Characterization of Foxp3 chromatin-binding modes during Treg cell activation. (A) rTreg (CD44^loCD62L^hi) and aTreg (CD44^hiCD62L^lo) cells were sorted to examine Foxp3–chromatin binding with CUT&RUN-seq or CUT&Tag-seq. Treg cells were gated to show CD44 and CD62L expression as well as Foxp3 protein levels after sorting. (B) A heatmap of Foxp3 CUT&RUN-seq showing three types of differential Foxp3 binding in aTreg and rTreg cells: increased (Up; P < 0.05, FC ≥ 2), constitutive (Cons; P > 0.5, 0.95 < FC < 1.05), and decreased (Down; P < 0.05, FC ≤ −2). Peak intensities were merged from two biological replicates. (C) Genomic distribution of Foxp3 peaks. Dis, distal regions (50 kb 5′ upstream or 3′ downstream); TES, transcription end sites. (D) Numbers of genes (with examples) associated with constitutive, increased (Up), and decreased (Down) Foxp3 binding in aTreg versus rTreg cells. These genes are defined by the nearest Foxp3 peaks to their transcription start sites. (E) Cross-comparison of gene expression (mRNA) and Foxp3–chromatin binding in aTreg and rTreg cells. Genes with significant changes (FC ≥ 2, FDR < 0.05) of both expression and Foxp3 binding are highlighted. Data were derived from two replicates per condition. (F) Distributions of Foxp3-binding modes (Up, Cons, Down, Other undetermined, and No binding) linked to differentially expressed genes (DEGs) in aTreg and rTreg cells defined in E. (G–I) Distribution of DEGs in aTreg and rTreg cells linked to Foxp3-binding modes: Cons (G), Up (H), and Down (I). For simplicity, only Treg-specific genes (i.e., P < 0.05 and |log₂FC| > 0.58 between rTreg versus Tn cells or between aTreg versus Te cells) related to different Foxp3-binding modes are shown.