Separation-of-function mutants reveal the NF-κB-independent involvement of IκBα in the regulation of intestinal stemness [RNAseq Knock-ins IKBa]

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-κBrelated (SOF DeltaNF-κB) or chromatin-related (SOF DeltaH2A,H4) activities. Expression of IκBα SOF DeltaNF-κB, but not SOF DeltaH2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by IκBα deficiency. . By ChIP assay we demonstrated IκBα binding to several stem cell genes that are transcriptionally repressed following IκBα SOF DeltaNF-κB induction. Our data indicate that SOF mutants represent an exclusive tool for studying IκBα functions in physiology and disease.