In vivo massive parallel reporter assay to analyze the function of cis-regulatory elements and variants implicated in substance use disorders

Project Summary Substance use disorders (SUD) are highly heritable. Genome-wide association studies (GWAS) have begun to identify genetic variants associated with SUD, but their functional role remains obscure as most of these variants lie in non-coding genomic regions, for which it is difficult to infer function. The primary goal of this proposal is to establish and deploy a massively parallel reporter assay (MPRA) in mouse brains in vivo after chronic exposure to fentanyl to measure regulatory activity and the effect of opioid addiction-associated human genetic variants. The MPRA leverages an innovative design (paired-TSS-MPRA) that captures transcription initiation from both candidate regulatory sequences and a reporter gene promoter, enabling us to probe enhancer transcription and enhancer function simultaneously at single-nucleotide resolution. This assay will allow us to characterize the regulatory grammar of elements that are differentially regulated in addiction settings, while also providing a platform for us to assess the regulatory potential of human GWAS-derived disease-risk variants in vivo. The R21 phase will ensure the rigor and reproducibility of our method by designing and validating an addiction-targeted paired-TSS-MPRA method in primary neurons and brain tissues of mice. The R33 phase will functionally characterize regulatory elements and human SUD-related GWAS variants in mice models of chronic fentanyl exposure (Aim 1) and validate the role of key transcription factors in regulatory mechanisms associated with fentanyl addiction-like behaviors (Aim 2). This knowledge will enable us to determine the precise mechanisms by which genetic variants will impact cis-regulatory activity and will provide a conceptual and analytical framework for studying the functional role of genetic variants associated with SUD.