Redox effector factor-1 regulates the activity of thyroid transcription factor 1 by controlling the redox state of the N transcriptional activation domain

Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcriptional regulator responsible for the activation of thyroid- and lung-specific genes. It has been demonstrated that its DNA binding activity is redox-regulated in vitro through the formation of dimers and oligomeric species. In this paper, we demonstrate that the redox regulation mainly involves a Cys residue (cys⁸⁷), which resides out of the DNA binding domain, belonging to the N-transactivation domain. In fact, the oxidized form of a truncated TTF-1 (containing the N-transactivation domain and the DNA-binding domain, here called TTF-1N-HD) looses specific DNA binding activity. Since most of the oxidized TTF-1N-HD is in a monomeric form, these data indicate that the redox state of Cys⁸⁷ may control the DNA-binding function of the homeodomain, suggesting that Cys⁸⁷ could play an important role in determining the correct folding of the homeodomain. By using gel retardation and transient transfection assays, we demonstrate that the redox effector factor-1 (Ref-1) mediates the redox effects on TTF-1N-HD binding and that it is able to modulate the TTF-1 transcriptional activity. Glutathione S-transferase pulldown experiments demonstrate the occurrence of interaction between Ref-1 and TTF-1N-HD. Having previously demonstrated that Ref-1 is able to modulate the transcriptional activity of another thyroid-specific transcription factor (Pax-8), our data suggest that Ref-1 plays a central role in the regulation of thyroid cells.