Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4+ T cell fate and interact with Runx3 to silence Cd4 in CD8+ T cells

Nature Immunology volume 15, pages 646–656 (2014)Cite this article

Subjects

Abstract

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4+CD8+ double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4+ T cells and redirected CD4+ T cells to a CD8+ T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8+ lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4+ T cell fate and establish CD8+ T cell identity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CD4-Cre–mediated deletion of TCF-1 alone or of both TCF-1 and LEF-1 impairs the development of CD4+ SP thymocytes.
Figure 2: The CD8*4 mature thymocytes in Tcf7−/−Lef1−/− mice belong to the CD8+ lineage.
Figure 3: Deficiency in TCF-1 alone or in both TCF-1 and LEF-1 redirects CD4+ T cells to the CD8+ lineage.
Figure 4: TCF-1 deficiency decreases Thpok expression but increases Runx3d expression in bipotent precursor cells.
Figure 5: Ectopic expression of Th-POK rectifies the defects in CD4+ T cell differentiation caused by loss of TCF-1.
Figure 6: TCF-1 acts through the GTE in the Thpok locus.
Figure 7: Deletion of Runx3 does not rectify the defects in differentiation into the CD4+ lineage caused by the loss of TCF-1 and LEF-1.
Figure 8: TCF-LEF and Runx factors act together in silencing Cd4 in CD8+ T cells.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Rothenberg, E.V., Moore, J.E. & Yui, M.A. Launching the T-cell-lineage developmental programme. Nat. Rev. Immunol. 8, 9–21 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Yang, Q., Jeremiah Bell, J. & Bhandoola, A. T-cell lineage determination. Immunol. Rev. 238, 12–22 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Singer, A., Adoro, S. & Park, J.H. Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Rev. Immunol. 8, 788–801 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Collins, A., Littman, D.R. & Taniuchi, I. RUNX proteins in transcription factor networks that regulate T-cell lineage choice. Nat. Rev. Immunol. 9, 106–115 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang, L. & Bosselut, R. CD4–CD8 lineage differentiation: Thpok-ing into the nucleus. J. Immunol. 183, 2903–2910 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Wang, L. et al. Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4+ T cells. Nat. Immunol. 9, 1122–1130 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Maurice, D., Hooper, J., Lang, G. & Weston, K. c-Myb regulates lineage choice in developing thymocytes via its target gene Gata3. EMBO J. 26, 3629–3640 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. He, X. et al. The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826–833 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Aliahmad, P. & Kaye, J. Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245–256 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Setoguchi, R. et al. Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development. Science 319, 822–825 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Egawa, T. & Littman, D.R. ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage. Nat. Immunol. 9, 1131–1139 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. He, X. et al. CD4–CD8 lineage commitment is regulated by a silencer element at the ThPOK transcription-factor locus. Immunity 28, 346–358 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Taniuchi, I. et al. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development. Cell 111, 621–633 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Staal, F.J., Luis, T.C. & Tiemessen, M.M. WNT signalling in the immune system: WNT is spreading its wings. Nat. Rev. Immunol. 8, 581–593 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Xue, H.H. & Zhao, D.M. Regulation of mature T cell responses by the Wnt signaling pathway. Ann. NY Acad. Sci. 1247, 16–33 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Weber, B.N. et al. A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476, 63–68 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Germar, K. et al. T-cell factor 1 is a gatekeeper for T-cell specification in response to Notch signaling. Proc. Natl. Acad. Sci. USA 108, 20060–20065 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yu, S. et al. The TCF-1 and LEF-1 transcription factors have cooperative and opposing roles in T cell development and malignancy. Immunity 37, 813–826 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Okamura, R.M. et al. Redundant regulation of T cell differentiation and TCRα gene expression by the transcription factors LEF-1 and TCF-1. Immunity 8, 11–20 (1998).

