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.