Tibetans are well adapted to the hypoxic environments at high altitude, yet the molecular mechanism of this adaptation remains elusive. We reported comprehensive genetic and functional analyses of EPAS1, a gene encoding hypoxia inducible factor 2α (HIF-2α) with the strongest signal of selection in previous genome-wide scans of Tibetans. We showed that the Tibetan-enriched EPAS1 variants down-regulate expression in human umbilical endothelial cells and placentas. Heterozygous EPAS1 knockout mice display blunted physiological responses to chronic hypoxia, mirroring the situation in Tibetans. Furthermore, we found that the Tibetan version of EPAS1 is not only associated with the relatively low hemoglobin level as a polycythemia protectant, but also is associated with a low pulmonary vasoconstriction response in Tibetans. We propose that the down-regulation of EPAS1 contributes to the molecular basis of Tibetans’ adaption to high-altitude hypoxia.

Yak is one of the largest native mammalian species at the Himalayas, the highest plateau area in the world with an average elevation of >4,000 m above the sea level. Yak is well adapted to high altitude environment with a set of physiological features for a more efficient blood flow for oxygen delivery under hypobaric hypoxia. Yet, the genetic mechanism underlying its adaptation remains elusive. We conducted a cross-tissue, cross-altitude, and cross-species study to characterize the transcriptomic landscape of domestic yaks. The generated multi-tissue transcriptomic data greatly improved the current yak genome annotation by identifying tens of thousands novel transcripts. We found that among the eight tested tissues (lung, heart, kidney, liver, spleen, muscle, testis, and brain), lung and heart are two key organs showing adaptive transcriptional changes and >90% of the cross-altitude differentially expressed genes in lung display a nonlinear regulation. Pathways related to cell survival and proliferation are enriched, including PI3K-Akt, HIF-1, focal adhesion, and ECM–receptor interaction. These findings, in combination with the comprehensive transcriptome data set, are valuable to understanding the genetic mechanism of hypoxic adaptation in yak.

We present a high-quality de novo genome assembly (rheMacS) of the Chinese rhesus macaque (Macaca mulatta) using long-read sequencing and multiplatform scaffolding approaches. Compared to the current Indian rhesus macaque reference genome (rheMac8), rheMacS increases sequence contiguity 75-fold, closing 21,940 of the remaining assembly gaps (60.8 Mbp). We improve gene annotation by generating more than two million full-length transcripts from ten different tissues by long-read RNA sequencing. We sequence resolve 53,916 structural variants (96% novel) and identify 17,000 ape-specific structural variants (ASSVs) based on comparison to ape genomes. Many ASSVs map within ChIP-seq predicted enhancer regions where apes and macaque show diverged enhancer activity and gene expression. We further characterize a subset that may contribute to ape- or great-ape-specific phenotypic traits, including taillessness, brain volume expansion, improved manual dexterity, and large body size. The rheMacS genome assembly serves as an ideal reference for future biomedical and evolutionary studies.

Human genetic adaptation to high altitudes (>2500 m) has been extensively studied over the last few years, but few functional adaptive genetic variants have been identified, largely owing to the lack of deep-genome sequencing data available to previous studies. Here, we build a list of putative adaptive variants, including 63 missense, 7 loss-of-function, 1,298 evolutionarily conserved variants and 509 expression quantitative traits loci. Notably, the top signal of selection is located in TMEM247, a transmembrane protein-coding gene. The Tibetan version of TMEM247 harbors one high-frequency (76.3%) missense variant, rs116983452 (c.248C > T; p.Ala83Val), with the T allele derived from archaic ancestry and carried by >94% of Tibetans but absent or in low frequencies (<3%) in non-Tibetan populations. The rs116983452-T is strongly and positively correlated with altitude and significantly associated with reduced hemoglobin concentration (p = 5.78 × 10−5), red blood cell count (p = 5.72 × 10−7) and hematocrit (p = 2.57 × 10−6). In particular, TMEM247-rs116983452 shows greater effect size and better predicts the phenotypic outcome than any EPAS1 variants in association with adaptive traits in Tibetans. Modeling the interaction between TMEM247-rs116983452 and EPAS1 variants indicates weak but statistically significant epistatic effects. Our results support that multiple variants may jointly deliver the fitness of the Tibetans on the plateau, where a complex model is needed to elucidate the adaptive evolution mechanism.

