53BP1 plays distinct roles in the recombination of RAG-cleaved on- and off-targets [ATAC-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE270087 | GEO | 2026/04/09
REPOSITORIES: GEO
Similar Datasets
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270086 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270090 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270088 | GEO
Project description:RAG endonuclease initiates V(D)J recombination by cleaving recombination signal sequences (RSSs), subsequently repaired by classical nonhomologous end-joining (c-NHEJ). However, 53BP1, as a key DNA damage response (DDR) factor promoting c-NHEJ, is dispensable for V(D)J recombination. Here, we report that 53BP1 orchestrates sequence strength of RAG targets to shape end-joining features during V(D)J recombination. Deficiency of 53BP1, rather than other DDR factors, specifically increases junctional microhomology (MH) of cryptic RSS (cRSS) recombination instead of RSS recombination. Depletion of RIF1 or Shieldin, downstream factors of 53BP1, partially mimics the phenotypes of 53BP1-deficiency. Notably, loss of RNF168, rather than other classical resection and alternative end-joining factors, is sufficient to fully rescue 53BP1-deficiency-mediated increase of junctional MH during cRSS recombination. Remarkably, RAG mutant and RSS score analyses indicate that sequence strength of RAG targets determines the unique roles of 53BP1 in balancing end-joining outcomes. Our findings provide deeper mechanistic insights into DNA damage repair for RAG cleaved on- and off-targets during V(D)J recombination.
2026-04-09 | GSE270092 | GEO
Project description:The shieldin (SHLD) complex, composed of SHLD1, SHLD2, SHLD3 and MAD2L2/REV7, acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end-resection and promote non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and for repair of RAG-induced DSBs in XLF-deficient cells, the role of SHLD in these activities remains elusive. Here, we report that contrary to 53BP1, SHLD1 is dispensable for lymphocyte development and V(D)J recombination, even in the sensitized XLF-deficient background or for the joining of distant V(D)J segments. By contrast, SHLD1 restricts resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR. Finally, we show that this end-protection function is required for orientation-specific joining of AID-initiated DSBs. We propose that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms; the synapsis of V(D)J segments and switch regions within chromatin independently of SHLD and the protection of AID-DSB ends against resection mediated by SHLD.
2022-05-11 | GSE202567 | GEO
Project description:In developing B lymphocytes, V(D)J recombination assembles IgH and Igk variable region exons from hundreds of gene segments clustered across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination center (RC). JH-RSS orientation programs RAG to scan upstream D- and VH-containing chromatin linearly presented by cohesin-mediated loop extrusion. During Igh scanning, RAG robustly utilizes only D- or VH-RSSs in convergent ("deletional") orientation with JH-RSSs. However, for Vk-to-Jk joining, RAG utilizes Vk-RSSs from deletional- and inversional-oriented clusters, inconsistent with linear scanning. Here, we elucidate the Vk-to-Jk joining mechanism. Igk undergoes robust primary and secondary rearrangements, which confounds scanning assays. Thus, we engineered cells to undergo only primary Vk-to-Jk rearrangements and found that RAG-scanning from the primary Jk-RC terminates just 8kb upstream within the CTCF-Site-based Sis element. While Sis and the Jk-RC barely interacted with the Vk locus, the CTCF-site-based Cer element, 4kb upstream of Sis, interacted with various loop-extrusion impediments across the locus. Like VH-locus inversion, DJH inversion abrogated VH-to-DJH joining; yet Vk-locus or Jk inversion allowed robust Vk-to-Jk joining. Together, these experiments implicated loop extrusion in bringing Vks near Cer for short-range diffusion-mediated capture by RC-based RAG. To elucidate key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vk-to-JH and D-to-Jk rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vk and Jk RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed ability of Igk versus Igh RSSs to promote robust diffusional joining. We propose that Igk evolved strong RSSs to mediate diffusional Vk-to-Jk joining; while Igh evolved weaker RSSs requisite for modulating VH joining by RAG scanning impediments.
