Alcohol Research Center

Chronic alcohol consumption results in brain gene expression changes that contribute to the risk of developing alcohol use disorder (AUD). Gene expression is regulated by epigenetic and chromatin remodeling mechanisms that alter transcription factor binding to DNA at specific genomic regions. This proposal focuses on the mammalian (m)SWI/SNF chromatin remodeling complex, which has previously been linked to AUD in human genetic studies and to ethanol tolerance in worms by the VCU-ARC. We will study two mutually exclusive core enzymatic subunits of the mSWI/SNF complex, SMARCA2 and SMARCA4, for their roles in regulating gene expression, ethanol consumption, and behavioral responses to ethanol in mice. We hypothesize that ethanol-induced increases in SMARCA2 and SMARCA4 in the ventral hippocampus result in increased occupancy of SMARCA2 and SMARCA4 at specific genomic locations to subsequently alter gene expression, and that these two members of the mSWI/SNF complex differentially regulate gene expression and behavioral responses to ethanol. In Specific Aim 1, we will use sensitive, state of the art methods to identify genomic regions at which SMARCA2 and SMARCA4 bind to regulate chromatin states and gene expression in specific cell types. The first experiment will employ cleavage under targets and release using nuclease (CUT&RUN) followed by massively parallel sequencing to determine the genome-wide targets of SMARCA2 and SMARCA4 under ethanol-naïve conditions and after chronic binge-like ethanol consumption. In the second experiment, we will perform single-nuclei RNA sequencing (snRNA-Seq) to identify genes in specific cell types that are altered by chronic binge- like ethanol consumption. Integration of the CUT&RUN and snRNA-Seq data will identify cell type-specific genes regulated by SMARCA2 and SMARCA4. Finally, we will use fluorescent in situ hybridization (RNAscope) to determine the ventral hippocampal regional specificity and cell types where SMARCA2, SMARCA4, and selected target genes, are altered following chronic binge-like drinking. In Specific Aim 2, in collaboration with the Behavior Core, we will use pharmacological and genetic tools to determine the roles of SMARCA2 and SMARCA4 in ethanol consumption, ethanol-induced locomotor activation and anxiolysis, and ethanol sedation sensitivity and tolerance. In the first experiment, we will employ dual SMARCA2/SMARCA4 small-molecule inhibitors and in the second experiment, we will conditionally knockout Smarca2 and Smarca4 in a Cre-dependent manner in the ventral hippocampus using a viral approach. This project will provide important functional information to VCU- ARC Projects 1, 2, 4, and 5. The overall impact of this study will be greater knowledge of the molecular mechanisms driving brain gene expression and behavioral alterations following chronic alcohol drinking and identification of a potential new pharmacotherapeutic target for AUD.