Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation

David E. Nowak, Bing Tian, Allan R. Brasier

Research output: Contribution to journalArticle

184 Citations (Scopus)

Abstract

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor KB (NF-κB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF- KB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-κB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-κB family binding to a variety of validated NF-κB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

Original languageEnglish (US)
Pages (from-to)715-724
Number of pages10
JournalBioTechniques
Volume39
Issue number5
DOIs
StatePublished - 2005

Fingerprint

Gene Regulatory Networks
Chromatin Immunoprecipitation
Chromatin
Genes
DNA
Transcription Factors
DNA-Binding Proteins
Proteins
Formaldehyde
Assays
Cytokines
STAT3 Transcription Factor
Pathogens
Antigen-Antibody Complex
Cellular Immunity
Esters
Inflammation

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Two-step cross-linking method for identification of NF-κB gene network by chromatin immunoprecipitation. / Nowak, David E.; Tian, Bing; Brasier, Allan R.

In: BioTechniques, Vol. 39, No. 5, 2005, p. 715-724.

Research output: Contribution to journalArticle

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