidal activity comparable to that of sulfentrazone even at a concentration of 37.5 g ai/ha. In addition, among six tested crops, wheat exhibited high tolerance to compound 1b even at a dosage of 300 g ai/ha. These results indicated that compound 1b might have the potential to be developed as a new herbicide for weed control of wheat field.

Assessment of the aroma impact of major odor-active thiols in pan-roasted white sesame seeds by calculation of odor activity values.

PubMed

Tamura, Hitoshi; Fujita, Akira; Steinhaus, Martin; Takahisa, Eisuke; Watanabe, Hiroyuki; Schieberle, Peter

2011-09-28

Eleven odor-active thiols, namely, 2-methyl-1-propene-1-thiol, (Z)-3-methyl-1-butene-1-thiol, (E)-3-methyl-1-butene-1-thiol, (Z)-2-methyl-1-butene-1-thiol, (E)-2-methyl-1-butene-1-thiol, 2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-mercapto-3-pentanone, 4-mercapto-3-hexanone, 3-mercapto-3-methylbutyl formate, and 2-methyl-3-thiophenethiol, recently identified in an extract prepared from white sesame seeds, were quantitated in sesame using stable isotope dilution analyses. For that purpose, the following deuterium-labeled compounds were synthesized and used as internal standards in the quantitation assays: [2H6]-2-methyl-1-propene-1-thiol, [2H3]-(E)- and [2H3]-(Z)-2-methyl-1-butene-1-thiol, [2H3]-2-methyl-3-furanthiol, [2H2]-3-mercapto-2-pentanone, [2H3]-4-mercapto-3-hexanone, [2H6]-3-mercapto-3-methylbutyl formate, and [2H3]-2-methyl-3-thiophenethiol. On the basis of the results obtained, odor activity values (OAVs) were calculated as ratio of the concentration and odor threshold of the individual compounds in cooking oil. According to their high OAVs, particularly the 3-methyl-1-butene-1-thiols (OAV: 2400) and the 2-methyl-1-butene-1-thiols (OAV: 960) were identified as the most odor-active compounds in pan-roasted white sesame seeds. These compounds were therefore suggested to be mainly responsible for the characteristic but rather unstable sulfury aroma of freshly pan-roasted white sesame seeds.

Store Separations From a Supersonic Cone

DTIC Science & Technology

2006-03-01

L DK/9H|; 21W-0< ,.LMD >DK/@HX½1¢, H|3*]�&¢DK>4ɜH|C H�I21WDK, > L@C 14L95 :H|3 > 3 ;=,.L H|L9C H|; .H 1 HJ3K1 ,.L ? ODK /9H...4; @I)DKH¯C > .I9D ,.> L@C{143 >4I@L@5 -J> ; .H 5¢L21W; , - HJ> ;=HJDK36,.HJC|8QA236> ; 14LH -J,.HJL@-C6D01WL@5 :> ,.LMD? ODK /9H 3...H 93 H|C6C I@36H > ]�[)8 [ ` I@C6,.L $ �I21^DK,.>4L@CQ\\ D /@3 > I /a º` ` [ t8 P½C½>4L@HU-14LC HJH4? ODK /@H

Reaction CH3 + OH studied over the 294-714 K temperature and 1-100 bar pressure ranges.

PubMed

Sangwan, Manuvesh; Chesnokov, Evgeni N; Krasnoperov, Lev N

2012-08-30

Reaction of methyl radicals with hydroxyl radicals, CH(3) + OH → products (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 294-714 K temperature and 1-100 bar pressure ranges (bath gas He). Methyl radicals were produced by photolysis of acetone at 193.3 nm. Hydroxyl radicals were generated in reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N(2)O at 193.3 nm, with H(2)O. Temporal profiles of CH(3) were recorded via absorption at 216.4 nm using xenon arc lamp and a spectrograph; OH radicals were monitored via transient absorption of light from a dc discharge H(2)O/Ar low pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light inside the reactor was determined by an accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The results of this study indicate that the rate constant of reaction 1 is pressure independent within the studied pressure and temperature ranges and has slight negative temperature dependence, k(1) = (1.20 ± 0.20) × 10(-10)(T/300)(-0.49) cm(3) molecule(-1) s(-1).

Observing Organic Molecules in Interstellar Gases: Non Equilibrium Excitation.

NASA Astrophysics Data System (ADS)

Wiesenfeld, Laurent; Faure, Alexandre; Remijan, Anthony; Szalewicz, Krzysztof

2014-06-01

In order to observe quantitatively organic molecules in interstellar gas, it is necessary to understand the relative importance of photonic and collisional excitations. In order to do so, collisional excitation transfer rates have to be computed. We undertook several such studies, in particular for H_2CO and HCOOCH_3. Both species are observed in many astrochemical environments, including star-forming regions. We found that those two molecules behave in their low-lying rotational levels in an opposite way. For cis methyl-formate, a non-equilibrium radiative transfer treatment of rotational lines is performed, using a new set of theoretical collisional rate coefficients. These coefficients have been computed in the temperature range 5 to 30 K by combining coupled-channel scattering calculations with a high accuracy potential energy surface for HCOOCH_3 -- He. The results are compared to observations toward the Sagittarius B2(N) molecular cloud. A total of 2080 low-lying transitions of methyl formate, with upper levels below 25 K, were treated. These lines are found to probe a cold (30 K), moderately dense (n ˜ 104 cm-3) interstellar gas. In addition, our calculations indicate that all detected emission lines with a frequency below 30 GHz are collisionally pumped weak masers amplifying the background of Sgr B2(N). This result demonstrates the generality of the inversion mechanism for the low-lying transitions of methyl formate. For formaldehyde, we performed a similar non-equilibrium treatment, with H_2 as the collisional partner, thanks to the accurate H_2CO - H_2 potential energy surface . We found very different energy transfer rates for collisions with para-H_2 (J=0) and ortho-H_2 (J=1). The well-known absorption against the cosmological background of the 111→ 101 line is shown to depend critically on the difference of behaviour between para and ortho-H_2, for a wide range of H_2 density. We thank the CNRS-PCMI French national program for continuous support and the CHESS Herschel KP program for travel supports. Discussions with C. Ceccarelli, P. Hily-Blant and S. Maret are acknowledged.

Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa.

PubMed

Erkek, Serap; Hisano, Mizue; Liang, Ching-Yeu; Gill, Mark; Murr, Rabih; Dieker, Jürgen; Schübeler, Dirk; van der Vlag, Johan; Stadler, Michael B; Peters, Antoine H F M

2013-07-01

In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG-rich sequences that lack DNA methylation. Residual nucleosomes are largely composed of the histone H3.3 variant and are trimethylated at Lys4 of histone H3 (H3K4me3). Canonical H3.1 and H3.2 histones are also enriched at CpG-rich promoters marked by Polycomb-mediated H3K27me3, a modification predictive of gene repression in preimplantation embryos. Histone variant-specific nucleosome retention in sperm is strongly associated with nucleosome turnover in round spermatids. Our data show evolutionary conservation of the basic principles of nucleosome retention in mouse and human sperm, supporting a model of epigenetic inheritance by nucleosomes between generations.

Dipotassium tetra­aqua­bis­[3,5-bis­(dicyano­methyl­ene)cyclo­pentane-1,2,4-trionato(1−)-κN]cobaltate(II)

PubMed Central

Chagas, Luciano Honorato; Janczak, Jan; Machado, Flavia C.; de Oliveira, Luiz Fernando C.; Diniz, Renata

2010-01-01

The title structure, K2[Co(C11N4O3)2(H2O)4], is isotypic with K2[Fe(C11N4O3)2(H2O)4]. The CoII atom is in a distorted octa­hedral CoN2O4 geometry, forming a dianionic mononuclear entity. Each dianionic unit is associated with two potassium cations and inter­acts with adjacent units through O—H⋯N and O—H⋯O hydrogen bonds. PMID:21589332

Smooth, an hnRNP-L Homolog, Might Decrease Mitochondrial Metabolism by Post-Transcriptional Regulation of Isocitrate Dehydrogenase (Idh) and Other Metabolic Genes in the Sub-Acute Phase of Traumatic Brain Injury.

PubMed

Sen, Arko; Gurdziel, Katherine; Liu, Jenney; Qu, Wen; Nuga, Oluwademi O; Burl, Rayanne B; Hüttemann, Maik; Pique-Regi, Roger; Ruden, Douglas M

2017-01-01

Traumatic brain injury (TBI) can cause persistent pathological alteration of neurons. This may lead to cognitive dysfunction, depression and increased susceptibility to life threatening diseases, such as epilepsy and Alzheimer's disease. To investigate the underlying genetic and molecular basis of TBI, we subjected w 1118 Drosophila melanogaster to mild closed head trauma and found that mitochondrial activity is reduced in the brains of these flies 24 h after inflicting trauma. To determine the transcriptomic changes after mild TBI, we collected fly heads 24 h after inflicting trauma, and performed RNA-seq analyses. Classification of alternative splicing changes showed selective retention (RI) of long introns (>81 bps), with a mean size of ~3,000 nucleotides. Some of the genes containing RI showed a significant reduction in transcript abundance and are involved in mitochondrial metabolism such as Isocitrate dehydrogenase (Idh), which makes α-KG, a co-factor needed for both DNA and histone demethylase enzymes. The long introns are enriched in CA-rich motifs known to bind to Smooth (Sm), a heterogeneous nuclear ribonucleoprotein L (hnRNP-L) class of splicing factor, which has been shown to interact with the H3K36 histone methyltransferase, SET2, and to be involved in intron retention in human cells. H3K36me3 is a histone mark that demarcates exons in genes by interacting with the mRNA splicing machinery. Mutating sm ( sm 4 /Df) resulted in loss of both basal and induced levels of RI in many of the same long-intron containing genes. Reducing the levels of Kdm4A, the H3K36me3 histone demethylase, also resulted in loss of basal levels of RI in many of the same long-intron containing genes. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) for H3K36me3 revealed increased levels of this histone modification in retained introns post-trauma at CA-rich motifs. Based on these results, we propose a model in which TBI temporarily decreases mitochondrial activity in the brain 24 h after inflicting trauma, which decreases α-KG levels, and increases H3K36me3 levels and intron retention of long introns by decreasing Kdm4A activity. The consequent reduction in mature mRNA levels in metabolism genes, such as Idh, further reduces α-KG levels in a negative feedback loop. We further propose that decreasing metabolism after TBI in such a manner is a protective mechanism that gives the brain time to repair cellular damage induced by TBI.

