R Di Lauro

Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity.

The cDNA for TTF‐1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF‐1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF‐1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK‐2 homeodomain. TTF‐1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF‐1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF‐1 gene is not located within previously described clusters of homeobox‐containing genes.

Role of the thyroid-stimulating hormone receptor signaling in development and differentiation of the thyroid gland

The thyroid-stimulating hormone/thyrotropin (TSH) is the most relevant hormone in the control of thyroid gland physiology in adulthood. TSH effects on the thyroid gland are mediated by the interaction with a specific TSH receptor (TSHR). We studied the role of TSH/TSHR signaling on gland morphogenesis and differentiation in the mouse embryo using mouse lines deprived either of TSH (pitdw/pitdw) or of a functional TSHR (tshrhyt/tshrhyt and TSHR-knockout lines). The results reported here show that in the absence of either TSH or a functional TSHR, the thyroid gland develops to a normal size, whereas the expression of thyroperoxidase and the sodium/iodide symporter are reduced greatly. Conversely, no relevant changes are detected in the amounts of thyroglobulin and the thyroid-enriched transcription factors TTF-1, TTF-2, and Pax8. These data suggest that the major role of the TSH/TSHR pathway is in controlling genes involved in iodide metabolism such as sodium/iodide symporter and thyroperoxidase. Furthermore, our data indicate that in embryonic life TSH does not play an equivalent role in controlling gland growth as in the adult thyroid.

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5′‐TAAT‐3′. The homeodomain of thyroid transcription factor 1 (TTF‐1HD) binds to sequences containing a 5′‐CAAG‐3′ core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF‐1HD by both structural and functional approaches. As already suggested by the three‐dimensional structure of TTF‐1HD, the DNA binding specificities of the TTF‐1, Antennapedia and Engrailed homeodomains, either wild‐type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3′ end of the core motif 5′‐NAAN‐3′. The nucleotide at the 5′ position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF‐1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins.

Vertebrate Hox homeobox genes are transcription factors which regulate antero‐posterior axial identity in embryogenesis, presumably through activation and/or repression of downstream target genes. Some of these targets were reported to code for molecules involved in cell‐cell interactions, whereas no relationship has yet been demonstrated between Hox genes and other transcription factors involved in determining and/or maintaining tissue specificity. The thyroid transcription factor‐1 (TTF‐1) is a homeodomain‐containing protein required for expression of thyroid‐specific genes. A 862 bp 5′ genomic fragment of the rat TTF‐1 gene, conferring thyroid‐specific expression to a reporter gene, was sufficient to mediate transactivation by the human HOXB3 gene in co‐transfection assay in both NIH3T3 or HeLa cells. HOXB3 is expressed in early mammalian embryogenesis in the anterior neuroectoderm, branchial arches and their derivatives, including the area of the thyroid primordia and thyroid gland. Transcription of the TTF‐1 promoter is induced only by HOXB3, while its paralogous gene HOXD3 or other Hox genes expressed more posteriorly (HOXA4, HOXD4, HOXC5, HOXC6, HOXC8 and Hoxd‐8) have no effect. Transactivation by HOXB3 is mediated by two binding sites containing an ATTA core located at ‐100 and +30 from the transcription start site. DNase I footprinting experiments show that the two sites bind HOXB3 protein synthesized in both Escherichia coli and eukaryotic cells, as well as nuclear factor(s) present in protein extracts obtained from mouse embryonic tissues which express group 3 Hox genes and TTF‐1. Some of the DNA‐protein complexes formed by the embryonic extracts are indistinguishable from those generated by HOXB3.(ABSTRACT TRUNCATED AT 250 WORDS)