Guidance of regulatory T cell development by Satb1-dependent super-enhancer establishment

  • An Erratum to this article was published on 22 March 2017
  • An Addendum to this article was published on 18 October 2017

Abstract

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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Figure 1: Identification of Treg-specific SEs.
Figure 2: Establishment of Treg-specific SEs in developing Treg cells.
Figure 3: Satb1 expression in Treg precursor cells and binding to Treg-SEs.
Figure 4: Potential roles of Satb1 in activating Treg-SEs.
Figure 5: Indispensable roles of Satb1 in tTreg cell development.
Figure 6: Induction of autoimmunity by T cell–specific deletion of Satb1.
Figure 7: Loss of Treg differentiation potential in Satb1-deficient tTreg precursor cells.
Figure 8: Satb1-dependent Treg-SE establishment and control of transcriptional changes in developing tTreg cells.

Change history

  • 23 January 2017

    In the version of this article initially published, the labels above the plots in Figure 4b were incorrect (with 'Open chromatin' above the first two columns and 'Closed chromatin' above the second two columns). The correct labeling is 'Open chromatin' above the first column, 'Closed chromatin' above the second column, 'Open chromatin' above the third column and 'Closed chromatin' above the fourth column. The error has been corrected in the HTML and PDF versions of the article.

  • 17 April 2017

    ChIP-seq, RNA-seq, MBD-seq and ATAC-seq data sets associated with this article were originally deposited in the DNA Data Bank of Japan under accession numbers DRA003955, DRA004738 and DRA005202. The sequencing data sets have now also been submitted to the NCBI SRA database. The SRA accession number is DRP003376, and all the data (109 samples in total) are accessible at https://www.ncbi.nlm.nih.gov/sra/?term=DRP003376.

References

  1. 1

    Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22, 531–562 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).

    CAS  Google Scholar 

  3. 3

    Fontenot, J.D., Gavin, M.A. & Rudensky, A.Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).

    CAS  Google Scholar 

  4. 4

    Khattri, R., Cox, T., Yasayko, S.A. & Ramsdell, F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat. Immunol. 4, 337–342 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Floess, S. et al. Epigenetic control of the Foxp3 locus in regulatory T cells. PLoS Biol. 5, e38 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Ohkura, N. et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity 37, 785–799 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Lee, H.M. & Hsieh, C.S. Rare development of Foxp3+ thymocytes in the CD4+CD8+ subset. J. Immunol. 183, 2261–2266 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Lio, C.W. & Hsieh, C.S. A two-step process for thymic regulatory T cell development. Immunity 28, 100–111 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Toker, A. et al. Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus. J. Immunol. 190, 3180–3188 (2013).

    Article  CAS  PubMed  Google Scholar 

  10. 10

    Waddington, C.H. The Strategy of the Genes: A Discussion of Some Aspects of Theoretical Biology (Allen and Unwin, 1957).

  11. 11

    Davidson, E.H. Emerging properties of animal gene regulatory networks. Nature 468, 911–920 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Whyte, W.A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. 15

    Adam, R.C. et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521, 366–370 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Vahedi, G. et al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature 520, 558–562 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Huehn, J. & Beyer, M. Epigenetic and transcriptional control of Foxp3+ regulatory T cells. Semin. Immunol. 27, 10–18 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. 20

    Kagey, M.H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Magnani, L., Eeckhoute, J. & Lupien, M. Pioneer factors: directing transcriptional regulators within the chromatin environment. Trends Genet. 27, 465–474 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. 22

    Zaret, K.S. & Carroll, J.S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Yasui, D., Miyano, M., Cai, S., Varga-Weisz, P. & Kohwi-Shigematsu, T. SATB1 targets chromatin remodelling to regulate genes over long distances. Nature 419, 641–645 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Cai, S., Lee, C.C. & Kohwi-Shigematsu, T. SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes. Nat. Genet. 38, 1278–1288 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Feng, Y. et al. A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance. Nature 528, 132–136 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Hao, B. et al. An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development. J. Exp. Med. 212, 809–824 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Yang, S., Fujikado, N., Kolodin, D., Benoist, C. & Mathis, D. Immune tolerance. Regulatory T cells generated early in life play a distinct role in maintaining self-tolerance. Science 348, 589–594 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Yadav, M. et al. Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J. Exp. Med. 209, 1713–1722 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Weiss, J.M. et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. J. Exp. Med. 209, 1723–1742 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Singh, K., Hjort, M., Thorvaldson, L. & Sandler, S. Concomitant analysis of Helios and neuropilin-1 as a marker to detect thymic derived regulatory T cells in naïve mice. Sci. Rep. 5, 7767 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Thiault, N. et al. Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat. Immunol. 16, 628–634 (2015).

