Nuclear Receptor Target Gene Discovery Using High-Throughput Chromatin Immunoprecipitation

Nuclear receptors are master transcription factors that regulate the development, physiology, and homeostasis of whole organisms through the direct control of gene expression in response to diverse ligands and hormonal stimuli. Nuclear receptors regulate the expression of their target genes through...

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Veröffentlicht in:Methods in Enzymology 2003, Vol.364, p.339-350
Hauptverfasser: Laganière, Josée, Deblois, Geneviève, Giguère, Vincent
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Deblois, Geneviève
Giguère, Vincent
description Nuclear receptors are master transcription factors that regulate the development, physiology, and homeostasis of whole organisms through the direct control of gene expression in response to diverse ligands and hormonal stimuli. Nuclear receptors regulate the expression of their target genes through association with specific DNA regulatory elements. While a significant number of nuclear receptor target genes have been identified to date, it is believed that these genes represent only a small fraction of the regulatory units likely to be under the control of nuclear receptors. Most nuclear receptor target genes identified so far were characterized through ‘‘gene oriented’’ approaches that study the regulation of one candidate gene at a time, and these studies are usually limited to the promoter region. To understand the complex nuclear receptor-driven transcriptional networks that operate in a living organism, a whole genome approach is required. This chapter describes a powerful ‘‘nuclear receptor/whole genome-oriented’’ approach to identify and more accurately study nuclear receptor regulatory networks. Prior to performing the actual Chromatin Immunoprecipitation (ChIP), it is important to remove and freeze an aliquot of the diluted fragmented chromatin corresponding to 10% of the total amount used for one IP. The development of high-throughput ChIP technology now permits a whole-genome analysis of gene regulation by nuclear receptors. The efficient cloning of new regulatory elements harboring high-affinity-binding sites for specific nuclear receptors can now be efficiently achieved, and allows for the identification of new target genes within a particular cell context.
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subjects Cell Line, Tumor
Chromatin - metabolism
Cloning, Molecular - methods
Genetic Techniques
Genome, Human
Humans
Precipitin Tests
Receptors, Cytoplasmic and Nuclear - genetics
Reverse Transcriptase Polymerase Chain Reaction
Time Factors
title Nuclear Receptor Target Gene Discovery Using High-Throughput Chromatin Immunoprecipitation
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