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Results for “DNA Demethylation”

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At least 19 recordsLinked to original sources
PubMed2005

Preventing transcriptional gene silencing by active DNA demethylation.

DNA methylation is important for stable transcriptional gene silencing. DNA methyltransferases for de novo as well as maintenance methylation have been well characterized. However, enzymes responsible for active DNA demethylation have been elusive and several reported mechanisms of active demethylation have been controversial. There has been a critical need for genetic analysis in order to firmly establish an in vivo role for putative DNA demethylases. Mutations in the bifunctional DNA glycosylase/lyase ROS1 in Arabidopsis cause DNA hypermethylation and transcriptional silencing of specific genes. Recombinant ROS1 protein has DNA glycosylase/lyase activity on methylated but not unmethylated DNA substrates. Therefore, there is now strong genetic evidence supporting a base excision repair mechanism for active DNA demethylation. DNA demethylases may be critical factors for genome wide hypomethylation seen in cancers and possibly important for epigenetic reprogramming during somatic cell cloning and stem cell function.

Amino Acid Sequence↗
PubMed2006

Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation.

DNA methylation is a stable epigenetic mark for transcriptional gene silencing in diverse organisms including plants and many animals. In contrast to the well characterized mechanism of DNA methylation by methyltransferases, the mechanisms and function of active DNA demethylation have been controversial. Genetic evidence suggested that the DNA glycosylase domain-containing protein ROS1 of Arabidopsis is a putative DNA demethylase, because loss-of-function ros1 mutations cause DNA hypermethylation and enhance transcriptional gene silencing. We report here the biochemical characterization of ROS1 and the effect of its overexpression on the DNA methylation of target genes. Our data suggest that the DNA glycosylase activity of ROS1 removes 5-methylcytosine from the DNA backbone and then its lyase activity cleaves the DNA backbone at the site of 5-methylcytosine removal by successive beta- and delta-elimination reactions. Overexpression of ROS1 in transgenic plants led to a reduced level of cytosine methylation and increased expression of a target gene. These results demonstrate that ROS1 is a 5-methylcytosine DNA glycosylase/lyase important for active DNA demethylation in Arabidopsis.

Arabidopsis↗
PubMed1989

Reactivation of hprt on the inactive X chromosome with DNA demethylating agents.

The C86 line of female embryonal carcinoma cells contains one active and one inactive X chromosome. Following methylnitrosourea mutagenesis, a clone called C86AGM2 was isolated that carries a mutated hprt gene on the active X chromosome. This hprtm allele encodes an HPRT enzyme that has less than 1% normal enzyme activity, is thermolabile, and has an altered isoelectric point. Following treatment with drugs that demethylate DNA, the hprt+ gene from the inactive X chromosome in C86AGM2 cells became active as determined by the appearance of HPRT activity with the thermodenaturation and electrofocusing characteristics of the normal enzyme. No expression of this hprt+ gene occurred if C86AGM2 cells were induced to differentiate prior to DNA demethylation. Stable lines of C86AGM2 cells expressing both the hprtm and hprt+ genes did not inactivate either gene following differentiation.

Animals↗
PubMed2005

DNA demethylation and carcinogenesis.

DNA methylation plays an important role in the establishment and maintenance of the program of gene expression. Tumor cells are characterized by a paradoxical alteration of DNA methylation pattern: global DNA demethylation and local hypermethylation of certain genes. Hypermethylation and inactivation of tumor suppressor genes are well documented in tumors. The role of global genome demethylation in carcinogenesis is less studied. New data provide evidence for independence of DNA hypo- and hypermethylation processes in tumor cells. These processes alter expression of genes that have different functions in malignant transformation. Recent studies have demonstrated that global decrease in the level of DNA methylation is related to hypomethylation of repeated sequences, increase in genetic instability, hypomethylation and activation of certain genes that favor tumor growth, and increase in their metastatic and invasive potential. The recent data on the role of DNA demethylation in carcinogenesis are discussed in this review. The understanding of relationships between hypo- and hypermethylation in tumor cells is extremely important due to reversibility of DNA methylation and attempts to utilize for anti-tumor therapy the drugs that modify DNA methylation pattern.

