Assessing sites of NF-κB DNA binding using chromatin immunoprecipitation

Methods Mol Biol. 2015:1280:47-59. doi: 10.1007/978-1-4939-2422-6_4.

Abstract

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.

MeSH terms

  • Animals
  • Binding Sites*
  • Cell Line
  • Chromatin / genetics
  • Chromatin / metabolism
  • Chromatin Immunoprecipitation / methods*
  • DNA / genetics*
  • DNA / metabolism*
  • Immunoprecipitation / methods
  • Mice
  • NF-kappa B / metabolism*
  • Polymerase Chain Reaction / methods
  • Protein Binding

Substances

  • Chromatin
  • NF-kappa B
  • DNA