    Article  CAS  PubMed  Google Scholar 

  20. Yu, S. & Xue, H.H. TCF-1 mediates repression of Notch pathway in T lineage-committed early thymocytes. Blood 121, 4008–4009 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tiemessen, M.M. et al. The nuclear effector of wnt-signaling, tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas. PLoS Biol. 10, e1001430 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Verbeek, S. et al. An HMG-box-containing T-cell factor required for thymocyte differentiation. Nature 374, 70–74 (1995).

    Article  CAS  PubMed  Google Scholar 

  23. Egawa, T., Tillman, R.E., Naoe, Y., Taniuchi, I. & Littman, D.R. The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells. J. Exp. Med. 204, 1945–1957 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Grusby, M.J., Johnson, R.S., Papaioannou, V.E. & Glimcher, L.H. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253, 1417–1420 (1991).

    Article  CAS  PubMed  Google Scholar 

  25. Goux, D. et al. Cooperating pre-T-cell receptor and TCF-1-dependent signals ensure thymocyte survival. Blood 106, 1726–1733 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Karlsson, L., Surh, C.D., Sprent, J. & Peterson, P.A. A novel class II MHC molecule with unusual tissue distribution. Nature 351, 485–488 (1991).

    Article  CAS  PubMed  Google Scholar 

  27. Zijlstra, M. et al. β2-microglobulin deficient mice lack CD4−8+ cytolytic T cells. Nature 344, 742–746 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Muroi, S. et al. Cascading suppression of transcriptional silencers by ThPOK seals helper T cell fate. Nat. Immunol. 9, 1113–1121 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Sun, G. et al. The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 6, 373–381 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Hattori, N., Kawamoto, H., Fujimoto, S., Kuno, K. & Katsura, Y. Involvement of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus. J. Exp. Med. 184, 1137–1147 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Li, L. et al. A far downstream enhancer for murine Bcl11b controls its T-cell specific expression. Blood 122, 902–911 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kohu, K. et al. Overexpression of the Runx3 transcription factor increases the proportion of mature thymocytes of the CD8 single-positive lineage. J. Immunol. 174, 2627–2636 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Taniuchi, I., Sunshine, M.J., Festenstein, R. & Littman, D.R. Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation. Mol. Cell 10, 1083–1096 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Araki, K. et al. mTOR regulates memory CD8 T-cell differentiation. Nature 460, 108–112 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lotem, J. et al. Runx3-mediated transcriptional program in cytotoxic lymphocytes. PLoS ONE 8, e80467 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Ito, K. et al. RUNX3 attenuates β-catenin/T cell factors in intestinal tumorigenesis. Cancer Cell 14, 226–237 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Yarmus, M. et al. Groucho/transducin-like enhancer-of-split (TLE)-dependent and -independent transcriptional regulation by Runx3. Proc. Natl. Acad. Sci. USA 103, 7384–7389 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Woolf, E. et al. Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis. Proc. Natl. Acad. Sci. USA 100, 7731–7736 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rothenberg, E.V. Decision by committee. new light on the CD4/CD8-lineage choice. Immunol. Cell Biol. 87, 109–112 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Park, J.H. et al. Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells. Nat. Immunol. 11, 257–264 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. McCaughtry, T.M. et al. Conditional deletion of cytokine receptor chains reveals that IL-7 and IL-15 specify CD8 cytotoxic lineage fate in the thymus. J. Exp. Med. 209, 2263–2276 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Yu, Q., Erman, B., Park, J.H., Feigenbaum, L. & Singer, A. IL-7 receptor signals inhibit expression of transcription factors TCF-1, LEF-1, and RORγt: impact on thymocyte development. J. Exp. Med. 200, 797–803 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Taniuchi, I. & Ellmeier, W. Transcriptional and epigenetic regulation of CD4/CD8 lineage choice. Adv. Immunol. 110, 71–110 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Zou, Y.R. et al. Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage. Nat. Genet. 29, 332–336 (2001).

