Thyroid transcription factor 1 and Pax8 synergistically activate the promoter of the human thyroglobulin gene

Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves a s the matrix for thyroid hormone biosynthesis. To obtain new insights in th e regulation of Tg gene expression, we investigated the interaction of the human Tg promoter with the thyroid-specific transcription factors TTF-1 and Pax8. A reporter gene, containing a 202 bp fragment from the human Tg 5 ' -flanking region including the promoter sequence and the transcriptional st art site, and expression vectors containing the cDNAs for human TTF-1 and P ax8 were used in cotransfection experiments, in the non-thyroidal cell line s COS-7 and HeLa. Pax8 increased the specific transcriptional activity of t he Tg promoter about threefold, whereas cotransfection with the homeodomain -containing protein TTF-1 stimulated promoter activity from six- to tenfold . The simultaneous expression of both factors stimulated the Tg promoter ac tivity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding ele ments corresponded to sites A and C in the rat Tg promoter. Site-directed m utagenesis of three nucleotides in each binding element inhibited binding o f TTF-1 to the two oligonucleotides. In cotransfection experiments, the mut ant site C decreased TTF-1 transactivation to 26% of the wild-type, whereas an additional mutation in the site A reduced this value to almost zero, th us proving the physiological relevance of these sites. The present results demonstrate that the activity of the human Tg promoter is closely dependent on the function of TTF-1 and Pax8, opening the field for further investiga tions of pathological alterations of Tg gene expression.