SP1 activates the MDR1 promoter through one of two distinct G-rich regions that modulate promoter activity

Abstract

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.