    Article  CAS  PubMed  Google Scholar 

  46. Wu, J.Q. et al. Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line. PLoS Genet. 8, e1002565 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Naito, T., Gomez-Del Arco, P., Williams, C.J. & Georgopoulos, K. Antagonistic interactions between Ikaros and the chromatin remodeler Mi-2beta determine silencer activity and Cd4 gene expression. Immunity 27, 723–734 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Xue, H.H. et al. GA binding protein regulates interleukin 7 receptor α-chain gene expression in T cells. Nat. Immunol. 5, 1036–1044 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Machanick, P. & Bailey, T.L. MEME-ChIP: motif analysis of large DNA datasets. Bioinformatics 27, 1696–1697 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hertz, G.Z. & Stormo, G.D. Identifying DNA and protein patterns with statistically significant alignments of multiple sequences. Bioinformatics 15, 563–577 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank R. Bosselut (National Cancer Institute of the US National Institutes of Health) for mice expressing the transgene encoding Th-POK; S.-C. Bae (Chungbuh National University) for the Myc-tagged Runx3 expression plasmid; B.J. Fowlkes for input and discussions; Y. Wakabayashi and Y. Luo (NHLBI) for high-throughput sequencing and data processing; T. Zhao for animal husbandry; the Flow Cytometry Core facility at the University of Iowa (J. Fishbaugh, H. Vignes and G. Rasmussen) for support for cell sorting; and Radiation Core facility at the University of Iowa (A. Kalen) for mouse irradiation. Supported by the American Cancer Society (RSG-11-161-01-MPC to H.-H.X.) and the US National Institutes of Health (HL095540 and AI105351 to H.-H.X.; HG006130 to K.T.; AI007485 (for support of F.C.S.); and P30CA086862 and 1S10 RR027219 to the Flow Core Facility at the University of Iowa). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

Author information

Author notes

  1. Bo Zhou

    Present address: Present address: Department of Nephrology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.,

  2. Farrah C Steinke and Shuyang Yu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Farrah C Steinke, Bo Zhou & Hai-Hui Xue

  2. Interdisciplinary Immunology Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Farrah C Steinke & Hai-Hui Xue

  3. State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China

    Shuyang Yu

  4. Insitute of Immunology, Third Military Medical University, Chongqing, China

    Xinyuan Zhou

  5. Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Bing He

  6. Developmental Biology Center, NHLBI, NIH, Bethesda, Maryland, USA

    Wenjing Yang & Jun Zhu

  7. Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

    Hiroshi Kawamoto

  8. Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Kai Tan

Authors
  1. Farrah C Steinke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shuyang Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xinyuan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bing He
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenjing Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bo Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hiroshi Kawamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jun Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kai Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Hai-Hui Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.C.S. and S.Y. did experiments and analyzed the data; X.Z. and B.Z. did the coimmunoprecipitation experiments; B.H. and W.Y. analyzed the ChIP-Seq data under the supervision of K.T. and J.Z.; H.K. provided anti-TCF-1; H.-H.X. designed and supervised the study and, with F.C.S. and S.Y., wrote the paper.

Corresponding author

Correspondence to Hai-Hui Xue.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conditional targeting of Tcf7.

