Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence

Abstract

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study.

Keywords: Alcohol-use disorder; DNA methylation; Epigenetics; Histone acetylation; Histone arginine acetylation; Histone lysine methylation; Histone ubiquitination.

Fig. 1. Hierarchical clustering of expression differences in factors modifying chromatin structure after chronic ethanol exposure (EXP), chronic ethanol exposure followed by 8-h withdrawal (WD), and chronic ethanol exposure followed by 21 days of abstinence (ABS) shows similarity of response to ethanol at EXP and WD

Genes identified as regulated by ethanol (main effect of treatment; q < 0.05, *; q < 0.01, **) were only identified at EXP and WD time points, and the expression pattern between EXP and WD were very similar. Each column in the heat-map represents the expression at each time, with red indicating up-regulation by ethanol and blue indicating down-regulation by ethanol. For each column, the selected line data (i.e., WSP and WSR) were collapsed, as we identified no significant line × treatment interactions (see Supplemental Tables 1, 2, and 3).

Fig. 2. Network of gene expression changes and their association to chromatin modifications after chronic ethanol exposure and withdrawal

Interaction networks of genes whose expression is significantly altered by ethanol at EXP and/or WD were created in Cytoscape 3.4 and organized based on the known chromatin modifications they target (designated by the edge color pointing to the affected histone or DNA structure) as well as on their molecular function (designated by the shape of the node). Upregulated expression in EXP or WD compared to their respective controls was designated by the colors pink (q < 0.05) or red (q < 0.01); downregulation of expression was designated by the colors azure (q < 0.05) or blue (q < 0.01).

Fig. 3

Summary of ethanol-induced changes in expression of genes involved in modifying chromatin structure and expected post-translational modification and transcriptional outcomes.