Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.
Gene
; 228(1-2): 169-79, 1999 Mar 04.
Article
em En
| MEDLINE
| ID: mdl-10072770
ABSTRACT
Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between -2500 and -1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type -2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA-protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fenobarbital
/
Esteroide Hidroxilases
/
Fatores de Transcrição
/
DNA
/
Hidrocarboneto de Aril Hidroxilases
/
Proteínas Estimuladoras de Ligação a CCAAT
/
Sistema Enzimático do Citocromo P-450
Tipo de estudo:
Prognostic_studies
Limite:
Animals
/
Female
/
Humans
/
Male
Idioma:
En
Revista:
Gene
Ano de publicação:
1999
Tipo de documento:
Article
País de afiliação:
Estados Unidos