Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice

Gene. 1999 Mar 4;228(1-2):169-79. doi: 10.1016/s0378-1119(98)00612-x.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aryl Hydrocarbon Hydroxylases*
  • Binding Sites / genetics
  • CCAAT-Enhancer-Binding Proteins*
  • Chloramphenicol O-Acetyltransferase / genetics
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Cytochrome P-450 Enzyme System / genetics*
  • DNA / drug effects*
  • DNA / genetics
  • DNA / metabolism
  • DNA Footprinting
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Deoxyribonuclease I
  • Female
  • Gene Expression Regulation / drug effects
  • Growth Hormone / genetics
  • Humans
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Mutation
  • NFI Transcription Factors
  • Nuclear Proteins
  • Phenobarbital / pharmacology*
  • Protein Binding
  • Rats
  • Rats, Sprague-Dawley
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Steroid Hydroxylases / genetics*
  • Transcription Factors*
  • Y-Box-Binding Protein 1


  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • NFI Transcription Factors
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Y-Box-Binding Protein 1
  • YBX1 protein, human
  • Growth Hormone
  • DNA
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Aryl Hydrocarbon Hydroxylases
  • steroid 16-beta-hydroxylase
  • Chloramphenicol O-Acetyltransferase
  • Deoxyribonuclease I
  • Phenobarbital