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation

PubMed Central

Serrano, Lourdes; Martínez-Redondo, Paloma; Marazuela-Duque, Anna; Vazquez, Berta N.; Dooley, Scott J.; Voigt, Philipp; Beck, David B.; Kane-Goldsmith, Noriko; Tong, Qiang; Rabanal, Rosa M.; Fondevila, Dolors; Muñoz, Purificación; Krüger, Marcus; Tischfield, Jay A.; Vaquero, Alejandro

2013-01-01

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 deposition through the deacetylation of H4K16Ac (acetylation of H4K16) and determines the levels of H4K20me2/3 throughout the cell cycle. SirT2 binds and deacetylates PR-Set7 at K90, modulating its chromatin localization. Consistently, SirT2 depletion significantly reduces PR-Set7 chromatin levels, alters the size and number of PR-Set7 foci, and decreases the overall mitotic deposition of H4K20me1. Upon stress, the interaction between SirT2 and PR-Set7 increases along with the H4K20me1 levels, suggesting a novel mitotic checkpoint mechanism. SirT2 loss in mice induces significant defects associated with defective H4K20me1–3 levels. Accordingly, SirT2-deficient animals exhibit genomic instability and chromosomal aberrations and are prone to tumorigenesis. Our studies suggest that the dynamic cross-talk between the environment and the genome during mitosis determines the fate of the subsequent cell cycle. PMID:23468428

Complex methyl group and hydrogen-bonded proton motions in terms of the Arrhenius and Schrödinger equations.

PubMed

Latanowicz, L

2008-01-01

Equations for the temperature dependence of the spectral densities J(is)(m)(momega(I) +/-omega(T)), where m=1, 2, omega(I) and omega(T) are the resonance and tunnel splitting angular frequencies, in the presence of a complex motion, have been derived. The spin pairs of the protons or deuterons of the methyl group perform a complex motion consisting of three component motions. Two of them involve mass transportation over the barrier and through the barrier. They are characterized by k((H)) (Arrhenius) and k((T)) (Schrödinger) rate constants, respectively. The third motion causes fluctuations of the frequencies (nomega(I)+/-omega(T)) and it is related to the lifetime of the methyl spin at the energy level influenced by the rotor-bath interactions. These interactions induce rapid transitions, changing the symmetry of the torsional sublevels either from A to E or from E(a) to E(b). The correlation function for this third motion (k((omega)) rate constant) has been proposed by Müller-Warmuth et al. The spectral densities of the methyl group hindered rotation (k((H)), k((T)) and k((omega)) rate constants) differ from the spectral densities of the proton transfer (k((H)) and k((T)) rate constants) because three compound motions contribute to the complex motion of the methyl group. The recently derived equation [Formula: see text] , where [Formula: see text] and [Formula: see text] are the fraction and energy of particles with energies from zero to E(H), is taken into account in the calculations of the spectral densities. This equation follows from Maxwell's distribution of thermal energy. The spectral densities derived are applied to analyse the experimental temperature dependencies of proton and deuteron spin-lattice relaxation rate in solids containing the methyl group. A wide range of temperatures from zero Kelvin up to the melting point is considered. It has been established that the motion characterized by k((omega)) influences the spin-lattice relaxation up to the temperature T(tun) only. This temperature is directly determined by the equation C(p)T=E(H) (thermal energy=activation energy), where C(p) is the molar heat capacity. Probably the cessation of the third motion is a result of the de Broglie wavelength related to this motion becoming too short. As shown recently, the potential barrier can be an obstacle for the de Broglie wave. The theoretical equations derived in this paper are compared to those known in the literature.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Experimental Study of Combustion and Emissions Characteristics of Methyl Oleate, as a Surrogate for Biodiesel, in a Direct injection Diesel Engine

USDA-ARS?s Scientific Manuscript database

This study evaluates the combustion and emissions characteristics of methyl oleate (C19H36O2 CAS# 112-62) produced by transesterification from oleic acid, one of the main fatty acid components of biodiesel. The ignition delay of ultra-low sulfur diesel#2 (ULSD) and its blends with methyl oleate (O20...

A comparison of N-methyl-D-aspartate-evoked release of adenosine and ( sup 3 H)norepinephrine from rat cortical slices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoehn, K.; Craig, C.G.; White, T.D.

1990-10-01

Tetrodotoxin reduced N-methyl-D-aspartate (NMDA)-evoked release of adenosine by 35% but virtually abolished (3H)norepinephrine release. Although (3H)norepinephrine release from rat cortical slices evoked by 500 microM NMDA was abolished by 1.2 mM Mg++, which produces a voltage-sensitive, uncompetitive block of NMDA-channels, adenosine release was increased in the presence of Mg++. Partial depolarization with 12 mM K+ relieved the Mg++ block of 500 microM NMDA-evoked (3H)norepinephrine release but did not affect adenosine release, indicating that a Mg++ requirement for the adenosine release process per se cannot account for this discrepancy. NMDA was 33 times more potent in releasing adenosine than (3H)norepinephrine. Atmore » submaximal concentrations of NMDA (10 and 20 microM), adenosine release was augmented in Mg+(+)-free medium. Although a high concentration of the uncompetitive NMDA antagonist MK-801 ((+)-5-methyl-10,11,dihydro-5H-dibenzo(a,d)cyclohepten-5-10-imine maleate) (3 microM) blocked NMDA-evoked release of (3H)norepinephrine and adenosine, a lower concentration (300 nM) decreased NMDA-evoked (3H)norepinephrine release by 66% without affecting adenosine release. These findings suggest that maximal adenosine release occurs when relatively few NMDA receptors are activated, raising the possibility that spare receptors exist for NMDA-evoked adenosine release. Rather than acting as a protectant against excessive NMDA excitation, released adenosine might provide an inhibitory threshold which must be overcome for NMDA-mediated neurotransmission to proceed.« less

A selected ion flow tube study of the reactions of H3O+, NO+ and O2+ with some oxygenated biogenic volatile organic compounds

NASA Astrophysics Data System (ADS)

Amelynck, C.; Schoon, N.; Kuppens, T.; Bultinck, P.; Arijs, E.

2005-12-01

The rate constants and product ion distributions of the reactions of H3O+, NO+ and O2+ with 2-methyl-3-buten-2-ol, cis-3-hexen-1-ol, cis-3-hexenyl acetate, 1,8-cineole, 6-methyl-5-hepten-2-one, camphor and linalool have been determined at 150 Pa and 297 K using a selected ion flow tube (SIFT). All reactions were found to proceed at a rate close to the collision rate, calculated with the Su and Chesnavich model, using the polarizability and electric dipole moment of the compounds derived from B3LYP/aug-cc-pVDZ quantum chemical calculations. Additionally the product ion distributions of the reactions of these three ions with the terpenoid alcohols nerol and geraniol have been obtained.

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy

PubMed Central

Aronsen, Jan Magnus; Ferrini, Arianna; Brien, Patrick; Alkass, Kanar; Tomasso, Antonio; Agrawal, Asmita; Bergmann, Olaf; Reik, Wolf; Roderick, Hywel Llewelyn

2016-01-01

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217–mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217–mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease. PMID:27893464

Crystal structure of azilsartan methyl ester ethyl acetate hemisolvate.

PubMed

Li, Zhengyi; Liu, Rong; Zhu, Meilan; Chen, Liang; Sun, Xiaoqiang

2015-02-01

The title compound, C26H22N4O5 (systematic name: methyl 2-eth-oxy-1-{4-[2-(5-oxo-4,5-di-hydro-1,2,4-oxa-diazol-3-yl)phenyl]benz-yl}-1H-1,3-benzo-diazole-7-carboxyl-ate ethyl acetate hemisolvate), was obtained via cyclization of methyl (Z)-2-eth-oxy-1-{(2'-(N'-hy-droxy-carbamimido-yl)-[1,1'-biphen-yl]-4-yl)meth-yl}-1H-benzo[d]imidazole-7-carboxyl-ate with diphen-yl carbonate. There are two independent mol-ecules (A and B) with different conformations and an ethyl acetate solvent mol-ecule in the asymmetric unit. In mol-ecule A, the dihedral angle between the benzene ring and its attached oxa-diazole ring is 59.36 (17); the dihedral angle between the benzene rings is 43.89 (15) and that between the benzene ring and its attached imidazole ring system is 80.06 (11)°. The corres-ponding dihedral angles in mol-ecule B are 58.45 (18), 50.73 (16) and 85.37 (10)°, respectively. The C-O-C-Cm (m = meth-yl) torsion angles for the eth-oxy side chains attached to the imidazole rings in mol-ecules A and B are 93.9 (3) and -174.6 (3)°, respectively. In the crystal, the components are linked by N-H⋯N and C-H⋯O hydrogen bonds, generating a three-dimensional network. Aromatic π-π stacking inter-actions [shortest centroid-centroid separation = 3.536 (3)Å] are also observed.

Electron-spin-resonance studies of 12CH3F + , 13CH3F + , and 12CH2DF + in neon matrices at 4 K: Comparison with theoretical calculations

NASA Astrophysics Data System (ADS)

Knight, Lon B., Jr.; Gregory, Brian W.; Hill, Devon W.; Arrington, C. A.; Momose, Takamasa; Shida, Tadamasa

1991-01-01

Various isotopic forms of the methyl fluoride cation 12CH3F+, 13CH3F+, and 12CH2DF+ have been generated by photoionization at 16.8 eV and separately by electron bombardment at 50 eV. The first electron-spin-resonance (ESR) results are reported for this radical cation which was isolated in neon matrices at 4 K. The measured A tensors or nuclear hyperfine parameters were compared with the results obtained from various computational approaches. Surprising observations were the large amounts of spin density on the methyl group, especially the hydrogen atoms, and the extreme differences in the deuterated spectra compared to the nondeuterated case. The presence of a single D atom apparently acts to prevent dynamic Jahn-Teller averaging which makes the methyl hydrogens equivalent on the ESR time scale. Such a dramatic Jahn-Teller effect has been previously observed for the similar methane cations CH+4 and CH2D+2. The magnetic parameters for CH2DF+ in neon at 4 K are gX=2.0032(5), gY=2.0106(8), and gZ=2.0120(5); for H: AX = 483(1), AY=476(1), and AZ=483(1) MHz; for D: ‖AX‖=5.0(3), ‖AY‖<3, and ‖AZ‖=7.1(3) MHz; for 19F : AX=965(1), AY=-130(2), and AZ=-166(1) MHz. For CH3F+, the g tensor and 19F A tensor were similar to those above but the H atoms were equivalent with values of AX=317(1), AY=323(2), and AZ=312 MHz.

