Regulated the AEJ -mediated V(D)J recombination by 53BP1 and ATM
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1015627 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR26040900_1.fastq.gz | Fastqsanger.gz | |||
| SRR26040900_2.fastq.gz | Fastqsanger.gz | |||
| SRR26041000_1.fastq.gz | Fastqsanger.gz | |||
| SRR26041000_2.fastq.gz | Fastqsanger.gz | |||
| SRR26041100_1.fastq.gz | Fastqsanger.gz |
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Project description:The critical role of the alternative end joining (AEJ) repair mechanism in V(D)J recombination for B and T lymphocyte development, particularly in the absence of the key non-homologous end joining (NHEJ) component Ku70, is not well understood. AEJ involves functionally redundant factors such as Parp1 vs Polθ and Lig3 vs Lig1, making it unclear which factors are responsible for repairing V(D)J recombination. Additionally, the regulatory mechanism of AEJ remains enigmatic. In this study, we utilized the murine Igκ antigen locus as a model and employed gene editing and HTGTS-Seq pipelines to systematically evaluate potential factors involved in V(D)J recombination. We assessed various aspects of recombination, including efficiency, V gene usage, end resection, joining fidelity, repair patterns, and inter-chromosome translocation. Our findings highlight the Parp1-XRCC1-Lig3 axis as a key mediator of V(D)J recombination, regulated by 53BP1 and ATM. This study advances our understanding of AEJ in DNA damage repair and contributes to our knowledge of lymphocyte development in immunology.
2023-12-31 | GSE242952 | GEO
Project description:ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination
| PRJNA1032082 | ENA
Project description:The critical role of the alternative end joining (AEJ) repair mechanism in V(D)J recombination for B and T lymphocyte development, particularly in the absence of the key non-homologous end joining (NHEJ) component Ku70, is not well understood. AEJ involves functionally redundant factors such as Parp1 vs Polθ and Lig3 vs Lig1, making it unclear which factors are responsible for repairing V(D)J recombination. Additionally, the regulatory mechanism of AEJ remains enigmatic. In this study, we utilized the murine Igκ antigen locus as a model and employed gene editing and HTGTS-Seq pipelines to systematically evaluate potential factors involved in V(D)J recombination. We assessed various aspects of recombination, including efficiency, V gene usage, end resection, joining fidelity, repair patterns, and inter-chromosome translocation. Our findings highlight the Parp1-XRCC1-Lig3 axis as a key mediator of V(D)J recombination, regulated by 53BP1 and ATM. This study advances our understanding of AEJ in DNA damage repair and contributes to our knowledge of lymphocyte development in immunology.
2023-12-31 | GSE246239 | GEO
Project description:53BP1 is a well-established DNA damage repair factor that has recently been shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.
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Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNAs that target ATM, ATR, CHK1, CHK2, and 53BP1 genes were transfected in MCF-10A non-transformed breast cell line by lentiviral particles and selected stable ATM, ATR, CHK1, CHK2, and 53BP1 knockdown MCF-10A cells. Scrambled control shRNA-transfected and wild type MCF-10A cells were applying as control. All knockdown and control MCF-10A cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in MCF-10A medium and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three biological replicates were applied.
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Project description:RAG endonuclease initiates immunoglobulin heavy chain (Igh) V(D)J recombination by cleaving V, D and J gene segments flanked by recombination signal sequences (RSSs), subsequently joined exclusively by classical nonhomologous end joining (c-NHEJ). While 53BP1, a key DNA damage response (DDR) factor promoting c-NHEJ, was reported to be dispensable for Igh V(D)J recombination. Here, we report that 53BP1 is a unique DDR factor playing unrecognized roles in joining RAG-initiated DNA breaks. Both RAG-cleaved RSSs and cryptic RSSs (cRSSs) are joined by c-NHEJ. Deficiency of 53BP1, rather than other DDR factors, slightly decreased the proximal V(D)J recombination without affecting c-NHEJ of RAG-initiated RSS breaks, but significantly increased microhomology (MH)-mediated alternative end joining (A-EJ) of RAG-initiated cRSS breaks, indicating that 53BP1 is specifically required for c-NHEJ of RAG-initiated cRSS breaks. Thus, our findings provide new insights into the distinct roles of 53BP1 in joining RAG-cleaved on- and off-targets during exclusively c-NHEJ-mediated V(D)J recombination
2026-04-09 | GSE270087 | 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.
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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.
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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.
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