HNF-4 plays a pivotal role in the liver-specific transcription of thechipmunk HP-25 gene

The gene for chipmunk hibernation-specific protein HP-25 is expressed speci fically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its str uctural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determine d by primer extension analysis, is located at 113 bp upstream of the transl ation initiation codon. Transient transfection studies in HepG2 cells revea led that the 80 bp 5' flanking sequence was sufficient for the liver-specif ic promoter activity. In a gel retardation assay using HepG2 nuclear extrac ts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDN A clones isolated by a yeast one-hybrid system for a protein capable of bin ding 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 chipmun k liver nuclear extracts that bound to this sequence was HNF-4. When transf ected into HeLa cells, HNF-4 transactivated transcription from the HP-25 ge ne promoter, and mutation of the HNF-4 binding site abolished transactivati on by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene ex pression.