THE CYP2B2 PHENOBARBITAL RESPONSE UNIT CONTAINS AN ACCESSORY FACTOR ELEMENT AND A PUTATIVE GLUCOCORTICOID RESPONSE ELEMENT ESSENTIAL FOR CONFERRING MAXIMAL PHENOBARBITAL RESPONSIVENESS

Hepatic cytochrome P450s play a critical role in the metabolism of hyd rophobic xenobiotics. One of the major unsolved problems in xenobiotic metabolism is the molecular mechanism whereby phenobarbital induces h epatic enzymes, particularly CYP2B1 and CYP2B2 in rat liver, By using primary rat hepatocytes for transfection analyses, we previously ident ified in the CYP2B2 5'-flank a 163-base pair Sau3AI fragment that conf ers phenobarbital inducibility on a cat reporter gene and that has the properties of a transcriptional enhancer, Transfection experiments wi th sub-regions of the Sau3AI fragment now indicate that a central core together with an upstream or downstream accessory element within the fragment can confer phenobarbital responsiveness. One such accessory e lement, AF1, was identified and localized, DNase I footprinting analys is revealed the presence of a footprint overlapping this AF1 element. It also identified three other major protected regions, two of which a re putative recognition sites for known transcription factors, Site-di rected mutagenesis indicated that a putative glucocorticoid response e lement as well as a nuclear factor 1 site and an associated nuclear re ceptor hexamer half-site are essential for conferring maximal phenobar bital inducibility, Taken together, the results indicate that phenobar bital induction of CYP2B2 requires interactions among multiple regulat ory proteins and cis-acting elements constituting a phenobarbital resp onse unit.