SUMMARY Chronic and excessive alcohol use can lead to alcohol dependence, a relapsing and remitting condition that ultimately costs lives, disrupts families and places a burden on health-care and judicial systems. Despite the increasing prevalence of alcohol use disorders (AUDs), there are still a limited number of pharmacological treatment options available, and they are largely ineffective at reducing drinking levels and preventing relapse in the majority of AUD patients. We, and others, propose that to design more effective treatments, a better understanding of the molecular mechanisms underlying AUDs is required. Towards this goal, our prior studies have focused on the nucleus accumbens (NAc), a brain region that processes motivated behaviors and that contributes to learning and memory. Chronic alcohol use has been associated with several neuroadaptations in the NAc that are posited to promote alcohol dependence, including alterations in dendritic spine plasticity and glutamate and dopamine signaling, among others. Thus, understanding of the specific molecular mechanisms associated with alcohol use in the NAc will provide insight into the candidate targets underlying these observed neuroadaptations and aid in the development of more targeted treatments. Recently we conducted a genome- wide screen for DNA methylation signatures in the NAc, specifically differentially methylated CpGs (DMCs) and regions (DMRs), identifying signals that distinguished heavy and light drinking rhesus macaques. In addition, the comparison of our findings to those reported in a similar methylation study of prefrontal cortex (PFC) from human control and AUD subjects identified some of the same genes, despite the studies being conducted in two different primate species and in two different brain regions. Specifically, some of the DMRs detected in the NAc and human PFC mapped to genes involved in Wnt and GABA receptor signaling, among other relevant pathways. Accordingly, this R03 small grant application aims to conduct genome-wide DNA methylation (GWDM) analysis of NAc tissue obtained from the same 32 AUD and control subjects previously used for DNA methylation analysis of human PFC (both PFC and NAc were obtained from the New South Wales Tissue Resource Center). This design enables within-subject comparison of human NAc and PFC, providing insight into the commonalities and differences in alcohol-associated signals across brain regions. The comparison of the human NAc DMCs/DMRs with those previously identified in rhesus macaque NAc following long-term alcohol use will identify alcohol-associated genes and mechanisms that are conserved in humans and primates. Finally, by comparing DMCs/DMRs obtained from males and females, we will be able to identify alcohol-associated sex-effects in the NAc. In total, this study will identify mechanisms associated with alcohol- linked neuroadaptions, informing the design of novel AUD therapies, and the translational value of testing them in nonhuman primates.