Ethanol deregulates Mecp2/MeCP2 in differentiating neural stem cells via interplay between 5-methylcytosine and 5-hydroxymethylcytosine at the Mecp2 regulatory elements

Methyl CpG Binding Protein 2 (MeCP2) is an important epigenetic factor in the brain. MeCP2 expression is affected by different environmental insults including alcohol exposure. Accumulating evidence supports the role of aberrant MeCP2 expression in ethanol exposure-induced neurological symptoms. However, the underlying molecular mechanisms of ethanol-induced MeCP2 deregulation remain elusive. To study the effect of ethanol on Mecp2/MeCP2 expression during neurodifferentiation, we established an in vitro model of ethanol exposure, using differentiating embryonic brain-derived neural stem cells (NSC). Previously, we demonstrated the impact of DNA methylation at the Mecp2 regulatory elements (REs) on Mecp2/MeCP2 expression in vitro and in vivo. Here, we studied whether altered DNA methylation at these REs is associated with the Mecp2/MeCP2 misexpression induced by ethanol. Binge-like and continuous ethanol exposure upregulated Mecp2/MeCP2, while ethanol withdrawal downregulated its expression. DNA methylation analysis by methylated DNA immunoprecipitation indicated that increased 5-hydroxymethylcytosine (5hmC) and decreased 5-methylcytosine (5mC) enrichment at specific REs were associated with upregulated Mecp2/MeCP2 following continuous ethanol exposure. The reduced Mecp2/MeCP2 expression upon ethanol withdrawal was associated with reduced 5hmC and increased 5mC enrichment at these REs. Moreover, ethanol altered global DNA methylation (5mC and 5hmC). Under the tested conditions, ethanol had minimal effects on NSC cell fate commitment, but caused changes in neuronal morphology and glial cell size. Taken together, our data represent an epigenetic mechanism for ethanol-mediated misexpression of Mecp2/MeCP2 in differentiating embryonic brain cells. We also show the potential role of DNA methylation and MeCP2 in alcohol-related neurological disorders, specifically Fetal Alcohol Spectrum Disorders.