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Molecular Cloning of Rat Hepatocyte Nuclear Factor 1 Gene and Analysis of Its Promotor

大鼠肝细胞核因子1基因的分子克隆及其启动子分析

基本信息

批准号：
02670108
负责人：
MIURA Naoyuki
金额：
$ 1.47万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

I screened a rat genomic libray using the rat hepatocyte nuclear factor(HNF)1cDNA as a probe and got 11 positive clones. All of 11 clones A, erc found to contain HNF1 gene. I focused on the promotor and 5' Ranking region of the HNFI gene. First, I determined the nucleotide sequence in the promotor region of the gene and found that the transcriptional start site is about 220 base upstream from the translation initiation site. When the promotor and 5' flanking region of the gene was connected to the reporter gene, luciferase, and was transfected into HepG2 cells, P19 cells and L cells, the data showed that the DNA fragment is transcriptionally active inHepG2 cellsibut not active in P19 cells and L cells. By 5'- deletion analysis of the promotor region of the gene, I found that the region from -118 to -36 was critically important for the tissue-specific expression of the HNF1 gene. Next I found ihat the region from -60 to -40 was protected in a DNase I foot printing analysis. The oligonucleotide that had the sequence from -61 to -36 was connected to the minimal promotor, TKCAT, and was transfected into HepG2 cells, it increased the transcriptional activity by a factor of 3. When the oligonucleotide %A, as incubated with a liver nuclear extract, it fon-ned a DNA-protein complex in a gel retardation assay. After inspecting the sequence of the region, I found that it is a similar sequence that transcription factor HNF4 can bind to. So I added tha oligonucleotide that had the sequence of HNF4-binding site in the ApoCIII gene as a competitor in the gel shift assay system mentioned above. The result showed that the factor that binds to the -61/-36 oligonucleotide is HNF4 protein. In summary, the HNF1 gene is regulated positively by the HNF4 gene.

以大鼠肝细胞核因子(HNF)1cDNA为探针，筛选大鼠基因组文库，得到11个阳性克隆。11个克隆A、ERC均含有HNF1基因。我重点研究了HNFI基因的启动子和5‘端排序区。首先，我测定了该基因启动子区域的核苷酸序列，发现转录起始点在翻译起始点上游约220个碱基。将该基因的启动子和5‘侧翼区连接到报告基因荧光素酶上，并将其导入肝癌细胞、P19细胞和L细胞，结果表明该片段在HepG2细胞中具有转录活性，而在P19细胞和L细胞中不具有转录活性。通过对该基因启动子区域的5‘-缺失分析，发现-118~-36区域对于HNF1基因的组织特异性表达至关重要。接下来，我发现在DNA酶I足迹分析中，从-60到-40的区域是受保护的。将序列从-61到-36的寡核苷酸连接到最小启动子TKCAT上，并将其导入HepG2细胞，其转录活性提高了3倍。当该寡核苷酸与肝细胞核提取液孵育时，在凝胶滞留试验中发现了DNA-蛋白质复合体。在检查了该区域的序列后，我发现它是转录因子HNF4可以结合的相似序列。因此，我在上述凝胶位移分析体系中加入了APOCIII基因中含有HNF4结合位点序列的寡核苷酸作为竞争对手。结果表明，与-61/-36寡核苷酸结合的因子为HNF4蛋白。综上所述，HNF1基因受HNF4基因正向调控。