Two activator protein-1 elements in the matrix metalloproteinase-1 promoter have different effects on transcription and bind Jun D, c-Fos, and Fra-2.
Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabolism as the only enzyme capable of degrading interstitial collagens at neutral pH. We used fragments of the rabbit collagenase promoter ranging from 1800 to 182 bp to measure transcriptional activity of the activator protein-1 (AP-1) site at -77. Mutation at -77 in this sequence greatly reduced basal transcription in all constructs. However, mutant constructs with at least 321 bp of promoter responded to phorbol myristate acetate, similar to their native counterparts, implicating upstream regions in mediating this response. Through mutagenesis and analysis of DNA-protein interactions, we also identified and characterized a novel AP-1 site at -186. Mutation at -186 in 321 bp of promoter modestly lowered basal activity but, in contrast to mutation at -77, reduced phorbol responsiveness by 50%. Mobility shift assays demonstrated specific inducible binding at both sites. DNA/protein complexes at both AP-1 sites contain c-Fos and Jun D proteins, while Fra-2 is present only at the -77 site. These studies (1) demonstrate cooperativity between these two AP-1 sites, (2) implicate the -186 site in phorbol inducibility and (3) identify specific members of the Fos and Jun families binding to these sites.
White
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Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA.
Brinckerhoff
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051921:Fos-Related Antigen-2; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D004247:DNA; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D013755:Tetradecanoylphorbol Acetate