HUMAN THYROXINE-BINDING GLOBULIN GENE - COMPLETE SEQUENCE AND TRANSCRIPTIONAL REGULATION

T4-binding globulin (TBG) is a glycoprotein of hepatic origin which tr ansports thyroid hormone in serum. To characterize the human TBG (hTBG ) gene, we studied its genomic organization, promoter activity, and re gulation. To this purpose, we isolated from liver a complete hTBG cDNA clone containing the 5'-untranslated region and localized the transcr iption start site (TSS). The analysis of genomic clones revealed that the hTBG gene consists of five exons and that its exon-intron organiza tion is similar to that of other members of the serine protease inhibi tor family. The first exon (exon 0) is a short noncoding sequence loca ted 1.62 kilobase pairs (kbp) upstream from exon 1. Potential cis-acti ng transcriptional regulatory elements including a TATA box, a CAAT bo x, 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'-flanki ng region, including exon 0, was inserted upstream of the bacterial ch loramphenicol acetyltransferase gene showed significant activity when transfected into a hepatoblastoma-derived (HepG2) cell line. The phorb ol ester, 12-O-tetradecanoylphorbol-13-acetate, down-regulated the pro moter activity by more than 80% and completely inhibited hTBG synthesi s, whereas thyroid hormone, glucocorticoid, estrogen, and nicotinic ac id 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 acety ltransferase construct. When nonhepatocyte-derived cell lines (CV-1 an d 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 ac tivity in HepG2 cells but not in CV-1 or CHO cells. On the other hand, mutational disruption of the putative hepatocyte nuclear factor-1 sit e (located 65 bp upstream of the TSS) completely abolished the promote r activity in all cell lines, indicating that this site is absolutely required for the transcription of the hTBG gene.