Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Demethylation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1984

Induction of EBV DNA demethylation and of EBV-specific transcription in Daudi cells treated with TPA and n-butyrate.

The Epstein-Barr virus (EBV) abortive producer cell line Daudi may be induced to an incomplete cycle of viral production by chemicals such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-butyrate. These chemicals greatly increase expression of the early antigen but not that of viral capsid antigen. DNA from these cells was cleaved with various restriction enzymes specific for the mCG sites. Viral sequences (mostly in an episomal form) were tested for their methylation patterns and compared to the methylation pattern of the producer cell line B95-8. The BamHI C, V, and H EBV DNA fragments appeared to be nonmethylated in B95-8 whether or not induced by TPA. The same fragments were partially methylated in noninduced Daudi cells. Treatment of the latter with TPA and n-butyrate increased the amount of viral DNA per cell, and hypomethylation of some EBV DNA sites occurred in parallel. Transcription changes were noted only at the level of the BamHI H fragment; cytoplasmic RNA molecules specific for this region were detected only after induction.

Burkitt Lymphoma↗
PubMed2005

Stage- and site-specific DNA demethylation during neural cell development from embryonic stem cells.

Activation of the transcription factor STAT3 is important for astrocyte differentiation during neural development. Demethylation of the methyl-CpG dinucleotide in the STAT3 binding site in the promoter of the glial fibrillary acidic protein (GFAP) gene, a marker for astrocytes, was previously shown to be a crucial cue for neural progenitors to express this gene in response to astrogenic signals during brain development. In this study, we analyzed the methylation status of the STAT3 binding site in the GFAP gene promoter during neural cell development from mouse embryonic stem (ES) cells in vitro. The CpG dinucleotide in the STAT3 binding site in the GFAP gene promoter exhibited a high incidence of cytidine-methylation in undifferentiated pluripotent ES cells. The high incidence of methylation of this particular cytidine was maintained in ES cell-derived neuroectoderm-like cells, but it underwent demethylation when the neural lineage cells became competent to express GFAP in response to a STAT3 activation signal. In contrast, hypermethylation of the CpG site was maintained in non-neural cells generated from the same ES cells. Progressive demethylation of the STAT3 binding site in the GFAP gene promoter was also observed in primary embryonic neuroepithelial cells during in vitro culture, whereas non-neural cells maintained hypermethylation of this site even after culture. Taken together, these results demonstrate that the astrocyte gene-specific cytidine-demethylation is programmed when neural progenitors from pluripotent cells are committed to a neural lineage that is capable of producing astrocytes.

Animals↗
PubMed1997

The role of DNA demethylation during development.

The somatic genomic methylation pattern which plays a role in the suppression of basal gene activity is established anew in each generation through developmentally regulated de novo and demethylation steps. Demethylation appears to be carried out by a nucleotide exchange reaction which may involve RNA molecules, and is directed to specific loci in the genome through interactions between cis acting elements and trans acting factors.

Animals↗
PubMed2002

Quantitative analysis of DNA demethylation and transcriptional reactivation of the FMR1 gene in fragile X cells treated with 5-azadeoxycytidine.

In fragile X syndrome, hypermethylation of the expanded CGG repeat and of the upstream promoter leads to transcriptional silencing of the FMR1 gene. Absence of the FMR1 protein results in mental retardation. We previously proved that treatment with 5-azadeoxycytidine (5-azadC) of fragile X cell lines results in reactivation of the FMR1 gene. We now show that this treatment causes passive demethylation of the FMR1 gene promoter. We employed the bisulfite-sequencing technique to detect the methylation status of individual CpG sites in the entire promoter region, upstream of the CGG repeat. Lymphoblastoid cell lines of fragile X males with full mutations of different sizes were tested before and after treatment with 5-azadC at various time points. We observed that individual cells are either completely unmethylated or not, with few relevant exceptions. We also investigated the extent of methylation in the full mutation (CGG repeat) itself by Southern blot analysis after digestion with methylation-sensitive enzymes Fnu4HI and McrBC and found that the CGG repeat remains at least partially methylated in many cells with a demethylated promoter. This may explain the quantitative discrepancy between the large extent of promoter demethylation and the limited levels of FMR1 transcriptional reactivation estimated by quantitative real-time fluorescent RT-PCR analysis.

Azacitidine↗
PubMed1983

Ultraviolet radiation-induced metallothionein-I gene activation is associated with extensive DNA demethylation.

Ultraviolet irradiation (UV) of cadmium-sensitive S49 mouse cells induces a large increase in cadmium-resistant variants. About 30%-40% of these variants make metallothionein (MT)-I mRNA while S49 cells do not. S49 cells contain two copies of the MT-I gene; both alleles are heavily methylated but can be conveniently distinguished by the methylation status of a single Hpa II site. In lines expressing MT-I, one allele becomes completely demethylated at all methylation-sensitive restriction sites examined over at least a 2.5 kb region spanning the MT-I gene. Activation of a quiescent gene by UV has implications for understanding the initiation of carcinogenesis.

Animals↗
PubMed1995

Mechanism of active DNA demethylation during embryonic development and cellular differentiation in vertebrates.

Incubation of hemimethylated and labelled oligodeoxynucleotides with nuclear extracts from differentiating chicken embryos and mouse myoblasts resulted in the replacement of m5C by C. One of the enzymes involved is m5CpG endonuclease. It cleaves only m5CpG and not, m5CpT, m5CpA, m5CpC or m6ApT. The enzyme is not sequence specific and catalyses the reaction in the presence of high concentrations of EDTA or EGTA.

Animals↗
PubMed1998

Induction of XIST expression from the human active X chromosome in mouse/human somatic cell hybrids by DNA demethylation.

X chromosome inactivation occurs early in mammalian development to transcriptionally silence one of the pair of X chromosomes in females. The XIST RNA, a large untranslated RNA that is expressed solely from the inactive X chromosome, is implicated in the process of inactivation. As previous studies have shown that the XIST gene is methylated on the active X chromosome, we have treated a mouse/human somatic cell hybrid retaining an active human X chromosome with demethylating agents to determine whether expression of the human XIST gene could be induced. Stable expression of XIST was observed after several rounds of demethylation and stability of XIST expression correlated with the loss of methylation at the three sites analysed. We conclude that methylation is sufficient to inhibit expression of the XIST gene in somatic cell hybrids. No loss of expression was detected for eight other X-linked genes from the active X chromosome that was expressing XIST , suggesting that additional developmental or species-specific factors are required for the inactivation process.

Animals↗
PubMed1999

DNA demethylation during the differentiation of 3T3-L1 cells affects the expression of the mouse GLUT4 gene.

GLUT4 is the major glucose transporter in adipose tissue and skeletal and cardiac muscles. We examined the mechanisms underlying GLUT4 gene expression in 3T3-L1 cells, which express the gene during their differentiation from preadipocytes to adipocytes. In transient transfections, the activity of a mouse GLUT4 promoter extending to -100 bp in the 5'-flanking region did not differ significantly between 3T3-L1 preadipocytes and adipocytes. Promoter activity up to -590 bp in preadipocytes and adipocytes showed a 70% lower and 228% higher activity, respectively, than promoter activity extending to -100 bp. We also examined methylation status of the GLUT4 promoter. Up to -100 bp, there were five CpG sites at -11, -30, -58, -63, and -75 bp. Two CpG sites at -11 and -30 bp were highly methylated in preadipocytes (60 and 92%, respectively) and highly demethylated in adipocytes (28.6 and 25%, respectively). Conversely, three CpG sites at -58, -63, and -75 bp were highly demethylated in both preadipocytes and adipocytes (<12%). In gel mobility-shift assays, a fragment extending from -40 to -1 bp generated a methylation-sensitive band with nuclear extracts from both preadipocytes and adipocytes when the CpG sites were methylated. Southwestern analysis identified a protein of approximately 55 kDa that bound strongly to the methylated probe. Furthermore, methylation of the CpG sites inhibited promoters extending to -50 or -70 bp. These results suggest that in addition to cell type-specific transcription factor, methylation of specific CpG sites and the methylation-sensitive transcription factor contribute to GLUT4 gene regulation during 3T3-L1 differentiation.

