title: Involvement of hepatocyte nuclear factor-4 in the expression of the growth hormone receptor 1A messenger ribonucleic acid in bovine liver.
abstract: The GH receptor 1A mRNA (GHR 1A mRNA) is one of the major GHR mRNA variants that differ in the 5'-untranslated region. The GHR 1A mRNA is unique because it is exclusively expressed in liver. The objective of the present study was to understand the mechanism for the liver-specific expression of the GHR 1A mRNA in the bovine. Twenty-six kilobases of 5'-flanking region of the bovine GHR gene was cloned and sequenced. The first exon (exon 1A) that corresponded to the 5'-untranslated region of the GHR 1A mRNA was 15,250 bp upstream from exon 2 in the GHR gene. The major transcription start site for the GHR 1A mRNA was 19 bp downstream from a putative TATA box. Transient transfection analyses of the 5'-flanking region of exon 1A in liver cell lines vs. nonliver cell lines did not reveal a positively regulatory region responsible for the liver-specific expression of the GHR 1A mRNA perhaps because the liver cell lines do not recapitulate the in vivo hepatic environment. A putative regulatory region was then found by deoxyribonuclease I footprinting analyses of the proximal 5'-flanking region of exon 1A with nuclear extracts from bovine liver tissue. This regulatory region contained a putative binding site for the liver-enriched transcription factor hepatocyte nuclear factor-4 (HNF-4). Binding of HNF-4 in bovine liver to this putative HNF-4 binding site was confirmed by electrophoretic mobility shift assays. Overexpression of HNF-4 enhanced the transcriptional activity of the 5'-proximal region of exon 1A in various cell lines. Mutation of the HNF-4 binding site abolished the transactivation. In addition, the HNF-4 mRNA was found to be primarily expressed in liver and absent in most nonhepatic tissues in the bovine. Collectively, these observations suggest that the liver-enriched transcription factor HNF-4 plays a role in the expression of GHR 1A mRNA in bovine liver.
authors.affiliation: Animal Sciences Research Center, Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. jiangh@missouri.edu
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.forename: M C
authors.initials: MC
authors.lastname: Lucy
mesh_terms: D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D011986:Receptors, Somatotropin; D014157:Transcription Factors