Phenobarbital responsiveness conferred by the 5′-flanking region of the rat CYP2B2 gene in transgenic mice
Introduction
Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s (CYP), aldehyde dehydrogenase, NADPH–P-450 oxidoreductase, and individual phase II enzyme systems (Frueh et al., 1997, Waxman and Azaroff, 1992). The PB induction response occurs in many species, including humans (Hansen et al., 1989, Honkakoski and Lang, 1989, Morel et al., 1990, Wen and Fulco, 1987). The rat is a well-characterized animal model of gene induction. In particular, the rat CYP2B1 and CYP2B2 genes exhibit basal expression levels that are extremely low, but are induced markedly in the liver within a few hours after PB treatment (Omiecinski et al., 1985). The PB induction response is transcriptionally mediated (Hardwick et al., 1983); however, the molecular mechanisms underlying the activation process are not clearly defined.
A 17 bp “barbie box” sequence was identified within the promoter regions of several PB-inducible genes and implicated in barbiturate regulation of the cytochrome P-450BM-1 gene in Bacillus megatarium (He and Fulco, 1991, Shaw and Fulco, 1993). However, the role of this P-450BM-1 element and the corresponding Bm1P1 protein regulator in the induction process has recently been questioned (Shaw et al., 1998). An accumulating body of evidence suggests that the barbie box is not a critical regulator of PB induction in mammalian liver cells (Honkakoski and Negishi, 1997, Park et al., 1996, Stoltz et al., 1998). Studies in transgenic mice were the first to indicate that the PB response region resides upstream of −800 bp in the 5′-flanking region of the CYP2B2 gene (Ramsden et al., 1993). Further evidence from both rat and mouse experiments implicated a PB responsive unit (PBRU) well upstream of the barbie box element as critical in modulating PB activation. Using gene transfection of primary rat hepatocytes, Anderson and co-workers (Trottier et al., 1995) initially delineated a PBRU as residing between −2318 bp and −2155 bp upstream of the transcription start site of the CYP2B2 gene. Similar findings were obtained in transfection assays performed in situ in rat liver (Park et al., 1996). In transfected mouse hepatocytes, an analogous PB-responsive enhancer module (PBREM) encompassing 51 bp from −2456 to −2250 of the Cyp2b10 gene was identified with transfected gene constructs as conferring PB responsiveness (Honkakoski et al., 1998a).
In the mouse hepatocyte studies, the PBREM was characterized as containing several critical binding motifs, including a nuclear receptor site and another for nuclear factor 1 (NF-1) (Honkakoski and Negishi, 1997, Honkakoski et al., 1998a, Honkakoski et al., 1998b). This PBREM region exhibits 91% sequence identity with the corresponding area of the rat CYP2B2 gene PBRU, where an NF-1 interaction also has been implicated in regulating PB induction (Stoltz et al., 1998). However, transfection efficiencies in primary hepatocytes are likely to be extremely low (<1%) (Ourlin et al., 1997) and levels of PB induction for transfected DNA constructs in hepatocytes do not approach that of the endogenous liver genes (Honkakoski and Negishi, 1997, Trottier et al., 1995). These considerations, coupled with the general extrapolation uncertainties with respect to results generated from in vitro experiments, provide a requirement for testing the basic features of the PB response domain model with in vivo techniques.
In this study, we used transgenic mouse approaches to assess the functional consequences of specific deletions and site-specific mutations within the region 2.5 kb upstream of the PB responsive rat CYP2B2 gene. Protein–DNA interactions at the PBRU domain were also characterized. Using the transgenic models, we demonstrate that sequences between −2500 and −1700 bp of the CYP2B2 gene are critical for PB induction, consistent with interpretations of several in vitro investigations (Honkakoski and Negishi, 1997, Park et al., 1996, Trottier et al., 1995). Based on several lines of evidence implicating the NF-1 element of the PBRU as a PB response modulator, we constructed transgenic mice with mutated and non-functional NF-1 binding sites within the CYP2B2 PBRU. The transgenes in these mice remained highly PB inducible. We conclude that the NF-1 regulatory component of the PBRU is not a critical contributor to the PB induction response.
