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Yi Zhang - One of the best experts on this subject based on the ideXlab platform.
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TET-mediated active DNA Demethylation: mechanism, function and beyond
Nature Reviews Genetics, 2017Co-Authors: Xiaoji Wu, Yi ZhangAbstract:In mammals, DNA methylation in the form of 5-methylcytosine (5mC) can be actively reversed to unmodified cytosine (C) through TET dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution or thymine DNA glycosylase (TDG)-dependent base excision repair. In the past few years, biochemical and structural studies have revealed mechanistic insights into how TET and TDG mediate active DNA Demethylation. Additionally, many regulatory mechanisms of this process have been identified. Technological advances in mapping and tracing the oxidized forms of 5mC allow further dissection of their functions. Furthermore, the biological functions of active DNA Demethylation in various biological contexts have also been revealed. In this Review, we summarize the recent advances and highlight key unanswered questions. Active DNA Demethylation in mammals is achieved through TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), followed by replication-dependent dilution of oxidized 5mC or thymine DNA glycosylase (TDG)-mediated excision of 5fC and 5caC coupled with base excision repair. Active DNA Demethylation is regulated at various levels, including substrate and cofactor availability, post-transcriptional and post-translational regulation of TET and TDG, and genomic localization of the Demethylation machinery. Studies of tissue distribution, genomic distribution and the dynamics of oxidized 5mC provide insights into the mechanism and function of active DNA Demethylation as well as the potential roles of oxidized 5mC. Active DNA Demethylation and oxidized 5mC are involved in pre-implantation embryo development, primordial germ cell development, pluripotency and differentiation, as well as neuronal functions. In certain biological contexts, such as in pre-implantation embryos, the biological meaning of TET-mediated oxidation is not fully understood. In some other biological contexts, such as neurons, the extent of active DNA Demethylation and its function require further study. TET may function in a catalytic-activity-independent manner. Further analysis is needed to distinguish the functions of the TET proteins themselves from the function of active DNA Demethylation. Emerging evidence suggests an interplay between TET, active DNA Demethylation and genomic instability and the DNA damage response. A key mode of regulating DNA methylation is through active Demethylation driven by TET-mediated oxidation of 5-methylcytosine (5mC). This Review discusses our latest understanding of the mechanisms and regulation of active DNA Demethylation, and the roles of active Demethylation (and the oxidized 5mC intermediates) in gene regulation, genome stability, development and disease.
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tet mediated active DNA Demethylation mechanism function and beyond
Nature Reviews Genetics, 2017Co-Authors: Xiaoji Wu, Yi ZhangAbstract:A key mode of regulating DNA methylation is through active Demethylation driven by TET-mediated oxidation of 5-methylcytosine (5mC). This Review discusses our latest understanding of the mechanisms and regulation of active DNA Demethylation, and the roles of active Demethylation (and the oxidized 5mC intermediates) in gene regulation, genome stability, development and disease.
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aid apobec deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways.
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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways. Activation-induced deaminase (AID)/APOBEC–family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA Demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for Demethylation have been described, pathways involving deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of deamination-coupled Demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA Demethylation.
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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways.
Guoliang Xu - One of the best experts on this subject based on the ideXlab platform.
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DNA Demethylation pathways additional players and regulators
BioEssays, 2017Co-Authors: Agnieszka Kolano, Matthias Bochtler, Guoliang XuAbstract:DNA Demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA Demethylation. Here, we review these new developments.
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a crispr based approach for targeted DNA Demethylation
Cell discovery, 2016Co-Authors: Xingxing Xu, Guoliang Xu, Lei Zhang, Xufang Li, Kangcheng Ruan, Feng Wang, Ronggui HuAbstract:In mammalian cells, DNA methylation critically regulates gene expression and thus has pivotal roles in myriad of physiological and pathological processes. Here we report a novel method for targeted DNA Demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Initially, modified single guide RNAs (sgRNAs) (sgRNA2.0) were constructed by inserting two copies of bacteriophage MS2 RNA elements into the conventional sgRNAs, which would facilitate the tethering of the Tet1 catalytic domain (Tet-CD), in fusion with dCas9 or MS2 coat proteins, to the targeted gene loci. Subsequently, such system was shown to significantly upregulate transcription of the target genes, including RANKL, MAGEB2 or MMP2, which was in close correlation to DNA Demethylation of their neighboring CpGs in the promoters. In addition, the dCas9/sgRNA2.0-directed Demethylation system appeared to afford efficient Demethylation of the target genes with tenuous off-target effects. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits.
