A pleiotropic hypoxia-sensitive EPAS1 enhancer is disrupted by adaptive alleles in Tibetans [mouse RNA-seq]

Background & Aims: A major focus of human genetics research has been to identify locally adapted alleles in global populations. In Tibetans, noncoding alleles in EPAS1 – who’s protein product HIF-2 is a key driver of the response to hypoxia – carry some of the strongest signatures of positive selection found in humans, yet their functional mechanism has never been systematically examined. Here we report the discovery of three enhancers within EPAS1, whose activity is significantly disrupted by Tibetan alleles in one or more key organs (endothelium, kidney, and heart). We further characterize one of these enhancers (ENH5) whose activity was found to be not only allele-specific, but also hypoxia-dependent in all three cell types. Deletion of this enhancer results in downregulation of EPAS1 and HIF-2 targets in acute hypoxia as well as a blunting of the transcriptional response to sustained hypoxia. In vivo deletion of the orthologous ENH5 in mice results in dysregulation of gene expression across multiple tissues. We propose that pleiotropic adaptive effects of the Tibetan alleles in EPAS1 underlie the strong selective signal at this gene. Mouse RNA-seq: At age 9 weeks the mice were placed in pairs, one KO and one WT, into a custom hypobaric hypoxia chamber as described in ref [17] . The pressure was set to 280-290 mmHg which is equivalent to ~8% O2 and the mice were left overnight (~16.5 hours). The following morning, both mice were removed together and euthanized one at a time in a randomly selected order via urethane injection as described in [18]. Mice were rapidly dissected and left kidney, right adrenal gland, left lung, left and right atria, and left and right ventricle were all independently flash frozen in either RLT Plus buffer (kidney, adrenal gland, lung) or Trizol (all heart tissues) and stored at -80C degrees. Once all tissues had been collected from all three pairs of mice, tissues were removed from -80C and homogenized. RNA was extracted one tissue at a time using RNeasy Plus Mini kit (Qiagen, 74134) in a Qiagen Qiacube for all samples stored in RLT buffer and by hand using RNeasy plus mini kit with Qiagen Dnase treatment (RNase-Free DNase Set, cat#79254). RNA was extracted and quality was assessed on an Agilent BioAnalyzer. The RIN was recorded for each sample. RNA-seq library preparation was performed using the TruSeq RNA Library Preparation Kit v2, Set B (Illumina, RS-122-2002). RNA-seq libraries were submitted to the University of Chicago Genomics Core where they were sequenced across 7 lanes of the Illumina HiSeq4000 (one organ per lane, balanced by genotype) with an average of 69.6M 50bp single end reads per sample.