Section snippets
In vitro DNase I footprinting
A pBluescript clone containing CYP2B2 5′ flanking gene sequence encompassing −2271/−2186 was used for in vitro DNase I footprinting. HindIII (sense strand) or XbaI (anti-sense strand) digestions were followed by a DNA polymerase I (Klenow fragment) fill-in reaction in the presence of [α-32P]dCTP. Plasmids were then digested with XbaI (sense strand) or HindIII (anti-sense strand) followed by separation of the CYP2B2 probe from vector by non-denaturing acrylamide gel electrophoresis. Footprinting
Results
Transgenic mice were developed that included 2500 bp of 5′ flanking sequence of the CYP2B2 gene fused to the entire CYP2B2 structural gene. Four out of the five −2500/2B2 lines derived were highly responsive to PB in the liver (Fig. 1); the remaining line exhibited no expression of the transgene in either untreated or PB-treated animals (data not shown), indicating that this gene was likely integrated into a silent chromosomal region. When the rat CYP2B2 gene was used as its own reporter,
Discussion
Earlier studies from our laboratory using transgenic mouse models of CYP2B2 gene expression indicated that a region critical to the PB regulatory process was located upstream of −800 bp in the 5′-flanking sequence of the rat gene (Ramsden et al., 1993). Reports from other investigators have now provided evidence for the importance of a PBRU, or PBREM, as a PB transcriptional activator in mammalian systems (Honkakoski and Negishi, 1997, Stoltz et al., 1998, Trottier et al., 1995). In both rat and
Acknowledgements
This project was supported by a grant from the National Institute of General Medical Sciences (GM32281) and a National Institute of Environmental Health Sciences Center grant (ES07033). C.J.O. is a Burroughs Wellcome Fund Toxicology Scholar.
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2005, Journal of Biological ChemistryCitation Excerpt :Thus the –1.8-kbp DNA fragment can be synergistically regulated by hCAR. After the discovery of the distal PBREM and the orphan nuclear receptor CAR, the mechanism of transcriptional activation of the CYP2B genes by PB and PB-type inducers became simplified when both enhancer and receptor were shown to be essential for the induction (4, 5, 8). Yet several important questions remain unanswered, including the following.
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2002, Comparative Biochemistry and Physiology - C Toxicology and PharmacologyFunctional analysis of the phenobarbital-responsive unit in rat CYP2B2
2001, Biochemical PharmacologyCitation Excerpt :Mutations with the most severe effects were those of NF-1 (Fig. 3), which is consistent with results in both transfected mouse and rat primary hepatocytes [6,8]. The in vivo role of NF-1 was recently called into question, since mutation of the NF-1 site did not reduce expression after PB treatment in three independent transgenic strains compared to two wild type transgenic strains [15]. The mutated transgenes, however, were expressed at higher levels in the untreated animal so that the fold-increase was reduced in the mutants.
Chromatin Assembly Enhances Binding to the CYP2B1 Phenobarbital-responsive Unit (PBRU) of Nuclear Factor-1, which Binds Simultaneously with Constitutive Androstane Receptor (CAR)/Retinoid X Receptor (RXR) and Enhances CAR/RXR-mediated Activation of the PBRU
2001, Journal of Biological ChemistryCitation Excerpt :The similar effects of mutation of the NF-1 site in the PBRU on PB induction in hepatocytes and CAR-activated expression in continuously cultured cells provide additional supporting evidence that CAR mediates the PB response and that the increase mediated by NF-1 is an enhancement of the trans-activation by CAR. Differences in the effects of mutations of the NF-1 site in transgenes (14) compared with transient transfections might be explained if assembly of the PBRU into chromatin, as in transgenes, alters the binding of NF-1. However, as with naked DNA, the NR-1, NR-2, and NF-1 sites were occupied simultaneously and largely independently in reassembled chromatin so that this explanation is not correct.