J Mol Evol (2011) 73:325–336 DOI 10.1007/s00239-011-9479-7
Two Polymorphic Residues Account for the Differences in DNA Binding and Transcriptional Activation by NF-jB Proteins Encoded by Naturally Occurring Alleles in Nematostella vectensis Francis S. Wolenski • Sushil Chandani • Derek J. Stefanik • Ning Jiang • Emma Chu John R. Finnerty • Thomas D. Gilmore
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Received: 7 October 2011 / Accepted: 8 December 2011 / Published online: 25 December 2011 Ó Springer Science+Business Media, LLC 2011
Abstract The NF-jB family of transcription factors is activated in response to many environmental and biological stresses, and plays a key role in innate immunity across a broad evolutionary expanse of animals. A simple NF-jB pathway is present in the sea anemone Nematostella vectensis, an important model organism in the phylum Cnidaria. Nematostella has previously been shown to have two naturally occurring NF-jB alleles (Nv-NF-jB-C and Nv-NF-jB-S) that encode proteins with different DNAbinding and transactivation abilities. We show here that polymorphic residues 67 (Cys vs. Ser) and 269 (Ala vs. Glu) play complementary roles in determining the DNAbinding activity of the NF-jB proteins encoded by these two alleles and that residue 67 is primarily responsible for the difference in their transactivation ability. Phylogenetic analysis indicates that Nv-NF-jB-S is the derived allele, consistent with its restricted geographic distribution. These results define polymorphic residues that are important for the DNA-binding and transactivating activities of two naturally occurring variants of Nv-NF-jB. The implications for the appearance of the two Nv-NF-jB alleles in natural populations of sea anemones are discussed. Keywords NF-jB Nematostella Polymorphism Evolution DNA binding Transactivation
Electronic supplementary material The online version of this article (doi:10.1007/s00239-011-9479-7) contains supplementary material, which is available to authorized users. F. S. Wolenski S. Chandani D. J. Stefanik N. Jiang E. Chu J. R. Finnerty T. D. Gilmore (&) Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA e-mail:
[email protected]
Introduction From insects to humans, the NF-jB signaling pathway is activated in response to environmental (chemicals, ultraviolet light, oxidative stress) and biological (dsRNA and pathogens) stresses (Oeckinghaus et al. 2011; Gilmore and Wolenski 2012). Activated NF-jB alters the expression of target genes to bring about cellular and organismal responses. The repertoire of genes activated by NF-jB depends on the stress and the cell type, but can include anti-oxidizing enzymes (superoxide dismutase, nitric oxide synthase) as well as anti-apoptotic (Bcl-xL, IAPs, TRAFs) and immune response (cytokines, anti-microbial peptides) proteins. The sea anemone Nematostella vectensis (phylum Cnidaria; class Anthozoa) is the first diploblastic animal shown to possess an NF-jB gene (Sullivan et al. 2007). NF-jB homologs were subsequently identified in other cnidarians (Meyer et al. 2009; Lange et al. 2011; Shinzato et al. 2011) and sponges (Gauthier and Degnan 2008). Recently, NFjB was identified in a non-metazoan, the unicellular holozoan Capsaspora owczarzaki (Sebe´-Pedro´s et al. 2011). C. owczarzaki, which was isolated from the hemolymph of a freshwater snail (Hertel et al. 2002), appears to be closely related to the clade comprising animals and choanoflagellates (Shalchian-Tabrizi et al. 2008; Sebe´-Pedro´s et al. 2011; Torruella et al. 2011). The presence of NF-jB in C. owczarzaki implies that the NF-jB gene arose approximately 1,000 million years ago, which is when holozoans appeared (Chernikova et al. 2011). However, the biological processes controlled by the NF-jB pathway have only been characterized in triploblastic animals (Gilmore and Wolenski 2012). In most mammalian cells, NF-jB is present as an inactive cytoplasmic complex that is bound by the inhibitor protein IjB. In response to an appropriate signal, IjB is
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phosphorylated by an IjB kinase (IKK) and targeted for degradation. Free NF-jB then translocates to the nucleus and binds to jB-sites in the enhancers of target genes to affect transcription. Nematostella has a single NF-jB gene, two genes encoding IjB proteins (IjB and Bcl-3) and two for IKK (Gilmore and Wolenski 2012); proteins in this core NFjB pathway have been shown to possess molecular activities that are similar to their vertebrate counterparts (Wolenski et al. 2011). The biological function of the NF-jB signaling pathway in Nematostella is not known. However, the Nematostella NF-jB protein (Nv-NF-jB) is expressed in the cytoplasm of a subset of ectodermal cells in juvenile and adult animals, and several Nematostella homologs of human NF-jB target genes are predicted to have upstream binding sites for Nv-NF-jB (Wolenski et al. 2011). Taken together, these data suggest that Nv-NF-jB is regulated in a manner similar to higher animals and that it may be activated in response to environmental stimuli in Nematostella. Members of the mammalian NF-jB family of transcription factors (p50, p52, c-Rel, RelA, RelB) have a conserved DNA-binding and dimerization domain called the Rel homology domain (RHD) (Gilmore 2006). Nematostella NF-jB is a 440 amino acid (aa) protein essentially consisting of the RHD and a short glycine-rich C-terminal region (Sullivan et al. 2007). By aa sequence and phylogenetic comparison to human proteins, Nv-NFjB appears to be most similar to the NF-jB p50 protein (Sullivan et al. 2007). Nematostella was previously shown to have two naturally occurring allelic variants of Nv-NFjB that differ at 10 residues (Sullivan et al. 2009). For simplicity, we will refer to these two variants as Nv-NFjB-C (Cys at aa 67) and Nv-NF-jB-S (Ser at aa 67). These two alleles show geographically distinct distributions, with the gene encoding Nv-NF-jB-C being the predominant allele in most natural populations (Sullivan et al. 2009). The two Nv-NF-jB proteins encoded by these allelic variants also differ in their DNA-binding and transactivation abilities, and their DNA-binding activity can be affected by reciprocal changes at polymorphic residue 67 (Sullivan et al. 2009), which lies in a peptide loop necessary for jB-site recognition on DNA (Ghosh et al. 1995). Additionally, the DNA-binding activity of Nv-NF-jB-C is more sensitive to thiol-reactive compounds and to redox conditions than is Nv-NF-jB-S (Sullivan et al. 2009). In this paper, we have further characterized residues responsible for differences in the activities of the proteins encoded by the two Nv-NF-jB alleles. We demonstrate that a second polymorphic residue, located at aa 269, affects the DNA-binding activity of Nv-NF-jB. We also show that residue 67, but not 269, contributes to the difference in transactivation abilities between the two variants. Finally, we present phylogenetic evidence suggesting that Nv-NF-jB-S is a derived allele.