Animals↗
PubMed2003

Valproate induces replication-independent active DNA demethylation.

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.

Blotting, Southern↗
PubMed2000

5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Amino Acid Sequence↗
PubMed1993

Induction of cholinergic and adrenergic differentiation in N-18 cells by differentiation agents and DNA demethylating agents.

Effects of various differentiating agents and DNA demethylating agents on the expression of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), marker enzymes for cholinergic and adrenergic differentiation, respectively, were examined in N-18 neuroblastoma cells. Retinoic acid (RA) and a medium conditioned over C6-glioma cells (GCM), which have been shown to enhance the ChAT activity of PC12 cells, NG108-15 cells and fetal rat brain cells, did not induce ChAT activity of N-18 cells. Treatment of the cells with the DNA demethylating agents alone also did not affect ChAT activity. But after pretreatment of the cells with the DNA demethylating agents, ChAT activity of N-18 cells was greatly increased by either RA or GCM. TH activity of N-18 cells was enhanced by forskolin, an activator of adenylate cyclase. The pretreatment of the cells with the DNA demethylating agents greatly enhanced the induction of TH activity by forskolin. Levels of ChAT and TH messenger RNA were altered in accordance with changes in ChAT and TH activities. Possible mechanisms of the actions of the demethylating agents on cholinergic and adrenergic differentiation are discussed.

Acetylcholine↗
PubMed2024

Transgenerational increases in DNA methylation in Arabidopsis plants defective in active DNA demethylation.

Spontaneous gain or loss of DNA methylation occurs in plant and animal genomes, and DNA methylation changes can lead to meiotically stable epialleles that generate heritable phenotypic diversity. However, it is unclear whether transgenerational epigenetic stability may be regulated by any cellular factors. Here, we examined spontaneously occurring variations in DNA methylation in wild-type and ros1 mutant Arabidopsis plants that were propagated for ten generations from single-seed descent. We found that the ros1 mutant, which is defective in active DNA demethylation, showed an increased transgenerational epimutation rate. The ros1 mutation led to more spontaneously gained methylation than lost methylation at individual cytosines, compared to the wild type which had similar numbers of spontaneously gained and lost methylation cytosines. Consistently, transgenerational differentially methylated regions were also biased toward hypermethylation in the ros1 mutant. Our results reveal a genetic contribution of the ROS1 DNA demethylase to transgenerational epigenetic stability and suggest that ROS1 may have an unexpected surveillance function in preventing transgenerational DNA methylation increases.

Arabidopsis↗
PubMed1986

DNA demethylation induced by 5-azacytidine does not affect fragile X expression.

Experiments were performed to determine the role of DNA demethylation in fragile X expression. Fragile X positive lymphoblastoid cells were treated with 5-azacytidine and harvested for analysis of fragile X expression both directly following treatment and after a recovery period in the absence of the drug. The effectiveness of 5-azacytidine treatment in inducing DNA demethylation was concurrently monitored by analysis of methylation changes at random autosomal loci in isolated DNA from treated cells. Under conditions where 5-azacytidine was found to inhibit fragile X expression, no DNA demethylation was observed. At the time when demethylation did occur, fragile X expression was not affected. These results strongly indicate that DNA demethylation is not involved in fragile X expression.

Azacitidine↗
PubMed1997

The RNA moiety of chick embryo 5-methylcytosine- DNA glycosylase targets DNA demethylation.