(a) Targeting strategy. The Tcf7 gene was conditionally targeted by the International Knockout Mouse Consortium (IKMC, project 37596). Depicted on top is partial structure of the Tcf7 gene with filled rectangles in yellow denoting exons (all numbered). The exon 4 of Tcf7 was flanked by two LoxP sites, and deletion of this exon results in a nonsense frame-shift mutation. Also marked are key enzyme sites and relative locations of 5' and 3' probes used in Southern blotting. Shown in the middle is the structure of Tcf7-targeted allele, highlighting the targeting arms, locations of inserted LoxP sites (filled triangles in red), Frt sites (open triangles in blue), β-galactosidase-neomycin resistant gene (LacZ-Neo) cassettes. Note that two extra BamHI sites were embedded in the LacZ-Neo cassette, and these two sites were used to facilitate detection of the targeted allele by Southern blotting. By crossing with Rosa26-Flippase knock-in mice, the Frt site-flanked LacZ-Neo cassette was excised, giving rise to the Tcf7-floxed allele. (b) Identification of targeted mice. Genomic DNA was extracted from tails of targeted mice, digested with BamHI, and Southern-blotted with the 5' or 3' probes. Both probes detect the WT allele at approximately 13.4 kb. The 5'-probe detects the targeted allele at ∼ 5.2 kb (top panel), and the 3' probe detects the targeted allele at ∼8.7 kb (bottom). The probes were amplified with the following primers: 5' probe: 5'-agggtgggcacagagatatg and 5'-gccagagctcagctgctaat; 3' probe: 5'-agccaaggtcattgctgagt and 5'-ccttcctgtgttgaggtggt.

Supplementary Figure 2 Deficiency in both TCF-1 and LEF-1 does not affect TCR-dependent induction of the expression of GATA-3 and Tox.

Although CD69 expression was reduced in TCRβhi thymocytes from Tcf7-/-Lef1-/- mice (Fig. 1c), the combination of CD69 and CD24 was sufficient to distinguish immature and mature subsets within the TCRβhi population. The surface-stained thymocytes from Tcf7-/-Lef1-/- and littermate controls were sorted for 3 subsets, pre-select DP (PreDP), post-select DP (PostDP), and CD4+8lo intermediate (IM). Gata3 and Tox expression was measured as an end outcome of TCR signaling in positively selected DP subsets. The relative expression of each gene was normalized to Hprt1. Data are pooled results from 3 independent experiments and shown as means ± s.d. (n ≥ 3). Gata3 and Tox expression between control and Tcf7-/-Lef1-/- within each subset was not statistically different (p>0.4). Note that the post-select DP thymocytes from Tcf7-/-Lef1-/- mice may contain a fraction of CD8+ T cells that had derepressed expression of CD4 (the CD8*4 cells). Because Gata3 and Tox were less abundantly expressed in CD8+ T cells compared with DP thymocytes, CD8*4 cells unlikely contributed to elevating Gata3 and Tox expression in Tcf7-/-Lef1-/- post-select DP thymocytes.

Supplementary Figure 3 Lack of TCF-1 and LEF-1 diminishes CD4+ T cell output independently of their role in thymocyte survival.

(a) and (b) Loss of TCF-1 or both TCF-1 and LEF-1 diminishes CD4+ T cell output in the periphery. Splenocytes were surface-stained, and TCRβ+ cells were analyzed for CD4+ and CD8+ lineage distribution. Representative and cumulative data are shown in a and b, respectively. (c) Loss of TCF-1 and LEF-1 reverses the CD4/CD8 ratio in the periphery. The CD4+ to CD8+ ratio is calculated from b. Data are from ≥ 4 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001. (d) Germline deletion of TCF-1 results massive cell death in TCRβhi thymocytes. Thymocytes from germline-targeted TCF-1 knockout mice and littermate controls were harvested, and Caspase-3&7 activation was measured in the TCRβhi subset. (e) Late deletion of TCF-1 and LEF-1 alleviates death of thymocytes. TCRβhi thymocytes were analyzed as in d. The frequency of Caspase3&7-positive subset is shown. Data are representative from ≥ 3 experiments.(f) Deficiency in TCF-1 and LEF-1 does not cause preferential death of CD4+ SP T cells. CD4+ or CD8+ TCRβhi thymocytes were analyzed for Caspase activation. Cumulative data from 3 experiments are shown.

Supplementary Figure 4 The redirected CD8+ T cells in B2m–/– chimeras reconstituted with Tcf7–/–Lef1–/– BM exhibit true CD8+ T cell characteristics.

