Most Foxp3+ regulatory T (Treg) cells develop in the thymus as a functionally mature T cell subpopulation specialized for immune suppression. Their cell fate appears to be determined before Foxp3 expression; yet molecular events that prime Foxp3− Treg precursor cells are largely obscure. We found that Treg cell–specific super-enhancers (Treg-SEs), which were associated with Foxp3 and other Treg cell signature genes, began to be activated in Treg precursor cells. T cell–specific deficiency of the genome organizer Satb1 impaired Treg-SE activation and the subsequent expression of Treg signature genes, causing severe autoimmunity due to Treg cell deficiency. These results suggest that Satb1-dependent Treg-SE activation is crucial for Treg cell lineage specification in the thymus and that its perturbation is causative of autoimmune and other immunological diseases.
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We thank Y. Nakamura for DNA sequencing support and assistance with RNA-seq experiments, S. Kojo for providing technical advice regarding ChIP-seq experiments, and K. Chen for reading the manuscript. Bioinformatics analyses were conducted using the computer system at the Genome Information Research Center of the Research Institute for Microbial Diseases at Osaka University. This work was supported by Grants-in-Aid for Japanese Society for the Promotion of Science (JSPS) Fellows 261560 from the JSPS to Y.K. and Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency to S.S. and JSPS Grants-in-Aid for Scientific Research B 15H04744 to N.O.
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Integrated supplementary information
(a) Patterns of indicated transcription factor-binding at SEs and surrounding regions in Treg and Tconv cells. Average normalized ChIP-seq signals of global Treg-SEs, common-SEs or Tconv-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 Treg and Tconv cells. (c) H3K27ac, H3K4me1 and H3K27me3, ATAC-seq, and MBD-seq signals at global common-SE and Tconv-SE regions and H3K4me3 signal around transcription start sites (TSS) of common- or Tconv-SE-associated genes in Treg and Tconv cells. Average normalized ChIP-seq density of common- or Tconv-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 Treg-SE, common-SE or Tconv-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 Tconv-SEs (19 genes) or -TEs (146 genes) (right) in Treg and Tconv cells. Average of two independent RNA-seq experiments are plotted in box-and-whisker plot (median and 10-90 percentiles are shown). ns P > 0.05, * P ≤ 0.1, *** P ≤ 0.001, and **** P ≤ 0.0001 (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).
(a) Heatmaps showing various epigenetic modifications at individual Treg-SE regions in DP, immature CD4SP (imCD4SP), tTreg precursor (pre-tTreg), thymic Treg (thyTreg) and peripheral Treg cells. Individual Treg-SEs (row) are listed in the same order in all sections and normalized ChIP-seq signals are shown for Treg-SE regions with merged ends ± 20 kb. Overall status of Treg-SEs suggested by combination of histone marks is indicated (right). (b) H3K27ac signals at Treg-SE and Treg-TE regions and associated gene expression during Treg cell development. Average of normalized ChIP-seq signals per kb at 66 Treg-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 Tconv-SE regions and H3K4me3 signal around transcription start sites (TSS) of common- or Tconv-SE-associated genes in indicated cells. Average normalized ChIP-seq density of common- or Tconv-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).
(a-c) Changes in various epigenetic modifications, mRNA transcription and transcription factor binding during Treg cell development around representative Treg-SE regions at the Ctla4, Il2ra and Ikzf2 loci (a), common-SE region at the Ets1 locus (b) and Tconv-SE region at the Tcf7 locus (c). H3K27ac, H3K4me1, H3K27me3, ATAC-seq, MBD-seq and RNA-seq signals in DP, immature CD4SP (imCD4SP), tTreg precursor (pre-tTreg), thymic Treg (thyTreg) and peripheral Treg cells, binding of various transcription factors in DP and Treg 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).
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 Tconv-SEs occupied by Satb1 in indicated cell populations. (b) Enrichment of transcription factor-binding motifs in Satb1-binding sites at open chromatin (left) and at closed chromatin (right) within Treg-SEs at the DP stage. Data are from one experiment (a,b).
Supplementary Figure 5 Effects of T cell–specific Satb1 deletion on characteristics of Treg cells and on thymic negative selection.
(a) Suppressive function of peripheral CD4+CD25+GFP+ cells from Satb1fl/+Cd4Cre+Foxp3GFP and CD4+CD25+GFP+ and CD4+CD25–GFP+ cells from Satb1fl/flCd4Cre+Foxp3GFP mice. Percentages of divided naive CD4+ T cells when cultured at indicated ratio with Treg cells are shown (mean ± SEM). (b) Scatter plot displaying global gene expression in CD4+CD25+GFP+ cells from Satb1fl/+Cd4Cre+Foxp3GFP and Satb1fl/flCd4Cre+Foxp3GFP mice. Average normalized fragments per kilobase of transcript per million reads mapped (FPKM) values from two independent RNA-seq experiments are plotted and Treg 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 Treg cell signature genes in thymic and peripheral CD4+CD25+Foxp3+ T cells from Satb1fl/+Cd4Cre+ and Satb1fl/flCd4Cre+ mice, and peripheral CD4+CD25–Foxp3+ T cells from Satb1fl/flCd4Cre+ mice. (e) Flow cytometry of CD25+Foxp3+ Treg cells from 4-week-old Satb1fl/+Cd4Cre+ and Satb1fl/flCd4Cre+ 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 ≤ 0.0001 (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 Satb1fl/+Cd4Cre+ and Satb1fl/flCd4Cre+ mice, for the expression of Treg cell signature and activation-associated molecules. (g) Flow cytometry of Va2+ thymocytes from Satb1fl/+Cd4Cre+OTII, Satb1fl/+Cd4Cre+OTII-RIP-mOVA, Satb1fl/flCd4Cre+OTII, and Satb1fl/flCd4Cre+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).