High-altitude adaptation of Tibetans represents a remarkable case of natural selection during recent human evolution. Previous genome-wide scans found many non-coding variants under selection, suggesting a pressing need to understand the functional role of non-coding regulatory elements (REs). Here, we generate time courses of paired ATAC-seq and RNA-seq data on cultured HUVECs under hypoxic and normoxic conditions. We further develop a variant interpretation methodology (vPECA) to identify active selected REs (ASREs) and associated regulatory network. We discover three causal SNPs of EPAS1, the key adaptive gene for Tibetans. These SNPs decrease the accessibility of ASREs with weakened binding strength of relevant TFs, and cooperatively down-regulate EPAS1 expression. We further construct the downstream network of EPAS1, elucidating its roles in hypoxic response and angiogenesis. Collectively, we provide a systematic approach to interpret phenotype-associated noncoding variants in proper cell types and relevant dynamic conditions, to model their impact on gene regulation.

Tibetans are well adapted to high-altitude environments. Among the adaptive traits in Tibetans, the relatively low hemoglobin level is considered a blunted erythropoietic response to hypoxic challenge. Previously, EPAS1 and EGLN1, the major upstream regulators in the hypoxic pathway, were reportedly involved in the hemoglobin regulation in Tibetans. In this study, we report a downstream gene (HMOX2) involved in heme catabolism, which harbors potentially adaptive variants in Tibetans. We first resequenced the entire genomic region (45.6 kb) of HMOX2 in Tibetans, which confirmed the previously suspected signal of positive selection on HMOX2 in Tibetans. Subsequent association analyses of hemoglobin levels in two independent Tibetan populations (a total of 1,250 individuals) showed a male-specific association between the HMOX2 variants and hemoglobin levels. Tibetan males with the derived C allele at rs4786504:T>C displayed lower hemoglobin level as compared with the T allele carriers. Furthermore, our in vitro experiments indicated that the C allele of rs4786504 could increase the expression of HMOX2, presumably leading to a more efficient breakdown of heme that may help maintain a relatively low hemoglobin level at high altitude. Collectively, we propose that HMOX2 contributes to high-altitude adaptation in Tibetans by functioning as a modifier in the regulation of hemoglobin metabolism.

Structural variants (SVs) may play important roles in human adaptation to extreme environments such as high altitude but have been under-investigated. Here, combining long-read sequencing with multiple scaffolding techniques, we assembled a high-quality Tibetan genome (ZF1), with a contig N50 length of 24.57 mega-base pairs (Mb) and a scaffold N50 length of 58.80 Mb. The ZF1 assembly filled 80 remaining N-gaps (0.25 Mb in total length) in the reference human genome (GRCh38). Markedly, we detected 17 900 SVs, among which the ZF1-specific SVs are enriched in GTPase activity that is required for activation of the hypoxic pathway. Further population analysis uncovered a 163-bp intronic deletion in the MKL1 gene showing large divergence between highland Tibetans and lowland Han Chinese. This deletion is significantly associated with lower systolic pulmonary arterial pressure, one of the key adaptive physiological traits in Tibetans. Moreover, with the use of the high-quality de novo assembly, we observed a much higher rate of genome-wide archaic hominid (Altai Neanderthal and Denisovan) shared non-reference sequences in ZF1 (1.32%–1.53%) compared to other East Asian genomes (0.70%–0.98%), reflecting a unique genomic composition of Tibetans. One such archaic hominid shared sequence—a 662-bp intronic insertion in the SCUBE2 gene—is enriched and associated with better lung function (the FEV1/FVC ratio) in Tibetans. Collectively, we generated the first high-resolution Tibetan reference genome, and the identified SVs may serve as valuable resources for future evolutionary and medical studies.

CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect and on-target complex mutations remain a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, to our knowledge no comprehensive in vivo off-target and on-target assessment has been conducted in NHPs. Here, we perform whole genome trio sequencing of Cas9-treated rhesus monkeys. We only find a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected off-target mutations (OTMs) were detected. Furthermore, the long-read sequencing data does not detect large structural variants in the target region.