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uracil DNA glycosylase ung promotes tet mediated DNA Demethylation
Journal of Biological Chemistry, 2016Co-Authors: Guifang Xu, Tianpeng Gu, Zhimei Xu, Magnar Bjørås, Guoliang Xu, G. Chen, Hans E. Krokan, Yarui DuAbstract:In mammals, active DNA Demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active Demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA Demethylation. To search for other factors that might contribute to Demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA Demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA Demethylation.
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Oxidative DNA Demethylation mediated by Tet enzymes
National Science Review, 2015Co-Authors: Guoliang Xu, Jiemin WongAbstract:DNA modification, methylation of cytosine (5mC), and oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) can have profound effects on genome function in animals. These modifications are intricately involved in DNA methylation reprograming dynamics during mammalian development. Together, they contribute to cell lineage restriction and maintenance, while also undergoing dynamic changes during cellular transitions and induced reprograming. The last five years have seen an intense research focus on enzymatic DNA Demethylation, triggered by the discovery of 5hmC and Tet dioxygenases. In this review, we evaluate recent findings that have provided new insights into the mechanisms underlying DNA Demethylation and its effect on developmental regulation.
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gadd45a promotes DNA Demethylation through tdg
Nucleic Acids Research, 2015Co-Authors: Zheng Li, Tianpeng Gu, Guoliang Xu, Alain R. Weber, Primo Schär, Jiazhen Shen, Binzhong Li, Fuchou Tang, Hailin WangAbstract:Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA Demethylation in vertebrates. However, it remained unclear how they contribute to the Demethylation process. Here, we demonstrate that Gadd45a promotes active DNA Demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative Demethylation. The connection of Gadd45a with oxidative Demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the Demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated Demethylation pathway.
Hongjun Song - One of the best experts on this subject based on the ideXlab platform.
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Active DNA Demethylation and 5-Hydroxymethylcytosine
Epigenetic Regulation in the Nervous System, 2020Co-Authors: Guo Li Ming, Hongjun SongAbstract:Cytosine C5-methylation (5mC) is the best characterized epigenetic modification in eukaryotic cells. In the mammalian nervous system, both DNA methylation and Demethylation have been shown to play important roles in dynamic regulation of gene expression programs under normal and pathological conditions. In this chapter, we review our current understanding of the molecular mechanisms underlying active DNA Demethylation, with a focus on the role of the recently identified DNA base 5-hydroxymethylcytosine (5hmC) in this process. We also discuss the potential role of active DNA Demethylation in the nervous system.
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emerging roles of tet proteins and 5 hydroxymethylcytosines in active DNA Demethylation and beyond
Cell Cycle, 2011Co-Authors: Yijing Su, Guo Li Ming, Chun Zhong, Hongjun SongAbstract:Cytosine methylation is the major epigenetic modification of metazoan DNA. Although there is strong evidence that active DNA Demethylation occurs in animal cells, the molecular details of this process are unknown. The recent discovery of the TET protein family (TET1–3) 5-methylcytosine hydroxylases has provided a new entry point to reveal the identity of the long-sought DNA demethylase. Here, we review the recent progress in understanding the function of TET proteins and 5-hydroxymethylcytosine (5hmC) through various biochemical and genomic approaches, the current evidence for a role of 5hmC as an early intermediate in active DNA Demethylation and the potential functions of TET proteins and 5hmC beyond active DNA Demethylation. We also discuss how future studies can extend our knowledge of this novel epigenetic modification.
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hydroxylation of 5 methylcytosine by tet1 promotes active DNA Demethylation in the adult brain
Cell, 2011Co-Authors: Yijing Su, Guo Li Ming, Chun Zhong, Hongjun SongAbstract:Cytosine methylation is the major covalent modification of mammalian genomic DNA and plays important roles in transcriptional regulation. The molecular mechanism underlying the enzymatic removal of this epigenetic mark, however, remains elusive. Here, we show that 5-methylcytosine (5mC) hydroxylase TET1, by converting 5mCs to 5-hydroxymethylcytosines (5hmCs), promotes DNA Demethylation in mammalian cells through a process that requires the base excision repair pathway. Though expression of the 12 known human DNA glycosylases individually did not enhance removal of 5hmCs in mammalian cells, Demethylation of both exogenously introduced and endogenous 5hmCs is promoted by the AID (activation-induced deaminase)/APOBEC (apolipoprotein B mRNA-editing enzyme complex) family of cytidine deaminases. Furthermore, Tet1 and Apobec1 are involved in neuronal activity-induced, region-specific, active DNA Demethylation and subsequent gene expression in the dentate gyrus of the adult mouse brain in vivo. Our study suggests a TET1-induced oxidation-deamination mechanism for active DNA Demethylation in mammals.
