Effects Of HIF-2? Mutation On Environmental Adaptation Of Plateau Zokor And Blind Mole Rat

In recent years,with the improvement of sequencing technology,the mechanism of hypoxia adaptation in plateau animals has been preliminarily revealed by the analysis of whole genome and candidate genes.It was found that a series of related genes play a role in plateau adaptation.It has been confirmed that HIF-2a is a key regulator of hypoxia signaling pathway,and its variation is closely related to hypoxia adaptation of Tibetans.Plateau zokor and spalax are small rodents,living in hypoxic underground environment.Plateau zokor and spalax formed a series of adaptation mechanisms to adapt the long-term hypoxic environment.We found that the conserved threonine in vertebrates was replaced by serine at position 480 of HIF-2a in plateau zokor and there are 4 SNPs in the promoter region of the spalax HIF-2a.Since it has been reported that the variation of HIF-2a was closely related for Tibetan population to the adaptation of the plateau hypoxic environment,we hypothesized that the variation in HIF-2a of plateau zokor and spalax are associated with their environmental adaptation.In order to verify the assumption,we utilized qRT-PCR,luciferase reporter gene,Western Blot,EMSA and other experimental techniques to study the influence of HIF-2a.The main contents of this study are shown as follows:1.Analysis of HIF-2a phosphorylated modification in Plateau zokor The gene fragment coding the amino acids sequence of HIF-2a from 420 to 540 ofHIF-2a was cloned by PCR,and used to construct a recombinant prokaryotic-expression vector pET32.Two amino acid replacements(SER480-THR and SER480-ALA)were carried out by site-directed mutagenesis.The purified soluble fusion proteins(TRX-S480,TRX-S480A and TRX-S480T)were purified by nickel column.The in vitro phosphorylation was performed using the purified fusion proteins.The results of Western Blot showed that the phosphorylation was enhanced when Ser was replaced by Thr.2.Effects of S480T on the transcriptional activity of HIF-2a in plateau zokorThe HIF-2a eukaryotic expression vector and the HRE reporter gene vector were co-transfected in 293 cell.The transcriptional activity was analyzed by dual luciferase reporter assay and the expression was analyzed by Western Blot.The results showed that Ser480 inhibit the phosphorylation of HIF-2a and increased protein stability and transcriptional activity of HIF-2a in plateau zokor.3.Different HIF-2a expression in basalt spalax and chalk spalaxThe expression of HIF-l?,HIF-2a and their target genes in different soils were analyzed by qRT-PCR.The results showed that there was no significant difference of mRNA levels of HIF-l? and its target genes between basalt and chalk animals.The mRNA level of HIF-2a in basalt spalax was significantly higher than that of chalk spalax,but there was no significant difference of mRNA levels of HIF-2a target genes between basalt and chalk animals.Reporter gene vector was constructed by the cloned spalax HIF-2a promoter.The effect of SNP site on the transcriptional activity was analyzed by site-directed mutagenesis and double luciferase reporter assay.The results showed that-325 T>C and-496 G>A did not affect its transcriptional activity,meanwhile the transcriptional activity of-1810T and-2023A,which is significantly more frequent in basalt spalax,was lowest,.The result of qRT-PCR and double luciferase reporter assay showed that the 1810 A>T and-2023 G>A SNP significantly inhibited the hypoxia induction of the HIF-2a promoter in the basaltic soil,so as to avoid the over expression of HIF-2 target genes.4.SNP affect the binding of specific transcription factorsAnalysis of software showed that-1810 A>T and-2023 G>A SNP may affect the recognition and binding of B-Myb and HNF4G.The eukaryotic expression vectors of B-Myb and HNF4G eukaryotic expression vectors were constructed,and the nucleoproteins were extracted for EMSA assay.We found that-2023 G abolish the binding of B-MYB in the upstream regulatory region of HIF-2a,and-1810A increased the binding of HNF4G with HIF-2 promoter.B-Myb acts as a transcriptional activator to activate transcription,and the heterodimer of HNF4G and HNF4a reduce transcriptional activity,consistent with the results reported gene assay.