Figure S5.
NFAT and AP-1 regulate Foxp3–chromatin binding. (A) DNA sequence motifs for transcription factors enriched at regions with significantly decreased Foxp3 binding (P < 0.05, FC > 2) in Treg cells from CsA-treated mice. (B) Peaks of Foxp3, Batf, and ATAC-seq that are significantly increased (Up; P < 0.05, log2FC ≥ 1) in aTreg versus rTreg cells. Unpaired, two-sample Wilcoxon test. (C) Co-immunoprecipitation of Batf and Foxp3 in HEK 293T cells ectopically expressing Batf and Foxp3. (D) Schematic procedures for Batf CRISPR deletion in nTreg cells. nTreg cells were sorted from RosaCas9Foxp3gfp mice and transduced with sgNC or sgBatf after in vitro activation; 7 days later, cells were restimulated by TCR agonists for 3 h before being harvested for CUT&RUN-seq and ATAC-seq. (E) Assessment of Batf CRISPR depletion in Treg cells by flow cytometry. (F) Foxp3 and Batf peaks and chromatin accessibility (ATAC-seq) at the Il2ra locus in control (NC) or Batf-depleted (sgBatf) Treg cells. Arrowhead indicates reduced binding of Foxp3. Data represent two replicates. (G) DNA sequence motifs for transcription factors enriched at the regions with reduced Foxp3 binding in Batf CRISPR knockout (sgBatf) nTreg cells. (H and I) Differential gene expression resulting from Batf KO (WT versus Batf KO) (Xu et al., 2021) and Foxp3 CRISPR deletion (this study) (H) or Foxp3 depletion (aTreg versus aWannabe) (van der Veeken et al., 2020) (I). Representative genes involved in Treg cell function are labeled. (J) Comparison of the effects of Batf KO and Foxp3 CRISPR deletion on the expression (RNA-seq) of Ctla4, Tnfrsf9, and Il10. Data were derived from two replicates per condition. Two-tailed, unpaired t tests; *P < 0.05, **P < 0.01, ****P < 0.0001. (K) Batf binding in Treg, Th2, and Th17 cells at the regions with increased Foxp3 binding in aTreg and rTreg cells (defined in Fig. 1 B). Batf ChIP-seq data in Th2 cells are from Iwata et al. (2017) and those in Th17 cells from Ciofani et al. (2012). (L and M) Batf and Irf4 binding in the regions of aTreg and rTreg cells with different Foxp3-binding modes. Irf4 ChIP-seq data are from Vasanthakumar et al. (2017). Source data are available for this figure: SourceData FS5.

NFAT and AP-1 regulate Foxp3–chromatin binding. (A) DNA sequence motifs for transcription factors enriched at regions with significantly decreased Foxp3 binding (P < 0.05, FC > 2) in Treg cells from CsA-treated mice. (B) Peaks of Foxp3, Batf, and ATAC-seq that are significantly increased (Up; P < 0.05, log2FC ≥ 1) in aTreg versus rTreg cells. Unpaired, two-sample Wilcoxon test. (C) Co-immunoprecipitation of Batf and Foxp3 in HEK 293T cells ectopically expressing Batf and Foxp3. (D) Schematic procedures for Batf CRISPR deletion in nTreg cells. nTreg cells were sorted from RosaCas9Foxp3gfp mice and transduced with sgNC or sgBatf after in vitro activation; 7 days later, cells were restimulated by TCR agonists for 3 h before being harvested for CUT&RUN-seq and ATAC-seq. (E) Assessment of Batf CRISPR depletion in Treg cells by flow cytometry. (F) Foxp3 and Batf peaks and chromatin accessibility (ATAC-seq) at the Il2ra locus in control (NC) or Batf-depleted (sgBatf) Treg cells. Arrowhead indicates reduced binding of Foxp3. Data represent two replicates. (G) DNA sequence motifs for transcription factors enriched at the regions with reduced Foxp3 binding in Batf CRISPR knockout (sgBatf) nTreg cells. (H and I) Differential gene expression resulting from Batf KO (WT versus Batf KO) (Xu et al., 2021) and Foxp3 CRISPR deletion (this study) (H) or Foxp3 depletion (aTreg versus aWannabe) (van der Veeken et al., 2020) (I). Representative genes involved in Treg cell function are labeled. (J) Comparison of the effects of Batf KO and Foxp3 CRISPR deletion on the expression (RNA-seq) of Ctla4, Tnfrsf9, and Il10. Data were derived from two replicates per condition. Two-tailed, unpaired t tests; *P < 0.05, **P < 0.01, ****P < 0.0001. (K) Batf binding in Treg, Th2, and Th17 cells at the regions with increased Foxp3 binding in aTreg and rTreg cells (defined in Fig. 1 B). Batf ChIP-seq data in Th2 cells are from Iwata et al. (2017) and those in Th17 cells from Ciofani et al. (2012). (L and M) Batf and Irf4 binding in the regions of aTreg and rTreg cells with different Foxp3-binding modes. Irf4 ChIP-seq data are from Vasanthakumar et al. (2017). Source data are available for this figure: SourceData FS5.

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