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:

Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5

Nature Immunology volume 9, pages 203–215 (2008)Cite this article

Abstract

Alternative lineage restriction and B cell fate commitment require the transcription factor Pax5, but the function of early B cell factor (EBF) in these processes remains mostly unexplored. Here we show that in the absence of EBF, 'expandable' and clonal lymphoid progenitor cells retained considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitor cells directed B cell generation at the expense of myeloid cell fates. EBF induced Pax5 and antagonized expression of genes encoding the transcription factors C/EBPα, PU.1 and Id2. Notably, sustained expression of EBF in Pax5−/− hematopoietic progenitor cells was sufficient to block their myeloid and T lineage potential in vivo. Furthermore, in Pax5−/− pro–B cells, higher EBF expression repressed alternative lineage genes. Thus, EBF can restrict alternative lineage 'choice' and promote commitment to the B cell fate independently of Pax5.

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: 'Expandable' and clonal Ebf1−/− lymphoid progenitor cells retain not only T lymphoid but also myeloid developmental potential.
Figure 2: Ebf1−/− lymphoid progenitor cells maintained in vitro in B lymphoid conditions show T cell, NK cell and myeloid developmental potential in vivo.
Figure 3: Higher expression of EBF in MPPs induces B cell development.
Figure 4: EBF promotes B cell fate in MPPs at the expense of myeloid cell fate options.
Figure 7: Constitutive expression of EBF blocks the myeloid lineage differentiation of Pax5−/− pro–B cells.
Figure 5: EBF induces the generation of B cell precursors from myeloid progenitor cells.
Figure 6: EBF inhibits non–B cell fates independently of Pax5.
Figure 8: EBF binds to the Id2 promoter region both in vitro and in vivo.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Orkin, S.H. Diversification of haematopoietic stem cells to specific lineages. Nat. Rev. Genet. 1, 57–64 (2000).

    Article  CAS  Google Scholar 

  2. Singh, H., Medina, K.L. & Pongubala, J.M. Contingent gene regulatory networks and B cell fate specification. Proc. Natl. Acad. Sci. USA 102, 4949–4953 (2005).

    Article  CAS  Google Scholar 

  3. Warren, L.A. & Rothenberg, E.V. Regulatory coding of lymphoid lineage choice by hematopoietic transcription factors. Curr. Opin. Immunol. 15, 166–175 (2003).

    Article  CAS  Google Scholar 

  4. Nutt, S.L. & Kee, B.L. The transcriptional regulation of B cell lineage commitment. Immunity 26, 715–725 (2007).

    Article  CAS  Google Scholar 

  5. Adolfsson, J. et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295–306 (2005).

    Article  CAS  Google Scholar 

  6. Lai, A.Y. & Kondo, M. Asymmetrical lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors. J. Exp. Med. 203, 1867–1873 (2006).

    Article  CAS  Google Scholar 

  7. Yoshida, T., Ng, S.Y., Zuniga-Pflucker, J.C. & Georgopoulos, K. Early hematopoietic lineage restrictions directed by Ikaros. Nat. Immunol. 7, 382–391 (2006).

    Article  CAS  Google Scholar 

  8. Scott, E.W., Simon, M.C., Anastasi, J. & Singh, H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573–1577 (1994).

    Article  CAS  Google Scholar 

  9. Zhang, D.E. et al. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein α-deficient mice. Proc. Natl. Acad. Sci. USA 94, 569–574 (1997).

    Article  CAS  Google Scholar 

  10. Laslo, P. et al. Multilineage transcriptional primming and stabilization of alternate hematopoietic cell fates. Cell 126, 755–766 (2006).

    Article  CAS  Google Scholar 

  11. DeKoter, R.P., Lee, H.J. & Singh, H.P.U. 1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16, 297–309 (2002).

    Article  CAS  Google Scholar 

  12. Medina, K.L. et al. Assembling a gene regulatory network for specification of the B cell fate. Dev. Cell 7, 607–617 (2004).

    Article  CAS  Google Scholar 

  13. Lin, H. & Grosschedl, R. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376, 263–267 (1995).

    Article  CAS  Google Scholar 

  14. Zhuang, Y., Soriano, P. & Weintraub, H. The helix-loop-helix gene E2A is required for B cell formation. Cell 79, 875–884 (1994).

