Abstract
Phenobarbital induction of CYP2B genes is mediated by a complex phenobarbital-responsive enhancer (PBRU), which contains a binding site for nuclear factor-1 (NF-1) flanked by two DR-4 nuclear receptor (NR) binding sites for a heterodimer of constitutive androstane receptor (CAR) and retinoid X receptor (RXR). To examine potential interactions between NF-1 and CAR/RXR, binding of purified recombinant proteins to DNA, or to chromatin assembled using Drosophila embryo extract, was examined. NF-1 and CAR/RXR bound simultaneously and independently to the overlapping NF-1 and NR-1 sites; binding of CAR/RXR to the NR-2 site was modestly increased by NF-1 binding; and CAR/RXR bound to a new site in the PBRU region, designated NR-3. Assembly of plasmid DNA into chromatin using Drosophila extract resulted in linearly phased nucleosomes in the PBRU region. The apparent binding affinity of NF-1 was increased by about 10-fold in assembled chromatin compared with DNA, whereas CAR/RXR binding was decreased. As observed for DNA, however, simultaneous, largely independent, binding to the NF-1 and NR sites was observed. CAR-mediated transactivation of the PBRU in cultured cells of hepatic origin was inhibited by mutations in the NF-1 site, and overexpression of NF-1 increased CAR transactivation in HepG2 cells. These studies demonstrate that NF-1 and CAR/RXR can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking NR sites were occupied after phenobarbital treatment. CAR-mediated trans-activation of the PBRU was increased by NF-1, analogous to NF-1 effects on phenobarbital induction in previous transient transfection studies and consistent with mediation of phenobarbital induction by CAR.
Highlights
Cytochromes P450 (P450s)1 play an important role in the Considerable progress has been made in understanding the molecular mechanisms of PB induction of CYP2B genes
These studies demonstrate that nuclear factor-1 (NF-1) and constitutive androstane receptor (CAR)/ retinoid X receptor (RXR) can both bind to the PBRU at the same time and that chromatin assembly increases NF-1 binding, which is consistent with previous in vivo footprinting studies in which the NF-1 site was occupied in untreated animals and the NF-1 and flanking nuclear receptor (NR) sites were occupied after phenobarbital treatment
It has been demonstrated that binding of CAR to the coactivator steroid receptor coactivator-1 is increased after incubation with the PB-like inducer, chrome P450 gene; PB, phenobarbital; PBRU, PB-responsive unit; NR, nuclear receptor; NF-1, nuclear factor-1; CAR, constitutive androstane receptor; RXR, retinoid X receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; DTT, dithiothreitol; DMS, dimethyl sulfate; MNase, micrococcal nuclease; MMTV, murine mammary tumor virus; kb, kilobase(s); bp, base pair(s); Ni-NTA, nickel-nitrilotriacetic acid
Summary
P450, cytochrome P450; CYP, cytometabolism of xenobiotics and in the biosynthesis of endogenous compounds. It has been demonstrated that binding of CAR to the coactivator steroid receptor coactivator-1 is increased after incubation with the PB-like inducer, chrome P450 gene; PB, phenobarbital; PBRU, PB-responsive unit; NR, nuclear receptor; NF-1, nuclear factor-1; CAR, constitutive androstane receptor; RXR, retinoid X receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; DTT, dithiothreitol; DMS, dimethyl sulfate; MNase, micrococcal nuclease; MMTV, murine mammary tumor virus; kb, kilobase(s); bp, base pair(s); Ni-NTA, nickel-nitrilotriacetic acid. CAR-dependent activation of the PBRU in cultured cells is decreased by mutations of the NF-1 site and coexpression of NF-1 with CAR enhances the CAR-dependent activation in HepG2 cells just as mutation of the NF-1 site inhibits PB induction in transiently transfected hepatocytes in primary culture or in situ
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