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Materials and Methods Plasmid Construction and Genomic Sequencing The cDNAs encoding Nv-NF-jB-C (ADQ57372.1), Nv-NF-jB-S (ABU48530.1), Nv-IjB (EU092641.1) and Nv-Bcl-3 (ADQ57373.1) have been described previously (Sullivan et al. 2009; Wolenski et al. 2011). Gene-specific primers were used to subclone these cDNAs into various pcDNA3.1 (?) (Invitrogen), pcDNA-FLAG or pcDNAMyc expression vectors. Point mutations of the two NvNF-jB alleles were made by overlapping PCR and are written as the wild-type allele and the relevant mutation (e.g., the Cys-to-Ser mutation in the Nv-NF-jB-C protein is Nv-NF-jB-C67S). A complete list of primers and details about plasmid constructions are included in supplemental material. Genotyping of Nv-NF-jB was performed following a previously described approach (Sullivan et al. 2009). Briefly, a fragment of the Nv-NF-jB gene encompassing exon 3 (containing codon 67) was amplified from genomic DNA by PCR with specific forward (50 -CACMGAGCCCTACCTAG AAA-30 , where M is A or C) and reverse primers (50 -TCGC TGTCATGTGTTGATCC-30 ). The amplified 753-bp product contains 460 nucleotides of intronic and 293 nucleotides of exonic sequence. Samples were electrophoresed on a 1% agarose gel and the PCR product was gel-purified using the QIAquick Gel Extraction kit (Qiagen). Fragments were sequenced at Promega using the reverse primer. Sequence chromatograms were analyzed to determine the identity of the codon (Cys or Ser) for residue 67. Alignment of NF-jB Proteins For Fig. 1a, the RHD sequences of NF-jB proteins from multiple species were aligned at sequences flanking the DNA recognition loop and at sequences flanking the L3 linker region (Ghosh et al. 1995). GenBank accession numbers for NF-jB proteins are as follows: C. owczarzaki, ADX60055.1; Amphimedon queenslandica, ABW76682.1; N. vectensis, ADQ57372.1 (Nv-NF-jB-C) and ABU48530.1 (Nv-NF-jBS); Hydra magnipapillata, ADU79237.1; Strongylocentrotus purpuratus, NP_999819.1; Drosophila melanogaster, AAF20135.1; and Homo sapiens, AAA36361.1. The Acropora digitifera sequence (aug_v2a.04467.t1) is available at http://marinegenomics.oist.jp/acropora_digitifera. Structural Modeling of Nv-NF-jB The RHDs of human p50 and Nv-NF-jB have 52% aa identity, which includes many aa positions known to be involved in dimerization and DNA binding (Sullivan et al. 2007). Because of this high conservation in aa sequence,
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the crystal structure of a p50 homodimer bound to DNA (Ghosh et al. 1995) was used as a template to model the structure of the Nv-NF-jB dimer. Coordinates for the p50 crystal structure (PDB ID:1NFK) were downloaded from the Protein Data Bank and a structural model of the RHD of Nv-NF-jB-S was generated by molecular modeling techniques following established approaches (Zhang 2007; Karplus 2009). The initial structure was modified through the Loop Refinement routine in SWISS-MODEL (Guex and Peitsch 1997). Simulations of the molecular dynamics of the model were performed using the CHARMM force field (release c30b) (Brooks et al. 2009). The aa substitutions C67/E269, S67/A269, and C67/A269 were also made in the Nv-NF-jB-S (S67/E269) structural model. These four models were prepared for CHARMM using InsightII (Accelrys, Inc.). Of note, the Nv-NF-jB-S model was chosen over Nv-NF-jB-C because of greater predicted stability in initial simulations. Once structural models were established, modifications were made to create a more accurate model of Nv-NF-jB bound to DNA. To keep these models consistent with biochemical experiments, the double-stranded DNA sequence (50 -GGGGAATCCCC-30 ) matched the jB-site in the probe used in EMSA experiments (Sullivan et al. 2009). The interaction of the Nv-NF-jB protein with the jB-site was refined using defined parameters (Foloppe et al. 2001). The protein–nucleic acid complexes were modeled in explicit water, sodium atoms were added to bring the net charge of the ensemble to zero, and structures were optimized using Steepest Descent minimization for 500 iterations. The minimized structures were heated to 275 K in 50 ps in molecular dynamics simulations using the Velocity Verlet integration method, and were equilibrated at this temperature for 300 ps. A 1-ns simulation at constant temperature was carried out for the four modeled variants of Nv-NF-jB. Interaction energies of aa 67 and 269 with the jB-site were measured using the INTe subroutine in CHARMM on structures extracted at 1-ps intervals over the last 200 ps of the simulations. All simulations were performed on an IBM p690 computer. Cell Culture and Transfection A293 human kidney carcinoma cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Biologos), 50 U/ml penicillin, and 50 lg/ml streptomycin. Transfection of human A293 cells with expression plasmids was performed using polyethylenimine (Polysciences) essentially as described previously (Sullivan et al. 2009). Briefly, on the day of transfection, cells were incubated with plasmid DNA and polyethylenimine at a ratio of 1:6 (DNA:polyethylenimine). Media was changed
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24 h post-transfection, and whole-cell lysates were prepared 24 h later. Western Blotting and Immunoprecipitation For western blotting, whole-cell lysates from A293 cells or whole animals were prepared in AT buffer (20 mM HEPES, pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20% w/v glycerol, 1% w/v Triton X-100, 20 mM NaF, 1 mM Na4P2O7, 1 mM dithiothreitol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml leupeptin, 1 lg/ml pepstatin A, 10 lg/ml aprotinin) as described previously (Wolenski et al. 2011). To create whole animal lysates from Nematostella, adult animals were pulverized with a pestle in AT lysis buffer and sonicated twice for 10 s in a 550 Sonic Dismembrator (ThermoFisher Scientific). Sodium chloride was added to the lysates to a final concentration of 150 mM and extracts were centrifuged at 16,0009g for 20 min at 4°C. The pellet was resuspended in AT buffer and then used in western blots. Primary antisera were as follows: rabbit anti-FLAG (2368, Cell Signaling; 1:1,000 dilution), rabbit anti-Nv-NF-jB (epitope PADFLQQGVFSTQNPSNM, Open Biosystems; 1:1,000) (Wolenski et al. 2011), and mouse anti-Myc (sc-40, Santa Cruz Biotechnology; 1:100). Nitrocellulose membranes were incubated with either an anti-rabbit (FLAG and Nv-NFjB) or anti-mouse (Myc) horseradish peroxidase-linked secondary antiserum, and complexes were detected with SuperSignal West Dura Extended Duration Substrate (Pierce). When using the anti-Nv-NF-jB antiserum, filters were blocked overnight at 4°C in phosphate-buffered saline containing 8% milk, 5% normal goat serum (Gibco), and 0.05% Tween 20. Co-immunoprecipitations were performed essentially as described previously (Wolenski et al. 2011). A293 cells were transfected with 5 lg of pcDNA 3.1 (?) or expression plasmids for the indicated FLAG- and Myc-tagged Nv-NF-jB, Nv-IjB, or Nv-Bcl-3 proteins. Whole-cell lysates were prepared in AT buffer 48 h post-transfection. Protein concentration was determined using the Bio-Rad protein assay reagent, 1% of total protein was removed as an input control, and the remaining lysate was immunoprecipitated with anti-Myc antiserum. Input controls and immunoprecipitates were subjected to western blotting for both FLAG- and Myc-tagged proteins. Electrophoretic Mobility Shift Assays (EMSAs) EMSAs were performed as described previously, using a 32P end-labeled jB-site from the Nv-IjB gene (50 -TCGAGAGGTCGGGGAATCCCCCCCCG-30 ; jB-site is underlined) (Sullivan et al. 2009). Whole-cell extracts were prepared in AT buffer and were subjected to western
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blotting with anti-Nv-NF-jB antiserum to normalize for equal expression of Nv-NF-jB proteins. Normalized extracts were incubated with the radiolabeled probe (100,000 cpm) in a 50 ll reaction volume for 30 min at room temperature. Samples were electrophoresed on a 5% non-denaturing polyacrylamide gel and protein-DNA complexes were detected by autoradiography. Band intensities were quantified using ImageJ (Abramoff et al. 2004) and were normalized to the amount of binding seen with Nv-NF-jB-C. Luciferase Reporter Gene Assays A293 cells were co-transfected with 2 lg of FLAG-NvNF-jB expression vectors and 0.5 lg of a luciferase reporter plasmid containing multimerized jB-sites from the human MHC-1 promoter (Mitchell and Sugden 1995). Two days after transfection, lysates were prepared and subjected to western blotting with anti-Nv-NF-jB antiserum to normalize for equal expression of FLAG-Nv-NF-jB proteins. Luciferase activity of normalized lysates was measured with the Luciferase Assay System (Promega) as described previously (Sullivan et al. 2009; Wolenski et al. 2011). Animal Collection and Culture Surface sediment was collected from estuaries in Sippewissett, Massachusetts and Peggy’s Cove, Nova Scotia in the summer of 2009 and anemones were extracted with transfer pipettes as they emerged from the sediment (Supplementary Fig. S4). The anemones were stored in artificial seawater (Instant Ocean, Aquarium Systems, Inc) with salinities commensurate to that of their respective collection sites (*30 ppt for Peggy’s Cove and *12 ppt for Sippewisset). In the laboratory, Nematostella were fed freshly hatched Artemia nauplii twice weekly. For western blotting (Fig. 5b), the homozygous Nv-NF-jB-C anemone was from Carlstadt, NJ, the Nv-NF-jB-S animal was from Peggy’s Cove, Nova Scotia and the heterozygous Nv-NFjB-C/S animal was from Sippewissett, Massachusetts. Statistical Test for Hardy–Weinberg Equilibrium The numbers for the three genotypes of Nv-NF-jB (Cys/ Cys, Cys/Ser, or Ser/Ser) for animals sequenced from the Sippewissett (32 animals) and Peggy’s Cove (44 animals) populations were used to calculate allele frequencies. Observed allele frequencies were compared to the expected allele frequencies calculated under the assumption of Hardy–Weinberg equilibrium. Departure from Hardy– Weinberg equilibrium was calculated by a Chi-square test (1 degree of freedom) (Rodriguez et al. 2009).
