The molecular mechanism regulating 24-hour rhythm of CYP2E1 expression in the mouse liver

Cytochrome P450 2E1 (CYP2E1) is clinically and toxicologically important and exhibits 24-hour periodicity in its activity. In the present study, we investigated whether hepatic nuclear factor-1alpha (HNF-1alpha) and clock genes with a striking 24-hour rhythm in mouse liver contributed to the 24-hour regulation of CYP2E1 expression. The results demonstrated that the expression of CYP2E1 messenger RNA (mRNA) in the liver was affected by HNF-1alpha and the circadian organization of molecular clocks. The mRNA levels of CYP2E1 in the liver increased from the late light phase to the early dark phase. Luciferase reporter gene analysis revealed that HNF-1alpha activated CYP2E1 promoter activity, which was restricted by CRY1, a member of the circadian organization of molecular clocks. Repressor activity of CRY1 was observed on the HNF-1alpha binding site of the CYP2E1 promoter region with mutated E-box. Serum shock induced approximately 24-hour oscillation in CYP2E1 mRNA in HepG2. Transfection of HNF-1alpha and CRY1 small interfering RNA dampened the oscillation of CYP2E1 mRNA in HepG2. Chromatin immunoprecipitation assay in the CYP2E1 promoter indicated that HNF-1alpha binding to the CYP2E1 promoter increased from the late light phase to the early dark phase. Using the chromatin immunoprecipitation reimmunoprecipitation assay, time-dependent differences were demonstrated for CRY1 protein interaction with HNF-1alpha transcriptional complexes, including coactivator p300 on the HNF-1alpha binding site in the CYP2E1 promoter.

Our results suggest that the transcription activator of HNF-1alpha acts periodically and the negative limbs of molecular clocks periodically inhibit CYP2E1 transcription, resulting in the 24-hour rhythm of its mRNA expression.