Comparison of activities of transcription factor NF-κB from two jellyfish models

• The immune transcription factor NF-κB pathway is present in jellyfish. • Activities and regulation of two jellyfish NF-κB proteins are compared and contrasted. • Alphafold3-based structures of jellyfish NF-κB proteins are generated. • Expression of NF-κB mRNA is characterized through development and in single cell analysis. Herein, transcription factor Nuclear Factor-kappaB (NF-κB) is characterized as the downstream effector of putative innate immune signaling pathways from two model jellyfish, Aurelia aurita ( Aa ) and Clytia hemisphaerica ( Ch ). Both jellyfish NF-κB proteins consist primarily of the N-terminal DNA-binding/dimerization domain, and they lack the C-terminal ankyrin repeat inhibitory domain found in vertebrate NF-κB proteins. Both jellyfish NF-κB proteins bind to a consensus mammalian NF-κB binding site, and their AlphaFold3-predicted structures on DNA are similar to that of mouse NF-κB p50. Neither Aa - nor Ch -NF-κB activated transcription of an NF-κB-site reporter gene in human cells; however, Aa -NF-κB did activate transcription from a GAL4-site reporter gene in yeast, whereas Ch -NF-κB did not. Aa - and Ch -NF-κB were both constitutively located in the nucleus when expressed in vertebrate cells. Homologs of IκB-like proteins from both species interacted with their corresponding NF-κB proteins in co-immunoprecipitation assays in HEK 293T cells. These IκB-like proteins influenced the subcellular localization of their cognate NF-κB proteins in vertebrate cells, and their ankyrin repeat domains were predicted to interact with Ch -NF-κB in a manner similar to mammalian IκB and NF-κB. RNA-sequencing data from Ch animals indicate that Ch -NF-κB is expressed at high levels in early developmental stages, when Ch -IκB expression is low, suggesting that active Ch -NF-κB controls an early developmental process. In contrast, in adult animals the expression of Ch -IκB is high, suggesting that Ch -NF-κB requires a signal in order to become active. Overall, these results provide comparative information on the structure, activity, and mRNA expression of jellyfish NF-κB pathway proteins among jellyfish, and they suggest roles for NF-κB in developing and adult jellyfish.