Mechanisms of RNF168 nucleosome recognition and ubiquitylation.

RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.

Adolescent-onset multisystem proteinopathy due to a novel VCP variant.

Valosin-containing protein (VCP) pathogenic variants are the most common cause of multisystem proteinopathy presenting with inclusion body myopathy, amyotrophic lateral sclerosis/frontotemporal dementia, and Paget disease of bone in isolation or in combination. We report a patient manifesting with adolescent-onset myopathy caused by a novel heterozygous VCP variant (c.467G > T, p.Gly156Val). The myopathy manifested asymmetrically in lower limbs and extended to proximal, axial, and upper limb muscles, with loss of ambulation at age 35. Creatine kinase value was normal. Alkaline phosphatase was elevated. Electromyography detected mixed low amplitude, short duration and high amplitude, long duration motor unit potentials. Muscle biopsy showed features of inclusion body myopathy, which in combination with newly diagnosed Paget disease of bone, supported the VCP variant pathogenicity. In conclusion, VCP-multisystem proteinopathy is not only a disease of adulthood but can have a pediatric onset and should be considered in differential diagnosis of neuromuscular weakness in the pediatric population.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C.

Pathogenic protein-truncating variants of RAD51C, which plays an integral role in promoting DNA damage repair, increase the risk of breast and ovarian cancer. A large number of RAD51C missense variants of uncertain significance (VUS) have been identified, but the effects of the majority of these variants on RAD51C function and cancer predisposition have not been established. Here, analysis of 173 missense variants by a homology-directed repair (HDR) assay in reconstituted RAD51C-/- cells identified 30 nonfunctional (deleterious) variants, including 18 in a hotspot within the ATP-binding region. The deleterious variants conferred sensitivity to cisplatin and olaparib and disrupted formation of RAD51C/XRCC3 and RAD51B/RAD51C/RAD51D/XRCC2 complexes. Computational analysis indicated the deleterious variant effects were consistent with structural effects on ATP-binding to RAD51C. A subset of the variants displayed similar effects on RAD51C activity in reconstituted human RAD51C-depleted cancer cells. Case-control association studies of deleterious variants in women with breast and ovarian cancer and noncancer controls showed associations with moderate breast cancer risk [OR, 3.92; 95% confidence interval (95% CI), 2.18-7.59] and high ovarian cancer risk (OR, 14.8; 95% CI, 7.71-30.36), similar to protein-truncating variants. This functional data supports the clinical classification of inactivating RAD51C missense variants as pathogenic or likely pathogenic, which may improve the clinical management of variant carriers. SIGNIFICANCE: Functional analysis of the impact of a large number of missense variants on RAD51C function provides insight into RAD51C activity and information for classification of the cancer relevance of RAD51C variants.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

Hidden tricks in MATH: Hypermorphic mutations in SPOP tumor suppressor explained by cryo-EM.

Loss-of-function mutations in SPOP E3 ubiquitin ligase drive multiple cancers. However, carcinogenic gain-of-function SPOP mutations have been a major puzzle. In this issue of Molecular Cell, Cuneo et al.1 show that several mutations map to SPOP oligomerization interfaces. Additional questions remain about SPOP mutations in malignancy.

METTL16 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the 'writers' of RNA N6-methyladenosine modification, may benefit from poly-(ADP-ribose)-polymerase inhibitor (PARPi) treatment. Mechanistically, METTL16 interacts with MRE11 through RNA and this interaction inhibits MRE11's exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 resulting in a conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression show increased sensitivity to PARPi, especially when combined with gemcitabine. Thus, our findings reveal a role for METTL16 in homologous recombination repair and suggest that a combination of PARPi with gemcitabine could be an effective treatment strategy for PDAC with elevated METTL16 expression.

A novel missense HNRNPA1 variant in the PY-NLS domain in a patient with late-onset distal myopathy.