BM cells from Tcf7-/-, Tcf7-/-Lef1-/-, or littermate controls were transplanted into lethally irradiated CD45.1+ congenic β2m-/- mice. Six weeks later, splenocytes were isolated from the BM chimeras and used for downstream analysis. (a) and (b) The redirected CD8+ T cells in the absence of TCF-1 or both TCF-1 and LEF-1 persist in the periphery. Donor-derived CD45.2+TCRβ+ splenocytes were analyzed for CD4+ and CD8+ lineage distribution. Representative contour plots (a) are from 4 independent experiments with ≥ 4 recipients analyzed in each experiment. Numbers of mature CD4+ and CD8+ splenocytes in the BM chimeras are shown in b as means ± s.d. (n ≥ 14). *, p<0.05; **, p<0.01***; p<0.001. (c) The redirected Tcf7-/-Lef1-/- CD8+ T cells express CD8+ T cell-characteristic genes. CD4+ and CD8+ splenic T cells were sorted from WT C57BL/6 mice, and CD8+CD4– and CD8*4 CD45.2+TCRβ+ splenocytes were sorted from the Tcf7-/-Lef1-/--reconstituted β2m-/- BM chimeras (Tcf7-/-Lef1-/- BM chimeras), followed by gene expression analysis. (d) The redirected Tcf7-/-Lef1-/- CD8+ T cells proficiently produce granzyme B and interferon-γ upon stimulation. Splenic T cells were isolated from WT B6 mice or Tcf7-/-Lef1-/- BM chimeras, and then activated by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody in the presence of IL-2. Three days later, the cells were stimulated with PMA/Ionomycin in the presence of Golgi plug, and then surface-stained for CD40L, intracellularly stained for granzyme B, interferon-γ, and IL-2. For c and d, similar results were obtained for redirected Tcf7-/- CD8+ T cells (not shown).

Supplementary Figure 5 Expression of a transgene encoding a TCR alters the timing of CD4-Cre–mediated deletion of Tcf7 and Lef1.

(a) Expression of the OT-II TG greatly diminished total thymocyte numbers in Tcf7-/- and Tcf7-/-Lef1-/- mice compared with littermate controls. Also compare with Fig. 1b.(b) CD4-Cre initiates deletion of Tcf7 and Lef1 at the DN stage in the presence of OT-II TG. Thymocytes from Tcf7-/-Lef1-/- and littermate controls with or without the OT-II TG were isolated and surface-stained. Lineage-negative CD4–CD8– thymocytes were sorted as DN, and TCRβ+CD69–CD4+CD8+ cells sorted as pre-select DP (PreDP) subsets. The expression of Tcf7 and Lef1 was measured by quantitative RT-PCR. Data are duplicate measurements of two samples. *, p<0.05; **, p<0.01; ***, p<0.001. Note that without the OT-II TG, CD4-Cre did not excise Tcf7 and Lef1 at the DN stage but initiated the deletion from the pre-select DP stage, consistent with our Western blot data in Fig. 1a. In contrast, in the presence of the OT-II TG, CD4-Cre initiated deletion of both Tcf7 and Lef1 from the DN stage. Because TCF-1 is critical for survival of early thymocytes (as seen in Supplementary Fig. 3d), early deletion of TCF-1 and LEF-1 in the presence of OT-II TG at least partly account for more severe reduction of total thymocytes as shown in (a). (c) OT-II TG T cells adopt CD8+ T cell fate in the absence of TCF-1 or both TCF-1 and LEF-1. The numbers of CD4+ or CD8+ SP thymocytes were calculated from the mature Vα2+TCRβhiCD24– thymic subset. Data are means ± s.d. (n ≥ 5-10). In spite of reduced total thymic cellularity upon deletion of TCF-1 or both TCF-1 and LEF-1, the mature OT-II+ thymocytes were predominantly CD8+.

