摘要
缺氧在胚胎发育、生理和病理反应中起着重要作用。机体和细胞为应对缺氧胁迫,形成了大量蛋白参与的,精巧、复杂的反应机制。在整个应答体系中,缺氧诱导因子(HIF)是最关键的调控者,居于核心位置。HIF家族包括HIF一1,一2和一3三个成员;每个成员都含有受氧调控的a亚基(HIF一1a,一2a和一3a)和组成性表达的B亚基。目前,我们对HIF家族的认识主要来自HIF—la和HIF一2a,对HIF一3a却知之甚少。本研究克隆得到斑马鱼,通过体内、体外实验,首次证实H_f-3a是转录因子,同时发现一个不受氧气调控的Hif-3a转录本(H3—513)并研究它的活性位点和体内功能。
本研究从模式生物斑马鱼中克隆得到-3A基因,它位于15号染色体,cDNA全长2,196 bp,ORF为l,881 bp,含15个外显子,编码626个氨基酸。与HIF—la相比,Hif-3a含有N一端保守结构域(bHLH和PAS)和氧应答结构域0DD,缺失C一端C—TAD结构域,新增LZIP结构域。斑马鱼呈现全身性表达模式,在胚胎发育初期含量很低,从4 hpf开始有明显表达,随后逐渐升高并维持在稳定水平;在成鱼肾脏和卵巢中3a表达最高,脑、鳃、脾脏、心脏、肠胃和精巢都有明显表达。转录水平, 3amRNA的表达受氧气浓度调控,成鱼或胚胎经过缺氧处理后, 3amRNA显著升高。翻译水平,Hif-3a也受氧气调控,0DD结构域中两个保守脯氨酸基序介导了I{if-3 a常氧条件下的降解,点突变该基序核心氨基酸后,斑马鱼Hif-3a的蛋白水平和活性都显著提高。
亚细胞定位显示Hif_3a是核蛋白,说明H.f_3 a具有作为转录因子的特征。本研究从体外和体内两个层面,证明Hif_3Ⅱ是转录因子。首先,利用荧光素酶报告体系,发现斑马鱼Hif_3d能显著促进报告基因p2.1的表达;Hif-3a与dominam netive Hif_3a( 3a)共转染,则抑制报告基因的表达,这说明Hif.3a在体外细胞系中具有转录活性。向胚胎显微注射Hif-3amRNA,能显著上调报告基因,同时发现,内源基因igfbp一16和rtp801的表达水平也明显上升;将dnI-{if_3 a与Hif.3a共注射胚胎,则检测不到-b和rtp801的变化;注射dnt{if_3a能抑制缺氧对bp一6和rtpS01的上调作用;这表明Hif_3a能够作为转录因子调节内源基因的表达,是转录因子。此外,实验显示Hif-3a具有独立的转录活性,且不影响Hif.1a活性。
本研究还克隆得到斑马鱼Hif-3a一个新的转录本H3—513。它的ORF长513 bp,含5个外显子,编码170个氨基酸,包括TAD和LZIP两个结构域,在时空表达模式上与Hif.3a类似。qRT.PCR检测发现,缺氧处理成鱼和胚胎,不影响h3一513 mRNA的表达;常氧下H3—513的蛋白稳定存在,表明H3.513的mRNA和蛋白部不受氧气调控。无论在细胞系还是活体胚胎细胞,H3.513都定位在细胞核中。体外转染不同细胞系,H3—513均能促进报告基因表达;胚胎注射H3—513mRNA显著增强内源基因igfbp一-la的表达,说明H3—513具有转录活性;通过删除突变证实,TAD结构域中保守的脯氨酸残基(Pr037)决定着H3—513的活性。过量表达H3.513导致胚胎出现一系列畸形,这说明它在体内具有重要作用。本研究克隆得到斑马鱼Hif_3a并证明它是转录因子:首次发现不受氧气调控的HIF—a蛋白.H3—513。这些结果为深入研究Hif:3a的功能奠定基础,对全面认识和理解HIF一a家族具有重要价值。
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
引文
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