Critical residues of the homeodomain involved in contacting DNA bases alsospecify the nuclear accumulation of thyroid transcription factor-1

The N-terminal end of thyroid transcription factor-1 (TTF-1) homeodomain is composed of a stretch of five basic amino-acids that is conserved in both POU- and NK2-class homeodomains and constitutes a functional nuclear locali zation signal. By analyzing the cellular distribution of fusion proteins, c omposed of a jellyfish green fluorescent variant and different parts of TTF -1, we show hers that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we iden tified a second region located in the center of the DNA recognition helix o f the homeodomain that is also able to specify a predominantly nuclear loca lization of the chimeric proteins, independently of the presence of the bas ic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumul ation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of t he homeodomain that are involved in nuclear targeting also encompass critic al amino-acids responsible for DNA binding site recognition, as evidenced b y the loss of DNA binding activity in vitro upon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in h omeodomain proteins, suggesting that this part of the homeodomain could pla y a general role in the nuclear localization of members of this family of p roteins.