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Regulation of Tcrb recombination ordering by c-Fos-dependent RAG deposition

Nature Immunology volume 9, pages 794–801 (2008)Cite this article

Abstract

Antigen receptor variable-(diversity)-joining (V(D)J) recombination at the locus encoding the T cell antigen receptor-β (Tcrb) is ordered, with Dβ-to-Jβ assembly preceding Vβ-to-DJβ joining. The molecular mechanism underlying this 'preferred' order of rearrangement remains unclear. Here we show that the Dβ 23–base pair recombination signal sequence (Dβ 23-RSS) contains a specific AP-1 transcription factor–binding site bound by AP-1 and its component c-Fos expressed at a specific stage. Cell-based recombination assays suggested that c-Fos interacted directly with the RAG recombinase and enhanced its deposition to Dβ 23-RSSs, thus conferring the priority of DJβ recombination. Loss of c-Fos decreased Tcrb recombination efficiency and disrupted recombination ordering in vivo. Our results show an unexpected function for c-Fos as a direct regulator of Tcrb recombination, rather than its usual function as a transcription regulator, and provide new insight into the mechanisms of recombination ordering.

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Figure 1: Identification of c-Fos as a potential regulator of the order of Tcrb recombination.
Figure 2: Binding of c-Fos to the AP-1-binding site in the Dβ 3′ 23-RSS.
Figure 3: Interaction of c-Fos with RAG enhances the deposition of RAG proteins to the Dβ 23-RSS by recruitment.
Figure 4: Enhancement of the recombination of Dβ 23-RSS–containing substrates by c-Fos–AP-1.
Figure 5: Effect of c-Fos mutant proteins on the recombination of Dβ 23-RSS–containing substrates.
Figure 6: Suppression of Vβ-to-Dβ recombination by c-Fos in pJH290-VD-RSS but not in pJH290-VD-RSSm.
Figure 7: Impairment of DJβ and VDJβ recombination and 'violation' of Tcrb recombination ordering in c-Fos-deficient mice.

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References

  1. Schlissel, M.S. Regulating antigen-receptor gene assembly. Nat. Rev. Immunol. 3, 890–899 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Bassing, C.H., Swat, W. & Alt, F.W. The mechanism and regulation of chromosomal V(D)J recombination. Cell 109, S45–S55 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Osipovich, O. et al. Essential function for SWI-SNF chromatin-remodeling complexes in the promoter-directed assembly of Tcrb genes. Nat. Immunol. 8, 809–816 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Skok, J.A. et al. Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nat. Immunol. 8, 378–387 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. van Gent, D.C., Ramsden, D.A. & Gellert, M. The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination. Cell 85, 107–113 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Eastman, Q.M., Leu, T.M.J. & Schatz, D.G. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85–88 (1996).

    Article  CAS  PubMed  Google Scholar 

  7. Swanson, P.C. The bounty of RAGs: recombination signal complexes and reaction outcomes. Immunol. Rev. 200, 90–114 (2004).

    Article  CAS  PubMed  Google Scholar 

  8. Sleckman, B.P. et al. Mechanisms that direct ordered assembly of T cell receptor beta locus V, D, and J gene segments. Proc. Natl. Acad. Sci. USA 97, 7975–7980 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Oettinger, M.A. How to keep V(D)J recombination under control. Immunol. Rev. 200, 165–181 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Tillman, R.E. et al. Cutting edge: targeting of Vβ to Dβ rearrangement by RSSs can be mediated by the V(D)J recombinase in the absence of additional lymphoid-specific factors. J. Immunol. 170, 5–9 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Khor, B. & Sleckman, B.P. Intra- and inter-allelic ordering of T cell receptor β chain gene assembly. Eur. J. Immunol. 35, 964–970 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Mostoslavsky, R., Alt, F.W. & Rajewsky, K. The lingering enigma of the allelic exclusion mechanism. Cell 118, 539–544 (2004).

