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 dosedependent 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 DNAbinding 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.DNA methylation is a modification of DNA whereby methyl groups are added as part of the covalent structure of the genome, thus providing an extra layer of epigenetic information. A well documented relationship exists between DNA methylation, chromatin structure, and gene expression (1) such that methylated genes are generally transcriptionally silent. Two mechanisms have been proposed to explain this repression: the first is that methylation causes interference in the binding of transcription factors and has been shown for several proteins such as AP2 (2) and c-Myc (3). The second mechanism involves the recruitment of various repressor complexes to methylated DNA via the binding of methylated DNA-binding proteins (MBDs). 1 These complexes contain proteins that have histone deacetylase and chromatin remodeling activities, leading to the formation of a more compact and transcriptionally inactive chromatin (4).Valproic acid/Valproate/2-n-propylpentanoic acid (VPA) has been used for decades in the treatment of epilepsy and is also effective as a mood stabilizer and in migraine therapy. Recent data suggest that this drug, in addition to its other known classical actions, can modulate the epigenome by inhibiting histone deacetylases (HDACs) (5, 6), similar to agents such as trichostatin A (TSA) and n-butyrate, thus triggering an increase in gene expression. Other studies also support a role for VPA in the regulation of methylated genes. It was demonstrated that the reelin gene, which encodes a neuron...

Valproate (VPA)(1) has been used for decades in the treatment of epilepsy, and is also effective as a mood stabilizer and in migraine therapy. It has been shown that VPA is also a histone deacetylase (HDAC) inhibitor. We have previously shown that VPA could trigger active demethylation of ectopically methylated transiently transfected DNA in HEK 293 cells. We therefore tested whether VPA treatment could bring about stable changes in the epigenome by causing changes in the state of DNA methylation of genomic DNA. Using a microarray gene expression analysis we identified the genes whose expression is induced by VPA treatment in HEK 293 cells. We found that a subset of these genes could also be induced by the classical DNA methylation inhibitor 5-aza-2'-deoxy-cytidine (5-aza-CdR) suggesting that VPA can alter the state of expression of genes, which are stably suppressed by DNA methylation. We mapped the state of methylation of three of these genes, MELANOMA ANTIGEN B2 GENE (MAGEB2), METALLOPROTEINASE 2 (MMP2) and WIF1, which are involved in tumor growth and metastasis. A chromatin immunoprecipitation (ChIP) assay revealed that VPA treatment caused as expected a change in the state of acetylation of these genes. Our data supports the concept that chromatin acetylation and DNA methylation are found in a dynamic interrelation and that the consequences of HDAC inhibitors are not limited to changes in histone acetylation but that they also bring about a change in the state of modification of DNA. The implications of our results on the future therapeutic utilities of VPA in cancer will be discussed.