Pathogenic HNRNPA1 variants underlying myopathy have been reported only in the prion-like domain of the heterogenous nuclear ribonucleoproteins A1, while two variants in the nuclear localization (PY-NLS) domain were described in ALS. Here we report a 61-year-old man who presented with 1-year history of bilateral foot drop without Paget disease or dementia. Examination revealed severe asymmetric distal weakness, predominantly affecting tibialis anterior and toe extensors. Creatine kinase was 1,013 U/L (normal <308). Alkaline phosphatase was normal. EMG demonstrated small polyphasic motor unit potentials and fibrillation potentials. Muscle biopsy showed numerous fibers containing rimmed vacuoles and occasional fibers harboring congophilic inclusions, or p62/TDP-43/hnRNPA1-immunoreacted aggregates. Next generation sequencing identified a novel heterozygous (c.959A>T, p. Asn320Ile) variant in HNRNPA1, affecting a highly conserved amino acid in PY-NLS domain. Muscle MRI showed abnormalities, consistent with HNRNPA1-myopathy. This patient expands the phenotypic spectrum of hnRNPA1-opathy due to a PY-NLS domain variant to include isolated distal myopathy.

SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition.

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.

Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.

The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

ATM-phosphorylated SPOP contributes to 53BP1 exclusion from chromatin during DNA replication.

53BP1 activates nonhomologous end joining (NHEJ) and inhibits homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Dissociation of 53BP1 from DSBs and consequent activation of HR, a less error-prone pathway than NHEJ, helps maintain genome integrity during DNA replication; however, the underlying mechanisms are not fully understood. Here, we demonstrate that E3 ubiquitin ligase SPOP promotes HR during S phase of the cell cycle by excluding 53BP1 from DSBs. In response to DNA damage, ATM kinase-catalyzed phosphorylation of SPOP causes a conformational change in SPOP, revealed by x-ray crystal structures, that stabilizes its interaction with 53BP1. 53BP1-bound SPOP induces polyubiquitination of 53BP1, eliciting 53BP1 extraction from chromatin by a valosin-containing protein/p97 segregase complex. Our work shows that SPOP facilitates HR repair over NHEJ during DNA replication by contributing to 53BP1 removal from chromatin. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects and chromosomal instability.

TIRR inhibits the 53BP1-p53 complex to alter cell-fate programs.

53BP1 influences genome stability via two independent mechanisms: (1) regulating DNA double-strand break (DSB) repair and (2) enhancing p53 activity. We discovered a protein, Tudor-interacting repair regulator (TIRR), that associates with the 53BP1 Tudor domain and prevents its recruitment to DSBs. Here, we elucidate how TIRR affects 53BP1 function beyond its recruitment to DSBs and biochemically links the two distinct roles of 53BP1. Loss of TIRR causes an aberrant increase in the gene transactivation function of p53, affecting several p53-mediated cell-fate programs. TIRR inhibits the complex formation between the Tudor domain of 53BP1 and a dimethylated form of p53 (K382me2) that is poised for transcriptional activation of its target genes. TIRR mRNA expression levels negatively correlate with the expression of key p53 target genes in breast and prostate cancers. Further, TIRR loss is selectively not tolerated in p53-proficient tumors. Therefore, we establish that TIRR is an important inhibitor of the 53BP1-p53 complex.

A rare germline CDKN2A variant (47T>G; p16-L16R) predisposes carriers to pancreatic cancer by reducing cell cycle inhibition.

Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.

Ubiquitin Phosphorylation at Thr12 Modulates the DNA Damage Response.

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein.

Dynamic protein interaction networks such as DNA double-strand break (DSB) signaling are modulated by post-translational modifications. The DNA repair factor 53BP1 is a rare example of a protein whose post-translational modification-binding function can be switched on and off. 53BP1 is recruited to DSBs by recognizing histone lysine methylation within chromatin, an activity directly inhibited by the 53BP1-binding protein TIRR. X-ray crystal structures of TIRR and a designer protein bound to 53BP1 now reveal a unique regulatory mechanism in which an intricate binding area centered on an essential TIRR arginine residue blocks the methylated-chromatin-binding surface of 53BP1. A 53BP1 separation-of-function mutation that abolishes TIRR-mediated regulation in cells renders 53BP1 hyperactive in response to DSBs, highlighting the key inhibitory function of TIRR. This 53BP1 inhibition is relieved by TIRR-interacting RNA molecules, providing proof-of-principle of RNA-triggered 53BP1 recruitment to DSBs.

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18.

The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using nuclear magnetic resonance (NMR) spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1, and it ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169, and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy.

A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.