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Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding

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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Figure 1: Examining NF-κB dimer binding by custom NF-κB PBMs.
Figure 2: Dimer-specific binding to traditional and nontraditional κB sites.
Figure 3: Preferences for flanking DNA bases and κB site length.
Figure 4: Comparison of c-Rel, RelA/N3,4 and RelA homodimer DNA-binding specificity.
Figure 5: Enrichment of PBM-determined κB sites in published data set of p50-bound genomic regions from LPS-stimulated human macrophages33.

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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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Contributions

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.

Corresponding author

Correspondence to Martha L Bulyk.

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The authors declare no competing financial interests.

Supplementary information

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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