Critical residues of the homeodomain involved in contacting DNA bases also specify 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 localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF-1, we show here that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic 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 accumulation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino-acids responsible for DNA binding site recognition, as evidenced by 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 homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.
Christophe-Hobertus
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C
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Faculté de Médecine, Belgique.
Duquesne
V
V
Pichon
B
B
Roger
P P
PP
Christophe
D
D
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016296:Mutagenesis; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D000074482:Thyroid Nuclear Factor 1; D014157:Transcription Factors; D049452:Green Fluorescent Proteins