Alcohol abuse disorders (AUD) are a major health hazard that affects millions of people every year in the United States. Risk factors for AUD include initial resistance to the intoxicating effects of alcohol, as well as the development of tolerance upon repeat exposure. AUDs also have significant genetic etiology, and many genes have been implicated from human genome-wide association studies (GWAS), including by our studies. A detailed molecular understanding of many of these genes is still lacking, though. “Regulation of the actin cytoskeleton” has been implicated by numerous mammalian alcohol transcriptomics studies, and major regulators of actin dynamics include the Rho family of GTPases, and their regulators. Our findings, supported by prior funding periods of this competitive renewal grant, have shown that SNPs in the Rho GTPase regulator RSU1 are associated with alcohol dependence and drinking, underscoring the translational significance of our studies. Our overall goal is to better understand the molecular mechanisms and neural tissue-specificity of Rho GTPase regulators using the powerful genetic toolkit of Drosophila. Based on our prior findings and extensive preliminary data, we hypothesize that different Rho GTPase regulators have distinct alcohol response phenotypes, depending on the neural circuits they are manipulated in. We will first determine the circuits and neurotransmitter systems that require regulators of the Rac1 GTPase from the Rho family for normal alcohol-induced sedation and tolerance. Second, we will determine the role in alcohol sedation and tolerance of 11 additional members of Rho GTPase signaling, including 8 orthologs of genes associated with addiction/alcohol phenotypes in human GWAS studies. These candidates have been suggested by alcohol- induced changes in accessibility of their DNA regulatory regions, as determined by our ATAC-seq experiments. The data also highlight four transcription factors, based on the overrepresentation of their binding motifs in alcohol-induced changes of accessible DNA regulatory elements. In a third aim, we will determine the role of two of these potential master regulators of alcohol-induced changes in gene regulation and in behavioral alcohol responses. The use of ATAC-seq to determine DNA regulatory elements in a tissue specific manner highlights a major innovative approach of our application. Taken together, these Aims are in line with NIAAA priorities focusing on the “Genetics of Alcohol Sensitivity and Tolerance”PA-18-660, which emphasize specific areas of research interest including “translational research in model organisms…for in vivo validation of candidate genes…from studies in humans”, and “studies of candidate genes…for tissue-specific gene expression, and for contributions to biological pathways”. Many prior publications linked to this grant, together with extensive preliminary data demonstrate the expertise of the investigator and the feasibility of the study. Our prov...