PROJECT SUMMARY The epigenome refers to covalent modifications of nuclear histone proteins and their associated DNA, which function to regulate gene expression. There is a wealth of data implicating epigenetic remodeling in neurobiological function and behavior, especially in the context of reward pathologies, such as addiction and depression. However, due to the promiscuity of the factors involved, previous studies have failed to distinguish between the mere presence and the functional relevance of a given chromatin modification at a specific gene. This limits the elucidation of the precise molecular mechanisms by which neuroepigenetic remodeling regulates transcription. To address this, we utilize a multidisciplinary approach that involves (1) analysis of chromatin immunoprecipiation (ChIP)- and RNA-sequencing (seq) datasets from brain reward regions following volitional reward behavior and (2) direct manipulation of such modifications, using an innovative strategy of gene-targeted epigenetic editing using engineered transcription factors (ETFs). The current proposal tests the hypothesis that histone posttranslational modifications (HPTMs), specifically histone H3 lysine 36 methylation (H3K36me3), directly functions in reward-mediated pre-mRNA alternative splicing. The rationale for this hypothesis includes recently published data that both alternative splicing and H3K36me3 enrichment are highly regulated by cocaine exposure, and our preliminary finding that there is a significant correlation between genome-wide H3K36me3 enrichment and the splicing complexity of expressed alternative isoforms. While chromatin-mediated alternative splicing is well established in cell- culture systems, it has not yet been described as a mechanism in brain, despite the prevalence of both widespread chromatin remodeling and alternative splicing in neurons. This proposal outlines a novel strategy to demonstrate neuronal H3K36me3-mediated alternative splicing, and the functional significance of this transcriptional mechanism to motivated behavior. In particular, we will analyze neuronal changes in mouse brain reward regions following drug or natural reward self-administration. This work will establish a strategy through which we can examine additional mechanisms of chromatin-mediated alternative splicing in various brain regions, expanding beyond the initial hypotheses of the current proposal.