Elsevier

Biochemical Pharmacology

Volume 57, Issue 9, 1 May 1999, Pages 1073-1076
Biochemical Pharmacology

Gene Expression and Development
Positive regulation of the rat CYP2B2 phenobarbital response unit by the nuclear receptor hexamer half-site·nuclear factor 1 complex

https://doi.org/10.1016/S0006-2952(98)00367-0Get rights and content

Abstract

Abstract. A distal 163-bp fragment mediates phenobarbital responsiveness of the rat CYP2B2 gene. Multiple cis-acting elements in this fragment cooperate to form a phenobarbital response unit (PBRU). A nuclear factor 1 binding site and an associated nuclear receptor hexamer half-site are present in both the rat CYP2B2 PBRU and the homologous mouse Cyp2b10 sequence. Based on mutational analyses, the hexamer half-site has been reported to act positively in CYP2B2 and negatively in Cyp2b10. However, the specific mutations introduced into the rat and mouse hexamer half-sites were different, raising the possibility that the different roles attributed to the element may be a consequence of the different mutations used. We introduced into the rat CYP2B2 hexamer half-site the specific mutational change previously introduced into the Cyp2b10 sequence, where its effect was to increase the basal level of expression and to abolish phenobarbital responsiveness. In the rat context, this mutation reduced but did not abolish phenobarbital responsiveness and decreased, rather than increased, the basal level of expression. The residual phenobarbital responsiveness of the hexamer half-site mutant, as well as that of nuclear factor 1 mutants, indicates that these elements behave as positive accessory sites, suggesting that factors binding to them function as activators of phenobarbital-dependent transcription.

Section snippets

Materials and methods

Rat hepatocytes were isolated, cultured, transfected, and treated with PB as previously described [6], except that Matrigel was omitted. CAT activity was assayed by the method of Gorman et al.[11]. The following oligos were synthesized by Life Technologies: NF1dm3, 5′-ACTTTCCTGACCTTTTCACAGTGCTTCCAT-3′; and HXm2, 5′-ACTCTGTACTTTCCTCTGGTTGGCACAGTGC CACCA-3′. Substitutions in the wild-type sequence are indicated by bold characters. To generate the construct HXm2, the pSa-Sa163 plasmid [6] was

Results and discussion

As previously mentioned, based on mutational analyses, the HX half-site seems to act positively in rat CYP2B2 and negatively in mouse Cyp2b10. To determine whether this apparent difference might be due to the different mutations introduced into the rat and mouse sequences, we generated an HXm2 mutant that carries the exact 4-bp substitution (AGGTCA → ACCAGA) introduced into the mouse half-site [5]. As we found previously with the HXm and HXm-NF1dm2 mutations [6], the HXm2 mutation reduced but

Acknowledgements

This work was supported by the Medical Research Council. We thank Eric Trottier for careful reading of the manuscript, Pierre Paquin and Guy Langlois for photographic work, Frances Sladek for the HNF-4 cDNA, and Luc Bélanger for the FTF expression vector.

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  • Identification and functional characterization of a conserved, nuclear factor 1-like element in the proximal promoter region of CYP1A2 gene specifically expressed in the liver and olfactory mucosa

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    NF-1-like elements containing the highly conserved TGG motif have been found in the promoters and enhancers of many genes. Of particular interest, an NF-1 site is present in the phenobarbital-responsive enhancer module in mouse Cyp2b10 and rat CYP2B2 genes and is important for the magnitude of the induction response (51-53). An NF-1 element was also found to be a functional component of the mouseCyp1a1 promoter in a study with mouse hepatoma cells (54); a similar site is present in rat and human CYP1A1 promoter and was found to be involved in the down-regulation of CYP1A1gene expression by oxidative stress (55).

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