A role of direct Foxp3-DNA binding in Foxp3–chromatin interaction. (A) Magnified structure of Foxp3^ΔN-DNA complex (PDB: 7TDX) highlighting the amino acid residues interacting with DNA. Mutations in Foxp3 M2, M4, and Δα-Helix are shown. Purified recombinant proteins were resolved by SDS-PAGE and visualized by Coomassie staining. (B and C) EMSA of recombinant Foxp3^ΔN proteins (0.4 and 0.8 µM) after incubation with DNA probes (0.4 µM) of inverted-repeat FKHM (B) or (T₃G)₆ (C). Gel was visualized by SYBR Gold nucleic acid stain. *, DNA-BMP-Foxp3^ΔN complexes. (D) Representative plots showing CD2 and Foxp3 expression in activated CD4 Tn (Th0) cells transduced with full-length WT and mutant Flag-Foxp3-IRES-CD2 retrovirus. (E) Foxp3 expression levels of Foxp3⁺ cells described in D. n = 4 technical replicates. Data represent more than two experiments. Unpaired, two-tailed t tests; ***P < 0.001, ****P < 0.0001. (F and G) Comparison of CD25 and Foxp3 expression in Th0 cells transduced with full-length WT and mutant Flag-Foxp3-IRES-CD2 retrovirus. Anti-Flag antibody was used to assess Foxp3 expression. No, no transduction. Isotype, FITC-isotype antibody. n = 4 technical replicates. (H and I) Relationship between CTLA-4 and Foxp3 expression in Th0 cells expressing full-length WT and mutant Flag-Foxp3-IRES-CD2. n = 4 technical replicates. (J) Comparison of Foxp3 binding by CUT&RUN-seq in Th0 cells expressing full-length WT or M4 mutant Flag-Foxp3. Two replicates were merged for analysis. (K–M) Foxp3 peaks at the Il2ra (K), Ctla4 (L), and Tnfrsf9 (M) loci in Th0 cells expressing full-length WT or M4 mutant Flag-Foxp3. Data represent one of two replicates. Source data are available for this figure: SourceData F6.