Stepwise neofunctionalization of the NF-κB family member c-Rel during vertebrate evolution

Abstract

Adaptive immunity and the five vertebrate NF-{kappa}B/Rel family members first appeared in cartilaginous fish, suggesting that divergence and specialization within the NF-{kappa}B family helped facilitate the evolution of adaptive immunity. One specialized function of the NF-{kappa}B c-Rel protein in macrophages is the activation of Il12b, which encodes a key regulator of T-cell development. We found that c-Rel is a far more potent regulator of Il12b than of any other inducible genes in macrophages, with c-Rel regulation of Il12b dependent on its heightened intrinsic DNA-binding affinity. c-Rel homodimers regulate Il12b transcription in part via motifs with little resemblance to canonical NF-{kappa}B motifs. ChIP-seq experiments further defined distinct c-Rel DNA-binding preferences genome-wide, and X-ray crystallography of a c-Rel/RelA chimeric protein identified key amino acid changes that support the unique c-Rel properties. Unexpectedly, these changes, along with the c-Rel/RelA binding affinity differences, were largely restricted to mammalian species. Together, our findings reveal how a transcription factor family member can undergo a structural transition at a late stage of vertebrate evolution, resulting in an increased intrinsic DNA binding affinity and with clear functional consequences, presumably to support the increasing complexity of immune regulation.