Phenobarbital responsiveness conferred by the 5 '-flanking region of the rat CYP2B2 gene in transgenic mice

Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochr ome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-speci fic mutations within the 2.5 kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between - 2500 and - 1 700 bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800 bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity e nrichment techniques and immunoblotting and electromobility shift assays we re used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200 bp in the PB responsive unit (PBRU) of CYP2B2. To t est the functional contribution of NF-1 in PB activation, we introduced spe cific mutations within the PBRU NF-1 element and demonstrated that these mu tations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type - 250 0;CYP2B2 transgene maintained full PB responsiveness. these results indicat e that, despite the avidity of the respective DNA-protein interaction withi n the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo. (C) 1999 Published by Elsevier Scien ce B.V. All rights reserved.