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

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. Capture Hi-C: five million HAECs were grown in normoxia for 24 hours, then harvested and counted, then corsslinked with 1% formaldehyde treatment. Cross-linked chromatin was digested with MboI endonuclease (New England Biolabs, R0147). Subsequently, the restriction fragment overhangs were filled in and the DNA ends were marked with biotin-14-dATP (Life Technologies, 19524-016). The biotin-labeled DNA was sheared and pulled down using Dynabeads MyOne Stretavidin T1 beads (Life Technologies, 65602). The in situ Hi-C library was amplified directly off of the T1 beads with six cycles of PCR, using Illumina primers and protocol (Illumina, 2007). Promoter capture was performed as described[8].The in situ Hi-C library was hybridized to 81,735 biotinylated 120-bp custom RNA oligomers (CustomArray, Inc. www.customarrayinc.com)) targeting promoter regions (four probes/ RefSeq transcription start sites). After hybridization, post-capture PCR (eight amplification cycles) was performed on the DNA bound to the beads via biotinylated RNA. Each library was sequenced on a full lane of an Illumina HiSeq 4000 machine. Standard promoter capture Hi-C analyses were performed using HOMER.