Examining post-translational modification-mediated protein–protein interactions using a chemical proteomics approach

PubMed Central

Li, Xiang; Foley, Emily A; Kawashima, Shigehiro A; Molloy, Kelly R; Li, Yinyin; Chait, Brian T; Kapoor, Tarun M

2013-01-01

Post-translational modifications (PTM) of proteins can control complex and dynamic cellular processes via regulating interactions between key proteins. To understand these regulatory mechanisms, it is critical that we can profile the PTM-dependent protein–protein interactions. However, identifying these interactions can be very difficult using available approaches, as PTMs can be dynamic and often mediate relatively weak protein–protein interactions. We have recently developed CLASPI (cross-linking-assisted and stable isotope labeling in cell culture-based protein identification), a chemical proteomics approach to examine protein–protein interactions mediated by methylation in human cell lysates. Here, we report three extensions of the CLASPI approach. First, we show that CLASPI can be used to analyze methylation-dependent protein–protein interactions in lysates of fission yeast, a genetically tractable model organism. For these studies, we examined trimethylated histone H3 lysine-9 (H3K9Me3)-dependent protein–protein interactions. Second, we demonstrate that CLASPI can be used to examine phosphorylation-dependent protein–protein interactions. In particular, we profile proteins recognizing phosphorylated histone H3 threonine-3 (H3T3-Phos), a mitotic histone “mark” appearing exclusively during cell division. Our approach identified survivin, the only known H3T3-Phos-binding protein, as well as other proteins, such as MCAK and KIF2A, that are likely to be involved in weak but selective interactions with this histone phosphorylation “mark”. Finally, we demonstrate that the CLASPI approach can be used to study the interplay between histone H3T3-Phos and trimethylation on the adjacent residue lysine 4 (H3K4Me3). Together, our findings indicate the CLASPI approach can be broadly applied to profile protein–protein interactions mediated by PTMs. PMID:23281010

Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer

PubMed Central

Nagasaka, Takeshi; Koi, Minoru; Kloor, Matthias; Gebert, Johannes; Vilkin, Alex; Nishida, Naoshi; Shin, Sung Kwan; Sasamoto, Hiromi; Tanaka, Noriaki; Matsubara, Nagahide; Boland, C. Richard; Goel, Ajay

2008-01-01

Background Colorectal cancers (CRCs) with the CpG island methylator phenotype (CIMP) often associate with epigenetic silencing of hMLH1 and an activating mutation in the BRAF gene. However, the current CIMP criteria are ambiguous, and often result in an underestimation of CIMP frequencies in CRCs. Since BRAF and KRAS belong to same signaling pathway, we hypothesized that not only mutations in BRAF, but mutant KRAS, may also associate with CIMP in CRC. Methods We determined the methylation status of a panel of 14 markers (7 canonical CIMP-related loci, and 7 new loci), MSI status, and BRAF/KRAS mutations in a cohort of 487 colorectal tissues that included both sporadic and Lynch syndrome patients. Results Methylation analysis of seven CIMP-related markers revealed that the mean number of methylated loci was highest in BRAF mutated CRCs [3.6], versus KRAS-mutated [1.2; P<0.0001] or BRAF/KRAS wild-type tumors [0.7; P<0.0001]. However, analyses with seven additional markers showed that the mean number of methylated loci in BRAF mutant tumors [4.4] was the same as in KRAS mutant CRCs [4.3; P=0.8610]. Although sporadic MSI-H tumors had the most average number of methylated markers [8.4], surprisingly Lynch syndrome CRCs also demonstrated frequent methylation [5.1]. Conclusions CIMP in CRC may result from activating mutations in either BRAF or KRAS, and the inclusion of additional methylation markers that correlate with mutant KRAS may help clarify CIMP in future studies. Additionally, aberrant DNA methylation is a common event not only in sporadic CRC, but also in Lynch syndrome CRCs. PMID:18435933

A transposon-directed epigenetic change in ZmCCT underlies quantitative resistance to Gibberella stalk rot in maize.

PubMed

Wang, Chao; Yang, Qin; Wang, Weixiang; Li, Yipu; Guo, Yanling; Zhang, Dongfeng; Ma, Xuena; Song, Wei; Zhao, Jiuran; Xu, Mingliang

2017-09-01

A major resistance quantitative trait locus, qRfg1, significantly enhances maize resistance to Gibberella stalk rot, a devastating disease caused by Fusarium graminearum. However, the underlying molecular mechanism remains unknown. We adopted a map-based cloning approach to identify the resistance gene at qRfg1 and examined the dynamic epigenetic changes during qRfg1-mediated maize resistance to the disease. A CCT domain-containing gene, ZmCCT, is the causal gene at the qRfg1 locus and a polymorphic CACTA-like transposable element (TE1) c. 2.4 kb upstream of ZmCCT is the genetic determinant of allelic variation. The non-TE1 ZmCCT allele is in a poised state, with predictive bivalent chromatin enriched for both repressive (H3K27me3/H3K9me3) and active (H3K4me3) histone marks. Upon pathogen challenge, this non-TE1 ZmCCT allele was promptly induced by a rapid yet transient reduction in H3K27me3/H3K9me3 and a progressive decrease in H3K4me3, leading to disease resistance. However, TE1 insertion in ZmCCT caused selective depletion of H3K4me3 and enrichment of methylated GC to suppress the pathogen-induced ZmCCT expression, resulting in disease susceptibility. Moreover, ZmCCT-mediated resistance to Gibberella stalk rot is not affected by photoperiod sensitivity. This chromatin-based regulatory mechanism enables ZmCCT to be more precise and timely in defense against F. graminearum infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

High resolution quantum cascade laser studies of the ν3 band of methyl fluoride in solid para-hydrogen.

PubMed

McKellar, A R W; Mizoguchi, Asao; Kanamori, Hideto

2011-09-28

Spectra of solid para-H(2) doped with CH(3)F at 1.8 K are studied in the ν(3) region (~1040 cm(-1)) using a quantum cascade laser source. As shown previously, residual ortho-H(2) in the sample (~1000 ppm) gives rise to distinct spectral features due to clusters of the form CH(3)F-(ortho-H(2))(N), with N = 0, 1, 2, 3, etc. Brief annealing at 7 K is found to give narrower spectral lines (≥0.006 cm(-1)) than conventional (5 K) annealing, and causes the N = 3 and 4 lines to fragment into two or more components. The N = 3 line is observed to be particularly stable and persistent. The N = 0 line (no ortho-H(2) neighbors) is resolved into two closely spaced (≈0.007 cm(-1)) components which are assigned to the K = 0 and 1 states of CH(3)F rotating around its C(3v) symmetry axis (ortho- and para-CH(3)F, respectively). Similar K-structure is also evident for other lines. Weak but persistent features ("N = 1/2 lines") are observed mid way between N = 0 and 1. © 2011 American Institute of Physics

Temperature Dependence of the Rate Constant for the CH3 Recombination Reaction: A Loss Process in Outer Planet Atmospheres

NASA Technical Reports Server (NTRS)

Cody, R. J.; Payne, W. A.; Thorn, R. P., Jr.; Romani, P. N.; Stief, L. J.; Nesbitt, F. L.; Iannone, M. A.; Tardy, D. C.

2002-01-01

The methyl free radical (CH3) has been observed in the atmospheres of Saturn and Neptune by the ISO satellite. There are discrepancies between the column densities for the CH3 radical derived from the ISO observations and the column densities derived from atmospheric photochemical models. For Neptune the model column density is 1.5 times that derived from ISO. For Saturn the model is 6 times that from ISO. The recombination of methyl radicals is the major loss process for methyl in these atmospheres. The serious disagreement between observed and calculated levels of CH3 has led to suggestions that the atmospheric models greatly underestimated the loss of CH3 due to poor knowledge of the rate of the reaction (1) CH3 + CH3 + M goes to C2H6 + M at the low temperatures and pressures of these atmospheric systems. Although the reaction CH3 + CH3 + M goes to C2H6 + M has been extensively studied both theoretically and experimentally, the laboratory conditions have been, with only a few exceptions, higher temperatures (T greater than 298K), higher pressures (P greater than or equal to 10 Torr - 13.3 mbar) or M=Ar rather than H2 or He as the bath gas.

COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CLEAR CELL RENAL CELL CARCINOMA

PubMed Central

2013-01-01

Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (e.g. VHL) and the maintenance of chromatin states (e.g. PBRM1). We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes. The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 and GRB10. Remodeling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment. PMID:23792563

piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells

PubMed Central

Pezic, Dubravka; Manakov, Sergei A.; Sachidanandam, Ravi; Aravin, Alexei A.

2014-01-01

Transposable elements (TEs) occupy a large fraction of metazoan genomes and pose a constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small noncoding piwi-interacting RNAs (piRNAs) recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments. PMID:24939875

Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers.

PubMed

DeVries, Avery; Wlasiuk, Gabriela; Miller, Susan J; Bosco, Anthony; Stern, Debra A; Lohman, I Carla; Rothers, Janet; Jones, Anya C; Nicodemus-Johnson, Jessie; Vasquez, Monica M; Curtin, John A; Simpson, Angela; Custovic, Adnan; Jackson, Daniel J; Gern, James E; Lemanske, Robert F; Guerra, Stefano; Wright, Anne L; Ober, Carole; Halonen, Marilyn; Vercelli, Donata

2017-08-01

The timing and mechanisms of asthma inception remain imprecisely defined. Although epigenetic mechanisms likely contribute to asthma pathogenesis, little is known about their role in asthma inception. We sought to assess whether the trajectory to asthma begins already at birth and whether epigenetic mechanisms, specifically DNA methylation, contribute to asthma inception. We used the Methylated CpG Island Recovery Assay chip to survey DNA methylation in cord blood mononuclear cells from 36 children (18 nonasthmatic and 18 asthmatic subjects by age 9 years) from the Infant Immune Study (IIS), an unselected birth cohort closely monitored for asthma for a decade. SMAD3 methylation in IIS (n = 60) and in 2 replication cohorts (the Manchester Asthma and Allergy Study [n = 30] and the Childhood Origins of Asthma Study [n = 28]) was analyzed by using bisulfite sequencing or Illumina 450K arrays. Cord blood mononuclear cell-derived IL-1β levels were measured by means of ELISA. Neonatal immune cells harbored 589 differentially methylated regions that distinguished IIS children who did and did not have asthma by age 9 years. In all 3 cohorts methylation in SMAD3, the most connected node within the network of asthma-associated, differentially methylated regions, was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. Moreover, SMAD3 methylation in IIS neonates with maternal asthma was strongly and positively associated with neonatal production of IL-1β, an innate inflammatory mediator. The trajectory to childhood asthma begins at birth and involves epigenetic modifications in immunoregulatory and proinflammatory pathways. Maternal asthma influences epigenetic mechanisms that contribute to the inception of this trajectory. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Microwave Spectroscopy of Trans-Ethyl Methyl Ether in the Ground State

