Characterization of phenobarbital-inducible mouse Cyp2b10 gene transcription in primary hepatocytes.
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.
Honkakoski
P
P
Pharmacogenectics Section, Laboratory of Reproductive and Development Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Moore
R
R
Gynther
J
J
Negishi
M
M
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007438:Introns; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics*; D010634:Phenobarbital / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D016385:TATA Box; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D017931:DNA Primers; D011725:Pyridines; D011994:Recombinant Proteins; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D015500:Chloramphenicol O-Acetyltransferase; D010634:Phenobarbital