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IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments. We want to thank the Bigas’ and Espinosa’s lab members for constructive discussions and suggestions and technical support. This work has been funded by Instituto de Salud Carlos III FEDER (PI19/0013), Agencia Estatal de Investigación, Spain (PID2019-104695RB-I00) to A.B., NIH/GM085490 to G.G., BIO2017-83650-P to JVF and Generalitat de Catalunya 2017SGR135. LM is a predoctoral fellow of 2015FI-B00806 and 2016FI-B1 00110 and MF has financial support by the Universitat de Vic-Universitat Central de Catalunya PhD fellowships program. The authors thankfully acknowledge the computer resources at Pirineus and the technical support provided by the Spanish Supercomputer Network (BCV-2020-1-0001).

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISCs). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, the main regulator of NF‐κB, exerts alternative nuclear functions as cytokine sensor in a subset of PRC2‐regulated genes. Here, we show that nuclear IκBα is present in the ISC compartment. Mice deficient for IκBα show altered intestinal cell differentiation with persistence of a fetal‐like ISC phenotype, associated with aberrant PRC2 activity at specific loci. Moreover, IκBα‐deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2‐dependent fetal‐like transcriptional signature. DSS treatment, which induces acute damage in the colonic epithelium of mice, results in a temporary loss of nuclear P‐IκBα and its subsequent accumulation in early CD44‐positive regenerating areas. Importantly, IκBα‐deficient mice show higher resistance to damage, likely due to the persistent fetal‐like ISC phenotype. These results highlight intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.