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Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes

Nature Immunology volume 8, pages 378–387 (2007)Cite this article

Abstract

Reversible contraction of immunoglobulin loci juxtaposes the variable (V) genes next to the (diversity)-joining-constant ((D)JC) gene domain, thus facilitating V-(D)J recombination. Here we show that the T cell receptor β (Tcrb) and T cell receptor αδ (Tcra-Tcrd) loci also underwent long-range interactions by looping in double-negative and double-positive thymocytes, respectively. Contraction of the Tcrb and Tcra loci occurred in rearranging thymocytes and was reversed at the next developmental stage. Decontraction of the Tcrb locus probably prevented further Vβ-DJβ rearrangements in double-positive thymocytes by separating the Vβ genes from the DJCβ domain. In most double-negative cells, one Tcrb allele was recruited to pericentromeric heterochromatin. Such allelic positioning may facilitate asynchronous Vβ-DJβ recombination. Hence, pericentromeric recruitment and locus 'decontraction' seem to contribute to the initiation and maintenance of allelic exclusion at the Tcrb locus.

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Figure 1: Structure of the Tcrb locus.
Figure 2: Reversible contraction of the Tcrb locus during T cell development.
Figure 3: Looping of the Tcrb locus in DN thymocytes.
Figure 4: Long-range interactions in the Tcrb locus during T cell development.
Figure 5: Interaction of the Vβ5 gene region with other gene segments of the Tcrb locus.
Figure 6: Contraction of the Tcra-Tcrd locus in DP thymocytes.
Figure 7: Looping of the Tcra-Tcrd locus in DP thymocytes.
Figure 8: Pericentromeric recruitment of Tcrb and Tcra-Tcrd loci during T cell development.

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Acknowledgements

We thank P. Ferrier (Centre d'Immunologie de Marseille-Luminy) for Tcra-transgenic Rag2−/− mice; L. Klein (Research Institute of Molecular Pathology) for Tcra-Tcrb-transgenic Rag2−/− mice; A. Souabni for help with the OP9-DL1 differentiation system; and G. Stengl for flow cytometry sorting. Supported by Boehringer Ingelheim, the Austrian GEN-AU initiative (financed by the Bundesminsterium für Bildung, Wissenschaft und Kultur), the Wellcome Trust (J.A.S.) and the Swedish Research Council (R.G.).

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Author notes

  1. Jane A Skok

    Present address: Present address: Department of Pathology, New York University School of Medicine, New York, New York 10016, USA.,

  2. Jane A Skok and Ramiro Gisler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Molecular Pathology, Division of Infection and Immunity, University College London, London, W1T 4JF, UK

    Jane A Skok & Deborah Farmer

  2. Research Institute of Molecular Pathology, Vienna Biocenter, A-1030, Vienna, Austria

    Ramiro Gisler, Maria Novatchkova & Meinrad Busslinger

  3. Department of Cell Biology and Genetics, Erasmus Medical Centre, 3000, Rotterdam, CA, The Netherlands

    Wouter de Laat

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Contributions

J.A.S. did most of the FISH experiments and did all confocal analyses of the FISH data; D.F. contributed to some FISH experiments and prepared DNA-FISH probes; R.G. did 3C analyses, in vitro differentiation experiments and flow cytometry sorting; W.d.L. provided training and supervision for the 3C experiments; M.N. did the bioinformatical analyses of the TCR loci; and M.B. planned the experimental outline and wrote the manuscript.

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Correspondence to Ramiro Gisler.

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Supplementary information

Supplementary Fig. 1

FACS analysis of the lymphoid cells used for 3C analysis. (PDF 217 kb)

Supplementary Fig. 2

Extended configuration of the Tcrb and Tcra/d loci in pre-B cells. (PDF 292 kb)

Supplementary Fig. 3

Additional pictures with looping configurations of the Tcrb and Tcra/d alleles. (PDF 500 kb)

Supplementary Fig. 4

Representative PCR analysis of a 3C experiment. (PDF 217 kb)

Supplementary Fig. 5

Linearity of the PCR assay and BglII digestion of the crosslinked chromatin. (PDF 243 kb)

Supplementary Fig. 6

Interactions of the proximal Dβ1 region with distal Vβ genes of the Tcrb locus in DN pro-T cells. (PDF 181 kb)

Supplementary Fig. 7

Peripheral and pericentromeric location of the Tcrb and Tcra/d loci. (PDF 191 kb)

Supplementary Table 1

Separation of Tcrb and Tcra/d probe signals in developing T cells. (PDF 32 kb)

Supplementary Table 2

Subnuclear location and monoallelic pericentromeric recruitment of the Tcrb and Tcra/d loci in thymocytes and peripheral T cells (PDF 28 kb)

Supplementary Table 3

Predominant recruitment of the Tcrb and Tcra/d loci to pericentromeric heterochromatin. (PDF 74 kb)

Supplementary Table 4

PCR primers used for 3C analysis of the Tcrb locus. (PDF 27 kb)

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Skok, J., Gisler, R., Novatchkova, M. et al. Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nat Immunol 8, 378–387 (2007). https://doi.org/10.1038/ni1448

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