Fig. 1. FoxP3 binds to active enhancers around both T_reg -up and -down loci.

(a) FoxP3 binding peaks (replicated in ChIPseq dataset ^26,32) in the vicinity of genes over- or under-expressed in T_reg vs T_conv cells, or random neutral genes. (b) Density of chromatin marks in a 10 kb window around FoxP3 binding sites chromatin for T_reg -up- or –down genes (per a). (c) Correlation of ChIPseq signal intensity for FoxP3 vs different chromatin marks in the vicinity of T_reg -up- or –down genes (Pearson r and p-value). (d) 48 hrs after transduction of activated CD4⁺ T cells with a retroviral vector encoding FoxP3, or an empty vector control, transcripts were quantitated by Nanostring profiling. WT/EV expression FoldChange is plotted vs t.test p.value. Transcripts belonging to the classic T_reg -up or - down signatures ⁷ are highlighted. (e) Ratio of H3K27ac intensity in T_reg vs T_conv cells (genomewide data ³¹); x-axis: ratio in resting cells; y-axis: ratio in activated cells; red and blue highlights represent the positions of enhancers within 50 kb of genes that belong to the FoxP3-induced and -repressed genes defined by FoxP3 transduction. (f) Binding of FoxP3 around IL2ra and Pde3b in CD4⁺ T cells transduced with EV or FoxP3, with traces from ex vivo T_reg cells ³¹ for reference (bottom row). (g) Genomewide comparison of FoxP3-binding in our FoxP3-transduced CD4⁺ T cells vs ex vivo T_reg cells ³¹. Representative of biological duplicates. (h) Changes induced by FoxP3 transduction (same plot as d), where green highlights denote genes that bind FoxP3 (ChIPseq analysis in transduced cells); the average of biological quadruplicates.