Derivation of the consensus DNA-binding sequence for p63 reveals unique requirements that are distinct from p53.
p63 is a member of the p53 family of proteins and plays an important role in epithelial development and differentiation. Although some p63 binding sites in the regulatory elements of epithelial genes have been identified, the optimal DNA-binding sequence has not been ascertained for this transcription factor. Here, we identify the preferred DNA-binding site of p63 by performing in vitro DNA selection experiments. Our analysis shows that the optimal p63 DNA-binding consensus motif consists of a CA(T)TG core and an AT-rich 5' and 3' flanking sequence. Gel shift and competition experiments demonstrate that there are specific sequence requirements that confer high DNA-binding affinity for p63 and that significant deviation from the consensus sequences result in poor or no binding. This pattern of DNA-binding is similar for both recombinant p63 and the endogenous protein present in keratinocyte nuclear extracts. Furthermore, we show that the consensus sequence is distinctly different from that of p53, particularly in the flanking sequences. Identification of the p63 consensus DNA-binding sequence will facilitate the validation of in vivo p63-responsive elements that mediate transcriptional regulation of a wide variety of target genes.
Ortt
Kori
K
Department of Biochemistry, State University of New York at Buffalo, 14214, USA.
Sinha
Satrajit
S
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D016415:Sequence Alignment; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D010750:Phosphoproteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; C486419:Trp63 protein, mouse; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004247:DNA