Functional Genomics of Tibetan Adaptations

摘要 虽然缺氧是人类生理上的主要压力，但一些种群已经适应了缺氧。 高海拔（HA）低氧环境。其中，藏族人被研究得最多 广泛地，并且已经显示它们具有相对低的血红蛋白（Hb）水平。作为 结果，他们严重缺氧，他们的生理如何应对缺氧仍然很差 明白通过全基因组关联研究的独特组合， 多基因适应和功能体外试验，迪里恩佐实验室已经取得了两个主要的 观察结果：1）Hb的最强关联信号和最强选择性扫描 基因组中位于EPAS 1基因座的信号影响内皮细胞中的增强子活性 (ECs)（2）自然选择有利于藏族人脉脉低。这些发现提高了 西藏人的心血管功能是否以及如何改变， 适应缺氧。为了回答这些问题，我们提出以下具体目标： 目标1。开发一个独特的资源，用于西藏适应性的功能表征。我们 将为24名西藏人（TBC）生成淋巴母细胞样细胞系并进行基因分型，并将购买24 来自1000基因组计划的汉族人（CHB）的LCL。这些LCL将是 重编程为诱导多能干细胞（iPSC），然后平行分化为 心肌细胞（CM）和EC。 目标二。测试假设，心血管功能是适应缺氧的目标， 藏人所有的CM和EC将在常氧和缺氧下培养; RNA-seq数据将被记录。 收集并分析，以鉴定基因和共表达模块， 转录响应缺氧之间的TBC和CHB。顺式调节变体， 解释人群间对缺氧反应的差异将使用 RNA-seq数据，反过来，将测试西藏人多基因适应的证据。 目标3。通过编辑iPSC研究适应性等位基因的影响。我们将使用编辑 删除10个增强子的技术，包括EPAS 1基因座上的增强子和其他在 目标二。我们将通过精确的基因组编辑替换等位基因。我们还将培育出转基因小鼠 在人类中缺失EPAS 1增强子和具有相应的增强子等位基因。 总之，我们将描述心血管适应HA缺氧的机制 通过一种独特的方法组合来阐明多基因的因果关系， 适应一个重要的环境压力，在非欧洲人口。

ABSTRACT Although hypoxia is a major stress on human physiology, several populations have adapted to high altitude (HA) hypoxic environments. Among them, Tibetans have been studied most extensively and they have been shown to have relatively low hemoglobin (Hb) levels. As a result, they are severely hypoxemic and how their physiology copes with hypoxia remains poorly understood. Through a unique combination of genome-wide association studies, tests for polygenic adaptation and functional in vitro assays, the Di Rienzo lab has made two major observations: 1) the strongest association signals for Hb and the strongest selective sweep signals in the genome, found at the EPAS1 locus, influence enhancer activity in endothelial cells (ECs), and 2) lower pulse was favored by natural selection in Tibetans. These findings raise the questions of whether and how cardiovascular functions changed in Tibetans as a result of adaptations to HA hypoxia. To answer them, we propose the following specific aims: Aim 1. Develop a unique resource for the functional characterization of Tibetan adaptations. We will generate and genotype lymphoblastoid cell lines for 24 Tibetans (TBC) and will purchase 24 LCLs from Han Chinese (CHB) from the 1000 genome project. These LCLs will be reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated in parallel into cardiomyocytes (CMs) and ECs. Aim 2. Test the hypothesis that cardiovascular functions were targets of adaptation to hypoxia in Tibetans. All the CMs and ECs will be cultured in normoxia and hypoxia; RNA-seq data will be collected and analyzed to identify genes and co-expression modules that differ in the transcriptional response to hypoxia between TBC and CHB. Cis-regulatory variants that may account for between-population differences in response to hypoxia will be identified using the RNA-seq data and, in turn, will be tested for evidence of polygenic adaptations in Tibetans. Aim 3. Investigate the effects of adaptive alleles by editing iPSCs. We will use editing technologies to delete 10 enhancers including those at the EPAS1 locus and others identified in Aim 2. We will replace alleles by precise genome editing. We will also generate transgenic mice with deletion of EPAS1 enhancer and with the corresponding enhancer alleles in humans. In summary, we will characterize the mechanisms of cardiovascular adaptations to HA hypoxia through a unique combination of approaches to elucidate the causal basis of a polygenic adaptation to an important environmental stressor, in a non-European population.