Functional Studies Of Key Genes EPAS1 And EGLN1 In The Tibetan Plateau

1.Purpose and significance: Recent studies showed that Tibetan people have been successfully settled in the Qinghai-Tibet Plateau as early as 30,000 years ago and have developed physiological adaptation.However,the molecular mechanism of the adaptation to hypoxic environments at high aititute remains unclear.Our previous studies and those from other laboratories suggested that hypoxia-inducible factor 2α(HIF2α,code by EPAS1)and its negative regulatory factor,PHD2(coded by EGLN1),may play a key role in the long-term adaptation of Tibetans to high altitude hypoxia though the functioinal consequence and the underlying molecular mechanism are yet to be revealed.We previously found that the allele frequencies of two highly conserved non-synonymous sites D4 E and C127 S of the EGLN1 gene were significantly higher in Tibetans(70.9%)than in other lowland populations(<1%),including Han Chinese,and there was a positive correlation with the low hemoglobin concentration in Tibetans,suggesting that the two mutations are likely functional.However,how these two loci affect the functional of EGLN1 is not clear.In order to systematically study the functional effects of the key genes and their interactions,we performed functional experiments using artificial hypoxia induction of Tibetan umbilical cord endothelial cells and transgenic mice.The aim of this study was to elucidate the molecular mechanism of the functional loci of HIF-2αand PHD2 in Tibetans.The results of this study will help reveal the molecular mechanism of human adaptation to hypoxic environments at high altitude,providing important genetic data for understanding high altitude adaptation of Tibetans.2.Methods: In this study,we employed a non-linear model in the analysis of the key phenotype—lower hemoglobin concentration in Tibetans.We combined different approaches,including genotype-phenotypic association analysis,comparative genomics,cell biology,ATAC-seq,CRISPR-Cas9 gene editing and artificial hypoxia induction experiment.We intend to explore the molecular mechanism of HIF-2αand PHD2 in adaptation to high altitute hypoxia,and to dissect the functional consequence of the EPAS1 and EGLN1 mutations in Tibetans.3.Results: With the use of a nonlinear model,we showed that the elevation limit for Tibetans was around 4500 m.Based on the systematic study of EPAS1,we found that the Tibetan-enriched EPAS1 mutations cause a down-regulation of its expression in umbilical cord endothelial cells and placenta.Furthermore,heterozygous EPAS1 knockout mice showed a blunted response to chronic hypoxia,similar to that seen in Tibetans.In addition,the genetic association analysis suggested that besides hemoglobin,EPAS1 may also contribute to other physiological traits in Tibetans,such as pulmonary artery pressure,which eventually help them to achieve long-term adaptation.The artificial hypoxia induction expereiment indicated that the largest change of transcriptome and chromatin openness occurred during the 1-3 days period,and compared with wildtype controls,the cell lines with adaptive genotypes showed relatively less overall transcriptome and chromatin openness change.The differentally expressed genes are enriched in neurodevelopmental and neural cell differentiation pathways.Finally,we successfully established the EGLN1D4 E gene knock-in mouse model.4.Novelty: We utilized a non-linear model and identified an altitude limit for high altitude adaptation in Tibetans.Using combined approaches including cell line and transgenic mice hypoxia inductions,as well as genetic association analysis of large-scale Tibetan populations,we presented by far the most systematic analysis of the molecular mechanisms of the two key genes involved in Tibetans’ adaptation to high altitude hypoxia.The findings will be useful for drug design,as well as for prevention and treatment of high altitude diseases.