    Article  CAS  PubMed  Google Scholar 

  13. Khor, B. & Sleckman, B.P. Allelic exclusion at the TCRβ locus. Curr. Opin. Immunol. 14, 230–234 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Jung, D., Giallourakis, C., Mostoslavsky, R. & Alt, F.W. Mechanism and control of V(D)J recombination at the immunoglobulin heavy chain locus. Annu. Rev. Immunol. 24, 541–570 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Olaru, A., Petrie, H.T. & Livak, F. Beyond the 12/23 rule of VDJ recombination independent of the RAG proteins. J. Immunol. 174, 6220–6226 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Jung, D. et al. Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted V(D)J recombination in nonlymphoid cells. Immunity 18, 65–74 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Stanhope-Baker, P., Hudson, K.M., Shaffer, A.L., Constantinescu, A. & Schlissel, M.S. Cell type-specific chromatin structure determines the targeting of V(D)J recombinase activity in vitro. Cell 85, 887–897 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Schlissel, M.S. & Stanhope-Baker, P. Accessibility and the developmental regulation of V(D)J recombination. Semin. Immunol. 9, 161–170 (1997).

    Article  CAS  PubMed  Google Scholar 

  19. Mathieu, N., Hempel, W.M., Spicuglia, S., Verthuy, C. & Ferrier, P. Chromatin remodeling by the T cell receptor (TCR)-β gene enhancer during early T cell development: implications for the control of TCR-β locus recombination. J. Exp. Med. 192, 625–636 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jackson, A.M. & Krangel, M.S. Turning T-cell receptor β recombination on and off: more questions than answers. Immunol. Rev. 209, 129–141 (2006).

    Article  PubMed  Google Scholar 

  21. Zhang, Z. et al. Transcription factor Pax5 (BSAP) transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin genes. Nat. Immunol. 7, 616–624 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Chien, Y.H. et al. T-cell receptor δ gene rearrangements in early thymocytes. Nature 330, 722–727 (1987).

    Article  CAS  PubMed  Google Scholar 

  23. Farina, A.R. et al. Transcriptional regulation of intercellular adhesion molecule 1 by phorbol ester in human neuroblastoma cell line SK-N-SH involves jun- and fos-containing activator protein 1 site binding complex(es). Cell Growth Differ. 8, 789–800 (1997).

    CAS  PubMed  Google Scholar 

  24. Borner, C., Hollt, V. & Kraus, J. Involvement of activator protein-1 in transcriptional regulation of the human μ-opioid receptor gene. Mol. Pharmacol. 61, 800–805 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Spicuglia, S. et al. Promoter activation by enhancer-dependent and -independent loading of activator and coactivator complexes. Mol. Cell 10, 1479–1487 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Fugmann, S.D., Villey, I.J., Ptaszek, L.M. & Schatz, D.G. Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Mol. Cell 5, 97–107 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Liang, H.E. et al. The “dispensable” portion of RAG2 is necessary for efficient V-to-DJ rearrangement during B and T cell development. Immunity 17, 639–651 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Hesse, J.E., Lieber, M.R., Gellert, M. & Mizuuchi, K. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals. Cell 49, 775–783 (1987).

    Article  CAS  PubMed  Google Scholar 

  29. Tsai, C.L., Drejer, A.H. & Schatz, D.G. Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination. Genes Dev. 16, 1934–1949 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. West, K.L. et al. A direct interaction between the RAG2 C terminus and the core histones is required for efficient V(D)J recombination. Immunity 23, 203–212 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Matthews, A.G. RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination. Nature 450, 1106–1110 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. McBride, K.F.C., Lefebvre, C. & Nemer, M. Interaction with GATA transcription factors provides a mechanism for cell-specific effects of c-Fos. Oncogene 22, 8403–8412 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Brown, H.J., Sutherland, J.A., Cook, A., Bannister, A.J. & Kouzarides, T. An inhibitor domain in c-Fos regulates activation domains containing the HOB1 motif. EMBO J. 14, 124–131 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, Z.Q. et al. Bone and haematopoietic defects in mice lacking c-fos. Nature 360, 741–745 (1992).

    Article  CAS  PubMed  Google Scholar 

  35. Dudley, E.C., Petrie, H.T., Shah, L.M., Owen, M.J. & Hayday, A.C. T cell receptor β chain gene rearrangement and selection during thymocyte development in adult mice. Immunity 1, 83–93 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Michie, A.M. & Zuniga-Pflucker, J.C. Regulation of thymocyte differentiation: pre-TCR signals and β-selection. Semin. Immunol. 14, 311–323 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Uematsu, Y. et al. In transgenic mice the introduced functional T cell receptor β gene prevents expression of endogenous β genes. Cell 52, 831–841 (1988).

