Decoding regulatory functions of genetic variants associated with substance use disorders

项目摘要 物质使用障碍（SUD）具有高度遗传性。全基因组关联研究（GWAS） 已经开始鉴定与SUD相关的遗传变异，但它们的功能作用仍然不清楚， 这些变异体中的大多数位于非编码基因组区域，很难推断其功能。 该提案的主要目标是开发和验证一种创新的高通量方法， 将有助于解释遗传变异的功能作用。有强有力的证据 支持非编码变体在转录调控中的作用，因为它们富含推定的 顺式调节元件，如细胞类型或刺激特异性增强子和启动子。然而，在这方面， 我们对顺式调控密码的理解仍然是初步的，因此很难预测给定的 变异将单独从序列影响基因调控功能。为了解决这个问题，我们 开发了一种新的方法，以单核苷酸的精度从一个转录起始位点进行分析。 大规模平行报告基因测定（TSS-MPRA）。与其他可用的遗传筛查方法不同 变体，该方法将提供新生转录的直接读数，揭示了频率 以及RNA聚合酶II（RNAPII）募集到转录起始位点（TSS）的精确位置。 这些知识将使我们能够确定遗传变异的精确机制， 影响顺式调节活性（包括它们影响的特定调节剂或染色质特征）。 在这里，我们建议建立新开发的TSS-MPRA在相关的实验系统， SUD研究（目标1），并证明其解码神经元调控变体功能的能力 系统（目标2）。因此，本提案的总体目标是提供一个概念性和分析性的 研究与SUD相关的遗传变异的功能作用的框架。

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.