Human thyroxine-binding globulin gene: complete sequence and transcriptional regulation.
T4-binding globulin (TBG) is a glycoprotein of hepatic origin which transports thyroid hormone in serum. To characterize the human TBG (hTBG) gene, we studied its genomic organization, promoter activity, and regulation. To this purpose, we isolated from liver a complete hTBG cDNA clone containing the 5'-untranslated region and localized the transcription start site (TSS). The analysis of genomic clones revealed that the hTBG gene consists of five exons and that its exon-intron organization is similar to that of other members of the serine protease inhibitor family. The first exon (exon 0) is a short noncoding sequence located 1.62 kilobase pairs (kbp) upstream from exon 1. Potential cis-acting transcriptional regulatory elements including a TATA box, a CAAT box, and a hepatocyte nuclear factor-1 binding motif were identified in the upstream region. A reporter gene in which 3.2 kbp of the 5'-flanking region, including exon 0, was inserted upstream of the bacterial chloramphenicol acetyltransferase gene showed significant activity when transfected into a hepatblastoma-derived (HepG2) cell line. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, down-regulated the promoter activity by more than 80% and completely inhibited hTBG synthesis, whereas thyroid hormone, glucocorticoid, estrogen, and nicotinic acid had little, if any, effect. A series of 5'-deletions revealed that the fragment -218 to +4 from the TSS had the highest promoter activity, nearly 1000-fold greater than the promoterless chloramphenicol acetyltransferase construct. When nonhepatocyte-derived cell lines (CV-1 and CHO) were tested, promoter activity was reduced by a factor of 100, showing that the promoter works in liver-specific manner. The region -218 to -102 contains liver-specific enhancer elements, since deletion to nucleotide -101 resulted in a profound reduction of the promoter activity in HepG2 cells but not in CV-1 or CHO cells. On the other hand, mutational disruption of the putative hepatocyte nuclear factor-1 site (located 65 bp upstream of the TSS) completely abolished the promoter activity in all cell lines, indicating that this site is absolutely required for the transcription of the hTBG gene.
Hayashi
Y
Y
Department of Medicine, University of Chicago, Illinois 60637-1470.
Mori
Y
Y
Janssen
O E
OE
Sunthornthepvarakul
T
T
Weiss
R E
RE
Takeda
K
K
Weinberg
M
M
Seo
H
H
Bell
G I
GI
Refetoff
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D005796:Genes; D018197:Hepatoblastoma; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D005810:Multigene Family; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D009525:Niacin / Q000494:pharmacology; D009687:Nuclear Proteins; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013975:Thyroxine-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D013975:Thyroxine-Binding Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D009525:Niacin; D004958:Estradiol; D003907:Dexamethasone; D013755:Tetradecanoylphorbol Acetate