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 alcohol and other drugs of abuse exert consistently note a disconnect 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 if 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 impact 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 changes in chromatin structure cannot unequivocally explain dysgenesis in isolation.

Epigenetic Impacts of Early Life Stress in Fetal Alcohol Spectrum Disorders Shape the Neurodevelopmental Continuum

“…In a murine cell culture model, there is a dose-dependent increase in Dnmt3a , Dnmt3b , and Dnmt3l expression, with increased DNMT3A protein abundance (Miozzo et al, 2018 ). While others have found dose-dependent decreased expression of Dnmt1 (Veazey et al, 2017 ). Moreover, increased gene expression of Dnmt1 and Dnmt3a in male rat hippocampus following early PAE (Lucia et al, 2019 ) or continuous PAE (Gangisetty et al, 2015 ) has been observed.…”

Programmed suppression of oxidative phosphorylation and mitochondrial function by gestational alcohol exposure correlate with widespread increases in H3K9me2 that do not suppress transcription

“…Di-methylation and tri-methylation of lysine nine are recognized components of heterochromatin that, along with HP1 and histone H4 lysine 20 methylation, play an essential role in transcriptional silencing and genome stability [ 19 ]. To determine if alcohol selectively impacts only the dimethylated state of H3K9, we returned to our previously published primary neurosphere model of exposure [ 12 , 13 ]. Here, we exposed cultured cells to 0, 160, and 240 mg/dL of alcohol for 3 days, then passaged exposed cells without alcohol for a 4-day recovery phase.…”

“…While exposure to drugs of abuse, including cocaine, morphine, and alcohol, all increase global acetylation and influence H3K9 methylation, these changes in chromatin structure scarcely correlate with altered gene expression, especially on a genome-wide scale [ 13 , 14 ]. In contrast, each of these toxicants also associates with the development of oxidative stress, which induces protective alterations in chromatin structure [ 15 , 16 ].…”

Programmed increases in LXRα induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity

“…We performed chromatin Immunoprecipitations (ChIP) using a previously described protocol [15,16], with the following modifications. Briefly, 50 mg of liver tissue were thawed on ice in cold PBS and dispersed into single cell suspensions using a 100 μm cell strainer (catalog no.…”