Supplementary Figure 6 Effects of mature CD4+ T cell–specific and Treg-specific Satb1 deletion on Treg development, phenotype and Treg-type DNA hypomethylation.
(a) Schematic diagram illustrating the timing of Satb1 deletion in Satb1fl/flCd4Cre+, Satb1fl/flThpokCre+, and Satb1fl/flFoxp3Cre+ mice. Satb1 expression level is indicated by color code. (b) Flow cytometry of peripheral CD4+CD25+Foxp3+ T cells from Satb1fl/+ThpokCre+ and Satb1fl/flThpokCre+ mice, and CD4+CD25–Foxp3+ T cells from Satb1fl/flThpokCre+ mice, for the expression of Treg cell signature molecules. (c) Scatter plot displaying global gene expression in CD4+CD25– cells from wild-type (WT) and Satb1fl/flThpokCre+ mice. Average normalized fragments per kilobase of transcript per million reads mapped (FPKM) values from two independent RNA-seq experiments are plotted and Treg 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 Satb1fl/+ThpokCre+ and Satb1fl/flThpokCre+ mice, and CD4+CD25–Foxp3+ from Satb1fl/flThpokCre+ mice. Each block indicates CpG residues within amplicons. (e) H3K27ac modification in indicated cell types and Satb1 binding in WT Tconv 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 Satb1fl/+ThpokCre+ and Satb1fl/flThpokCre+ mice, for the expression of Nrp1 and Helios, and summary data showing the percentages of Nrp1+ tTreg and Nrp1– pTreg 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–CD45RBhi T cells from WT (CD45.1+) and Satb1fl/flThpokCre+ (CD45.2+) mice, for identification of Treg 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 Satb1fl/+Foxp3Cre+ and Satb1fl/flFoxp3Cre+ mice, for identification of Treg cells by the expression of Foxp3 and CD25. (j) Percentages of CD25+Foxp3+ Treg cells among CD4SP thymocytes and CD4+ splenocytes in 4-day-old Satb1fl/+Foxp3Cre+ (n = 5) and Satb1fl/flFoxp3Cre+ mice (n = 8). Horizontal line indicates mean. ns P > 0.05 (two-tailed t-test). (k) Flow cytometry of peripheral CD4+CD25+Foxp3+ Treg cells from 4-week-old Satb1fl/+Foxp3Cre+ and Satb1fl/flFoxp3Cre+ mice, for Treg 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).
Supplementary Figure 7 Phenotypic and epigenetic characteristics of Satb1-deficient tTreg precursor cells.
(a) Flow cytometry of tTreg precursor (pre-tTreg) cells from Satb1fl/+Cd4Cre+ and Satb1fl/flCd4Cre+ mice, for the expression of Treg cell signature molecules. (b) Percentages of CD25–Foxp3+ CD4SP thymocytes in 4-week-old Satb1fl/+Cd4Cre+ and Satb1fl/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 Treg-SE regions in DP and immature CD4SP (imCD4SP) thymocytes from wild-type (WT) and Satb1fl/flCd4Cre+ mice. Average normalized ChIP-seq density of Treg-SEs is plotted for merged Treg-SE regions ± 20 kb. Merged ends of Treg-SEs are marked as S (start) and E (end). (e) Categorization of Treg-SEs by the chromatin state of initial Satb1 binding at the DP stage and effects of Satb1 deletion on their activation. Treg-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 Treg-SE loci (middle panel) and H3K27ac, H3K4me1 and gene transcription at representative Treg-SE during Treg cell development in WT and Satb1fl/flCd4Cre+ mice (bottom) are shown for each category. Bars indicate 10 kb. (f) Summary data showing the effects of Satb1 deletion on Treg-SEs categorized in e, shown as log2 fold-change (log2FC) of H3K27ac signal at Treg-SEs between pre-tTreg cells of WT and Satb1fl/flCd4Cre+ mice (mean ± SEM). Dots indicate individual Treg-SEs. (g) Heatmap showing relative H3K27ac signals at individual Treg-SE, common-SE, and Tconv-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 Tconv-SE regions in DP, imCD4SP and pre-tTreg cells from WT and Satb1fl/flCd4Cre+ mice. Average normalized ChIP-seq density of common-SEs or Tconv-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).
(a) Volcano plot showing the differential gene expression between wild-type (WT) and Satb1fl/flCd4Cre+ CD24+CD25+GITR+ CD4SP thymocytes (mixture of immature thymic Treg (thyTreg) and tTreg precursor (pre-tTreg) cells). Genes associated with Treg-SEs, common-SEs and Tconv-SEs are highlighted. (b) H3K27ac of individual Treg-SE regions at the pre-tTreg cell stage (top) and associated gene expression in CD24+CD25+GITR+ CD4SP thymocytes (bottom) in WT and Satb1fl/flCd4Cre+ mice, shown as ratios to the average of two populations. Treg-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 Treg cell development. Treg-SE regions are poised at least from the DP stage and bound by Satb1. Upon receiving the signal to direct Treg cell differentiation, Treg-SE regions undergo Satb1-dependent activation, likely through the recruitment of epigenetic modifying enzymes, and become Treg lineage-committed precursor cells. As Treg cell development proceeds, enhancer-promoter looping facilitates the expression of associated Treg cell signature genes, including Foxp3, as well as other epigenetic modifications such as Treg-specific DNA demethylation, histone modification of promoter regions and chromatin loosening of enhancer and promoter regions. Once Treg 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).
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Kitagawa, Y., Ohkura, N., Kidani, Y. et al. Guidance of regulatory T cell development by Satb1-dependent super-enhancer establishment. Nat Immunol 18, 173–183 (2017). https://doi.org/10.1038/ni.3646
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