Although alcohol dependence may result from neuroadaptation to chronic alcohol consumption involving changes in the expression of multiple genes (Melendez et al., 2012
), the precise biological mechanisms are not well known. Our data indicate that subacute alcohol consumption may possibly lead to epigenetic changes (e.g., DNA methylation), potentially impacting gene transcription. Because investigation of DNA methylation levels in human subjects does not allow assessment of the causal role of alcohol consumption on epigenetic changes, or comparison between DNA methylation in peripheral blood and brain, we used a subacute mouse drinking model to estimate the validity of EtOH-induced HTR3A
promoter methylation changes in the peripheral blood of human alcoholics and deduce the effects of chronic alcohol consumption on HTR3A
methylation and expression in human brain.
First, we observed that the methylation levels of 2 CpG sites (CpG−27 and CpG+54) in the mouse gene Htr3a
promoter region and the mean methylation level of the 8 Htr3a
promoter CpGs were significantly higher in the blood of EtOH-exposed mice when compared to that of water-drinking mice ( and ). This result is consistent with our recent finding that an HTR3A
promoter CpG was hypermethylated in the peripheral blood of alcohol dependent human subjects (Zhang et al., in press
). These data indicate that chronic EtOH consumption can causally alter HTR3A
promoter methylation status and produce the hypermethylation observed in the peripheral blood of human alcoholics. Nevertheless, this conclusion should be made with caution, as the mouse Htr3a
promoter DNA sequence analyzed in this study was not homologous to the human HTR3A
promoter DNA sequence and, of course, the subacute EtOH consumption in the DID model does not recapitulate consumption patterns in human alcoholics.
Additionally, we used the mouse model to investigate EtOH-induced methylation changes of mouse Htr3a
promoter CpGs in 9 different brain regions to model DNA methylation changes caused by chronic EtOH consumption in the human brain (which is not easily accessible) and to assess whether peripheral changes are indicative of DNA methylation levels in the brain. Here, we investigated EtOH-induced DNA methylation changes in mouse prefrontal cortex, striatum, VTA, AMY, and HIPPO, all of which play important roles in reward and motivational processes because of their involvement in dopaminergic and glutamatergic neurotransmission and thus mediate reinforcing effects of alcohol or drugs of abuse (Jentsch and Taylor, 1999
; Lobo and Nestler, 2011
; Pascual et al., 2009
). The tissue from the CBL, which mediates many of alcohol’s effects on motor control, was also collected. As shown in , EtOH drinking resulted in either hypermethylation (in the HIPPO) or hypomethylation (in the DMPFC and the DMSTR) of certain CpGs in mouse Htr3a
promoter region. The altered methylation of HTR3A
promoter region caused by alcohol consumption may have a significant effect on HTR3A
transcription and ultimately influence vulnerability to alcohol dependence by impacting serotonin signaling at the 5-HT3
Several brain regions (including CBL) did not show significantly altered DNA methylation levels of Htr3a in EtOH-exposed mice. These findings suggest that EtOH-induced DNA methylation changes are tissue specific and that gene expression in certain brain regions may be more greatly impacted by EtOH treatment than other brain regions. Moreover, the baseline mean methylation level of 8 mouse Htr3a promoter CpGs in the 9 mouse brain regions varied from about 10% (in CBL) to about 50% (in the VSTR) (). This result suggests a native tissue-specific DNA methylation pattern in the brain of mice that were not exposed to EtOH. It may also help explain the tissue-specific gene expression phenomenon in human and animal brains, given the intimate correlation between gene expression levels and gene promoter methylation levels.
Additionally, EtOH consumption-associated Htra3
expression changes were examined in mouse brain DMSTR tissue samples. As 2 CpGs (CpG−96 and CpG−27) in the promoter of Htr3a
showed hypomethylation in brain DMSTR of EtOH-drinking mice, we hypothesized that Htr3a
expression in the DMSTR would be increased as previous studies have demonstrated an inverse correlation between methylation and gene expression levels in alcoholic subjects (Bleich et al., 2006
; Bonsch et al., 2006
). Expression levels of Htr3a
in the DMSTR were measured by real-time PCR and compared in a separate cohort of 14 EtOH-drinking and 14 water-drinking male CD-1 mice using the comparative CT
expression level in the DMSTR was found to be 1.43-fold higher in alcohol-drinking mice than in water-drinking mice (p
= 0.044). Our findings further suggest that promoter methylation levels may be reversely correlated with gene expression in specific brain regions such as the DMSTR. Brain tissues in which methylation changes were not in Htr3a
promoter region were not analyzed as these methylation changes are not expected to impact expression levels.
Importantly, the use of the 5-day DID approach in CD-1 mice allows mice to drink a maximum amount of EtOH in a nonstressful environment (Rhodes et al., 2007
), therefore reducing an impact of stress on DNA methylation levels and enabling specific detection of EtOH-induced DNA methylation changes in mouse blood and brain. Moreover, CD-1 mice were drawn from a large breeding population that has accumulated many recombination events. The CD-1 genome displays similar patterns of linkage disequilibrium and heterogeneity as those of wild-caught mice (Agrawal et al., 2010
). Hence, the observations in CD-1 mice are applicable to a broad range of genetic studies. It is important to consider that EtOH-induced DNA methylation alterations could be species specific. Although a mouse model has been used to study cocaine-induced histone epigenetic modifications (but not DNA methylation) in the brain (Maze et al., 2011
), there are no such published studies using an animal model to investigate EtOH-induced DNA or histone epigenetic alterations in the brain. It is unknown whether EtOH-induced DNA methylation changes in the outbred CD-1 mice appropriately correlate to that in human alcoholics. It would be useful to confirm these findings with additional models. In addition, although these data indicate that a 5-day EtOH treatment is sufficient to induce detectable DNA methylation changes, a longitudinal study to measure EtOH-induced DNA methylation changes at different time points may provide additional information about the effects of chronic EtOH consumption on alterations in DNA methylation, and the effects of EtOH on Htr3a
Taken together, we employed a mouse model to validate changes in DNA methylation seen in human alcoholics (Zhang et al., in press
). Importantly, our data indicate a causal relationship between EtOH consumption and changes in DNA methylation and gene expression, which has not been possible to investigate in human populations. In addition, our data indicate a lack of a simple relationship between peripheral blood DNA methylation levels with those seen in the brain, although hypermethylation of HTR3A
in peripheral blood could potentially serve as a biomarker reflecting the situation in specific brain regions such as the HIPPO. This information is critical for the investigation of epigenetic changes in the population and highlights the importance of using animal models to validate work with human subjects. The findings from this study contribute to our understanding of the epigenetic mechanisms that underlie the development of alcohol dependence and may help in the effort to identify strategies to modify DNA methylation patterns of genes to prevent or treat alcohol dependence.