title: Unveiling a new essential cis element for the transactivation of the CYP3A4 gene by xenobiotics.
abstract: Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor alpha (RXRalpha) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at -7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them alpha-eta half-sites. Introduction of a mutation into either an alpha or beta half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRalpha heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with alpha and beta half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.
authors.affiliation: Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Toriyabe
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Nagata
authors.forename: Tomonari
authors.initials: T
authors.lastname: Takada
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Aratsu
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Matsubara
authors.forename: Kouichi
authors.initials: K
authors.lastname: Yoshinari
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Yamazoe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008875:Middle Aged; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*; D015262:Xenobiotics / Q000494:pharmacology*
chemical_list: D015262:Xenobiotics; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human