    Article  CAS  PubMed  Google Scholar 

  38. Suzuki, D., Wang, L., Senoo, M. & Habu, S. The positional effect of Eβ on Vβ genes of TCRβ chain in the ordered rearrangement and allelic exclusion. Int. Immunol. 17, 1553–1560 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Chowdhury, D. & Sen, R. Stepwise activation of the immunoglobulin μ heavy chain gene locus. EMBO J. 20, 6394–6403 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chen, L., Glover, J.N.M., Hogan, P.G., Rao, A. & Harrison, S.C. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42–48 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Xu, W. et al. STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation. Am. J. Physiol. Lung Cell. Mol. Physiol. 285, L137–L148 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Difilippantonio, M.J., McMahan, C.J., Eastman, Q.M., Spanopoulou, E. & Schatz, D.G. RAG1 mediates signal sequence recognition and recruitment of RAG2 in V(D)J recombination. Cell 87, 253–262 (1996).

    Article  CAS  PubMed  Google Scholar 

  43. Arbuckle, J.L., Fauss, L.J., Simpson, R., Ptaszek, L.M. & Rodgers, K.K. Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination. J. Biol. Chem. 276, 37093–37101 (2001).

    Article  CAS  PubMed  Google Scholar 

  44. Peak, M.M., Arbuckle, J.L. & Rodgers, K.K. The central domain of core RAG1 preferentially recognizes single-stranded recombination signal sequence heptamer. J. Biol. Chem. 278, 18235–18240 (2003).

    Article  CAS  PubMed  Google Scholar 

  45. Liu, X. et al. Restricting Zap70 expression to CD4+CD8+ thymocytes reveals a T cell receptor-dependent proofreading mechanism controlling the completion of positive selection. J. Exp. Med. 197, 363–373 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sun, Z. et al. PKC-θ is required for TCR-induced NF-κB activation in mature but not immature T lymphocytes. Nature 404, 402–407 (2000).

    Article  CAS  PubMed  Google Scholar 

  47. Ardouin, L., Ismaili, J., Malissen, B. & Malissen, M. The CD3-γ δ ε and CD3-ζ /ηmodules are each essential for allelic exclusion at the T cell receptor β locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-β, -γ, and -δ Loci. J. Exp. Med. 187, 105–116 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank D. Schatz (Yale University), S. Desiderio (Johns Hopkins University) and M. Gellert (National Institutes of Health) for recombination plasmids; R. Bosselut, D. Li and S. Li for instructive comments on the manuscript; Q. Yuan for animal husbandry; and Z. Tan for cell sorting and flow cytometry analysis. Supported by the National Natural Science Foundation of China (30671913 and 30623003), the Ministry of Science and Technology (2006CB504303 and 2007CB815802), the National High-Tech Research and Development Program of China (2007AA02Z167), the National Basic Research Program of China (2007CB914504), the Chinese Academy of Sciences (KSCX1-YW-R-43 and KSCX2-YW-R-10) and the Council of Shanghai Municipal Government for Science and Technology (05QMX1460 and 06DZ22032).

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Authors and Affiliations

  1. Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Xiaoming Wang, Gang Xiao, Yafeng Zhang, Xiaomin Wen & Xiaolong Liu

  2. Model Animal Research Center, Nanjing University, Nanjing, 210093, China

    Xiang Gao

  3. Division of Hematopoiesis Center for AIDS Research, Kumamoto University, Kumamoto, 860-0811, Japan

    Seiji Okada

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  1. Xiaoming Wang
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Contributions

X.Wa. designed and did most experiments, analyzed data and prepared the manuscript; G.X. and X.We. constructed plasmids; Y.Z. did the coimmunoprecipitation and GST precipitation assays; X.G. and S.O. provided materials; and X.L. conceptualized the research, directed the study and prepared the manuscript.

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Correspondence to Xiaolong Liu.

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Wang, X., Xiao, G., Zhang, Y. et al. Regulation of Tcrb recombination ordering by c-Fos-dependent RAG deposition. Nat Immunol 9, 794–801 (2008). https://doi.org/10.1038/ni.1614

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