SpringerProtocols: Abstract: Experimental Approaches for the Study of Cytochrome P450 Gene Regulation

Experimental Approaches for the Study of Cytochrome P450 Gene Regulation

By: Hollie I. Swanson², Susan D. Kraner², Soma S. Ray³, Martin Hoagland³, Earl D. Thompson², Xinyu Zheng², Yanan Tian⁴

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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.

Affiliation(s): (2) Department of Molecular and Biomedical Pharmacology, University of Kentucky Medical Center, Lexington, KY
(3) University of Kentucky Medical Center, Lexington, KY
(4) Department of Veterinary Physiology and Pharmacology, Texas A & M University, College Station, TX

Book Title: Drug Metabolism and Transport: Molecular Methods and Mechanisms

Series: Methods in Pharmacology and Toxicology | Pub. Date: Sep-24-2004 | Page Range: 19-44 | DOI: 10.1385/1-59259-832-3:017

Subject: Pharmacology/Toxicology

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

References

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