HNF-4 plays a pivotal role in the liver-specific transcription of the chipmunk HP-25 gene.
The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5' flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.
Kojima
M
M
Hibernation Control Project, Kanagawa Academy of Science and Technology, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan.
Takamatsu
N
N
Ishii
T
T
Kondo
N
N
Shiba
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001798:Blood Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000378:metabolism; D004268:DNA-Binding Proteins; D017930:Genes, Reporter; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012589:Sciuridae; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001798:Blood Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C081699:HP-25 protein, Tamias asiaticus; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D014157:Transcription Factors; D008156:Luciferases