Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Correspondence
  • Published:

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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., Saintamand, A., Saad, F., Carrion, C., Lecardeur, S. & Cogné, M. 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. Pham, P., Afif, S. A., Shimoda, M., Maeda, K., Sakaguchi, N. & Pedersen, L. C. et al. Structural analysis of the activation-induced deoxycytidine deaminase required in immunoglobulin diversification. DNA Repair. 43, 48–56 (2016).

    Article  CAS  Google Scholar 

  4. Kohli, R. M., Maul, R. W., Guminski, A. F., McClure, R. L., Gajula, K. S. & Saribasak, H. et al. Local sequence targeting in the AID/APOBEC family differentially impacts retroviral restriction and antibody diversification. J. Biol. Chem. 285, 40956–40964 (2010).

    Article  CAS  Google Scholar 

  5. Silvas, T. V. & Schiffer, C. A. APOBEC3s: DNA-editing human cytidine deaminases. Protein Sci. 28, 1552–1566 (2019).

    CAS  PubMed  Google Scholar 

  6. Buisson, R., Langenbucher, A., Bowen, D., Kwan, E. E., Benes, C. H. & Zou, L. et al. Passenger hotspot mutations in cancer driven by APOBEC3A and mesoscale genomic features. Science 364, eaaw2872 (2019).

    Article  CAS  Google Scholar 

  7. Petljak, M., Alexandrov, L. B., Brammeld, J. S., Price, S., Wedge, D. C. & Grossmann, S. et al. Characterizing mutational signatures in human cancer cell lines reveals episodic APOBEC mutagenesis. Cell 176, 1282–1294 (2019).

    Article  CAS  Google Scholar 

  8. Koning, F. A., Newman, E. N., Kim, E. Y., Kunstman, K. J., Wolinsky, S. M. & Malim, M. H. Defining APOBEC3 expression patterns in human tissues and hematopoietic cell subsets. J. Virol. 83, 9474–9485 (2009).

    Article  CAS  Google Scholar 

  9. 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 

  10. Arudchandran, A., Bernstein, R. M. & Max, E. E. Single-stranded DNA breaks adjacent to cytosines occur during Ig gene class switch recombination. J. Immunol. 173, 3223–3229 (2004).

    Article  CAS  Google Scholar 

  11. Seidl, T., Whittall, T., Babaahmady, K. & Lehner, T. B-cell agonists up-regulate AID and APOBEC3G deaminases, which induced IgA and IgG antibodies and anti-viral function. Immunology 135, 207–215 (2011).

    Article  Google Scholar 

  12. Guo, K., Halemano, K., Schmitt, K., Katuwal, M., Wang, Y. & Harper, M. S. et al. Immunoglobulin VH gene diversity and somatic hypermutation during SIV infection of rhesus macaques. Immunogenetics 67, 355–370 (2015).

    Article  CAS  Google Scholar 

  13. Halemano, K., Guo, K., Heilman, K. J., Barrett, B. S., Smith, D. S. & Hasenkrug, K. J. et al. Immunoglobulin somatic hypermutation by APOBEC3/Rfv3 during retroviral infection. Proc. Natl Acad. Sci. USA 111, 7759–7764 (2014).

    Article  CAS  Google Scholar 

  14. Boyer, F., Boutouil, H., Dalloul, I., Dalloul, Z., Cook-Moreau, J. & Aldigier, J. C. 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 

  15. 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 

  16. 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 

  17. Issaoui, H., Ghazzaui, N., Ferrad, M., Boyer, F. & Denizot, Y. Class switch recombination junctions are not affected by the absence of the immunoglobulin heavy chain Eμ enhancer. Cell Mol. Immunol. 16, 671–673 (2019).

    Article  CAS  Google Scholar 

  18. 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 

  19. Okeoma, C. M., Lovsin, N., Peterlin, M. & Ross, S. R. APOBEC3 inhibits mouse mammary tumour virus replication in vivo. Nature 445, 927–930 (2007).

    Article  CAS  Google Scholar 

  20. Stavrou, S., Crawford, D., Blouch, K., Browne, E. P., Kohli, R. M. & Ross, S. R. Different modes of retrovirus restriction by human APOBEC3A and APOBEC3G in vivo. Plos Pathog. 10, e1004145 (2014).

    Article  Google Scholar 

  21. Saintamand, A., Rouaud, P., Saad, F., Rios, G., Cogné, M. & Denizot, Y. Elucidation of IgH 3’ region regulatory role during class switch recombination via germline deletion. Nat. Commun. 6, 7084 (2015).

    Article  CAS  Google Scholar 

  22. Issaoui, H., Ghazzaui, N., Saintamand, A., Carrion, C., Oblet, C. & Denizot, Y. 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 

  23. Chen, J. & MacCarthy, T. The preferred nucleotide contexts of the AID/ APOBEC cytidine deaminases have differential effects when mutating retrotransposon and virus sequences compared to host genes. PLOS Comput Biol. 13, e1005471 (2017).

    Article  Google Scholar 

Download references

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.

Author information

Author notes

  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

Authors
  1. Hussein Issaoui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mélissa Ferrad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nour Ghazzaui
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sandrine Lecardeur
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jeanne Cook-Moreau
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. François Boyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yves Denizot
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to François Boyer or Yves Denizot.

Ethics declarations

Competing interests

The authors declare no competing interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41423-019-0301-6

Search

Advanced search

Quick links