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

本研究检测了雌性小鼠肝脏中Cyp3a基因组成表达的周期性变化。在检测的5个小鼠Cyp3a基因中，一些在肝脏中特异性表达雌性的基因表现出动情周期和时间依赖性调节。Cyp3a41和Cyp3a16基因在阴道发情期的表达量是发情前期的2-3倍。这两个基因在午夜的表达量都是正午的2倍，并且与富肝核转录因子HNF4alpha的变化有很好的相关性。利用原代培养的雌性小鼠肝细胞和Cyp3a41基因的5'侧区进行报告基因检测表明，参与组成型和糖皮质激素诱导表达的元件位于-1631+61区域。在该区域发现了一个预测的HNF4alpha结合序列，该位点的突变导致糖皮质激素诱导表达和组成表达的减少。为了证实这一结果，使用流体动力DNA递送进行了体内报告基因检测。结果，在雌性小鼠中观察到-1631+61区域驱动的报告活性，而在雄性小鼠中没有。预测HNF4alpha结合位点的突变完全抑制了报告基因的活性。将HNF4alpha的表达质粒转染到肝细胞后，Cyp3a41基因得以表达，且在女性肝细胞中表达更为明显，同时用合成糖皮质激素地塞米松处理可增强Cyp3a41基因的表达。ChIP实验表明，HNF4alpha与Cyp3a41基因启动子结合，在女性肝细胞中更为显著。综上所述，本研究揭示了HNF4 α结合Cyp3a41基因启动子近端区域激活转录，HNF4 α可能是导致Cyp3a41基因表达昼夜调节的一个因素。

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.