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Uracil-DNA glycosylase is not implicated in the choice of the DNA repair pathway during B-cell class switch recombination

Cellular & Molecular Immunology volume 16, pages 93–95 (2019)Cite this article

Mature B-cells express membrane IgM and IgD (of same specificity) through alternative splicing of a pre-mRNA encompassing constant (C)μ and Cδ genes. After encountering antigen, B-cells undergo class switch recombination (CSR) that substitutes the Cμ gene with Cγ, Cε, or Cα, thereby generating IgG, IgE, and IgA antibodies with the same antigenic specificity but new effector functions. 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, 2 CSR is controlled in cis by IgH locus super-enhancers3 and in trans by a wide spectrum of enzymes and proteins.1, 2 Among them, the role of the uracil DNA glycosylase (UNG) remains controversial. UNG is a key enzyme of base excision repair, which carries out faithful repair. Some authors estimate that during CSR, the UNG enzymatic activity removes the AID-induced dC to dU converted base of single-strand DNA, generating abasic sites and leading to DNA strand breaks.1 For other authors, the role of UNG is to stabilize the S–S synapse and to recruit DNA repair factors that facilitate the end-joining process.4, 5 Thus, the classical non-homogenous end joining pathway would be increased over the alternative end joining (A-EJ) pathway in UNG-deficient mice,4 suggesting an intriguing role of UNG in promoting the A-EJ pathway. These results were based on the analysis of several Sμ–Sγ1 sequences obtained in UNG-deficient conditions due to technical limitations (conventional Sanger sequencing) to investigate S–S junction molecular signatures. We recently reported a new computational tool (CSReport) for automatic analysis of CSR junctions sequenced by high-throughput sequencing.6 We used this tool to analyze the rare Sμ–σδ junctions formed during IgD CSR7, 8 and Sμ–Sγ3, Sμ–Sγ1, and Sμ–Sα junctions in wild-type (wt) mice.9 We thus used CSReport and high-throughput sequencing to analyze the molecular signature of Sμ–Sγ3, Sμ–Sγ1, and Sμ–Sα junctions in UNG-deficient mice in detail.

Our research has been approved by our local ethics committee review board (Comité Régional d’Ethique sur l’Expérimentation Animale du Limousin, Limoges, France) and carried out according to the European guidelines for animal experimentation. Wt mice and UNG-deficient mice (a gift from Dr. Tomas Lindahl, UK) were used. Single-cell suspensions of spleen cells were cultured for 4 days at 1 × 106 cells/ml in RPMI 1640 with 10% fetal calf serum and 5 μg/ml LPS, with (CSR toward IgG1) or without (CSR toward IgG3) addition of 20 ng/ml IL-4 or 2 ng/ml TGFβ (PeproTech, Rocky Hill, NJ) (CSR toward IgA).3, 9, 10 Splenocytes were washed in PBS and stained with various antibodies: anti-B220-PC5, anti-CD138-APC, anti-IgM PECy7, anti-IgG3-FITC, anti-IgG1-FITC, and anti-IgA-FITC. Cells were analyzed on a Fortessa LSR2 (Beckman Coulter). In parallel experiments, stimulated splenocyte DNA was extracted for investigation of Sμ–Sγ3, Sμ–Sγ1, and Sμ–Sα junctions. As previously described in detail,6 junctions were amplified with PCR. Libraries of 200 bp were prepared from the 1–2 kb PCR products of Sμ–Sγ1, Sμ–Sγ3, and Sμ–Sα amplified for Ion Proton sequencing (“GénoLim platform” of the Limoges University, France). Sequenced reads were then mapped to the Sμ, Sγ1, Sγ3, and Sαregions using BLAST algorithms. The computational tool developed for experiments performs junction assembly; identifies breakpoints in Sμ, Sγ1, Sγ3, and Sα; identifies junction structure (blunt, micro-homology, large-homology, or junction with insertions) and outputs a statistical summarization of the identified junctions.

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Acknowledgements

We thank Dr. Tomas Lindahl for sending us the UNG-deficient mice. This work was supported by ANR (projet EpiSwitch-3’RR 2016). N.G. was supported by a grant from the 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. These authors contributed equally: Nour Ghazzaui and Hussein Issaoui.

Authors and Affiliations

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

    Nour Ghazzaui, Hussein Issaoui, Yves Denizot & François Boyer

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

    Alexis Saintamand

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Contributions

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

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Correspondence to Yves Denizot.

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Ghazzaui, N., Issaoui, H., Saintamand, A. et al. 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). https://doi.org/10.1038/s41423-018-0034-y

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