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J. Biol. Chem., Vol. 279, Issue 29, 29902-29910, July 16, 2004

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CCAAT/Enhancer-binding Protein (C/EBP) Activates Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1) through the Interaction with DNA-bound NF-Y^*

Qin-Shi Zhu, Bin Qian, and Daniel Levy

From the Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033

Microsomal epoxide hydrolase (mEH) plays a central role in xenobiotic metabolism as well as mediating the sodium-dependent uptake of bile acids into the liver, where these compounds regulate numerous biological processes such as cholesterol metabolism and hepatocyte signaling pathways. Little is known, however, about the factors that control the constitutive and inducible expression of the mEH gene (EPHX1) that is altered during development and in response to numerous xenobiotics. In previous studies we have established that GATA-4 binding to the EPHX1 core promoter is critical for EPHX1 expression. The –80/+25 bp core promoter also contained a reversed CCAAT box (–5/–1 bp), integrity of which was required for maximal basal EPHX1 transcription in HepG2 cells. Transient transfection of CCAAT/enhancer-binding protein (C/EBP) substantially stimulated EPHX1 promoter activity. Electrophoretic mobility shift assays, however, revealed that nuclear factor Y (NF-Y), but not C/EBP, directly bound to this site although increased expression of NF-Y had no effect on EPHX1 promoter activity. These results suggested that C/EBP activated EPHX1 expression through its interaction with NF-Y bound to the CCAAT box. The existence of a C/EBP[NF-Y] complex was supported by electrophoretic mobility shift assays using antibodies against NF-Y and C/EBP as well as by the ability of a dominant-negative NF-Y expression vector to inhibit promoter activity. The interaction between these transcription factors was established by co-immunoprecipitation analysis and glutathione S-transferase pull-down assays, whereas the association of the two factors and the interaction of NF-Y with the CCAAT box in vivo was confirmed by chromatin immunoprecipitation assays. C/EBP-dependent EPHX1 activation was also supported by reconstitution studies in HeLa cells that lack this protein. These results establish that EPHX1 expression is regulated by C/EBP interacting with DNA-bound NF-Y.

Received for publication, January 15, 2004 , and in revised form, May 10, 2004.

^* This work was supported by Grant R01 DK 25836 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 2011 Zonal Ave., Los Angeles, CA 90033. Tel.: 323-442-1525; Fax: 323-442-1224; E-mail: dlevy{at}usc.edu.

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