In parallel with analysis of the genome-wide DNA methylation, it was next of interest to examine the effect of alcohol on the cellular distribution of DNA methylation marks, including DNA methylation mark 5-methyl cytidine (5-MeC) and DNA methyltransferase (DNMT). In undifferentiated NSC, 5-MeC immunostaining (im) was moderately intense and distributed throughout the nucleus. However, in the control-undifferentiated group, increased 5-MeC-im intensity was seen in the nucleus, but preferentially distributed near the nuclear membrane into differentiation. A redistribution of 5-MeC-im throughout most of the nucleus was observed in alcohol-treated NSC during the differentiation stage (Figure 2A-C). A redistribution of DNMT1-im was also evident in the three treatment groups. The DNMT1 was distributed similarly to 5-MeC-im--low and throughout the nucleus--but translocated into cytoplasm in differentiated NSC. Alcohol exposure prevented this redistribution in a subpopulation of the NSC during differentiation (Figure 2D-E). The significance of these translocations may be pertinent to euchromatic vs heterochromatic distribution of epigenetic marks associated genes.
