SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.


March 2021
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All studies must disclose on these points even when the disclosure is negative. No statistical methods were used to predetermine sample size for all experiments. Sample sizes were chosen based on previous studies in this field that used similar sample sizes to generate reproducible results (Lescale C et al, Nat Com, 2016;Dev H et al, Nature Cell Biology, 2018). The number of independent experiments are indicated in the legend of each Figure. No data was excluded from this study.
All experiments were repeated by at least two biological replicates with two or three isogenetic clones with consistent results. All attempts at replication were successful.
Sex-and age-matched mice were used. Randomization is not relevant because we did not use different experimental groups in our study.
No experiments were blinded, since subjective rating of data was not involved. Investigators were not blinded to allocation during experiments and outcome assessment. Blinding was not possible as investigators need to verify the control and matched mutant mouse strains as well as cell lines before each experiment. For many other approaches used in the manuscript, including Western blots, Flow Cytometry and IP, blinding was not feasible.
CRISPR-Cas9 edited v-Abl/Bcl2 pro-B cell clones were generated as previously described ( CRISPR-Cas9 edited CH12F3 cell clones were generated as previously described (Dev H, et al, Nature Cell Biology, 2018). The authors thank F. Alt (Harvard University, USA) for CH12F3 cells and 53bp1 knockout CH12F3 cell clones and T. Honjo (Kyoto University, Japan) for permission to use the CH12F3 cell line.
List of cell lines used in this study: v-abl Pro-B cell lines: 12095: WT, murine-derived pro-B cells (Lescale C. et al., Nat Com. 2016) 12096: WT, murine-derived pro-B cells (Lescale C. et al., Nat Com. 2016 Note that full information on the approval of the study protocol must also be provided in the manuscript.