DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, ...and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing ...activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.
Synopsis
Proteomic interaction and CRISPR/Cas9 doxorubicin sensitivity screens identify shieldin proteins as the sought‐after effectors through which the 53BP1/RIF1/REV7 axis antagonizes homologous recombination (HR) and promotes non‐homologous end‐joining (NHEJ) repair of DNA double strand breaks (DSB).
Mass spectrometry identifies SHLD2/FAM35A as REV7 interactor.
SHLD2 is recruited to DSBs and promotes their repair.
SHLD2 promotes NHEJ and antagonizes HR by inhibiting DNA end resection.
SHLD2 is part of a larger multi‐protein complex composed of REV7 and SHLD1/C20orf196.
SHLD2 is a biomarker for the prognosis of TNBC/basal‐like BC.
Proteomic interaction and CRISPR/Cas9 doxorubicin sensitivity screens identify the sought‐after effectors through which the 53BP1/RIF1/REV7 axis antagonizes DNA break resection and promotes non‐homologous end‐joining repair.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection ...and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.
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► 53BP1 inhibits BRCA1 recruitment to DSB sites in G1 ► RIF1 is the effector of 53BP1 during DSB repair ► Class-switch recombination requires RIF1 ► RIF1 recruitment to DSB sites in S/G2 is inhibited by BRCA1-CtIP
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break ...(DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
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IJS, NUK, SBMB, UL, UM, UPUK
Genes that modulate the effectiveness of chemotherapy in acute myeloid leukemia (AML), in particular the response to cytarabine (Ara-C), are not completely understood. Problematically, the standard ...of care, Ara-C-containing chemotherapy is curative in only∼30% of AML patients. Using genome-wide CRISPR-Cas9 screening, we identified the E3 ubiquitin ligase HERC1 as novel modulator of Ara-C response in two murine AML cell lines. Furthermore, we found that HERC1 is overexpressed in several human hematological cancer cell lines, and that higher expression levels of HERC1 in human AML patients is associated with shorter overall survival. CRISPR-based targeting of HERC1 in murine and human AML cells enhanced their sensitivity to pyrimidine and purine analogues, suggesting a key role of HERC1 in modulating sensitivity to this class of drugs. Treatment with Ara-C led to a more sustained cell cycle arrest in HERC1-depleted cell lines, thereby triggering a stronger apoptotic response. To understand if HERC1 could target key mediators of Ara-C response for ubiquination and degradation, comparative transcriptomic and proteomic analyses were performed between Herc1-depleted and wild-type murine AML cells. These analysis identified 106 protein candidates regulated at the post-transcriptional level, including deoxycytidine kinase (DCK), which phosphorylates Ara-C to promote its incorporation into DNA. Taken together, our findings identify HERC1 as a driver of resistance to nucleoside analogues in hematological malignancies and suggest that HERC1 may regulate the abundance of proteins that regulate Ara-C metabolism and response.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract The pogo transposable element derived zinc finger protein, POGZ, is notably associated with neurodevelopmental disorders through its role in gene transcription. Many proteins involved in ...neurological development are often dysregulated in cancer, suggesting a potential role for POGZ in tumor biology. Here, we provided experimental evidence that POGZ influences the growth and metastatic spread of triple negative breast cancers (TNBC). In well-characterized models of TNBC, POGZ exerted a dual role, both as a tumor promoter and metastasis suppressor. Mechanistically, loss of POGZ potentiated TGFβ pathway activation to exert cytostatic effects while simultaneously increasing the mesenchymal and migratory properties of breast tumors. Whereas POGZ levels are elevated in human breast cancers, the most aggressive forms of TNBC tumors, including those with increased mesenchymal and metastatic properties, exhibit dampened POGZ levels, and low POGZ expression was associated with inferior clinical outcomes in these tumor types. Taken together, these data suggest that POGZ is a critical suppressor of the early stages of the metastatic cascade.
Defects in DNA repair genes have been extensively associated with cancer susceptibility. Germline pathogenic variants (GPV) in genes involved in homologous recombination repair pathways predispose to ...cancers arising mainly in the breast and ovary, but also other tissues. The RAD51 paralogs RAD51C and RAD51D were included in this group 10 years ago when germline variants were associated with non‐BRCA1/2 familial ovarian cancer. Here, we have reviewed the landscape of RAD51C and RAD51D germline variants in cancer reported in the literature during the last decade, integrating this list with variants identified by in‐house patient screening. A comprehensive catalog of 341 variants that have been classified applying ACMG/AMP criteria has been generated pinpointing the existence of recurrent variants in both genes. Recurrent variants have been extensively discussed compiling data on population frequencies and functional characterization if available, highlighting variants that have not been fully characterized yet to properly establish their pathogenicity. Finally, we have complemented this data with relevant information regarding the conservation of mutated residues among RAD51 paralogs and modeling of putative hotspot areas, which contributes to generating an exhaustive update on these two cancer predisposition genes.
Germline Pathogenic Variants, including likely pathogenic and pathogenic variants, reported in the last decade in RAD51C and RAD51D.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Highlights ► AID protein levels limit antibody diversification, off-target mutations and cancer. ► AID subcellular localization and stability regulation define the optimal AID levels. ► These ...regulatory mechanisms could operate redundantly in a complex network. ► AID is expressed in cancer and normal non-B cells at very different levels. ► Whether AID levels are regulated equally in these versus normal B cells is unclear.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes ...the presence of HP1 at DNA double‐strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA‐damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology‐directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1‐dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+/delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+/delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology‐directed DNA repair both in vitro and in vivo.
Synopsis
The zinc finger protein POGZ facilitates homologous recombination, irrespective of cell type, by retaining the HP1‐gamma‐BARD1‐BRCA1 complex at DSB.
POGZ is required for template‐dependent DNA repair (HR/SSA).
POGZ deficiency reduces the recruitment of HP1‐gamma and the BRCA1/BARD1 complex to sites of DNA damage.
Heterozygous loss of POGZ results in growth defects, abnormal behavior and aberrant DSBs in multiple tissues.
Pogz+/delta B cells show reduced class switch recombination.
The zinc finger protein POGZ facilitates homologous recombination, irrespective of cell type, by retaining the HP1‐gamma‐BARD1‐BRCA1 complex at DSB.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes ...(DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that
-deficient B cells exhibit greater Ca
mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P
, which manifests
as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.
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