    Article  CAS  Google Scholar 

  15. Bain, G. et al. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79, 885–892 (1994).

    Article  CAS  Google Scholar 

  16. Urbanek, P., Wang, Z.Q., Fetka, I., Wagner, E.F. & Busslinger, M. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79, 901–912 (1994).

    Article  CAS  Google Scholar 

  17. Murre, C. Helix-loop-helix proteins and lymphocyte development. Nat. Immunol. 6, 1079–1086 (2005).

    Article  CAS  Google Scholar 

  18. Hagman, J., Belanger, C., Travis, A., Turck, C.W. & Grosschedl, R. Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression. Genes Dev. 7, 760–773 (1993).

    Article  CAS  Google Scholar 

  19. Engel, I. & Murre, C. The function of E- and Id proteins in lymphocyte development. Nat. Rev. Immunol. 1, 193–199 (2001).

    Article  CAS  Google Scholar 

  20. O'Riordan, M. & Grosschedl, R. Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 11, 21–31 (1999).

    Article  CAS  Google Scholar 

  21. Hesslein, D.G. et al. Pax5 is required for recombination of transcribed, acetylated, 5′ IgH V gene segments. Genes Dev. 17, 37–42 (2003).

    Article  CAS  Google Scholar 

  22. Nutt, S.L., Urbanek, P., Rolink, A. & Busslinger, M. Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev. 11, 476–491 (1997).

    Article  CAS  Google Scholar 

  23. Nutt, S.L., Heavey, B., Rolink, A.G. & Busslinger, M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 (1999).

    Article  CAS  Google Scholar 

  24. Rolink, A.G., Nutt, S.L., Melchers, F. & Busslinger, M. Long–term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401, 603–606 (1999).

    Article  CAS  Google Scholar 

  25. Mikkola, I., Heavey, B., Horcher, M. & Busslinger, M. Reversion of B cell commitment upon loss of Pax5 expression. Science 297, 110–113 (2002).

    Article  CAS  Google Scholar 

  26. Delogu, A. et al. Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells. Immunity 24, 269–281 (2006).

    Article  CAS  Google Scholar 

  27. Cobaleda, C., Schebesta, A., Delogu, A. & Busslinger, M. Pax5: the guardian of B cell identity and function. Nat. Immunol. 8, 463–470 (2007).

    Article  CAS  Google Scholar 

  28. Dias, S., Silva, H., Jr., Cumano, A. & Vieira, P. Interleukin-7 is necessary to maintain the B cell potential in common lymphoid progenitors. J. Exp. Med. 201, 971–979 (2005).

    Article  CAS  Google Scholar 

  29. Kikuchi, K., Lai, A.Y., Hsu, C.L. & Kondo, M. IL-7 receptor signaling is necessary for stage transition in adult B cell development through up-regulation of EBF. J. Exp. Med. 201, 1197–1203 (2005).

    Article  CAS  Google Scholar 

  30. Seet, C.S., Brumbaugh, R.L. & Kee, B.L. Early B cell factor promotes B lymphopoiesis with reduced interleukin 7 responsiveness in the absence of E2A. J. Exp. Med. 199, 1689–1700 (2004).

    Article  CAS  Google Scholar 

  31. Maier, H. et al. Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription. Nat. Immunol. 5, 1069–1077 (2004).

    Article  CAS  Google Scholar 

  32. Fuxa, M. & Busslinger, M. Reporter gene insertions reveal a strictly B lymphoid-specific expression pattern of Pax5 in support of its B cell identity function. J. Immunol. 178, 3031–3037 (2007).

    Article  CAS  Google Scholar 

  33. Schmitt, T.M. & Zuniga-Pflucker, J.C. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 (2002).

    Article  CAS  Google Scholar 

  34. Wiesmann, A. et al. Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity 12, 193–199 (2000).

    Article  CAS  Google Scholar 

  35. Medina, K.L. et al. Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen. Nat. Immunol. 2, 718–724 (2001).

    Article  CAS  Google Scholar 

  36. Hagman, J. & Lukin, K. Early B-cell factor 'pioneers' the way for B-cell development. Trends Immunol. 26, 455–461 (2005).

