Nature Immunology: doi: /ni Supplementary Figure 1. Characteristics of SEs in T reg and T conv cells.
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1 Supplementary Figure 1 Characteristics of SEs in T reg and T conv cells. (a) Patterns of indicated transcription factor-binding at SEs and surrounding regions in T reg and T conv cells. Average normalized ChIPseq signals of global T reg -SEs, common-ses or T conv -SEs were plotted for merged SE regions ± 20 kb. Merged ends of SEs are marked as S (start) and E (end). (b) Frequency of sites co-occupied by Smc1a and Med1 within indicated regions in T reg and T conv cells. (c) H3K27ac, H3K4me1 and H3K27me3, ATAC-seq, and MBD-seq signals at global common-se and T conv -SE regions and H3K4me3 signal around transcription start sites (TSS) of common- or T conv -SE-associated genes in T reg and T conv cells. Average normalized ChIPseq density of common- or T conv -SEs is plotted for merged SE regions ± 20 kb or TSS ± 5 kb. Merged ends of SEs are marked as S (start) and E (end). (d) Heatmap showing the expression of genes associated with T reg -SE, common-se or T conv -SE in indicated immune cells. LT-HSC: long-term hematopoietic stem cell, ST-HSC: short-term hematopoietic stem cell, MPP: multipotent progenitor, CMP: common myeloid progenitor, MEP: megakaryocyte-erythrocyte progenitor, GMP: granulocyte-monocyte progenitor, and CLP: common lymphoid progenitor. (e) Expression of genes associated with common-ses (334 genes) or -TEs (3516 genes) (left) and with T conv -SEs (19 genes) or -TEs (146 genes) (right) in T reg and T conv cells. Average of two independent RNA-seq experiments are plotted in box-and-whisker plot (median and percentiles are shown). ns P > 0.05, * P 0.1, *** P 0.001, and **** P (Kruskal- Wallis test, followed by Dunn s multiple comparisons test). Data are from one experiment (transcription factor ChIP-seq, ATAC-seq, H3K4me1 and H3K27me3 ChIP-seq), representative of two independent experiments (H3K27ac ChIP-seq, H3K4me3 ChIP-seq and MBD-seq) (a-c), or are average of two independent experiments (RNA-seq) (d,e).
2 Supplementary Figure 2 Chromatin configuration changes at SE regions in developing tt reg cells. (a) Heatmaps showing various epigenetic modifications at individual T reg -SE regions in DP, immature CD4SP (imcd4sp), tt reg precursor (pre-tt reg ), thymic T reg (thyt reg ) and peripheral T reg cells. Individual T reg -SEs (row) are listed in the same order in all sections and normalized ChIP-seq signals are shown for T reg -SE regions with merged ends ± 20 kb. Overall status of T reg -SEs suggested by combination of histone marks is indicated (right). (b) H3K27ac signals at T reg -SE and T reg -TE regions and associated gene expression during T reg cell development. Average of normalized ChIP-seq signals per kb at 66 T reg -SEs or average FPKM of associated genes (± SEM) are plotted. (c) H3K27ac, H3K4me1 and H3K27me3, ATAC-seq, and MBD-seq signals at global common-se and T conv -SE regions and H3K4me3 signal around transcription start sites (TSS) of common- or T conv -SE-associated genes in indicated cells. Average normalized ChIP-seq density of common- or T conv -SEs is plotted for merged SE regions ± 20 kb or TSS ± 5 kb. Merged ends of SEs are marked as S (start) and E (end). Data in a c are from one experiment (ATAC-seq, H3K4me1 and H3K27me3 ChIP-seq), representative of two independent experiments (H3K27ac ChIP-seq, H3K4me3 ChIP-seq and MBD-seq), or average of two independent experiments (RNA-seq).
3 Supplementary Figure 3 Establishment of individual SEs in developing tt reg cells. (a-c) Changes in various epigenetic modifications, mrna transcription and transcription factor binding during T reg cell development around representative T reg -SE regions at the Ctla4, Il2ra and Ikzf2 loci (a), common-se region at the Ets1 locus (b) and T conv -SE region at the Tcf7 locus (c). H3K27ac, H3K4me1, H3K27me3, ATAC-seq, MBD-seq and RNA-seq signals in DP, immature CD4SP (imcd4sp), tt reg precursor (pre-tt reg ), thymic T reg (thyt reg ) and peripheral T reg cells, binding of various transcription factors in DP and T reg cells, and vertebrate conservation score are shown. Bars indicate 25 kb. Data are from one experiment (ATAC-seq, H3K4me1 and H3K27me3 ChIP-seq), representative of two independent experiments (H3K27ac ChIP-seq, H3K4me3 ChIP-seq and MBD-seq), or average of two independent experiments (RNA-seq).
