Decoding regulatory functions of genetic variants associated with substance use disorders

解码与物质使用障碍相关的遗传变异的调节功能

• 批准号: 10467696
• 负责人: Christopher W Benner
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10467696
• 关键词: Address; Animal Model; Biological; Biological Assay; Cells; Cerebrum; Chromatin; Code; Communities; Complement; Computer Analysis; DNA; Data; Development; Disease; Disease model; Elements; Enhancers; Ensure; Epigenetic Process; Frequencies; Future; Gene Expression; Gene Expression Profile; Genetic Transcription; Genetic screening method; Genomic Segment; Goals; Heritability; Knowledge; Length; Location; Measures; Mediating; Membrane; Methods; Mus; Neurons; Nucleosomes; Nucleotides; Organoids; Pattern; Positioning Attribute; RNA Polymerase II; Regulation; Regulator Genes; Regulatory Element; Reporter; Reproducibility; Research; Role; Series; Signal Transduction; Site; Stimulus; Substance Use Disorder; Substance abuse problem; Substance of Abuse; System; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Untranslated RNA; Variant; addiction; cell type; design; experimental study; expression vector; genetic variant; genome wide association study; improved; in vivo; induced pluripotent stem cell; innovation; next generation sequencing; promoter; recruit; therapeutic target; therapeutically effective

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.

项目摘要 药物使用障碍（SUD）是高度遗传的。全基因组关联研究（GWAS） 已经开始识别与SUD相关的遗传变异，但它们的功能作用仍然晦涩难懂 这些变体中的大多数在于非编码基因组区域，很难推断功能。 该提案的主要目的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。有强烈的证据 支持非编码变体在转录调节中的作用，因为它们在推定中富含 顺式调节元素，例如细胞类型或刺激特异性增强子和启动子。然而， 我们对顺式调节法规的理解仍然是基本的，因此很难预测给定的 变体将单独影响基因调节功能。为了解决这一限制，我们有 开发了一种新方法来介绍具有单核苷酸精度的转录起始位点 大规模并行的记者测定法（TSS-MPRA）。与其他可用方法筛选遗传不同 变体，此方法将直接读取新生的转录，以揭示频率 RNA聚合酶II（RNAPII）募集到转录起始位点（TSSS）的精确位置。 这些知识将使我们能够确定遗传变异的确切机制 影响顺式调节活性（包括其影响的特定调节剂或染色质特征）。 在这里，我们建议在相关的实验系统中建立新开发的TSS-MPRA SUD研究（AIM 1），并证明其解码神经元中调节变体功能的能力 系统（目标2）。因此，该提案的总体目标是提供概念和分析 研究与SUD相关的遗传变异的功能作用的框架。