Identification of a Novel AP-2 Consensus DNA Binding Site

Abstract Activator Protein (AP)-2 is a transcription factor that is required for mouse development. AP-2 activates expression of positive and negative growth regulators including erbB-2 and p21 WAF1/CIP1 . Induction of p21 has been correlated with cell cycle and growth inhibition of human cancer cells. Because several endogenous AP-2 binding sites do not fit the known consensus sequences well, we sought to define AP-2's interaction with DNA more precisely. Using C yclic A mplification and S election of T argets (CAST'ing) of random oligonucleotide sequences and recombinant human AP-2 protein, we identified 17 novel AP-2 binding sites. Mobility shift assays showed significant AP-2 binding of the novel sites as compared to p21, erbB-2 and hMtIIa sites. Several sites that bound with high specificity and affinity did not fit known AP-2 consensus sequences. A sequence comparison based on several of the novel sequences yielded a putative consensus binding sequence of 5′-TAGAAAGNYCYNG-3′. These DNA binding sites may help identify novel targets of AP-2 and aid in further understanding AP-2 function.