Functional inactivation of Thyroid Transcription Factor-1 in PCCl3 thyroid cells

Thyroid Transcription Factor-1 (TTF-1) is a key regulator of thyroid development and function. In order to identify the genes whose expression depends on TTF-1 transcriptional activity within the thyrocyte we analyzed the consequence of the functional inactivation of this factor in PCCl3 cells. The expression of a fusion protein composed of the DNA binding domain of TTF-1 and of the strong repressive domain of the Engrailed protein (Engr HD) resulted in a dramatic loss of epithelial cell morphology and in proliferation arrest. These changes were reversed when the inhibition of endogenous TTF-1 was relieved. No change was observed when a similar fusion protein containing point mutations abolishing DNA binding activity (Engr HDm) was produced in the cells. Microarrays hybridizations were performed on PCCl3 cells expressing a TTF1 antagonist (Engr HD –Dox) versus non-expressing cells (Engr HD +Dox), and on cells expressing the control protein (Engr HDm – Dox) versus non-expressing cells (Engr HDm + Dox). After amplification and labelling, sample pairs were hybridized onto Rat MI ReadyArray (Microarrays Inc., Alabama USA). The oligonucleotides set consists of 34,717 70-mer transcript probes representing genes and alternative splice products from the Operon Biotechnologies' Array Ready Oligo Set version 3.0 for Rattus norvegicus. Hybridizations were replicated with dye swap.