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) β1 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 TGFβ1. 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.