We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB 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-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB 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.

Heterotrophy has been shown to mitigate coral–algal dysbiosis (coral bleaching) under heat challenge, but the molecular mechanisms underlying this phenomenon remain largely unexplored. Here, we quantified coral physiology and gene expression of fragments from 13 genotypes of symbiotic Oculina arbuscula after a 28-d feeding experiment under (1) fed, ambient (24 °C); (2) unfed, ambient; (3) fed, heated (ramp to 33 °C); and (4) unfed, heated treatments. We monitored algal photosynthetic efficiency throughout the experiment, and after 28 d, profiled coral and algal carbohydrate and protein reserves, coral gene expression, algal cell densities, and chlorophyll-a and chlorophyll-c2 pigments. Contrary to previous findings, heterotrophy did little to mitigate the impacts of temperature, and we observed few significant differences in physiology between fed and unfed corals under heat challenge. Our results suggest the duration and intensity of starvation and thermal challenge play meaningful roles in coral energetics and stress response; future work exploring these thresholds and how they may impact coral responses under changing climate is urgently needed. Gene expression patterns under heat challenge in fed and unfed corals showed gene ontology enrichment patterns consistent with classic signatures of the environmental stress response. While gene expression differences between fed and unfed corals under heat challenge were subtle: Unfed, heated corals uniquely upregulated genes associated with cell cycle functions, an indication that starvation may induce the previously described, milder “type B” coral stress response. Future studies interested in disentangling the influence of heterotrophy on coral bleaching would benefit from leveraging the facultative species studied here, but using the coral in its symbiotic and aposymbiotic states.

In this report, we investigate the evolution of transcription factor NF-κB by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-κB has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-κB rather than Rel proteins. As with mammalian NF-κB proteins, removal of the ANK repeats is required for Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. We also show that three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, and lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-κB in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor.