FoxP3 associates with enhancer-promoter loops to regulate Treg-specific gene expression.

Gene expression programs are specified by higher-order chromatin structure and enhancer-promoter loops (EPLs). T regulatory cell (T_reg) identity is dominantly specified by the transcription factor (TF) FoxP3, whose mechanism of action is unclear. We applied chromatin conformation capture with immunoprecipitation (HiChIP) in T_reg and closely related conventional CD4⁺ T cells (T_conv). EPLs identified by H3K27Ac HiChIP showed a range of connection intensity, with some superconnected genes. TF-specific HiChIP showed that FoxP3 interacts with EPLs at a large number of genes, including some not differentially expressed in T_reg versus T_conv, but enriched at the core T_reg signature loci that it up-regulates. FoxP3 association correlated with heightened H3K27Ac looping, as ascertained by analysis of FoxP3-deficient T_reg-like cells. There was marked asymmetry in the loci where FoxP3 associated at the enhancer- or the promoter-side of EPLs, with enrichment for different transcriptional cofactors. FoxP3 EPL intensity distinguished gene clusters identified by single-cell ATAC-seq as covarying between individual T_regs, supporting a direct transactivation model for FoxP3 in determining T_reg identity.