title: Chromatin immunoprecipitation analysis of NFκB transcriptional regulation by nuclear IκBα in human macrophages.
abstract: Transcription factor NFκB comprises a family of proteins that serve as crucial regulators of genes involved in host immune and inflammatory responses, cell survival, proliferation, and differentiation. Since transcription of NFκB-dependent genes is increased in numerous inflammatory disorders as well as in many types of cancer and leukemia, inhibition of NFκB-dependent transcription thus represents an important therapeutic target. We have previously shown that in human leukocytes, transcription of NFκB-dependent genes is inhibited by the nuclear translocation and accumulation of IκBα, which can be induced by an inhibitor of CRM1-dependent nuclear export, leptomycin B (LMB). In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the regulation of NFκB recruitment to NFκB-dependent promoters by nuclear IκBα induced by LMB. We show that in lipopolysaccharide (LPS)-stimulated human U-937 macrophages, recruitment of NFκB p65 and p50 proteins to NFκB-dependent promoters of IκBα and cIAP2 genes is suppressed by the LMB-induced nuclear IκBα. Even though in this study we use U-937 macrophages, this protocol should be readily adaptable to analyze the regulation of NFκB recruitment by nuclear IκBα also in other cell types.
authors: Department of Biological Sciences, St. John's University, Queens, NY, USA.
authors: Sitharam
authors: S
authors: Ramaswami
authors: Subrata
authors: S
authors: Manna
authors: Ashish
authors: A
authors: Juvekar
authors: Steven
authors: S
authors: Kennedy
authors: Ales
authors: A
authors: Vancura
authors: Ivana
authors: I
authors: Vancurova
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism
chemical_list: D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha