The transcription factor NF-κB regulates a broad range of biological processes including innate and adaptive immunity, cell growth, differentiation, apoptosis, and tumorigenesis 
. In addition, NF-κB is involved in mediating cellular responses to numerous environmental stressors. Stressors that can activate the NF-κB pathway in insects and vertebrates include pathogens, ultraviolet light, oxidative stress, and shear stress. Given that the same stressors can activate the NF-κB pathway in insects and vertebrates, the role of NF-κB in combating stress must predate the radiation of triploblastic animals, a process that was already well underway during the Cambrian explosion (542–525 million years ago).
The NF-κB signaling pathway is primarily controlled by subcellular location. In response to an appropriate stressor or stimulus, NF-κB is released from a latent cytoplasmic state and enters the nucleus to activate the transcription of a diverse set of effector genes including ones encoding antimicrobial peptides, mucin, heat-shock factors, and anti-oxidant proteins 
. Target genes of NF-κB contain DNA-binding sites (‘κB sites’) in their promoters/enhancers.
NF-κB binds to a κB site as a dimer via sequences in the conserved DNA-binding/dimerization domain called the Rel homology domain (RHD) 
. The presence of an RHD characterizes a superfamily of proteins that includes the Rel/NF-κB family and the NFAT family. Multiple RHD proteins have been identified in the genomes of individual protostomes and deuterostomes. The human genome encodes ten RHD proteins (NF-κB1, NF-κB2, Rel, RelA, RelB and NFAT1-5), and the Drosophila
genome encodes four RHD proteins (Dorsal, Dif, Relish, and NFAT). No RHD proteins are encoded in the sequenced genomes of fungi or the choanoflagellate Monosiga brevicolis
, suggesting that the RHD domain originated early in metazoan evolution 
. A single RHD-containing NF-κB-like protein was identified in the demosponge Amphimedon queenslandica 
; however, no NFAT-like protein has yet been found in Amphimedon
or any other sponge. The presence of distinct NF-κB and NFAT proteins in two cnidarians—the sea anemone Nematostella vectensis
and the coral Acropora millepora—
indicates that the RHD proteins had begun to diversify prior to the cnidarian-triploblast divergence, which occurred ~700 million years ago 
NF-κB dimers bind to DNA with high affinity and make multiple contacts with the κB site 
. One highly conserved element required for DNA binding by all NF-κB proteins is a DNA recognition loop, which has the consensus sequence RFRYXC
EG. Almost all known members of the Rel/NF-κB subfamily, including the NF-κBs of Amphimedon
, have Cys at position 6 of this sequence (; Figure S1
). Only the Relish protein in several insects and the previously reported Nematostella
NF-κB protein (Nv-NF-κB) have Ser at position 6 
. All known NFAT proteins have Thr at this position ().
Phylogenetic relationships among major metazoan lineages and sequence of the DNA recognition loop in Rel Homology Domain proteins.
In some human RHD-containing proteins, a Cys-to-Ser mutation at position 6 of the DNA recognition loop has functional consequences. A Cys-to-Ser mutation at this position in human RelA or c-Rel greatly increases the DNA-binding activity of each protein 
. In addition, certain thiol-reactive compounds can inhibit DNA binding by wild-type Cys-containing RelA or c-Rel, but the same compounds do not affect the corresponding Cys-to-Ser mutants 
. Furthermore, conversion of the Cys to Ser can render NF-κB proteins resistant to redox regulation. The thiol group in the Cys of the DNA recognition loop has been shown to be important for DNA binding 
, with this residue (Cys62 in human p50) being a known site of redox control 
In this paper, we show that two Nv-NF-κB alleles encoding proteins with different DNA-binding and transactivation properties are present in wild Nematostella populations. Both alleles are widely distributed along both the Atlantic and Pacific coasts of the US. This is the first demonstration of a highly prevalent functional polymorphism within an NF-κB protein in any species. The functional differences between these variants suggest that departures from Hardy-Weinberg equilibrium observed in natural populations are due, at least partially, to selection acting on this locus.