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DNA excision repair proteins and gadd45 as molecular players for active DNA Demethylation
Cell Cycle, 2009Co-Authors: Dengke K, Guo Li Ming, Hongjun SongAbstract:DNA cytosine methylation represents an intrinsic modification signal of the genome that plays important roles in heritable gene silencing, heterochromatin formation and certain transgenerational epigenetic inheritance. In contrast to the process of DNA methylation that is catalyzed by specific classes of methyltransferases, molecular players underlying active DNA Demethylation have long been elusive. Emerging biochemical and functional evidence suggests that active DNA Demethylation in vertebrates can be mediated through DNA excision repair enzymes, similar to the well-known repair-based DNA Demethylation mechanism in Arabidopsis. As key regulators, non-enzymatic Gadd45 proteins function to recruit enzymatic machineries and promote coupling of deamination, base and nucleotide-excision repair in the process of DNA Demethylation. In this article, we review recent findings and discuss functional and evolutionary implications of such mechanisms underlying active DNA Demethylation.
Rahul M Kohli - One of the best experts on this subject based on the ideXlab platform.
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aid apobec deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways.
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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways. Activation-induced deaminase (AID)/APOBEC–family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA Demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for Demethylation have been described, pathways involving deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of deamination-coupled Demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to deamination. Further, upon AID/APOBEC overexpression, the deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine deamination are one intrinsic barrier to the proposed function of deaminases in DNA Demethylation.
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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA Demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Li Shen, Yi Zhang, Yu Ye, Hana L Goldschmidt, James T Stivers, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA Demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA Demethylation pathways.
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demystifying DNA Demethylation
Science, 2011Co-Authors: Christopher S Nabel, Rahul M KohliAbstract:Variability and adaptability are necessary for overcoming the challenges of multicellular life. To address this need, nature has evolved a substantial enzymatic toolbox for altering cytosine within the genome. Methylation of the nucleotide cytosine (C) at the 5-position of the base has profound impacts on gene expression and cellular identity. The reverse of this process, DNA Demethylation, is equally important for cleaning the genomic slate during embryogenesis or achieving rapid reactivation of previously silenced genes. Although the mechanism of DNA methylation has been rigorously established, active DNA Demethylation in mammals has remained enigmatic, as disparate observations have failed to coalesce into a consistent model. Cytosine deamination, oxidation, and base excision repair enzymes have been proposed in a dizzying variety of combinations ( 1 ). Against this backdrop, two reports in this issue, by Ito et al. ( 2 ) on page 1300 and He et al. on page 1303 ( 3 ), help bring new clarity to the mechanistic model for DNA Demethylation.
Moshe Szyf - One of the best experts on this subject based on the ideXlab platform.
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DNA Demethylation induced by the methyl-CpG-binding domain protein MBD3.
Gene, 2008Co-Authors: Shelley E. Brown, Matthew Suderman, Michael Hallett, Moshe SzyfAbstract:Abstract The methyl-CpG binding domain protein MBD3 has been shown to be essential for embryonic development and differentiation, and to act by suppressing gene expression through the recruitment of co-repressor complexes. We have recently shown that MBD3 is also involved in maintaining the demethylated and active state of rRNA genes, and that depletion of MBD3 results in hypermethylation of rRNA promoters. The possibility that MBD3 could also trigger DNA Demethylation of RNA polymerase II-transcribed genes has not been addressed. In this study we used a gain-of-function approach to examine whether MBD3 expression alters DNA methylation states in a living cell and whether it has specific targets for DNA methylation or Demethylation in the genome. We used a combination of methylated DNA immunoprecipitation (mDIP) and hybridization to a human promoter tiling microarray to examine the landscape of DNA methylation patterns in response to MBD3 overexpression. We demonstrate that MBD3 induces genomic DNA Demethylation and that it has specific targets in the genome with which it associates. Demethylation is localized to promoter regions with intermediate CpG density, and promoters with predicted transcription factor binding sites for NF-Y were significantly affected. These data demonstrate a causal relationship between MBD3 and DNA Demethylation of genomic targets in cells.
