Multiple DSB Resection Activities Redundantly Promote Alternative End Joining-Mediated Class Switch Recombination.

Xikui Sun,Chengming Zhu,Hongman Xue,Jiejie Xu,Mingyu Gu,Xiaoli Xi,Chun Chen,Junchao Dong,Jingning Bai

doi:10.3389/fcell.2021.767624

Abstract

Alternative end joining (A-EJ) catalyzes substantial level of antibody class switch recombination (CSR) in B cells deficient for classical non-homologous end joining, featuring increased switch (S) region DSB resection and junctional microhomology (MH). While resection has been suggested to initiate A-EJ in model DSB repair systems using engineered endonucleases, the contribution of resection factors to A-EJ-mediated CSR remains unclear. In this study, we systematically dissected the requirement for individual DSB resection factors in A-EJ-mediated class switching with a cell-based assay system and high-throughput sequencing. We show that while CtIP and Mre11 both are mildly required for CSR in WT cells, they play more critical roles in mediating A-EJ CSR, which depend on the exonuclease activity of Mre11. While DNA2 and the helicase/HRDC domain of BLM are required for A-EJ by mediating long S region DSB resection, in contrast, Exo1’s resection-related function does not play any obvious roles for class switching in either c-NHEJ or A-EJ cells, or mediated in an AID-independent manner by joining of Cas9 breaks. Furthermore, ATM and its kinase activity functions at least in part independent of CtIP/Mre11 to mediate A-EJ switching in Lig4-deficient cells. In stark contrast to Lig4 deficiency, 53BP1-deficient cells do not depend on ATM/Mre11/CtIP for residual joining. We discuss the roles for each resection factor in A-EJ-mediated CSR and suggest that the extent of requirements for resection is context dependent.

Highlights

Mature B cells undergo immunoglobulin heavy chain (IgH) class switch recombination (CSR) to mediate different antibody effector functions
We continue to demonstrate that double-strand breaks (DSBs) resection factors together with Ataxia telangiectasia-mutated (ATM) play important roles in meditating Alternative end joining (A-EJ) in the absence of Lig4
Consistent with a prior report (Dinkelmann et al, 2009), the mild increase in resection and MH usage in Mre11-silenced cells that resembled ATM-deficient cells supports the notion that Mre11 has a minor role in meditating classical non-homologous end joining (c-NHEJ) in wild type B cells through activating ATM-dependent DNA damage response (DDR)

Summary

Introduction

Mature B cells undergo immunoglobulin heavy chain (IgH) class switch recombination (CSR) to mediate different antibody effector functions. CSR replaces the initially expressed μ constant gene (Cμ) with a downstream constant gene through genomic DNA recombination (Xu et al, 2012). Stimulating B cells with combinations of activators and cytokines directs CSR to particular CH genes by modulating germline transcription to recruit AID, which introduces into S regions multiple C to U mutations that are subsequently converted to staggered double-strand breaks (DSBs) by base excision and mismatch repair with yet unclear mechanisms (Hwang et al, 2015; Yu and Lieber, 2019). CSR is completed by joining donor Sμ and acceptor S region DSBs in a deletion-preferred fashion to promote antibody production (Dong et al, 2015)

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