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， 血管重塑全基因组关联研究（GWAS）已经定义了单个基因组之间的关联。 核苷酸多态性（SNP）和PAH。例如，非编码SNP rs 2856830，接近于HLA-SNP。 DPA 1与PAH风险和生存率增加有关。然而，这种“标签”SNP可能不是疾病的原因， 关联，因为它们通常与相邻的真正的“功能性”SNP处于连锁不平衡（LD）中 （fSNPs）驱动疾病。我们发现SNP rs 2856830与fSNP rs 9277336处于LD中，其具有增强子， 活性并以等位基因特异性结合转录因子ACTN 4。rs 9277336（G）等位基因与 PAH严重程度增加。fSNP rs 9277336通过ACTN 4的等位基因特异性结合调节HLA-DPA 1 与一个远端基因靶点ATF 6 B接触。因此，我假设rs 9277336（G）等位基因 通过降低ACTN 4的等位基因特异性结合来控制内皮功能障碍和PAH， 通过近端和远端染色质相互作用破坏HLA-DPA 1和ATF 6 B的调节。这 （1）确定SNP rs 9277336（G）等位基因是否表现出 减少与ACTN 4的结合。用寡核苷酸暴露于肺动脉内皮细胞（PAEC）核 在离体细胞提取物中，rs 9277336（G）等位基因表现出比（A）更低的与ACTN 4的结合。在这里，我将执行 确定ACTN 4是否显示与rs 9277336（G）相比（A）的等位基因特异性结合的更确定性实验 完整细胞中的等位基因，主要使用等基因诱导型多能干细胞衍生的内皮细胞（iPSC-EC） 携带单核苷酸单核苷酸编辑。(2)确定ACTN 4与SNP rs 9277336的结合是否调节HLA- DPA 1表达。我们发现，ACTN 4功能的丧失和获得可调节HLA-1的表达。 PAEC中的DPA 1。ACTN 4或HLA-DPA 1缺陷介导的血管生成和PAEC迁移。我将评估 ACTN 4与rs 9277336（G）等位基因结合的减少是否调节并依赖于HLA-DPA 1水平， 在iPSC-EC中的控制功能，从而证明了（G）等位基因的致病作用。(3)确定ATF 6 B是否 控制PAH中的病理性内皮功能。基于现有的Hi-C染色质接触图，我们发现 证据表明，ACTN 4与rs 9277336的结合可能通过长程相互作用调节ATF 6 B。ATF 6 B是一种同种型 ATF 6是一种已知的PAH介质。我将进行染色质确认捕获（3C），以确定rs 9277336是否 与ATF 6 B基因发生物理接触。我还将评估ATF 6 B功能丧失和获得是否会破坏PAEC 功能，从而建立了rs 9277336远端与ATF 6 B相互作用并介导病理性 内皮功能，除了其对HLA-DPA 1的影响。意义：由于风险（G）等位基因显著 在人群中富集（MAF=0.852），该SNP的致病活性的证据将定义为什么显著的 有更严重PAH风险的人群比例。这样的洞察力将对更好地 PAH诊断和治疗，从而为我作为一名医生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.