We have previously shown that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. RNA from enzyme purified by SDS-PAGE was isolated and cloned. The clones have an insert ranging from 240 to 670 bp and contained on average one CpG per 14 bases. All six clones tested had different sequences and did not have any sequence homology with any other known RNA. RNase-inactivated 5-MeC-DNA glycosylase regained enzyme activity when incubated with recombinant RNA. However, when recombinant RNA was incubated with the DNA substrate alone there was no demethylation activity. Short sequences complementary to the labeled DNA substrate are present in the recombinant RNA. Small synthetic oligoribonucleotides (11 bases long) complementary to the region of methylated CpGs of the hemimethylated double-stranded DNA substrate restore the activity of the RNase-inactivated 5-MeC-DNA glycosylase. The corresponding oligodeoxyribonucleotide or the oligoribonucleotide complementary to the non-methylated strand of the same DNA substrate are inactive when incubated in the complementation test. A minimum of 4 bases complementary to the CpG target sequence are necessary for reactivation of RNase-treated 5-MeC-DNA glycosylase. Complementation with double-stranded oligoribonucleotides does not restore 5-MeC-DNA glycosylase activity. An excess of targeting oligoribonucleotides cannot change the preferential substrate specificity of the enzyme for hemimethylated double-stranded DNA.

Animals↗
PubMed2000

A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells.

In the developing chicken embryo, active DNA demethylation requires both RNA and proteins (Nucleic Acids Res. 25, 2375-2380, 1997; ibid. 25, 4545-4550, 1997, FEBS Lett. 449, 251-254, 1999a). In vitro assays indicate that in the 5- and 12-day-old embryos the highest specific activity of 5-methylcytosine DNA glycosylase is found in the brain, the eyes and the skin. In situ hybridization with antisense CpG-rich RNA tightly associated to the DNA demethylation complex shows a restricted expression pattern only in proliferating tissues such as the neuroepithelia of the brain in 5-day-old embryos. The RNA is absent in differentiated tissues like the skeletal and heart muscle, liver and the crystallin-producing cells in the lens. The CpG-rich RNA is transcribed in a developmental stage-specific rather than in a cell-specific manner. In contrast transcripts of DNA methyltransferase are found in dividing and quiescent cells. In situ hybridization with a probe of a RNA helicase which is also associated with the DNA demethylation complex shows a very similar localization in mitotically active tissues as the CpG-rich RNA. The content of 5-methylcytosine in individual cells was determined with a specific monoclonal antibody and cytometric analysis on tissue sections. The results indicate that proliferating cells have on the average 15% more methylated cytosines than non-dividing cells. This represents roughly 3x10(6) more methylation sites per haploid genome.

Animals↗
PubMed2001

Glucocorticoid-induced DNA demethylation and gene memory during development.

Glucocorticoid hormones were found to regulate DNA demethylation within a key enhancer of the rat liver-specific tyrosine aminotransferase (Tat) gene. Genomic footprinting analysis shows that the glucocorticoid receptor uses local DNA demethylation as one of several steps to recruit transcription factors in hepatoma cells. Demethylation occurs within 2-3 days following rapid (< 1 h) chromatin remodeling and recruitment of a first transcription factor, HNF-3. Upon demethylation, two additional transcription factors are recruited when chromatin is remodeled. In contrast to chromatin remodeling, the demethylation is stable following hormone withdrawal. As a stronger subsequent glucocorticoid response is observed, demethylation appears to provide memory of the first stimulation. During development, this demethylation occurs before birth, at a stage where the Tat gene is not yet inducible, and it could thus prepare the enhancer for subsequent stimulation by hypoglycemia at birth. In vitro cultures of fetal hepatocytes recapitulate the regulation analyzed in hepatoma cells. There fore, demethylation appears to contribute to the fine-tuning of the enhancer and to the memorization of a regulatory event during development.

5-Methylcytosine↗
PubMed1995

Ras induces a general DNA demethylation activity in mouse embryonal P19 cells.

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.

Animals↗
PubMed1999

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

Animals↗
PubMed2006

Aberrant DNA demethylation in promoter region and aberrant expression of mRNA of PAX4 gene in hematologic malignancies.

The PAX4 gene, a member of the paired box (PAX) gene family, is thought to be involved in regulating the fate of beta-cells in the mammalian pancreas. We observed the aberrant expression of PAX4 mRNA in 10 of 15 hematologic cell lines analyzed by RT-PCR. The restoration of PAX4 gene expression after treatment with the demethylating agent 5-aza-2'-deoxycitidine, as well as bisulfite sequencing analysis, indicated that gene overexpression was caused by DNA demethylation at the promoter region. Such DNA demethylation also was observed in primary lymphoma (20 out of 45 patients) on combined bisulfite restriction assay (COBRA). Forced expression of the PAX4 gene in the HEK293 and SHSY/610 cell lines conferred positive effects on cell growth. This profile of PAX4 thus corresponds to that of a candidate oncogene in hematologic malignancies.