4 Supplementary Figure 4 Satb1 binding to SEs and transcription factor binding motifs of Satb1-binding sites at the DP stage. (a) Percentage of common-ses and T conv -SEs occupied by Satb1 in indicated cell populations. (b) Enrichment of transcription factorbinding motifs in Satb1-binding sites at open chromatin (left) and at closed chromatin (right) within T reg -SEs at the DP stage. Data are from one experiment (a,b).
5 Supplementary Figure 5 Effects of T cell specific Satb1 deletion on characteristics of T reg cells and on thymic negative selection. (a) Suppressive function of peripheral CD4 + CD25 + GFP + cells from Satb1 fl/+ Cd4Cre + Foxp3 GFP and CD4 + CD25 + GFP + and CD4 + CD25 GFP + cells from Satb1 fl/fl Cd4Cre + Foxp3 GFP mice. Percentages of divided naive CD4 + T cells when cultured at indicated ratio with T reg cells are shown (mean ± SEM). (b) Scatter plot displaying global gene expression in CD4 + CD25 + GFP + cells from Satb1 fl/+ Cd4Cre + Foxp3 GFP and Satb1 fl/fl Cd4Cre + Foxp3 GFP 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. (c) Flow cytometry of indicated cells after in vitro culture with TCR stimulation, with or without IL-2, for the expression of Foxp3. Numbers indicate percentages of Foxp3 + cells in live CD4 + T cells. (d) DNA methylation status of the T reg cell signature genes in thymic and peripheral CD4 + CD25 + Foxp3 + T cells from Satb1 fl/+ Cd4Cre + and Satb1 fl/fl Cd4Cre + mice, and peripheral CD4 + CD25 Foxp3 + T cells from Satb1 fl/fl Cd4Cre + mice. (e) Flow cytometry of CD25 + Foxp3 + T reg cells from 4-week-old Satb1 fl/+ Cd4Cre + and Satb1 fl/fl Cd4Cre + mice for the expression of Nrp1 and Helios, and summary graph showing the percentages of Nrp1 CD25 + Foxp3 + and Nrp1 + CD25 + Foxp3 + cells among CD4 + T cells (mean ± SEM, n = 4). ** P 0.01 and **** P (two-way ANOVA, followed by Holm-Sidak s multiple comparison test). (f) Flow cytometry of CD25 + Foxp3 + CD4SP cells found in the thymus of 4-week-old Satb1 fl/+ Cd4Cre + and Satb1 fl/fl Cd4Cre + mice, for the expression of T reg cell signature and activation-associated molecules. (g) Flow cytometry of Va2 + thymocytes from Satb1 fl/+ Cd4Cre + OTII, Satb1 fl/+ Cd4Cre + OTII-RIP-mOVA, Satb1 fl/fl Cd4Cre + OTII, and Satb1 fl/fl Cd4Cre + OTII-RIP-mOVA mice, for the examination of CD4SP thymocyte percentage. Data are representative or summary of three independent experiments with three or more mice (a,c-f), of two experiments with two mice (b), and of four experiments with four or more mice (g).
6 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/fl Cd4Cre +, Satb1 fl/fl ThpokCre +, and Satb1 fl/fl Foxp3Cre + 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/fl ThpokCre + mice, and CD4 + CD25 Foxp3 + T cells from Satb1 fl/fl ThpokCre + 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/fl ThpokCre + 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/fl ThpokCre + mice, and CD4 + CD25 Foxp3 + from Satb1 fl/fl ThpokCre + 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/fl ThpokCre + 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 (two-way ANOVA, followed by Holm-Sidak s multiple comparison test). (g) Flow cytometry of CD and CD CD4 + T cells from Rag2 / mice, which received CD4 + CD25 CD45RB hi T cells from WT (CD ) and Satb1 fl/fl ThpokCre + (CD ) mice, for identification of T reg cells by CD25 and Foxp3 expression. Summary graph shows percentages of Foxp3 + cells among CD or CD T cells (n = 6, mean ± SEM). **** P (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/fl Foxp3Cre + mice, for identification of T reg cells by the expression of Foxp3 and CD25. (j) Percentages of
7 CD25 + Foxp3 + T reg cells among CD4SP thymocytes and CD4 + splenocytes in 4-day-old Satb1 fl/+ Foxp3Cre + (n = 5) and Satb1 fl/fl Foxp3Cre + 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/fl Foxp3Cre + 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).
8 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/fl Cd4Cre + 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/fl Cd4Cre + mice. ** P 0.01 (two-tailed t-test). (c) Percentages of Foxp3 + cells after TCR and IL-2 stimulation of cells in b. *** P (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/fl Cd4Cre + 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/fl Cd4Cre + 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 foldchange (log2fc) of H3K27ac signal at T reg -SEs between pre-tt reg cells of WT and Satb1 fl/fl Cd4Cre + 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/fl Cd4Cre + 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).
9 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/fl Cd4Cre + 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/fl Cd4Cre + 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 lineagecommitted 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 features 1 and Satb1 transcription is repressed by Foxp3 2 ; 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, (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, (2011).
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d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
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