UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, 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.

REV7: Jack of many trades.

The HORMA domain protein REV7, also known as MAD2L2, interacts with a variety of proteins and thereby contributes to the establishment of different complexes. With doing so, REV7 impacts a diverse range of cellular processes and gained increasing interest as more of its activities became uncovered. REV7 has important roles in translesion synthesis and mitotic progression, and acts as a central component in the recently discovered shieldin complex that operates in DNA double-strand break repair. Here we discuss the roles of REV7 in its various complexes, focusing on its activity in genome integrity maintenance. Moreover, we will describe current insights on REV7 structural features that allow it to be such a versatile protein.

H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres.

The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.

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

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. 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, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes 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.

The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells.

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2.

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110ß isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110ß, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110ß via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110ß. Cells from mice carrying mutations in the p110ß RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110ß downstream of GPCRs.

Beyond KRAS mutation status: influence of KRAS copy number status and microRNAs on clinical outcome to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. METHODS: Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n = 17) or poor (n = 17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. RESULTS: Copy number loss of the KRAS locus was observed in the tumour of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild-type KRAS tumours were associated with a poor PFS. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good PFS. In wild-type KRAS patients, miRNA expression did not correlate with PFS in a multivariate model. CONCLUSIONS: Our results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of mCRC eligible for anti-EGFR therapy.

MicroRNAs in colorectal cancer metastasis.

Metastatic disease is the major cause of death in colorectal cancer (CRC) patients. The metastatic process is highly inefficient and comprises multiple sequential steps. While many genetic factors relevant in this process have already been identified, the epigenetic factors underlying each step still remain obscure. MicroRNAs (miRNAs) are key regulators in tumourigenesis, but their role in the development of cancer metastasis is poorly investigated. The majority of miRNAs involved in the metastatic process have been identified in breast cancer cell lines, and in CRC less data are available. We review the role of miRNAs in the metastatic pathway of CRC, including escape of apoptosis, epithelial-mesenchymal transition (EMT), angiogenesis, and invasion. Better understanding of the complex role of miRNAs in the development of CRC metastases may provide new insights that could be of therapeutic consequence.