The promoter competition assay (PCA): a new approach to identify motifs involved in the transcriptional activity of reporter genes.
Identifying particular motifs responsible for promoter activity is a crucial step toward the development of new gene-based preventive and therapeutic strategies. However, to date, experimental methods to study promoter activity remain limited. We present in this report a promoter competition assay designed to identify, within a given promoter region, motifs critical for its activity. This assay consists in co-transfecting the promoter to be analyzed and double-stranded oligonucleotides which will compete for the binding of transcription factors. Using the recently characterized SBEM promoter as model, we first delineated the feasibility of the method and optimized the experimental conditions. We then identified, within an 87-bp region responsible for a strong expression of the reporter gene, an octamer-binding site essential for its transcriptional regulation. The importance of this motif has been confirmed by site-directed mutagenesis. The promoter competition assay appears to be a fast and efficient approach to identify, within a given promoter sequence, sites critical for its activity.
Hube
Florent
F
Department of Pathology, University of Manitoba, Winnipeg R3E 0W3, Manitoba, Canada.
Myal
Yvonne
Y
Leygue
Etienne
E
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D019295:Computational Biology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005821:Genetic Techniques; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D009077:Mucins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1 / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D009077:Mucins; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors; D008156:Luciferases