Constitutive androstane receptor-responsive elements for mouse Cyp1a2 transcriptional activation induced by constitutive androstane receptor ligands

The mouse cytochrome P450 1A2 (Cyp1a2) gene is one of the constitutive androstane receptor (CAR, NR1I3) activator-inducible genes, and the regions involved in induction were examined herein. A reporter gene assay indicated the involvement of the -0.2-kb region in the induction of transcriptional activation by the mouse CAR agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Some putative nuclear receptor-binding elements were identified in this region, and mutations in the elements located at -160/-155 or -153/-148 abolished this induction. An electrophoretic mobility shift assay demonstrated that a fragment comprised of three elements was capable of binding to the CAR/retinoid X receptor alpha (RXRα) heterodimer. The three elements comprise the two elements indicated above and one located at -146/-141. A chromatin immunoprecipitation assay confirmed CAR binding to the region including these elements in chromatin after treatment with TCPOBOP. These results indicate that mouse Cyp1a2 is the direct target of CAR, and binding of the CAR/RXRα heterodimer to the newly identified region in the promoter may be involved in transcriptional activation. Binding motifs were estimated as ER1 (everted repeat with a spacing of 1 nucleotide, -160/-155 and -153/-148) and ER8 (everted repeat with a spacing of 8 nucleotides, formed with -160/-155 and -146/-141).