Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sμ and in a downstream acceptor S region, with a DSB in Sμ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sμ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.
IgH class switching exploits a general property of two DNA breaks to be joined in cis over long chromosomal distances
CHIARLE, Roberto;
2014-01-01
Abstract
Antibody class switch recombination (CSR) in B lymphocytes joins two DNA double-strand breaks (DSBs) lying 100-200 kb apart within switch (S) regions in the immunoglobulin heavy-chain locus (IgH). CSR-activated B lymphocytes generate multiple S-region DSBs in the donor Sμ and in a downstream acceptor S region, with a DSB in Sμ being joined to a DSB in the acceptor S region at sufficient frequency to drive CSR in a large fraction of activated B cells. Such frequent joining of widely separated CSR DSBs could be promoted by IgH-specific or B-cell-specific processes or by general aspects of chromosome architecture and DSB repair. Previously, we found that B cells with two yeast I-SceI endonuclease targets in place of Sγ1 undergo I-SceI-dependent class switching from IgM to IgG1 at 5-10% of normal levels. Now, we report that B cells in which Sγ1 is replaced with a 28 I-SceI target array, designed to increase I-SceI DSB frequency, undergo I-SceI-dependent class switching at almost normal levels. High-throughput genome-wide translocation sequencing revealed that I-SceI-generated DSBs introduced in cis at Sμ and Sγ1 sites are joined together in T cells at levels similar to those of B cells. Such high joining levels also occurred between I-SceI-generated DSBs within c-myc and I-SceI- or CRISPR/Cas9-generated DSBs 100 kb downstream within Pvt1 in B cells or fibroblasts, respectively. We suggest that CSR exploits a general propensity of intrachromosomal DSBs separated by several hundred kilobases to be frequently joined together and discuss the relevance of this finding for recurrent interstitial deletions in cancer.
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