SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination
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ABSTRACT:
PROVIDER: PRJNA836576 | ENA |
REPOSITORIES: ENA
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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:53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin—a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)— as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.
2018-07-26 | PXD009650 | Pride
Project description: Not available
| S-EPMC9240092 | biostudies-literature
Project description:53BP1 activity drives genome instability and embryonic lethality in BRCA1-deficient cells by inhibiting homologous recombination (HR).53BP1’s anti-recombinogenic functions require phosphorylation-dependent interactions with two effector complexes, PTIP and RIF1/Shieldin. While RIF1/Shieldin is thought to block 5’-3’ nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here we show that mutation of the PTIP interaction site in 53BP1 (S25A) increases Shieldin association with DNA damage. Despite excessive Shieldin “end-blocking” activity, the mutant protein allows end resection sufficient to rescue the lethality of BRCA1D11 mice. End resection in BRCA1D1153BP1S25A mice is rewired in a manner driven by DNA2 since Shieldin blocks EXO1-mediated nucleolytic processing. Despite ample resection, mutant cells fail to complete HR, as 53BP1/Shieldin also inhibits RNF168-mediated loading of PALB2/RAD51 onto ssDNA post-resection. As a result, BRCA1D1153BP1S25A mice exhibit hallmark features of HR insufficiency, including increased developmental neuronal apoptosis, premature aging and hypersensitivity to PARP inhibitors. Disruption of Shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study supports a model in which RIF1/Shieldin and PTIP associate independently with 53BP1 to regulate distinct end-resection pathways, and reveals a critical function of 53BP1/Shieldin post-resection that limits RNF168-mediated loading of RAD51.
2019-10-22 | GSE133806 | GEO
Project description:53BP1 activity drives genome instability and embryonic lethality in BRCA1-deficient cells by inhibiting homologous recombination (HR).53BP1’s anti-recombinogenic functions require phosphorylation-dependent interactions with two effector complexes, PTIP and RIF1/Shieldin. While RIF1/Shieldin is thought to block 5’-3’ nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here we show that mutation of the PTIP interaction site in 53BP1 (S25A) increases Shieldin association with DNA damage. Despite excessive Shieldin “end-blocking” activity, the mutant protein allows end resection sufficient to rescue the lethality of BRCA1D11 mice. End resection in BRCA1D1153BP1S25A mice is rewired in a manner driven by DNA2 since Shieldin blocks EXO1-mediated nucleolytic processing. Despite ample resection, mutant cells fail to complete HR, as 53BP1/Shieldin also inhibits RNF168-mediated loading of PALB2/RAD51 onto ssDNA post-resection. As a result, BRCA1D1153BP1S25A mice exhibit hallmark features of HR insufficiency, including increased developmental neuronal apoptosis, premature aging and hypersensitivity to PARP inhibitors. Disruption of Shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study supports a model in which RIF1/Shieldin and PTIP associate independently with 53BP1 to regulate distinct end-resection pathways, and reveals a critical function of 53BP1/Shieldin post-resection that limits RNF168-mediated loading of RAD51.
2019-10-22 | GSE133805 | GEO
Project description:BRCA1 deficiencies cause breast, ovarian and other cancers, and render tumours hypersensitive to PARP-inhibitors. To understand resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP-inhibitors. Thus, we identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, termed “Shieldin” (SHLD1/2), with FAM35A interacting with single-stranded DNA via its C-terminal OB-fold region. We establish that Shieldin promotes DNA double-strand break (DSB) end-joining, acting as the downstream effector of 53BP1/RIF1/MAD2L2 to restrict DSB resection and counteract homologous recombination in BRCA1-deficient cells by antagonising BRCA2/RAD51 loading. Notably, Shieldin inactivation further sensitises BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.
2018-05-31 | PXD009830 | Pride
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: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:We used homologous recombination based CRISPR Knock-in strategy to insert a SFB tag at C terminal of 53BP1 in 293T cells. We performed tandem affinity purification of endogenous 53BP1 in both soluble and chromatin fraction to uncover known and new 53BP1 binding proteins which might play roles in DNA damage response. We identified and examined FOXK1 for its role in DNA damage response through the interaction with 53BP1 mainly in soluble fraction.
2021-09-09 | PXD020090 | Pride