Abstract
BackgroundThe NMDA receptor represents a particularly important site of ethanol action in the CNS. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. Increasing evidence that epigenetic mechanisms are involved in dynamic and long-lasting regulation of gene expression in multiple neuroadaptive processes prompted us to investigate the role of DNA methylation in mediating CIE-induced up-regulation of NR2B gene transcription. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 5′-regulatory area following CIE treatment.MethodsPrimary cortical cultured neurons were subjected to ethanol treatment in a CIE paradigm. Bisulfite conversion followed by pyrosequencing was used for quantitative measurement and analysis of CpG methylation status within the 5′-regulatory area of the NR2B gene; chromatin immunoprecipitation (ChIP) assay was used to examine DNA levels associated with methylation and transcription factor binding. Electrophoretic mobility shift assay (EMSA) and in vitro DNA methylation assays were performed to determine the direct impact of DNA methylation on the interaction between DNA and transcription factor and promoter activity.ResultsAnalysis of individual CpG methylation sites within the NR2B 5′regulatory area revealed three regions with clusters of site-specific CpG demethylation following CIE treatment and withdrawal. This was confirmed by ChIP showing similar decreases of methylated DNA in the same regions. The CIE-induced demethylation is characterized by being located near certain transcription factor binding sequences, AP-1 and CRE, and occurred during treatment as well as after ethanol withdrawal. Furthermore, the increase in vitro of methylated DNA decreased transcription factor binding activity and promoter activity. An additional ChIP assay indicated that the CIE-induced DNA demethylation is accompanied by increased occupation by transcription factors.ConclusionsThese results suggest an important role of DNA demethylation in mediating CIE-induced NR2B gene up-regulation, thus implicating a novel molecular site of alcohol action.
Highlights
Ethanol exposure on a chronic intermittent regimen has been found to produce behavioral excitability, seizure susceptibility, and increased anxiety [1,2]
We have identified a molecular target of epigenetic regulation in alcohol addiction by focusing on the role of chronic intermittent ethanol (CIE)-induced changes in DNA methylation within the 59-regulatory area of NMDA receptor 2B (NR2B) gene
By focusing the affect of methylation on transcription activity, we looked into the relationships between the DNA methylation status of the NR2B promoter and transcription factor accessibility as well as gene expression
Summary
Ethanol exposure on a chronic intermittent regimen has been found to produce behavioral excitability, seizure susceptibility, and increased anxiety [1,2]. Repeated alcohol-withdrawal is known to induce long-lasting adaptations that underlie behavioral phenotypes associated with alcohol addiction It is not understood mechanistically, addiction is thought to depend on molecular and cellular adaptations that lead to persistent changes in transcription, translation, and synaptic morphology [3,4]. Increases in N-methyl-Daspartate receptor (NMDAR) subunit levels contributing to upregulation of glutamate transmission by ethanol exposure were suggested by recent work showing up-regulated binding, function, and expression following chronic ethanol treatment [7,8,9,10]. We recently reported that NMDA receptor 2B (NR2B) gene expression was persistently up-regulated following chronic intermittent ethanol (CIE) treatment. To dissect the changes of DNA methylation in the NR2B gene, we have screened a large number of CpG sites within its 59-regulatory area following CIE treatment
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