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Class switch recombination junctions are not affected by the absence of the immunoglobulin heavy chain Eμ enhancer

Cellular & Molecular Immunology volume 16, pages 671–673 (2019)Cite this article

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The mice were housed and the procedures were conducted in agreement with European Directive 2010/63/EU on animals used for scientific purposes applied in France as the « Décret n°2012–118 du 1er février 2013 relatif à la protection des animaux utilisés à des fins scientifiques ». Accordingly, the present project (APAFiS≠13855) was authorized by the « Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche ». Eμ-deficient and wt mice (housed in a conventional animal facility) were used. Single-cell suspensions of B-splenocytes were cultured for 4 days at 1 × 106 cells/ml in RPMI 1640 supplemented with 10% fetal calf serum and 5 μg/ml LPS, with (CSR toward IgG1) or without (CSR toward IgG3) the addition of 20 ng/ml IL-4 or 2 ng/ml TGFβ (PeproTech, Rocky Hill, NJ) (CSR toward IgA).14,16 Splenocytes were washed in PBS and stained with various antibodies: anti-B220-BV510 (BioLegends), anti-CD138-APC (Becton Dickinson Biosciences), anti-IgM PECy7 (ebiosciences), anti-IgG3-FITC (Becton Dickinson Biosciences), anti-IgG1-PE (Becton Dickinson Biosciences), and anti-IgA-FITC (Southern Biotech). The cells were analyzed on a Fortessa LSR2 (Beckman Coulter). In parallel experiments, stimulated B-cell splenocyte DNA was extracted for the investigation of Sμ-Sγ3, Sμ-Sγ1, and Sμ-Sα junctions. As previously described in detail,11 the junctions were PCR amplified. Libraries of 200 bp were prepared from the 1-2 kb PCR products of Sμ-Sγ3, Sμ-Sγ1, and Sμ-Sα amplification for Ion Proton sequencing (“GénoLim platform” of the Limoges University, France). The sequenced reads were then mapped to Sμ, Sγ1, Sγ3, and Sα regions using the BLAST algorithm. As previously reported,11 the computational tool developed for experiments performs junction assembly, identifies breakpoints in Sμ, Sγ1, Sγ3, and Sα, identifies junction structure (blunt, microhomology, large homology or insertion junctions) and outputs a statistical summary of the identified junctions.

Confirming previous studies,5,6,7 flow cytometry experiments indicated that compared to wt mice, the deletion of the Eμ enhancer had only a minor impact on CSR toward IgG3 (mean 5.0 vs 5.6%), IgG1 (mean 8.5 vs 19.3%), and IgA (mean 1.7 vs 3.5%) (Fig. 1b). We then extracted stimulated B-cell DNA to investigate the molecular signatures of these Sμ-Sγ3 (IgG3 CSR), Sμ-Sγ1 (IgG1 CSR), and Sμ-Sα (IgA CSR) junctions. When a few dozen CSR junctions are reported after cloning and subsequent Sanger sequencing, several hundred or thousand junctions can be documented using our computational tool of CSR junctions sequenced by high-throughput sequencing. The structural profiles (blunt, microhomology, large homology or junction with insertions) of 2443 Sμ-Sγ3, 8982 Sμ-Sγ1, and 617 Sμ-Sα Eμ-deficient junctions and 7209 Sμ-Sγ3, 14,700 Sμ-Sγ1, and 2639 Sμ-Sα wt junctions are reported in Fig. 1c. No significant difference (Mann–Whitney U-test) was found between Eμ-deficient mice and wt mice. In addition to the type of junctions, the distribution of IgG3, IgG1, and IgA junctions in terms of distance from the forward PCR primer in Sμ and from one of the reverse primers in Sγ3, Sγ1, and Sα was similar in Eμ-deficient and wt mice (Fig. 1d). The same results were found for the localization of the breakpoints within AID hotspots (AGCT, WRCY, RGYW) and other motifs (Fig. 1e) (displayed along specifically targeted segments within S regions).

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Fig. 1

References

  1. Stavnezer, J. & Schrader, C. E. IgH chain class switch recombination: mechanism and regulation. J. Immunol. 193, 5370–5378 (2014).

    Article  CAS  Google Scholar 

  2. Rouaud, P. et al. Elucidation of the enigmatic IgD class-switch recombination via germline deletion of the IgH 3’ regulatory region. J. Exp. Med. 211, 975–985 (2014).

    Article  CAS  Google Scholar 

  3. Saintamand, A. et al. Elucidation of IgH 3’ region regulatory role during class switch recombination via germline deletion. Nat. Commun. 6, 7084 (2015).

    Article  CAS  Google Scholar 

  4. Wuerffel, R. et al. S-S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase. Immunity 27, 711–722 (2007).

    Article  CAS  Google Scholar 

  5. Marquet, M. et al. The Eµ enhancer region influences H chain expression and B cell fate without impacting IgVH repertoire and immune response in vivo. J. Immunol. 193, 1171–1183 (2014).

