Abstract
Alterations in DNA methylation have been associated with alcohol exposure and proposed to contribute to continued alcohol use; however, the molecular mechanisms involved remain obscure. We investigated the escalating effects of alcohol use on DNA methylation, gene expression and predicted neural effects in the nucleus accumbens of rhesus macaques that self-administered 4% alcohol for over 12 months. Using an exploratory approach to identify CpG-rich regions, followed by bisulfite sequencing, the methylation levels of 2.7 million CpGs were compared between seven low-binge drinkers and nine heavy–very heavy drinking subjects. We identified 17 significant differential methylation regions (DMRs), including 14 with methylation levels that were correlated with average daily alcohol consumption. The size of the DMRs ranged from 29 to 158 bp (mean=63.7), included 4–19 CpGs per DMR (mean=8.06) and spanned a range of average methylation values from 5 to 34%. Eight of the DMRs mapped to genes implicated in modulating synaptic plasticity. Six of the synaptic genes have not previously been linked to alcohol use. Validation studies of these eight DMRs using bisulfite amplicon sequencing and an expanded set of 30 subjects confirmed the significant alcohol-dose-associated methylation of the DMRs. Expression analysis of three of the DMR-associated genes, LRP5, GPR39 and JAKMIP1, revealed significant correlations between DMR methylation and whole-gene or alternative transcript expression, supporting a functional role in regulating gene expression. Together, these studies suggest that alcohol-associated synaptic remodeling may be regulated and coordinated at the level of DNA methylation.
Highlights
Chronic and excessive alcohol use can lead to alcohol dependence, a relapsing and remitting condition that costs lives and disrupts families
Detail for each differential methylation regions (DMRs): chromosome position in the MacaM genome, genes located within 50 kb of the DMR, genomic context, CpG islands (CGI) context, the number of CpGs within the DMR, the average methylation difference between the comparison classes, corrected Sidak P-value for the DMR and the Pearson’s correlation coefficient and P-value between DMR average methylation level and the average g kg −1 per day ethanol consumed (P-values o 0.05 are shown in bold)
By focusing on clusters of differentially methylated CpGs in two alcohol-dose groups (L/binge drinkers (BDs) and H/VHD), we identified DMRs linked to genes of high relevance to alcohol use using a relatively small number of subjects
Summary
Chronic and excessive alcohol use can lead to alcohol dependence, a relapsing and remitting condition that costs lives and disrupts families. In an effort to understand the neuroadaptive changes associated with dependent or compulsive drinking, investigations have focused on brain regions that process motivated behaviors and on cellular mechanisms underlying learning and memory. In the NAc, chronic alcohol use has been linked to changes in dendritic structures[3] and neurotransmitter signaling[4] thought to contribute to alcohol tolerance, craving and withdrawal.[5] elucidating the molecular mechanisms that link alcohol use and these neural adaptations remains a challenge for fully understanding and treating alcohol dependence. The dependence of self-reported alcohol use in human studies severely limits the evaluation of alcohol dose effects on neural DNA methylation or associated changes in gene expression. Unknown nicotine or drug use, or comorbid psychiatric and medical conditions have the potential to contribute confounding effects
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