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

Alcohol (Fayetteville, N.Y.) (2017-04-24)
Kylee J Veazey, Haiqing Wang, Yudhishtar S Bedi, William M Skiles, Richard Cheng-An Chang, Michael C Golding
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.

MATERIALS
Product Number
Brand
Product Description

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Penicillin-Streptomycin, Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
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Heparin sodium salt from porcine intestinal mucosa, Grade I-A, ≥180 USP units/mg, powder, BioReagent, suitable for cell culture
SAFC
MEM Non-essential Amino Acid Solution (100×), without L-glutamine, liquid, sterile-filtered, BioReagent, suitable for cell culture
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Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose and sodium bicarbonate, without L-glutamine and sodium pyruvate, liquid, sterile-filtered, suitable for cell culture, suitable for hybridoma
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Bovine Serum Albumin, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, ≥98%
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Anti-trimethyl-Histone H3 (Lys4) Antibody, clone MC315, rabbit monoclonal, culture supernatant, clone MC315, Upstate®
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Anti-acetyl-Histone H3 (Lys9) Antibody, serum, Upstate®