Fig. 5.

Analysis of EtOH-induced changes in chromatin structure in a fully differentiated cell type. A) Alcohol-induced changes in H3K9 acetylation, H3K9 dimethylation H3K4 trimethylation, and H3K27 trimethylation in mouse embryonic fibroblasts. Fibroblast cells were treated with 160 mg/dl EtOH for 4 days followed by a 4-day recovery period in medium without EtOH. Samples were subjected to chromatin immunoprecipitation and enrichment assayed within the regulatory regions of the indicated genes. Fold changes for H3K9 acetylation, H3K9 dimethylation and H3K4 trimethylation relative to the control are displayed; no alterations in H3K27 trimethylation were observed. Within the three separate biological replicates (N = 3), three ChIPs were performed, and two qPCR replicates performed on each independent ChIP. Differences were determined using a two-way ANOVA. B) Transcript levels of candidate genes are not impacted by EtOH-induced changes in chromatin structure. Transcripts encoding the indicated candidate genes were quantified using RT-qPCR. C) EtOH-induced up-regulation of transcripts encoding Dnmt1, Ehmt2 and Ezh2 do not persist beyond window of exposure. Transcripts encoding Dnmt1, Eed, Ehmt1, Ehmt2, Ezh2 and Setdb1 were measured using qPCR both during the period of exposure and after the four-day recovery phase. In analyses using RT-qPCR, measured Ct values were normalized to the geometric mean of Gapdh, Hprt, and Ppia and graphed relative to the control treatment. Graphs represent three independent replicates (N = 3), with two independent RT reactions and three qPCR measurements for each RT. All data are reported as Mean ± SEM. Differences were assessed using an unpaired, two-tailed parametric Student’s t-test. * p<0.05, ** p<0.01 versus untreated control; n=4.* p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.