Azacitidine↗
PubMed1995

Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver.

Mouse Slp, a duplicate of the fourth complement component (C4) gene, exhibits EDTA-independent complement activity with a hepatic expression that is male specific. To provide an underlying mechanism for the male-specific expression, we have analyzed the promoter activity of the various 5'-flanking sequences and CpG demethylation of the Slp gene. Transient transfections using HepG2 cells indicate that the element TTCCGGGC (nt -124 to -117) regulates the promoter activity. Moreover, CpG at position -121 of this regulatory element is demethylated to a much higher degree in males than in females. This sexually dimorphic DNA demethylation is consistent with the male-specific expression of the Slp gene in DBA/2 males. The regulatory element binds to the different TTCCGGGC-specific nuclear proteins depending on the methylation of the CpG site. In contrast, the corresponding CpG at position -119 of the C4 gene, which is expressed in both males and females, is demethylated at equal and high levels in both sexes. We therefore propose that the DNA demethylation and methylation-sensitive transcription factors may be a part of the regulatory mechanism for the male-specific expression of the Slp gene.

Animals↗
PubMed2006

Chromosomal instability correlates with genome-wide DNA demethylation in human primary colorectal cancers.

DNA hypomethylation is a common trait of colorectal cancer. Studies in tumor cell lines and animal models indicate that genome-wide demethylation may cause genetic instability and hence facilitate or accelerate tumor progression. Recent studies have shown that DNA hypomethylation precedes genomic damage in human gastrointestinal cancer, but the nature of this damage has not been clearly established. Here, we show a thorough analysis of DNA methylation and genetic alterations in two series of colorectal carcinomas. The extent of DNA demethylation but not of hypermethylation (both analyzed by amplification of intermethylated sites in near 200 independent sequences arbitrarily selected) correlated with the cumulated genomic damage assessed by two different techniques (arbitrarily primed PCR and comparative genomic hybridization). DNA hypomethylation-related instability was mainly of chromosomal nature and could be explained by a genome-wide effect rather than by the concurrence of the most prevalent genetic and epigenetic alterations. Moreover, the association of p53 mutations with genomic instability was secondary to DNA hypomethylation and the correlation between DNA hypomethylation and genomic instability was observed in tumors with and without mutation in the p53 gene. Our data support a direct link between genome-wide demethylation and chromosomal instability in human colorectal carcinogenesis and are consistent with the studies in model systems demonstrating a role of DNA demethylation in inducing chromosomal instability.

Chromosomal Instability↗
PubMed1995

Expression of antisense to DNA methyltransferase mRNA induces DNA demethylation and inhibits tumorigenesis.

Many tumor cell lines overexpress DNA methyltransferase (MeTase) activity; however it is still unclear whether this increase in DNA MeTase activity plays a causal role in naturally occurring tumors and cell lines, whether it is critical for the maintenance of transformed phenotypes, and whether inhibition of the DNA MeTase in tumor cells can reverse transformation. To address these basic questions, we transfected a murine adrenocortical tumor cell line Y1 with a chimeric construct expressing 600 base pairs from the 5' of the DNA MeTase cDNA in the antisense orientation. The antisense transfectants show DNA demethylation, distinct morphological alterations, are inhibited in their ability to grow in an anchorage-independent manner, and exhibit decreased tumorigenicity in syngeneic mice. Ex vivo, cells expressing the antisense construct show increased serum requirements, decreased rate of growth, and induction of an apoptotic death program upon serum deprivation. 5-Azadeoxycytidine-treated cells exhibit a similar dose-dependent reversal of the transformed phenotype. These results support the hypothesis that the DNA MeTase is actively involved in oncogenic transformation.

Animals↗