3T3 Cells↗
PubMed2004

Transplacental exposure to the DNA demethylating agent, 5-AZA-CdR, affects the sexual behavior of CD-1 male mice.

Intrauterine exposure to 5-AZA-2'-deoxycytidine (5-AZA-CdR) alters gene expression causing malformations, abnormal post-natal growth and altered reproductive capacity. To elucidate whether the phenomenon observed in 5-AZA-CdR in utero exposed male mice was a behavioral alteration, at gestation day (GD) 10, CD-1 pregnant mice were administered 1mg/kg i.p. of 5-AZA-CdR or saline solution. After parturition, the number and sex of pups were recorded. While litter size was not affected, the ratio of male to female offspring was altered in treated mice. To determine whether the phenotypic observation of male gender corresponded to the appropriate genotype, presence of Sry gene in 5-AZA-CdR F1 males was determined. At 3 months of age, the male sexual behavior test outlined by Chubb was conducted. Presence of vaginal plug and pregnancy were determined in the natural breeding phase. Mount latency and number of mounts per mouse were assessed in the behavioral test phase. In utero exposed male mice resulted in diminished mating behavior (as measured by vaginal plug presence, mount latency and number of mounts) and reduced sexual interest while exposed to a receptive female. While normal presence of Sry gene was observed, mating behavior was altered in exposed males suggesting that the reproductive alteration could be attributed to a behavioral phenomenon.

Animals↗
PubMed2005

An epigenetic approach to the treatment of advanced MDS; the experience with the DNA demethylating agent 5-aza-2'-deoxycytidine (decitabine) in 177 patients.

During the last 10 years, three European phase II studies were performed to investigate the treatment of elderly patients with myelodysplastic syndrome (MDS) with low-dose 5-aza-2'-deoxycytidine (decitabine, DAC). All these European trial data were reviewed on the basis of the International Prognostic Scoring System (IPSS) risk criteria and the response criteria as recently published by an international working group. To investigate the results in a larger cohort of patients and to determine risk factors, all data were pooled with some observations from the PCH 95-06 US phase II study. The response rate in the 177 patients evaluated (median age 70 years) was 49%. The median response duration was 36 weeks, and the median survival was 15 months. Analysis of the data according to sex, age, French-American-British classification, percentage of blasts in the bone marrow, IPSS risk group, lactate dehydrogenase and cytogenetics did not reveal any factor predictive of response. Overall, 69% of patients benefited, including those with stable disease during therapy. Response duration was significantly shorter with increasing risk (according to the IPSS classification). Haemoglobin level and neutrophil count showed an inverse correlation to the IPSS classification. Univariate analysis showed a significantly inferior survival for elderly patients (>75 years of age) and for those with high levels of serum lactate dehydrogenase (LDH) (more than two times the normal values). Patients with high-risk cytogenetic abnormalities according to the IPSS risk criteria showed better overall survival than those with intermediate-risk abnormalities. When analysed according to the IPSS risk classification, high-risk patients had worse survival prospects following decitabine therapy than those with intermediate risk; however, compared to the originally reported IPPS outcomes for high-risk patients, they probably showed better survival. During the treatment period, 18% of the patients progressed towards acute leukaemia. Decitabine showed a rather low toxicity profile in this elderly patient group. In conclusion, low-dose decitabine is an active drug for the treatment of MDS patients, even for those older than 75 years with bad prognostic characteristics.

Adult↗
PubMed2003

Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells.

