Disconnect between alcohol-induced alterations in chromatin structure and gene transcription in a mouse embryonic stem cell model of exposure

Abstract

Alterations to chromatin structure induced by environmental insults have become an attractive explanation for the persistence of exposure effects into subsequent life stages. However, a growing body of work examining the epigenetic impact that alcohol and other drugs of abuse exert consistently notes a disconnection between induced changes in chromatin structure and patterns of gene transcription. Thus, an important question is whether perturbations in the 'histone code' induced by prenatal exposures to alcohol implicitly subvert gene expression, or whether the hierarchy of cellular signaling networks driving development is such that they retain control over the transcriptional program. To address this question, we examined the impact of ethanol exposure in mouse embryonic stem cells cultured under 2i conditions, where the transcriptional program is rigidly enforced through the use of small molecule inhibitors. We find that ethanol-induced changes in post-translational histone modifications are dose-dependent, unique to the chromatin modification under investigation, and that the extent and direction of the change differ between the period of exposure and the recovery phase. Similar to in vivo models, we find post-translational modifications affecting histone 3 lysine 9 are the most profoundly impacted, with the signature of exposure persisting long after alcohol has been removed. These changes in chromatin structure associate with dose-dependent alterations in the levels of transcripts encoding Dnmt1, Uhrf1, Tet1, Tet2, Tet3, and Polycomb complex members Eed and Ezh2. However, in this model, ethanol-induced changes to the chromatin template do not consistently associate with changes in gene transcription, impede the process of differentiation, or affect the acquisition of monoallelic patterns of expression for the imprinted gene Igf2R. These findings question the inferred universal relevance of epigenetic changes induced by drugs of abuse and suggest that changes in chromatin structure cannot unequivocally explain dysgenesis in isolation.

Keywords: Chromatin; Developmental programming; Embryonic stem cells; Environmental epigenetics; Epigenetic inheritance; Epigenetics; Fetal alcohol syndrome; Neurodevelopmental programming; Teratogen.

Fig. 1

Dose-dependent and histone modification-specific changes in chromatin structure. Murine embryonic stem cells were cultured in varying concentrations of ethanol (80 mg/dL, 160 mg/dL, or 240 mg/dL) for four days, followed by a no-ethanol recovery period for ten days. Samples were taken at Day-4, Day-8, and Day-14 and analyze for enrichment of H3K4me3, H3K9ac, H3K27me3, and H3K9me2 using chromatin immunoprecipitation. The enrichment of the indicated post-translational histone modifications was then analyzed within the promoter regions of candidate genes using qPCR. The heat map represents fold change compared to the control group. Within the three separate biological replicates (N = 3), three ChIPs were performed, and two qPCR replicates performed on each independent ChIP. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 2

Alterations in gene transcription arising during and after the window of EtOH exposure. Murine embryonic stem cells examined in Figure 1 were cultured in varying concentrations of ethanol (80 mg/dL, 160 mg/dL, or 240 mg/dL) for four days, followed by a no-ethanol recovery period for ten days. Samples were taken at Day-4, Day-8, and Day-14, RNA isolated and transcript levels encoding the candidate genes examined in Figure 1 assayed using quantitative RT-PCR. 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. Differences were measured using a one-way ANOVA, error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 3

Alterations in the genetic pathways controlling alcohol metabolism, oxidative stress response and the control of DNA methylation. Murine embryonic stem cells were cultured in varying concentrations of ethanol (80 mg/dL, 160 mg/dL, or 240 mg/dL) for four days, followed by a no-ethanol recovery period for ten days. Samples were taken at Day-4, Day-8, and Day-14, and transcripts encoding genes involved in A) alcohol metabolism and the oxidative stress response, B) H3K27, H3K9 and DNA methyltransferase enzymes, C) TET family of Fe(II) and a-KG- dependent dioxygenases, and D) histone demethylases examined using RT-qPCR. 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. Differences were measured using a one-way ANOVA, error bars represent SEM. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Fig. 4

EtOH exposure does not disrupt the ability of ESCs to acquire imprinted patterns of transcription during differentiation. A) ESCs were seeded into flasks and exposed to varying concentrations of ethanol (0 mg/dL, 80 mg/dL, 160 mg/dL, or 240 mg/dL) for four days, followed by a no-ethanol recovery period for ten days. Cells were then differentiated into neuroblasts using an established 5-day protocol. At Day-4 and Day -19, cells were monitored for changes in the expression of the surface markers CD90.2 and CD24a using flow cytometry. Graphs represent three separate biological replicates (N = 3) examining half a million cells in each replicate. B) Representative graphs examining allelic patterns of Igf2R transcripts through ESC differentiation using a polymophism-based restriction digest assay. In this assay, transcripts derived from the maternal C57BL/6J strain (upper band) are refractory to Taq1 digestion, while transcripts derived from the paternal Mus musculus castaneus strain (lower doublet) are susceptible. C) Densitometry analysis of Igf2R allelic expression. Graph represents three separate biological replicates (N = 3).

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.