CtIP is essential for early B cell proliferation and development in mice

B cell development requires efficient proliferation and successful assembly and modifications of the immunoglobulin gene products. CtIP is an essential gene implicated in end-resection and DNA repair. Here we show that CtIP is essential for early B cell development, while dispensable in naïve B cells. CtIP loss is well-tolerated in G1 arrested B cells and during V(D)J recombination. But, in proliferating B cells, CtIP loss leads to a progressive cell death characterized by ATM hyper-activation, G2/M arrest, genomic instability, and 53BP1 nuclear body formation, indicating that the essential role of CtIP during proliferation underscores its stage-specific requirement in B cells. B cell proliferation requires phosphorylation of CtIP at T847 presumably by CDK, but not its interaction with CtBP, Rb, nor its nuclease activity. CtIP phosphorylation by ATM/ATR at T859 (T855 in mice) promotes end-resection in G1 arrested cells, but dispensable for B cell development and class switch recombination, suggesting distinct roles for T859 and T847 phosphorylation in B cell development. Despite a moderate and transient delay at day 3, CtipT855A/T855A B cells undergo robust CSR upon activation in vitro. Consistent with the partial rescue in the reconstitution experiments, activated CtipT855A/T855A B cells show a moderate reduction in overall proliferation. Yet, the frequency of IgG1+ B % cells does not significantly differ in CtipT855A/T855A B cells vs the control (p=0.74) at each cell division, suggesting T855A-CtIP does not directly compromise IgG1 switching beyond cell proliferation. Moreover, high throughput sequencing analyses of over 10,000 CSR junctions between Sμ and downstream switch regions show that the usage of peripheral switch regions (an indicator of end-resection) and the number of micro-homologies at the junction (an indicator of Alt-EJ) are also not affected by the T855A mutation of CtIP. Taken together these findings indicate that ATR/ATM-mediated phosphorylation of CtIP at T859 promotes end-resection, but is largely dispensable for CSR in vivo.