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Phylogenetic Analysis of NF-jB Proteins For phylogenetic analyses, the RHD sequences of 14 NFjB and NFAT proteins from a broad range of taxa were used. The NFAT proteins have approximately 25% sequence identity to the RHD of Nv-NF-jB and were used as an outgroup. The sequences of these 14 RHD-containing proteins were manually trimmed to include the N-terminal DNA recognition loop and the C-terminal nuclear localization sequence. The sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) with the Gonnet protein weight matrix (gap opening penalty = 10; gap extension penalty = 0.1); the gapped alignment comprised 379 aa, contained 4,223 characters, and alignment gaps were treated as missing characters. Phylogenetic relationships among proteins were inferred from the full alignment using maximum-likelihood, Bayesian inference and neighbor-joining analyses. One hundred and twelve alternate models of aa substitution were compared using the program ProtTest 2.4 (Abascal et al. 2005) with the substitution process optimized along branch length and tree topology. The empirically determined top scoring model was the WAG matrix (Whelan and Goldman 2001). The overall likelihood of the model was improved by incorporating rate variation (the shape coefficient of the Gamma distribution, a = 0.305; the coefficient of rate variation among sites = 1/a1/2 = 1.811) as well as a portion of invariable aa among the sites (invariable proportion set to 0.04). The WAG matrix with the modifications of a gamma-distributed rate variation and an invariable proportion of aa among sites was specified in subsequent phylogenetic analyses. The phylogenetic relationships among RHD sequences were determined by three methods on two platforms. Two analyses were conducted using the Geneious v.5.5 portal (http://geneious.com): a maximum-likelihood analysis was performed with PhyML (Guindon and Gascuel 2003), and a Bayesian inference analysis was performed with MrBayes (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). For the third analysis, neighbor-joining was conducted using the MABL portal (Dereeper et al. 2008) with the pair-wise distances between sequences calculated with the Prodist program and tree topology determined with the BioNJ program (Gascuel 1997). Tree topologies were identical for the maximum-likelihood analysis with a gapped (Fig. 5a) and gap-free (Supplemental Fig. S7), and all presented phylogenies used a gapped alignment. For all analyses, 1,000 replicates of the bootstrap were performed to evaluate the support for specific clades, and trees were then visualized with FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Accession numbers for all sequences used can be found in Supplementary Table S2. Of note, the NF-jB proteins of
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Fig. 1 Nv-NF-jB-C and Nv-NF-jB-S have polymorphisms at amino c acid positions 67 and 269 that are predicted to influence the interaction energy of Nv-NF-jB with DNA. a Sequences surrounding the Nv-NF-jB polymorphisms at positions 67 and 269 were manually aligned with other NF-jB proteins. A black background indicates identical aa, whereas gray background indicates similar residues. Sequences are ordered from the earliest taxonomical branch (holozoan; Co) to the most recent (humans; Hs): Capsaspora owczarzaki, Co-NF-jB; Amphimedon queenslandica, Aq-NF-jB; Nematostella vectensis, Nv-NF-jB-C and Nv-NF-jB-S; Hydra magnipapillata, Hm-NF-jB; Acropora digitifera, Ad-NF-jB; Strongylocentrotus purpuratus, Sp-NF-jB; Drosophila melanogaster, Dm-Relish; and Homo sapiens, Hs-p50. Black arrows are above positions 67 and 269 of Nv-NF-jB. b A homodimer of Nv-NF-jB-S protein bound to a jB-site was computationally modeled using the crystal structure of the human NF-jB p50 homodimer as a template. Amino acid substitutions were made in the Nv-NF-jB-S protein model (S67/ E269) to create three other models (C67/E269, S67/A269, and C67/ A269). The interaction energy (kcal/mol) of residues 67 and 269 with the target DNA was determined. Measurements were calculated for the last 200 ps of a 1-ns simulation. For visual purposes, the average interaction energy in 5 ps intervals was graphed. c The mean and standard deviation (SD) of the interaction energies over the last 200 ps for the indicated proteins
C. owczarzaki and Hydra aligned poorly to other proteins and were not included in the phylogeny.
Results Identification of an Ala/Glu Polymorphism at aa 269 of Nv-NF-jB and Computational Modeling of Its Effect on DNA Binding The Nv-NF-jB-C and Nv-NF-jB-S alleles have 19 nucleotide differences within the 440 aa coding region. These nucleotide differences predict 10 aa changes (Supplementary Fig. S1), six of which are in the RHD (Supplementary Fig. S2). NF-jB proteins from many other species have a Cys residue at the position corresponding to aa 67 of Nv-NF-jB and this residue is part of a peptide loop that contacts DNA in human NF-jB p50 (Ghosh et al. 1995), which is the human protein most similar to Nv-NFjB. The Nv-NF-jB-C protein was previously shown to bind DNA more avidly than Nv-NF-jB-S (Sullivan et al. 2009). Reciprocal mutations that changed aa 67 to the corresponding residue from the other allele affected DNA binding: specifically, mutant Nv-NF-jB-C67S bound DNA stronger than the parental Nv-NF-jB-C protein and mutant Nv-NF-jB-S67C showed weaker binding than Nv-NF-jBS. Moreover, in several human NF-jB family proteins, a Cys-to-Ser change of the residue analogous to aa 67 increases DNA-binding activity (Garcı´a-Pineres et al. 2001; Liang et al. 2006b). Therefore, to explain the unexpected weaker DNA binding by full-length Nv-NFjB-S as compared to Nv-NF-jB-C, we hypothesized that a
second aa difference between the two Nv-NF-jB variants (in addition to the difference at aa 67) influences the strength of DNA binding. To identify a second candidate polymorphic residue that could affect DNA binding by Nv-NF-jB, we first aligned the aa sequences of the two Nv-NF-jB variants with NFjB proteins from other species. Based on this alignment, a second unusual polymorphic residue located at aa 269 of Nv-NF-jB was apparent (Fig. 1a). Most NF-jB proteins (e.g., Nv-NF-jB-C, sponge, coral, and human p50) have an Ala at the aa 269 position, and all of these proteins also have a Cys at residue 67. In contrast, the few NF-jB proteins with a residue other than Cys at the aa 67 position (Nv-NF-jB-S, Drosophila Relish [S], Hydra [T], and C. owczarzaki [S]) do not have an Ala at the aa 269 position,
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and indeed, they have no consistent residue at position 269. In the crystal structure of human p50, the Ala residue corresponding to Ala269 of Nv-NF-jB-C is part of a peptide loop that extends into the major groove of DNA (Ghosh et al. 1995). Seven of the ten aa of this peptide loop in human p50 are conserved in Nv-NF-jB-C, including the Ala at aa 269. None of the four other polymorphic residues in the RHD of Nv-NF-jB-C and Nv-NF-jB-S (Supplementary Fig. S2) is part of a region or sequence known to influence NF-jB activity. Therefore, based on these sequence analyses, we hypothesized that the Ala/Glu polymorphism at aa 269 would also affect DNA binding by Nv-NF-jB. We next used computational modeling to predict whether and how the Ala/Glu polymorphism at residue 269 of Nv-NF-jB might impact DNA binding. A solved crystal structure of the human p50 homodimer bound to DNA (Ghosh et al. 1995) was used as a template to model the Nv-NF-jB-S (S67/E269) protein bound to a jB-site. Amino acids were also substituted in Nv-NF-jB-S to generate protein models with the two single (Nv-NF-jBS67C and Nv-NF-jB-S-E269A) and one double (Nv-NFjB-S67C/E269A) substitutions. The energy of interaction of each of these four modeled proteins with DNA was then calculated for the last 200 ps of a 1-ns simulation (Fig. 1b). The interaction energy was lowest for Nv-NF-jB-SE269A, intermediate and similar for Nv-NF-jB-S and NvNF-jB-S67C/E269A, and highest for Nv-NF-jB-S67C (Fig. 1c). In this simulation, proteins with a low interaction energy are predicted to have a stronger DNA-binding ability (Pan and Nussinov 2011). Thus, these results predict that a Ser at aa 67 confers a stronger DNA-binding ability (as compared to Cys at 67), and that an Ala at aa 269 also imparts stronger DNA binding (as compared to Glu at aa 269). Moreover, the negative charge of a Glu residue at aa 269 likely causes an electrostatic repulsion with nucleotides in the major groove of DNA.
