HNF-1 regulates the liver-specific transcription of the chipmunk HP-20 gene.
The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.
Ono
M
M
Department of Biosciences, School of Science, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan.
Hosoe
Y
Y
Azuma
S
S
Shoji
M
M
Nara
K
K
Kondo
N
N
Shiba
T
T
Takamatsu
N
N
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000235:genetics*; D019556:COS Cells; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012589:Sciuridae / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D001798:Blood Proteins; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; C081698:HP-20 protein, Tamias asiaticus; D051538:Hepatocyte Nuclear Factor 1-alpha; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D008156:Luciferases