Widespread cryptic RAG-mediated recombination in developing T lymphocytes
- 1The Pirbright Institute
Abstract
V(D)J recombination generates antigen receptor diversity through the targeted activity of the RAG1/2 recombinase, but the extent to which RAG also engages cryptic genomic targets during normal lymphocyte development remains poorly defined. Here, we developed a targeted PCR-sequencing approach to detect and quantify rare RAG-mediated rearrangements in mouse thymocytes. We first examined the TCRβ locus and found that four of its twelve pseudogenes undergo detectable rearrangement in vivo, despite being considered non-functional components of the repertoire. Extending this analysis across the locus revealed 33 previously uncharacterized cryptic recombination sites involved in non-functional rearrangements with DJβ segments. These events occurred across both the Vβ region and the largely inaccessible Vβ30–Dβ1 intergenic region, with individual cryptic sites spanning a broad range of recombination frequencies. Cryptic sites were highly heterogeneous in sequence and chromatin context: neither RSS resemblance, predicted Z-DNA formation, local epigenetic features, nor chromosomal interactions reliably distinguished sites with detectable recombination from those at which recombination was not detected. We further identified additional cryptic RAG-mediated rearrangements at the Bcl11b locus, demonstrating that such events are not restricted to antigen receptor loci. Together, these findings reveal an unexpectedly broad landscape of low-frequency RAG-mediated DNA rearrangement in developing T lymphocytes and suggest that cryptic target selection cannot be explained solely by the local genetic and epigenetic features examined here.
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BibTeX
@misc{sobral2026widespread,
title = {{Widespread cryptic RAG-mediated recombination in developing T lymphocytes}},
author = {D. Sobral and J. Silva and D. Santos and J. Perreira Leal and J. Demengeot and M. Bonnet-Di Placido},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.01.748595},
url = {https://www.biorxiv.org/content/10.64898/2026.09.01.748595v1},
note = {Public domain},
}
RIS
TY - UNPB
TI - Widespread cryptic RAG-mediated recombination in developing T lymphocytes
AU - D. Sobral
AU - J. Silva
AU - D. Santos
AU - J. Perreira Leal
AU - J. Demengeot
AU - M. Bonnet-Di Placido
PY - 2026
DA - 2026/09/01
PB - bioRxiv
DO - 10.64898/2026.09.01.748595
UR - https://www.biorxiv.org/content/10.64898/2026.09.01.748595v1
AB - V(D)J recombination generates antigen receptor diversity through the targeted activity of the RAG1/2 recombinase, but the extent to which RAG also engages cryptic genomic targets during normal lymphocyte development remains poorly defined. Here, we developed a targeted PCR-sequencing approach to detect and quantify rare RAG-mediated rearrangements in mouse thymocytes. We first examined the TCRβ locus and found that four of its twelve pseudogenes undergo detectable rearrangement in vivo, despite being considered non-functional components of the repertoire. Extending this analysis across the locus revealed 33 previously uncharacterized cryptic recombination sites involved in non-functional rearrangements with DJβ segments. These events occurred across both the Vβ region and the largely inaccessible Vβ30–Dβ1 intergenic region, with individual cryptic sites spanning a broad range of recombination frequencies. Cryptic sites were highly heterogeneous in sequence and chromatin context: neither RSS resemblance, predicted Z-DNA formation, local epigenetic features, nor chromosomal interactions reliably distinguished sites with detectable recombination from those at which recombination was not detected. We further identified additional cryptic RAG-mediated rearrangements at the Bcl11b locus, demonstrating that such events are not restricted to antigen receptor loci. Together, these findings reveal an unexpectedly broad landscape of low-frequency RAG-mediated DNA rearrangement in developing T lymphocytes and suggest that cryptic target selection cannot be explained solely by the local genetic and epigenetic features examined here.
ER -