    Article  CAS  Google Scholar 

  6. Li, F., Yan, Y., Pieretti, J., Feldman, D. A. & Eckhardt, L. A. Comparison of identical and functional Igh alleles reveals a nonessential role for Eμ in somatic hypermutation and class-switch recombination. J. Immunol. 185, 6049–6057 (2010).

    Article  CAS  Google Scholar 

  7. Perlot, T., Alt, F. W., Bassing, C. H., Suh, H. & Pinaud, E. Elucidation of IgH introninc enhancer functions via germ-line deletion. Proc. Natl Acad. Sci. USA 102, 14362–14367 (2005).

    Article  CAS  Google Scholar 

  8. Chaudhuri, J. & Alt, F. W. Class-switch recombination: interplay of transcription, DNA deamination and DNA repair. Nat. Rev. Immunol. 4, 541–552 (2004).

    Article  CAS  Google Scholar 

  9. Chang, H. H. Y., Pannunzio, N. R., Adachi, N. & Lieber, M. R. Non-homologous DNA end joining and alternative pathways to double break repair. Nat. Rev. Mol. Cell. Biol. 18, 495506 (2017).

    Article  Google Scholar 

  10. Panchakshari, R. A. et al. DNA double-strand break response factors influence end-joining features of IgH class switch and general translocation junctions. Proc. Natl Acad. Sci. USA 115, 762–767 (2018).

    Article  CAS  Google Scholar 

  11. Boyer, F. et al. CSReport: a new computational tool designed for automatic analysis of class switch recombination junctions sequenced by high-throughput sequencing. J. Immunol. 198, 4148–4155 (2017).

    Article  CAS  Google Scholar 

  12. Ghazzaui, N., Issaoui, H., Saintamand, A., Boyer, F. & Denizot, Y. Analysis of IgD CSR junctions by high-throughput sequencing. Immunol. Lett. 188, 86–88 (2017).

    Article  Google Scholar 

  13. Issaoui, H., Ghazzaui, N., Saintamand, A., Denizot, Y. & Boyer, F. IgD class switch recombination is not controlled through the immunoglobulin heavy chain 3’ regulatory region super-enhancer. Cell. Mol. Immunol. 14, 871–874 (2017).

    Article  Google Scholar 

  14. Issaoui, H., Ghazzaui, N., Saintamand, A., Denizot, Y. & Boyer, F. High throughput sequencing reveals similar molecular signatures for class switch recombination junctions for the γ and α isotypes. Cell. Mol. Immunol. 16, 90–92 (2019).

    Article  CAS  Google Scholar 

  15. Ghazzaui, N., Issaoui, H., Saintamand, A., Denizot, Y. & Boyer, F. Uracil-DNA glycosylase is not implicated in the choice of the DNA repair pathway during B-cell class switch recombination. Cell. Mol. Immunol. 16, 93–95 (2019).

    Article  CAS  Google Scholar 

  16. Issaoui, H. et al. The IgH 3’ regulatory region super-enhancer does not control IgA class switch recombination in the B1 lineage. Cell. Mol. Immunol. 15, 289–291 (2018).

    Article  Google Scholar 

  17. Vincent-Fabert, C. et al. Genomic deletion of the whole IgH 3’ regulatory region (hs3a, hs1,2, hs3b, hs4) dramatically affects class switch recombination and Ig secretion to all isotypes. Blood 116, 1895–1898 (2010).

    Article  CAS  Google Scholar 

  18. Saintamand, A. et al. Eμ and 3’RR IgH enhancers show hierarchic unilateral dependence in mature B-cells. Sci. Rep 7, 442 (2017).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Authors are “Equipe Labellisée LIGUE 2018”. This work was supported by ANR (projet EpiSwitch-3’RR 2016). N.G. was supported by a grant from the “Société Française d’Hématologie”. H.I. and M.F. are supported by the University of Limoges and the “Région Nouvelle Aquitaine”. F.B. is supported by the Fondation Partenariale de l’Université de Limoges and ALURAD.

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  1. These author contributed equally: Hussein Issaoui, Nour Ghazzaui

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  1. UMR CNRS 7276, INSERM U1262, Equipe Labellisée LIGUE 2018, University of Limoges, CBRS, rue Pr. Descottes, 87025, Limoges, France

    Hussein Issaoui, Nour Ghazzaui, Mélissa Ferrad, François Boyer & Yves Denizot

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Contributions

Y.D. designed the research. N.G., H.I., and M.F. performed the research. F.B. and Y.D. analyzed the data. N.G., H.I., M.F., F.B., and Y.D. wrote the paper.

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Correspondence to Yves Denizot.

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Issaoui, H., Ghazzaui, N., Ferrad, M. et al. Class switch recombination junctions are not affected by the absence of the immunoglobulin heavy chain Eμ enhancer. Cell Mol Immunol 16, 671–673 (2019). https://doi.org/10.1038/s41423-019-0229-x

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