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.此前我们已证实，核因子κB（NF-κB）抑制剂IκBα（IkBa）可与多梳抑制复合体2（PRC2）结合至染色质，从而在炎症信号存在时赋予PRC2靶基因应答性。鉴于IκBα作为NF-κB负调控因子的经典功能已被广泛认知，其这一替代功能在生理及疾病状态下均长期难以被探明。本研究首次鉴定出IκBα上可用于构建功能分离突变体（separation-of-function, SOF）的特定氨基酸残基，此类突变体可分别丧失与NF-κB相关（SOF NF-κB）或染色质相关（SOF H2A,H4）的活性。仅过表达IκBα SOF NF-κB突变体（而非SOF H2A/H4突变体）即可负调控肠细胞内特定的干性程序，从而逆转IκBα缺失所导致的分化阻滞。通过染色质免疫沉淀（ChIP）实验，我们证实IκBα可结合多个干性基因，且在诱导IκBα SOF NF-κB突变体表达后，这些基因的转录会受到抑制。本研究数据表明，SOF突变体可为研究IκBα在生理及疾病状态下的功能提供专属研究工具。