Figure 5.
Tumor microenvironment remodels Foxp3–chromatin binding linked to enhanced Treg suppressive function. (A–C) CD44 and Klrg1 expression in Treg and Tcon cells isolated from spleen, MC38 tumor, and tumor-draining lymph nodes (dLN). n = 5. Data represent more than three experiments. Paired, two-tailed t tests; **P < 0.01. tuTreg cells were used for CUT&RUN-seq. (D) Regions with increased Foxp3 binding in aTreg versus rTreg cells from lymphoid organs and in tuTreg versus aTreg cells from lymphoid organs. Unique 1: increased Foxp3 binding in aTreg versus rTreg cells (P < 0.05, FC ≥ 2) but not in tuTreg versus aTreg cells; unique 2: increased Foxp3 binding in tuTreg versus aTreg cells (P < 0.05, FC ≥ 2) but not in aTreg versus rTreg cells; overlap: increased Foxp3 binding in both aTreg versus rTreg cells and in tuTreg versus aTreg cells. Two replicates were merged for analysis. (E) Regions with decreased Foxp3 binding. Unique 1: reduced Foxp3 binding in aTreg versus rTreg cells (P < 0.05, FC ≤ −2) but not in tuTreg versus aTreg cells; unique 2: reduced Foxp3 binding in tuTreg versus aTreg cells (P < 0.05, FC ≤ −2) but not in aTreg versus rTreg cells; overlap: reduced Foxp3 binding in both aTreg versus rTreg cells and in tuTreg versus aTreg cells. Differences between tuTreg and aTreg cells in unique 1 group are not statistically significant. (F and G) Peaks (F) and genes (G) linked to increased (Up) and decreased (Down) Foxp3 binding in aTreg versus rTreg cells and in tuTreg versus aTreg cells. Representative genes are shown. (H and I) Foxp3 peaks at the Klrg1 (H) and Runx2 (I) loci. Arrowheads indicate sites with increased Foxp3 binding in tuTreg cells. Data are representative of two replicates. (J) Comparison of genes linked to increased Foxp3 binding in aTreg versus rTreg, tuTreg versus aTreg, and rTreg cells after TCR stimulation in vitro. (K) DNA sequence motifs for transcription factors enriched at regions with increased Foxp3 binding in the overlap and unique groups defined in D. (L) DNA sequence motifs for transcription factors enriched at regions with decreased Foxp3 binding in aTreg versus rTreg cells and in tuTreg versus aTreg cells, respectively defined as unique 1 and unique 2 groups in E. (M) Percentages of canonical forkhead motif (FKHM) or TnG repeats enriched at Foxp3-binding sites defined in D compared with other regions (background).

Tumor microenvironment remodels Foxp3–chromatin binding linked to enhanced Treg suppressive function. (A–C) CD44 and Klrg1 expression in Treg and Tcon cells isolated from spleen, MC38 tumor, and tumor-draining lymph nodes (dLN). n = 5. Data represent more than three experiments. Paired, two-tailed t tests; **P < 0.01. tuTreg cells were used for CUT&RUN-seq. (D) Regions with increased Foxp3 binding in aTreg versus rTreg cells from lymphoid organs and in tuTreg versus aTreg cells from lymphoid organs. Unique 1: increased Foxp3 binding in aTreg versus rTreg cells (P < 0.05, FC ≥ 2) but not in tuTreg versus aTreg cells; unique 2: increased Foxp3 binding in tuTreg versus aTreg cells (P < 0.05, FC ≥ 2) but not in aTreg versus rTreg cells; overlap: increased Foxp3 binding in both aTreg versus rTreg cells and in tuTreg versus aTreg cells. Two replicates were merged for analysis. (E) Regions with decreased Foxp3 binding. Unique 1: reduced Foxp3 binding in aTreg versus rTreg cells (P < 0.05, FC ≤ −2) but not in tuTreg versus aTreg cells; unique 2: reduced Foxp3 binding in tuTreg versus aTreg cells (P < 0.05, FC ≤ −2) but not in aTreg versus rTreg cells; overlap: reduced Foxp3 binding in both aTreg versus rTreg cells and in tuTreg versus aTreg cells. Differences between tuTreg and aTreg cells in unique 1 group are not statistically significant. (F and G) Peaks (F) and genes (G) linked to increased (Up) and decreased (Down) Foxp3 binding in aTreg versus rTreg cells and in tuTreg versus aTreg cells. Representative genes are shown. (H and I) Foxp3 peaks at the Klrg1 (H) and Runx2 (I) loci. Arrowheads indicate sites with increased Foxp3 binding in tuTreg cells. Data are representative of two replicates. (J) Comparison of genes linked to increased Foxp3 binding in aTreg versus rTreg, tuTreg versus aTreg, and rTreg cells after TCR stimulation in vitro. (K) DNA sequence motifs for transcription factors enriched at regions with increased Foxp3 binding in the overlap and unique groups defined in D. (L) DNA sequence motifs for transcription factors enriched at regions with decreased Foxp3 binding in aTreg versus rTreg cells and in tuTreg versus aTreg cells, respectively defined as unique 1 and unique 2 groups in E. (M) Percentages of canonical forkhead motif (FKHM) or TnG repeats enriched at Foxp3-binding sites defined in D compared with other regions (background).

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