Supplementary Figure 6 ChIP-Seq analysis of the binding of TCF-1 to the Thpok and Cd4 loci

ChIP-Seq of TCF-1 in whole thymocytes was reported by Li L et al. (Blood 122, 902, 2013), and ChIP-Seq of Runx3 in CD8+ T cells was reported by Lotem J et al (PLoS One, 8, e80467, 2013). The data were downloaded and processed for peak calling using MACS. Using the same stringent criteria (detailed in Supplementary Fig. 8), wherein 2,827 TCF-1 binding peaks were identified in CD8+ T cells, we found 32,663 peaks in whole thymocytes. Possible reasons for the higher numbers of TCF-1 binding peaks in whole thymocytes include: 1) TCF-1 may regulate different target genes during thymocyte maturation stages. The binding events detected in whole thymocytes are a collection of all TCF-1 binding events at different stages; and 2) the ChIP-Seq control sample was from input DNA for peak calling, whereas ChIP-Seq of TCF-1 and Runx3 by us and Lotem J et al used IgG or non-immune serum-immunoprecipitated samples as control. The ChIP-Seq track wiggle files were uploaded to the UCSC genome browser for visualization of enriched binding by the transcription factors. For the select gene locus, the transcription start site (TSS) and orientation are marked by arrows. The horizontal bars over TCF-1 or Runx3 tracks indicate the enriched binding peaks identified by MACS. (a) shows enriched binding of TCF-1 at the Thpok GTE in whole thymocytes but not in CD8+ T cells. (b) shows co-occupancy of TCF-1 and Runx3 at the Cd4 silencer in all cell types. TCF-1 is also associated with Cd4 enhancer and weakly with Cd4 promoter in whole thymocytes, consistent with reported TCF-1 binding to these regions by Huang Z et al (J. Immunol. 176, 4880, 2006). Note that no TCF-1 binding to Cd4 enhancer and promoter in CD8+ T cells.

Supplementary Figure 7 The TCF-1-binding sites in the Thpok GTE are important for the GTE enhancer activity.

(a) The TCF-1 sites in the 473-bp Thpok GTE. The two TCF-1 sites are marked and mutant sequence aligned. (b) Conservation of TCF-1 sites across different species, with core sequence highlighted. (c) Mutation of TCF-1 sites in the Thpok GTE abrogates its enhancer activity. 293T cells were transfected with the indicated luciferase reporter constructs along with an internal control pRL-TK. Forty-eight hours later, luciferase activity was measured as in Fig. 6c.

Supplementary Figure 8 ChIP-Seq analysis of TCF-1 in CD8+ T cells.

(a) Numbers of TCF-1 peaks identified using stringent and permissive settings of the MACS algorithm. Under the stringent setting, 2,827 peaks were defined as stringent TCF-1 peaks, and under the permissive settings, 6,577 additional peaks were defined as permissive TCF-1 peaks. (b) Genomic distribution of all TCF-1 binding peaks. Promoter region is defined as “-5 kb to +1 kb” flanking the transcription start sites of known RefSeq genes. (c) Overlap of TCF-1 binding peaks with H3K4me3 and H3K27me3 peaks in human naïve CD8+ T cells. The homologous regions of the H3K4me3 and H3K27me3 peaks from human CD8+ T cells were identified in mouse genome using the LiftOver tool. TCF-1 binding peaks in different genomic regions were then assessed for peak overlapping. The criterion for overlapping is that the closest boundary-to-boundary distance of two peaks is within 400 bp. (d) TCF-LEF and (e) Runx motifs found in the permissive TCF-1 binding peaks. The motif logos are shown. Note that the motifs found in the permissive TCF-1 peaks are consistent with those found in the stringent TCF-1 peaks (Fig. 8c and 8d). (f) Venn diagram showing the motif distribution in the permissive TCF-1 peaks.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1581 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Steinke, F., Yu, S., Zhou, X. et al. TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4+ T cell fate and interact with Runx3 to silence Cd4 in CD8+ T cells. Nat Immunol 15, 646–656 (2014). https://doi.org/10.1038/ni.2897

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2897

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing