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 used a 24 hr application of 20 mM EtOH to allow for comparisons with the results of the genome-wide transcriptome studies. Examination of DNA methylation changes that were occurring on the promoters around transcription start site (TSS) or CpG islands (CGIs) revealed that EtOH treatment increased global DNA methylation in undifferentiated hESCs (Fig. 2B and C). Thus, plots of relative DNA methylation levels at the TSS and the CGIs after treatment with 20 mM EtOH (left panels) revealed EtOH-induced DNA hypermethylation at the TSS and the CGIs. Similarly, aligned probes plot of methylation showed that treatment of undifferentiated hESCs with 20 mM EtOH resulted in an increase in DNA methylation compared to non-treated control (middle panels) and log plots for methylation comparing control cells (x-axis) vs. cells treated with 20 mM EtOH (y-axis) also showed greater level of DNA methylation after EtOH treatment (right panels).
