Characterization of the Mouse Cyp1B1 Gene
IDENTIFICATION OF AN ENHANCER REGION THAT DIRECTS ARYL HYDROCARBON RECEPTOR-MEDIATED CONSTITUTIVE AND INDUCED EXPRESSION*
- From the Department of Pharmacology, Medical Science Center, University of Wisconsin, Madison, Wisconsin 53706
Abstract
Transcriptional activation of theCyp1B1 gene in rodents is stimulated by both polycyclic hydrocarbons and cAMP. The mouse Cyp1B1 gene structure contains three exons, of which the second nucleotide of exon 2 is the translation start site. Primer extension analysis identified a transcription start domain defining an exon 1 of 371 base pairs. The sequence 1.075 kilobases upstream of the transcription start site showed 11 xenobiotic-responsive elements (XRE) (TnGCGTG or GCGTG) that are putative aryl hydrocarbon receptor (AhR)-binding sites and three steroidogenic factor-1 motifs that are associated with cAMP-mediated transcriptional activation of genes. A transiently transfected Cyp1B1-luciferase construct, composed of exon 1 and 1.075 kilobases of 5′-flanking region, was induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10.0 ± 3.0-fold, n = 6) in C3H10T1/2 cells, which exclusively express Cyp1B1. The 90-base pair basal promoter contains two SP-1 sites, one SF-1 site, and a TATA-like box. TCDD induction and basal expression were dependent on positive regulatory elements present between −1075 and −810. Five XRE motifs localized in the enhancer region were completely conserved between mouse and humanCYP1B1 sequences. Similar inductions were seen in Hepa-1 cells, which express Cyp1A1 but not Cyp1B1. However, basal Cyp1B1 promoter activities were 4–10-fold higher in C3H10T1/2 cells providing the enhancer region was present, partially reproducing the in vivo cell-specific expression of Cyp1B1. Gel shift experiments established that TCDD stimulates AhR binding to the downstream XRE in the enhancer region. However, oligonucleotides that encompass two other XREs show a high affinity complex of similar size that is evident even without TCDD treatment and that does not contain either the AhR or AhR nuclear translocator. The fourth XRE is immediately adjacent to an E-box, and this oligonucleotide formed a smaller complex that was dependent on this E-box sequence. Negative regulatory sequences have been located between the promoter and TCDD-responsive enhancer regions. Constitutive expression of the Cyp1B1 gene was lost in AhR-deficient cells and was restored by transfected AhR cDNA. Reporter constructs function in a parallel manner, demonstrating the key role of the AhR in constitutive as well as TCDD-induced expression of Cyp1B1in mouse embryo fibroblasts.
Footnotes
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↵* This work was supported by National Institutes of Health Grant CA 16265.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, Medical Science Center, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-3128; Fax: 608-262-1257; E-mail:jefcoate{at}facstaff.wisc.edu.
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↵1 The abbreviations used are: AhR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; kb, kilobase(s); bp, base pair(s); Arnt, AhR nuclear translocator; XRE, xenobiotic-responsive element; DXE, diverse sequence xenobiotic-responsive element (36); PCR, polymerase chain reaction; SF-1, steroidogenic factor-1.
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↵2 J. Weisz, P. B. Brake, and C. R. Jefcoate, unpublished results.
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↵3 W. F. Greenlee, personal communication.
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↵4 A second experiment using different batches of Hepa-1 cells and media provided similar relative activities, but p1075/+150 provided only half the induction.
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↵5 D. L. Alexander and C. R. Jefcoate, unpublished results.
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↵6 Ü. Savas and C. R. Jefcoate, unpublished results.
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↵7 K. K. Bhattacharyya and C. R. Jefcoate, unpublished results.
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↵8 L. Zhang and J. B. Fagan, unpublished results.
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↵9 L. Zhang and C. R. Jefcoate, unpublished results.
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- Received February 18, 1997.
- Revision received November 26, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
