Assessment of the role of dynamic Foxp3–chromatin binding in regulating gene expression. (A) Ratios of gene expression levels (assessed by RNA-seq) in resting and activated wannabe and Treg cells (van der Veeken et al., 2020) for gene clusters defined in (Fig. 3 H). Genes whose expression was significantly changed in wannabe Treg cells are highlighted (red, decreased; blue, increased). (B) Ratios of gene expression levels in Treg cells with or without Foxp3 CRISPR deletion for gene clusters defined in (Fig. 3 H). Treg cells were treated with or without TCR agonists for 3 h before RNA-seq. Genes whose expression significantly changed after Foxp3 deletion are highlighted. Data are averages of two replicates. (C) Comparison of the expression levels of Gata1 and Pde3b in rTreg, aTreg, and resting and activated wannabe Treg cells (rWannabe and aWannabe). CPM, count per million. (D and E) DNA sequence motifs for transcription factors enriched at regions with increased (D) or decreased (E) Foxp3 binding in aTreg versus rTreg cells. (F and G) Comparison of Foxp3 binding and chromatin accessibility (ATAC-seq) in aTreg and rTreg cells. Data were merged from two replicates.