This research project aims at defining the precise template requirements and the role of post-translational modifications for TTF-1 activity, both in cultured cell lines and during mouse development.
TTF-1 needs to bind at more than one site to stimulate transcription of thyroglobulin and thyroperoxidase, two genes that are exclusively expressed in thyroid; in artificial promoters, at least 5 polymerized TTF-1 binding sites are required, while in the natural promoters of the two sites are sufficient to provide TTF-1 stimulation, indicating that in the natural promoters TTF-1 cooperates with other transcription factors.
TTF-1 has been demonstrated to be phosphorylated and the 7 serine residues where phosphorylation occurs have been identified. cDNA mutants encoding TTF-1 derivatives that cannot be phosphorylated have been constructed. Such phosphorylation deficient TTF-1 derivatives show no decrease in transcriptional activity. In keeping with this observation, it has been shown that in the dog thyroid system there is no difference in the phosphorylation state of TTF-1 between undifferentiated and differentiated cells. Conversely, Pax-8 mRNA is present at higher levels in differentiated cells, suggesting that this factor could contribute to expression of differentiated phenotype in this system.
TTF-1 DNA binding activity has been demonstrated to be sensitive to redox; it has been demonstrated that two specific cysteine residues are responsible for this type of regulation. It is conceivable that such mechanism play an important role also in vivo.
A new transcription factor, TTF-2, has been cloned. The TTF-2 mRNA is present, among several adult mouse tissues examined exclusively in the thyroid gland. In situ hybridization studies revealed expression of TTF-2 in the developing thyroid and in the pituitary. In both organs TTF-2 expression is transient and disappears at about day 10-12 p.c. This finding is of great interest since thyroid differentiation occurs just after disappearance of TTF-2 mRNA, suggesting that this transcription factor could, in the embryonic thyroid, function as a repressor of differentiation. Subsequently, TTF-2 mRNA reappears, as it is found in adult thyroid. Here, TTF-2 behaves as a transcriptional activator, since it has been shown to mediate transcriptional regulation by insulin and Thyroid stimulating hormone (TSH). TTF-2 mRNA level itself is under tight hormonal control. Mice homozygous for a disrupted TTF-2 allele have been obtained. These mice die shortly after birth, demonstrating that TTF-2 plays an essential role in controlling a vital function.
Expression of the orphan nuclear receptor NGFI-B was shown to be strongly induced in thyrocytes following stimulation of the cAMP cascade. Transactivation of a NGFI-B dependent promoter was also highest after cAMP stimulation, as compared to other stimuli. This suggests that NGFI-B plays the role of an early factor controlling the expression of genes involved in the differentiated functions and/or proliferation of the thyrocytes.
MAJOR SCIENTIFIC BREAKTHROUGHS:
TTF-1 is subjected to phosphorylation and to redox regulation; TTF-1 is able to recognize different templates, presumably by cooperation with other factors.
A new transcription factor (TTF-2), relevant in both thyroid and pituitary morphogenesis, has been cloned and characterized. TTF-2 is subjected to hormonal regulation and it appears to play, in mice, an essential role for survival.
NGFI-B, a transcription factor whose role has not been clearly ascertained, seems to play an important role in regulating thyroid function.
Funding SchemeCSC - Cost-sharing contracts