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

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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.