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

Abstract

To generate antibodies with different effector functions, B cells undergo Immunoglobulin Heavy Chain (IgH) class switch recombination (CSR). The ligation step of CSR is usually mediated by the classical nonhomologous end-joining (cNHEJ) pathway. In cNHEJ-deficient cells, a remarkable ∼25% of CSR can be achieved by the alternative end-joining (Alt-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 suppresses 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 interchromosomal translocations independent of end resection. Finally, temporal 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 favored substrates for MH-mediated end-joining contributing to the robustness and resection independence of A-EJ-mediated CSR.

Keywords: CtIP; alternative end-joining; class switch recombination.

Fig. 1.

CtIP deficiency or mutation did not reduce A-EJ-mediated CSR in Xrcc4-deficient B cells. (A) Representative flow cytometry analysis of purified splenic B cells, stimulated with IL-4 and anti-CD40, at day 3 or day 4, from control (WT or CD21^CreXrcc4^C/+CtIP^+/+(C)), CD21^CreXrcc4^C/CCtIP^+/+(C), CD21^CreXrcc4^C/C CtIP^C/C, and CD21^CreXrcc4^C/C CtIP^T855A/T855A mice with representative CTV labeling for cell proliferation upon stimulation. Cells that underwent two cell divisions were gated based on CTV dilution and plotted for IgG1+ frequency. (B) The percentage of live and IgG1+ cells at day 3 after stimulation. Two-tailed Mann–Whitney U test, ****P < 0.0001. (C) The relative (to control analyzed in parallel) percentage of live and IgG1+ cells at day 3 after stimulation in cells with two divisions, measured by CTV.

Fig. 2.

CtIP deficiency reduces end resection in Xrcc4-deficient B cells. (A) Diagram of the HTGTS assay with a single bait site at the 5′ Sμ region. The IgH locus is drawn in its native orientation on mouse chromosome 12. The solid and dashed lines represent junctions made in the (−) (normal internal deletion and CSR) and (+) (inversion) orientations, respectively. The different switch regions are color coded. The bait site is marked with a red arrow. The bait starts from 114,664,910 near the telomere of Chr. 12. (B) The distribution of prey sites inside (IgH all) and outside (non-IgH) the IgH locus. (C) The distribution of IgH prey sites by switch region—Sμ, Sγ1, Sε, and others. (D and E) Relative frequency of distal preys among the prey in each respective switch region. Two-tailed Mann–Whitney U test, *P < 0.05; ns is not marked. (D) for Sμ, distal = downstream/centromeric to Chr12: 114,662,100. (E) for Sγ1, distal = downstream/centromeric to Chr12:114,568,500. For (B) to (E), the data represent greater than or equal to three independent repeats of each genotype. Error bar = SD.

Fig. 3.

Diagram and relative distribution of Sμ preys in Xrcc4-deficient B cells. The diagram of ∼10 kb (chr12: 114,658,100–114,668,100, mm10) Sµ region and the counts of RGYW motifs per 50 bp by orientation ([+], blue and [−], red). The percentage of preys that fall into the distal region (

Fig. 4.

Diagram and relative distribution of Sγ1 preys in Xrcc4-deficient B cells. The diagram of ∼30 kb (chr12: 114,558,500–114,588,500, mm10) Sγ1 region and the counts of RGYW motifs per 150 bp by orientation ([+], blue and [−], red). The percentage of preys that fall into the distal region (

Fig. 5.

The MH usage of IgH junctions recovered from Xrcc4-deficient B cells. (A) The graphic distribution of all IgH preys by MH, blunt, and INS. (B) The percentage of IgH junctions with ≥4-nt MH by genotype. (C) The graphic distribution of all Sμ preys by MH, blunt, and INS. (D) The percentage of Sμ junctions with ≥4-nt MH by genotype. (E) The graphic distribution of all Sγ1 preys by MH, blunt, and INS. (F) The percentage of Sγ1 junctions with ≥4-nt MH by genotype. (A), (C) and (E) represent the pool of, at least, three independent libraries per genotype. For A, C, and E, Kolmogorov–Smirnov test, *P < 0.05, and ****P < 0.0001. In B, D, and F, the bar and error bars represent the average and the standard derivations. The P values in B, D, and F were calculated via unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.0005, and ****P < 0.0001.

Fig. 6.

The impact of ATM inhibition on A-EJ-mediated CSR. (A) Representative flow cytometry of Xrcc4^−/− B cells with or without ATMi (7.5 µM, KU55933) at day 4 of stimulation. (B) Quantification of live IgG1+% at day 4 after stimulation, with and without 7.5-μM ATMi (KU55933). Two-tailed Mann–Whitney U test, **P < 0.01, and *P < 0.05. (C) The percentage of IgH preys that fall into each switch region by genotype. (D) The quantification of the fraction of Sε preys among all IgH preys. (E) The ratio of Sμ preys that fall into (−) vs. (+) orientations. For B–E, the bars and error bars represent average and SDs. Two-tailed Mann–Whitney U test, **P < 0.01, and *P < 0.05.

Fig. 7.

CSR junction analyses by cell division. (A) The MH, blunt, and INS distributions of WT Sμ and Sγ1 junctions at day 3 or 4 by switch region (Sμ or Sγ1). Kolmogorov–Smirnov test, **P < 0.01. (B) Representative CTV labeling analyses of B cells from WT BL6 mice. The IgG1+% of cells with the defined number of cell divisions plotted below. The underlines marked the division groups used for sequence analyses. Cells with the least divisions were in group I and the most divisions in group IV. (C) The relative distribution of IgH preys among different switch regions by cell divisions. The bars and error bars represent average and SDs. (D) The MH, blunt, and INS usages among Sμ preys by cell division. Kolmogorov–Smirnov test, *P < 0.05, and **P < 0.01.