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. 2013 Apr 19;288(16):11546-54.
doi: 10.1074/jbc.M113.451153. Epub 2013 Mar 18.

Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor κB (NF-κB) Proteins

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Free PMC article

Cross-species Analysis Reveals Evolving and Conserved Features of the Nuclear Factor κB (NF-κB) Proteins

Grigory Ryzhakov et al. J Biol Chem. .
Free PMC article

Abstract

NF-κB is a key regulator of immune gene expression in metazoans. It is currently unclear what changes occurred in NF-κB during animal evolution and what features remained conserved. To address this question, we compared the biochemical and functional properties of NF-κB proteins derived from human and the starlet sea anemone (Nematostella vectensis) in 1) a high-throughput assay of in vitro preferences for DNA sequences, 2) ChIP analysis of in vivo recruitment to the promoters of target genes, 3) a LUMIER-assisted examination of interactions with cofactors, and 4) a transactivation assay. We observed a remarkable evolutionary conservation of the DNA binding preferences of the animal NF-κB orthologs. We also show that NF-κB dimerization properties, nuclear localization signals, and binding to cytosolic IκBs are conserved. Surprisingly, the Bcl3-type nuclear IκB proteins functionally pair up only with NF-κB derived from their own species. The basis of the differential NF-κB recognition by IκB subfamilies is discussed.

Figures

FIGURE 1.
FIGURE 1.
DNA binding profiles of Nematostella and human NF-κB dimers. A, NF-κB proteins were expressed in E. coli, and the soluble fractions of bacterial lysates (Input; separated from insoluble debris by centrifugation) were incubated for 2 h at room temperature with streptavidin-agarose attached to biotinylated DNA oligonucleotides containing NF-κB-binding (from the 3′-region of the TNF gene) or control sequences. The bound proteins were eluted with high-salt buffer, and the eluates were subjected to SDS-PAGE, followed by Coomassie Blue staining. B, a heat map of the binding profiles based on the microarray analysis of four NF-κB dimers (presented in columns) was generated using MultiExperiment Viewer (25, 26). The z-scores (supplemental Table 1) of each individual protein were used as input for MultiExperiment Viewer. Within the heat map, probes that contain the 803 11-mer sequences and represent k-mer space given by the consensus sequence RGGRNNHHYYB can be found as rows. A color gradient reflects the binding affinity z-scores of NF-κB dimers for a probe, where high-affinity probes (positive values) are shown in yellow, z-scores near zero are shown in black, and low-affinity probes (negative values) are shown in blue (see side bar). Hierarchical clustering was used to describe relationships between binding profiles of the different dimers (Euclidean distance correlation and complete linkage analysis). C, pairwise comparisons of the DNA binding profiles of Nematostella and human NF-κB dimers based on the array analysis. The value at the bottom of each graph is the correlation coefficient for the pair in the graph. These graphs were built for the z-score data sets. z-scores were obtained using log2-transformed intensities, and the median of replicates were calculated for each probe within every array. D, DNA barcodes of the NF-κB proteins based on the top 20 highest binding motifs for each individual protein were created using WebLogo online software. E, recruitment of human and Nematostella NF-κB p50 to human gene promoters. Plasmids encoding Myc-tagged HsNF-κB p50 and NvNF-κB p50 proteins were transfected into human 293ET cells. One day after transfection, cells were lysed, and the lysates were subjected to ChIP using anti-Myc (clone 9E10) or IgG control antibodies. The NF-κB recruitment to gene promoters was analyzed by qPCR of the precipitated DNA using specific primers to the human TNF and IL-10 gene promoters. The data are presented as -fold change over a negative IgG control.
FIGURE 2.
FIGURE 2.
Conservation of NF-κB protein/protein interactions. FLAG- and Renilla luciferase-tagged proteins were expressed as pairs in 293ET cells. The cell lysates were subjected to immunoprecipitation using FLAG-agarose, the proteins bound to the sorbent were eluted with FLAG peptide, and the luciferase activities were measured in the eluates and total lysates. The data are shown as the ratio of luciferase activity in eluates and lysates normalized against the control (empty vector).
FIGURE 3.
FIGURE 3.
Human IκBζ discriminates between human and Nematostella NF-κB. A, Renilla luciferase-tagged NF-κB p50 proteins coexpressed with either FLAG-tagged RelA or HsIκBζ. The protein complexes were pulled out from cell lysates using FLAG-agarose, the bound proteins were eluted with FLAG peptide, and the luciferase activities in the lysates and eluates were measured. The data are shown as -fold binding compared with the control (bacterial alkaline phosphatase). B, the deletions mutants of human NF-κB p50, N-terminally tagged with Renilla luciferase (left), were coexpressed with either bacterial alkaline phosphatase (control) or FLAG-tagged HsIκBζ. The protein/protein interactions were analyzed as described for A. Rel, RHD. C, the HsIκBζ-binding region of HsNF-κB p50 was aligned with the corresponding region of NvNF-κB p50 using ClustalW software. The NLS motif is underlined. D, LUMIER assay of selected NF-κB/IκB interactions. Renilla luciferase-tagged wild-type human and Nematostella NF-κB p50 or NLS mutant versions were coexpressed with either IRF3 (control) or FLAG-tagged IκB proteins in 293ET cells. The protein complexes were pulled out from cell lysates using FLAG-agarose, the bound proteins were eluted with FLAG peptide, and the luciferase activities in the lysates and eluates were measured. The data are shown as -fold binding compared with the control (IRF3). E, HsNF-κB p50 or NvNF-κB p50 was coexpressed with or without HsIκBζ and the NGAL/lcn2 promoter-based luciferase reporter. The luciferase activities were measured in cell lysates. The data are shown as -fold induction over the control (empty vector) as means ± S.D. of triplicate experiments. F, plasmids expressing either GFP or NF-κB proteins were delivered into nfkb1/2−/− murine embryonic fibroblasts using electroporation. Two days later, cells were stimulated with 1 μg/ml LPS for 1 and 4 h and subjected to RNA extraction. Induction of lcn2 mRNA synthesis was measured by qPCR. The data are shown as means ± S.D. of a representative experiment.
FIGURE 4.
FIGURE 4.
Distinct properties of Nematostella IκB and Bcl3 proteins. A, LUMIER assay of selected NF-κB/IκB interactions. The Renilla luciferase-tagged human and Nematostella NF-κB p50 proteins were coexpressed with one of the FLAG-tagged IκB proteins in 293ET cells. The protein complexes were pulled out from cell lysates using FLAG-agarose, the bound proteins were eluted with FLAG peptide, and the luciferase activities in the lysates and eluates were measured. The data are shown as -fold binding compared with the control (bacterial alkaline phosphatase). B, 293ET cells seeded in 96-well plates were transfected with 3 or 10 ng (lo and hi, respectively)/well NvBcl3- or NvIκB-encoding plasmid and 10 ng/well pNF-κB-luc reporter plasmid. One day later, the cells were stimulated for 6 h with 10 ng/ml TNF-α before collection. The luciferase activities were measured in cell lysates. The data are shown as -fold induction over the control (empty vector) as means ± S.D. of a triplicate experiment. C and D, the amino acid sequence alignments of human and Nematostella IκB proteins. Sequences were aligned using ClustalW software, and the ankyrin repeats (AR) were labeled according to a previously used annotation (30). Conserved residues are shown in boldface, and the predicted loop residues of HsBcl3 contacting HsNF-κB p50 are underlined.
FIGURE 5.
FIGURE 5.
Nematostella NF-κB activity is suppressed by Nematostella IκB proteins. A and B, 293ET cells seeded in 96-well plates were transfected for 24 h with either 10 ng/well pNF-κB-luc (A) or 10 ng of Nvbcl3 gene promoter-based (B) luciferase reporter plasmid and increasing amounts of the NvBcl3- or NvIκB-expressing constructs (2, 5, and 10 ng/well in A and 10 ng/well in B) or control plasmid (empty vector). One day later, the cells were lysed, and the luciferase activities were measured. The data are shown as -fold induction over the control (empty vector) as means ± S.D. of a triplicate experiment.

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