| Hypoxia,low oxygen tension,not only Occurs during normal emb~onic development and plays an important role in physiological progress,but also associates with a variety of pathological conditions.Animals and cells have developed elaborate mechanisms that allow adaptation to hypoxic environments.Hypoxia—inducible factors(HIFs)are key regulators of the physiological responses to hypoxia The HIF family consists of three members,i.e.,HIF-1,- 2 and·3.Each of them is composed of a unique oxygen—dependent a subunit(i.e.,HIF - 1a,-2a and - 3a)and a constitutively expressed B subunit.Knowledge about this important family is mostly derived from studies on HIF.1ct and to a tess degree on HIF- 2ct.However,our understanding of HIF - 3u is still limited.Here we cloned the zebrafish h/f-3a,and firstly confirmed its transcriptional factor role in vitro and in vivo.We also found a new transcript of Hif-3ct(H3—5 1 3),which was not regulated by oxygen.In this thesis,we cloned zebrafish h/f-3a gene.The full-len~h h/f-3a cDNA was 2,196 bp,which contained complete open reading frame of 1,881 bp(15 exons), encoding a protein of 626 amino acids In comparison with HIF—l a , Hif-3a had the conserved N . terminal domains(bHLH and PAS)and oxygen—dependent domain (ODD),but lacked the C—TAD.In addition,Hif-3ct had a new motif LZIP.During early development,zebrafish hif-3a mRNA was hardly detected until 4 hpf.The hif-3a mRNA levels gradually increased after 4 hpf and maintained at relatively high levels thereafter.In adult fish,the highest levels were observed in ovary and kidney, h/f-3a mRNA was also easily detected in testes,intestine and stomach,brain,gills, spleen,and heart.In the transcriptional level,hypoxia treatment could significantly increase h/f-3a mRNA in adult and embryos.In protein level,the fast degradation of Hif-3a was induced by two conserved Pro motifs in the ODD.When the Pro were mutated to Ala,the stabilty and activity ofHif-3ct were enhanced.The subcellular localization indicated that h/f-3a encoded a nuclear protein, which lead us to surmise that Hif-3et acted as a transcriptional factor in the nucleas So we designed experiments to confirm this hypothesis.First,we found zebrafish Hif-3a could upregulate the reporter gene p2.1.The activity of reporter gene was absent when co-transfection Hif-3a with dominant negative Hif-3a(dnHif-3a).These results indicated that Hif-3a had transcriptional activity in vitro.Next,significantly enhancement of p2.1 and endogenous genes(igflop—lb and rtp801)were detected in zebrafish embryos when we overexpressed the hif-3a mRNA.Co-injection of dnltif-3a and Hif-3a could restrain the upregulation of ig/bp—1b and rtp801.And dnHif-3a could inhibit the hypoxia inducible expression of igg,p-lb and rtp801. These results indicated that Hif-3a had transcriptional activity in vivo.In addition,we also found that Hif-3ct and Hif-l u did not exhibit additive or inhibitory effect. tt3—513,a new transcript of Hif-3a,was cloned in this thesis.The ORF of H3—513 was 5 1 3 bp,which contained 5 exons,encoding a protein of 1 70 amino acids.In comparison with Hif-3~t,E13—5 1 3 had only two domains:TAD and LZIP.The temporal and spatial expression patterns of H3—5 1 3 were similar to Hif-3a.Both the mRNA and protein levels Of H3-5 1 3 were not changed under different oygen concentration . which indicated that 1-13—5 1 3 was a consistent expression HIF—a.H3—5 1 3 encoded a nuclear protein in both cell lines(in vitro)and embryo cells(in vivo).H3—5 1 3 had HRE-dependent transcriptionalactivity in different cell lines ; in vivo , force-expression of H3—5 I 3 could upregulate the expression of endogenous gene(igfbp-la).These results disclosed that H3—5 1 3 also has transcriptional activity.Furthermore,we found the Pr037,a conserved residue in TAD domain,determined the activity of H3—513.In addition,the embryos displayed multi—abnormality when overexpression of H3—513.This finding suggested that H3-513 may play an important role in zebrafish embryogenesis.In this study,we cloned zebrafish Hif-3a and confirmed its transcriptional factor role.And we discovered a new HIF.a member which could exist under normoxia.These results will provide new information about HIF-a family and lay the foundations for the research of HIF - 3a... |
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