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Fig. 2 Nv-NF-jB aa 67 and 269 influence DNA binding. Lysates from A293 cells transfected with empty vector (-) or the indicated NvNF-jB proteins were used in an EMSA (top panel). Lysates are ordered for each allele (Nv-NF-jB-C: C67/A269, C67S/A269, C67/ A269E, and C67S/A269E; and Nv-NF-jB-S: S67/E269, S67C/E269, S67/E269A, and S67C/E269A). Relative DNA binding was determined by measuring the band intensities for each protein, and these values were then normalized to the value for Nv-NF-jB-C (100). The bottom panel shows an anti-Nv-NF-jB western blot of extracts used in the EMSA to confirm approximately equal expression of the indicated Nv-NF-jB proteins
vector. Extracts from transfected cells were then used in an EMSA with a jB-site probe from the Nv-IjB gene, which was shown previously to be bound by Nv-NF-jB (Sullivan et al. 2009; Wolenski et al. 2011). Similar to previous results, Nv-NF-jB-C (Fig. 2, top panel) bound DNA more avidly than Nv-NF-jB-S. The DNA-binding abilities of both Nv-NF-jB-C and Nv-NF-jB-S were strongest when the proteins had a Ser/Ala combination at positions 67/269 and were weakest with a Cys/Glu combination. Western blotting of extracts with anti-Nv-NF-jB antiserum confirmed equal expression of Nv-NF-jB proteins (bottom panel). These results indicate that Ser at aa 67 and Ala at aa 269 can independently and together confer increased DNA binding onto Nv-NF-jB.
Polymorphic Residues 67 and 269 Influence the DNABinding Activity of the Nv-NF-jB-C and Nv-NF-jB-S Proteins
Amino Acid Differences at Position 67 Affect jB-site Reporter Gene Activation by Nv-NF-jB
To determine experimentally whether residues 67 and 269 influence the DNA-binding activity of Nv-NF-jB proteins, a series of reciprocal single and double aa mutations were made (Fig. 2). Mutants of Nv-NF-jB-C (i.e., C67/A269) include C67S, A269E, and C67S/A269E. For Nv-NF-jB-S (i.e., S67/E269), mutants S67C, E269A, and S67C/E269A were created. Thus, for each Nv-NF-jB variant we had the protein encoded by the natural allele, as well as each single and double mutant at aa 67 and 269. The cDNAs encoding these eight Nv-NF-jB proteins were subcloned into pcDNA-FLAG expression vectors, and A293 cells were transfected with each expression
The eight Nv-NF-jB proteins described directly above were next analyzed in a jB-site luciferase reporter gene assay in A293 cells. The reporter values of the eight NvNF-jB proteins were normalized to the value of Nv-NFjB-C (1.0) (Fig. 3a, top panel). Similar to previous results (Sullivan et al. 2009), the Nv-NF-jB-S protein activated expression of the reporter gene 3.4-fold greater than NvNF-jB-C. Western blotting with anti-Nv-NF-jB antiserum showed that all proteins were expressed at approximately equal levels (Fig. 3a, bottom panel). To determine whether the aa differences at residues 67 and 269 have an effect on transcriptional activation,
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transactivation by these eight Nv-NF-jB proteins was compared based on the aa at residue 67 (Cys vs. Ser) or at residue 269 (Ala vs. Glu). Nv-NF-jB proteins with Ser at residue 67 activated transcription 2.5-fold stronger than those with a Cys at residue 67 (Fig. 3b). The presence of an Ala or a Glu at aa 269 did not correlate with the differences in the ability of Nv-NF-jB proteins to activate transcription (Fig. 3b). These results indicate that a Ser residue at aa 67 confers increased transactivation ability onto Nv-NF-jB as compared to Cys at this position.
Fig. 3 Nv-NF-jB residue 67 determines the strength of reporter gene activation. a A reporter assay using a multimeric jB-site luciferase reporter plasmid was performed in A293 cells. Cells were co-transfected with expression vectors for the indicated Nv-NF-jB proteins. The value above each bar is the average of three experiments performed with triplicate samples and is relative to the luciferase activity with the Nv-NF-jB-C (C67/A269) protein (1.0). The bottom panel shows an anti-Nv-NF-jB western blot of normalized extracts to confirm approximately equal expression of the indicated Nv-NF-jB proteins. b Relative luciferase values were compared based on the aa at residue 67 (Cys vs. Ser) or 269 (Ala vs. Glu) and were normalized to 1.0 for proteins with a Cys at aa 67 or Ala at aa 269. Error bars indicate standard error. Above each dashed line are values for a Student’s t test (two-tailed, paired) for differences in the activation values of the Nv-NF-jB proteins with changes at residues 67 or 269. *The difference in activation values of the Cys vs. Ser comparison at residue 67 was statistically significant (P = 5.5 9 10-14)
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Nv-NF-jB-C and Nv-NF-jB-S Show Equal Abilities to Form Dimers and Interact with the Inhibitor Proteins Nv-IjB and Nv-Bcl-3 To compare the abilities of the two natural Nv-NF-jB variants to form homo- and heterodimers, A293 cells were co-transfected with two types of expression plasmids: (1) Myc-Nv-NF-jB-C or Myc-Nv-NF-jB-S; and (2) FLAGNv-NF-jB-C or FLAG-Nv-NF-jB-S. Whole-cell lysates were immunoprecipiated with anti-Myc antiserum and samples were analyzed by western blotting with either antiFLAG or anti-Myc antiserum (Fig. 4a). Homodimers of
Fig. 4 Nv-NF-jB-C and Nv-NF-jB-S form homo- and heterodimers and interact with Nv-IjB and Nv-Bcl-3 with equal avidity. a A293 cells were transfected with the indicated Myc- or FLAG-tagged NvNF-jB expression vectors. Whole-cell lysates were immunoprecipitated (IP) with anti-Myc antiserum (left panels) and then western blotted (WB) with anti-FLAG (top panels) or anti-Myc (bottom panels) antiserum. The right panels show western blots of the input lysates from whole-cell extracts. b Myc-Nv-Nv-IjB and FLAG-NvNF-jB-C or FLAG-Nv-NF-jB-S were coexpressed in A293 cells and analyzed by co-immunoprecipitation assays (as in a). Lysates were immunoprecipitated with anti-Myc antiserum and then western blotted with anti-FLAG antiserum. c Myc-Nv-Bcl-3 and FLAG-NvNF-jB-C and FLAG-Nv-NF-jB-S were co-expressed in A293 cells and analyzed by co-immunoprecipitation assays (as in b)
