Figure 9.
NFAT and AP-1 regulate Foxp3–chromatin binding and tunable Treg function. (A–C) Foxp3 binding in Treg cells from oil and CsA treated mice (A). Foxp3gfp mice received oil or CsA (30 mg/kg body weight) i.p. every 12 h twice. Treg cells were then sorted from spleens and lymph nodes for Foxp3 CUT&RUN-seq. Data were merged from three replicates. Foxp3 expression level was assessed in Treg cells (B). Representative Foxp3 peaks at the Il10 locus in one of three replicates are shown (C). (D and E) Comparison of Batf and Foxp3 binding and chromatin accessibility in rTreg and aTreg cells. Data were merged from two replicates. (F) Genes linked to Foxp3 and Batf binding are upregulated in aTreg cells compared with rTreg cells. Two-sample Kolmogorov–Smirnov (K–S) test. (G) Coimmunoprecipitation of Batf and Foxp3 in nTreg cells expanded in vitro for 7 days. Cells were re-stimulated by TCR agonists for 15 h before experiment. (H and I) Batf and Foxp3 binding and chromatin accessibility in Treg cells after CRISPR deletion of Batf. Unpaired two-sample Wilcoxon test. Data were derived from two replicates. (J) Foxp3 binding, chromatin-accessibility, and Batf binding at the Tnfrsf9 locus in Treg cells that received retroviral sgBatf or sgNC. Data are representative of two replicates. (K) Batf and Foxp3 peaks in Th0 cells expressing Batf (“B”), Flag-Foxp3 (“F”), or both (“B+F”). Differential Foxp3 peaks (P < 0.05, FC > 2) are shown. Data were derived from two replicates. (L and M) Relationships between CD25 (L) or CTLA-4 (M) and Flag-Foxp3 levels in Th0 cells expressing full-length Flag-Foxp3, Batf, or both (Foxp3+Batf). Anti-Flag antibody was used to assess Flag-Foxp3 expression. n = 4 replicates. Data represent two experiments. Two-way ANOVA. (N) Comparison of Batf peaks in aTreg cells (this study) and Th2 and Th17 cells (Ciofani et al., 2012; Iwata et al., 2017). (O) A hypothetical model of dynamic Foxp3–chromatin interaction. In the resting state, Foxp3 associates with chromatin via preexistent DNA-binding proteins (e.g., Ets1) to confer Treg basal function by regulating genes such as Il2ra (CD25) and Ctla4. Upon stimulation or differentiation, induced DNA-binding proteins (e.g., NFAT and AP-1) recruit Foxp3 or facilitate Foxp3–chromatin binding to regulate genes (e.g., Il10, Ctla4, and Klrg1) that enhance Treg suppression of autoimmunity and antitumor response. Direct Foxp3-DNA binding stabilizes Foxp3–chromatin interaction, although it alone is insufficient to confer stable interaction with chromatin in physiological settings. When these induced proteins degrade, Foxp3–chromatin binding and Treg function are reset to the basal level. Foxp3 complex may also be actively displaced by undetermined mechanisms. For simplicity, other Foxp3-interacting proteins are not shown. Source data are available for this figure: SourceData F9.

NFAT and AP-1 regulate Foxp3–chromatin binding and tunable Treg function. (A–C) Foxp3 binding in Treg cells from oil and CsA treated mice (A). Foxp3gfp mice received oil or CsA (30 mg/kg body weight) i.p. every 12 h twice. Treg cells were then sorted from spleens and lymph nodes for Foxp3 CUT&RUN-seq. Data were merged from three replicates. Foxp3 expression level was assessed in Treg cells (B). Representative Foxp3 peaks at the Il10 locus in one of three replicates are shown (C). (D and E) Comparison of Batf and Foxp3 binding and chromatin accessibility in rTreg and aTreg cells. Data were merged from two replicates. (F) Genes linked to Foxp3 and Batf binding are upregulated in aTreg cells compared with rTreg cells. Two-sample Kolmogorov–Smirnov (K–S) test. (G) Coimmunoprecipitation of Batf and Foxp3 in nTreg cells expanded in vitro for 7 days. Cells were re-stimulated by TCR agonists for 15 h before experiment. (H and I) Batf and Foxp3 binding and chromatin accessibility in Treg cells after CRISPR deletion of Batf. Unpaired two-sample Wilcoxon test. Data were derived from two replicates. (J) Foxp3 binding, chromatin-accessibility, and Batf binding at the Tnfrsf9 locus in Treg cells that received retroviral sgBatf or sgNC. Data are representative of two replicates. (K) Batf and Foxp3 peaks in Th0 cells expressing Batf (“B”), Flag-Foxp3 (“F”), or both (“B+F”). Differential Foxp3 peaks (P < 0.05, FC > 2) are shown. Data were derived from two replicates. (L and M) Relationships between CD25 (L) or CTLA-4 (M) and Flag-Foxp3 levels in Th0 cells expressing full-length Flag-Foxp3, Batf, or both (Foxp3+Batf). Anti-Flag antibody was used to assess Flag-Foxp3 expression. n = 4 replicates. Data represent two experiments. Two-way ANOVA. (N) Comparison of Batf peaks in aTreg cells (this study) and Th2 and Th17 cells (Ciofani et al., 2012; Iwata et al., 2017). (O) A hypothetical model of dynamic Foxp3–chromatin interaction. In the resting state, Foxp3 associates with chromatin via preexistent DNA-binding proteins (e.g., Ets1) to confer Treg basal function by regulating genes such as Il2ra (CD25) and Ctla4. Upon stimulation or differentiation, induced DNA-binding proteins (e.g., NFAT and AP-1) recruit Foxp3 or facilitate Foxp3–chromatin binding to regulate genes (e.g., Il10, Ctla4, and Klrg1) that enhance Treg suppression of autoimmunity and antitumor response. Direct Foxp3-DNA binding stabilizes Foxp3–chromatin interaction, although it alone is insufficient to confer stable interaction with chromatin in physiological settings. When these induced proteins degrade, Foxp3–chromatin binding and Treg function are reset to the basal level. Foxp3 complex may also be actively displaced by undetermined mechanisms. For simplicity, other Foxp3-interacting proteins are not shown. Source data are available for this figure: SourceData F9.

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