NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKβ to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112−195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKβ (amino acids 701−745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKβ compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKβ. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.

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Biological and biochemical functions of immunity transcription factor NF-κB in basal metazoans are largely unknown. Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq), in the phylum Porifera. Structurally and phylogenetically, the Aq-NF-κB protein is most similar to NF-κB p100 and p105 among vertebrate proteins, with an Nterminal DNA-binding/dimerization domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile more similar to human NF-κB proteins than Rel proteins. Aq-NF-κB also resembles the mammalian NF-κB protein p100 in that C-terminal truncation results in translocation of Aq-NF-κB to the nucleus and increases its transcriptional activation activity. Overexpression of a human or sea anemone IκB kinase (IKK) can induce C-terminal processing of Aq-NF-κB in vivo, and this processing requires C-terminal serine residues in Aq-NF-κB. Unlike human NF-κB p100, however, the C-terminal sequences of Aq-NF-κB do not effectively inhibit its DNAbinding activity when expressed in human cells. Tissue of another demosponge, a black encrusting sponge, contains NF-κB site DNAbinding activity and an NF-κB protein that appears mostly processed and in the nucleus of cells. NF-κB DNA-binding activity and processing is increased by treatment of sponge tissue with LPS. By transcriptomic analysis of A. queenslandica we identified likely homologs to many upstream NF-κB pathway components. These results present a functional characterization of the most ancient metazoan NF-κB protein to date, and show that many characteristics of mammalian NF-κB are conserved in sponge NF-κB, but the mechanism by which NF-κB functions and is regulated in the sponge may be somewhat different.