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:

A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer

Nature Immunology volume 5, pages 443–450 (2004)Cite this article

Abstract

We have identified a silencer and an antisilencing element that interact at a distance of 85 kilobases to regulate expression of the recombination activating genes Rag1 and Rag2 in thymocytes. Transgenic experiments showed that Rag promoter-proximal cis elements directed tissue-specific expression and that a Runx-dependent intergenic silencer suppressed expression in developing T cells. Deletion of the antisilencing element from the genomic Rag locus unmasked the intergenic silencer and abrogated Rag expression in developing CD4+CD8+ T cells. We speculate that the Rag antisilencing element belongs to a class of cis elements that might be useful for genome diversification by activating genes encoded by otherwise silent transposable elements.

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: Transgenic mouse constructs.
Figure 2: Rag2 expression is regulated by a combination of promoter proximal elements, a silencer and a distal antisilencer.
Figure 3: The Rag intergenic silencer suppresses expression of Rag1 and Rag2 in DP T cells.
Figure 4: The Rag silencer is active on the CD2 promoter and is Runx dependent.
Figure 5: The Rag ASE is required for expression of Rag1 and Rag2 in DP T cells but is not sufficient to function as an LCR.
Figure 6: Targeted deletion of the ASE.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Schatz, D.G., Oettinger, M.A. & Baltimore, D. The V(D)J recombination activating gene, RAG-1. Cell 59, 1035–1048 (1989).

    Article  CAS  Google Scholar 

  2. Oettinger, M.A., Schatz, D.G., Gorka, C. & Baltimore, D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517–1523 (1990).

    Article  CAS  Google Scholar 

  3. McBlane, J.F. et al. Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395 (1995).

    Article  CAS  Google Scholar 

  4. Schluter, S.F. & Marchalonis, J.J. Cloning of shark RAG2 and characterization of the RAG1/RAG2 gene locus. FASEB J. 17, 470–472 (2003).

    Article  CAS  Google Scholar 

  5. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  Google Scholar 

  6. Shinkai, Y. et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867 (1992).

    Article  CAS  Google Scholar 

  7. Wilson, A., Held, W. & MacDonald, H. Two waves of recombinase gene expression in developing thymocytes. J. Exp. Med. 179, 1355–1360 (1994).

    Article  CAS  Google Scholar 

  8. Petrie, H. et al. Multiple rearrangements in T cell receptor α chain genes maximize the production of useful thymocytes. J. Exp. Med. 178, 615–622 (1993).

    Article  CAS  Google Scholar 

  9. Shinkai, Y. et al. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science 259, 822–825 (1993).

    Article  CAS  Google Scholar 

  10. von Boehmer, H. & Fehling, H.J. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15, 433–452 (1997).

    Article  CAS  Google Scholar 

  11. Turka, L.A. et al. Thymocyte expression of RAG-1 and RAG-2: termination by T cell receptor cross-linking. Science 16, 778–781 (1991).

    Article  Google Scholar 

  12. Borgulya, P., Kishi, H., Uematsu, Y. & von Boehmer, H. Exclusion and inclusion of α and β T cell receptor alleles. Cell 69, 529–537 (1992).

    Article  CAS  Google Scholar 

  13. Yannoutsos, N. et al. The role of recombination activating gene (RAG) reinduction in thymocyte development in vivo . J. Exp. Med. 194, 471–480 (2001).

    Article  CAS  Google Scholar 

  14. Guo, J. et al. Regulation of the TCRα repertoire by the survival window of CD4+CD8+ thymocytes. Nat. Immunol. 3, 469–476 (2002).

    Article  Google Scholar 

  15. Gay, D., Saunders, T., Camper, S. & Weigert, M. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177, 999–1008 (1993).

    Article  CAS  Google Scholar 

  16. Tiegs, S.L., Russell, D.M. & Nemazee, D. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177, 1009–1020 (1993).

    Article  CAS  Google Scholar 

  17. Yu, W. et al. Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization. Nature 400, 682–687 (1999).

    Article  CAS  Google Scholar 

  18. Monroe, R.J. et al. RAG2:GFP knockin mice reveal novel aspects of RAG2 expression in primary and peripheral lymphoid tissues. Immunity 11, 201–212 (1999).

