Functional Genomic Approaches to Dissect COPD GWAS Loci

描述(申请人提供)：慢性阻塞性肺疾病(COPD)是美国第三大致死原因，受吸烟和遗传易感性的影响很大。这项建议的主要目标是识别功能性遗传变异 目的：研究两个COPD全基因组关联基因座HHIP和FAM13A区域的功能变异，并确定这些功能变异改变COPD相关基因表达和细胞表型的分子机制。我们假设这两个基因座的功能变异是调节性变异，可以通过量化人支气管上皮细胞中的增强子活性来衡量。此外，我们假设功能变体通过与特定转录因子的差异结合来调节HHIP或FAM13A基因的表达，从而影响细胞对吸烟诱导的细胞损伤的敏感性。为了验证这些假设，在项目的R21阶段，我们将应用最近开发的大规模平行报告分析(MPRA)来识别在暴露于室内空气或香烟烟雾(CS)中的BEAS-2B细胞中显示等位基因特异性增强子活性的功能变体，随后在报告分析中进行验证。我们还将 使用FIRE确认原代人支气管上皮细胞(HBE)中等位基因特异性的开放染色质，我们将使用染色体构象捕获分析来评估已识别的调控元件与相关启动子的远程相互作用。在项目的R33阶段，我们将确定功能变体在吸烟诱导BEAS-2B细胞和HBE细胞死亡中的作用。随后，我们将应用针对1529个已知人类转录因子(TF)的siRNAs文库来鉴定与所鉴定的对遗传变体具有不同亲和力的增强子元件结合的TF(通过EMSA和CHIP)，改变增强子的活性(使用共转染和报告分子分析)，以及调节BEAS-2B细胞中的HHIP或FAM13A的表达(通过基因沉默后的RT-PCR)。为了证明这些机制研究的临床相关性，我们将在吸烟者的HBE细胞中进行体内验证，这些细胞来自已建立COPD对照的吸烟者。最终，这些功能特征将确定具有潜在治疗意义的生物途径。

DESCRIPTION (provided by applicant): Chronic obstructive pulmonary disease (COPD), the third leading cause of death in the U.S, is strongly influenced by cigarette smoking and genetic predisposition. The primary objectives of this proposal are to identify functional genetic variants in two COPD genome-wide association (GWAS) loci, HHIP and FAM13A regions, and to define the molecular mechanisms by which these functional variants alter gene expression and cellular phenotypes relevant to COPD. We hypothesize that functional variants in these two loci are regulatory variants that can be measured by quantifying enhancer activity in human bronchial epithelial cells. Furthermore, we hypothesize that functional variants, through differential bindin to specific transcription factors, regulate HHIP or FAM13A gene expression, thus affecting cellular sensitivity to smoke-induced cell injury. To test these hypotheses, in the R21 phase of the project, we will apply recently developed massively parallel reporter assays (MPRA) to identify functional variants that show allele-specific enhancer activity in Beas-2B cells exposed to either room air or cigarette smoke (CS), followed by validation in reporter assays. We will also confirm allele- specific open chromatin in primary human bronchial epithelial cells (HBE) using FAIRE, and we will assess long-range interactions of identified regulatory elements with the relevant promoters using chromosome conformation capture assays. In the R33 phase of the project, we will determine the role of the functional variants in smoke-induced cell death in both Beas-2B cells and HBE. Subsequently, we will apply an siRNAs library targeting 1529 known human transcription factors (TFs) to identify TFs that bind to the identified enhancer elements with differential affinity to genetic variants (by EMSA and ChIP), alter enhancer activity (using co-transfection and reporter assays), and regulate HHIP or FAM13A expression (by RT-PCR following gene silencing) in Beas-2B cells. To demonstrate the clinical relevance of these mechanistic studies, we will perform in vivo validation in HBE cells from smokers with established COPD vs. smoking controls. Ultimately, these functional characterizations will identify biological pathways with potential therapeutic implications.