    Article  CAS  Google Scholar 

  37. Akashi, K., Traver, D., Miyamoto, T. & Weissman, I.L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404, 193–197 (2000).

    Article  CAS  Google Scholar 

  38. DeKoter, R.P., Walsh, J.C. & Singh, H.P.U. 1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors. EMBO J. 17, 4456–4468 (1998).

    Article  CAS  Google Scholar 

  39. Walsh, J.C. et al. Cooperative and antagonistic interplay between PU.1 and GATA-2 in the specification of myeloid cell fates. Immunity 17, 665–676 (2002).

    Article  CAS  Google Scholar 

  40. Nutt, S.L., Metcalf, D., D'Amico, A., Polli, M. & Wu, L. Dynamic regulation of PU.1 expression in multipotent hematopoietic progenitors. J. Exp. Med. 201, 221–231 (2005).

    Article  CAS  Google Scholar 

  41. DeKoter, R.P. & Singh, H. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288, 1439–1441 (2000).

    Article  CAS  Google Scholar 

  42. Zhang, Z., Cotta, C.V., Stephan, R.P., deGuzman, C.G. & Klug, C.A. Enforced expression of EBF in hematopoietic stem cells restricts lymphopoiesis to the B cell lineage. EMBO J. 22, 4759–4769 (2003).

    Article  CAS  Google Scholar 

  43. Rumfelt, L.L., Zhou, Y., Rowley, B.M., Shinton, S.A. & Hardy, R.R. Lineage specification and plasticity in CD19- early B cell precursors. J. Exp. Med. 203, 675–687 (2006).

    Article  CAS  Google Scholar 

  44. Chen, H. et al. PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene 11, 1549–1560 (1995).

    CAS  PubMed  Google Scholar 

  45. Schaniel, C., Bruno, L., Melchers, F. & Rolink, A.G. Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-B I-cell clones. Blood 99, 472–478 (2002).

    Article  CAS  Google Scholar 

  46. Ikawa, T., Kawamoto, H., Wright, L.Y. & Murre, C. Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20, 349–360 (2004).

    Article  CAS  Google Scholar 

  47. Mansson, R. et al. Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors. Immunity 26, 407–419 (2007).

    Article  Google Scholar 

  48. Singh, H. Gene targeting reveals a hierarchy of transcription factors regulating specification of lymphoid cell fates. Curr. Opin. Immunol. 8, 160–165 (1996).

    Article  CAS  Google Scholar 

  49. Fuxa, M. et al. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18, 411–422 (2004).

    Article  CAS  Google Scholar 

  50. Roessler, S. et al. Distinct promoters mediate the regulation of Ebf1 gene expression by interleukin-7 and Pax5. Mol. Cell. Biol. 27, 579–594 (2007).

    Article  CAS  Google Scholar 

  51. Lundgren, M. et al. Transcription factor dosage affects changes in higher order chromatin structure associated with activation of a heterochromatic gene. Cell 103, 733–743 (2000).

    Article  CAS  Google Scholar 

  52. Souabni, A., Cobaleda, C., Schebesta, M. & Busslinger, M. Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1. Immunity 17, 781–793 (2002).

    Article  CAS  Google Scholar 

  53. Sun, X.H. Constitutive expression of the Id1 gene impairs mouse B cell development. Cell 79, 893–900 (1994).

    Article  CAS  Google Scholar 

  54. Djuretic, I.M. et al. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells. Nat. Immunol. 8, 145–153 (2007).

    Article  CAS  Google Scholar 

  55. Singh, H. Shaping a helper T cell identity. Nat. Immunol. 8, 119–120 (2007).

    Article  CAS  Google Scholar 

  56. Maeda, T. et al. Regulation of B versus T lymphoid lineage fate decision by the proto-oncogene LRF. Science 316, 860–866 (2007).

