A rat thyroglobulin promoter fragment, capable of directing thyroid‐specific transcription, binds at least three different factors, TTF‐1, TTF‐2 and UFA, which are all present in nuclear extracts of the differentiated rat thyroid cell line FRTL‐5. TTF‐1 and TTF‐2 are FRTL‐5 specific, as demonstrated by their absence in nuclear extracts prepared from cell lines that do not express any thyroid‐differentiated function, while UFA is present in all cell lines tested. TTF‐1 has been extensively purified. It binds to the rat thyroglobulin promoter at three different sites which share sequence homology. Mutations in two of the three sites decrease both binding of TTF‐1 in vitro and promoter function in vivo. This suggests that the tissue‐specific expression of the thyroglobulin genes is mediated, at least in part, by the presence of a transcription factor exclusively in thyroid cells.

The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.

Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid-specific expression to a heterologous gene is contained between positions -168 and + 39. DNA-binding studies have demonstrated that this region interacts with two thyroidspecific factors (TTF-1 and TTF-2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF-1 with the A and C regions, UFA with the A region, and TTF-2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF-1-binding site with a high-affinity TTF-1 binding site. There is a correlation between the presence of TTF-1 and TTF-2 DNA-binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF-1 and TTF-2.The follicular cells of the thyroid gland are required for the production and controlled release of the thyroid hormones, 3',3,5-triiodothyronine and thyroxin. There are at least four functions which are good markers for the differentiated status of the follicular cells : expression of thyroglobulin, thyroid peroxid,ase, a Na+/l-symport, and the thyroid-stimulating hormone (TSH) receptor [l, 21. Of these functions, only the genes encoding thyroglobulin and thyroid peroxidase have been cloned [3 -61. Both thyroglobulin and thyroid peroxidase mRNA are only detectable in thyroid tissues. The 5' flanking DNA from the rat thyroglobulin gene is able to confer thyroid-specific expression to a heterologous gene, which shows that the thyroid-specific expression of the gene is mediated, at least in part, by transcriptional control [7]. The shortest region of the DNA that can act as a thyroid-specific promoter is located between nucleotides -168 and + 39 [7]. This sequence is thought to interact with the transcriptional apparatus to mediate the expression of thyroglobulin in thyroid cells. We have investigated the interaction between nuclear factors from thyroid and non-thyroid cells with the thyroglobulin promoter [S]. A DNA sequence, which is repeated three times, specifically interacts with a thyroid-specific DNA-binding activity, TTF-1. A second thyroid-specific DNA-binding activity, TTF-2, and an ubiquitous DNA-bindCorrespondence to R. Di Lauro, EMBL, Meyerhof Strasse 1, W-6900 Heidelberg, Federal Republic of GermanyAbbreviations. TTF-1; thyroid transcription factor 1 ; TTF-2, thyroid transcription factor 2; UFA, ubiquitous factor A; RSV, Rous sarcoma virus; CAT, chloramphenichol acetyltransferase; TSH, thyroid-stimulating hormone.Enzymes. Restriction endonucleases (EC 3.1.21.4); DNA polymerase I (EC 2.7.7.7); chloramphenicol acetyltransferase (EC 2.3.1.28); thymidine kinase (EC 2.7.1....

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.

The TSH receptor (TSHr) is one of the most important thyroid differentiation markers. The binding of the TSH hormone to its receptor is an essential step in the modulation of thyroid function and differentiation. Here we report that the thyroid transcription factor 1 (TTF1), a transcription factor essential for thyroid-specific gene expression, binds to the TSHr minimal promoter. The promoter, when mutated at this binding site, shows a decreased activity in thyroid cells. In cotransfection experiments in nonthyroid cells, TTF1 is able to trans-activate the TSHr minimal promoter. This finding strengthens the importance of TTF1 in the maintenance of thyroid differentiation. The promoters of the main thyroid differentiation markers thyroglobulin, thyroperoxidase, and now TSHr, are regulated by TTF1.