Constitutive Foxp3–chromatin binding regulates the basal function of Treg cells. (A) Expression patterns of genes with comparable expression levels in aTreg and rTreg cells defined in Fig. 1 G. Z scores of two biological replicates across samples are shown. (B) Ratios of gene expression levels of indicated clusters in published “wannabe” Treg cells and WT Treg cells isolated from heterozygous female mice (van der Veeken et al., 2020). Genes whose expression significantly changed (P ≤ 0.05) in wannabe Treg cells are highlighted: red, decreased; blue, increased. Ctla4 and Il2ra serve as controls. (C) Schematic of Foxp3 CRISPR deletion in nTreg cells. Treg cells were sorted from Foxp3gfpRosaCas9 mice. Cells were harvested on day 7 for RNA-seq. (D) Foxp3 expression in Treg cells transduced with negative control (NC) or Foxp3 sgRNAs. Data represent more than three experiments. (E) Ratios of gene expression of indicated clusters in sgFoxp3- and sgNC-transduced Treg cells. Genes whose expression significantly changed (P ≤ 0.05) are highlighted. Data were derived from two replicates per condition. Ctla4 and Il2ra are controls. (F) Foxp3 peaks at the Ikzf4 and Lrrc32 loci in rTreg and aTreg cells. Con., DNA sequence conservation in placental mammals. Data are representative of two replicates. (G) Functional annotation of selected genes (clusters C1–C3) linked to constitutive Foxp3 binding. (H) Foxp3 peaks at the Il2ra and Ctla4 loci. Empty arrowheads indicate constitutive Foxp3 binding, and filled arrowheads indicate increased Foxp3 binding in aTreg versus rTreg cells. Data are representative of two replicates. (I) DNA sequence motifs of transcription factors enriched at constitutive Foxp3-binding sites. (J and K) Comparison of Foxp3 and Ets1 peaks. Ets1 ChIP-seq data in bulk Treg cells are from Samstein et al. (2012). (L) Coimmunoprecipitation of Ets1 and Foxp3 in nTreg cells expanded in vitro for 7 days. Note: IgG and Ets1 bands partially overlap. Source data are available for this figure: SourceData F2.