Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression (original) (raw)
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Molecular and Cellular Biology, 2007
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. Thes...
Molecular and Cellular Biology, 2000
We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.
Proceedings of the National Academy of Sciences, 2011
Methylation on lysine 9 of histone H3 (H3K9me) and DNA methylation play important roles in the transcriptional silencing of specific genes and repetitive elements. Both marks are detected on class I and II endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs). Recently, we reported that the H3K9-specific lysine methyltransferase (KMTase) Eset/Setdb1/KMT1E is required for H3K9me3 and the maintenance of silencing of ERVs in mESCs. In contrast, G9a/Ehmt2/KMT1C is dispensable, despite the fact that this KMTase is required for H3K9 dimethylation (H3K9me2) and efficient DNA methylation of these retroelements. Transcription of the exogenous retrovirus (XRV) Moloney murine leukemia virus is rapidly extinguished after integration in mESCs, concomitant with de novo DNA methylation. However, the role that H3K9 KMTases play in this process has not been addressed. Here, we demonstrate that G9a, but not Suv39h1 or Suv39h2, is required for silencing of newly integrated Moloney murine leukemia virus-based vectors in mESCs. The silencing defect in G9a −/− cells is accompanied by a reduction of H3K9me2 at the proviral LTR, indicating that XRVs are direct targets of G9a. Furthermore, de novo DNA methylation of newly integrated proviruses is impaired in the G9a −/− line, phenocopying proviral DNA methylation and silencing defects observed in Dnmt3a-deficient mESCs. Once established, however, maintenance of silencing of XRVs, like ERVs, is dependent exclusively on the KMTase Eset. Taken together, these observations reveal that in mESCs, the H3K9 KMTase G9a is required for the establishment, but not for the maintenance, of silencing of newly integrated proviruses. epigenetics | covalent histone modification | long terminal repeat R etroviruses have colonized all classes of vertebrates and are responsible for a range of pathologies in mammals, including cancer in distantly related species and acquired immunodeficiency syndrome in humans. Although productive retroviral infection by a subset of retroviruses is cytopathic, proviral elements of the acuteand slow-transforming classes induce tumorigenesis by expressing viral oncogenes and perturbing the expression of cellular genes, respectively. Given the potential deleterious effects of retroviral infection, a number of cell autonomous pathways that act at the transcriptional or post-transcriptional stages of the retroviral replicative cycle have evolved to inhibit retroviral expression (1, 2).
DNA methylation directs a time-dependent repression of transcription initiation
Current Biology, 1997
Background: The regulation of DNA methylation is required for differential expression of imprinted genes during vertebrate development. Earlier studies that monitored the activity of the Herpes simplex virus (HSV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-dependent repression of gene activity. Here, we examine the mechanism of methylation-dependent HSV tk gene regulation by direct determination of nucleoprotein organization during the establishment of a transcriptionally silenced state after microinjection of templates with defined methylation states into Xenopus oocyte nuclei. Results: The transcriptional silencing conferred by a methylated DNA segment was not immediate, as methylated templates were initially assembled into active transcription complexes. The eventual loss of DNase I hypersensitive sites and inhibition of transcription at the HSV tk promoter only occurred after several hours. Flanking methylated vector DNA silenced the adjacent unmethylated HSV tk promoter, indicative of a dominant transmissible repression originating from a center of methylation. The resulting repressive nucleoprotein structure silenced transcription in the presence of activators that are able to overcome repression of transcription by nucleosomes. Conclusions: Silencing of transcription by DNA methylation is achieved at the level of transcription initiation and involves the removal of transcriptional machinery from active templates. This transcriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which are able to inhibit transcription more effectively than nucleosomes alone.
Journal of Biological …, 2008
The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.
