Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins

• Published in: Biochemical pharmacology Vol. 51; no. 3; pp. 345 - 356
• Main Authors: Luc, Phuong-Van T., Adesnik, Milton, Ganguly, Sabya, Shaw, Peter M.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 09.02.1996; Elsevier Science
• Subjects: Animals; Aryl Hydrocarbon Hydroxylases; Binding Sites; Biological and medical sciences; C/EBPα; CCAAT-Enhancer-Binding Proteins; Cell Nucleus - enzymology; CYP2B1; CYP2B2; Cytochrome P-450 Enzyme System - genetics; cytochrome P450; Deoxyribonuclease I; DNA Footprinting; DNA-Binding Proteins - pharmacology; Enzymes. Coenzymes. Vitamins. Pigments; Fundamental and applied biological sciences. Psychology; gene; Liver - enzymology; Liver - ultrastructure; Metabolisms and neurohumoral controls; Nuclear Proteins - pharmacology; Oligonucleotides - pharmacology; Phenobarbital; Plasmids; promote; Promoter Regions, Genetic; Rats; Steroid Hydroxylases - genetics; transcription; Transcription, Genetic - drug effects; Transfection; Vertebrates: anatomy and physiology, studies on body, several organs or systems; C/EBPα; C/EBP; transcription; SRE; FT; ds; promote; EMSA; CYP2B2; DSE; CYP2B1; and TE; CYP; IL; gene; AH; ARNT; cytochrome P450; PIPES; CMV; PB; STAT; PMSF; CAT; TAE; PCR; Promoter; Regulation(control); Gene; Transcription; Cytochrome P450; Gene expression; In vitro
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/0006-2952(95)02190-6

Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 ( CYP2B) genes. One site, which maps to a region in the 5′-flanking region between −2.2 and −2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between −0.05 and −0.15 kb. DNase 1 footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between −42 and −66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBPα bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBPα could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBPα.