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Molecular analysis of γ1, γ3, and α class switch recombination junctions in APOBEC3-deficient mice using high-throughput sequencing

Cellular & Molecular Immunology volume 17, pages 418–420 (2020)Cite this article

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Activation-induced deaminase (AID) is required for immunoglobulin (Ig) class switch recombination (CSR), in which the constant (C)μ gene of IgM is substituted with Cγ, Cε, or Cα, thereby generating IgG, IgE, and IgA antibodies, respectively, with new effector functions but the same antigenic specificity.1 AID targets specific DNA switch (S) regions preceding C regions except for Cδ.2 Sμ is usually the donor region, while Sγ,ε,α are the acceptor regions. AID deaminates C into U on single-stranded DNA by targeting the WRCY (W = A/T, R = A/G, and Y = C/T) hot motif and, to a lesser extent, the SYC (S = G/C, Y = C/T) cold motif.3,4 AID is a member of the apolipoprotein B editing complex (APOBEC) family. Among APOBEC genes, a family of evolutionarily conserved cytidine deaminases, APOBEC3 is implicated in diverse cell functions including innate immunity against retroviruses.4 The DNA-editing APOBEC3 enzymes have recently attracted attention due to their involvement in cancer and potential applications in gene editing.5,6,7 While a single copy of each APOBEC3 gene is present in rodents, seven copies of each APOBEC3 gene are found in humans. For example, APOBEC3A is expressed predominantly in monocytes, while high levels of APOBEC3G are found in B-cells. Both APOBEC3A and APOBEC3G expression is elevated in response to interferon-α.8 CSR is mediated by only AID attack, but some studies have suggested the putative involvement of APOBEC in the cleavage of some σδ and Sα DNAs.9,10 Furthermore, B-cell agonists that stimulate CSR upregulated both AID and APOBEC3G.11 APOBEC3G was also found to mutate VH genes during viral infection.12,13 APOBEC3 may contribute to a basal level of CSR, but its involvement was largely undetectable when a few dozen CSR junctions were reported after cloning and subsequent sequencing by the Sanger method. We recently reported a new computational tool (CSReport) for the automatic analysis of CSR junctions sequenced by high-throughput sequencing.14 The structural profiles (blunt, microhomology, large homology, or junctions with insertions) and locations of breakpoints within AID hotspots and other motifs can be analyzed for not just a few dozen but rather several hundreds or thousands of junctions. This computational tool has already been used to analyze the phenotypes of rare Sμ–Sδ junctions during IgD CSR9,15 and compare the structural profiles of Sμ–Sγ1/Sμ–Sγ3/Sμ–Sα junctions in uracil DNA glycosylase-deficient mice,16 IgH Eμ enhancer-deficient mice,17 and wild-type (wt) mice.18 In this study, we used high-throughput sequencing to analyze the molecular signature of Sμ–Sγ1/Sμ–Sγ3/Sμ–Sα junctions in mice expressing human APOBEC3A or APOBEC3G in an APOBEC3 knockout background for the first time.

Mice were housed and procedures were conducted in agreement with European Directive 2010/63/EU on the use of animals 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” and reviewed by the Ethics Committee of the University of Limoges (France). APOBEC3-deficient mice19 expressing human APOBEC3A (hAPOBEC3A) or hAPOBEC3G20 and C57BL6 wt mice (both types of mice were housed in a conventional animal facility) were used. B-splenocytes (1 × 106 cells/ml) were cultured for 4 days in RPMI 1640 with 10% fetal calf serum and 5 μg/ml LPS with (for IgG1 CSR) or without (for IgG3 CSR) 20 ng/ml IL-4 or 5 ng/ml TGFβ (for IgA CSR) (PeproTech, Rocky Hill, NJ).21,22 Splenocytes were stained with several labeled antibodies and analyzed on a Fortessa LSR2 (Beckman Coulter). B-cell splenocyte DNA was extracted, and Sμ–Sγ1, Sμ–Sγ3, and Sμ–Sα junctions were quantified as previously reported.14 Ion proton sequencing was performed in the “GénoLim platform” at the Limoges University (France). Sequenced reads were mapped to Sμ,γ1,γ3,α regions using the BLAST algorithm. The computational tool developed for the experiments performed junction assembly, identified breakpoints in S regions, identified junction structures (blunt, microhomology, large homology, or junctions with insertions) and output a statistical summary of the identified junctions.

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Acknowledgements

The 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 “Société Française d’Hématologie” and ANR (projet EpiSwitch-3’RR 2016). H.I. and M.F. are supported by the University of Limoges and “Région Nouvelle Aquitaine”. F.B. is supported by Fondation Partenariale de l’Université de Limoges and ALURAD.

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  1. These authors contributed equally: Hussein Issaoui, Mélissa Ferrad, Nour Ghazzaui

Authors and Affiliations

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

    Hussein Issaoui, Mélissa Ferrad, Nour Ghazzaui, Sandrine Lecardeur, Jeanne Cook-Moreau, François Boyer & Yves Denizot

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  1. Hussein Issaoui
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Contributions

Y.D. designed the research and obtained grants and the agreement of the Ethics Committee. N.G., H.I., M.F., and S.L. performed the research. F.B., J.C.M., and Y.D. analyzed the data. N.G., H.I., M.F., J.C.M., F.B., and Y.D. wrote the paper.

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Correspondence to François Boyer or Yves Denizot.

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Issaoui, H., Ferrad, M., Ghazzaui, N. et al. Molecular analysis of γ1, γ3, and α class switch recombination junctions in APOBEC3-deficient mice using high-throughput sequencing. Cell Mol Immunol 17, 418–420 (2020). https://doi.org/10.1038/s41423-019-0301-6

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