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Shieldin/CST-dependent and independent functions of 53BP1 control DNA end-joining and repair pathway choice

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Robert Parker

LAB HEAD: J. Ross Chapman

PROVIDER: PXD045534 | Pride | 2024-08-27

REPOSITORIES: Pride

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			Action	DRS
	Ash2_SHLD.mgf	Mgf
	HFX0062_IPP183_C1_GST_1.raw	Raw
	HFX0062_IPP183_C1_GST_2.raw	Raw
	HXF0062_IPP183_C1_SHLD1_1.raw	Raw
	HXF0062_IPP183_C1_SHLD1_2.raw	Raw

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RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity

Project description:The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

2022-02-04 | PXD031311 | Pride

ASF1 acts with RIF1 to promote DNA end-joining in BRCA1 deficient cells

Project description:RIF1 acts downstream of 53BP1 to coordinate DNA double strand break repair pathway choice between non-homologous end joining (NHEJ) and homologous recombination (HR). Here we identified ASF1 as an endogenous RIF1-associated protein. We showed that ASF1 forms complex with RIF1 and regulates RIF1-dependent functions in DNA damage response.

2022-06-09 | PXD027727 | Pride

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination (END-seq)

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

53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination (ChIP-seq)

2019-10-22 | GSE133805 | GEO

53BP1 cooperation with the REV7–shieldin complex underpins DNA structure-specific NHEJ

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

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Project description:Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage responsefactor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice, and rescues HR deficiency, as measured by hypersensitivity to PARP (polyADPribose polymerase) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA cross-link repair that is distinct from HR. Disruption of the non-homologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi Anemia pathway for ICL repair. Brca1 therefore has two separate roles in ICL repair, whereas FANCD2 provides a key activity that can not be bypassed by ablation of 53BP1 or Ku. B cells were stimulated to undergo class switch recombination in vitro. Chromatin from B cells was harvested 72 hours post-stimulation and used for RPA ChIP to study the extent of resection of DNA DSBs.

2012-09-30 | E-GEOD-35698 | biostudies-arrayexpress

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

2012-09-30 | GSE35698 | GEO

SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination

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

Shieldin complex promotes DNA end joining and counters homologous recombination in BRCA1-null cells

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

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair

Project description:MAD2L2 (also known as REV7) functions in a diverse range of processes. It facilitates DNA lesion bypass through translesion synthesis (TLS), aids in interstrand crosslink repair and contributes to timely mitotic progression. Moreover, MAD2L2 plays an important role in DNA repair at DNA double strand breaks (DSBs) and uncapped telomeres. As the first identified member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice between non-homologous end-joining (NHEJ) and homologous recombination (HR) by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization surface, HORMA domain containing protein MAD2L2 has the extraordinary ability to wrap its C-terminal tail around an interacting peptide motif in SHLD3, thereby likely creating a very stable complex. We show that appropriate function of MAD2L2 in DNA repair as a member of the shieldin complex requires it to form a dimer that is mediated by SHLD2 and accelerates MAD2L2-SHLD3 interaction. Dimerization defective MAD2L2 impairs assembly of shieldin and fails to promote NHEJ at telomeres and during immunoglobulin class switch recombination (CSR). Moreover, dimerization of MAD2L2, along with the presence of SHLD3, allows the shieldin complex to interact with the AAA+ family ATPase TRIP13, known to drive topological switches in HORMA domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

2021-09-21 | PXD026912 | Pride

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