Ethanol-induced Htr3a Promoter Methylation Changes in Mouse Blood and Brain

Background: Abnormal DNA methylation has been observed in promoter regions of a number of genes in human alcoholics. It is unclear whether DNA methylation changes in alcoholics result directly from alcohol consumption or predated the occurrence of alcohol abuse or dependence and whether altered DNA methylation influences gene expression.

Methods: We investigated ethanol (EtOH)-induced DNA methylation changes in mouse serotonin receptor 3a gene (Htr3a). A 5-day drinking-in-the-dark paradigm was applied to 28 male outbred CD-1 mice (15 EtOH-drinking and 13 water-drinking). The Sequenom MassARRAY approach was used to quantify methylation levels of 8 CpGs around Htr3a transcription start site in trunk blood and 9 brain regions (dorsomedial prefrontal cortex [DMPFC], ventromedial prefrontal cortex, ventral tegmental area, dorsolateral striatum, dorsomedial striatum [DMSTR], ventral striatum, amygdala, hippocampus [HIPPO], and cerebellum). DNA methylation differences between the 2 groups of mice (EtOH- and water-drinking) were analyzed using multivariate analysis of covariance with consideration of EtOH consumption amount. Expression levels of Htr3a in the DMSTR were measured by real-time PCR in 14 EtOH-drinking and 14 water-drinking male CD-1 mice.

Results: EtOH drinking increased methylation levels of specific Htr3a promoter CpGs in mouse blood (CpG-27: p = 0.028; CpG+54: p = 0.044) and HIPPO (CpG+151: p = 0.012) but reduced methylation levels of specific Htr3a promoter CpGs in mouse DMSTR (CpG-96: p = 0.020; CpG-27: p = 0.035) and DMPFC (CpG+138: p = 0.011; CpG+151: p = 0.040). Nevertheless, methylation levels of Htr3a promoter CpGs in 6 other brain regions were not significantly altered by EtOH consumption. Additionally, the expression level of Htr3a in the DMSTR was 1.43-fold higher in alcohol-drinking mice than in water-drinking mice (p = 0.044).

Conclusions: Our findings indicate that alcohol consumption may induce tissue-specific DNA methylation changes and further suggest that Htr3a promoter methylation levels may be reversely correlated with Htr3a expression levels in specific brain regions such as DMSTR.