Molecular cloning of the rat thyroxine-binding globulin gene and analysis of its promoter activity

We cloned the rat T4-binding globulin (rTBG) gene, characterized its organization, and studied its promoter activity and regulation. A genomic DNA library was constructed and screened using an rTBG complementary DNA (cDNA) as a probe. An 8.6-kilobase pair (kbp) clone was partially sequenced and compared with the sequence of the previously cloned cDNA. It helped complete the cDNA sequence and identify the first noncoding exon (exon 0). The transcription start site was identified using an RNase protection assay. The rTBG genomic clone contained 1.2 kbp 5'-flanking and 1.7 kbp 3'-flanking regions. The sizes of exons and introns of the rTBG gene are similar to those of the human TBG gene, belonging to the serine protease inhibitor family. The 5'-flanking region contains a TATA box, a CAAT box, and a consensus sequence for the hepatocyte nuclear factor 1-binding site. We tested the promoter activity of the 1.2-kbp 5'-flanking region using a luciferase reporter plasmid. When transfected into a hepatocyte-derived cell line (HepG2), the plasmid construct containing the fragment -1227 to +11 (transcription start site, +1) showed a 9-fold increase in luciferase activity compared with that of a promoterless luciferase vector. No promoter activity was detected in a nonhepatocyte-derived cell line (COS1). Serial 5'-deletion revealed that the construct containing the fragment -180 to +11 had 40% of the maximal promoter-induced luciferase activity. And that containing the fragment -53 to +11 showed no significant increase of luciferase activity. These results suggest that the region -180 to -53, containing hepatocyte nuclear factor 1-binding site, is essential to the liver-specific expression. We previously reported down-regulation of rTBG messenger RNA by T3 in vivo. The present study failed to show T3's effect on the promoter activity of the 1.2-kbp 5'-flanking region of the rTBG gene.