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的变异-- 相关特征。这些相关变异大多存在于基因间隔区，有人认为 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 表达与受影响基因相邻的数量性状基因座(cis-eQTL)，以及许多序列变体 与个体基因的表达和与疾病相关的特征密切相关。然而， 破译潜在的分子机制一直是一个挑战。传统的实验室方法， 例如荧光素酶报告构建或凝胶迁移率改变分析，以及最近染色质 分析(DNA酶超敏反应图谱，ATAC-Seq)清楚地表明了对疾病的功能影响- 相关的启动子变体，但没有有效的方法来识别与这些变体结合的调控蛋白 变异位点及其在基因表达调控中的中介作用。因此，替代方法 需要更有效地鉴定这些未知的调控蛋白，这些蛋白的启动子结合和 互作受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)，它允许识别与特定目标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。 HyCCAPP靶向荧光素酶报告质粒的技术进展及原理验证 应用于与基因表达变化相关的启动子变异，将建立一种强大的新的和 用于研究调节序列变体改变结合的机制的有效工具 调节蛋白。目前没有其他技术允许对DNA结合进行有效的从头识别 受序列变异影响的蛋白质。常用荧光素酶报告质粒的使用方法 新的HyCCAPP技术将有助于揭示基因表达调控的新机制 促进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.