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High-throughput sequencing reveals similar molecular signatures for class switch recombination junctions for the γ and α isotypes

Cellular & Molecular Immunology volume 16, pages 90–92 (2019)Cite this article

After encountering an antigen, B cells undergo class switch recombination (CSR), which substitutes the Cμ gene with Cγ, Cε, or Cα to generate IgG, IgE, and IgA antibodies with the same antigenic specificity but new effector functions.1 The DNA-editing enzyme activation-induced deaminase (AID) is essential for CSR by targeting switch (S) regions preceding Cμ (namely, the Sμ donor region) and the Cγ, Cε, and Cα genes (namely, the Sγ,ε,α acceptor regions).1 Cis-and trans-controlled DNA double strand beaks are generated during this process.2,3,4,5,6 Recruitment of DNA repair factors that facilitate the end-joining process is a crucial step of class switch recombination. Two pathways are implicated in this end joining. The classical non-homogenous end joining (c-NHEJ) pathway ligates DNA ends with no or little homology. By contrast, the alternative end joining (A-EJ) pathways is used to ligate DNA ends that have microhomology.3,4,5,6 Previous reports have suggested that IgG and IgA CSR might be differentially regulated, with preferential use of c-NHEJ for γ CSR and A-EJ for α CSR.7, 8 We recently reported a computational tool (CSReport) for automatic analysis of CSR junctions sequenced by high-throughput sequencing9 and used it to analyze the rare Sμ-σδ junctions that formed during IgD CSR.10, 11 We thus used CSReport and high-throughput sequencing to analyze the molecular signature of Sμ-Sγ3, Sμ-Sγ1, and Sμ-Sα junctions in wt mice in greater detail.

Our research was approved by our local ethics committee review board (Comité Régional d’Ethique sur l’Expérimentation Animale du Limousin, Limoges, France) and conducted according to the European guidelines for animal experimentation. Single-cell suspensions of spleen cells from wild-type (wt) 129 mice were cultured 4 days at 1 × 106 cells/ml in RPMI 1640 with 10% fetal calf serum (FCS) and 5 μg/ml LPS, with or without the addition of 20 ng/ml IL-4 or 2 ng/ml TGFβ (PeproTech, Rocky Hill, NJ). Splenocyte DNA was then extracted for investigation of Sμ-Sγ3, Sμ-Sγ1, and Sμ-Sα junctions. As previously described in detail,9 the junctions were PCR amplified. Libraries of 200 bp were prepared from the 1 to 2 kb PCR products of Sμ-Sγ1, Sμ-Sγ3, and Sμ-Sα amplification for Ion Proton sequencing (“GénoLim platform” of Limoges University, France). The sequenced reads were then mapped to Sμ and acceptor Sγ1, Sγ3, and Sα regions using the BLAST algorithm. The computational tool developed for the experiments performs junction assembly, identifies breakpoints in Sμ, Sγ1, Sγ3 Sα, identifies the junction structure (blunt, micro-homology or junction with insertions), and outputs a statistical summary of the identified junctions.

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Acknowledgements

This work was supported by ANR (project EpiSwitch-3’RR 2016). N.G. was supported by a grant from Association de Spécialisation et d’Orientation Scientifique (Lebanon) and the municipality of Khiam (Lebanon). H.I. is supported by CORC (FJA/NP 2015-109) and the University of Limoges. The authors are “Equipe Labellisée LIGUE 2018”. We thank the GénoLim platform and the Cytometry platform of the University of Limoges for sequencing and cell sorting. F.B. is supported by Fondation Partenariale de l’Université de Limoges and ALURAD.

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    1. UMR CNRS 7276, INSERM U1262, CBRS, Limoges University, rue Pr. Descottes, Limoges, 87025, France

      Hussein Issaoui, Nour Ghazzaui, Yves Denizot & François Boyer

    2. INSERM U1236, Rennes 1 University, Rennes, France

      Alexis Saintamand

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    Contributions

    H.I. and N.G. contributed equally to this work. Y.D. designed the research. N.G. and H.I. performed the research. A.S., F.B., and Y.D. analyzed the data. N.G., H.I., F.B., and Y.D. wrote the paper.

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    Correspondence to Yves Denizot.

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    Issaoui, H., Ghazzaui, N., Saintamand, A. et al. 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). https://doi.org/10.1038/s41423-018-0025-z

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