    Article  CAS  Google Scholar 

  57. Allman, D. et al. Thymopoiesis independent of common lymphoid progenitors. Nat. Immunol. 4, 168–174 (2003).

    Article  CAS  Google Scholar 

  58. Schwarz, B.A. & Bhandoola, A. Circulating hematopoietic progenitors with T lineage potential. Nat. Immunol. 5, 953–960 (2004).

    Article  CAS  Google Scholar 

  59. Sitnicka, E. et al. Complementary signaling through flt3 and interleukin-7 receptor α is indispensable for fetal and adult B cell genesis. J. Exp. Med. 198, 1495–1506 (2003).

    Article  CAS  Google Scholar 

  60. Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D. & Hayakawa, K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225 (1991).

    Article  CAS  Google Scholar 

  61. Schlissel, M.S., Corcoran, L.M. & Baltimore, D. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J. Exp. Med. 173, 711–720 (1991).

    Article  CAS  Google Scholar 

  62. Ehlich, A., Martin, V., Muller, W. & Rajewsky, K. Analysis of the B-cell progenitor compartment at the level of single cells. Curr. Biol. 4, 573–583 (1994).

    Article  CAS  Google Scholar 

  63. Corcoran, A.E., Riddell, A., Krooshoop, D. & Venkitaraman, A.R. Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 391, 904–907 (1998).

    Article  CAS  Google Scholar 

  64. Li, C. & Wong, W.H. Model-based analysis of oligonucleotide arrays:model validation, design issues and standard error application. Genome Biology 2, 31.1–32.11 (2001).

    Google Scholar 

  65. Bertolino, E. et al. Regulation of interleukin 7–dependent immunoglobulin heavy-chain variable gene rearrangements by transcription factor STAT5. Nat. Immunol. 6, 836–843 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J.C. Zuniga-Pflucker (University of Toronto) for OP9 and OP9-DL1 cells; M. Busslinger (Institute of Molecular Pathology, Vienna) for Pax5−/− mice and pro–B cells; B. Kee (University of Chicago) for the E2A retroviral constructs; J. Hagman (National Jewish Medical and Research Center) for the epitope-tagged EBF construct; S. Firner (Max Planck Institute of Immunobiology) for doing 'ChIP-on-ChIP' chromatin immunnoprecipitation with EBF; the Immunology Applications Core Facility and the Functional Genomics Facility of the University of Chicago for advice on cell sorting and microarray analysis, respectively; and members of the Singh lab for suggestions and critical comments. Supported by the Irvington Institute (K.L.M.), the Leukemia Lymphoma Society (M.T. and D.A.), the Howard Hughes Medical Institute (H.S.), the National Institute of Allergy and Infectious Diseases and National Institute on Aging of the National Institutes of Health (D.A.) and the German Research Foundation (Deutsche Forschungsgemeinschaft; R.G.).

Author information

Author notes

  1. Kay L Medina

    Present address: Present address: Department of Immunology, The Mayo Clinic, Rochester, Minnesota 55905, USA.,

  2. Jagan M R Pongubala and Daniel L Northrup: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, The University of Chicago, Chicago, 60637, Illinois, USA

    Jagan M R Pongubala, David W Lancki, Kay L Medina, Eric Bertolino & Harinder Singh

  2. Department of Pathology and Laboratory of Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA

    Daniel L Northrup, Matthew Thomas & David Allman

  3. Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology, Freiburg, 79108, Germany

    Thomas Treiber & Rudolf Grosschedl

Authors
  1. Jagan M R Pongubala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel L Northrup
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David W Lancki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kay L Medina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas Treiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eric Bertolino
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Matthew Thomas
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rudolf Grosschedl
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David Allman
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Harinder Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M.R.P. designed and did experiments, analyzed data and wrote the manuscript; D.L.N. and D.W.L. designed and did experiments and analyzed data; K.L.M., T.T, M.T. and E.B. did experiments; D.A. and R.G. designed and supervised research and contributed to the writing; and H.S. designed and supervised research and wrote the manuscript.

Corresponding authors

Correspondence to David Allman or Harinder Singh.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–2 (PDF 1003 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pongubala, J., Northrup, D., Lancki, D. et al. Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5. Nat Immunol 9, 203–215 (2008). https://doi.org/10.1038/ni1555

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1555

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