Dynamic Foxp3 binding regulates tunable gene expression in aTreg cells. (A) Expression patterns of genes that are upregulated and linked to increased Foxp3–chromatin binding in aTreg versus rTreg cells are defined in Fig. 1 H. (B) Functional annotation of genes in clusters C3–C5. Neg, negative. Proc, process. (C) Ratios of gene expression levels in resting and activated wannabe Treg and WT Treg cells (van der Veeken et al., 2020). (D) Schematic procedures of Foxp3 CRISPR deletion and restimulation of Treg cells by anti-CD3 and anti-CD28 antibodies. Treg cells were sorted from Foxp3^gfpRosa^Cas9 mice. (E) Ratios of gene expression levels in Cas9-expressing Treg cells that received retroviral sgFoxp3 and sgNC with or without TCR/costimulation. Data are averages of two replicates. (F) Normalized expression levels of Klrg1, Tnfrsf8, and Tnfrsf9 in rTreg cells, aTreg cells, and activated and resting wannabe Treg cells (van der Veeken et al., 2020). CPM, count per million. Unpaired, two-tailed t tests; ***P < 0.001, ****P < 0.0001. n = 3 replicates. (G) Expression levels of indicated genes in sgNC- and sgFoxp3-transduced Treg cells with or without TCR restimulation. n = 2 replicates. (H) Cross-comparison of Treg and Tcon cells for genes that are downregulated and linked to decreased Foxp3–chromatin binding in aTreg cells versus rTreg cells defined in Fig. 1 I. (I) Foxp3 binding at the Gata1 locus in rTreg and aTreg cells. Data are representative of two replicates. Filled arrowhead indicates decreased Foxp3 binding in aTreg cells. (J) Comparison of Foxp3–chromatin binding and ATAC-seq in rTreg and aTreg cells. Data were derived from two replicates; P values represent unpaired, two-sample Wilcoxon tests.