Supplementary Figure 6: Effects of mature CD4⁺ T cell–specific and T_reg-specific Satb1 deletion on T_reg development, phenotype and T_reg-type DNA hypomethylation.

(a) Schematic diagram illustrating the timing of Satb1 deletion in Satb1^fl/flCd4Cre⁺, Satb1^fl/flThpokCre⁺, and Satb1^fl/flFoxp3Cre⁺ mice. Satb1 expression level is indicated by color code. (b) Flow cytometry of peripheral CD4⁺CD25⁺Foxp3⁺ T cells from Satb1^fl/+ThpokCre⁺ and Satb1^fl/flThpokCre⁺ mice, and CD4⁺CD25^–Foxp3⁺ T cells from Satb1^fl/flThpokCre⁺ mice, for the expression of T_reg cell signature molecules. (c) Scatter plot displaying global gene expression in CD4⁺CD25^– cells from wild-type (WT) and Satb1^fl/flThpokCre⁺ mice. Average normalized fragments per kilobase of transcript per million reads mapped (FPKM) values from two independent RNA-seq experiments are plotted and T_reg up and down signature genes are highlighted. (d) DNA methylation status of the Foxp3 CNS2 region in peripheral CD4⁺CD25^–Foxp3^–, CD4⁺CD25⁺Foxp3⁺Nrp1⁺ and CD4⁺CD25⁺Foxp3⁺Nrp1^– cells from Satb1^fl/+ThpokCre⁺ and Satb1^fl/flThpokCre⁺ mice, and CD4⁺CD25^–Foxp3⁺ from Satb1^fl/flThpokCre⁺ mice. Each block indicates CpG residues within amplicons. (e) H3K27ac modification in indicated cell types and Satb1 binding in WT T_conv cells at the Foxp3 locus. Vertebrate conservation score and CNS0 region are shown. Bar indicates 5 kb. (f) Flow cytometry of peripheral CD25⁺Foxp3⁺ T cells in Satb1^fl/+ThpokCre⁺ and Satb1^fl/flThpokCre⁺ mice, for the expression of Nrp1 and Helios, and summary data showing the percentages of Nrp1⁺ tT_reg and Nrp1^– pT_reg cells in peripheral CD4⁺ T cells (mean ± SEM, n = 6). ns P > 0.05 and **** P ≤ 0.0001 (two-way ANOVA, followed by Holm-Sidak’s multiple comparison test). (g) Flow cytometry of CD45.1⁺ and CD45.2⁺ CD4⁺ T cells from Rag2^–/– mice, which received CD4⁺CD25^–CD45RB^hi T cells from WT (CD45.1⁺) and Satb1^fl/flThpokCre⁺ (CD45.2⁺) mice, for identification of T_reg cells by CD25 and Foxp3 expression. Summary graph shows percentages of Foxp3⁺ cells among CD45.1⁺ or CD45.2⁺ T cells (n = 6, mean ± SEM). **** P ≤ 0.0001 (two-tailed t-test). (h) DNA methylation status of 6 CpG residues within the Foxp3 CNS2 in WT and Satb1-deficient CD4⁺ T cells isolated on day 17 in the experiment described in g. CpGs from 5’ end are numbered as 1-6 (column) and amplicons are ordered from the most demethylated ones (row). Demethylated or methylated status is indicated by color code. 0-50% of fully methylated amplicons are omitted for presentation, as the wavy lines indicate (upper panel). Top 3.75% of total reads, when ordered from the most demethylated clones, are zoomed in (bottom). Summary data of two independent experiments, showing the percentage of fully demethylated amplicons, are also shown (right). (i) Flow cytometry of CD4SP thymocytes and CD4⁺ splenocytes from 4-day-old Satb1^fl/+Foxp3Cre⁺ and Satb1^fl/flFoxp3Cre⁺ mice, for identification of T_reg cells by the expression of Foxp3 and CD25. (j) Percentages of CD25⁺Foxp3⁺ T_reg cells among CD4SP thymocytes and CD4⁺ splenocytes in 4-day-old Satb1^fl/+Foxp3Cre⁺ (n = 5) and Satb1^fl/flFoxp3Cre⁺ mice (n = 8). Horizontal line indicates mean. ns P > 0.05 (two-tailed t-test). (k) Flow cytometry of peripheral CD4⁺CD25⁺Foxp3⁺ T_reg cells from 4-week-old Satb1^fl/+Foxp3Cre⁺ and Satb1^fl/flFoxp3Cre⁺ mice, for T_reg cell signature molecule expression. Data are representative or summary of three independent experiments with three or more mice (b,d,f,g,i-k), average of two independent RNA-seq experiments (c), representative of two independent experiments (e), or representative and summary of two independent experiments (h).