Abstract
The unique DNA-binding properties of distinct NF-κB dimers influence the selective regulation of NF-κB target genes. To more thoroughly investigate these dimer-specific differences, we combined protein-binding microarrays and surface plasmon resonance to evaluate DNA sites recognized by eight different NF-κB dimers. We observed three distinct binding-specificity classes and clarified mechanisms by which dimers might regulate distinct sets of genes. We identified many new nontraditional NF-κB binding site (κB site) sequences and highlight the plasticity of NF-κB dimers in recognizing κB sites with a single consensus half-site. This study provides a database that can be used in efforts to identify NF-κB target sites and uncover gene regulatory circuitry.
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References
Baldwin, A.S. Jr. Series introduction: the transcription factor NF-κB and human disease. J. Clin. Invest. 107, 3–6 (2001).
Tak, P.P. & Firestein, G.S. NF-κB: a key role in inflammatory diseases. J. Clin. Invest. 107, 7–11 (2001).
Zhang, G. & Ghosh, S. Toll-like receptor-mediated NF-κB activation: a phylogenetically conserved paradigm in innate immunity. J. Clin. Invest. 107, 13–19 (2001).
Hiscott, J., Kwon, H. & Genin, P. Hostile takeovers: viral appropriation of the NF-κB pathway. J. Clin. Invest. 107, 143–151 (2001).
Natoli, G., Saccani, S., Bosisio, D. & Marazzi, I. Interactions of NF-κB with chromatin: the art of being at the right place at the right time. Nat. Immunol. 6, 439–445 (2005).
Hoffmann, A., Natoli, G. & Ghosh, G. Transcriptional regulation via the NF-κB signaling module. Oncogene 25, 6706–6716 (2006).
Natoli, G. Tuning up inflammation: how DNA sequence and chromatin organization control the induction of inflammatory genes by NF-κB. FEBS Lett. 580, 2843–2849 (2006).
Bonizzi, G. & Karin, M. The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25, 280–288 (2004).
Hayden, M.S. & Ghosh, S. Signaling to NF-κB. Genes Dev. 18, 2195–2224 (2004).
Gerondakis, S. et al. Unravelling the complexities of the NF-κB signalling pathway using mouse knockout and transgenic models. Oncogene 25, 6781–6799 (2006).
Hoffmann, A., Leung, T.H. & Baltimore, D. Genetic analysis of NF-κB/Rel transcription factors defines functional specificities. EMBO J. 22, 5530–5539 (2003).
Chen, F.E. & Ghosh, G. Regulation of DNA binding by Rel/NF-κB transcription factors: structural views. Oncogene 18, 6845–6852 (1999).
Kunsch, C., Ruben, S.M. & Rosen, C.A. Selection of optimal κB/Rel DNA-binding motifs: interaction of both subunits of NF-κB with DNA is required for transcriptional activation. Mol. Cell. Biol. 12, 4412–4421 (1992).
Udalova, I.A., Mott, R., Field, D. & Kwiatkowski, D. Quantitative prediction of NF-κB DNA-protein interactions. Proc. Natl. Acad. Sci. USA 99, 8167–8172 (2002).
Hoffmann, A. & Baltimore, D. Circuitry of nuclear factor κB signaling. Immunol. Rev. 210, 171–186 (2006).
Bonizzi, G. et al. Activation of IKKα target genes depends on recognition of specific κB binding sites by RelB-p52 dimers. EMBO J. 23, 4202–4210 (2004).
Sanjabi, S. et al. A c-Rel subdomain responsible for enhanced DNA-binding affinity and selective gene activation. Genes Dev. 19, 2138–2151 (2005).
Senftleben, U., Li, Z.W., Baud, V. & Karin, M. IKKβ is essential for protecting T cells from TNFα-induced apoptosis. Immunity 14, 217–230 (2001).
Xiao, G., Harhaj, E.W. & Sun, S.C. NF-κB-inducing kinase regulates the processing of NF-κB2 p100. Mol. Cell 7, 401–409 (2001).
Fusco, A.J. et al. NF-κB p52:RelB heterodimer recognizes two classes of κB sites with two distinct modes. EMBO Rep. 10, 152–159 (2009).
Britanova, L.V., Makeev, V.J. & Kuprash, D.V. In vitro selection of optimal RelB/p52 DNA-binding motifs. Biochem. Biophys. Res. Commun. 365, 583–588 (2008).
Berger, M.F. & Bulyk, M.L. Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat. Protoc. 4, 393–411 (2009).
Berger, M.F. et al. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat. Biotechnol. 24, 1429–1435 (2006).
Mukherjee, S. et al. Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays. Nat. Genet. 36, 1331–1339 (2004).
Linnell, J. et al. Quantitative high-throughput analysis of transcription factor binding specificities. Nucleic Acids Res. 32, e44 (2004).
Berger, M.F. & Bulyk, M.L. Protein binding microarrays (PBMs) for rapid, high-throughput characterization of the sequence specificities of DNA binding proteins. Methods Mol. Biol. 338, 245–260 (2006).
Bulyk, M.L., Huang, X., Choo, Y. & Church, G.M. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. Proc. Natl. Acad. Sci. USA 98, 7158–7163 (2001).
Chen, Y.Q., Sengchanthalangsy, L.L., Hackett, A. & Ghosh, G. NF-κB p65 (RelA) homodimer uses distinct mechanisms to recognize DNA targets. Structure 8, 419–428 (2000).
Grilli, M., Chiu, J.J. & Lenardo, M.J. NF-κB and Rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143, 1–62 (1993).
Li, Q. & Verma, I.M. NF-κB regulation in the immune system. Nat. Rev. Immunol. 2, 725–734 (2002).
Lim, C.A. et al. Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited by NF-κB upon TLR4 activation. Mol. Cell 27, 622–635 (2007).
Kasowski, M. et al. Variation in transcription factor binding among humans. Science 328, 232–235 (2010).
Schreiber, J. et al. Coordinated binding of NF-κB family members in the response of human cells to lipopolysaccharide. Proc. Natl. Acad. Sci. USA 103, 5899–5904 (2006).
Wang, J. et al. Distinct roles of different NF-κB subunits in regulating inflammatory and T cell stimulatory gene expression in dendritic cells. J. Immunol. 178, 6777–6788 (2007).
Merika, M., Williams, A.J., Chen, G., Collins, T. & Thanos, D. Recruitment of CBP/p300 by the IFNβ enhanceosome is required for synergistic activation of transcription. Mol. Cell 1, 277–287 (1998).
Fujita, T., Nolan, G.P., Ghosh, S. & Baltimore, D. Independent modes of transcriptional activation by the p50 and p65 subunits of NF-κB. Genes Dev. 6, 775–787 (1992).
Leung, T.H., Hoffmann, A. & Baltimore, D. One nucleotide in a κB site can determine cofactor specificity for NF-κB dimers. Cell 118, 453–464 (2004).
Cheng, C.S. et al. The specificity of innate immune responses is enforced by repression of interferon response elements by NF-κB p50. Sci. Signal. 4, ra11 (2011).
Chen, Y.Q., Ghosh, S. & Ghosh, G. A novel DNA recognition mode by the NF-κB p65 homodimer. Nat. Struct. Biol. 5, 67–73 (1998).
Mauxion, F., Jamieson, C., Yoshida, M., Arai, K. & Sen, R. Comparison of constitutive and inducible transcriptional enhancement mediated by κB-related sequences: modulation of activity in B cells by human T-cell leukemia virus type I tax gene. Proc. Natl. Acad. Sci. USA 88, 2141–2145 (1991).
Wong, D. et al. Extensive characterization of NF-κB binding uncovers noncanonical motifs and advances the interpretation of genetic functional traits. Genome Biol. 12, R70 (2011).
Fordyce, P.M. et al. De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis. Nat. Biotechnol. 28, 970–975 (2010).
Rucker, P., Torti, F.M. & Torti, S.V. Recombinant ferritin: modulation of subunit stoichiometry in bacterial expression systems. Protein Eng. 10, 967–973 (1997).
Field, S., Udalova, I. & Ragoussis, J. Accuracy and reproducibility of protein-DNA microarray technology. Adv. Biochem. Eng. Biotechnol. 104, 87–110 (2007).
Gordân, R., Narlikar, L. & Hartemink, A.J. Finding regulatory DNA motifs using alignment-free evolutionary conservation information. Nucleic Acids Res. 38, e90 (2010).
Workman, C.T. et al. enoLOGOS: a versatile web tool for energy normalized sequence logos. Nucleic Acids Res. 33, W389–392 (2005).
Acknowledgements
This work was funded by US National Institutes of Health (NIH) grant R01 HG003985 to M.L.B., HFSP grant RGY0085/2005-C to M.L.B., NIH grant R01 AI073868 to S.T.S., FP7 Collaborative Project Model-In grant 222008 to J.R. and I.U., support from the UK Medical Research Council to J.R. and I.U. and support from Wellcome Trust grant 075491/Z/04 to J.R. A.C. was funded by NIH grant T32 CA009120, and B.A. was funded by the i2b2/HST Summer Institute in Bioinformatics and Integrative Genomics NIH grant U54 LM008748. We thank G. Natoli, M. Pasparakis and L. Giorgetti for helpful discussions.
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T.S. designed and carried out PBM experiments and carried out ChIP data analysis. T.S. and B.A. carried out PBM data analyses. A.B.C. and K.J.W. made mouse protein samples. A.B.C. carried out SPR experiments. A.T., D.W., J.R. and I.A.U. provided human protein samples. The manuscript was written by T.S., A.B.C., S.T.S. and M.L.B.
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Supplementary Text and Figures
Supplementary Discussion, Supplementary Methods, Supplementary Figures 1–7 and Supplementary Tables 1–3 (PDF 9502 kb)
Supplementary Spreadsheet 1
PBM probeset and dataset. (DNA probes sequences used in the PBM experiments, and PBM-derived z-scores for all described experiments are provided.) (XLSX 1508 kb)
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Siggers, T., Chang, A., Teixeira, A. et al. Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding. Nat Immunol 13, 95–102 (2012). https://doi.org/10.1038/ni.2151
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DOI: https://doi.org/10.1038/ni.2151
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