Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

Abstract

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.

Diversification of Transcription Factor NF-κB in Protists

“…Protist NF-κB proteins vary in domain structure and choanoflagellates show evidence of gene duplication Suga et al (4) reported the presence of a single gene encoding an NF-κB-like protein in C. owczarzaki (Co). The overall protein structure of Co-NF-κB is similar to most other basal NF-κB proteins known to date, in that it has an N-terminal RHD, followed by a glycine-rich region (GRR), and five C-terminal ANK repeats (15)(16)(17)(18). However, Co-NF-κB is larger than other NF-κB homologs, due primarily to additional residues C-terminal to the ANK repeats (Fig.…”

“…That is, in most metazoans, the activity of NF-κB is regulated at the posttranscriptional level, whereas in Aiptasia and corals, we have found that NF-κB mRNA levels and DNA-binding activity appear to be coordinately regulated, suggesting transcriptional regulation. That is, in Aiptasia, thermal bleaching causes transcriptional upregulation of NF-κB, which also results in increased protein expression of nuclear, (15)(16)(17). Moreover, induction of NF-κB DNA-binding activity and protein levels by loss of symbiosis in Aiptasia appears to be primarily a result of increased transcription of NF-κB and not due to induced ANK repeat degradation.…”

“…For example, while some cnidarians contain homologs to TLRs, other cnidarians, some sponges, and choanoflagellates contain only TIR-domain proteins that lack the important extracellular components of the TLR (5,11,15,16,28). Furthermore, Capsaspora does not contain any homologs to TLRs, ILR-1 or TNFRs (4).…”

“…That analysis showed that this set of 305 genes was predicted to be involved in several biological processes, including 14 genes that encode proteins associated with developmental and immune system processes ( Fig. 5C), which are biological processes regulated by NF-κB in many more complex organisms and suggested to be regulated by NF-κB in several basal organisms (15)(16)(17)(20)(21)(22)(23). Although 14 genes may seem low, the total database for human GO analysis of immune system and developmental processes genes is approximately 2200 genes, but the number of annotated homologs that exist in Capsaspora in these two GO categories is only 66 genes (Supplemental Table 4).…”

“…In sponge and some cnidarian NF-κBs, removal of C-terminal ANK-repeat sequences are required for nuclear localization in vertebrate cell-based assays (15)(16)(17).…”