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

Richard Ramsden, Nancy B. Beck, Karen M. Sommer, Curtis John Omiecinski

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 2.5 kb 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 -1700 bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800 bp 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 -2200 bp 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.

Original languageEnglish (US)
Pages (from-to)169-179
Number of pages11
JournalGene
Volume228
Issue number1-2
DOIs
StatePublished - Mar 4 1999

Fingerprint

5' Flanking Region
Phenobarbital
Transgenic Mice
NFI Transcription Factors
Genes
Mutation
Transcriptional Activation
DNA
Cytochromes
steroid 16-beta-hydroxylase
Transgenes
Reporter Genes
Immunoblotting
Proteins

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Ramsden, Richard ; Beck, Nancy B. ; Sommer, Karen M. ; Omiecinski, Curtis John. / Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice. In: Gene. 1999 ; Vol. 228, No. 1-2. pp. 169-179.
@article{bd5ba05ba72546dd9b22efe3cfcef7bd,
title = "Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice",
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 2.5 kb 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 -1700 bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800 bp 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 -2200 bp 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.",
author = "Richard Ramsden and Beck, {Nancy B.} and Sommer, {Karen M.} and Omiecinski, {Curtis John}",
year = "1999",
month = "3",
day = "4",
doi = "10.1016/S0378-1119(98)00612-X",
language = "English (US)",
volume = "228",
pages = "169--179",
journal = "Gene",
issn = "0378-1119",
publisher = "Elsevier",
number = "1-2",

}

Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice. / Ramsden, Richard; Beck, Nancy B.; Sommer, Karen M.; Omiecinski, Curtis John.

In: Gene, Vol. 228, No. 1-2, 04.03.1999, p. 169-179.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice

AU - Ramsden, Richard

AU - Beck, Nancy B.

AU - Sommer, Karen M.

AU - Omiecinski, Curtis John

PY - 1999/3/4

Y1 - 1999/3/4

N2 - 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 2.5 kb 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 -1700 bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800 bp 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 -2200 bp 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.

AB - 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 2.5 kb 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 -1700 bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800 bp 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 -2200 bp 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.

UR - http://www.scopus.com/inward/record.url?scp=0033522186&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033522186&partnerID=8YFLogxK

U2 - 10.1016/S0378-1119(98)00612-X

DO - 10.1016/S0378-1119(98)00612-X

M3 - Article

C2 - 10072770

AN - SCOPUS:0033522186

VL - 228

SP - 169

EP - 179

JO - Gene

JF - Gene

SN - 0378-1119

IS - 1-2

ER -