PT - JOURNAL ARTICLE AU - Takayoshi Toriyabe AU - Kiyoshi Nagata AU - Tomonari Takada AU - Yusuke Aratsu AU - Tsutomu Matsubara AU - Kouichi Yoshinari AU - Yasushi Yamazoe TI - Unveiling a New Essential Cis Element for the Transactivation of the <em>CYP3A4</em> Gene by Xenobiotics AID - 10.1124/mol.108.050575 DP - 2009 Mar 01 TA - Molecular Pharmacology PG - 677--684 VI - 75 IP - 3 4099 - http://molpharm.aspetjournals.org/content/75/3/677.short 4100 - http://molpharm.aspetjournals.org/content/75/3/677.full SO - Mol Pharmacol2009 Mar 01; 75 AB - Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor α (RXRα) 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 α-η half-sites. Introduction of a mutation into either an α or β half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXRα heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with α and β 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. The American Society for Pharmacology and Experimental Therapeutics