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FLAG-Nv-NF-jB-C (upper left panel, lane 2) and FLAGNv-NF-jB-S (upper left panel, lane 4) were detected at approximately equal levels in the appropriate anti-Myc immunoprecipitates. Heterodimers of Nv-NF-jB-C and Nv-NF-jB-S (upper left panel, lanes 3 and 5) were also detected at approximately equal levels. A western blot of whole-cell lysates used as input controls confirmed that Myc- and FLAG-tagged Nv-NF-jB proteins were expressed in the appropriate cell extracts (right panels). FLAG-Nv-NF-jB was detected only in anti-Myc immunoprecipitates in which Myc-Nv-NF-jB was co-expressed (Supplementary Fig. S3). Nv-IjB has previously been shown to interact with NvNF-jB-S, to sequester it in the cytoplasm, and to block its ability to activate expression of a jB-site reporter gene in A293 cells (Wolenski et al. 2011). To determine whether Nv-IjB can also interact with Nv-NF-jB-C, A293 cells were co-transfected with expression vectors for Myc-NvIjB and for either FLAG-Nv-NF-jB-C or FLAG-Nv-NFjB-S. Lysates were immunoprecipitated with anti-Myc antiserum and samples were analyzed by western blotting with either anti-FLAG or anti-Myc antiserum. FLAG-NvNF-jB-C and FLAG-Nv-NF-jB-S were detected at approximately equal levels in the appropriate the anti-Myc (IjB) immunoprecipitates (Fig. 4b, upper left panel), and western blots of whole-cell lysates confirmed that the MycIjB and FLAG-Nv-NF-jB proteins were expressed in the appropriate cell extracts (right panels). Like Nv-IjB, Nv-Bcl-3 has been shown to interact with and inhibit the transactivation ability of Nv-NF-jB-S (Wolenski et al. 2011). To compare the abilities of Nv-NFjB-S and Nv-NF-jB-C to interact with Nv-Bcl-3, coimmunoprecipitations were performed as described above for Nv-IjB. A293 cells were co-transfected with expression vectors for Myc-Nv-Bcl-3 and either FLAG-Nv-NFjB-C or FLAG-Nv-NF-jB-S. By anti-FLAG western blotting, FLAG-Nv-NF-jB-C and FLAG-Nv-NF-jB-S were detected at approximately equal levels in the antiMyc (Nv-Bcl-3) immunoprecipitates (Fig. 4c, upper left panel). The results presented in this section indicate that NvNF-jB-C and Nv-NF-jB-S do not differ in their abilities to form dimers or to interact with the Nv-IjB and Nv-Bcl-3 inhibitor proteins. The Distribution of the Nematostella NF-jB-C and NF-jB-S Alleles is Geographically Uneven A fragment of the Nv-NF-jB gene from 403 animals collected on the Atlantic and Pacific coasts of North America as well as the southern coast of Great Britain was previously genotyped by sequencing of genomic DNA (Sullivan et al. 2009). Of these genotyped animals, approximately
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77% were homozygous Nv-NF-jB-C, 6% were homozygous Nv-NF-jB-S, and 17% were heterozygous. However, in specific geographic areas, these allele ratios varied significantly from the overall allelic distribution. For example, 94% of anemones collected from Sippewissett, Massachusetts were homozygous for Nv-NF-jB-C, whereas 89% of anemones from Peggy’s Cove, Nova Scotia were homozygous for Nv-NF-jB-S. To further investigate this skewed distribution of alleles, additional anemones were collected from these two estuaries (Supplementary Fig. S4) and were genotyped. As shown in Table 1, over 95% (31/ 32) of the newly collected animals from Sippewissett were homozygous for Nv-NF-jB-C, with a single heterozygote identified. In contrast, animals from Peggy’s Cove were almost exclusively homozygous for Nv-NF-jB-S (43/44), with one homozygous Nv-NF-jB-C anemone present. Only the observed frequencies of Nv-NF-jB alleles in the Peggy’s Cove population significantly depart from the frequencies expected under Hardy–Weinberg equilibrium. Phylogenetic Analysis Suggests that Nv-NF-jB-S is a Derived Allele Previous phylogenetic analysis of NF-jB homologs across a broad range of species indicated that Nv-NF-jB-S was basal to vertebrate NF-jB proteins (Sullivan et al. 2007). Here, we performed maximum-likelihood (Fig. 5a), Bayesian inference (Supplementary Fig. S5), and neighborjoining (Supplementary Fig. S6) analyses with additional NF-jB sequences including the Nematostella NF-jB-C allele and homologs from sponge and other cnidarians. The NFAT proteins, including a single Nematostella sequence, were used as an outgroup for the NF-jB phylogenetic analysis. By the maximum-likelihood and Bayesian inference analyses, NF-jB of the sponge A. queenslandica appears as the sister branch to the cnidarian/vertebrate NFjB groups (Fig. 5a; Supplementary Fig. S5). However, in Table 1 Uneven distribution of Nv-NF-jB alleles in two locations along the northeastern coast of North America Genotype
Allele frequencies
Location
n
Cys/Cys
Cys/Ser
Sippewissett
32
31
1
Peggy’s Cove*
44
1
0
Ser/Ser
Cys
Ser
0
0.98
0.02
43
0.02
0.98
Anemones were collected from estuaries in Sippewissett, Massachusetts, and Peggy’s Cove, Nova Scotia in August 2009. A PCR product of the Nv-NF-jB gene surrounding codon 67 was sequenced to determine genotype * By a Chi-square test (1 degree of freedom), the observed distribution of Nv-NF-jB alleles in the Peggy’s Cove population departs from the expected distribution under Hardy–Weinberg equilibrium (P \ 0.001)
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the six polymorphic residues of Nv-NF-jB within the RHD to other cnidarian NF-jB proteins shows that the Nv-NFjB-C variant has four residues that are conserved in all other cnidarians NF-jB proteins whereas the Nv-NF-jB-S allele has only one (Supplementary Table S3). Expression of cDNAs for the two Nv-NF-jB alleles in A293 cells results in proteins with distinct mobilities on SDS-polyacrylamide gels, with Nv-NF-jB-C migrating more slowly than Nv-NF-jB-S [(Sullivan et al. 2009); see also Fig. 2)]. Western blotting confirmed that anemones homozygous or heterozygous for the Nv-NF-jB alleles express proteins that co-migrate with proteins synthesized by the corresponding cDNAs in A293 cells (Fig. 5b).
