Identification of sequences involved in both basal expression and phenobarbital induction of a CYP2B2 gene using in vitro transcription system.

Cytochrome P450 2B2 and 2B1 are induced in mammalian liver in response to barbiturates such as phenobarbital. The induction of the proteins is mediated at the transcriptional level. Owing to the lack of a suitable phenobarbital-responsive cell line to investigate the molecular mechanism responsible for the induction, we adopted an alternative approach involving the use of in vitro transcription system, DNA constructs containing a G-free cassette under the control of various section of the 5' flanking sequence of rat CYP2B2 gene were transcribed in nuclear protein extracts isolated from the livers of phenobarbital-treated or untreated rats. The results of these experiments indicated the presence within the CYP2B2 promoter of two positive DNA elements, one located between -178 and -368 and the other between -368 and -984, that are involved in regulating basal transcription of the gene. In addition, a negative regulatory element is present between -2880 and -5600. A DNA sequence involved in regulating the induction of expression of the gene in response to phenobarbital is located between -178 and -368. The use of synthetic oligonucleotides as competitor in vitro transcription assays indicated the importance for transcriptional control of the CYP2B2 gene of sequences located between -183 and -199 and -31 and -72 that were identified previously [1] as binding rat liver nuclear proteins that are enriched or activated in vivo by phenobarbital.