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 \((C_H)\) region exons. End-Joining of DSBs in the upstream donor S region (Sm) to DSBs in a downstream acceptor S region \((S_{acc})\) replaces the initial set of \(C_H\) exons, Cm, with a set of downstream \(C_H\) exons, leading to Ig class switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). In addition to joining to 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 Sm but rarely in \(S_{acc}s\), suggesting that AID targeting to \(S_{acc}s\) requires prior recruitment to Sm. To test this hypothesis, we assessed CSR and ISDs in B cells lacking Sm and found that AID frequently targets downstream \(S_{acc}s\) independently of Sm. 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 junctions between I-SceI meganuclease-generated 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.
| Identifer | oai:union.ndltd.org:harvard.edu/oai:dash.harvard.edu:1/10336910 |
| Date | January 2011 |
| Creators | Zhang, Tingting |
| Contributors | Alt, Frederick W. |
| Publisher | Harvard University |
| Source Sets | Harvard University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Rights | open |
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