Discussion
Fig. 5 Phylogenetic analysis and functional characterization of the Nv-NF-jB-C and Nv-NF-jB-S alleles. a The aa sequences of 14 proteins were aligned and a maximum-likelihood phylogenetic tree was built using PhyML. PhyML determined the best tree based on the log -6723.45607. The values for the maximum likelihood tree are shown above the branched nodes. A Bayesian inference was also performed (see Supplemental Fig. S5) and values for this analysis are shown below the branched nodes. One thousand bootstraps were performed for each analysis. The NFATs were used as an outgroup to root the tree. Ad, Acropora digitifera; Am, Acropora millepora; Aq, Amphimedon queenslandica; Cr, Carcinoscorpius rotundicauda; Dm, Drosophila melanogaster; El, Edwardsiella lineata; Hs, Homo sapiens; Mm, Mus musculus; Nv, Nematostella vectensis; Sp, Strongylocentrotus purpuratus. For the scale bar, the length of a horizontal branch is proportional to the number of aa substitutions that have occurred along that branch. The NF-jB transcript in the lined sea anemone Edwardsiella was identified through a tblastn search of mRNA contigs using Nv-NF-jB-C and Nv-NF-jB-S as query sequences. Details of sequencing and assembly of the Edwardsiella transcriptome are being published elsewhere. b AntiNv-NF-jB western blotting of A293 cells transfected with empty vector (lane 1), pcDNA-Nv-NF-jB-C (lane 2), pcDNA-Nv-NF-jB-S (lane 3) or extracts from adult Nematostella genotyped as either homozygous Nv-NF-jB-C (lane 4), homozygous Nv-NF-jB-S (lane 5) or heterozygous Nv-NF-jB-C/Nv-NF-jB-S (lane 6). Nv-NF-jB proteins are indicated by an arrow. Nv-NF-jB-C has reduced migration as compared to the Nv-NF-jB-S protein
all three analyses, the two Nv-NF-jB alleles group with the other cnidarian homologs and form a distinct clade separate from the vertebrate NF-jB proteins that has strong bootstrap support (99.8%). Although the difference is small, by maximum-likelihood analysis the distance (aa substitutions per site) between A. queenslandica NF-jB and Nv-NF-jBC (0.685) is shorter than to Nv-NF-jB-S (0.696), suggesting that the Nv-NF-jB-C aa sequence is more similar to that of the ancestral NF-jB. Furthermore, comparison of
In this work, we have characterized polymorphic aa residues that are responsible for the differences in activities between naturally occurring variants of the Nematostella NF-jB protein. Our results show that polymorphic residues at positions 67 and 269 appear to be the primary residues that account for the DNA-binding differences between the two proteins. For strong DNA binding, a Ser residue at aa 67 is preferred over Cys, while Ala is preferred over Glu at aa 269. The Cys67/Ala269 configuration is the one that occurs in the majority of NF-jB proteins across many species, including human p50, and is the combination that is found in the most common allele in natural populations of Nematostella (Sullivan et al. 2009). Each natural NvNF-jB variant has one residue at position 67 or 269 that increases DNA binding and one polymorphic residue that decreases it, such that the overall DNA-binding activities of the Nv-NF-jB-C and Nv-NF-jB-S proteins are at a similar intermediate level. Thus, the combination of residues that are present at residues 67 and 269 might be necessary for proper biological function. Among the six polymorphic aa in the RHD of Nv-NFjB, only those at positions 67 and 269 occur in areas of high sequence conservation (Supplementary Fig. S2). All NF-jB proteins that have a Cys at aa 67 also have an Ala at aa 269 (human, sea urchin S. purpuratus, Nv-NF-jB-C, coral A. digitifera, sponge A. queenslandica). In contrast, NF-jB proteins with a Ser or Thr at aa 67 do not have Ala at aa 269 (Drosophila, Nv-NF-jB-S, H. magnipapillata, C. owczarzaki). Therefore, NF-jB proteins with a Ser or Thr at the 67 position appear to require a second compensatory change at the 269 residue, perhaps to maintain DNAbinding activity in a moderate range. The presence of the Cys/Ala combination at the 67/269 positions in most NFjB proteins and the occurrence of compensatory changes at these positions in a select few NF-jB proteins suggest a conserved interplay of these two residues through
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evolution. Of note, the p52/p100 type of NF-jB proteins uniformly have Cys/Ser at these two positions (Supplementary Table S1) and the phosphorylation state of the Ser residue affects the dimerization ability of p52 (Barre´ and Perkins 2010). The residues in human p50 that are analogous to aa 67/269 in Nv-NF-jB are part of peptide loops that interact with DNA (Ghosh et al. 1995). Cys-to-Ser changes at positions analogous to aa 67 have also been shown to increase DNA-binding activity in certain human NF-jB family proteins, including RelA and c-Rel (Garcı´a-Pineres et al. 2001; Liang et al. 2006a, b). However, we are the first to characterize the effects on DNA binding of mutations at the residue analogous to Ala269 in Nv-NF-jB. In human p50, residues flanking the analogous Ala (conserved Lys268 and Pro270 in Nv-NF-jB) make direct contact with DNA (Ghosh et al. 1995). Thus, a Glu at aa 269 in Nv-NFjB may disrupt the function of this peptide loop by creating a steric hindrance or an electrostatic repulsion. In our computational model of Nv-NF-jB, the Cys/Glu configured protein has a positive interaction energy (Fig. 1c) and is not predicted to form strong interactions with DNA. This prediction is supported by our EMSA data, wherein NvNF-jB-C/A269E binds DNA poorly (Fig. 2a). Reporter gene assays with wild-type and mutant Nv-NFjB proteins indicate that residue 67 influences transactivation. Nv-NF-jB proteins with a Ser at position 67 activated the reporter gene 2.5-fold greater than those with a Cys. In contrast, the polymorphism at residue 269 did not significantly affect transcriptional activation (Fig. 3b), even though residue 269 influences DNA binding. We have three hypotheses to explain why Ser at aa 67 enables Nv-NF-jB to activate transcription more strongly than Nv-NF-jB proteins with a Cys at this position: (1) Ser is favored for interaction with a co-factor involved in transcriptional activation; (2) Ser at aa 67 causes Nv-NF-jB to have a conformation on DNA that is more favorable for transactivation; and/or (3) Ser at aa 67 changes some property of DNA binding (e.g., on–off rate) that influences transactivation. The DNAbinding affinity of transcription factors is not necessarily correlated with transactivation ability (Fujita et al. 1992; Perkins 1997). Furthermore, the conformation of p50 dimers on DNA has been shown to affect their ability to activate transcription (Fujita et al. 1992). Of note, Relish also has a Ser at the residue analogous to aa 67, and Relish homodimers, like Nv-NF-jB homodimers, are effective activators of transcription (Ertu¨rk-Hasdemir et al. 2009; Wolenski et al. 2011), whereas human NF-jB p50 and p52 homodimers (with Cys at this position) do no activate transcription. The aa differences between the two Nv-NF-jB variants do not appear to affect the ability of the Nv-NF-jB-C and Nv-NF-jB-S proteins to form homodimers or heterodimers (Fig. 4a) or to bind the Nv-IjB (Fig. 4b) and Nv-Bcl-3