    Article  CAS  Google Scholar 

  19. Monroe, R.J., Chen, F., Ferrini, R., Davidson, L. & Alt, F.W. RAG2 is regulated differentially in B and T cells by elements 5′ of the promoter. Proc. Natl. Acad. Sci. USA 96, 12713–12718 (1999).

    Article  CAS  Google Scholar 

  20. Yu, W. et al. Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5′ of RAG2. Science 285, 1080–1084 (1999).

    Article  CAS  Google Scholar 

  21. Hsu, L.Y. et al. A conserved transcriptional enhancer regulates RAG gene expression in developing B cells. Immunity 19, 105–117 (2003).

    Article  CAS  Google Scholar 

  22. Fuller, K. & Storb, U. Identification and characterization of the murine Rag1 promoter. Mol. Immunol. 34, 939–954 (1997).

    Article  CAS  Google Scholar 

  23. Miranda, G.A. et al. Combinatorial regulation of the murine RAG-2 promoter by Sp1 and distinct lymphocyte-specific transcription factors. Mol. Immunol. 38, 1151–1159 (2002).

    Article  CAS  Google Scholar 

  24. Li, Q., Peterson, K.R., Fang, X. & Stamatoyannopoulos, G. Locus control regions. Blood 100, 3077–3086 (2002).

    Article  CAS  Google Scholar 

  25. Muller, H. Types of visible variations induced by X-rays in Drosophila. J. Genet. 22, 299–334 (1930).

    Article  Google Scholar 

  26. Wakimoto, B.T. Beyond the nucleosome: epigenetic aspects of position-effect variegation in Drosophila. Cell 93, 321–324 (1998).

    Article  CAS  Google Scholar 

  27. Grosveld, F., van Assendelft, G.B., Greaves, D.R. & Kollias, G. Position-independent, high-level expression of the human β-globin gene in transgenic mice. Cell 51, 975–985 (1987).

    Article  CAS  Google Scholar 

  28. Bonifer, C., Yannoutsos, N., Kruger, G., Grosveld, F. & Sippel, A.E. Dissection of the locus control function located on the chicken lysozyme gene domain in transgenic mice. Nucleic Acids Res. 22, 4202–4210 (1994).

    Article  CAS  Google Scholar 

  29. Lakso, M. et al. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860–5865 (1996).

    Article  CAS  Google Scholar 

  30. Festenstein, R. et al. Locus control region function and heterochromatin-induced position effect variegation. Science 271, 1123–1125 (1996).

    Article  CAS  Google Scholar 

  31. de Boer, J. et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314–325 (2003).

    Article  CAS  Google Scholar 

  32. 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  Google Scholar 

  33. 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  Google Scholar 

  34. Rajewsky, N., Socci, N.D., Zapotocky, M. & Siggia, E.D. The evolution of DNA regulatory regions for proteo-γ bacteria by interspecies comparisons. Genome Res. 12, 298–308 (2002).

    Article  CAS  Google Scholar 

  35. Zavolan, M., Rajewsky, K., Socci, N.D. & Gaasterland, T. SMASHing regulatory sites in DNA by human-mouse sequence comparisons. Proc. Comput. Systems Bioinformatics CSB2003, 277–286 (2003).

    Google Scholar 

  36. Dillon, N. & Festenstein, R. Unravelling heterochromatin: competition between positive and negative factors regulates accessibility. Trends Genet. 18, 252–258 (2002).

    Article  CAS  Google Scholar 

  37. Milot, E. et al. Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription. Cell 87, 105–114 (1996).

    Article  CAS  Google Scholar 

  38. Kioussis, D. & Festenstein, R. Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Curr. Opin. Genet. Dev. 7, 614–619 (1997).

    Article  CAS  Google Scholar 

  39. Baxter, J., Merkenschlager, M. & Fisher, A.G. Nuclear organisation and gene expression. Curr. Opin. Cell Biol. 14, 372–376 (2002).

    Article  CAS  Google Scholar 

  40. Francastel, C., Walters, M.C., Groudine, M. & Martin, D.I. A functional enhancer suppresses silencing of a transgene and prevents its localization close to centrometric heterochromatin. Cell 99, 259–269 (1999).

    Article  CAS  Google Scholar 

  41. 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 

  42. Flajnik, M.F. Comparative analyses of immunoglobulin genes: surprises and portents. Nat. Rev. Immunol. 2, 688–698 (2002).

    Article  CAS  Google Scholar 

  43. Agrawal, A., Eastman, Q.M. & Schatz, D.G. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394, 744–751 (1998).

    Article  CAS  Google Scholar 

  44. Hiom, K., Melek, M. & Gellert, M. DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations. Cell 94, 463–470 (1998).

    Article  CAS  Google Scholar 

  45. Misulovin, Z., Yang, X.W., Yu, W., Heintz, N. & Meffre, E. A rapid method for targeted modification and screening of recombinant bacterial artificial chromosome. J. Immunol. Methods 257, 99–105 (2001).

    Article  CAS  Google Scholar 

  46. Bunting, M., Bernstein, K.E., Greer, J.M., Capecchi, M.R. & Thomas, K.R. Targeting genes for self-excision in the germ line. Genes Dev. 13, 1524–1528 (1999).

    Article  CAS  Google Scholar 

  47. Jareborg, N., Birney, E. & Durbin, R. Comparative analysis of noncoding regions of 77 orthologous mouse and human gene pairs. Genome Res. 9, 815–824 (1999).

    Article  CAS  Google Scholar 

  48. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  Google Scholar 

  49. Nagaoka, H., Gonzalez-Aseguinolaza, G., Tsuji, M. & Nussenzweig, M.C. Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production. J. Exp. Med. 191, 2113–2120 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Velinzon for flow cytometry cell sorting; members of the Nussenzweig laboratory and D. Dorsett for discussions; E. Besmer for help with the manuscript, and M. Zavolan for discussions and help with customizing SMASH; and D. Kioussis for the CD2 cassette. Supported in part by National Institutes of Health and Howard Hughes Medical Institute (M.C.N.).

Author information

Author notes

  1. Ziva Misulovin

    Present address: Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, 63110, USA

  2. Wong Yu & Nikolaus Rajewsky

    Present address: Department of Biology, New York University, Washington Square, New York, New York, 10003-6688, USA

  3. Bernardo R Peixoto

    Present address: Molecular Genetic and Pathology Unit, Hospital Divino Espirito Santo, 9500, Azores, Portugal

Authors and Affiliations

  1. Laboratory of Molecular Immunology, Rockefeller University, New York, 10021, New York, USA

    Nikos Yannoutsos, Ziva Misulovin, Anna Gazumyan, Wong Yu, Bernardo R Peixoto, Thomas Eisenreich & Michel C Nussenzweig

  2. Howard Hughes Medical Institute, Rockefeller University, New York, 10021, New York, USA

    Vasco Barreto, Ziva Misulovin, Anna Gazumyan, Thomas Eisenreich & Michel C Nussenzweig

  3. Center of Studies in Physics and Biology, Rockefeller University, New York, 10021, New York, USA

    Nikolaus Rajewsky

  4. Stanford University Medical School, Stanford, California, 94305-5323, USA

    Wong Yu

Authors
  1. Nikos Yannoutsos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vasco Barreto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ziva Misulovin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna Gazumyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wong Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nikolaus Rajewsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bernardo R Peixoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Thomas Eisenreich
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michel C Nussenzweig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Nikos Yannoutsos or Michel C Nussenzweig.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yannoutsos, N., Barreto, V., Misulovin, Z. et al. A cis element in the recombination activating gene locus regulates gene expression by counteracting a distant silencer. Nat Immunol 5, 443–450 (2004). https://doi.org/10.1038/ni1053

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1053

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