NASA Astrophysics Data System (ADS)

Kobayashi, Kaori; Sakai, Yusuke; Tsunekawa, Shozo; Miyamoto, Taihei; Fujitake, Masaharu; Ohashi, Nobukimi

2013-06-01

The trans-ethyl methyl ether molecule (CH_3CH_2OCH_3) has two inequivalent methyl group internal rotors which corresponds to the two vibrational motions, ν_{28} and ν_{29}. Due to these internal rotations, a rotational transition could be split into maximum five components. The skeletal torsion (ν_{30}) is another low-lying state (ν_{30}) that interacts with the ν_{28} and ν_{29} modes. The microwave spectra of the trans-ethyl methyl ether molecule in the ν_{28} = 1, ν_{29} = 1, and ν_{30} = 1, 2 and 3 have been extensively studied by using Hougen's tunneling matrix formalism. The microwave spectroscopy in the ground state was studied by several groups. The splitting due to the ν_{28} mode (C-CH_3 internal rotation) is small in the ground state and was not fully resolved in most of the previous studied rotational transitions. In this paper, we report the results of the pulsed nozzle-jet Fourier transform microwave spectroscopy so as to measure the fully resolved spectra. The submillmeter wave spectroscopy was also carried out. Our analysis including the previously reported transitions would be useful for astronomical observations. K. Kobayashi, T. Matsui, N. Mori, S. Tsunekawa, and N. Ohashi J. Mol. Spectrosc. {269}, 242 2011. K. Kobayashi, T. Matsui, S. Tsunekawa, and N. Ohashi J. Mol. Spectrosc. {255}, 164 2009. K. Kobayashi, T. Matsui, N. Mori, S. Tsunekawa, and N. Ohashi J. Mol. Spectrosc.{251}, 301 2008. K. Kobayashi, K. Murata, S. Tsunekawa, and N. Ohashi Int. Symposium on Mol. Spectrosc., 65th Meeting TH15 2010.} M. Hayashi, and K. Kuwada J. Mol. Structure {28}, 147 1975. M. Hayashi, and M. Adachi J. Mol. Structure {78}, 53 1982. S. Tsunekawa, Y. Kinai, Y. Kondo, H. Odashima, and K. Takagi Molecules {8}, 103 2003. U. Fuchs, G. Winnewisser, P. Groner, F. C. De Lucia, and E. Herbst Astrophys. J. Suppl. {144}, 277 2003.

Ion dipole capture cross sections at low ion and rotational energies - Comparison of integrated capture cross sections with reaction cross sections for NH3 and H2O parent-ion collisions.

NASA Technical Reports Server (NTRS)

Dugan, J. V., Jr.; Canright, R. B., Jr.

1972-01-01

The numerical capture cross section is calculated from the capture ratio, defined as the fraction of trajectories reaching a prescribed minimum separation of 3 A. The calculated capture cross sections for a rotational temperature of 77 K suggest large reaction cross sections in 80 K experiments for the large dipole-moment target, methyl cyanide.

The Bromodomain of Gcn5 Regulates Site Specificity of Lysine Acetylation on Histone H3*

PubMed Central

Cieniewicz, Anne M.; Moreland, Linley; Ringel, Alison E.; Mackintosh, Samuel G.; Raman, Ana; Gilbert, Tonya M.; Wolberger, Cynthia; Tackett, Alan J.; Taverna, Sean D.

2014-01-01

In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain “reader” function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential “histone/epigenetic code.” MS data are available via ProteomeXchange with identifier PXD001167. PMID:25106422

Shifting from Ziegler-Natta to Philips-type catalyst? A simple and safe access to reduced titanium systems for ethylene polymerization.

PubMed

Popoff, Nicolas; Espinas, Jeff; Gouré, Eric; Boyron, Olivier; Le Roux, Erwan; Basset, Jean-Marie; Gauvin, Régis M; De Mallmann, Aimery; Taoufik, Mostafa

2011-12-01

Silica-supported titanium(IV) chloride is readily reduced by Mashima and co-workers' reagent (1-methyl-3,6-bis(trimethylsilyl)-1,4-cyclohexadiene) to afford materials active in ethylene polymerisation without need of aluminum alkyl cocatalyst. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MET-2-Dependent H3K9 Methylation Suppresses Transgenerational Small RNA Inheritance.

PubMed

Lev, Itamar; Seroussi, Uri; Gingold, Hila; Bril, Roberta; Anava, Sarit; Rechavi, Oded

2017-04-24

In C. elegans, alterations to chromatin produce transgenerational effects, such as inherited increase in lifespan and gradual loss of fertility. Inheritance of histone modifications can be induced by double-stranded RNA-derived heritable small RNAs. Here, we show that the mortal germline phenotype, which is typical of met-2 mutants, defective in H3K9 methylation, depends on HRDE-1, an argonaute that carries small RNAs across generations, and is accompanied by accumulated transgenerational misexpression of heritable small RNAs. We discovered that MET-2 inhibits small RNA inheritance, and, as a consequence, induction of RNAi in met-2 mutants leads to permanent RNAi responses that do not terminate even after more than 30 generations. We found that potentiation of heritable RNAi in met-2 animals results from global hyperactivation of the small RNA inheritance machinery. Thus, changes in histone modifications can give rise to drastic transgenerational epigenetic effects, by controlling the overall potency of small RNA inheritance. Copyright © 2017 Elsevier Ltd. All rights reserved.

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE PAGES

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand; ...

2015-10-17

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Progressive Chromatin Condensation and H3K9 Methylation Regulate the Differentiation of Embryonic and Hematopoietic Stem Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ugarte, Fernando; Sousae, Rebekah; Cinquin, Bertrand

Epigenetic regulation serves as the basis for stem cell differentiation into distinct cell types, but it is unclear how global epigenetic changes are regulated during this process. Here, we tested the hypothesis that global chromatin organization affects the lineage potential of stem cells and that manipulation of chromatin dynamics influences stem cell function. Using nuclease sensitivity assays, we found a progressive decrease in chromatin digestion among pluripotent embryonic stem cells (ESCs), multipotent hematopoietic stem cells (HSCs), and mature hematopoietic cells. Quantitative high-resolution microscopy revealed that ESCs contain significantly more euchromatin than HSCs, with a further reduction in mature cells. Increasedmore » cellular maturation also led to heterochromatin localization to the nuclear periphery. Functionally, prevention of heterochromatin formation by inhibition of the histone methyltransferase G9A resulted in delayed HSC differentiation. Lastly, our results demonstrate global chromatin rearrangements during stem cell differentiation and that heterochromatin formation by H3K9 methylation regulates HSC differentiation.« less

Chaetocin reactivates the lytic replication of Epstein-Barr virus from latency via reactive oxygen species.

PubMed

Zhang, Shilun; Yin, Juan; Zhong, Jiang

2017-01-01

Oxidative stress, regarded as a negative effect of free radicals in vivo, takes place when organisms suffer from harmful stimuli. Some viruses can induce the release of reactive oxygen species (ROS) in infected cells, which may be closely related with their pathogenicity. In this report, chaetocin, a fungal metabolite reported to have antimicrobial and cytostatic activity, was studied for its effect on the activation of latent Epstein-Barr virus (EBV) in B95-8 cells. We found that chaetocin remarkably up-regulated EBV lytic transcription and DNA replication at a low concentration (50 nmol L -1 ). The activation of latent EBV was accompanied by an increased cellular ROS level. N-acetyl-L-cysteine (NAC), an ROS inhibitor, suppressed chaetocin-induced EBV activation. Chaetocin had little effect on histone H3K9 methylation, while NAC also significantly reduced H3K9 methylation. These results suggested that chaetocin reactivates latent EBV primarily via ROS pathways.

Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakravarty, R.; Rice, R.H.

1989-01-05

The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction ofmore » cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.« less

A computational model for histone mark propagation reproduces the distribution of heterochromatin in different human cell types.

PubMed

Schwämmle, Veit; Jensen, Ole Nørregaard

2013-01-01

Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.

Comparative Studies on Performance Characteristics of CI Engine Fuelled with Neem Methyl Ester and Mahua Methyl Ester and Its Respective Blends with Diesel Fuel.

PubMed

Ragit, S S; Mohapatra, S K; Kundu, K

2014-01-01

In the present investigation, neem and mahua methyl ester were prepared by transesterification using potassium hydroxide as a catalyst and tested in 4-stroke single cylinder water cooled diesel engine. Tests were carried out at constant speed of 1500 rev/min at different brake mean effective pressures. A series of tests were conducted which worked at different brake mean effective pressures, OkPa, 1kPa, 2kPa, 3kPa, 4kPa, 5kPa, 6kPa and 6.5kPa. The performance and exhaust emission characteristics of the diesel engine were analyzed and compared with diesel fuel. Results showed that BTE of NME was comparable with diesel and it was noted that the BTE of N0100 is 63.11% higher than that of diesel at part load whereas it reduces 11.2% with diesel fuel at full load. In case of full load, NME showed decreasing trend with diesel fuel. BTE of diesel was 15.37% and 36.89% at part load and full load respectively. The observation indicated that BTE for MME 100 was slightly higher than diesel at part loads. The specific fuel consumption (SFC) was more for almost all blends at all loads, compared to diesel. At part load, the EGT of MME and its blends were showing similar trend to diesel fuel and at full load, the exhaust gas temperature of MME and blends were higher than diesel. Based on this study, NME could be a substitute for diesel fuel in diesel engine.

Curcumin modulates the effect of histone modification on the expression of chemokines by type II alveolar epithelial cells in a rat COPD model.

PubMed

Gan, Lixing; Li, Chengye; Wang, Jian; Guo, Xuejun

2016-01-01

Studies have suggested that histone modification has a positive impact on various aspects associated with the progression of COPD. Histone deacetylase 2 (HDAC2) suppresses proinflammatory gene expression through deacetylation of core histones. To investigate the effect of histone modification on the expression of chemokines in type II alveolar epithelial cells (AEC II) in a rat COPD model and regulation of HDAC2 expression by curcumin in comparison with corticosteroid. The rat COPD model was established by cigarette smoke exposure and confirmed by histology and pathophysioloy. AEC II were isolated and cultured in vitro from the COPD models and control animals. The cells were treated with curcumin, corticosteroid, or trichostatin A, and messenger RNA (mRNA) expression of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2α (MIP-2α) was assessed by quantitative real-time polymerase chain reaction (RT-PCR). The expression of HDAC2 was measured by Western blot. Chromatin immunoprecipitation was used to detect H3/H4 acetylation and H3K9 methylation in the promoter region of three kinds of chemokine genes (IL-8, MCP-1, and MIP-2α). Compared to the control group, the mRNAs of MCP-1, IL-8, and MIP-2α were upregulated 4.48-fold, 3.14-fold, and 2.83-fold, respectively, in the AEC II from COPD model. The protein expression of HDAC2 in the AEC II from COPD model was significantly lower than from the control group ( P <0.05). The decreased expression of HDAC2 was negatively correlated with the increased expression of IL-8, MCP-1, and MIP-2α mRNAs (all P <0.05). The level of H3/H4 acetylation was higher but H3K9 methylation in the promoter region of chemokine genes was lower in the cells from COPD model than from the control group (all P <0.05). Curcumin downregulated the expression of MCP-1, IL-8, and MIP-2α, and the expression was further enhanced in the presence of corticosteroid. Moreover, curcumin restored HDAC2 expression, decreased the levels of H3/H4 acetylation, and increased H3K9 methylation in the promoter region of chemokine in the presence or absence of dexamethasone (all P <0.05). Curcumin may suppress chemokines and restore corticosteroid resistance in COPD through modulating HDAC2 expression and its effect on histone modification.

