Abstract
The mouse phenobarbital (PB)-inducible Cyp2b10 gene promoter has been isolated and sequenced, and control of its expression has been characterized. The 1405-base pair (bp) Cyp2bl0 promoter sequence is 83% identical to the corresponding region from the rat CYP2B2 gene. In addition to the lack of CA repeats, differences include insertion of 42 base pairs (-123/-82 bp) into the middle of a consensus sequence to the so-called "Barbie box." In this report, we have developed a primary mouse hepatocyte culture system in which endogenous 2B10 mRNA as well as Cyp2b10-driven CAT activity were induced by PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), but not by the 3-chloro derivative of TCPOBOP. Deletion analysis of the Cyp2b10 promoter identified a basal transcription element at -64/-34 bp and a negative element at -971/-775 bp. Sequences contained within the -1404/-971 bp region are responsible for the induced CAT activity. DNase I protection and gel shift assays detected five major protein binding sites within the -1404/-971 bp fragment, one of which shared high sequence identity with a portion of a regulatory element in CYP2B2 gene (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene 158, 263-268). Our results indicate that sequences important for PB-induced transcription of Cyp2b10 gene are located in the distal promoter.
Highlights
Many P450 genes are induced by exogenous chemicals including drugs, environmental toxins, and carcinogens
As for acid glycoprotein (AGP) gene, the kinetics of PB induction are much slower than for CYP2B (Fournier et al, 1994), other studies do not demonstrate any protein binding to Barbie boxlike sequences in rat or mouse AGP promoter (Ratajczak et al, 1992; Lee et al, 1993), and basal and dexamethasone-dependent expression are governed by another elements at Ϫ155/143 and Ϫ120/Ϫ105 bp (Ingrassia et al, 1994)
The other 21 genomic clones did not contain any portions of the Cyp2b10 gene, and they could be classified into seven groups, confirming the large size of Cyp2b gene family (Lakso et al, 1991)
Summary
Vol 271, No 16, Issue of April 19, pp. 9746 –9753, 1996 Printed in U.S.A. Characterization of Phenobarbital-inducible Mouse Cyp2b10 Gene Transcription in Primary Hepatocytes*. As for AGP gene, the kinetics of PB induction are much slower than for CYP2B (Fournier et al, 1994), other studies do not demonstrate any protein binding to Barbie boxlike sequences in rat or mouse AGP promoter (Ratajczak et al, 1992; Lee et al, 1993), and basal and dexamethasone-dependent expression are governed by another elements at Ϫ155/143 and Ϫ120/Ϫ105 bp (Ingrassia et al, 1994). These conflicting data indicate that the role of Barbie box in CYP2B regulation is not yet resolved.
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