Supplementary Figure 7: Phenotypic and epigenetic characteristics of Satb1-deficient tT_reg precursor cells.

(a) Flow cytometry of tTreg precursor (pre-tT_reg) cells from Satb1^fl/+Cd4Cre⁺ and Satb1^fl/flCd4Cre⁺ mice, for the expression of T_reg cell signature molecules. (b) Percentages of CD25^–Foxp3⁺ CD4SP thymocytes in 4-week-old Satb1^fl/+Cd4Cre⁺ and Satb1^fl/flCd4Cre⁺ mice. ** P ≤ 0.01 (two-tailed t-test). (c) Percentages of Foxp3⁺ cells after TCR and IL-2 stimulation of cells in b. *** P ≤ 0.001 (two-tailed t-test). (d) H3K4me1 signals at global T_reg-SE regions in DP and immature CD4SP (imCD4SP) thymocytes from wild-type (WT) and Satb1^fl/flCd4Cre⁺ mice. Average normalized ChIP-seq density of T_reg-SEs is plotted for merged T_reg-SE regions ± 20 kb. Merged ends of T_reg-SEs are marked as S (start) and E (end). (e) Categorization of T_reg-SEs by the chromatin state of initial Satb1 binding at the DP stage and effects of Satb1 deletion on their activation. T_reg-SEs are divided into those with at least one Satb1-binding site at closed chromatin (left), with Satb1-binding sites at open chromatin only (middle), or with no Satb1 binding at the DP stage. Schematic diagram illustrating the categorization by Satb1 binding type (top panel), examples of T_reg-SE loci (middle panel) and H3K27ac, H3K4me1 and gene transcription at representative T_reg-SE during T_reg cell development in WT and Satb1^fl/flCd4Cre⁺ mice (bottom) are shown for each category. Bars indicate 10 kb. (f) Summary data showing the effects of Satb1 deletion on T_reg-SEs categorized in e, shown as log2 fold-change (log2FC) of H3K27ac signal at T_reg-SEs between pre-tT_reg cells of WT and Satb1^fl/flCd4Cre⁺ mice (mean ± SEM). Dots indicate individual T_reg-SEs. (g) Heatmap showing relative H3K27ac signals at individual T_reg-SE, common-SE, and T_conv-SE regions in indicated cell populations. Ratio to the maximum value among the listed cell populations is shown. (h) H3K27ac signals at global common-SE and T_conv-SE regions in DP, imCD4SP and pre-tT_reg cells from WT and Satb1^fl/flCd4Cre⁺ mice. Average normalized ChIP-seq density of common-SEs or T_conv-SEs is plotted for merged SE regions ± 20 kb. Merged ends of SEs are marked as S (start) and E (end). Data are representative of three independent experiments with three or more mice (a-c), from one experiment (d), or representative of two independent experiments (e-h).