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

ACTN4 与功能性 SNP rs9277336 结合控制肺动脉中的基因组结构和内皮病理表型

• 批准号: 10750069
• 负责人: Anna Kirillova
• 金额: $ 5.27万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-26 至 2027-03-25
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750069
• 关键词: ATF6 gene; Alleles; Angiogenesis Inhibition; Antigen-Presenting Cells; Architecture; Behavior; Binding; Biological Assay; Blood Vessels; Cardiovascular system; Cell Extracts; Cell physiology; Cells; Chromatin; Clinical; DNA; Data; Diagnosis; Diagnostic; Disease; Distal; Endothelial Cells; Endothelium; Enhancers; Exhibits; Exposure to; Gene Targeting; Genes; Genetic; Genome; Genotype; Hematological Disease; Histocompatibility Antigens Class II; Human; Immune; Inflammatory; Link; Linkage Disequilibrium; Lung; Lung diseases; Maps; Mediating; Mediator; Mutation; Nuclear; Nucleotides; Oligonucleotides; Pathogenicity; Pathologic; Patients; Peripheral Blood Mononuclear Cell; Persons; Phenotype; Physicians; Population; Populations at Risk; Process; Property; Protein Isoforms; Regulation; Risk; Role; SNP genotyping; Scientist; Severities; Severity of illness; Single Nucleotide Polymorphism; Specificity; Testing; Therapeutic; Training; Untranslated RNA; Variant; Vascular remodeling; angiogenesis; career; cell motility; cell type; chromatin immunoprecipitation; clinical translation; cohort; diagnostic tool; endothelial dysfunction; experimental study; gain of function; genome wide association study; induced pluripotent stem cell; insight; loss of function; migration; mortality; novel diagnostics; physical model; prognostic tool; pulmonary arterial hypertension; pulmonary artery endothelial cell; pulmonary vasoconstriction; transcription factor; vasoconstriction

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.

项目摘要 背景：肺动脉高压（PAH）的特征是肺部血管收缩和 血管重塑。全基因组关联研究（GWAS）已确定了单一的关联 核苷酸多态性（SNP）和PAH。例如，非编码SNP RS2856830，与HLA- DPA1与PAH风险增加和生存有关。但是，这种“标签” SNP可能不负责疾病 关联，因为它们通常与相邻的真实“功能性” SNP处于连锁二序（LD） （FSNP）驱动疾病。我们证明SNP RS2856830在LD中使用FSNP RS9277336，它具有增强剂 活性并以等位基因特异性结合转录因子ACTN4。 RS9277336（G）等位基因是关联的 随着PAH的严重程度增加。 FSNP RS9277336通过ACTN4的等位基因特异性结合调节HLA-DPA1 与远端基因靶标ATF6B接触。那我假设RS9277336（G）等位基因 通过降低ACTN4的等位基因特异性结合来控制内皮功能障碍和PAH，因此 通过近端和远端染色质相互作用破坏HLA-DPA1和ATF6B的调节。这 假设将以以下特定目的进行测试：（1）确定SNP RS9277336（g）等位基因是否显示 减少了与ACTN4的结合。寡核苷酸暴露于肺动脉内皮细胞（PAEC）核苷酸 细胞提取物在体内，rs9277336（g）等位基因与ACTN4的结合低于（a）。在这里，我将执行 更确定的实验以确定ACTN4是否显示了与RS9277336（g）vs（a）的同等等位基因特异性结合 完整细胞中的等位基因，主要使用异基因诱导多能干细胞衍生的内皮细胞（IPSC-EC） 携带单个SNP核苷酸编辑。 （2）确定ACTN4是否与SNP rs9277336结合来调节HLA- DPA1表达。我们发现ACTN4的功能损失和功能相互调控的HLA-表达 DPA1在PAECS中。 ACTN4或HLA-DPA1缺乏介导的血管生成和PAEC迁移。我会评估 降低了ACTN4与RS9277336（G）等位基因的结合是否调节并依赖于HLA-DPA1水平 IPSC-EC中的控制功能，因此提供了（G）等位基因的致病作用。 （3）确定ATF6B是否 控制PAH中的病理内皮功能。根据现有的Hi-C染色质触点图，我们发现 ACTN4与RS9277336结合的证据可以通过远程相互作用调节ATF6B。 ATF6B是同工型 ATF6，是PAH的已知中介。我将执行染色质确认捕获（3C），以确定RS9277336是否 物理接触ATF6B基因。我还将评估ATF6B的损失和功能收益是否破坏PAEC 功能，从而建立了一个范式，rs9277336与ATF6B相互作用并介导病理学 内皮功能除了对HLA-DPA1的影响。意义：由于风险（G）等位基因显着 该SNP的致病活性富含人口（MAF = 0.852），将定义为什么有重要的 比例的人面临更严重的PAH的风险。这种见解会对更好的影响产生重大影响 PAH诊断和疗法，从而为我做好了长期作为身体科学家的准备。