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| Status |
Public on Sep 15, 2015 |
| Title |
On-target and Off-target Activities of RAG Are Restricted by Topological Chromatin Domains |
| Organism |
Mus musculus |
| Experiment type |
Other
|
| Summary |
RAG initiates V(D)J recombination in developing lymphocytes by generating "on-target" DNA double-stranded breaks at bona fide recombination signal sequence (RSS) pairs. We now employ RAG-generated DNA breaks in endogenous or ectopically-inserted RSS pairs as bait to identify huge numbers of RAG "off-target" sites. These off-target DNA breaks occur across convergent CTCF-binding element (CBE)-flanked loop domains containing the bait RSS pairs. Such off-target cleavage occurs at the simple CAC motif that defines the RSS cleavage-site and shows orientation-dependence best explained by a two-dimensional tracking mechanism. Deletion of the CBE-based IGCR1 IgH regulatory element disrupts antibody IgH recombination domains and, correspondingly, alters distributions of RAG on- and off-targets across the IgH locus. RAG off-targets frequently involved in chromosomal translocations occur in convergent RSS pairs at enhancer regions within a loop. Our findings reveal how RAG is developmentally focused and re-focused in lymphocytes and implicate mechanisms by which chromatin domains harness biological processes within them.
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| Overall design |
We performed high-throughput genome-wide translocation sequencing (HTGTS) in progenitor B cells or cell lines to study signatures of RAG on and off-targeting activity using RAG-generated breaks in endogenous or ectopically-inserted paired bona fide RSSs as bait. Sequenceing was dong by Illumina Miseq.
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| Contributor(s) |
Alt FW, Hu J, Zhang Y |
| Citation(s) |
26593423 |
| Submission date |
Sep 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jiazhi Hu |
| E-mail(s) |
heartlead@gmail.com
|
| Organization name |
Children's Hospital Boston
|
| Department |
PCMM
|
| Lab |
Frederick Alt
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
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| Samples (40)
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| Relations |
| BioProject |
PRJNA295978 |
| SRA |
SRP063873 |
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