Functional analyses of EPAS1 alleles from populations adapted to high altitude

适应高海拔人群的 EPAS1 等位基因的功能分析

• 批准号: 9761277
• 负责人: Olivia Anne Gray
• 金额: $ 4.5万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2020-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761277
• 关键词: ATAC-seq; Affect; Alleles; Altitude; Altitude Sickness; Architecture; Asthma; Biological Assay; Blood; CRISPR/Cas technology; Cardiovascular Diseases; Cardiovascular system; Cell Respiration; Cellular Assay; Characteristics; Chromatin; Chromatin Structure; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Cultured Cells; Data; Data Set; Dimensions; Disease; Down-Regulation; Embryo; Endothelial Cells; Endothelium; Enhancers; Environment; Epigenetic Process; Evolution; Functional disorder; Genes; Genetic; Genetic Enhancer Element; Genetic Population Study; Goals; Haplotypes; Hematopoietic; Hemoglobin; Hemoglobin concentration result; Heritability; Homeostasis; Human; Hypoxemia; Hypoxia; Hypoxia-Inducible Factor Pathway; Indigenous; Inherited; LacZ Genes; Lead; Light; Link; Luciferases; Lung; Modernization; Molecular; Mus; Myocardial Ischemia; Natural Selections; Outcome; Oxygen; Pathway interactions; Phenotype; Physiological; Physiological Adaptation; Population; Proteins; Pulmonary Hypertension; Reporter; Reporter Genes; Role; Sea; Severity of illness; Signal Transduction; Source; Spatial Distribution; Stress; Technology; Testing; Tissues; Transcript; Transgenic Mice; Transgenic Organisms; Translating; Validation; Variant; angiogenesis; causal variant; cell type; combat; expectation; experience; genetic variant; genome wide association study; genome-wide; human disease; in vitro Assay; in vivo; mouse model; reproductive; response; survival outcome; targeted treatment

PROJECT SUMMARY For over 10,000 years Tibetan populations have lived at altitudes above 3500m resulting in a modern population of over 3 million indigenous people adapted to thrive in an environment with just 60% the sea level oxygen concentration. This environment parallels the hypoxia that characterizes or complicates many cardiovascular and hematopoietic disorders. Tibetans display many unique phenotypes, including unelevated hemoglobin levels. Despite the low hemoglobin levels, Tibetans do not have higher oxygen saturation than lowlanders at the same altitude and are therefore severely hypoxemic. Intriguingly, they also experience better reproductive outcomes, and protection against hypoxic pulmonary hypertension and chronic mountain sickness compared to lowlanders at high altitude. Population genetic studies have repeatedly shown strong signals of positive selection and association with unelevated hemoglobin levels in the endothelial PAS domain 1 (EPAS1) gene, whose protein product, Hif-2α, is a central regulator of the hypoxia inducible factors (HIF) pathway. The Di Rienzo lab has conducted a genome-wide association study for hemoglobin levels in Tibetans that allowed narrowing down the region of association to a 17kb segment containing epigenetic signatures of active enhancer elements in endothelial cells, a cell type which has been implicated in both normal hypoxic response and in diseases characterized by hypoxic tissues and HIF pathway dysfunction, such as asthma and pulmonary hypertension. This project seeks to comprehensively examine the effects of these genetic variants on endothelial function and how these genetic effects translate into physiological adaptations to hypoxia. To achieve this goal, in Aim 1, I will examine the changes in chromatin architecture in endothelial cells cultured in normoxia and hypoxia using capture HiC and ATAC-seq. These results will not only help inform my expectations for the EPAS1 locus, but will also provide an invaluable dataset on the impacts of hypoxia on endothelial chromatin architecture. In Aim 2, I will focus on identifying causal SNPs in EPAS1 by using reporter gene assays and CRISPR-cas9 gene editing technology followed by functional and molecular assays of cellular phenotypes. This will allow me to both identify causal SNPs at the EPAS1 locus, and definitively connect them to EPAS1 expression and physiological outcomes in an endothelial context. In Aim 3, having validated causal alleles with differential enhancer activity, I will examine their effect in vivo by performing transient LacZ transgenic assays in mice. This will broaden the scope of our understanding of the adaptive alleles by indicating their spatial distribution and the sufficiency of high and low-altitude alleles to drive expression in the endothelium.

项目总结 一万多年来，藏族人民一直生活在海拔3500米以上的地区，形成了现代的 300多万土著人民适应了在仅有60%海平面的环境中茁壮成长 氧气浓度。这种环境与低氧类似，低氧是许多人的特征或复杂性 心血管和血液系统疾病。藏族人表现出许多独特的表型，包括不起眼的 血红蛋白水平。尽管藏族人的血红蛋白水平较低，但他们的血氧饱和度并不比 在同一海拔的低地居民，因此严重缺氧症。有趣的是，他们的体验也更好 生殖结局以及对缺氧性肺动脉高压和慢性高原反应的保护 与高海拔的低地人相比。种群遗传学研究一再显示出强烈的信号 阳性选择及其与内皮PAS结构域1(EPAS1)未升高的血红蛋白水平的关系 基因，其蛋白产物HIF-2α是低氧诱导因子途径的中央调节因子。这个 Di Rienzo实验室对藏族人的血红蛋白水平进行了一项全基因组关联研究，这使得 将关联区域缩小到包含Active的表观遗传特征的17kb片段 内皮细胞中的增强子元件，一种与正常缺氧反应有关的细胞类型 在以缺氧组织和HIF途径功能障碍为特征的疾病中，如哮喘和肺 高血压。 该项目旨在全面研究这些基因变异对内皮细胞的影响。 以及这些遗传效应如何转化为对低氧的生理适应。为了实现这一目标， 在目标1中，我将研究在常氧和低氧条件下培养的内皮细胞染色质结构的变化。 用捕捉法和ATAC-seq法进行缺氧。这些结果不仅将帮助我了解我对 EPAS1基因座，但也将提供关于缺氧对内皮细胞染色质影响的宝贵数据集 建筑。在目标2中，我将重点利用报告基因分析和基因分析来确定EPAS1中的因果SNPs CRISPR-Cas9基因编辑技术，随后进行细胞表型的功能和分子分析。 这将使我能够识别EPAS1基因座的因果SNP，并最终将它们与EPAS1联系起来 在内皮环境中的表达和生理结果。在目标3中，验证了因果等位基因 不同的增强子活性，我将通过瞬时LacZ转基因检测来检验它们在体内的效果 在老鼠身上。这将通过指示适应等位基因的空间来拓宽我们对其理解的范围 高海拔和低海拔等位基因的分布及其在内皮细胞中驱动表达的充分性。