The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module

Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed i n adult human liver, is subject to transcriptional induction by a variety o f structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4- luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of th e reporter gene was observed only with the longest construct, which encompa ssed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive regio n was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-induci ble expression on a heterologous promoter. Further deletion mutants localiz ed the induction to bases -7836 to -7607. In vitro DNase I footprint analys is of this region revealed four protected sites (FP1, FP2, FP3, and FP4). T wo of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682 ), overlapped binding motifs for the orphan human pregnane X receptor (hPXR ). Cotransfection of responsive constructs with a hPXR expression vector su bstantially increased the rifampicin-inducibility to similar to 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between el ements within the distal enhancer region and cis-acting elements in the pro ximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, wh ich mediates the transcriptional induction of CYP3A4 by activators of hPXR.