Study of periodic change of the expression of constitutively expressing drug-metabolizing cytochrome P450 genes

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In this Study, periodical change of the constitutive expression of Cyp3a genes in the liver of female mice was examined. Among five mouse Cyp3a genes examined, some genes expressing female-specifically in the liver showed estrous cycle- and time- dependent modulation. Expression of Cyp3a41 and Cyp3a16 genes in vaginal estrus is 2-3-fold higher than that in proestrus. Expression of both genes in midnight was 2-fold higher than that in noon, and it was well correlated to the change of HNF4alpha, one of liver-enriched nuclear transcription factors. Reporter gene assay using female mouse hepatocytes in primary culture and 5'-flanking region of the Cyp3a41 gene indicated that the element involved in constitutive- and glucocorticoid hormone-inducible expression locates within -1631+61 region. In this region, one predictive HNF4alpha binding sequence was found, and mutation of the site caused reduction of glucocorticoid hormone-inducible expression as well as constitutive expression In order to confirm this result, in vivo reporter gene assay using the hydrodynamic DNA delivery was carried out. As a result, reporter activity driven from -1631+61 region was observed in female mice, and not in male mice. Mutation at the predictive HNF4alpha binding site completely suppressed reporter gene activity. Transfection of the expression plasmid for HNF4alpha into hepatocytes caused expression of Cyp3a41 gene, more prominently in female hepatocytes, and this expression was enhanced by simultaneous treatment with synthetic glucocorticoid hormone dexamethasone. ChIP assay indicated that HNF4alpha bound to the Cyp3a41 gene promoter, more remarkable in female hepatocytes. In conclusion, this study revealed that HNF4 alpha binds to proximal region of the Cyp3a41 gene promoter to activate transcription, and HNF4 alpha might be a factor to cause circadian modulation of Cyp3a41 gene expression.