Disrupted FOXP3‐EZH2 Interaction is a Molecular Feature of Impaired Regulatory T Cells

Abstract

BackgroundWhile FOXP3+ regulatory T cell (Treg) dysfunction has been linked to human inflammatory bowel disease (IBD), molecular mechanisms for disease pathophysiology are unclear. Tregs play a vital role in maintaining self‐tolerance and homeostasis by suppressing pathological immune responses. We recently documented that the transcription factor FOXP3 via histone methyltransferase EZH2 prevents multi‐organ inflammation in mice. Here, we tested the hypothesis that a physical FOXP3‐EZH2 protein interaction is essential for gene co‐repressive function critical for human Treg physiological functions.MethodsHuman FOXP3 mutations clinically relevant to IBD and Immune dysregulation, polyendocrinopathy, enteropathy, X‐linked [IPEX] syndrome pathogenesis were generated by site‐directed mutagenesis. T cells were isolated from human blood donors and lamina propria of Crohn's disease (CD) patients. Proximity Ligation Assay (PLA), confocal microscopy, and co‐immunoprecipitation (co‐IP) studies were performed for biochemical analysis. Dual Luciferase Reporter (DLR) Assay, Chromatin‐immunoprecipitation (ChIP) and RT‐PCR were performed to study gene regulation.ResultsPLA, confocal imaging and co‐IP studies revealed that constitutive FOXP3‐EZH2 interaction was uniquely abrogated by FOXP3 cysteine 232 to glycine mutation (C232G) that was previously associated with early‐onset IBD in contrast to IPEX‐inducing FOXP3 variants and wild‐type. As demonstrated by DLR experiment, C232 mutant consequently displayed impaired repression of model IL2 and IL17 promoters compared to wild‐type FOXP3. Establishing a mechanistic link between disrupted FOXP3‐EZH2 interaction and EZH2‐mediated gene repression, ChIP‐PCR revealed that C232 mutant lacked the capacity to recruit EZH2 to the IFNG locus as evident by diminished tri‐methylation of Histone 3 at lysine 27. Therefore, the impaired gene repressive function of C232 mutant is indicative of compromised Treg‐physiologic function. Exploring a generalizable mechanism, IL‐6 impaired FOXP3‐EZH2 interaction in a manner that correlated with increased tyrosine phosphorylation of FOXP3. Notably, IL‐6‐induced effects were reversed by a clinically available JAK1/2 inhibitor ruxolitinib. Remarkably, in lamina propria‐derived CD4+ T cells, reduced FOXP3‐EZH2 complex was a feature of CD patients in contrast to non‐CD control subjects.ConclusionsDestabilized FOXP3‐EZH2 protein interaction and consequent impairment of EZH2‐mediated co‐repressor function may drive the pathological characteristics of intestinal inflammation. Our study indicates potential therapeutic approaches and mechanisms for improving Treg function during IBD pathogenesis via stabilizing FOXP3‐EZH2 interaction.Support or Funding InformationThis work was supported by NIDDK training grant T32DK007198 and the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567) (to AOB), the National Institutes of Health NIDDK RO1 grant DK52913 (to RAU), NIAID RO1 grant AI089714 and the Leona Helmsley Charitable Trust (to WAF).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.