Administrative Supplement - pHyCCAPP

在过去的十年里，全基因组关联研究和全面的全基因组 测序分析发现了常见和罕见的变异，这些变异与广泛的 与疾病相关的特征。这些相关变异中的大多数位于基因间隔区，而且一直是 这表明它们可以调节单个基因的表达。事实上，基因分析已经确定了一种 与患病基因相邻的大量表达数量性状基因座(cis-eQTL)，以及许多 序列变异既与单个基因的表达密切相关，也与疾病相关 特征。然而，破译潜在的分子机制一直是具有挑战性的。传统型 实验室方法，如荧光素酶报告构建或电泳迁移率改变分析 由于最近的染色质分析(DNA酶超敏反应图谱，ATAC-Seq)清楚地表明 对疾病相关启动子变体的影响，但目前还没有有效的方法来识别调控 与这些变异位点结合的蛋白质，并介导它们对基因表达的调节作用。 因此，需要其他方法来更有效地识别这些未知的调节蛋白。 其启动子结合和相互作用受eQTL变异的影响。 我们最近开发了一种新的方法，杂交捕获染色质相关蛋白 蛋白质组学(HyCCAPP)，允许通过以下方式鉴定与特定目标染色质区域结合的所有蛋白质 质谱学。我们建议采用HyCCAPP方法来分析荧光素酶报告 通常用于评估序列变体对启动子活性的影响的质粒构建体，并应用 用于分析选定的eQTL启动子变体的技术。我们假设HyCCAPP 技术将发现新的调控蛋白，介导启动子变异对基因的影响 表达，揭示了eQTL潜在的新的分子机制。我们将追求三个目标 具体目标：1)优化HyCCAPP用于荧光素酶报告质粒分析；2)分析选择 利用质粒HyCCAPP在eQTL区域的启动子变异，以及3)验证蛋白质结合对 CRISPR-Cas9编辑细胞系的体内基因表达。面向目标的HyCCAPP技术发展 荧光素酶报告质粒，以及对与基因相关的启动子变体的原理证明应用 表达变化，将建立一个强大的新的和有效的工具，通过 哪些调控序列变体改变了调控蛋白的结合。目前没有其他技术允许 受序列变异影响的DNA结合蛋白的有效从头识别。常用的用法 使用荧光素酶报告质粒方法与新的HyCCAPP技术将有助于揭示新的 基因表达调控在人类发育进步中的作用机制 精神错乱。

Over the past decade, genome-wide association studies and comprehensive whole-genome sequencing analyses have uncovered both common and rare variants that are associated with a wide range of disease related traits. The majority of these associated variants lie in intergenic regions, and it has been suggested that they modulate the expression of individual genes. Indeed, genetic analyses have identified a large number of expression quantitative trait loci adjacent to the affected genes (cis-eQTL), and many sequence variants are strongly associated with both the expression of individual genes and disease-related traits. However, deciphering the underlying molecular mechanisms has been challenging. Traditional laboratory approaches, such as luciferase reporter constructs or electrophoretic mobility shift assays, as well as recent chromatin analyses (DNase hypersensitivity mapping, ATAC-Seq) clearly suggest a functional impact for disease associated promoter variants, but no effective methods exist to identify the regulatory proteins binding to these variant sites and mediating their effect on the regulation of gene expression. Therefore, alternative approaches are required to more efficiently identify these unknown regulatory proteins whose promoter binding and interaction is affected by eQTL variants. We recently developed a novel approach, Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP), allows the identification of all proteins bound to a specific target chromatin region by mass spectrometry. We propose to adapt the HyCCAPP approach for the analysis of luciferase reporter plasmid constructs commonly used to assess the impact of sequence variants on promoter activity, and apply the technology to the analysis of selected eQTL promoter variants. We hypothesize that the HyCCAPP technology will uncover novel regulatory proteins mediating the effect of promoter variants on gene expression, revealing potentially novel molecular mechanisms underlying eQTLs. We will pursue three Specific Aims: 1) optimize HyCCAPP for the analysis of luciferase reporter plasmids, 2) analyze select promoter variants in eQTL regions using plasmid HyCCAPP, and 3) validate the impact of protein binding on gene expression in vivo in CRISPR-Cas9 edited cell lines. The technology development of HyCCAPP to target luciferase reporter plasmids, and a proof-of-principle application to promoter variants associated with gene expression changes, will establish a powerful new and effective tool for the investigation of the mechanisms by which regulatory sequence variants alter binding of regulatory proteins. No other current technology allows the effective de novo identification of DNA binding proteins affected by sequence variants. The use of commonly used luciferase reporter plasmid approaches with the new HyCCAPP technology will help reveal new mechanisms of gene expression regulation contributing to the development and progression of human disorders.