Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells.

In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells. However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells. In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold. The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection. This demethylation may be targeted to the promoter and extend into the gene. By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants. High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.     

Virion protein 16 induces demethylation of DNA integrated within chromatin in a novel mammalian cell model

DNA methylation and demethylation play important roles in mediating epigenetic regulation. So far, the mechanism of DNA demethylation remains elusive and controversial. Here, we constructed a plasmid, named with pCBS-luc, that contained an artificial CpG island, eight Gal4 DNA-binding domain binding site, an SV40 promoter, and a firefly luciferase reporter gene. The linearized pCBS-luc plasmid was methylated in vitro by DNA methyltransferase, and transfected into the HEK293 cells. The stable HEK293 transfectants with methylated pCBS-luc (me-pCBS-luc) were selected and obtained. The methylation status of the selected stable cell lines were confirmed by bisulfite sequencing polymerase chain reaction amplification. The methylation status could be maintained even after 15 passages. The virion protein 16 (VP16) was reported to enhance DNA demethylation around its binding sites of the promoter region in Xenopus fertilized eggs. Using our me-pCBS-luc model, we found that VP16 also had the ability to activate the expression of methylated luciferase reporter gene and induce DNA demethylation in chromatin DNA in mammalian cells. Altogether, we constructed a cell model stably integrated with the me-pCBS-luc reporter plasmid, and in this model we found that VP16 could lead to DNA demethylation. We believe that this cell model will have many potential applications in the future research on DNA demethylation and dynamic process of chromatin modification.     

Control of organ-specific demethylation by an element of the T-cell receptor-alpha locus control region

DNA methylation is important for mammalian development and the control of gene expression. Recent data suggest that DNA methylation causes chromatin closure and gene silencing. During development, tissue specifically expressed gene loci become selectively demethylated in the appropriate cell types by poorly understood processes. Locus control regions (LCRs), which are cis-acting elements providing stable, tissue-specific expression to linked transgenes in chromatin, may play a role in tissue-specific DNA demethylation. We studied the methylation status of the LCR for the mouse T-cell receptor alpha/delta locus using a novel assay for scanning large distances of DNA for methylation sites. Tissue-specific functions of this LCR depend largely on two DNase I-hypersensitive site clusters (HS), HS1 (T-cell receptor alpha enhancer) and HS1'. We report that these HS induce lymphoid organ-specific DNA demethylation in a region located 3.8 kilobases away with little effect on intervening, methylated DNA. This demethylation is impaired in mice with a germline deletion of the HS1/HS1' clusters. Using 5'-deletion mutants of a transgenic LCR reporter gene construct, we show that HS1' can act in the absence of HS1 to direct this tissue-specific DNA demethylation event. Thus, elements of an LCR can control tissue-specific DNA methylation patterns both in transgenes and inside its native locus.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A-MuLV)-transformed cell lines, which were able to differentiate from the mu- to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A-MuLV-transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM-bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.     

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.     

Feedback regulation of DNA methyltransferase gene expression by methylation.

This paper tests the hypothesis that expression of the DNA methyltransferase, dnmt1, gene is regulated by a methylation-sensitive DNA element. Methylation of DNA is an attractive system for feedback regulation of DNA methyltransferase as the final product of the reaction, methylated DNA, can regulate gene expression in cis. We show that an AP-1-dependent regulatory element of dnmt1 is heavily methylated in most somatic tissues and in the mouse embryonal cell line, P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1. dnmt1 is highly over expressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19 cells by 5-azadeoxycytidine results in demethylation of the AP-1 regulatory region and induction of dnmt1 expression in P19cells, but not Y1 cells. We propose that this regulatory region of dnmt1 acts as a sensor of the DNA methylation capacity of the cell. These results provide an explanation for the documented coexistence of global hypomethylation and high levels of DNA methyltransferase activity in many cancer cells and for the carcinogenic effect of hypomethylating diets.     

Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation

We report here genome-wide mapping of DNA methylation patterns at proximal promoter regions in mouse embryonic stem (mES) cells. Most methylated genes are differentiation associated and repressed in mES cells. By contrast, the unmethylated gene set includes many housekeeping and pluripotency genes. By crossreferencing methylation patterns to genome-wide mapping of histone H3 lysine (K) 4/27 trimethylation and binding of Oct4, Nanog, and Polycomb proteins on gene promoters, we found that promoter DNA methylation is the only marker of this group present on approximately 30% of genes, many of which are silenced in mES cells. In demethylated mutant mES cells, we saw upregulation of a subset of X-linked genes and developmental genes that are methylated in wild-type mES cells, but lack either H3K4 and H3K27 trimethylation or association with Polycomb, Oct4, or Nanog. Our data suggest that in mES cells promoter methylation represents a unique epigenetic program that complements other regulatory mechanisms to ensure appropriate gene expression.     

Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes.

In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation.     

Differentiation of two mouse cell lines is associated with hypomethylation of their genomes.

Methyl-accepting assays and a sensitive method for labeling specific CpG sites have been used to show that the DNA of F9 embryonal carcinoma cells decreases in 5-methylcytosine content by ca. 9% during retinoic acid-induced differentiation, whereas the DNA of dimethyl sulfoxide-induced Friend murine erythroleukemia (MEL) cells loses ca. 3.8% of its methyl groups. These values correspond to the demethylation of 2.2 X 10(6) and 0.9 X 10(6) 5'-CpG-3' sites per haploid genome in differentiating F9 and MEL cells, respectively. Fluorography of DNA restriction fragments methylated in vitro and displayed on agarose gels showed that demethylation occurred throughout the genome. In uninduced F9 cells, the sequence TCGA tended to be more heavily methylated than did the sequence CCGG, whereas this tendency was reversed in MEL cells. The kinetics of in vitro DNA methylation reactions catalyzed by MEL cell DNA methyltransferase showed that substantial numbers of hemimethylated sites accumulate in the DNA of terminally differentiating F9 and MEL cells, implying that a partial loss of DNA-methylating activity may accompany terminal differentiation in these two cell types.