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

 Title: Elucidating Mechanisms of IgH Class Switch Recombination Involving Switch Regions and Double Strand Break Joining Author: Zhang, Tingting Citation: Zhang, Tingting. 2011. Elucidating Mechanisms of IgH Class Switch Recombination Involving Switch Regions and Double Strand Break Joining. Doctoral dissertation, Harvard University. Full Text & Related Files: Zhang_gsas.harvard_0084L_10047.pdf (9.298Mb; PDF) Abstract: 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. Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:10336910 Downloads of this work:

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