Abstract We previously demonstrated that the NF-κB inhibitor IκBα binds the chromatin together with PRC2 to regulate a subset of developmental- and stem cell-related genes. This alternative function has been elusive in both physiological and disease conditions because of the predominant role of IκBα as a negative regulator of NF-κB. We here uniquely characterize specific residues of IκBα that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-κB-related (SOF ΔNF-κB ) or chromatin-related (SOF ΔH2A,H4 ) activities. Expression of IκBα SOF ΔNF-κB , but not SOF ΔH2A/H4 , is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by IκBα deficiency. In contrast, full IκBα activity is required for regulating clonogenic/tumor-initiating activity of colorectal cancer cells. Our data indicate that SOF mutants represent an exclusive tool for studying IκBα functions in physiology and disease, and identified IκBα as a robust prognosis biomarker for human cancer.

Separation-of-function mutants reveal the NF-κB-independent involvement of IκBα in the regulation of stem cell and oncogenic programs