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

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. Sustain Hypoxia RNA-seq (teloHAECs): For sustained hypoxia experiments, three independent clonal isolates of ENH-5 KO and WT control teloHAECs were plated at very low confluence in two sets of 6-well plates and allowed to adhere overnight. The following day, one set of plates were placed in a humidified incubator contained within the box, and the entire box was maintained at 37C, 5% CO2, 1%O2. The cells were fed every two days for 14 days and then harvested. RNA-seq library prep and sequencing were performed at the University of Chicago Genomics core and run together on the Illumina NovaSEQ (PE 100bp).