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(Fig. 4c) inhibitor proteins. Thus, we would not expect animals heterozygous for the two Nv-NF-jB alleles to have different regulation of NF-jB. Nevertheless, we did not find any heterozygous animals in Peggy’s Cove, suggesting that heterozygous animals are selected against in some situations or that sexual reproduction is limited in this small estuary. There is a preponderance of animals homozygous for the rare Nv-NF-jB-S allele in Peggy’s Cove, Nova Scotia, where the ratio of the two alleles also deviates significantly from Hardy–Weinberg equilibrium (Table 1). This suggests at least three possibilities: (1) that animals from Peggy’s Cove were collected in a non-random manner; (2) that most animals in Peggy’s Cove were derived from a rare founder effect possibly coupled with genetic drift (and animals do not easily mix sexually); and/or (3) that animals homozygous for the Nv-NF-jB-S allele were selected due to a local environmental condition. In terms of animal collection, buckets of sediment were collected from five distinct pools at Peggy’s Cove and anemones were collected as they emerged from the sediment. Thus, we believe that animals were collected randomly from Peggy’s Cove. Therefore, it is likely that the preponderance of the Nv-NF-jB-S allele in Peggy’s Cove is due either to a founder effect or to an environmental selective pressure. A founder effect has been described in Nematostella populations along the southern coast of England (Reitzel et al. 2008), demonstrating that such an effect can occur in natural populations. In terms of an environmental pressure that might have led to selection of the Nv-NF-jB-S allele, the DNA-binding ability of the Nv-NF-jB-S is less sensitive to inhibition by thiol-reactive compounds and to oxidative conditions than the Nv-NF-jB-C protein (Sullivan et al. 2009). Thus, anemones with the Nv-NF-jB-S allele may have tolerated unfavorable environmental conditions better than those with the Nv-NF-jB-C allele. The sponge A. queenslandica belongs to the earliest branch of multicellular animals known to have NF-jB. By phylogeny, the A. queenslandica NF-jB forms a sister branch to the cnidarian and vertebrate groups (Fig. 5a). A. queenslandica NF-jB is more similar to Nv-NF-jB-C than to Nv-NF-jB-S by both sequence homology and the conservation of residues 67 and 269. This suggests that NvNF-jB-C is the ancestral form of the Nematostella protein and thus, that Nv-NF-jB-S is the derived allele. By maximum likelihood, the distance between the two Nv-NF-jB alleles (0.011) is comparable to that between the NF-jB p50 proteins of mouse and human (0.018). Indeed, there are six aa differences between the RHDs of the two Nematostella NF-jB variants, which is identical to the number of aa differences between the two mammalian NF-jB p50 proteins, even though mice and humans are
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thought to have diverged 100 million years ago (Nei et al. 2001). Nevertheless, there are 110 nucleotide differences between the mouse and human p50 RHDs, whereas the two Nematostella alleles have only 12 nucleotide differences. The greater number of nucleotide differences between the mouse and human p50 RHD sequences is consistent with the larger evolutionary distance between mice and humans as compared to individual animals within the same Nematostella species. Additionally, the large number of synonymous nucleotide changes between the mouse and human p50 coding sequences suggests a requirement for a high level of aa conservation between the two mammalian NF-jB proteins. The relatively low ratio of nucleotide to aa differences (12:6) between the two Nv-NF-jB alleles is more consistent with rapid aa changes being favored as an adaptation to an environmental condition(s) than the more gradual synonymous nucleotide changes that would be expected to occur due to genetic drift (Yang and Bielawski 2000).
Conclusions Our results show that polymorphic residues in two Nematostella NF-jB allelic variants affect their DNA-binding and transcriptional activation abilities. Specifically, we show that residues 67 and 269 have compensatory effects on DNA binding in the natural Nv-NF-jB-C and Nv-NF-jB-S proteins. The limited distribution of the Nv-NF-jB-S allele in nature, the variation of this allele from Hardy–Weinberg equilibrium, and its phylogenetic position as the derived allele are all consistent with either a founder effect or selective pressure leading to expansion of the Nv-NF-jB-S allele in local populations. Future studies will be aimed at identifying biological processes controlled by Nv-NF-jB, which may shed light on the influences that led to the appearance of the two alleles in nature. Acknowledgments This research was supported by grant MCB0920461 from the National Science Foundation (J.R.F., T.D.G.) and ARRA supplement CA047763-22S3 (to T.D.G.). F.S.W. was supported by predoctoral grant by the Superfund Basic Research Program at Boston University 5 P42 ES07381, and F.S.W. and D.J.S. were supported by Warren-McLeod Fellowships. N.J. was supported by funds from the Boston University Undergraduate Research Opportunities Program. We thank Tristan Lubinski and Lauren Friedman for help with bioinformatic analyses and helpful discussions.
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