Dynamic Foxp3-chromatin interaction controls tunable Treg cell function [RNA-seq]

Nuclear factor Foxp3 determines regulatory T (Treg) cell fate and function via mechanisms that remain unclear. Here we investigate the nature of Foxp3-mediated gene regulation in suppressing autoimmunity and antitumor immune response. Contrasting with previous models, we find that Foxp3-chromatin binding is regulated by Treg activation states, tumor microenvironment, and antigen and cytokine stimulations. Proteomics studies uncovered dynamic proteins within the Foxp3 proximity upon TCR or IL-2 receptor signaling in vitro, reflecting intricate interactions among Foxp3, signal transducers, and chromatin. Pharmacological inhibition and genetic knockdown experiments indicate that NFAT and AP-1 protein Batf are required for enhanced Foxp3-chromatin binding in activated Treg cells and tumor-infiltrating Treg cells to modulate target gene expression. Furthermore, mutations at Foxp3 DNA-binding domain destabilize Foxp3-chromatin association. These representative settings delineate context-dependent Foxp3-chromatin interaction, suggesting that Foxp3 associates with chromatin by hijacking DNA-binding proteins resulting from Treg activation or differentiation, which is stabilized by direct Foxp3-DNA binding, to dynamically regulate Treg cell function according to immunological contexts. We developed a comparative proteomics method by projecting the spatial information (PSI) of nuclear proteins with peroxidase–mediated proximity ligation, thus revealing the protein components of the cis-regulatory elements bound by Treg lineage–specifying factor Foxp3. We performed RNA sequencing for naïve CD4 T cells (Tn, CD4+ GFP- CD25- CD44low CD62Lhigh), effector CD4 T cells (aTe, CD4+ GFP- CD44high CD62Llow), resting Treg cells (rTreg, CD4+ GFP+ CD44low CD62Lhigh), and activated Treg cells (aTreg, CD4+ GFP+ CD44high CD62Llow) sorted from 8-10 week old male Foxp3-gfp knockin mice. To reveal signal-dependent transcriptional regulation, we first induced Treg cells in vitro (iTreg) from CD4 naïve T cells isolated from male Foxp3-gfp mice with TCR agonists, Interleukion-2 (IL-2), TGF-beta, and ascorbic acid and then stimulated them with IL-2 for 30 min or with plate-bound anti-CD3 and anti-CD28 antibodies for 3 hours. After stimulation, cells were immediately lysed with the TRIzol reagent for RNA extraction and sequencing.