THE NUCLEAR ORPHAN RECEPTOR CAR-RETINOID-X RECEPTOR HETERODIMER ACTIVATES THE PHENOBARBITAL-RESPONSIVE ENHANCER MODULE OF THE CYP2B GENE

PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding si tes, NR1 and NR2. Consistent with the finding that anti-retinoid X rec eptor (RXR) could supershift the NR1-nuclear protein complex, DNA affi nity chromatography with NR1 oligonucleotides enriched the nuclear orp han receptor RXR from the hepatic nuclear extracts of phenobarbital-tr eated mice. In addition to RXR, the nuclear orphan receptor CAR was pr esent in the same enriched fraction. Ln the phenobarbital-treated mice , the binding of both CAR and RXR was rapidly increased before the ind uction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR, PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells whe n the NR1 site was functional. A CAR-RXR heterodimer has thus been cha racterized as a tans-acting factor for the phenobarbital-inducible Cyp 2b10 gene.