    Article  CAS  PubMed  Google Scholar 

  32. 32

    Mucida, D. et al. Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II–restricted cytotoxic T lymphocytes. Nat. Immunol. 14, 281–289 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Tai, X. et al. Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals. Immunity 38, 1116–1128 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Mousavi, K. et al. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol. Cell 51, 606–617 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Gavin, M.A. et al. Foxp3-dependent programme of regulatory T-cell differentiation. Nature 445, 771–775 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. 36

    Morikawa, H. et al. Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell–specific transcriptional regulation. Proc. Natl. Acad. Sci. USA 111, 5289–5294 (2014).

    Article  CAS  Google Scholar 

  37. 37

    Beyer, M. et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat. Immunol. 12, 898–907 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Mahmud, S.A. et al. Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells. Nat. Immunol. 15, 473–481 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Cheng, G., Yu, A., Dee, M.J. & Malek, T.R. IL-2R signaling is essential for functional maturation of regulatory T cells during thymic development. J. Immunol. 190, 1567–1575 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Asano, M., Toda, M., Sakaguchi, N. & Sakaguchi, S. Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation. J. Exp. Med. 184, 387–396 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Beecham, A.H. et al. Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis. Nat. Genet. 45, 1353–1360 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Kim, J.M., Rasmussen, J.P. & Rudensky, A.Y. Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat. Immunol. 8, 191–197 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Lee, P.P. et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15, 763–774 (2001).

    Article  CAS  Google Scholar 

  44. 44

    Wing, K. et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science 322, 271–275 (2008).

    Article  CAS  Google Scholar 

  45. 45

    Ito, Y. et al. Detection of T cell responses to a ubiquitous cellular protein in autoimmune disease. Science 346, 363–368 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Kurts, C. et al. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J. Exp. Med. 184, 923–930 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Barnden, M.J., Allison, J., Heath, W.R. & Carbone, F.R. Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).

    Article  CAS  Google Scholar 

  48. 48

    Feng, J., Liu, T., Qin, B., Zhang, Y. & Liu, X.S. Identifying ChIP-seq enrichment using MACS. Nat. Protoc. 7, 1728–1740 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. 49

    Shao, Z., Zhang, Y., Yuan, G.C., Orkin, S.H. & Waxman, D.J. MAnorm: a robust model for quantitative comparison of ChIP-Seq data sets. Genome Biol. 13, R16 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Lara-Astiaso, D. et al. Immunogenetics. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Mingueneau, M. et al. The transcriptional landscape of αβ T cell differentiation. Nat. Immunol. 14, 619–632 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

Y. Kitagawa designed, performed and analyzed most experiments, including flow cytometric analyses, in vivo and in vitro experiments, ChIP-seq, library preparation for sequencing and bioinformatics analyses. N.O. performed ATAC-seq and MBD-seq, Y. Kidani assisted with bioinformatical analyses and performed immunoblotting. A.V. and K.H. provided crucial advice. R.K. performed H3K4me3 ChIP-seq. K.Y. assisted with histological analysis. D.M. and S.N. performed amplicon sequencing. I.T. and T.K.-S. provided helpful suggestions. T.K.-S. and M.K. provided Satb1 conditional knockout mouse. I.T. provided Thpok-Cre mouse. Y. Kitagawa and S.S. wrote the manuscript, and all authors reviewed it. T.K.-S. and N.O. critically read the manuscript and provided advice. S.S. supervised the project.

Corresponding author

Correspondence to Shimon Sakaguchi.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characteristics of SEs in Treg and Tconv cells.

(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).

Supplementary Figure 2 Chromatin configuration changes at SE regions in developing tTreg cells.

(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 ac 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 3 Establishment of individual SEs in developing tTreg cells.

(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+CD25GFP+ 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+CD25Foxp3+ 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 Nrp1CD25+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+CD25Foxp3+ 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+CD25Foxp3, CD4+CD25+Foxp3+Nrp1+ and CD4+CD25+Foxp3+Nrp1 cells from Satb1fl/+ThpokCre+ and Satb1fl/flThpokCre+ mice, and CD4+CD25Foxp3+ 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+CD25CD45RBhi 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 CD25Foxp3+ 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).

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

(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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Treg-SEs, Tconv-SEs and their associated gene list. (XLSX 41 kb)

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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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