Deuterium and oxygen-18 isotope effects on nucleophilic displacement by monomeric water in aprotic solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurz, J.L.; Lee, J.

1980-07-30

The H/sub 2/O/D/sub 2/O rate-constant ratio for nucleophilic attack by water at a methyl carbon (eq 1) is reduced to very near unity when the water is a dilute solute in L/sub 2/O+CH/sub 3/X..-->..L/sub 2/O/sup +/CH/sub 3/+X/sup -/ a dipolar aprotic solvent. This effect was observed for three leaving groups (CH/sub 3/X equals 1-methylthiophenium ion (MeTh/sup +/), methyl perchlorate (MeOCl/sub 3/), and methyl trifluoromethanesulfonate (MeOTf)) and for two aprotic solvents acetonitrile (MeCn), and tetrahydrothiophene 1,1-dioxide (TMSO:'';sulfolane). It was also observed that the corresponding H/sub 2//sup 16/O/H/sub 2//sup 18/O rate-constant ratio is 1.002 +- 0.004 (95% confidence limits) for the reactionmore » of MeTh/sup +/ with dilute H/sub 2/O in TMSO/sub 2/ at 35/sup 0/C. Although alternative explanations exist (vide infra), these observations are consistent with a mechanism in which no significant positive charge is present on the L/sub 2/O oxygen in the rate-determining transition state and thus in which the rate-determining process does not involve (and is not preceded by) any significant covalent-bonding interaction between the nucleophile and the methyl carbon. All reactions were followed by uv spectroscopy in a Cary 16K spectrophotometer.« less

Acute dietary zinc deficiency before conception compromises oocyte epigenetic programming and disrupts embryonic development.

PubMed

Tian, X; Diaz, F J

2013-04-01

Recent findings show that zinc is an important factor necessary for regulating the meiotic cell cycle and ovulation. However, the role of zinc in promoting oocyte quality and developmental potential is not known. Using an in vivo model of acute dietary zinc deficiency, we show that feeding a zinc deficient diet (ZDD) for 3-5 days before ovulation (preconception) dramatically disrupts oocyte chromatin methylation and preimplantation development. There was a dramatic decrease in histone H3K4 trimethylation and global DNA methylation in zinc deficient oocytes. Moreover, there was a 3-20 fold increase in transcript abundance of repetitive elements (Iap, Line1, Sineb1, Sineb2), but a decrease in Gdf9, Zp3 and Figla mRNA. Only 53% and 8% of mature eggs reached the 2-cell stage after IVF in animals receiving a 3 and 5 days ZDD, respectively, while a 5 day ZDD in vivo reduced the proportion of 2-cells to 49%. In vivo fertilized 2-cell embryos cultured in vitro formed fewer (38%) blastocysts compared to control embryos (74%). Likewise, fewer blastocyst and expanded blastocyst were collected from the reproductive tract of zinc deficient animals on day 3.5 of pregnancy. This could be due to a decrease in Igf2 and H19 mRNA in ZDD blastocyst. Supplementation with a methyl donor (SAM) during IVM restored histone H3K4me3 and doubled the IVF success rate from 17% to 43% in oocytes from zinc deficient animals. Thus, the terminal period of oocyte development is extremely sensitive to perturbation in dietary zinc availability. Copyright © 2013 Elsevier Inc. All rights reserved.

A family of silver(I) complexes built with 2-sulfoterephthalic acid monosodium salt and different aminopyridine ligands: Syntheses, structures and properties

NASA Astrophysics Data System (ADS)

Zhang, Jie; Tan, Gai-Xiu; Liu, Bao-Lin; Dai, Yu-Bei; Xu, Na; Wen, Wei-Fen; Cao, Chong; Xiao, Hong-Ping

2017-05-01

Five Ag(I) coordination complexes, namely, [Ag6(2-stp)2(3-methyl-2-apy)3·H2O]n (1), [Ag3(2-stp)(4-methyl-2-apy)3]n (2), [Na2Ag18(2-stp)4(2-Hstp)4(5-methyl-2-apy)16 (H2O)4·11H2O]n (3), Ag3(2-stp)(6-methy-2-apy)4·H2O (4), and [Ag6(2-stp)2(6-methyl-2-apy)8(H2O)2·H2O]n (5) (2-NaH2stp = 2-sulfoterephthalic acid monosodium salt, 3-methyl-2-apy = 3-methyl-2-aminopyridine, 4-methyl-2-apy = 4-methyl-2-aminopyridine, 5-methyl-2-apy = 5-methyl-2-aminopyridine, 6-methyl-2-apy = 6-methyl-2-aminopyridine), have been synthesized and structurally characterized. Complexes 1 and 2 show two-dimensional network. In complex 3, the adjacent Ag10 units are bridged by 5-methyl-2-apy ligands to form a 2D infinite undulated sheet. Adjacent 2D sheets are linked by coordinative bonds between carboxylic oxygen atoms and Na(I) ions to form a 3D coordination polymer. Complex 4 is a 0-D discrete trinuclear molecule, and the self-complementary the Osbnd H⋯O and Nsbnd H⋯O hydrogen bonds incorporating hydrogen bond motifs extend these molecules into a 2D supramolecular framework. Compound 5 exhibits 1D-chain structure. However, complex 5 shows 3D supramolecular structure results from the linkage of neighboring layers through a rich hydrogen-bonding between uncoordinated sulfonates, amino groups and coordinated carboxylates. The thermogravimetric analyses and photoluminescence of the complexes were also investigated.

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

PubMed

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

Glucostatic regulation of (+)-(/sup 3/H)amphetamine binding in the hypothalamus: correlation with Na/sup +/, K/sup +/-ATPase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angel, I.; Hauger, R.L.; Luu, M.D.

1985-09-01

Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (37/sup 0/C) resulted in a time-dependent decrease in specific (+)-(/sup 3/H)amphetamine binding in the crude synaptosomal fraction prepared from these slices. The addition of D-glucose resulted in a dose- and time-dependent stimulation of (+)-(/sup 3/H)amphetamine binding, whereas incubations with L-glucose, 2-deoxy-D-glucose, or 3-O-methyl-D-glucose failed to increase the number of (+)-(/sup 3/H)amphetamine binding sites. Ouabain potently inhibited the glucose-induced stimulation of (+)-(/sup 3/H)amphetamine binding, suggesting the involvement of Na/sup +/, K/sup +/-ATPase. Preincubation of hypothalamic slices with glucose also resulted in an increase in Na/sup +/,K/sup +/-ATPase activity and the number ofmore » specific high-affinity binding sites for (/sup 3/H)ouabain, and a good correlation was observed between the glucose-stimulated increase in (+)-(/sup 3/H)amphetamine and (/sup 3/H)ouabain binding. These data suggest that the (+)-(/sup 3/H)amphetamine binding site in hypothalamus, previously linked to the anorectic actions of various phenylethylamines, is regulated both in vitro and in vivo by physiological concentrations of glucose. Glucose and amphetamine appear to interact at common sites in the hypothalamus to stimulate Na/sup +/,K/sup +/-ATPase activity, and the latter may be involved in the glucostatic regulation of appetite.« less

Reduced Histone H3 Lysine 9 Methylation Contributes to the Pathogenesis of Latent Autoimmune Diabetes in Adults via Regulation of SUV39H2 and KDM4C.

PubMed

Liu, Xi-Yu; Li, Hong

2017-01-01

Aims . Latent autoimmune diabetes in adults (LADA) is an autoimmune disease of which the mechanism is not clear. Emerging evidence suggests that histone methylation contributes to autoimmunity. Methods . Blood CD4 + T lymphocytes from 26 LADA patients and 26 healthy controls were isolated to detect histone H3 lysine 4 and H3 lysine 9 methylation status. Results . Reduced global H3 lysine 9 methylation was observed in LADA patients' CD4 + T lymphocytes, compared to healthy controls ( P < 0.05). H3 lysine 4 methylation was not statistically different. The reduced H3 lysine 9 methylation was associated with GADA titer but not correlated with glycosylated hemoglobin (HbA1c). When the LADA patient group was divided into those with complication and those without, relatively reduced global H3 lysine 9 methylation was observed in LADA patients with complication ( P < 0.05). The expression of histone methyltransferase SUV39H2 for H3 lysine 9 methylation was downregulated in LADA patients, and the expression of histone demethylase KDM4C which made H3 lysine 9 demethylation was upregulated. Conclusion . The reduction of histone H3 lysine 9 methylation which may due to the downregulation of methyltransferase SUV39H2 and the upregulation of demethylase KDM4C was found in CD4 + T lymphocytes of LADA patients.

Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass

PubMed Central

Williams, Lucy H.; Kalantry, Sundeep; Starmer, Joshua; Magnuson, Terry

2011-01-01

Repression of Xist RNA expression is considered a prerequisite to reversal of X-chromosome inactivation (XCI) in the mouse inner cell mass (ICM), and reactivation of X-linked genes is thought to follow loss of Xist RNA coating and heterochromatic markers of inactivation, such as methylation of histone H3. We analyzed X-chromosome activity in developing ICMs and show that reactivation of gene expression from the inactive-X initiates in the presence of Xist coating and H3K27me3. Furthermore, depletion of Xist RNA coating through forced upregulation of NANOG does not result in altered reactivation kinetics. Taken together, our observations suggest that in the ICM, X-linked gene transcription and Xist coating are uncoupled. These data fundamentally alter our perception of the reactivation process and support the existence of a mechanism to reactivate Xp-linked genes in the ICM that operates independently of loss of Xist RNA and H3K27me3 from the imprinted inactive-X. PMID:21471155

BRCC36, A Novel Subunit of a BRCA1/2 E3 Ubiquitin Ligase Complex: Candidate Breast Cancer Susceptibility Gene

DTIC Science & Technology

2005-09-01

Hancock, A.K. Godwin, And Anthony T. Yeung. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation. In Molecular Diagnostics (Ed. G...R.G.H. Cotton, L. Hancock, A.K. Godwin, And Anthony T. Yeung. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation. In Molecular ... Diagnostics (Ed. G. Patrinos and W Ansorge) in press 2005. 9 Godwin, A.K., Ph.D. E-CONCLUSIONS: E.1. "BRCC36, a Novel Subunit of a BRCA1/2 E3 Ubiquitin

Adolescent alcohol exposure alters lysine demethylase 1 (LSD1) expression and histone methylation in the amygdala during adulthood.

PubMed

Kyzar, Evan J; Zhang, Huaibo; Sakharkar, Amul J; Pandey, Subhash C

2017-09-01

Alcohol exposure in adolescence is an important risk factor for the development of alcoholism in adulthood. Epigenetic processes are implicated in the persistence of adolescent alcohol exposure-related changes, specifically in the amygdala. We investigated the role of histone methylation mechanisms in the persistent effects of adolescent intermittent ethanol (AIE) exposure in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 days on/off) or intermittent n-saline (AIS) during postnatal days (PND) 28-41 and used for behavioral and epigenetic studies. We found that AIE exposure caused a long-lasting decrease in mRNA and protein levels of lysine demethylase 1(Lsd1) and mRNA levels of Lsd1 + 8a (a neuron-specific splice variant) in specific amygdaloid structures compared with AIS-exposed rats when measured at adulthood. Interestingly, AIE increased histone H3 lysine 9 dimethylation (H3K9me2) levels in the central nucleus of the amygdala (CeA) and medial nucleus of the amygdala (MeA) in adulthood without producing any change in H3K4me2 protein levels. Acute ethanol challenge (2 g/kg) in adulthood attenuated anxiety-like behaviors and the decrease in Lsd1 + 8a mRNA levels in the amygdala induced by AIE. AIE caused an increase in H3K9me2 occupancy at the brain-derived neurotrophic factor exon IV promoter in the amygdala that returned to baseline after acute ethanol challenge in adulthood. These results indicate that AIE specifically modulates epizymes involved in H3K9 dimethylation in the amygdala in adulthood, which are possibly responsible for AIE-induced chromatin remodeling and adult psychopathology such as anxiety. © Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

The tumor suppressor SirT2 regulates cell cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation

USDA-ARS?s Scientific Manuscript database

The establishment of the epigenetic mark H4K20me1 (monomethylation of H4K20) by PR-Set7 during G2/M directly impacts S-phase progression and genome stability. However, the mechanisms involved in the regulation of this event are not well understood. Here we show that SirT2 regulates H4K20me1 depositi...

Let-7e inhibits TNF-α expression by targeting the methyl transferase EZH2 in DENV2-infected THP-1 cells.

PubMed

Zhang, Yingke; Zhang, Qianqian; Gui, Lian; Cai, Yan; Deng, Xiaohong; Li, Cheukfai; Guo, Qi; He, Xiaoshun; Huang, Junqi

2018-05-16

Tumor necrosis factor α (TNFα), an important inflammatory cytokine, is associated with dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), a severe pathological manifestation of dengue virus (DENV) infection. However, the regulatory mechanism of microRNA on TNFα is currently unknown. Our study showed that the TNFα expression increased immediately and then later decreased, while a marked increase for the miRNA let-7e was detected in dengue virus type 2 (DENV2)-infected peripheral blood mononuclear cells (PBMCs). From this study, we found that let-7e was able to inhibit TNFα expression, but bioinformatics analysis showed that the enhancer of zeste homolog 2 (EZH2) was the potential direct target of let-7e instead of TNFα. EZH2 methyl transferase can produce H3K27me3 and has a negative regulatory role. Using a dual-luciferase reporter assay and Western blotting, we confirmed that EZH2 was a direct target of let-7e and found that siEZH2 could inhibit TNFα expression. In the further study of the regulatory mechanism of EZH2 on TNFα expression, we showed that siEZH2 promoted EZH1 and H3K4me3 expression and inhibited H3K27me3 expression. More importantly, we revealed that siEZH2 down-regulated NF-κB p65 within the nucleus. These findings indicate that the let-7e/EZH2/H3K27me3/NF-κB p65 pathway is a novel regulatory axis of TNFα expression. In addition, we determined the protein differences between siEZH2 and siEZH2-NC by iTRAQ and found a number of proteins that might be associated with TNFα. © 2018 Wiley Periodicals, Inc.

Runx2 mediates epigenetic silencing of the bone morphogenetic protein-3B (BMP-3B/GDF10) in lung cancer cells

PubMed Central

2012-01-01

Background The Runt-related transcription factor Runx2 is essential for bone development but is also implicated in progression of several cancers of breast, prostate and bone, where it activates cancer-related genes and promotes invasive properties. The transforming growth factor β (TGF-β) family member bone morphogenetic protein-3B (BMP-3B/GDF10) is regarded as a tumor growth inhibitor and a gene silenced in lung cancers; however the regulatory mechanisms leading to its silencing have not been identified. Results Here we show that Runx2 is highly expressed in lung cancer cells and downregulates BMP-3B. This inverse relationship between Runx2 and BMP-3B expression is further supported by increased expression of BMP-3B in mesenchymal cells from Runx2 deficient mice. The ectopic expression of Runx2, but not DNA binding mutant Runx2, in normal lung fibroblast cells and lung cancer cells resulted in suppression of BMP-3B levels. The chromatin immunoprecipitation studies identified that the mechanism of Runx2-mediated suppression of BMP-3B is due to the recruitment of Runx2 and histone H3K9-specific methyltransferase Suv39h1 to BMP-3B proximal promoter and a concomitant increase in histone methylation (H3K9) status. The knockdown of Runx2 in H1299 cells resulted in decreased histone H3K9 methylation on BMP-3B promoter and increased BMP-3B expression levels. Furthermore, co-immunoprecipitation studies showed a direct interaction of Runx2 and Suv39h1 proteins. Phenotypically, Runx2 overexpression in H1299 cells increased wound healing response to TGFβ treatment. Conclusions Our studies identified BMP-3B as a new Runx2 target gene and revealed a novel function of Runx2 in silencing of BMP-3B in lung cancers. Our results suggest that Runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells. PMID:22537242

Distributions of methyl group rotational barriers in polycrystalline organic solids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beckmann, Peter A., E-mail: pbeckman@brynmawr.edu, E-mail: wangxianlong@uestc.edu.cn; Conn, Kathleen G.; Division of Education and Human Services, Neumann University, One Neumann Drive, Aston, Pennsylvania 19014-1298

We bring together solid state {sup 1}H spin-lattice relaxation rate measurements, scanning electron microscopy, single crystal X-ray diffraction, and electronic structure calculations for two methyl substituted organic compounds to investigate methyl group (CH{sub 3}) rotational dynamics in the solid state. Methyl group rotational barrier heights are computed using electronic structure calculations, both in isolated molecules and in molecular clusters mimicking a perfect single crystal environment. The calculations are performed on suitable clusters built from the X-ray diffraction studies. These calculations allow for an estimate of the intramolecular and the intermolecular contributions to the barrier heights. The {sup 1}H relaxation measurements,more » on the other hand, are performed with polycrystalline samples which have been investigated with scanning electron microscopy. The {sup 1}H relaxation measurements are best fitted with a distribution of activation energies for methyl group rotation and we propose, based on the scanning electron microscopy images, that this distribution arises from molecules near crystallite surfaces or near other crystal imperfections (vacancies, dislocations, etc.). An activation energy characterizing this distribution is compared with a barrier height determined from the electronic structure calculations and a consistent model for methyl group rotation is developed. The compounds are 1,6-dimethylphenanthrene and 1,8-dimethylphenanthrene and the methyl group barriers being discussed and compared are in the 2–12 kJ mol{sup −1} range.« less

Destruction of C2H4O2 isomers in ice-phase by X-rays: Implication on the abundance of acetic acid and methyl formate in the interstellar medium

NASA Astrophysics Data System (ADS)

Rachid, Marina G.; Faquine, Karla; Pilling, S.

2017-12-01

The C2H4O2 isomers methyl formate (HCOOCH3), acetic acid (CH3COOH) and glycoaldehyde (HOCH2CHO) have been detected in molecular clouds in the interstellar medium, as well as, hot cores, hot corinos and around protostellar objects. However, their abundances are very different, being methyl formate more abundant than the other two isomers. This fact may be related to the different destruction by ionizing radiation of these molecules. The goal of this work is experimentally study the photodissociation processes of methyl formate and acetic acid ices when exposed to broadband soft X-ray from 6 up to 2000 eV. The experiments were performed coupled to the SGM beamline in the Brazilian Synchrotron Light Source (LNLS/CNPEM) at Campinas, Brazil. The simulated astrophysical ices (12 K) were monitored throughout the experiment using infrared vibrational spectroscopy (FTIR). The analysis of processed ices allowed the determination of the effective destruction cross sections of the parent molecules as well as the effective formation cross section of daughter molecular species such as CO, CO2, H2O, CH4 and H2CO (only for methyl formate) and the hydrocarbons C2H6 and C5H10 (only for acetic acid). The half-lives of molecules at ices toward young stellar objects (YSOs) and inside molecular clouds (e.g. Sgr B2 and W51) due to the presence of incoming soft X-rays were estimated. We determined the effective formation rate and the branching ratios for assigned daughter species after the establishment of a chemical equilibrium. The main product from photodissociation of both methyl formate and acetic acid is CO, that can be formed by recombination of ions, formed during the photodissociation, in the ice surface. The relative abundance between methyl formate and acetic acid (NCH3COOH/NHCOOCH3) in different astronomical scenarios and their column density evolution in the presence of X-rays were calculated. Our results suggest that such radiation field can be one of the factors that explain the difference in the C2H4O2 isomers abundances.

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

PubMed Central

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

2012-01-01

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 (PHD4–6) 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 PHD4–6 reduce PHD4–6's binding ability and MLL4's catalytic activity. PHD4–6'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 PHD4–6's binding activity and is a novel epigenetic mechanism that underlies opposing effects of MLL4 and PRMT7 on cellular differentiation. PMID:23249737

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.

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

PubMed

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

PubMed Central

Li, Xiaojie; Qian, Weiqiang; Zhao, Yusheng; Wang, Chunlei; Shen, Jie; Zhu, Jian-Kang; Gong, Zhizhong

2012-01-01

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression. PMID:22733760

Reduced 3,4'-bipyrazoles from a simple pyrazole precursor: synthetic sequence, molecular structures and supramolecular assembly.

PubMed

Cuartas, Viviana; Insuasty, Braulio; Cobo, Justo; Glidewell, Christopher

2017-10-01

The reaction of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde and N-benzylmethylamine under microwave irradiation gives 5-[benzyl(methyl)amino]-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde, C 19 H 19 N 3 O, (I). Subsequent reactions under basic conditions, between (I) and a range of acetophenones, yield the corresponding chalcones. These undergo cyclocondensation reactions with hydrazine to produce reduced bipyrazoles which can be N-formylated with formic acid or N-acetylated with acetic anhydride. The structures of (I) and of representative examples from this reaction sequence are reported, namely the chalcone (E)-3-{5-[benzyl(methyl)amino]-3-methyl-1-phenyl-1H-pyrazol-4-yl}-1-(4-bromophenyl)prop-2-en-1-one, C 27 H 24 BrN 3 O, (II), the N-formyl derivative (3RS)-5'-[benzyl(methyl)amino]-3'-methyl-1',5-diphenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazole]-2-carbaldehyde, C 28 H 27 N 5 O, (III), and the N-acetyl derivative (3RS)-2-acetyl-5'-[benzyl(methyl)amino]-5-(4-methoxyphenyl)-3'-methyl-1'-phenyl-3,4-dihydro-1'H,2H-[3,4'-bipyrazole], which crystallizes as the ethanol 0.945-solvate, C 30 H 31 N 5 O 2 ·0.945C 2 H 6 O, (IV). There is significant delocalization of charge from the benzyl(methyl)amino substituent onto the carbonyl group in (I), but not in (II). In each of (III) and (IV), the reduced pyrazole ring is modestly puckered into an envelope conformation. The molecules of (I) are linked by a combination of C-H...N and C-H...π(arene) hydrogen bonds to form a simple chain of rings; those of (III) are linked by a combination of C-H...O and C-H...N hydrogen bonds to form sheets of R 2 2 (8) and R 6 6 (42) rings, and those of (IV) are linked by a combination of O-H...N and C-H...O hydrogen bonds to form a ribbon of edge-fused R 2 4 (16) and R 4 4 (24) rings.

Association of H3K9me3 and H3K27me3 repressive histone marks with breast cancer subtypes in the Nurses' Health Study.

PubMed

Healey, Megan A; Hu, Rong; Beck, Andrew H; Collins, Laura C; Schnitt, Stuart J; Tamimi, Rulla M; Hazra, Aditi

2014-10-01

Repressive histone tail modifications have been associated with molecular breast cancer subtypes. We investigated whether histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) were associated with tumor features and subtypes while adjusting for prospectively collected reproductive and lifestyle breast cancer risk factors. We have tissue microarray data with immunohistochemical marker information on 804 incident cases of invasive breast cancer diagnosed from 1976-2000 in the Nurses' Health Study. Tissue microarray sections were stained for global H3K9me3 and H3K27me3, and scored into four categories. Multivariate odds ratios (OR) and 95 % confidence intervals (CI) were calculated using logistic regression models for tumor features and subtypes, adjusting for breast cancer risk factors. While there were no significant associations between H3K9me3 and tumor features, H3K27me3 was significantly associated with lower grade tumors compared to high grade tumors in the multivariate model (OR = 1.95, 95 % CI 1.35-2.81, p = 0.0004). H3K27me3 was suggestively associated with estrogen receptor-positive (ER+) tumors (OR = 1.47, 95 % CI 0.97-2.23, p = 0.07). In subtype analyses, H3K27me3 was positively associated with the luminal A subtype compared to all other subtypes (OR = 1.42, 95 % CI 1.14-1.77, p = 0.002), and was inversely associated with HER2-type (OR = 0.58, 95 % CI 0.37-0.91, p = 0.02) and basal-like breast cancer (OR = 0.52, 95 % CI 0.36-0.76, p = 0.0006). In the largest immunohistochemical examination of H3K9me3 and H3K27me3 in breast cancer, we found that H3K27me3 positivity, but not H3K9me3, was associated with lower grade tumors and the luminal A subtype after adjusting for reproductive and lifestyle breast cancer risk factors.

[Ex-situ remediation of PAHs contaminated site by successive methyl-beta-cyclodextrin enhanced soil washing].

PubMed

Sun, Ming-Ming; Teng, Ying; Luo, Yong-Ming; Li, Zhen-Gao; Jia, Zhong-Jun; Zhang, Man-Yun

2013-06-01

Polycyclic aromatic hydrocarbon (PAH) polluted sites caused by abandoned coking plants have attracted great attentions. This study investigated the feasibility of using methyl-beta-cyclodextrin (MCD) solution to enhance ex situ soil washing for extracting PAHs. Treatment with elevated temperature (50 degrees C) in combination with ultrasonication (35 kHz, 30 min) at 100 g x L(-1) was effective. It was found that 96.7% +/- 2.4% of 3-ring PAH, 89.7% +/- 3.2% of 4-ring PAH, 76.3% +/- 2.2% of 5 (+6)-ring PAH and 91.3% +/- 3.1% of total PAHs were removed from soil after five successive washing cycles. The desorption kinetics of PAHs from contaminated soil was determined before and after successive washings. The 400 h Tenax extraction of PAHs from soil was decreasing gradually with increasing washing times. Furthermore, the F(r), F(sl), k(r), k(sl) and k(vl) were significantly lower than those of CK (P < 0.01). Therefore, considering the removal efficiency and potential environmental risk after soil )ashing, successive washing three times was selected as a reasonable parameter. These results have practical implications for site risk assessment and cleanup strategies.

A novel ''donor-π-acceptor'' type fluorescence probe for sensing pH: mechanism and application in vivo.

PubMed

Chao, Jianbin; Wang, Huijuan; Zhang, Yongbin; Yin, Caixia; Huo, Fangjun; Song, Kailun; Li, Zhiqing; Zhang, Ting; Zhao, Yaqin

2017-11-01

A novel pH fluorescent probe 1-(pyren-1-yl)-3-(6-methoxypridin-3-yl)-acrylketone, (PMPA), which had a pyrene structure attached to methoxypyridine, was synthesized for monitoring extremely acidic and alkaline pH. The pH titrations indicated that PMPA displayed a remarkable emission enhancement with a pK a of 2.70 and responded linearly to minor pH fluctuations within the extremely acidic range of 1.26-3.97. Interestingly, PMPA also exhibited strong pH-dependent characteristics with pK a 9.32 and linear response to extreme-alkalinity range of 8.54-10.36. In addition, PMPA displayed a good selectivity, excellent photostability and large Stokes shift (167nm). Furthermore, the probe PMPA had excellent cell membrane permeability and was applied successfully to rapidly detect pH in living cells. pH value in these organs was closely related to many diseases, so these findings suggested that the probe had potential application in pH detecting for disease diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Vacuum ultraviolet and infrared spectra of condensed methyl acetate on cold astrochemical dust analogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sivaraman, B.; Nair, B. G.; Mason, N. J.

2013-12-01

Following the recent report of the first identification of methyl acetate (CH{sub 3}COOCH{sub 3}) in the interstellar medium (ISM), we have carried out vacuum ultraviolet (VUV) and infrared (IR) spectroscopy studies on methyl acetate from 10 K until sublimation in an ultrahigh vacuum chamber simulating astrochemical conditions. We present the first VUV and IR spectra of methyl acetate relevant to ISM conditions. Spectral signatures clearly showed molecular reorientation to have started in the ice by annealing the amorphous ice formed at 10 K. An irreversible phase change from amorphous to crystalline methyl acetate ice was found to occur between 110more » K and 120 K.« less

Spatial distribution of epigenetic modifications in Brachypodium distachyon embryos during seed maturation and germination.

PubMed

Wolny, Elzbieta; Braszewska-Zalewska, Agnieszka; Hasterok, Robert

2014-01-01

Seed development involves a plethora of spatially and temporally synchronised genetic and epigenetic processes. Although it has been shown that epigenetic mechanisms, such as DNA methylation and chromatin remodelling, act on a large number of genes during seed development and germination, to date the global levels of histone modifications have not been studied in a tissue-specific manner in plant embryos. In this study we analysed the distribution of three epigenetic markers, i.e. H4K5ac, H3K4me2 and H3K4me1 in 'matured', 'dry' and 'germinating' embryos of a model grass, Brachypodium distachyon (Brachypodium). Our results indicate that the abundance of these modifications differs considerably in various organs and tissues of the three types of Brachypodium embryos. Embryos from matured seeds were characterised by the highest level of H4K5ac in RAM and epithelial cells of the scutellum, whereas this modification was not observed in the coleorhiza. In this type of embryos H3K4me2 was most evident in epithelial cells of the scutellum. In 'dry' embryos H4K5ac was highest in the coleorhiza but was not present in the nuclei of the scutellum. H3K4me1 was the most elevated in the coleoptile but absent from the coleorhiza, whereas H3K4me2 was the most prominent in leaf primordia and RAM. In embryos from germinating seeds H4K5ac was the most evident in the scutellum but not present in the coleoptile, similarly H3K4me1 was the highest in the scutellum and very low in the coleoptile, while the highest level of H3K4me2 was observed in the coleoptile and the lowest in the coleorhiza. The distinct patterns of epigenetic modifications that were observed may be involved in the switch of the gene expression profiles in specific organs of the developing embryo and may be linked with the physiological changes that accompany seed desiccation, imbibition and germination.

World Trade Center (WTC) dust exposure in mice is associated with inflammation, oxidative stress and epigenetic changes in the lung

PubMed Central

Sunil, Vasanthi R.; Vayas, Kinal N.; Fang, Mingzhu; Zarbl, Helmut; Massa, Christopher; Gow, Andrew J.; Cervelli, Jessica A.; Kipen, Howard; Laumbach, Robert J.; Lioy, Paul J.; Laskin, Jeffrey D.; Laskin, Debra L.

