Nuclear Factor-1 Motif and Redundant Regulatory Elements Comprise Phenobarbital-Responsive Enhancer inCYP2B1/2

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Abstract
Although the induction of drug-metabolizing systems by phenobarbital has been recognized for about 40 years, the mechanism by which cytochrome P450 gene expression is increased is still not well understood. A 163-bp fragment at about -2.2 Kb in CYP2B2 has been shown to mediate phenobarbital induction in primary rat hepatocytes (Trottier, et al. [1995] Gene 158:263-268) and by an in situ transient transfection assay in rat liver (Park, Y., et al. [1996]. J. Biol. Chem. 271:23725-23728). Deletion mutations of this fragment indicated that the 88-bp stretch from -2258 to -2170 was the minimal sequence that could mediate phenobarbital induction in the in situ system if single copies of the deleted fragments fused to the CYP2C1 proximal promoter were assayed. If three copies of the fragments were present, 5′ and 3′ deletions defined a minimal 37-bp core fragment, which, although necessary for phenobarbital responsiveness, was not sufficient unless additional sequence was present at either end, suggesting that redundant elements were present in the two flanking regions. Site-specific mutagenesis of an NF-1 site within the 88-bp fragment and linker scanning mutagenesis across the fragment indicated that the NF-1 site and a region to the 5′ side of the site contributed to the magnitude of the response, but neither the NF-1 mutations nor any of the linker scanning mutations eliminated the response to phenobarbital. Mutation in a region 3′ of the NF-1 site resulted in elevated basal expression without substantial effects on phenobarbital-induced expression. Binding of NF-1 to the 37-bp core fragment was established by gel-shift competition studies and by supershifts of the protein-DNA complexes by antisera to NF-1. Additional protein-DNA complexes were detected in the regions flanking the NF-1 site. These studies indicate that the CYP2B2 phenobarbital-responsive enhancer contains multiple constitutive and phenobarbital-responsive elements. Binding of nuclear proteins from control or phenobarbital-treated animals in vitro to this region was very similar. The only difference detected was a complex that was substantially reduced by phenobarbital treatment and mapped to the 3′ side of the NF-1 site.