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

Abstract

Thyroglobulin (Tg) is an essential thyroid-specific protein, which serves as the matrix for thyroid hormone biosynthesis. To obtain new insights in the 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 start site, and expression vectors containing the cDNAs for human TTF-1 and Pax8 were used in cotransfection experiments, in the non-thyroidal cell lines COS-7 and HeLa. Pax8 increased the specific transcriptional activity of the 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 activity in a multiplicative manner up to 25-fold. TTF-1 binding sites could be localized precisely by lectron mobility shift assay. The two binding elements corresponded to sites A and C in the rat Tg promoter. Site-directed mutagenesis of three nucleotides in each binding element inhibited binding of TTF-1 to the two oligonucleotides. In cotransfection experiments, the mutant 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, thus 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 investigations of pathological alterations of Tg gene expression.

Thyroid hormone metabolism may depend on dietary fat

“…The endogenous ligand of peroxisome proliferator activated receptor γ (PPARγ) -15-deoxy-Δ 12,14 -PGJ 2 (15d PGJ 2 ) -a product of C20:4 metabolism (Forman et al, 1995) was shown to facilitate the synthesis of thyroglobulin in a functional rat epithelial cell line (Kasai et al, 2000). Taking into consideration the similar mode of regulation of thyroglobulin and TPO gene transcription (Espinoza et al, 2001), 15dPGJ 2 might represent an intermediate in the signalling pathway by which PUFA affect TPO level.…”

Poly(ADP-ribose) polymerase 1 binds to Pax8 and inhibits its transcriptional activity

“…In fact, Pax8 knockout mice have a very small thyroid gland, which is deprived of follicular cells (Mansouri et al 1998). Recently, it has been demonstrated that Pax8 cooperates with the transcription factor NKX2-1 (Espinoza et al 2001, Miccadei et al 2002, and our laboratory specifically demonstrated that Pax8 and NKX2-1 form a functional heterocomplex responsible for the synergistic transcriptional activation of the Tg promoter in differentiated thyroid cells (Di Palma et al 2003). The relevance of this interaction has been further confirmed by the analysis of the in vivo mouse model bearing a partial deficiency of Ttf1 and Pax8 genes.…”

14 Genetics of Congenital Hypothyroidism

“…68 Pax-8 activates transcription of TPO, TG, and NIS, and acts synergistically with TTF-1 to activate the promoter of human TG gene. [70][71][72] Dominant negative properties of the mutant allele or haploinsufficiency are not considered likely underlying mechanisms for pathogenicity of Pax-8 mutations. The exact mechanism is still unclear but one possibility is that the human PAX-8 locus is imprinted, with selective expression of the mutant allele leading to disease.…”