Enhancer elements in the mouse Cypla2 gene for constitutive expression

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Abstract

CYP1A2 is one of the major hepatic cytochrome P450s that is involved in the metabolism of many drugs, as well as in the activation of chemical carcinogens. To elucidate the transcriptional regulation of the constitutive expression of the mouse Cypla2 gene, the 4.8-kbp 5-flanking region of the gene was analyzed for transcriptional activity using a primary cultured mouse hepatocyte system. With 5- and 3-deletion analysis, two enhancer elements, i.e., a 20-bp DNA fragment (E1) from −4401 to −4382 and a 9-bp (E2) from −4300 to −4292, were identified. E1 and E2 contain a phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and TRE-like element, respectively. Electrophoretic mobility shift assay confirmed specific binding between these two enhancer elements and nuclear proteins. Site-directed mutagenesis assay suggested that the TRE element in E1 is essential for constitutive expression of the mouse Cypla2 gene.

Section snippets

Materials and methods

Chemicals. Materials for culturing hepatocytes were purchased from Wako Pure Chemical Industries (Osaka, Japan), Gibco BRL (Grand Island, NY), and Sigma Chemical (St. Louis, MO). Percoll was obtained from Amersham Pharmacia Biotech (Uppsala, Sweden). TaKaRa LA-Taq and native Pfu DNA polymerase were from TaKaRa Biochemicals (Shiga, Japan) and Stratagene (La Jolla, CA), respectively. BigDye terminator cycle sequencing ready reaction kit was from Perkin–Elmer Applied Biosystems (Foster city, CA)

Results and discussion

To investigate the transcriptional activity of the isolated mouse Cyp1a2 gene, we constructed a reporter gene (−4795/+27Luc) fusing 4.8 kbp of the 5-flanking region of mouse Cypla2 with the 5-end of the luciferase structure gene. This reporter gene was transiently transfected into mouse hepatocytes by electroporation, showing that −4795/+27Luc had prominent transcriptional activity. To further identify the regulatory region in the 5-flanking 4.8 kbp sequence, various 5-deletion mutants were

Acknowledgements

This work was partly supported by Grants-in-Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and from the Smoking Research Foundation.

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    Abbreviations: AhR, aryl hydrocarbon receptor; Arnt, AhR nuclear translocator; AP-1, activator protein 1; (k)bp, (kilo)base pairs; EMSA, electrophoretic mobility shift assay; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, phorbol-12-O-tetradecanoate-13-acetate; TRE, TPA-responsive element; cTRE, consensus TRE; XRE, xenobiotic-responsive element.

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