MULTIPLE, FUNCTIONAL DBP SITES ON THE PROMOTER OF THE CHOLESTEROL 7-ALPHA-HYDROXYLASE P450 GENE, CYP7 - PROPOSED ROLE IN DIURNAL REGULATIONOF LIVER GENE-EXPRESSION

Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regu lated in vivo at the protein and the mRNA level in response to multipl e physiological factors, including liver cholesterol synthesis, bile a cid feedback inhibition, and diurnal rhythm. In the present study we i nvestigated whether the liver transcription factor DBP (albumin promot er D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning similar to 100:1), con tributes to the diurnal regulation of CYP7 gene expression. DNase I fo otprinting analysis using bacterially expressed DEP and a cloned 5'-fl anking DNA segment of the rat CYP7 gene revealed five distinct DBP-bin ding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter- chloramphenicol acetyltransferase reporter was activated up to 12-fold upon cotransfection of a DBP expression vector, whereas an HNF-1 alph a expression vector did not stimulate CYP7 gene activity. 5'-Deletion analyses and site-specific mutagenesis revealed that this stimulating effect of DBP can in part be ascribed to its functional interaction wi th DBP binding sites B (nts -115/-125), C (nts -172/-195), and D (nts -214/-230). C/EBP beta (LAP), another liver enriched basic-leucine zip per transcription factor, bound to these same sites but effected a mor e modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes less than or equal to 2-fold diur nal change in rat liver, and the diurnally regulated DBP is proposed t o determine the relative rates of basal versus diurnally regulated CYP 7 gene transcription and thus may be a primary mechanism for setting t he 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhance d expression and the onset of the diurnal pattern of CYP7 enzyme level s at this stage of development.