Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP

To study the molecular mechanisms of circadian gene expression, we have sou ght to identify genes whose expression in mouse liver is regulated by the t ranscription factor DBP (albumin D-site-binding protein). This PAR basic le ucine 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 15 alpha-hydroxylase, is a nove l circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone a nd estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displ ays circadian kinetics indistinguishable from those of the highly related C YP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also displ ay dairy variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprin ting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 ge nes are indeed regulated by DBP. These conclusions are corroborated by gene tic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs an d protein expression in the liver was significantly impaired in a mutant mo use strain homozygous for a dbp null allele. These experiments strongly sug gest that DBP is a major factor controlling circadian expression of the Cyp 2a4 and Cyp2a5 genes in the mouse liver.