The DNA methylation pattern is tightly correlated with chromatin structure in that transcriptionally active chromatin domains are hypomethylated, whereas inactive regions are hypermethylated (3). However, the mechanisms defining the relationship between histone hypoacetylation and DNA hypermethylation are not clear. While it is well accepted that DNA methylation can promote chromatin deacetylation and inactivation (4), a number of studies suggest that chromatin status can also alter the pattern of DNA methylation. Genome wide demethylation was shown to be induced by n-butyrate, an inhibitor of histone acetylation (5). Tichostatin A (TSA), 1 an inhibitor of histone deacetylase, was shown to induce selective loss of DNA methylation in Neurospora (6), and very recently disruption of histone methylation was shown to eliminate DNA methylation in Neurospora (7). We have recently shown that TSA can induce demethylation of ectopically methylated genes (1). Hypermethylation of CpG islands has attracted considerable attention as a mechanism responsible for gene silencing during tumor suppression. It is clear that methylation of tumor suppressor genes cannot be explained by the increase in the general level of DNA methyltransferase activity observed in cancer cells. Significant effort has been therefore directed toward identification of factors that recruit DNA methyltransferases to specific promoters. In accordance with this hypothesis, a recent publication demonstrated that the oncogenic transcription factor PML-RAR fusion protein recruits DNMT1 to the retinoic acid ␤II promoter, resulting in methylation and silencing of this promoter (8).An alternative potential mechanism of bringing about DNA hypermethylation is inhibition of demethylase activity. This possibility was not previously explored, since it has long been believed that the DNA methylation pattern is controlled exclusively by DNA methyltransferases and that the DNA methylation reaction is irreversible. Nevertheless, we have recently extracted DNA demethylase activity from human lung cancer cell line A549 (9) and have shown that a human embryonal kidney cell line HEK293 possesses active demethylase activity (1).The DNA methylation state of a gene in a living cell represents a steady state, and it is hard to determine whether it is a consequence of increased DNA methylation or reduced demethylation. Moreover, it is hard to determine whether demethylation of a gene in a proliferating cell is passive, resulting from inhibition of DNA methylation during DNA synthesis or an active true removal of a methyl group. We have therefore recently developed a transient transfection system that enables studying demethylase activity in cancer cells. A reporter CMV-GFP construct is methylated in vitro by a CpG methyltransferase mSssI and introduced into human transformed HEK293 cells. The plasmid is extracted 96 h after transfection, and its state of methylation is determined by either methylation sensitive restriction enzyme analysis or bisulfite mapping. Since we have demonst...

MBD2 is the only member of a family of methyl-CpGbinding proteins that has been reported to be both a transcriptional repressor and a DNA demethylase (dMTase). To understand the apparently contradictory function of MBD2/dMTase, we studied the effects of dMTase overexpression on the activity of various in vitro methylated promoters transiently transfected into HEK293 cells. We found that forced expression of a MBD2/dMTase expression vector (His-dMTase) differentially activated two methylated reporters, pSV40-CAT (the SV40 enhancerless promoter adjacent to the chloramphenicol acetyltransferase (CAT) reporter gene) and pGL2T؉I4xTBRE (a region of the p21 promoter next to the luciferase reporter gene), in a time-and dose-dependent manner. His-dMTase increased pSV40-CAT expression by 3-10-fold after 96 h, while pGL2T؉I4xTBRE expression was increased by 2-3-fold after only 48 h and did not further increase at 96 h. Gene activation was not universal because no effect was seen with the p19-ARF promoter. We then assessed whether activation might be due to demethylation within the promoter region. Using bisulfite mapping, we found that exogenous expression of His-dMTase induced demethylation at 8 of the 10 CpG sites within the SV40 promoter. The observation that His-dMTase increases the demethylase activity in the cells was also confirmed using an in vitro CpG demethylase assay with a mC32pG oligonucleotide substrate and purified Q-Sepharose fractions from HEK293 cells transfected with His-dMTase or empty pcDNA3.1His vector. We propose that a single protein possessing both repressor and demethylase functions has evolved to coordinate a program that requires suppression of some methylated genes and activation of others.The epigenome consists of an additional component that is part of the covalent structure of the genome, a coating of methyl groups. In vertebrates, 80% of cytosine residues within the dinucleotide sequence CpG are modified by methylation in a pattern that is tissue-specific and that is formed during development and maintained in somatic cells (1). It has been well established that the DNA methylation pattern is maintained exclusively by DNA methyltransferase activities, but we have recently proposed that DNA demethylase activities might also participate in the process (2-4) and that the methylation pattern is a steady state balance of reversible methylationdemethylation reactions (5, 6). We have shown that histone acetylation promotes active demethylation of ectopically methylated genes (3) and that inhibitors of histone acetylation inhibit demethylation (4).It is well documented that the state of activity of a gene, the chromatin structure, and DNA methylation are correlated (7) such that areas of the genome that are methylated are usually less expressed. One molecular mechanism that explains this relationship has recently been elucidated. Repressor complexes are recruited to methylated DNA via the binding of methylCpG binding domain proteins (MBDs).1 These complexes contain proteins that have histone deacet...

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