SELEX and missing phosphate contact analyses reveal flexibility within the AP-2α protein:DNA binding complex

The AP-2 family of transcription factors are defined by the presence of a novel DNA binding domain, termed a 'basic helix-span-helix' motif, The AP-2 genes regulate important aspects of vertebrate embryogenesis and have also been linked to the control of cell proliferation and tumorigenesis, but the cellular targets that the AP-2 proteins control are largely undefined. In particular, since only a limited number of sequences have previously been utilized to define the nature of the AP-2 binding site, the range of DNA sequences recognized by the AP-2 proteins remains unknown. We have therefore utilized a SELEX analysis to identify multiple new AP-2 alpha binding sites, Moreover, we have devised a novel missing phosphate and nucleotide competition analysis to characterize the residues in the binding site required for AP-2 alpha protein:DNA contact. These studies suggest that the AP-2 alpha protein:DNA complex is flexible and indicate that AP-2 alpha can bind three related sequence motifs: GCC N3 GGC, GCC N4 GGC and GCC N3/4 GGG, The availability of these more refined consensus sequences should assist in the identification of target genes for this critical transcription factor.