Residues flanking the HOX YPWM motif contribute to cooperative interactions with PBX.
Hox genes encode transcription factors that are major determinants of embryonic patterning. Recently, we and others have shown that specific recognition of target sites in DNA is partly achieved through cooperative interaction with the extradenticle/pre-B-cell transformation-related gene (EXD/PBX) family of homeodomain-containing proteins. This interaction is mediated by the YPWM motif present N-terminal to the homeodomain in HOX proteins. In the present study, we use YPWM peptides to confirm the importance of this motif for mediating HOX/PBX interactions. We also used a novel monoclonal antibody directed against the YPWM to show that occlusion of this motif abrogates cooperativity with PBX. In addition, we present evidence that residues flanking the YPWM, both N-terminally and C-terminally, stabilize the HOX.PBX cooperative complex. Because these flanking residues are also conserved among paralogs, they are likely to help distinguish the specificity of HOX/PBX interactions. Our data further show that the relative importance of individual residues within and flanking the YPWM is dependent on the identity of position 6 of the cooperative binding site (TGATTNATGG). These results suggest that interactions between PBX and the YPWM motif are modified by a base pair predicted to contact the N-terminal arm of the HOX homeodomain.
Shanmugam
K
K
McGill Cancer Centre, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Featherstone
M S
MS
Saragovi
H U
HU
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D000099261:Homeobox A1 Protein
D000409:Alanine; D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D010446:Peptide Fragments; D000075062:Pre-B-Cell Leukemia Transcription Factor 1; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D000099261:Homeobox A1 Protein; C489520:Hoxd4 protein, mouse; C083400:PBX1 protein, human; C072064:HOXD10 protein, human