Assessing sites of NF-κB DNA binding using chromatin immunoprecipitation.
The NF-κB transcription factor is in fact a family of related proteins which dimerize to form at least 12 distinct complexes which regulate the expression of hundred of genes of importance to a range of physiological and pathological processes. The binding of NF-κB to the regulatory regions and promoters of target genes is influenced by a number of factors including the sequence of DNA-binding sites, the posttranslational modification of NF-κB, and the interaction of cofactors and co-regulators of transcription. In addition, the binding of NF-κB to promoters is highly dynamic and the recruitment of specific subunits to specific binding sites may occur with distinct kinetics. Moreover, genome-wide analysis of NF-κB chromatin binding indicates that the majority of DNA-binding events are not associated with changes in transcriptional activity. Thus, the analysis of NF-κB recruitment and activity at specific binding sites is of critical importance in understanding the regulation of transcription. In this chapter we describe a chromatin immunoprecipitation assay to investigate the in situ binding of NF-κB to specific sites in the genome.
Institute of Infection, Immunology and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Sir Graeme Davies Building, 120 University Place, Glasgow, G12 8TA, UK.
Amy
A
Colleran
Patricia E
PE
Collins
Ruaidhrí J
RJ
Carmody
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding
D002843:Chromatin; D016328:NF-kappa B; D004247:DNA