Elucidating Mechanisms of IgH Class Switch Recombination Involving Switch Regions and Double Strand Break Joining

During IgH class switch recombination (CSR) in mature B lymphocytes, activation-induced cytidine deaminase (AID) initiates DNA double strand breaks (DSBs) within switch (S) regions flanking different sets of the IgH locus (IgH) constant (CH) region exons. End-Joining of DSBs in the upstream donor S region (S&mu) to DSBs in a downstream acceptor S region (Sacc) replaces the initial set of CH exons, C&mu, with a set of downstream CH exons, leading to Ig class switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). In addition to joining to AID-induced DSBs within another S region, AID-induced DSBs within a given S region are often rejoined or joined to other DSBs in the same S region to form internal switch deletions (ISDs). ISDs were frequently observed in S&mu but rarely in Saccs, suggesting that AID targeting to Saccs requires prior recruitment to S&mu. To test this hypothesis, we assessed CSR and ISDs in B cells lacking S&mu and found that AID frequently targets downstream Saccs independently of S&mu. These studies also led us to propose an alternative pathway of "downstream" IgE class switching that involves joining of DSBs within the downstream S&gamma1 and S&epsilon regions as a first step before joining of S&mu to the hybrid downstream S region. To further elucidate the CSR mechanism, we addressed the long-standing question of whether S region DSBs during CSR involves a direction-specific mechanism similar to joining of RAG1/2 endonuclease-generated DSBs during V(D)J recombination. We used an unbiased high throughput method to isolate and sequence junctions between I-SceI meganucleasegenerated DSBs at a target site that replaces the IgH S&gamma1 region and other genomic DSBs of endogenous origin. Remarkably, we found that the I-SceI-generated DSBs were joined to both upstream DSBs in S&mu and downstream DSBs in S&epsilon predominantly in orientations associated with joining during productive CSR. This process required the DSB response factor 53BP1 to maintain the orientation-dependence, but not the overall levels, of joining between these widely separated IgH breaks. We propose that CSR exploits a mechanism involving 53BP1 to enhance directional joining of DSBs within IgH in an orientation that leads to productive CSR.