Protein and DNA contact surfaces that mediate the selective action of the Phox1 homeodomain at the c-fos serum response element.
The human homeodomain protein Phox1 can impart serum-responsive transcriptional activity to the c-fos serum response element (SRE) by interacting with serum response factor (SRF). This activity is shared with other Paired class homeodomains but not with more distantly related homeodomains. To understand the mechanism of action of Phox1 at the SRE and the basis for the selective activity of Paired class homeodomains in this context, we performed a detailed mutagenesis of the Phox1 homeodomain. We found that amino acid residues that contact the major groove of the DNA are required for SRE activation in vivo, suggesting an in vivo requirement for major-groove DNA contact by the homeodomain. In contrast, substitution of a lysine residue in the N-terminal arm of the Phox1 homeodomain appeared to abolish DNA binding without affecting activity in vivo. Certain substitutions on the exposed surfaces of helices 1 and 2, not required for DNA binding, abolished activity in vivo, suggesting that these surfaces contact an accessory protein(s) required for this activity. We also found that transfer of a single amino acid residue from the surface of Phox1 helix 1 to the corresponding position in the distantly related Deformed (Dfd) homeodomain imparts to Dfd the ability to activate the SRE in vivo. We propose that Phox1 interacts with one or more factors at the SRE, in addition to SRF, and that the specificity of this interaction is determined by residues on the surfaces of helices 1 and 2.
Simon
K J
KJ
Graduate Program in Molecular and Cellular Biology, State University of New York at Stony Brook, 11794, USA.
Grueneberg
D A
DA
Gilman
M
M
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D029721:Drosophila Proteins; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D026362:Serum Response Factor; D014162:Transfection
D004268:DNA-Binding Proteins; C066676:Dfd protein, Drosophila; D029721:Drosophila Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; C082157:PRRX1 protein, human; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D000409:Alanine