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Acetylation-induced transcription is required for active DNA Demethylation in methylation-silenced genes.
Molecular and Cellular Biology, 2007Co-Authors: Ana C. D'alessio, Ian C.g. Weaver, Moshe SzyfAbstract:A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent Demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA Demethylation. Histone acetylation precedes but is not sufficient to trigger DNA Demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, α-amanitin, or CDK7-specific small interfering RNA inhibits DNA Demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after Demethylation. TSA-induced Demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA Demethylation follows rather than precedes early transcription and point towards a novel function for DNA Demethylation as a memory of actively transcribed genes.
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Transcription is required for active DNA Demethylation
Cancer Research, 2007Co-Authors: Ana C. D'alessio, Ian C.g. Weaver, Shelley E. Brown, Moshe SzyfAbstract:1052 A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA) induced replication-independent Demethylation assay in HEK 293 cells and chromatin immuno-precipitation assays with pertinent histone modification antibodies and bisulfite mapping, we study the temporal and causal relationship between chromatin and active DNA Demethylation. We show that RNA transcription is required for DNA Demethylation. Histone acetylation precedes but is not sufficient to trigger DNA Demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, a-amanitin, or CDK7 siRNA inhibits DNA Demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the CMV promoter only after Demethylation. TSA induced Demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA Demethylation follows rather than precedes early transcription and points towards a novel function for DNA Demethylation - a memory of actively transcribed genes. We are currently investigating if SWI/SNF chromatin remodeling factors associated with transcription, which have been shown to set a platform for histone eviction by allowing a more open configuration of the chromatin, may favor DNA demethylase access to the DNA. Supported by a grant from the National Cancer Institute of Canada to MS. Shelley E. Brown is supported by a CIHR fellowship.
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valproate induces replication independent active DNA Demethylation
Journal of Biological Chemistry, 2003Co-Authors: Nancy Detich, Veronica Bovenzi, Moshe SzyfAbstract:In this report, we demonstrate that valproic acid (VPA), a drug that has been used for decades in the treatment of epilepsy and as a mood stabilizer, triggers replication-independent active Demethylation of DNA. Thus, this drug can potentially reverse DNA methylation patterns and erase stable methylation imprints on DNA in non-dividing cells. Recent discoveries support a role for VPA in the regulation of methylated genes; however, the mechanism has been unclear because it is difficult to dissociate active Demethylation from the absence of DNA methylation during DNA synthesis. We therefore took advantage of an assay that measures active DNA Demethylation independently from other DNA methylation and DNA replication activities in human embryonal kidney 293 cells. We show that VPA induces histone acetylation, DNA Demethylation, and expression of an ectopically methylated CMV-GFP plasmid in a dose-dependent manner. In contrast, valpromide, an analogue of VPA that does not induce histone acetylation, does not induce Demethylation or expression of CMV-GFP. Furthermore, we illustrate that methylated DNA-binding protein 2/DNA demethylase (MBD2/dMTase) participates in this reaction since antisense knockdown of MBD2/dMTase attenuates VPA-induced Demethylation. Taken together, our data support a new mechanism of action for VPA as enhancing intracellular demethylase activity through its effects on histone acetylation and raises the possibility that DNA methylation is reversible independent of DNA replication by commonly prescribed drugs.
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Ras Induces a General DNA Demethylation Activity in Mouse Embryonal P19 Cells
Journal of Biological Chemistry, 1995Co-Authors: Moshe Szyf, Johanne Theberge, Vera BozovicAbstract:Abstract We demonstrate that expression of v-Ha-ras in mouse embryonal P19 cells results in genome-wide Demethylation. Analysis of the pattern of methylation of specific genes reveals that different types of genes are demethylated in the ras transfectants: skeletal muscle specific genes, a gene specifically expressed in the adrenal cortex (c21), ubiquitous genes, and exogenously introduced sequences. Transient transfection and in vitro Demethylation assays reveal that the ras transfectants express high levels of a general DNA Demethylation activity. This demonstrates that the general DNA Demethylation activity in mouse embryonal cells is controlled by an important cellular signal transducer and that DNA demethylase is a potential downstream effector of Ras.