TRANSCRIPTIONAL REGULATION OF THE MURINE MULTIDRUG-RESISTANCE GENE MDR1B BY PROGESTERONE OCCURS VIA AN INDIRECT MECHANISM

The murine multidrug resistance gene mdr1b is highly induced in the en dometrium during pregnancy. Evidence suggests that induction occurs ma inly as a result of progesterone action. To study the molecular mechan isms involved in this induction, 5'-flanking sequences between -540 an d +97 of the mdr1b gene were fused to the reporter gene, bacterial chl oramphenicol acetyltransferase (p540CAT). Unlike most progesterone-res ponsive genes, mdr1b is preferentially activated by the A form of the progesterone receptor. We now report that activation is not observed w ith a DNA-binding domain mutant of progesterone receptor A (PRA) sugge sting that induction occurs at the transcriptional level. Time course experiments demonstrated that induction was first observed 12 hr after hormone addition, suggestive of a secondary (or late) response gene, Sequence comparison highlighted the region M1 (-234 to -206), which co ntains a partially conserved progesterone response element, Its functi onal significance was evaluated by expression assays and gel shift ana lysis, Reporter plasmids with modifications of this element were trans fected into HeLa cells. Constructs containing the native M1 element, o r a mutated element (M1mt) that eliminated any similarity to a progest erone response element, were induced four-fold by progesterone whereas an element containing a consensus progesterone response element (M1PR E) was induced eight-fold. In addition, by gel shift analysis, the M1 element did not bind the progesterone receptor or any other factors. T his suggested that the M1 region does not participate in the response to pro-gesterone. 5' Nested deletion analysis, used to identify other regions of the upstream regulatory region that contributed to inductio n by progesterone, demonstrated that enhancer sequences between -122 a nd -65, which contain binding sites for C/EBP beta and NF-Y, were impo rtant. Mutations in the binding sites for these factors decreased indu ction by progesterone. On the basis of our studies using 540 bp of ups tream sequence, mdr1b is activated transcriptionally by progesterone, in an indirect manner dependent on basal factors.