The human multidrug resistance gene: sequences upstream and downstream of the initiation site influence transcription

To identify the DNA sequences required for multidrug resistance (MDR1) gene transcription, we have optimized conditions for transcription of the MDR1 proximal promoter in vitro. Using HeLa cell nuclear extracts, the direction, initiation, and RNA polymerase II dependence of transcription in vitro accurately reflect events in cells. The DNA template concentration, reaction temperature, and MgCl2 concentration were critical parameters of the in vitro system. Using conditions optimized for these parameters, the effect of deletions in the 5' flanking region and deletions in sequences downstream of the initiation site were examined. We found that deletion of sequences 5' and 3' of the transcription initiation site modulated the level of transcription. Of particular interest was the deletion of exon 1 sequences +5 to +127, which completely inhibited accurately initiated MDR1 transcription. Reconstitution of the +5 site, used for initiation in vivo and in vitro, did not reverse the inhibition. MDR1 transcription was specifically inhibited by an oligonucleotide corresponding to sequences +46 to +58. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the efficiency of the MDR1 proximal promoter.