How T cells regulate interleukin 4 (IL-4) expression is not completely understood. We show here that single-positive thymocytes express IL-4, but attenuate GATA-3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. Type 2 polarization blocks Dnmt1 recruitment, enhances histone H3 Lys4 methylation (indicative of accessible chromatin) and initiates DNA demethylation of the locus. Dnmt1-/- CD4 and CD8 T cells derepress IL-4 expression considerably, demethylate DNA and increase H3 Lys4 methylation without affecting GATA-3 expression, demonstrating that Dnmt1 and DNA methylation are essential for proper Il4 regulation. These results indicate that Dnmts, DNA and histone methylation, and transcription factors 'collaborate' to determine appropriate Il4 expression patterns.

Animals↗
PubMed2006

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks.

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3' to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

Animals↗
PubMed2004

5-Aza-2'-deoxycytidine stimulates inducible nitric oxide synthase induction in C6 astrocytoma cells.

The influence of a nucleoside analog 5-aza-2'-deoxycytidine (5-AzadC) on inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) production in various rat cell types was investigated. In C6 astrocytoma cell line and primary astrocytes, 5-AzadC enhanced proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-1)-triggered NO synthesis in a time- and dose-dependent manner. In contrast, 5-AzadC did not potentiate NO production in IFN-gamma-stimulated macrophages, fibroblasts, or endothelial cells. Blockade of transcription or translation in C6 cells abolished the observed effect, suggesting the iNOS gene expression, rather than its catalytic activity, as a target for the drug action. Accordingly, 5-AzadC upregulated IFN-gamma-induced expression of iNOS mRNA in C6 astrocytes. The effect of 5-AzadC on astrocyte NO release was blocked by the inhibitor of p44/42 mitogen activated protein kinase-dependent signaling. Finally, the observed stimulatory effect of 5-AzadC on iNOS expression was apparently independent of DNA demethylation, as DNA digestion with methylation-sensitive restriction enzyme HpaII showed that 5-AzadC failed to demethylate cellular DNA in conditions used for iNOS induction.

Animals↗
PubMed1997

Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA.

We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.

Animals↗
PubMed2005

Epigenetic treatment of hematopoietic malignancies: in vivo targets of demethylating agents.

Although the first studies using DNA demethylating agents at low doses in hematologic neoplasia and hemoglobinopathies were initiated more than 20 years ago, development of this type of nonintensive treatment has only been spurred in the last 6 to 8 years by the discovery of many genes that are specifically hypermethylated in cancer. These provide a powerful rationale for using azanucleosides (and other small molecules being developed for DNA demethylation) as a novel means of pharmacologic targeting of cancer cells that is distinct from low-dose chemotherapy. Encouraging response rates of about 50% in myelodysplasia with 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine or DAC) have resulted in a number of phase III studies being initiated in this disorder. The development of such drugs for the treatment of acute myeloid leukemia (AML) is ongoing. While the specificity of DNA demethylation has been delineated by studying distinct genes or sets of genes, and proof-of-principle studies of in vivo methylation report demethylation and reactivation of genes like p15/INK4b and gamma-globin, responses to demethylating agents may be more complex. Specifically, so-called cancer testis antigens (CTAs) are intriguing targets for demethylation, since they are silenced in many hematopoietic disorders and may be reactivated by epigenetic therapy. Thus, demethylating agents and histone deacetylase inhibitors may also induce a T-cell-mediated antileukemic or antitumor effect.

Antigens, Neoplasm↗
PubMed1996

Promoter-specific modulation of insulin-like growth factor II genomic imprinting by inhibitors of DNA methylation.

