Disruption of FOXP3–EZH2 Interaction Represents a Pathobiological Mechanism in Intestinal Inflammation

• Authors: Adebowale O. Bamidele, Mayo Clinic; Phyllis A. Svingen, Mayo Clinic; Mary R. Sagstetter, Mayo Clinic; Olga F. Sarmento, Mayo Clinic; Michelle Gonzalez, Mayo Clinic; Manuel B. Braga Neto, Mayo Clinic; Subramaniam Kugathasan, Emory University; Gwen Lomberk, Medical College of Wisconsin; Raul A. Urrutia, Medical College of Wisconsin; William A. Faubion Jr., Mayo Clinic
• Journal Title: Cellular and Molecular Gastroenterology and Hepatology
• Volume: Volume 7, Number 1
• Publisher: Elsevier: Creative Commons Attribution Non-Commercial No-Derivatives License | 2019-01-01, Pages 55-71
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1016/j.jcmgh.2018.08.009
• Subjects: Health Sciences, Pathology; Health Sciences, Medicine and Surgery
• Keywords: C232, cysteine 232; CD, Crohn’s disease; ChIP, chromatin-immunoprecipitation; Crohn’s Disease; EED, embryonic ectoderm development; EZH2, enhancer of zeste homolog 2; Epigenetics; FCS, fetal calf serum; FOXP3, forkhead domain-containing X-chromosome–encoded protein; H3K27me3, trimethylated histone H3 at lysine 27; IBD, inflammatory bowel disease; IL, interleukin; IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked; JAK, Janus kinase; LZ, leucine zipper; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PLA, proximity ligation assay; PMA, phorbol 12-myristate 13-acetate; PRC2, polycomb repressive complex 2; Proinflammatory Cytokine; Regulatory T Cells; STAT, signal transducer and activator of transcription; SUZ12, suppressor of zeste; Th, T helper; Treg, regulatory T cell; WT, wild-type; co-IP, co-immunoprecipitation

Abstract

Background & Aims: Forkhead box protein 3 (FOXP3)+ regulatory T cell (Treg) dysfunction is associated with autoimmune diseases; however, the mechanisms responsible for inflammatory bowel disease pathophysiology are poorly understood. Here, we tested the hypothesis that a physical interaction between transcription factor FOXP3 and the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is essential for gene co-repressive function. Methods: Human FOXP3 mutations clinically relevant to intestinal inflammation were generated by site-directed mutagenesis. T lymphocytes were isolated from mice, human blood, and lamina propria of Crohn's disease (CD) patients and non-CD controls. We performed proximity ligation or a co-immunoprecipitation assay in FOXP3-mutant+, interleukin 6 (IL6)-treated or CD-CD4+ T cells to assess FOXP3–EZH2 protein interaction. We studied IL2 promoter activity and chromatin state of the interferon γ locus via luciferase reporter and chromatin-immunoprecipitation assays, respectively, in cells expressing FOXP3 mutants. Results: EZH2 binding was abrogated by inflammatory bowel disease–associated FOXP3 cysteine 232 (C232) mutation. The C232 mutant showed impaired repression of IL2 and diminished EZH2-mediated trimethylation of histone 3 at lysine 27 on interferon γ, indicative of compromised Treg physiologic function. Generalizing this mechanism, IL6 impaired FOXP3–EZH2 interaction. IL6-induced effects were reversed by Janus kinase 1/2 inhibition. In lamina propria–derived CD4+T cells from CD patients, we observed decreased FOXP3–EZH2 interaction. Conclusions: FOXP3–C232 mutation disrupts EZH2 recruitment and gene co-repressive function. The proinflammatory cytokine IL6 abrogates FOXP3–EZH2 interaction. Studies in lesion-derived CD4+ T cells have shown that reduced FOXP3–EZH2 interaction is a molecular feature of CD patients. Destabilized FOXP3–EZH2 protein interaction via diverse mechanisms and consequent Treg abnormality may drive gastrointestinal inflammation.