Valproate Induces Replication-independent Active DNA Demethylation

Abstract

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...

The social environment and the epigenome

“…However, other groups disputed this finding [Ng et al, 1999]. Later data from our laboratory further supported the demethylation activity of MBD2 [Detich et al, , 2003a[Detich et al, ,2003a. A very recent Environmental and Molecular Mutagenesis.…”

“…DOI 10.1002/em Indeed, in contrast to DNMTs, which are recruited by chromatin silencing enzymes such as SUV39 [Fuks et al, 2003a] and EZH2 [Vire et al, 2006;, demethylation is facilitated by histone acetylation [Cervoni and Szyf, 2001;Cervoni et al, 2002]. Pharmacological acetylation using HDAC inhibitors such as TSA [Cervoni and Szyf, 2001] or valproic acid [Detich et al, 2003a] trigger replication-independent active demethylation of transiently transfected in vitro methylated plasmids and causes genomic demethylation [Milutinovic et al, 2007;Ou et al, 2007]. Interestingly TSA facilitates DNA demethylation in adult hippocampal neurons as well [Weaver et al, 2004], suggesting that the activity of TSA is not limited to cells in culture or to cycling cells.…”

Environmental regulation of the neural epigenome

“…The form of the re-packaging, including potential re-modeling of epigenetic marks, could influence the probability of subsequent transcriptional activity. There is evidence that alterations to chromatin structure can re-define DNA methylation states, and that chromatin alterations can affect both de novo DNA methylation or demethylation [74][75][76][77]. Increasing histone acetylation leads to transcription factor binding at previously methylated sites, and to demethylation.…”

Valproic Acid: Second Generation

“…We believe that, with the utilization of second-generation VPA drugs in addition to epilepsy research, pharmacological research in the next decade will proceed (at least in part) along the following paths: 1) A new mechanism of action for VPA as an additional inducer of replication-independent active DNA demethylation in living cells; 63 2) the effectiveness of VPA and its derivatives in regulating the growth differentiation and apoptosis of tumor cells;…”