Comparison of NF-kappa B from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

Transcription factor NF-kappa B is a key regulator of immunity in mammals, but its function in protists like Capsaspora and choanoflagellates is not known. Here, Leah Williams et al. characterize and compare the structure, activity, and regulation of NF-kappa B from Capsaspora and one choanoflagellate, providing further insight into the origins of NF-kappa B. We provide a functional characterization of transcription factor NF-kappa B in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-kappa B in two protists using phylogenetic, cellular, and biochemical techniques. NF-kappa B of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-kappa B proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-kappa B to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-kappa B is not induced by I kappa B kinases in human cells. Overexpressed Co-NF-kappa B localizes to the cytoplasm in Co cells. Co-NF-kappa B mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-kappa B. Three NF-kappa B-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-kappa B proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.