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Experimental Approaches for the Study of Cytochrome P450 Gene Regulation

  • Hollie I. Swanson
  • Susan D. Kraner
  • Soma S. Ray
  • Martin Hoagland
  • Earl D. Thompson
  • Xinyu Zheng
  • Yanan Tian
Protocol
Part of the Methods in Pharmacology and Toxicology book series (MIPT)

Abstract

This chapter uses cytochrome P450 1A1 (CYP1A1) as a model “xenobiotic inducible” gene and the aryl hydrocarbon receptor (AHR) as a regulator of this gene product to illustrate how studies may be performed to understand the relationships between the regulator protein and its target genes. The methodology described was developed to study transcriptional activation. Because transcriptional repression occurs nearly as frequently as activation, the assays described may require modifications to accommodate both mechanisms. Approaches typically used to determine whether changes in gene expression induced by a particular drug or xenobiotic are a result of an increase in gene transcription mediated by specific protein/DNA interactions are described. These approaches and assays include the use of inhibitors of transcription and translation, nuclear runoffs, promoter analysis, and reporter assays to determine the role of specific DNA sequences, determination of specific DNA–protein interactions both in vitro and in vivo using electrophoretic gel mobility shift assays, UV-crosslinking, Southwestern blotting, yeast one-hybrid screening, DNA footprinting, site affinity amplification binding, and the chromatin immunoprecipitation (ChIP) assay, and studies to determine functional roles for specific transcription factors.

Key Words

Aryl hydrocarbon receptor aryl hydrocarbon receptor nuclear translocator chromatin immunoprecipitation assay CYP1A1 cytochrome P450 dioxin responsive element DNA footprinting DNA microarrays electromobility shift assays gene regulation luciferase activity nuclear runoff promoter analysis protein–DNA interactions reporter analysis reverse transcription-polymerase chain reaction site-affinity amplification binding small inhibitory RNA Southwestern blot, 2,3,7,8-tetrachlorodibenzo-p-dioxin transfection transient transfection stable transfection viral UV-crosslinking yeast one-hybrid screening 

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Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Hollie I. Swanson
    • 1
  • Susan D. Kraner
    • 1
  • Soma S. Ray
    • 2
  • Martin Hoagland
    • 2
  • Earl D. Thompson
    • 1
  • Xinyu Zheng
    • 1
  • Yanan Tian
    • 3
  1. 1.Department of Molecular and Biomedical PharmacologyUniversity of Kentucky Medical CenterLexington
  2. 2.University of Kentucky Medical CenterLexington
  3. 3.Department of Veterinary Physiology and PharmacologyTexas A & M UniversityCollege Station

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