Ethanol-induced methylation of cell cycle genes in neural stem cells

Ethanol inhibits the proliferation of neural precursors by altering mitogenic and anti-mitogenic growth factor signaling and can affect global methylation activity in the fetus. We tested the hypothesis that epigenetic modification of specific cell cycle genes underlies the ethanol-induced inhibition of growth factor-regulated cell cycle progression. Monolayer cultures of neural stem cells (NSCs) were treated with fibroblast growth factor 2 or transforming growth factor (TGF) beta 1 in the absence or presence of ethanol. Ethanol increased the total length of the cell cycle by elongating the amount of time spent in the gap 1 (G1) and synthesis (S) phases of the cell cycle. Ethanol induced the hypermethylation of multiple cell cycle genes associated with the G1/S and gap 2/mitotic phase (G2/M) checkpoints and increased the expression and activity of DNA methyltransferases. These changes were most pronounced in the presence of TGF beta 1. Epigenetic alterations paralleled the down-regulation of associated transcripts and other checkpoint-related mRNAs both in vitro (NS-5 cell culture) and in vivo (fetal mouse cortex). Ethanol-induced hypermethylation was accompanied by decreases in the proportion of NSCs expressing associated cell cycle proteins. Thus, ethanol disrupts growth factor-related cell cycle progression by inducing checkpoint restriction at the G1/S transition through a feed-forward system involving the methylation of G2/M regulators.