To determine the extent of EtOH's epigenetic effects on the molecular regulation in hESCs, we initially measured the effect of EtOH on global DNA methylation. Recent studies have reported changes in methylation status upon EtOH treatment (Sanchez-Alvarez et al., 2013; Zhou et al., 2011). It has been shown that alcohol exposure alters migration and important basic processes of neural stem cells (Zhou et al., 2011). Their studies also showed that alcohol exposure altered the methylation potential of genes that were initially in a quiescent state (Zhou et al., 2011). To follow up on such studies using hESCs, we performed a biochemical assay for DNA methylation, which revealed a significant increase in global DNA methylation after 48 hrs of EtOH (20 or 50 mM) treatment (Fig. 2A). Global DNA methylation was slightly, but not significantly higher with 50 mM compared to 20 mM EtOH treatment. To further study the molecular effect of EtOH on DNA methylomic signatures in hESCs, we performed a genome-wide DNA methylation analysis in undifferentiated or differentiated hESCs with or without EtOH treatment. For these studies, we again
