Circadian Expression of the Steroid 15 α-Hydroxylase (Cyp2a4) and Coumarin 7-Hydroxylase (Cyp2a5) Genes in Mouse Liver Is Regulated by the PAR Leucine Zipper Transcription Factor DBP

Abstract

To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15α-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver.

ACKNOWLEDGMENTS

Luis Lopez-Molina and Daniel J. Lavery contributed equally to this study and should both be considered primary authors.

We are grateful to Nicolas Roggli for preparation of the artwork, to Frederic Bancel for purified P450 standards 2A4 and 2A5, and to Christian Larroque and Reinhard Lange for the anti-mouse P450 2A antibody.

This work was supported by the Swiss National Science Foundation, the State of Geneva, and Glaxo Wellcome Experimental Research, Geneva, Switzerland.