from 3 independent experiments with three replicates in each experiment. of amnion, fetus growth and high-altitude adaptation of Tibetans. Lack of oxygen occurs in a number of processes, e.g., embryonic development1, postnatal maturation2, and tumorigenesis3. Under hypoxia condition, hypoxia-inducible factors (HIFs) are activated and function as transcriptional regulators of genes involved in the hypoxic response4,5,6. HIFs are heterodimers consisting of an oxygen-labile subunit (HIF) and a stable subunit (HIF). HIF in mammals includes three isoforms, of which HIF1 and HIF2 are the best characterized7. In environments with sufficient oxygen, HIF subunits are hydroxylated at conserved proline residues by prolyl hydroxylases (PHDs) and rapidly degraded by ubiquitin-proteosome system. While in the hypoxic environment, activity of PHDs is diminished, and Levonorgestrel as a consequence, HIF subunits become stabilized. The resulting stabilized HIF proteins dimerize with HIF, and activate the transcription of the target genes8. HIF1 is expressed ubiquitously, while the expression of HIF2 is more restricted, with highest expression levels in heart, lung and placenta9. HIF2 is encoded by endothelial PAS domain protein 1 (EPAS1) gene. Levonorgestrel Studies onEPAS1inactivation revealed that mice with loss ofEPAS1developed a syndrome of multiple-organ pathology10and may die at mid-gestation11. Thus it is hypothesized that certainEPAS1expression levels are important for its proper functions in the course of embryo development. Genetic variation in the regulatory region ofEPAS1gene may influence the transcription levels ofEPAS1and may further contribute to the changes of the biological functions. Recently, genome-wide studies on high-altitude adaptation have suggested several SNPs in the regulatory region (intron and 3 downstream) ofEPAS1gene responsible for the genetic adaptation of high-altitude hypoxia in Tibetans12,13,14. However, whether there are variants in the promoter region ofEPAS1gene related to high-altitude adaptation awaits further studies. Functional studies focusing on the molecular mechanism need to investigate the direction and the magnitude of gene expression changes associated with the variants and figure out what tissues, developmental time points, and what the downstream target genes involved. Thus, we had three specific aims in this study. Firstly, we were interested to identify the variants in the promoter region with divergence between Tibetans and Han Chinese. Secondly, we wanted to explore the direction and the magnitude ofEPAS1gene expression changes associated with the variants. Thirdly, we intended to investigate the candidate targets Levonorgestrel that are regulated by EPAS1. == Results == == Eleven variants were identified in the promoter region ofEPAS1gene == As compared with the Rabbit polyclonal to OSBPL10 1000 Genome Data, we found that Tibetans have several specific variants in the promoter region ofEPAS1gene (Table 1), among which four variants have minor allele frequency (MAF) larger than 0.05. A 40-bp insertion was identified at 742 bp relative to the transcriptional start site ofEPAS1gene (NM_001430.4,Supplementary Fig. S1) in Tibetans but was absent in Chinese Han individuals (Supplementary Fig. S2). Another three variants newly identified in Tibetans lay at 706 bp, 138 bp and 129 bp, respectively, and two of them (138 bp and 129 bp) were in strong LD with the 742 indel polymorphism (R2= 1,Supplementary Fig. S3). Moreover, rs56721780:G>C and rs13428739:C>T lying at 886 bp and 607 bp, respectively, had much higher MAF in Tibetans than that of Han Chinese (0.372vs. 0.010 and 0.39vs. 0.010, allele frequency in Han Chinese was from 1000 Genomes Project) and were also in strong LD with the 742 indel polymorphism (R2= 0.8 and 0.95, Levonorgestrel respectively). The frequency of the haplotypes constructed by the three variants (rs56721780:G>C, 742 indel and rs13428739:C>T) in strong LD was listed inTable 2. == Table 1. Variants in the promoter region ofEPAS1gene identified in Tibetans. == Note:aNA, not available;bTSS, transcriptional start site;cthe second allele is minor allele;dINDEL, insertion and deletion. == Table 2. Information of the haplotypes defined by rs56721780:G>C, 742 indel and rs13428739:C>T. == == C allele of rs56721780:G>C decreased the binding affinity of IKZF1 to the cis-acting element inEPSA1promoter == To further analyze the function of the three variants used to construct haplotypes, function prediction was performed by TFSEARCH software. The Prediction results demonstrated that transcription factor IKZF1 isoform 3 and 5 (also known as Ik-2 an Ik-3, respectively15) bind to the cis-acting element with allele G at rs56721780:G>C site, while the binding effect is missing for allele C (Supplementary Fig. S1). In this study, EMSA experiment.