Acute IL-2 and TCR signaling induces dynamic Foxp3–chromatin binding. (A) Schematic procedures for IL-2 and TCR stimulations of ex vivo isolated Treg cells. The rTreg cells were FACS-sorted from lymphoid organs of Foxp3^gfp-DTR mice and stimulated with plate-bound anti-CD3 and anti-CD28 antibodies (1 µg/ml each) or recombinant IL-2 (500 U/ml) for 3 h before Foxp3 CUT&RUN-seq. Flow cytometry plot is reused (Fig. 1 A). (B) Principal component analysis (PCA) of Foxp3 CUT&RUN-seq results. n = 2 replicates. (C and D) Changes in Foxp3–chromatin binding in rTreg cells after 3 h of TCR (C) or IL-2 (D) stimulation. Increased (Up) and decreased (Down) Foxp3 binding is defined by P < 0.05, FC ≥ 2; and constitutive (Cons.) Foxp3 binding, by P > 0.5, 0.95 < FC < 1.05. (E and F) Comparison of the peaks (E) and linked genes (F) of different Foxp3–chromatin binding modes in aTreg (versus rTreg) and rTreg cells after IL-2 or TCR stimulation. (G–I) Representative Foxp3 peaks at the Il10 and Ctla4 (G), Il1rl1 (H), and Bcl2 (I) loci. Arrowheads indicate sites with increased Foxp3 binding in aTreg versus rTreg cells or in rTreg cells after IL-2 or TCR stimulation. Data represent two replicates. (J and K) DNA sequence motifs for transcription factors enriched at regions with increased Foxp3 binding in rTreg cells upon TCR (J) or IL-2 (K) stimulation.