Linking DNA methylation and histone modification -patterns and paradigms
Although it is now accepted that chromatin structure has a large impact on the regulation of gene expression, little is known about how individual epigenetic marks are set up and then maintained through DNA replica-tion and cell division. Chemical modification of DNA or of chromatin-associated proteins, particularly histones, has a major influence on chromatin structure and gene expression. In animal cells, DNA can be modified by methylation of cytosine residues in CpG dinucleotides, and the N-terminal tails of histone proteins are subject to a wide range of different modifications, including acetylation, methylation, phosphorylation and ubiq-uitylation. All of these chemical changes seem to have a substantial influence on chromatin structure and gene function, which differs depending on the type and location of the modification. In this Review we take advantage of evidence from recent genetic, biochemical and microarray studies to explore the relationship between DNA methylation and histone modification, particularly focusing on methylation of histone H3 at lysine 9 (H3K9) and 27 (H3K27), which are important modifications for gene repression. Although DNA methylation and histone modification are carried out by different chemical reactions and require different sets of enzymes, there seems to be a biological relationship between the two systems that plays a part in modulating gene repression programming in the organism. We describe how DNA meth-ylation and specific histone modifications influence each other during mammalian development. It seems that the relationship can work in both directions: his-tone methylation can help to direct DNA methylation patterns, and DNA methylation might serve as a template for some histone modifications after DNA repli-cation. Recent evidence indicates that, at the molecular level, these connections might be accomplished through direct interactions between histone and DNA methyl-transferases. We then discuss how histone modification and DNA methylation can have different roles in gene silencing, with histone modifications providing labile transcriptional repression and DNA methylation being a highly stable silencing mark that is not easily reversed. Finally, we address how understanding the relationship between these two types of modification can help us to decipher the epigenetic blocks that inhibit cellular reprogramming and to understand mechanisms of gene repression in cancer. Generating modification patterns Generation of the basal bimodal DNA methylation pattern. The basic methylation pattern of the animal genome is bimodal: almost all CpG dinucleotides are methylated, except those located in CpG islands, which are to a large extent constitutively unmodified. The DNA methylation pattern is erased in the early embryo and then re-established in each individual at approximately the time of implantation 1,2. Differential methylation is established through two counteracting mechanisms: a wave of indiscriminate de novo methylation 3 and a mechanism for ensuring that CpG islands remain unmethylated. The precise details of how CpG islands are protected are not completely elucidated, but early studies using transgenic mice and transfection experiments in embryonic stem cells suggested that protection
Histones and DNA methylation in mammalian chromatin. Differential inhibition by histone H1
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1991
Histones (from calf thymus or from human placenta), if renatured in the presence of EDTA, caused a severe inhibition of in vitro methylation of doubie-stranded DNA (from M/crococcus /uteus) by human placenta DNA methyltransferase. The absence of EDTA during the histone renaturation procedure abolished -at least in the 'physiological' range of the histones/DNA ratio -the inhibition. The H t component was responsible for this inhibition, no effect being exerted by the other histones. H n preparations were more effective if renatured in the presence of EDTA -90% inhibition being reached at a 0.3:1 (w/w) HI/DNA ratio. It seems likely that the requirement for the presence of EDTA during the renaturation process is correlated to its ability to induce a fairly stable ordered conformation of the bistones, although this effect could also be shown with the 'inactive' H,., H~ and H 3 components, and was instead less evident with histone H n. The restriction to histone H t of the ability to inhibit enzymic DNA methylation may account for the lower methylation levels present in the internucleosomal DNA of mammalian chromatin.
Evidence for gene silencing by endogenous DNA methylation
Proceedings of the National Academy of Sciences, 1998
Transformed cells can spontaneously silence genes by de novo methylation, and it is generally assumed that this is due to DNA methyltransferase activity. We have tested the alternative hypothesis that gene silencing could be due to the uptake of 5-methyl-dCMP into DNA, via the di- and triphosphonucleotides. 5-Methyl-dCMP would be present in cells from the ongoing repair of DNA. We have isolated a strain of Chinese hamster ovary (CHO) cells, designated HAM − , which spontaneously silences two tested genes at a very high frequency. We have shown that this strain incorporates 5-[ 3 H]methyldeoxycytidine into 5-methylcytosine and thymine in DNA. It also has low 5-methyl-dCMP deaminase activity. Another HAM + strain has high deaminase activity and a very low frequency of gene silencing. The starting strain, CHO K1, has a phenotype intermediate between HAM − and HAM + .