Supplementary Figure 8: Effects of Satb1-dependent T_reg-SE activation on associated gene expression.

(a) Volcano plot showing the differential gene expression between wild-type (WT) and Satb1^fl/flCd4Cre⁺ CD24⁺CD25⁺GITR⁺ CD4SP thymocytes (mixture of immature thymic T_reg (thyT_reg) and tT_reg precursor (pre-tT_reg) cells). Genes associated with T_reg-SEs, common-SEs and T_conv-SEs are highlighted. (b) H3K27ac of individual T_reg-SE regions at the pre-tT_reg cell stage (top) and associated gene expression in CD24⁺CD25⁺GITR⁺ CD4SP thymocytes (bottom) in WT and Satb1^fl/flCd4Cre⁺ mice, shown as ratios to the average of two populations. T_reg-SEs are grouped into three categories by the effects of Satb1 deletion, as shown in Fig. 8e. (c) Hypothetical model of super-enhancer establishment and subsequent transcriptional regulation during thymic T_reg cell development. T_reg-SE regions are poised at least from the DP stage and bound by Satb1. Upon receiving the signal to direct T_reg cell differentiation, T_reg-SE regions undergo Satb1-dependent activation, likely through the recruitment of epigenetic modifying enzymes, and become T_reg lineage-committed precursor cells. As T_reg cell development proceeds, enhancer-promoter looping facilitates the expression of associated T_reg cell signature genes, including Foxp3, as well as other epigenetic modifications such as T_reg-specific DNA demethylation, histone modification of promoter regions and chromatin loosening of enhancer and promoter regions. Once T_reg cell development is complete, Foxp3 amplifies pre-established molecular features¹ and Satb1 transcription is repressed by Foxp3²; however, mediator, cohesin, and various transcription factors, including Foxp3, occupy the sites where Satb1 initially bound, maintaining the local chromatin structure. 1. Gavin, M.A. et al. Foxp3-dependent programme of regulatory T–cell differentiation. Nature 445, 771–775 (2007). 2. Beyer, M. et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nature immunology 12, 898–907 (2011).