Epigenetic Regulation of Stress-Potentiated Ethanol Drinking PROJECT SUMMARY/ABSTRACT A common mechanism that regulates both stress-sensitivity and alcohol use is epigenetic regulation of gene transcription. Of particular importance in substance-use disorders is G9a, a histone methyltransferase implicated in models of alcohol use disorder (AUD). G9a in the nucleus accumbens (NAc) regulates drinking, but its mechanisms are unknown. The long-term goal of this project is to elucidate the mechanisms that can lead to stress-potentiated drinking and to discover methods to block or reverse these changes. Because the dynorphin system plays a prominent role in stress and ethanol-related behaviors, and dynorphin is present in a major subset of NAc neurons, Aim 1 will test the hypothesis that NAc G9a’s effects on stress-potentiated ethanol drinking are mediated through dynorphin-positive neurons (NAcDyn+). This hypothesis will be tested by using a novel Cre- dependent shRNA viral vector and a novel Cre-dependent G9a over-expression viral vector in both dynorphin- Cre mice and enkephalin-Cre mice. Specifically, G9a will be knocked down or overexpressed in these NAc neuronal subsets, and the effects of G9a will be tested on two different forms of stress-potentiated drinking. We will also examine the NAc subregions involved in G9a’s effects. Next, Aim 2 will test the hypothesis that the mechanism underlying G9a’s effects on stress-potentiated ethanol drinking involve NAc intrinsic excitability by altering the expression of a specific potassium (K+) channel subunit. The hypothesis for Aim 2 is that the effects of G9a on this K+ channel subunit reduces stress-potentiated drinking. We will directly test G9a’s ability to regulate this K+ channel subunit with a CRISPR-fused G9a, and we will test G9a’s effects on transcription and chromatin state using single nuclei multiomics. Finally, Aim 3 will test the hypothesis that the effects of NAc G9a on stress-potentiated ethanol drinking ar