The insulin-like growth factor II (IGF-II) gene is maternally imprinted in most normal tissues with only the paternal allele being transcribed. In several human tumors, however, IGF-II is expressed from both parental alleles. To explore the underlying mechanism of IGF-II imprinting, we have examined the effect of DNA demethylation in cultured human and mouse astrocyte cells. An increased expression of IGF-II was observed when these cells were treated with the DNA demethylating agents, 5-azacytidine or 2-deoxy-5-azacytidine. Allelic analysis indicated that, following DNA demethylation, the increment in IGF-II mRNA was primarily derived from the normally suppressed maternal allele. Examination of promoter usage revealed that only the most proximal promoter (mP3 in mouse and hP4 in human) responded to DNA demethylating agents, whereas the expression of IGF-II from the other promoters remained unchanged. The enhanced expression of IGF-II from these promoters suggests the presence of a methylation-response element in or near mP3 and hP4. This study indicates that DNA demethylating agents increase IGF-II expression primarily by stimulating the normally imprinted allele through the activation of the most proximal IGF-II promoter.

Animals↗
PubMed1994

Exemption of satellite DNA from demethylation in immortalized differentiated derivatives of F9 mouse embryonal carcinoma cells.

DNA methylation in F9 embryonal carcinoma cells at various stages of retinoic acid-induced differentiation was compared to that in immortalized differentiated derivatives of this cell line. Different repetitive sequences, such as L1 LINE, GAPDH pseudogenes, and major and minor satellite DNA, lost their methylation to similar extents during F9 differentiation into parietal endoderm cells. In the immortalized derivatives 8a and P1, which phenotypically resemble F9 cells at intermediate stages of differentiation, methylation patterns diverged: Methylation of L1 and GAPDH sequences was strongly and moderately diminished, respectively, whereas methylation of satellite DNA was almost as high as that in stem cells. P19 embryonal carcinoma cells and D3 embryonic stem cells possessed methylation patterns similar to those of F9 stem cells. In immortalized cell lines with differentiated phenotypes derived from P19, methylation was diminished uniformly throughout the genome, but the overall level of methylation remained higher than that in terminally differentiated F9 cells. Treatment with the methylation inhibitor deoxyazacytidine halted the proliferation of 8a, P1, and F9 cells and elicited marked changes in morphology. However, markers of differentiation were not induced in F9 cells. The finding that all immortalized differentiated derivatives of embryonal carcinoma cells retained a higher level of DNA methylation--at least in parts of their genomes--than did terminally differentiated cells may indicate a function of demethylation of DNA and of satellite sequences in particular in terminal differentiation of extraembryonic tissues.

Animals↗
PubMed2002

The oncoprotein Set/TAF-1beta, an inhibitor of histone acetyltransferase, inhibits active demethylation of DNA, integrating DNA methylation and transcriptional silencing.

Histone hypoacetylation and DNA hypermethylation are hallmarks of gene silencing. Although a role for DNA methylation in regulating histone acetylation has been established, it is not clear how and whether epigenetic histone markings influence DNA modifications in transcriptional silencing. We have previously shown that induction of histone acetylation by trichostatin A promotes demethylation of ectopically methylated DNA (Cervoni, N., and Szyf, M. (2001) J. Biol. Chem. 276, 40778-40787). The oncoprotein Set/TAF-Ibeta is a subunit of the recently identified inhibitor of acetyltransferases complex that inhibits histone acetylation by binding to and masking histone acetyltransferase targets (Seo, S. B., McNamara, P., Heo, S., Turner, A., Lane, W. S., and Chakravarti, D. (2001) Cell 104, 119-130). We show here that the overexpression of Set/TAF-Ibeta, whose expression is up-regulated in multiple tumor tissues, inhibits demethylation of ectopically methylated DNA resulting in gene silencing. Overexpression of a mutant Set/TAF-Ibeta that does not inhibit histone acetylation is defective in inhibiting DNA demethylation. Taken together, these results are consistent with a novel regulatory role for Set/TAF-Ibeta, integrating epigenetic states of histones and DNA in gene regulation and provide a new mechanism that can explain how hypermethylation of specific regions might come about by inhibition of demethylation in cancer cells.

Acetyltransferases↗