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 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 15alpha-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.
Lavery
D J
DJ
Glaxo Wellcome Experimental Research, Institut de Biologie Cellulaire et de Morphologie, Université de Lausanne, CH1005 Lausanne, Switzerland.
Lopez-Molina
L
L
Margueron
R
R
Fleury-Olela
F
F
Conquet
F
F
Schibler
U
U
Bonfils
C
C
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002940:Circadian Rhythm; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D015971:Gene Expression Regulation, Enzymologic; D006720:Homozygote; D006801:Humans; D016350:Leucine Zippers; D051379:Mice; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
C063898:DBP protein, human; D004268:DNA-Binding Proteins; C498260:Dbp protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C460223:Cyp2a4 protein, mouse; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2; C050356:steroid 15-alpha-hydroxylase