Regulation of cytochrome P4502B2 gene expression

Biochemical SocietyTransactions ( 1 994) 22

125s I

Regulation of cytochrome P4502B2 gene expression LESLEY A. FORREST, AMAL SHERVINGTON, IAN R. PHILLIPS* and ELIZABETH A. SHEPHARD

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Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, U.K. and *Departmentof Biochemistry, Queen Mary and Westfield College, University of London, Mile h d Road, London El 4NS, U.K. Many cytochromes P450 (CYPs) are selectively inducible by the compounds that they metabolize. For instance, two members of the CYP2B subfamily, namely CYP2Bl and CYP2B2, are induced in mammalian liver by the barbiturate, phenobarbital [l31. The induction of these proteins is mediated by an increase in the corresponding mRNAs [2, 4-71, which in turn is due to an increase in the transcription of CYP2B genes I7-91. Because of the lack of a suitable phenobarbital-responsivecell-line we have taken an indirect approach to identify potential CYP2B2 gene regulatory sequences via their ability to interact with trans-acting nuclear proteins. DNA fragments spanning -1390 to +160 of a novel CYP2B2 gene were used as probes in gel retardation assays [ 101of nuclear protein extracts from the livers of phenobarbital-treated and untreated rats. Many protein-binding sites were identified, the majority of which bound proteins that were present in equal abundance in extracts from the livers of phenobarbital-treated or untreated rats. However, two fragments of the promoter, -3 1/-177 and -178/-364, were found to contain binding sites for protein(s) whose abundance or activity was greater in extracts from phenobarbital-treated,than from untreated, animals. To localize more precisely the binding sites for proteins that were enriched or activated in response to phenobarbital, subfragments of the two regions were used as probes or competitors in gel retardation assays. In this way, the distal binding site was found to be located between -178 and -217 (Fig. 1A), and the proximal site between -3 1 and -85 (Fig. 1B). The relative abundance, within liver nuclear extracts from phenobarbital-treated and untreated rats, of the proteins that interacted with these binding sites was investigated by incubating each DNA fragment with various amounts of the protein extracts. Analysis of the resulting autoradiograms by image densitometry revealed that the DNA-protein complexes formed with the - 1781217 and -31/-85 fragments were 4.0-fold and 3.8-fold more abundant, respectively, with extracts from phenobarbital-treated, than with those from untreated, rats. A double-stranded synthetic oligonucleotide specifying the sequence between -183 and -199, when used as a competitor in gel retardation assays, completely inhibited the binding of nuclear protein(s) to the -178/-217 fragment. When used as a probe, this 17 bp-oligonucleotide formed a DNA-protein complex that was about 4-fold more abundant with liver nuclear extracts from phenobarbital- treated rats compared with those from untreated animals. Thus, the DNA sequences responsible for binding of a phenobarbital-enrichedor activated protein to fragment - 1781-368 of the promoter are localized to a 17 bp-region between - 183 and 199. Gel retardation assays using fragments -3 1/45 and - 1781-217 as competitors showed that the binding of protein(s) to each fragment was competed by a 200-fold molar excess of unlabelled homologous DNA but not by a similar excess of the heterologous fragment, demonstrating that the two fragments interact with different nuclear proteins. Analysis of the CYP2B2 gene promoter by DNase footprinting revealed the presence, on the sense strand, of several protein binding sites. Two of the footprints were more prominent with nuclear protein extracts from phenobarbital-treated than from untreated rats, and their positions corresponded closely to those of the sequences identified by gel retardation as binding proteins enriched or activated in response to phenobarbital. On the template strand of the promoter, a very large footprint, extending from about -250 to -6. was obtained with extracts from phenobarbitdtreated rats but not with those from untreated animals. The first part of the footprint to develop, between -173 and -198, corresponds closely to the position of one of the sites identified by gel retardation as binding a phenobarbital-enriched protein. Thus, the binding of a protein to this site may be responsible for recruiting other regulatory proteins to adjacent regions of the

Fig. 1. Analysis of a CYP2B2 promoter by gel retardation. Radiolabelled DNA fragments -178/-217 (A) and -311-85 (B) were incubated with 5 pg of liver nuclear protein isolated from untreated (v) or phenobarbital-treated (PB) animals. promoter, hence giving rise to the large footpnnt. Although the identities of the transcription factors that bind to regions -183 to -199 and -31 to -85 of the CYP2B2 promoter remain to be established, the former region contains an 8 bp-long sequence which differs by only a single nucleotide from the consensus binding site for members of the octamer class of POUdomain regulatory proteins [ 1I], and computer analysis of the -31 to -85 region revealed the presence of a sequence with similarity to a C/EBP binding site [ 121. In conclusion, we have detected, by gel retardation and DNase footprinting, the presence within a CYP2B2 gene promoter of two DNA sequences which bind proteins that are more abundant or more active in liver nuclear extracts from phenobarbital-treated than from untreated rats. These sequences and their cognate binding proteins may well be involved in mediating the induction of expression of CYP2B2 genes in response to phenobarbital. This work was supported by a grant from the Cancer Research Campaign. 1. Thomas, P. E., Reik, D. E., Ryan, D. E.& Levin, W. (1981) J.

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