CtIP-mediated DNA resection is dispensable for IgH class switch recombination by alternative end-joining

To generate antibodies with different effector functions, B cells undergo Immunoglobulin class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical non-homologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ~25% CSR can be achieved by the alternative end-joining (A-EJ) pathway that preferentially uses microhomology (MH) at the junctions. While A-EJ mediated repair of endonuclease generated breaks requires DNA end-resection, we show that CtIP-mediated DNA end-resection is dispensable for A-EJ-mediated CSR using cNHEJ-deficient B cells. High-throughput sequencing analyses revealed that loss of ATM/ATR phosphorylation of CtIP at T855 or ATM kinase inhibition suppress resection without altering the MH-pattern of the A-EJ-mediated switch junctions. Moreover, we found that ATM kinase promotes Alt-EJ mediated CSR by suppressing inter-chromosomal translocations independent of end-resection. Finally, temperol analyses reveal that MHs are enriched in early internal deletions even in cNHEJ-proficient B cells. Thus, we propose that repetitive IgH switch regions represent favoriate substrates for MH-mediated end-joining contributing to the robustness and resection-indepndence of A-EJ-mediated CSR. Overall design: Studies of A-EJ-mediated CSR are complicated by the fact that junctional MHs, a characteristic feature of A-EJ, can also be generated by cNHEJ. Although the MHs of cNHEJ junctions are thought to be shorter than those generated by A-EJ, the cutoff is not absolute and junctions with short MHs (1-3nt) cannot be definitively attributed to either DSB repair pathway. To circumvent this ambiguity, we examined CSR in B cells that are genetically deficient for cNHEJ owing to loss of the core cNHEJ factor XRCC4, an obligatory partner of LIG4. The role of CtIP in A-EJ-mediated CSR in Xrcc4-/- murine B cells was then evaluated by either conditional inactivation of CtIP or non-phosphorylatable CtIP mutation (T855A). Unexpectedly, we found that isotype switching in Xrcc4-/- B cells was unaffected by either deletion or mutation (T855A) of CtIP. Moreover, despite a significant reduction in end-resection, the CSR junctions recovered from Xrcc4-deficient alone and Xrcc4-deficient & CtIP-T855A mutant cells displayed similar MH usage, suggesting that MH-mediated end-ligation can occur without extensive end-resection during CSR. Similarly, ATM kinase inhibitor (ATMi) also attenuated end-resection without affecting MH-usage during A-EJ-mediated CSR. The analysis of ATMi-treated Xrcc4-/- cells also identified an end-resection independent role for ATM in regulating the orientation of CSR by both cNHEJ and A-EJ. Finally, when CSR junctions were analyzed by cell division, MHs (4-15 bps) are significantly enriched in early internal deletion junctions even in cNHEJ-proficient B cells. Taken together, our findings showed that A-EJ-mediated CSR can occur independent of CtIP-mediated end-resection, suggesting that the repetitive IgH switch regions are uniquely prone to MH-mediated repair.