Separation-of-function mutants reveal the NF-?B-independent involvement of I?Ba in the regulation of intestinal stemness [RNAseq Knock-ins IKBa]
We previously demonstrated that the NF-?B inhibitor I?Ba 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 I?Ba as a negative regulator of NF-?B. We here uniquely characterize specific residues of I?Ba that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-?Brelated (SOF DeltaNF-?B) or chromatin-related (SOF DeltaH2A,H4) activities. Expression of I?Ba SOF DeltaNF-?B, but not SOF DeltaH2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by I?Ba deficiency. . By ChIP assay we demonstrated I?Ba binding to several stem cell genes that are transcriptionally repressed following I?Ba SOF DeltaNF-?B induction. Our data indicate that SOF mutants represent an exclusive tool for studying I?Ba functions in physiology and disease. Overall design: We performed RNA-seq on Knock-in I?Ba-SOF samples, in untreated and TNF-treated conditions: (2x conditions) 3x (replicates) I?Ba WT, (2x) 3x I?Ba N108 mutant and (2x) 3x I?Ba M3 mutant (triple H84, E85, E86 to A mutant). All samples are derived from HCT116 cell line.