SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity.
To define sequences in the human multidrug resistance (MDR1) promoter that influence transcription, we measured the activity of MDR1 promoter constructs using luciferase as a reporter gene. Deletion of promoter sequences to -121 (relative to the transcription initiation site) had very little effect on promoter activity in transiently transfected cells. Further deletion to -88 increased activity about 4-fold, while deletion to -51 decreased activity about 10-fold. The data indicated that between -121 and -88 and between -73 and -51 were sequences that modulated promoter activity. Because these two regions contain G-rich sequences, which are important for function in other promoters, the effect of mutation of the G-rich regions (-110 to -103 and -61 to -43) on MDR1 promoter activity was measured. Mutation of the -110 G-box (-110 to -103) resulted in a 6-fold increase in promoter activity and inhibited the formation of a specific nuclear protein complex, suggesting that this region functions as a transcriptional "repressor" binding site in cycling cells. In contrast, mutation of the -50 G-box (-61 to -43) reduced promoter activity 5-fold. DNase-I footprinting and electrophoretic mobility shift analysis indicated that specific but distinct protein-DNA complexes formed at each of these two G-rich regions. The -50 G-box contains overlapping sites to which both SP1 and EGR-1 bind specifically. Co-transfection of MDR1 promoter constructs with SP1 into cells that lack SP1 (Drosophila Schneider 2 cells) activated equivalently both the wild type MDR1 promoter and a synthetic promoter containing the MDR1 -50 GC box and MDR1 initiator element.
Cornwell
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MM
Clinical Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Smith
D E
DE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D004330:Drosophila; D004351:Drug Resistance / Q000235:genetics*; D051766:Early Growth Response Protein 1; D005786:Gene Expression Regulation; D006147:Guanine; D006801:Humans; D017874:Immediate-Early Proteins; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D006147:Guanine; D008156:Luciferases