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Received for publication October 19, 2004.
Revised March 17, 2005.
Accepted for publication March 18, 2005.
CCAAT/enhancer-binding proteins (C/EBPs) are key transcription factors involved in the constitutive expression of several cytochrome P450 genes in the liver. Their concentration and activity change in several pathophysiological conditions. For instance, during inflammation, released cytokines induce repressive C/EBP
-LIP, which antagonizes constitutive C/EBP transactivators (C/EBP
and C/EBP-LAP), down-regulating genes such as CYP3A4. However, the mechanism by which hepatic C/EBP factors modulate transcription of the CYP3A4 gene is not known. To elucidate the mechanism of action, we cotransfected luciferase reporter vectors, containing 5'-flanking deletions of the CYP3A4 gene, along with expression vectors for C/EBP-LAP, C/EBP-LIP and C/EBP, in hepatic (HepG2) and non-hepatic (HeLa) cells. Analysis of the -3557 to -6954 bp region demonstrated the existence of a 288 bp sequence at -5.95 kb, which showed maximal response to C/EBP-LAP (
30 fold increase in HepG2 cells). Co-expression of LAP with increasing amounts of LIP reduced the activating effect by ca. 70%. Site-directed mutagenesis of predicted C/EBP binding sites demonstrated the presence of four functional C/EBP responsive motifs within this distal flanking region. Further experiments using chromatin immunoprecipitation proved the binding of endogenous C/EBP to the -5.95 kb enhancer of the CYP3A4 gene in human hepatocytes. Expression of recombinant LAP and LIP by means of adenoviral vectors resulted in their binding to this region, which was followed by activation/repression of CYP3A4. Taken together, our results uncover a new distal enhancer site in the CYP3A4 gene where C/EBP-LAP binds and activates transcription while the truncated form, C/EBP-LIP, antagonizes LAP activity and causes gene repression.
Key words:
DNA binding sites, Promoter analysis, Regulation of gene expression, Cytochrome P450, Regulation - transcriptional, Regulation - xenobiotic
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