Protocol

Transcriptional Regulation

Volume 809 of the series Methods in Molecular Biology pp 121-134

Date:

Chromatin Immunoprecipitation Analysis of NFκB Transcriptional Regulation by Nuclear IκBα in Human Macrophages

  • Sitharam RamaswamiAffiliated withDepartment of Biological Sciences, St. John’s University
  • , Subrata MannaAffiliated withDepartment of Biological Sciences, St. John’s University
  • , Ashish JuvekarAffiliated withDepartment of Biological Sciences, St. John’s University
  • , Steven KennedyAffiliated withDepartment of Biological Sciences, St. John’s University
  • , Ales VancuraAffiliated withDepartment of Biological Sciences, St. John’s University
  • , Ivana VancurovaAffiliated withDepartment of Biological Sciences, St. John’s University Email author 

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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.

Key words

Chromatin immunoprecipitation IκBα Leptomycin B Macrophages NFκB Nuclear accumulation Nuclear transport Transcriptional regulation