Genome-wide analysis of the nucleus accumbens identifies DNA methylation signals differentiating low/binge from heavy alcohol drinking

Abstract

Alcohol-use disorders encompass a range of drinking levels and behaviors, including low, binge, and heavy drinking. In this regard, investigating the neural state of individuals who chronically self-administer lower doses of alcohol may provide insight into mechanisms that prevent the escalation of alcohol use. DNA methylation is one of the epigenetic mechanisms that stabilizes adaptations in gene expression and has been associated with alcohol use. Thus, we investigated DNA methylation, gene expression, and the predicted neural effects in the nucleus accumbens core (NAcc) of male rhesus macaques categorized as "low" or "binge" drinkers, compared to "alcohol-naïve" and "heavy" drinkers based on drinking patterns during a 12-month alcohol self-administration protocol. Using genome-wide CpG-rich region enrichment and bisulfite sequencing, the methylation levels of 2.6 million CpGs were compared between alcohol-naïve (AN), low/binge (L/BD), and heavy/very heavy (H/VHD) drinking subjects (n = 24). Through regional clustering analysis, we identified nine significant differential methylation regions (DMRs) that specifically distinguished ANs and L/BDs, and then compared those DMRs among H/VHDs. The DMRs mapped to genes encoding ion channels, receptors, cell adhesion molecules, and cAMP, NF-κβ and Wnt signaling pathway proteins. Two of the DMRs, linked to PDE10A and PKD2L2, were also differentially methylated in H/VHDs, suggesting an alcohol-dose independent effect. However, two other DMRs, linked to the CCBE1 and FZD5 genes, had L/BD methylation levels that significantly differed from both ANs and H/VHDs. The remaining five DMRs also differentiated L/BDs and ANs. However, H/VHDs methylation levels were not distinguishable from either of the two groups. Functional validation of two DMRs, linked to FZD5 and PDE10A, support their role in regulating gene expression and exon usage, respectively. In summary, the findings demonstrate that L/BD is associated with unique DNA methylation signatures in the primate NAcc, and that the methylation signatures identify synaptic genes that may play a role in preventing the escalation of alcohol use.

Keywords: Alcohol; DNA methylation; Differentially methylated regions; Exon-usage; Gene expression; Nonhuman primates.

Figure 1

Characterization of significant differentially methylated CpGs and regions (DMRs) in the NAc core. a) Kernel density estimation plot (R package, vR3.0.2) of the overall delta methylation rates of the significant CpGs discovered comparing AN and L/BD (left) and AN and H/VHD (right). b) Comparison of the average DMR methylation between AN (green, n=8) and L/BD (blue, n= 7); p-value (independent t-test) = 0.043. c) Distribution of chromatin states associated with the 9 NAcc DMRs, based on 7 brain regions reported in the Epigenomics Roadmap Database (Roadmap Epigenomics et al., 2015). TSSA indicates active transcription start site; TSSFlank indicates TSS flanking sequence; TSSFlnkU and TSSDFlnkD are upstream and downstream TSS flaking sequence respectively; EnhG is genomic enhancer; EnhA is active enhancer; Wk.Enh is weak enhancer; TxSt is strong transcriptional activity; TxWk is weak transcriptional activity; Het is associated heterochromatin; ReproPC and ReproPC are repressed polycomb region and weak polycomb region respectively; Quies is quiescent state. d) The average methylation rate for the 9 DMRs is shown for ANs (green), L/BDs (blue) and HDs (red). ANOVA; *p-value<0.05; **p-value<0.01.

Figure 2

Summary of DMR location and gene structure, CpG methylation and transcript expression for FZD5. a) The two exons are represented in grey. The translation start site is represented by a red line (ATG). The DMR (green line) is located in the promoter region, approximately 2.3kb upstream of the transcription start site (TSS). b) The average methylation rate for the 8 CpGs within the DMR is shown for ANs (green boxes) and L/BDs (blue boxes) (n_AN= 8, n_L/BD= 7, independent t-test, p_(AN-L/BD)= 0.016, c) Comparison of the average DMR methylation based on BSAS analysis: ANs shown in green (n=11), L/BDs shown in blue (n=14) and HDs shown in red (n= 12). One-way ANOVA with Bonferroni correction for multiple comparisons, p(FZD5)_ANvsL/BD= 0.031, p(FZD5)_L/BDvsH/VHD= 0.046. d) The relative expression of the single gene transcript is shown (n_AN= 8, n_L/BD= 9, n_HD= 8); One-way ANOVA test with Bonferroni correction for multiple comparisons, p_(AN-L/BD)= 0.022, p_(L/BD-HD)= 0.013). Error bars are mean +/− SEM. e) Correlation between the average amount of alcohol and the normalized expression of FZD5. Pearson’s coefficient r= −0.508, p-value= 0.031. f) Correlation between the average DMR methylation and the normalized expression of FZD5. Pearson’s coefficient r= −0.487, p-value= 0.022.

Figure 3

Summary of DMR location and gene structure, CpG methylation and transcript expression for PDE10A. a) The exons are represented in grey. The translation start site is represented by a red line (ATG). The DMR (green line) is located in the promoter region, approximately 3.0kb upstream of the transcription start site (TSS) of exon 1a. Exon 1b is an alternative first exon with an in-frame ATG codon. b) The average methylation rate for the 7 CpGs within the DMR is shown for ANs (green boxes) and L/BDs (blue boxes) (n_AN= 8, n_L/BD= 7, independent t-test, p_(AN-L/BD)= 0.016). c) Comparison of the average DMR methylation based on BSAS analysis: ANs shown in green (n=9), L/BDs shown in blue (n=11) and HDs shown in red (n= 12). One-way ANOVA with Bonferroni correction for multiple comparisons, p_(AN-L/BD)= 0.037; p_(L/BD-HD)=0.008. d) The relative expression of TVs using either exon 1a or 1b is shown (n_AN= 7, n_L/BD= 8, n_HD= 6; One-way ANOVA test with Bonferroni correction for multiple comparisons, exon 1b: p_(AN-L/BD)= 0.026, p_(AN-HD)= 0.015). Error bars are mean +/−SEM. e) Correlation between the average amount of alcohol and the normalized expression of PDE10A. Pearson’s coefficient r= −0.062, p-value= 0.596. f) Correlation between the average DMR methylation and the normalized expression of PDE10A. Pearson’s coefficient r= −0.579, p-value= 0.024.