title: Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.
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 kappaB (NF-kappaB) 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-kappaB 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-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-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.
authors.affiliation: The University of Texas Medical Branch, Galveston 77555-1060, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Nowak
authors.forename: Bing
authors.initials: B
authors.lastname: Tian
authors.forename: Allan R
authors.initials: AR
authors.lastname: Brasier
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000737:chemistry; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000032:analysis / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D007107:Immune System; D047468:Immunoprecipitation; D007249:Inflammation; D007700:Kinetics; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D013388:Succinimides / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C498143:RELA protein, human; D050796:STAT3 Transcription Factor; D013388:Succinimides; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; C001426:N-hydroxysuccinimide