Identification of amino acid residues of transcription factor AP-2 involved in DNA binding.
AP-2 is a cell-type specific, developmentally regulated transcription factor which has been described as a critical regulator of gene expression during vertebrate development and embryogenesis. Although the overall domains of this factor necessary for their activity have been identified, the exact identity of AP-2 amino acid residues responsible for its interaction with the DNA structure has not yet been described. Here, we describe the identification of a region of AP-2 which was protected by an oligonucleotide probe containing its binding site from trypsin digestion, monitored by peptide mapping by MALDI-TOF mass spectrometry. Furthermore, we analyzed the relative in vitro DNA-binding activity, the stimulatory potency on the AP-2-dependent APOE promoter, as well as the ability to inhibit the effect of the wild-type protein of each one of a set of single-site substitution AP-2 mutants spanning the identified region. Taken together, our data clearly demonstrate that the region between amino acid residues 252-260 of AP-2 is essential for its DNA-binding activity. Particularly, the individual substitution in any of the residues 253, 254, 255, 257 or 260 is sufficient for completely abolishing the interaction with DNA and the stimulation of APOE promoter activity. These results indicate a crucial role of this region in the formation 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.
García
M A
MA
Centro de Biología Molecular Severo Ochoa, CSIC-Universidad Autónoma de Madrid, 28049, Spain.
Campillos
M
M
Ogueta
S
S
Valdivieso
F
F
Vázquez
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001057:Apolipoproteins E / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020218:Response Elements / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D001057:Apolipoproteins E; D004268:DNA-Binding Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; D014357:Trypsin