The NF-kappaB signaling pathway of the sea anemone Nematostella vectensis: Functional characterization of core elements and two naturally occurring polymorphisms

The NF-kappaB family of eukaryotic transcription factors is activated in response to many environmental and biological stresses and is required for several important developmental processes. The sea anemone Nematostella vectensis (Nv) is the leading developmental and genomic model for the phylum Cnidaria, which includes anemones, hydras, jellyfish and corals. Sequencing of the Nematostella genome revealed sequences encoding NF-kappaB pathway proteins, including homologs of NF-kappaB, IkappaB, Bcl-3 and IKK. The goal of this research was to characterize molecular and biological functions of the Nv-NR-kappaB pathway proteins, in part because Nematostella is a model organism in which a framework for understanding the evolutionary origins of NF-kappaB signaling could be established.;In this thesis, cDNAs for Nv-NF-kappaB pathway proteins were cloned and characterized. Nv-NF-kappaB is shown to bind to kappaB sites and activate transcription of reporter genes containing multimeric kappaB sites. Nv-NF-kappaB is localized in the nucleus of cells in developing Nematostella embryos and is expressed in the cytoplasm of a subset of ectodermal cells in juvenile and adult Nematostella. When over-expressed in human A293 cells, Nv-IkappaB and Nv-Bcl-3 can both interact with Nv-NF-kappaB and block its ability to activate reporter gene expression. Nv-IKK is most similar to human IKK&egr;/TBK kinases, and can phosphorylate Nv-IkappaB in vitro. These results indicate that the core NF-kappaB pathway proteins in Nematostella are similar to their vertebrate homologs.;This thesis also describes two geographically dispersed, naturally occurring NF-kappaB alleles in Nematostella. Proteins encoded by these two alleles, Nv-NF-kappaB-C and Nv-NF-kappaB-S, are polymorphic at key amino acids and have differing activities. Polymorphic residue 67 contributes to the difference between the two Nv-NF-kappaB variants in reporter gene transactivation. Computational modeling and electrophoretic mobility shift assays indicate that polymorphic positions 67 and 269 are responsible for differences in DNA binding of the two NF-kappaB variants. Additionally, the DNA-binding activity of Nv-NF-kappaB-C is more sensitive to redox conditions and thiol-reactive compounds than is the activity of Nv-NP-kappaB-S. These results suggest that environmental conditions, such as oxidative stress, have driven the divergence of the two alleles.