2017-01-01

Exposure to World Trade Center (WTC) dust has been linked to respiratory disease in humans. In the present studies we developed a rodent model of WTC dust exposure to analyze lung oxidative stress and inflammation, with the goal of elucidating potential epigenetic mechanisms underlying these responses. Exposure of mice to WTC dust (20 μg, i.t.) was associated with upregulation of heme oxygenase-1 and cyclooxygenase-2 within 3 days, a response which persisted for at least 21 days. Whereas matrix metalloproteinase was upregulated 7 days post-WTC dust exposure, IL-6RA1 was increased at 21 days; conversely, expression of mannose receptor, a scavenger receptor important in particle clearance, decreased. After WTC dust exposure, increases in methylation of histone H3 lysine K4 at 3 days, lysine K27 at 7 days and lysine K36, were observed in the lung, along with hypermethylation of Line-1 element at 21 days. Alterations in pulmonary mechanics were also observed following WTC dust exposure. Thus, 3 days post-exposure, lung resistance and tissue damping were decreased. In contrast at 21 days, lung resistance, central airway resistance, tissue damping and tissue elastance were increased. These data demonstrate that WTC dust-induced inflammation and oxidative stress are associated with epigenetic modifications in the lung and altered pulmonary mechanics. These changes may contribute to the development of WTC dust pathologies. PMID:27986442

Structural, Optical, Electrical and Photoelectrical Properties of 2-Amino-4-(5-bromothiophen-2-yl)-5,6-dihydro-6-methyl-5-oxo-4 H-pyrano[3,2-c] quinoline-3-carbonitrile Films

NASA Astrophysics Data System (ADS)

Mansour, A. M.; El-Taweel, F. M. A.; Abu El-Enein, R. A. N.; El-Menyawy, E. M.

2017-12-01

2-Amino-4-(5-bromothiophen-2-yl)-5,6-dihydro-6-methyl-5-oxo-4 H-pyrano[3,2-c] quinoline-3-carbonitrile (ABDQC) powder was synthesized and showed thermal stability up to 535 K. ABDQC films were successfully prepared using thermal evaporation. X-ray diffraction showed that the prepared ABDQC powder had a polycrystalline structure, whereas the deposited film had an amorphous structure. The surface morphology of the films was characterized by using a transmission electron microscope. Optical absorption properties of ABDQC films were investigated by spectrophotometric measurements of the transmittance and reflectance in the wavelength range 200-2500 nm. The films were found to have indirect allowed optical band gap of 2.5 eV. Current-voltage characteristics of Au/ABDQC/ p-Si/Al were measured at different temperatures (300-420 K) in which the temperature dependence of the diode parameters has been discussed. Under illumination, the device showed open-circuit voltage and short-circuit current of 0.09 V and 3.26 × 10-4 A, respectively.

Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

PubMed Central

Gordon, Catherine A.; Hartono, Stella R.; Chédin, Frédéric

2013-01-01

Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease. PMID:23894490

Identification and Characterization of the Spodoptera Su(var) 3-9 Histone H3K9 trimethyltransferase and Its Effect in AcMNPV Infection

PubMed Central

Li, Binbin; Li, Sisi; Yin, Juan; Zhong, Jiang

2013-01-01

Histone H3-lysine9 (H3K9) trimethyltransferase gene Su(var) 3-9 was cloned and identified in three Spodoptera insects, Spodoptera frugiperda ( S . frugiperda ), S . exigua and S . litura . Sequence analysis showed that Spodoptera Su(var) 3-9 is highly conserved evolutionarily. Su(var) 3-9 protein was found to be localized in the nucleus in Sf9 cells, and interact with histone H3, and the heterochromatin protein 1a (HP1a) and HP1b. A dose-dependent enzymatic activity was found at both 27 °C and 37 °C in vitro, with higher activity at 27 °C. Addition of specific inhibitor chaetocin resulted in decreased histone methylation level and host chromatin relaxation. In contrast, overexpression of Su(var) 3-9 caused increased histone methylation level and cellular genome compaction. In AcMNV-infected Sf9 cells, the transcription of Su(var) 3-9 increased at late time of infection, although the mRNA levels of most cellular genes decreased. Pre-treatment of Sf9 cells with chaetocin speeded up viral DNA replication, and increased the transcription level of a variety of virus genes, whereas in Sf9 cells pre-transformed with Su(var) 3-9 expression vector, viral DNA replication slow down slightly. These findings suggest that Su(var) 3-9 might participate in the viral genes expression an genome replication repression during AcMNPV infection. It provided a new insight for the understanding virus–host interaction mechanism. PMID:23894480

Arabidopsis histone methyltransferase SET DOMAIN GROUP8 mediates induction of the jasmonate/ethylene pathway genes in plant defense response to necrotrophic fungi.

PubMed

Berr, Alexandre; McCallum, Emily J; Alioua, Abdelmalek; Heintz, Dimitri; Heitz, Thierry; Shen, Wen-Hui

2010-11-01

As sessile organisms, plants have to endure a wide variety of biotic and abiotic stresses, and accordingly they have evolved intricate and rapidly inducible defense strategies associated with the activation of a battery of genes. Among other mechanisms, changes in chromatin structure are thought to provide a flexible, global, and stable means for the regulation of gene transcription. In support of this idea, we demonstrate here that the Arabidopsis (Arabidopsis thaliana) histone methyltransferase SET DOMAIN GROUP8 (SDG8) plays a crucial role in plant defense against fungal pathogens by regulating a subset of genes within the jasmonic acid (JA) and/or ethylene signaling pathway. We show that the loss-of-function mutant sdg8-1 displays reduced resistance to the necrotrophic fungal pathogens Alternaria brassicicola and Botrytis cinerea. While levels of JA, a primary phytohormone involved in plant defense, and camalexin, a major phytoalexin against fungal pathogens, remain unchanged or even above normal in sdg8-1, induction of several defense genes within the JA/ethylene signaling pathway is severely compromised in response to fungal infection or JA treatment in mutant plants. Both downstream genes and, remarkably, also upstream mitogen-activated protein kinase kinase genes MKK3 and MKK5 are misregulated in sdg8-1. Accordingly, chromatin immunoprecipitation analysis shows that sdg8-1 impairs dynamic changes of histone H3 lysine 36 methylation at defense marker genes as well as at MKK3 and MKK5, which normally occurs upon infection with fungal pathogens or methyl JA treatment in wild-type plants. Our data indicate that SDG8-mediated histone H3 lysine 36 methylation may serve as a memory of permissive transcription for a subset of defense genes, allowing rapid establishment of transcriptional induction.

Modification of Tet1 and histone methylation dynamics in dairy goat male germline stem cells.

PubMed

Zheng, Liming; Zhai, Yuanxin; Li, Na; Wu, Chongyang; Zhu, Haijing; Wei, Zhuying; Bai, Chunling; Li, Guangpeng; Hua, Jinlian

2016-04-01

Tet (ten-eleven translocation) protein 1 is a key enzyme for DNA demethylation, which modulates DNA methylation and gene transcription. DNA methylation and histone methylation are critical elements in self-renewal of male germline stem cells (mGSCs) and spermatogenesis. mGSCs are the only type of adult stem cells able to achieve intergenerational transfer of genetic information, which is accomplished through differentiated sperm cells. However, numerous epigenetic obstacles including incomplete DNA methylation and histone methylation dynamics make establishment of stable livestock mGSC cell lines difficult. The present study was conducted to detect effects of DNA methylation and histone methylation dynamics in dairy goat mGSCs self-renewal and proliferation, through overexpression of Tet1. An immortalized dairy goat mGSC cell line bearing mouse Tet1 (mTet1) gene was screened and characteristics of the cells were assayed by quantitative real-time PCR (qRT-PCR), immunofluorescence assay, western blotting, fluorescence activated cell sorting (FACS) and use of the cell counting kit (CCK8) assay. The screened immortalized dairy goat mGSC cell line bearing mTet1, called mGSC-mTet1 cells was treated with optimal doxycycline (Dox) concentration to maintain Tet1 gene expression. mGSC-mTet1 cells proliferated at a significantly greater rate than wild-type mGSCs, and mGSCs-specific markers such as proliferating cell nuclear antigen (PCNA), cyclinD1 (CCND1), GDNF family receptor alpha 1 (Gfra1) and endogenic Tet1, Tet2 were upregulated. The cells exhibited not only reduction in level of histone methylation but also changes in nuclear location of that methylation marker. While H3K9me3 was uniformly distributed throughout the nucleus of mGSC-mTet1 cells, it was present in only particular locations in mGSCs. H3K27me3 was distributed surrounding the edges of nuclei of mGSC-mTet1 cells, while it was uniformly distributed throughout nuclei of mGSCs. Our results conclusively demonstrate that modification of mGSCs with mTet1 affected mGSC maintenance and seemed to promote establishment of stable goat mGSC cell lines. Taken together, our data suggest that Tet1 had novel and dynamic roles for regulating maintenance of pluripotency and proliferation of mGSCs by forming complexes with PCNA and histone methylation dynamics. This may provide new solutions for mGSCs stability and livestock mGSC cell line establishment. © 2016 John Wiley & Sons Ltd.

The induction of H3K9 methylation by PIWIL4 at the p16{sup Ink4a} locus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugimoto, Keiki; Kage, Hidenori; Aki, Naomi

The field of epigenetics has made progress by the identification of the small RNA-mediated epigenetic modification. However, little is known about the key proteins. Here, we report that the human PIWI-like family is a candidate protein that is involved in the pathway responsible for chromatin remodeling. The PIWI-like family proteins, expressed as the Flag-fusion proteins, formed a bulky body and localized to the nuclear periphery. Transient transfection of PIWI-like 4 (PIWIL4), only member of the PIWI-like family that was ubiquitously expressed in human tissues, induced histone H3 lysine 9 methylation at the p16{sup Ink4a} (CDKN2A) locus. The elevated level ofmore » histone methylation resulted in the downregulation of the p16{sup Ink4a} gene. These results suggest PIWIL4 plays important roles in the chromatin-modifying pathway in human somatic cells.« less

Nizatidine, N-[2-[[[2-[(dimethylamino)methyl]-4-thiazolyl]methyl]thio]ethyl]-N‧-methyl-2-nitro-1,1-ethenediamine

NASA Astrophysics Data System (ADS)

Stephenson, Gregory A.; Wozniak, Timothy J.; Stowell, Joseph G.; Byrn, Stephen R.

1996-06-01

C 12H 21N 5S 2O 2, Mr = 331.46, triclinic, P1¯, a = 7.3645(5), b = 8.8785(9), c = 25.678(4) Å, α = 89.35(1)°, β = 82.49(1)°, γ = 87.46(1)°, V = 1662.9(5) Å 3, Z = 4, D x = 1.324 g cm -3, λ(Cu Kα) = 1.54184 Å, μ = 29.57 cm -1, F(000) = 704.0, T = 293 K, final R = 0.057 for 4830 observed reflections. Nizatidine, Axid®, is a selective H 2-receptor antagonist used in the treatment of duodenal ulcers. The pharmaceutical nizatidine crystallizes with two independent molecules in the asymmetric unit. The two molecules have similar conformations, with the exception of the dimethylamino group. The bisalkylamino-2-nitroethene moiety differs from the structurally related hydrogen oxalate salt form of ranitidine in that the nitro group of nizatidine is the cis geometrical isomer with respect to the ethylamino group.