Cloning and expression of Nv-NF-κB, Nv-IκB, Nv-Bcl-3, and Nv-IKK proteins. (A) Structures of human (Homo sapiens [Hs]) and Nematostella vectensis (Nv) orthologs. Conserved domains are shaded in gray or black. Numbers indicate amino acid locations of domains and C termini. (B) A293 cells were transfected with vector alone (−) or FLAG-tagged expression plasmids for the indicated proteins, and lysates were analyzed by anti-FLAG (α-FLAG) Western blotting (35). Molecular mass markers (in kDa) are indicated to the right.

Nv-IκB interacts with Nv-NF-κB and inhibits Nv-NF-κB-dependent transactivation and nuclear localization. (A) A293 cells were transfected with the indicated FLAG-Nv-NF-κB or Myc-Nv-IκB expression vector. Whole-cell lysates were immunoprecipitated (IP) with an anti-Myc antibody (left panels) and then subjected to Western blotting (WB) with anti-FLAG or anti-Myc antiserum. The right panels show Western blots of 1% of the input from the whole-cell extracts. (B) A293 cells were cotransfected with a constant amount of Nv-NF-κB (1.0 μg), increasing amounts of Nv-IκB (0.031, 0.062, 0.125, and 0.25 μg, from left to right), and 0.5 μg of the 3×-κB reporter plasmid. A luciferase assay was then performed as described for Fig. 2B. (C to E) DF-1 chicken fibroblast cells were transfected with expression vectors for the indicated proteins. Indirect immunofluorescence was then performed using anti-Nv-NF-κB and anti-Myc primary antibodies, which were detected with FITC (Nv-NF-κB) or Texas Red (Myc-IκB). Nuclei were also stained with DAPI (4′,6-diamidino-2-phenylindole). Cells were transfected with expression plasmids for Nv-NF-κB (C) or Myc-Nv-IκB (D) or cotransfected with Nv-NF-κB and Myc-Nv-IκB (E). (F) Localization of Nv-NF-κB in cells expressing both Nv-NF-κB (FITC) and Myc-Nv-IκB (Texas Red) (percentages were determined from 58 cells counted). Nuc, nuclear; N/C, nuclear-cytoplasmic; Cyto, cytoplasmic.

Phylogenetic analysis and functional characterization of Nv-IKK. (A) The amino acid sequences of 38 proteins were aligned, cut to a final 209 residues, and used to build a maximum likelihood phylogenetic tree. RAxML determined the best tree based on the log (−8,505.833421). One hundred replicates of the bootstrap were performed to evaluate the support for specific clades. Bootstrap analysis was performed as support for specific clades. The MEK kinases were used as an outgroup to root the tree. Bf, Branchiostoma floridae; Bm, Brugia malayi; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Gg, Gallus gallus; Hm, Hydra magnipapillata; Hs, Homo sapiens; Mm, Mus musculus; Nv, Nematostella vectensis; Sp, Strongylocentrotus purpuratus; Tc, Tribolium castaneum; Tn, Tetraodon nigroviridis; Xl, Xenopus laevis. For the scale bar, the length of horizontal branches is proportional to the number of amino acid substitutions that have occurred along that branch. (B) FLAG-Nv-IKK was immunoprecipitated from transfected A293 cells, and immune complex kinase assays were performed with GST alone or the indicated GST-Nv-IκB fusion proteins (top gel row). The bottom gel row shows a Coomassie blue-stained gel as a control for GST protein expression. Above the gels is the general structure of Nv-IκB with the residues around S42 and S47, compared to the IKK phosphorylation motif of human IκBα. The asterisks above IκBα and Nv-IκB denote the locations of Ser residues phosphorylated in vitro by human TBK and Nv-IKK, respectively.

Computationally predicted Nv-NF-κB target genes. Shown at the top is a graph of the total number of human (gray bars) and predicted Nematostella (black bars) NF-κB target genes that fall into each indicated Gene Ontology biological process category. These genes were selected from 78 computationally predicted Nv-NF-κB target gene homologues (see the text for details). These four GO terms were the highest-scoring categories for the 78 Nematostella genes (with GO significance scores ranging from 2.3 × 10⁻⁹ to 1.5 × 10⁻⁶). At the bottom are lists of the human gene names that correspond to the predicted Nematostella homologues in each GO category.

