Identification of amino acid residues of transcription factor AP-2 involved in DNA binding

AP-2 is a cell-type specific, developmentally regulated transcription facto r which has been described as a critical regulator of gene expression durin g vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identified, the exact i dentity of AP-2 amino acid residues responsible for its interaction with th e DNA structure has not yet been described. Here, we describe the identific ation of a region of AP-2 which was protected by an oligonucleotide probe c ontaining its binding site from trypsin digestion, monitored by peptide map ping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependen t APOE promoter, as well as the ability to inhibit the effect of the wild-t ype protein of each one of a set of single-site substitution AP-2 mutants s panning the identified region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential f or its DNA-binding activity. Particularly, the individual substitution in a ny of the residues 253, 254, 255, 257 or 260 is sufficient for completely a bolishing the interaction with DNA and the stimulation of APOE promoter act ivity. These results indicate a crucial role of this region in the formatio n of an active DNA-binding domain and strongly suggest that these residues provide direct contacts with the DNA structure at the AP-2 binding site. (C ) 2000 Academic Press.