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IgH locus suicide recombination does not depend on NHEJ in contrast to CSR in B cells

Cellular & Molecular Immunology volume 16, pages 201–202 (2019)Cite this article

There are several pathways solicited for DSB reparation. The two major ones are NHEJ and homologous recombination (HR), but there is also the A-EJ pathway. Defects in NHEJ decrease CSR4,5 and underline the importance of NHEJ in this process. HR does not seem to be directly implicated in CSR but rather to compete with NHEJ9 and could contribute to DNA repair of unresolved CSR-induced DSB after the cell cycle S phase.10 A-EJ accounts for the residual CSR. A-EJ mediators are not well defined and several systems seem to be involved such as MMEJ (microhomology-mediated end joining) and TMEJ (DNA polymerase theta (Polθ)-mediated end joining). Among the actors identified in the MMEJ, PARP-1 has been implicated in CSR.11 DNA Polθ and TMEJ deficiency do not impact CSR.9 To gain insights into DSB resolution during LSR, we analyzed the recruitment of XRCC4, RAD51, PARP-1, and Polθ to the S and 3′RR regions. We performed chromatin immunoprecipitation (ChIP) analysis in 48 h in vitro lipopolysaccharide + interleukin-4-stimulated B cells using antibodies raised against XRCC4, RAD51, PARP-1, Polθ, or no antibody (mock condition), and we determined the relative enrichment of Sµ, Sγ1, hs1,2, or hs4. Sγ3 was used as a negative control (Fig. 1d). We observed an increased level of enrichment of XRCC4 and PARP-1 proteins at the Sμ region, which is consistent with the involvement of NHEJ and A-EJ. Surprisingly, we did not see enrichment of XRCC4 at the level of Sγ1. PARP-1 was also found to be associated with hs1,2 and hs4. The absence of XRCC4 protein recruitment at the level of Sγ1 is surprising, but loop conformation of the IgH locus and synapse of S regions during B-cell activation12 may allow the repair of DSB by DNA repair molecules present at Sμ. At the 3′RR segments, the absence of XRCC4 and the recruitment of PARP-1 suggests the orientation of DSB repair at these sites to A-EJ. No RAD51 recruitment was observed on the studied segments of the IgH locus consistent with its lack of involvement in CSR and this discards HR usage during LSR. Polθ does not seem to be recruited to the IgH locus as fold enrichment (FE) values for Sµ, Sγ1, hs1,2, or hs4 were not statistically different from that of Sγ3. Polθ has not been shown to be required during CSR, but some data from the literature led us to test its participation in LSR. Polθ expression is increased in germinal B cells13 and Polθ is responsible for insertions longer than 4 bp and microhomologies >2 bp in DSB repair products14 as particularly observed in LSR junctions. However, our data suggest no role for Polθ during LSR.

Finally, delineation of mechanisms controlling accurate repair of IgH locus DSB, which are a potential source of translocation, is primordial. Our results account for major involvement of the A-EJ pathway during DSB repair during LSR rather than NHEJ as in CSR and suggest that CSR and LSR IgH locus recombinations are controlled by different balances between the DSB repair pathways.

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Fig. 1

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Acknowledgements

This work was supported by grants from Conseil Régional du Limousin, Association pour la Recherche sur le Cancer, Ligue Contre le Cancer (Comité de la Haute-Vienne, Comité de la Corrèze). H.B. is supported by Région Limousin and Université de Limoges. F.B. is supported by Fondation Partenariale de l’Université de Limoges and Association Limousine pour l’Utilisation du Rein Artificiel à Domicile. M.C. is supported by Institut Universitaire de France and Fondation pour la Recherche Médicale. We acknowledge the Biologie Intégrative Santé Chimie Environnement (BISCEM) platform of the Limoges University (France) for sequencing.

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  1. CRIBL, UMR CNRS 7276 INSERM U1262, 87000, Limoges, France

    Hend Boutouil, François Boyer, Jeanne Cook-Moreau, Michel Cogné & Sophie Péron

  2. Institut Universitaire de France, 75000, Paris, France

    Michel Cogné

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H.B., F.B., J.C.-M., M.C., and S.P. designed research; H.B., F.B., and S.P. performed research and analyzed data. J.C.-M., M.C., and S.P. obtained financial grants and wrote the manuscript.

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Correspondence to Sophie Péron.

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Boutouil, H., Boyer, F., Cook-Moreau, J. et al. IgH locus suicide recombination does not depend on NHEJ in contrast to CSR in B cells. Cell Mol Immunol 16, 201–202 (2019). https://doi.org/10.1038/s41423-018-0172-2

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