Decoding regulatory functions of genetic variants associated with substance use disorders

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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 develop and validate an innovative high-throughput method that will facilitate the interpretation of the functional role of genetic variants. There is strong evidence supporting a role for non-coding variants in transcriptional regulation as they are enriched in putative cis-regulatory elements, such as cell type- or stimulus- specific enhancers and promoters. However, our understanding of the cis-regulatory code is still rudimentary, making it difficult to predict how a given variant will impact gene regulatory functions from sequence alone. To address this limitation, we have developed a new method to profile transcription initiation sites with single-nucleotide precision from a massively parallel reporter assay (TSS-MPRA). Unlike other available methods screening genetic variants, this method will provide a direct readout of nascent transcription revealing both the frequency and precise locations of RNA polymerase II (RNAPII) recruitment to Transcription Start Sites (TSSs). This knowledge will enable us to determine the precise mechanisms by which genetic variants will impact cis-regulatory activity (including the specific regulators or chromatin features they impact). Here we propose to establish the newly developed TSS-MPRA in a relevant experimental system for SUD research (Aim 1) and demonstrate its ability to decode regulatory variant functions in neuronal systems (Aim 2). Thus, the overarching goal of this proposal is to provide a conceptual and analytical framework for studying the functional role of genetic variants associated with SUD.