Elsevier

Gene

Volume 228, Issues 1–2, 4 March 1999, Pages 169-179
Gene

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

https://doi.org/10.1016/S0378-1119(98)00612-XGet rights and content

Abstract

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. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific 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 −1700 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 enrichment techniques and immunoblotting and electromobility shift assays were 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 test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type −2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA–protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.

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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