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.
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References
Baeuerle, P. A. and Baltimore, D. (1996) NFκB: ten years after. Cell 87, 13–20.
Baldwin, A. S. (2001) The transcription factor NFκB and human disease. J. Clin. Invest. 107, 3–6.
Yamamoto, Y. and Gaynor, R. B. (2001) Therapeutic potential of inhibition of the NFκB pathway in the treatment of inflammation and cancer. J. Clin. Invest. 107. 135–142.
Ghosh, S. and Karin, M. (2002) Missing pieces in the NFκB puzzle. Cell 109, S81–96.
Wan, F. and Lenardo, M. J. (2010) The nuclear signaling of NFκB: current knowledge, new insights, and future perspectives. Cell Res. 20, 24–33.
Prasad, S., Ravindran, J. and Aggarwal, B. B. (2010) NFκB and cancer: how intimate is the relationship. Mol. Cell. Biochem. 336, 25–37.
Li, Q., Withoff, S. and Verma, I. M. (2005) Inflammation-associated cancer: NFκB is the lynchpin. Trends Immunol. 26, 318–325.
Grivennikov, S. I., Greten, F. R. and Karin, M. (2010) Immunity, inflammation, and cancer. Cell 140, 883–899.
Ghosh, S. and Hayden, M. S. (2008) New regulators of NFκB in inflammation. Nature Rev. Immunol. 8, 837–848.
Fan, Y., Dutta, J., Gupta, N., Fan, G. and Gélinas, C. (2008) Regulation of programmed cell death by NFκB and its role in tumorigenesis and therapy. Adv. Exp. Med. Biol. 615, 223–250.
Arenzana-Seisdedos, F., Thompson, J., Rodriguez, M. S., Bachelerie, F., Thomas, D. and Hay, R. T. (1995) Inducible nuclear expression of newly synthesized IκBα negatively regulates DNA-binding and transcriptional activities of NF-κB. Mol. Cell Biol. 15, 2689–2696.
Arenzana-Seisdedos, F., Turpin, P., Rodriguez, M., Thomas, D., Hay, R. T., Virelizier, J. L. and Dargemont, C. (1997) Nuclear localization of IκBα promotes active transport of NF-κB from the nucleus to the cytoplasm. J. Cell Sci. 110, 369–378.
Turpin, P., Hay, R. T. and Dargemont, C. (1999) Characterization of IκBα nuclear import pathway. J. Biol. Chem. 274, 6804–6812.
Rodriguez, M., Thompson, J., Hay, R. T. and Dargemont, C. (1999) Nuclear retention of IκBα protects it from signal-induced degradation and inhibits NF-κB transcriptional activation. J. Biol. Chem. 274, 9108–9115.
Kunsch, C., Ruben, S. M. and Rosen, C. A. (1992) Selection of optimal kappa B/Rel DNA-binding motifs: interaction of both subunits of NFκB with DNA is required for transcriptional activation. Mol. Cell Biol. 12, 4412–4421.
Lin, R., Gewert, D. and Hiscott, J. (1995) Differential transcriptional activation in vitro by NFκB/Rel proteins. J. Biol. Chem. 270, 3123–3131.
Udalova, I. A., Mott, R., Field, D. and Kwiatkowski, D. (2002) Quantitative prediction of NFκB DNA-protein interactions. Proc. Natl. Acad. Sci.USA 99, 8167–8172.
Saccani, S., Pantano, S. and Natoli, G. (2001) Two waves of NFκB recruitment to target promoters. J. Exp. Med. 193, 1351–1359.
Saccani, S., Pantano, S. and Natoli, G. (2003) Modulation of NFκB activity by exchange of dimers. Mol. Cell 11, 1563–1574.
Saccani, S., Marazzi, I., Beg, A. A. and Natoli, G. (2004) Degradation of promoter-bound p65/RelA is essential for the prompt termination of the NFκB response. J. Exp. Med. 200, 107–113.
Vancurova, I., Miskolci, V. and Davidson, D. (2001) NF-κB activation in TNF-stimulated neutrophils is mediated by protein kinase C-δ: Correlation to nuclear IκBα. J. Biol. Chem. 276, 19746–19752.
Castro, S., Miskolci, V., Kalasapudi, B., Davidson, D. and Vancurova, I. (2002) NFκB regulation in human neutrophils by nuclear IκBα: Correlation to apoptosis. J. Immunol. 169, 3947–3953.
Ghosh, C. C., Vu, H. Y., Mujo, T. and Vancurova I. (2008) Analysis of nucleocytoplasmic shuttling of NFκB proteins in human leukocytes. Methods Mol. Biol. 457, 279–292.
Ghosh, C. C., Ramaswami, S., Juvekar, A., Vu, H. Y., Galdieri, L., Davidson, D. and Vancurova, I. (2010) Gene specific repression of proinflammatory cytokines in stimulated human macrophages by nuclear IκBα. J. Immunol. 185, 3685–3693.
Vu, H.Y., Juvekar, A., Ghosh, C., Ramaswami S., Le, D. H. and Vancurova, I. (2008) Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of ΙκΒα. Arch. Biochem. Biophys. 475, 156–163.
Yoshida, M., Nishikawa, M., Nishi, K., Abe, K., Horinouchi, S. and Beppu, T. (1990) Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells. Exp. Cell. Res. 187, 150–156.
Kudo, N., Wolff, B., Sekimoto, T., Schreiner, E. P., Yoneda, Y., Yanagida, M., Horinouchi, S. and Yoshida, M. (1998) Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp. Cell. Res. 242, 540–547.
Acknowledgments
This work was supported by NIH grants GM079581 and AI085497 to I. Vancurova.
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Ramaswami, S., Manna, S., Juvekar, A., Kennedy, S., Vancura, A., Vancurova, I. (2012). Chromatin Immunoprecipitation Analysis of NFκB Transcriptional Regulation by Nuclear IκBα in Human Macrophages. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_8
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DOI: https://doi.org/10.1007/978-1-61779-376-9_8
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