Nv-NF-κB can bind DNA and activate transcription. (A) Lysates from A293 cells transfected with empty vector (lane 1) or FLAG-Nv-NF-κB (lanes 2 to 4) or a lysate from adult Nematostella (lanes 5 and 6) was used in an EMSA. Where indicated, supershifts (SS) were performed with anti-FLAG or anti-Nv-NF-κB antiserum. The Nematostella extract was prepared from an adult animal that was taken from a population genotyped as homozygous for the Nv-NF-κB Cys allele (35). Lanes 5 and 6 are a darker exposure than lanes 1 to 4, from the same gel. The leftmost lane contains probe alone. (B) A reporter assay using the multimeric 3×-κB site luciferase reporter plasmid was performed with A293 cells. Cells were transfected with increasing amounts of the Nv-NF-κB plasmid (0.13, 0.25, 0.5, 1.0, and 2.0 μg, from left to right). Values are relative to that for the luciferase activity seen with the empty vector (−) (1.0) and are the averages of three experiments, each performed with triplicate samples. Error bars indicate standard errors (SE). (C) A293 cells were transfected with the 3×-κB reporter plasmid and 1.0 μg of empty vector, Myc-Nv-NF-κB, or Myc-Nv-NF-κB-ΔC (aa 3 to 393). Values are the averages from three experiments, each performed with triplicate samples. The bottom panel shows an anti-Myc Western blot of normalized extracts used in the reporter gene assay to confirm approximately equal expression of the two Nv-NF-κB proteins. *, by a Student's t test (two-tailed, paired), the reporter activity of Myc-Nv-NF-κB compared to that of Myc-Nv-NF-κB-ΔC is statistically different (P = 2.76 × 10⁻⁴). (D) The indicated GAL4-Nv-NF-κB fusion proteins were analyzed for their abilities to activate transcription from a GAL4 site-containing reporter in yeast. Values are relative to GAL4 alone (1.0) and are the averages of seven independent experiments performed with duplicate samples. *, by a t test (two-tailed, paired), the reporter gene activity induced by GAL4-Nv-NF-κB is statistically significantly different from those induced by GAL4-Nv-NF-κB-ΔC (P = 9.34 × 10⁻⁸) and GAL4-Nv-NF-κB C-terminal sequences (GAL4-Nv-NF-κB-Cterm) (P = 3.95 × 10⁻⁷).

Nv-Bcl-3 interacts with Nv-NF-κB, inhibits Nv-NF-κB-dependent transactivation, and alters Nv-NF-κB nuclear localization. (A) FLAG-Nv-NF-κB and Myc-Nv-Bcl-3 were expressed in A293 cells and then analyzed by coimmunoprecipitation assays (as in Fig. 3A). Whole-cell lysates were immunoprecipitated (IP) with an anti-Myc antibody (left panels) and then Western blotted (WB) with anti-FLAG or anti-Myc antiserum. The right panels show Western blots of 1% of the input from the whole-cell extracts. (B) A κB site luciferase reporter gene assay with a constant amount of Nv-NF-κB and increasing amounts of Nv-Bcl-3 was performed as described for Fig. 3B. (C) Nv-NF-κB and Myc-Nv-Bcl-3 were coexpressed in DF-1 chicken fibroblast cells and analyzed by indirect immunofluorescence (as in Fig. 3E). (D) Localization of Nv-NF-κB in DF-1 cells expressing both Nv-NF-κB (FITC) and Myc-Nv-Bcl-3 (Texas Red) (percentages were determined from 55 cells counted). Nuc, nuclear; N/C, nuclear-cytoplasmic; Cyto, cytoplasmic.

Nv-NF-κB is in the cytoplasm of a subset of ectodermal cells in Nematostella in both juvenile polyps and adult animals. (A) Four-week-old Nematostella polyp. (B) Whole-mount immunofluorescence was performed on polyps with preimmune serum or anti-Nv-NF-κB antiserum. (C) Immunofluorescence was performed with anti-Nv-NF-κB antiserum on a transverse section of a polyp. Fluorescence was observed primarily in a subset of cells in the outer ectoderm. The lower panel shows a higher magnification of a region containing NF-κB-positive cells; the white arrow indicates a cell with green (FITC) cytoplasm and a blue (DAPI) nucleus. (D) Adult Nematostella. Immunohistochemistry was performed with anti-Nv-NF-κB antiserum on transverse (E) and longitudinal (F) sections of an adult animal. The lower part of panel E is a higher magnification of the indicated region, as in panel C. (G) Anti-Nv-NF-κB Western blotting of A293 cells transfected with empty vector (lane 1) or pcDNA-Nv-NF-κB (Cys allele) (lane 2) or an extract from an adult Nematostella (Cys/Cys homozygote) (lane 3) from the same population that was analyzed by immunohistochemistry. Nv-NF-κB is indicated by the arrow, and molecular mass markers (in kDa) are indicated to the left of the panel.

Nv-NF-κB binds κB sites upstream of the Nv-ikb gene. (A) The Nv-ikb gene has two κB sites within 337 nucleotides upstream of the start codon (upper panel): κB1 (from −337 to −327) and κB2 (from −177 to −167). An EMSA (lower panel) was performed using lysates from A293 cells transfected with FLAG-Nv-NF-κB by using the indicated probes. The MHC probe contains a κB site from the human MHC-1 promoter. (B) Two hundred twenty-one nucleotides (from −358 to −138) of the Nv-ikb promoter that contained the two κB sites were subcloned upstream of a luciferase reporter gene (WT) and used in a reporter assay in A293 cells as described for Fig. 2B. Point mutations were made to disrupt the two κB sites (Mut). Values are relative to the luciferase activity seen with the empty vector (−) (1.0) and are the averages from three experiments, each performed with triplicate samples. Error bars indicate SE. *, by a t test (two-tailed, paired), the reporter activity of the WT reporter is statistically different (P = 3.6 × 10⁻⁷) from that of the Mut reporter.