HyCCAPP: A new method for the functional analysis of regulatory SNPs

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 分析发现了与多种疾病相关的常见和罕见变异， 相关特征。这些相关的变异体大多位于基因间区域，并且已经提出， 它们调节单个基因的表达。事实上，遗传分析已经确定了大量的 表达数量性状基因座（cis-eQTL），许多序列变异， 与单个基因的表达和疾病相关性状密切相关。但是，在这方面， 破译潜在的分子机制一直是具有挑战性的。传统的实验室方法， 如荧光素酶报告基因构建体或电泳迁移率变动测定，以及最近染色质 分析（DNA酶超敏反应图谱，ATAC-Seq）清楚地表明对疾病的功能影响- 相关的启动子变体，但是没有有效的方法来鉴定与这些变体结合的调节蛋白。 变异位点并介导它们对基因表达调控的作用。因此，替代方法 需要更有效地鉴定这些未知的调节蛋白，其启动子结合和 互作受eQTL变体的影响。 我们最近开发了一种新的方法，染色质相关蛋白的杂交捕获， 蛋白质组学（HyCCAPP），允许识别与特定靶染色质区域结合的所有蛋白质 通过质谱分析。我们建议采用HyCCAPP方法分析荧光素酶报告基因 质粒构建体通常用于评估序列变体对启动子活性的影响，并应用 该技术用于分析所选eQTL启动子变体。我们假设HyCCAPP 这项技术将揭示新的调节蛋白，介导启动子变体对基因的影响， 表达，揭示潜在的新的分子机制eQTL。我们将追捕三名 具体目的：1）优化HyCCAPP用于荧光素酶报告质粒的分析，2）分析选择 使用质粒HyCCAPP在eQTL区域中的启动子变体，和3）验证蛋白质结合对 CRISPR-Cas9编辑的细胞系中的体内基因表达。 HyCCAPP靶向荧光素酶报告质粒的技术开发和原理验证 应用于与基因表达变化相关的启动子变体，将建立一个强大的新的， 有效的工具，用于研究调控序列变异体改变 调节蛋白。目前没有其他技术可以有效地从头鉴定DNA结合 受序列变异影响的蛋白质。使用常用的荧光素酶报告质粒的方法 新的HyCCAPP技术将有助于揭示基因表达调控的新机制 有助于人类疾病的发展和进展。

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), that 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.