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A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires

Nature Immunology volume 16, pages 1085–1093 (2015)Cite this article

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Abstract

The locus encoding the T cell antigen receptor (TCR) α-chain and δ-chain (Tcra-Tcrd) undergoes recombination of its variable-diversity-joining (V(D)J) segments in CD4−CD8− double-negative thymocytes and CD4+CD8+ double-positive thymocytes to generate diverse TCRδ repertoires and TCRα repertoires, respectively. Here we identified a chromatin-interaction network in the Tcra-Tcrd locus in double-negative thymocytes that was formed by interactions between binding elements for the transcription factor CTCF. Disruption of a discrete chromatin loop encompassing the D, J and constant (C) segments of Tcrd allowed a single V segment to frequently contact and rearrange to D and J segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulated TCRδ diversity and indirectly regulated TCRα diversity.

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Figure 1: Long-range interaction network within the Tcra-Tcrd locus.
Figure 2: Generation of INT1-2-deficient mice.
Figure 3: Thymocyte development in INT1-2-deficient mice.
Figure 4: Restricted TCRδ repertoire in INT1-2-deficient mice.
Figure 5: Deletion of INT1-2 alters chromatin loop organization but not chromatin accessibility.
Figure 6: Generation of INT2M mice.
Figure 7: Partial redundancy between INT1 and INT2.
Figure 8: Restricted TCRα repertoire in INT1-2-deficient mice.

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Acknowledgements

We thank C. Bock for the production of mice with gene targeting; Y.-W. He (Duke University) for the targeting vector pGKneoF2L2DTA; O. Fedrigo for sequencing of 4C samples; D. Corcoran for advice on the processing of 4C sequencing data, L. Martinek, N. Martin and M. Cook for help with cell sorting and analysis; J. Liang for technical advice; Z. Huang for technical support; and E. Oltz and Y. Zhuang for comments on the manuscript. Supported by the US National Institutes of Health (R37 GM41052 to M.S.K.).

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Authors and Affiliations

  1. Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA

    Liang Chen, Zachary Carico, Han-Yu Shih & Michael S Krangel

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  1. Liang Chen
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  2. Zachary Carico
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Contributions

L.C. and M.S.K. planned the study; L.C., Z.C. and H.-Y.S. designed and performed the experiments; and L.C., Z.C. and M.S.K. analyzed the experiments and wrote the manuscript.

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Correspondence to Michael S Krangel.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Orientations of CBEs in the Tcra-Tcrd locus.

Top track, CTCF binding to the C57BL/6 Tcra-Tcrd locus in DN thymocytes14 (GEO accession number GSE41743); Central track, CBE orientation. Among 113 CBEs, 98 CBEs are oriented with the G-rich core on the top strand (blue lines) and 15 are oriented with the G-rich core on the bottom strand (red lines); Bottom track, Tcra-Tcrd gene segments.

Supplementary Figure 2 Vδ4 and Vδ6.3 use in γδ T cells from INT1-2-deficient mice.

Vδ4 and Vδ6.3 staining is shown for pregated gd ΤCR+ thymocytes from WT and INT1-2KO littermates. Percentages of Vδ4+ and Vδ6.3+ gd cells are indicated. Data are representative of two independent experiments.

Supplementary Figure 3 Regulation of Vδ and Vα rearrangements in INT1-2-deficient mice.

(a) Genomic DNA extracted from DP thymocytes from WT and INT1-2KO mice was analyzed for rearrangement of Trdv2-2 to different Jα segments by SYBR Green-qPCR, with normalization to Cd14. Data represent the mean ± SEM of 3 samples for each genotype, with each sample representing an individual mouse. (b) Genomic DNA extracted from adult DN3 thymocytes from WT and INT1-2KO mice was analyzed for rearrangement of Vδ-to-DD-Trdj1 or Vα- or Vδ-to-Traj61 or -Traj58 by SYBR Green-qPCR, with normalization to Cd14. Data represent the mean ± SEM of 3 samples for each genotype, with each sample representing an individual mouse. (c) Genomic DNA extracted from adult DN3 thymocytes from WT and INT1-2KO mice was analyzed for rearrangement of Trdv2-2-to-DD-Trdj1 or -Trdj2 by SYBR Green-qPCR, with normalization first to Cd14 and then to known quantities of plasmids containing cloned Trdv2-2-DD-Trdj1 or Trdv2-2-DD-Trdj2 rearrangements. Data represent the mean ± SEM of 3 samples for each genotype, with each sample representing a pool of 2-3 mice. Statistical significance was evaluated by 2-way ANOVA with Sidak’s multiple comparison test (a,b) or by unpaired Student’s t-test with Holm-Sidak correction for multiple comparisons. *, P<0.01; **, P<0.0001. ND, not detected.

Source data

Supplementary Figure 4 ChIP and quantitative PCR analysis of the binding of CTCF to wild-type alleles and alleles with deletion of INT1-2 in DN thymocytes.

WT and INT1-2KO alleles were on a Rag2–/– background. Trdv4 served as a negative control. Data represent the mean ± SEM of 4 WT and 3 INT1-2KO samples, with each sample representing a pool of 2-3 mice. Statistical significance was determined by unpaired Student’s t-test with Holm-Sidak correction for multiple comparisons.

Source data

Supplementary Figure 5 Chromatin looping in INT1-2-deficient mice facilitates Trdv2-2 contacts and rearrangement to Dδ and Jδ segments.

CBEs (red ovals) are depicted along with their orientations (embedded white arrowheads). In wild-type, stable looping between the TEA and INT2 CBEs is depicted by the thick red line, whereas heterogeneous looping involving the INT1 CBE is depicted by the multiple thin red arrows. In INT1-2KO, the TEA CBE loops to the INT3 and Trdv2-2 CBEs (thick and thin red lines, respectively). Within these loops, Trdv2-2 more frequently contacts Dδ and Jδ gene segments (thin gray lines).

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Supplementary Figures 1–5 and Supplementary Table 1 (PDF 947 kb)

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Chen, L., Carico, Z., Shih, HY. et al. A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires. Nat Immunol 16, 1085–1093 (2015). https://doi.org/10.1038/ni.3232

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