Separation-of-function mutants reveal the NF-kB-independent involvement of IkBα in the regulation of intestinal stemness

We have previously shown that the NF-κB inhibitor IkBa binds the chromatin together with PRC2 to confer responsiveness to PRC2 targets in the presence of inflammatory cues. This alternative function has been elusive in both physiological and disease conditions because of the predominant role of IkBa as a negative regulator of NF-κB. Here, we uniquely characterize the specific residues of IkBa that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-κB-related (SOF NF-κB) or chromatin-related (SOF H2A,H4) activities. Expression of IkBa SOF NF-κB, but not SOF H2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thereby rescuing the differentiation block imposed by IkBa deficiency. By ChIP assay, we demonstrated that IkBa binds to several stemness genes that are transcriptionally repressed upon IkBa SOF NF-κB induction. Our data suggest that SOF mutants provide an exclusive tool for studying IkBa functions in physiology and disease.