ACTN4 Binding to Functional SNP rs9277336 Controls the Genome Architecture and Endothelial Pathophenotypes in Pulmonary Arterial

项目摘要 背景：肺动脉高压(PAH)的特征是肺血管收缩和 血管重塑。全基因组关联研究(GWAS)已经定义了单个 核苷酸多态(SNPs)和PAH。例如，非编码SNP rs2856830，与人类白细胞抗原- DPA1与PAH风险和存活率的增加有关。然而，这种“标签”SNP可能并不是疾病的原因 关联，因为它们经常与邻近的真正的功能SNPs处于连锁不平衡(LD)状态 (FSNPs)导致疾病。结果表明，SNP rs2856830与带有增强子的fSNP rs9277336位于LD中 并结合具有等位基因特异性的转录因子ACTN4。Rs9277336(G)等位基因与 随着PAH严重程度的增加。FSNP rs9277336通过ACTN4和ACTN4的等位基因特异性结合调节人类白细胞抗原-DPA1 与远端基因靶点ATF6B接触。因此，我假设rs9277336(G)等位基因 通过减少ACTN4的等位基因特异性结合来控制内皮功能障碍和PAH，从而 通过近端和远端染色质相互作用扰乱人类白细胞抗原-DPA1和ATF6B的调节。这 验证假设的具体目的如下：(1)确定SNP rs9277336(G)等位基因是否显示 减少了对ACTN4的结合。与肺动脉内皮细胞(PAEC)核接触的寡核苷酸 体外细胞提取液中，rs9277336(G)等位基因与ACTN4的结合低于(A)。在这里，我将表演 更明确的实验以确定ACTN4是否显示出与rs9277336(G)vs(A)的等位基因特异性结合 完整细胞中的等位基因，主要使用同基因可诱导多能干细胞来源的内皮细胞(IPSC-EC) 携带单SNP核苷酸编辑的基因。(2)确定ACTN4与SNP rs9277336结合是否调节人类白细胞抗原-2 DPA1表达。我们发现ACTN4的功能丧失和功能获得相互调节了人类白细胞抗原-4的表达。 PAEC中的DPA1。ACTN4或HLADPA1缺陷介导血管生成和PAEC迁移。我会评估 ACTN4与rs9277336(G)等位基因结合减少是否调节并依赖于人类白细胞抗原-DPA1水平 从而证实了(G)等位基因在IPSC-ECs中的致病作用。(3)确定ATF6B是否 控制PAH的病理性内皮功能。根据现有的Hi-C染色质联系图，我们发现 有证据表明，ACTN4与rs9277336结合可能通过远程相互作用调节ATF6B。ATF6B是一种异构体 ATF6，已知的多环芳烃的介体。我将执行染色质确认捕获(3C)以确定rs9277336是否 身体接触ATF6B基因。我还将评估ATF6B功能的丧失和恢复是否会扰乱PAEC 功能，从而建立了rs9277336与ATF6B远端相互作用的范例，并介导了病理 内皮功能，以及它对人类白细胞抗原DPA1的影响。意义：由于风险(G)等位基因显著 在人群中丰富(MAF=0.852)，这种SNP致病活性的证据将定义为什么显著的 有一部分人有患上更严重的PAH的风险。这种洞察力将对更好地 PAH诊断和治疗学，从而为我作为一名内科科学家的长期职业生涯做好准备。

Project Summary Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling. Genome-wide association studies (GWAS) have defined associations between single nucleotide polymorphisms (SNPs) and PAH. For example, non-coding SNP rs2856830, in proximity to HLA- DPA1, is linked to increased PAH risk and survival. Yet, such “tag” SNPs may not be responsible for disease association, because they are often in linkage disequilibrium (LD) with neighboring true “functional” SNPs (fSNPs) that drive disease. We showed that SNP rs2856830 is in LD with fSNP rs9277336, which has enhancer activity and binds a transcription factor, ACTN4, with allele-specificity. The rs9277336 (G) allele is associated with increased PAH severity. fSNP rs9277336 regulates HLA-DPA1 via allele-specific binding of ACTN4 and comes into contact with a distal gene target, ATF6B. Thus, I hypothesize that the rs9277336 (G) allele controls endothelial dysfunction and PAH through reduced allele-specific binding of ACTN4, thus disrupting the regulation of HLA-DPA1 and ATF6B via proximal and distal chromatin interactions. This hypothesis will be tested with the following Specific Aims: (1) Determine if SNP rs9277336 (G) allele displays reduced binding to ACTN4. With oligonucleotides exposed to pulmonary artery endothelial cell (PAEC) nuclear cell extracts ex vivo, the rs9277336 (G) allele exhibited lower binding to ACTN4 than (A). Here, I will perform the more definitive experiment to determine if ACTN4 shows such allele-specific binding to the rs9277336 (G) vs (A) allele in intact cells, primarily using isogenic inducible pluripotent stem cell-derived endothelial cells (iPSC-EC) that carry single SNP nucleotide edits. (2) Determine if ACTN4 binding to SNP rs9277336 regulates HLA- DPA1 expression. We found that loss- and gain-of-function of ACTN4 reciprocally regulated expression of HLA- DPA1 in PAECs. ACTN4 or HLA-DPA1 deficiency mediated angiogenesis and PAEC migration. I will assess whether reduced ACTN4 binding to the rs9277336 (G) allele regulates and depends upon HLA-DPA1 levels to control function in iPSC-ECs, thus proving the pathogenic action of the (G) allele. (3) Determine if ATF6B controls pathologic endothelial function in PAH. Based on existing Hi-C chromatin contact maps, we found evidence that ACTN4 binding to rs9277336 may regulate ATF6B via long-range interaction. ATF6B is an isoform of ATF6, a known mediator of PAH. I will perform chromatin confirmation capture (3C) to determine if rs9277336 physically contacts the ATF6B gene. I will also assess if ATF6B loss- and gain-of-function disrupts PAEC function, thus establishing a paradigm by which rs9277336 distally interacts with ATF6B and mediates pathologic endothelial function, in addition to its effects on HLA-DPA1. Significance: Since the risk (G) allele is significantly enriched in the population (MAF=0.852), proof of this SNP’s pathogenic activity would define why a significant proportion of persons are at risk for more severe PAH. Such insight would have substantial impact on better PAH diagnostics and therapeutics, thus preparing me for a long career as a physician-scientist.