Functional Genomics of Tibetan Adaptations

摘要 尽管缺氧是人类生理的主要压力，但有几个群体已经适应了 高海拔(HA)缺氧环境。其中，研究最多的是藏族人 而且它们的血红蛋白(Hb)水平相对较低。作为一名 结果，他们严重缺氧症，他们的生理如何应对缺氧仍然很差。 明白了。通过全基因组关联研究的独特组合，测试 多基因适应和体外功能分析，Di Rienzo实验室已经做出了两个主要的 观测：1)Hb的最强关联信号和最强的选择性扫描 基因组中位于EPAS1基因座的信号影响内皮细胞的增强子活性 (2)藏族人的自然选择倾向于较低的脉搏。这些发现提高了 藏族人的心血管功能是否以及如何因此而发生变化 对HA低氧的适应。为了回答这些问题，我们提出了以下具体目标： 目的1.开发藏语适应功能表征的独特资源。我们 将为24名藏族人(TBC)创造淋巴母细胞系并进行基因分型，并将购买24 来自中国汉族人的LCLS(CHB)来自1000基因组计划。这些LCL将是 重新编程为诱导多能干细胞(IPSCs)，然后并行分化为 心肌细胞(CMS)和内皮细胞。 目的2.检验心血管功能是低氧适应靶点的假设 藏族人。所有的CMS和ECs将在常氧和低氧中培养；RNA-seq数据将 收集和分析以确定不同的基因和共表达模块 TBC和CHB对低氧的转录反应。顺式监管变体，可能 对缺氧反应的种群间差异的解释将使用 RNA-seq数据，然后，将测试藏族人多基因适应的证据。 目的3.通过编辑IPSCs来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，包括位于EPAS1位点的那些和在 目的2.我们将通过精确的基因组编辑取代等位基因。我们还将培育转基因小鼠 在人类中具有EPAS1增强子的缺失和相应的增强子等位基因。 综上所述，我们将描述心血管对HA低氧适应的机制 通过一种独特的方法组合来阐明多基因 在非欧洲人群中，对一个重要的环境应激源的适应。

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.