2024-04-08 | GSE254039 | GEO
Project description:In developing B lymphocytes, V(D)J recombination assembles IgH and Igk variable region exons from hundreds of gene segments clustered across mega-base Igh and Igk loci. V, D, and J segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination center (RC). JH-RSS orientation programs RAG to scan upstream D- and VH-containing chromatin linearly presented by cohesin-mediated loop extrusion. During Igh scanning, RAG robustly utilizes only D- or VH-RSSs in convergent ("deletional") orientation with JH-RSSs. However, for Vk-to-Jk joining, RAG utilizes Vk-RSSs from deletional- and inversional-oriented clusters, inconsistent with linear scanning. Here, we elucidate the Vk-to-Jk joining mechanism. Igk undergoes robust primary and secondary rearrangements, which confounds scanning assays. Thus, we engineered cells to undergo only primary Vk-to-Jk rearrangements and found that RAG-scanning from the primary Jk-RC terminates just 8kb upstream within the CTCF-Site-based Sis element. While Sis and the Jk-RC barely interacted with the Vk locus, the CTCF-site-based Cer element, 4kb upstream of Sis, interacted with various loop-extrusion impediments across the locus. Like VH-locus inversion, DJH inversion abrogated VH-to-DJH joining; yet Vk-locus or Jk inversion allowed robust Vk-to-Jk joining. Together, these experiments implicated loop extrusion in bringing Vks near Cer for short-range diffusion-mediated capture by RC-based RAG. To elucidate key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vk-to-JH and D-to-Jk rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vk and Jk RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed ability of Igk versus Igh RSSs to promote robust diffusional joining. We propose that Igk evolved strong RSSs to mediate diffusional Vk-to-Jk joining; while Igh evolved weaker RSSs requisite for modulating VH joining by RAG scanning impediments.
2024-04-08 | GSE263123 | GEO
Project description:IgH class switch recombination (CSR) in B lymphocytes switches IgH constant regions to change antibody functions. CSR is initiated by DNA double strand breaks (DSBs) within a donor IgH switch (S) region and a downstream acceptor S region. CSR is completed by fusing donor and acceptor S region DSB ends by classical non-homologous end-joining (C-NHEJ) and, in its absence, by alternative end-joining (A-EJ) that is more biased to use longer junctional micro-homologies (MHs). Deficiency for DSB response (DSBR) factors, including ATM and 53BP1, variably impair CSR end-joining, with 53BP1 deficiency having the greatest impact. However, studies of potential impact of DSBR factor deficiencies on MH-mediated CSR end-joining have been technically limited. We now use a robust DSB joining assay to elucidate impacts of deficiencies for DSBR factors on CSR and chromosomal translocation junctions in primary mouse B cells and CH12F3 B lymphoma cells. Compared to wild-type, CSR and c-Myc to S region translocation junctions in the absence of 53BP1, and to a lesser extent other DSBR factors, have increased MH-utilization; indeed, 53BP1-deficient MH-profiles resemble those associated with C-NHEJ deficiency. Yet, translocation junctions between c-Myc DSB and general DSBs genome-wide are not MH-biased in ATM-deficient versus wild-type CH12F3 cells and less biased in 53BP1- and C-NHEJ-deficient cells than CSR junctions or c-Myc to S region translocation junctions. We discuss potential roles of DSBR factors in suppressing increased MH-mediated DSB end-joining and features of S regions that may render their DSBs prone to MH-biased end-joining in the absence of DSBR factors.
2018-01-08 | GSE106922 | GEO
Project description:53BP1 regulates DNA end-joining in lymphocytes, diversifying immune antigen receptors. This involves nucleosome-bound 53BP1 at DNA double-stranded breaks (DSBs) recruiting RIF1 and shieldin, a poorly understood DNA-binding complex. The 53BP1-RIF1-shieldin axis is pathological in BRCA1-mutated cancers, blocking homologous recombination (HR) and driving illegitimate non-homologous end-joining (NHEJ). However, how this axis regulates DNA end-joining and HR suppression remains unresolved. We investigated shieldin and its interplay with CST, a complex recently implicated in 53BP1-dependent activities. Immunophenotypically, mice lacking shieldin or CST are equivalent, with class-switch recombination co-reliant on both complexes. DNA damage-responsive signalling promotes shieldin-CST interaction, demonstrating shieldin as a DSB-specific CST adaptor. Furthermore, DNA polymerase ζ functions downstream of shieldin, establishing DNA fill-in synthesis as the physiological function of shieldin-CST. Lastly, 53BP1 suppresses HR and promotes NHEJ in BRCA1-deficient cells independently of shieldin. These findings showcase the resilience of the 53BP1 pathway, achieved through the collaboration of chromatin-bound 53BP1 complexes and DNA end-processing effector proteins.
2024-08-27 | PXD045534 | Pride
Project description:The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSB) and promotes their resolution via the DNA repair pathways of Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). We and others have shown that DDR activation requires DROSHA. However, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment and how DROSHA influences DNA repair, remains poorly understood. Here we show that DROSHA associates to DSBs independently from transcription. Neither H2AX, nor ATM nor DNA-PK kinase activities are required for its recruitment to break site. Rather, DROSHA interacts with RAD50 and inhibition of MRN by Mirin treatment abolishes this interaction. MRN inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSB and, as a consequence, also 53BP1 recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increase the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and direct DNA repair toward NHEJ.
2021-03-15 | GSE166631 | GEO