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Establishing mutational outcomes after genome editing is of increasing importance with the advent of highly efficient genome-targeting tools. Next-generation sequencing (NGS) has become a vital method to investigate the extent of mutagenesis at specific target sites. Thus, robust and simple-to-use software that enables researchers to retrieve mutation profiles from NGS data is needed. Here, we present Sequence Interrogation and Quantification (SIQ), a tool that can analyse sequence data of any targeted experiment (e.g. CRISPR, I-SceI, TALENs) with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using novel and insightful tornado plot visualizations as outputs we illustrate that SIQ readily identifies differences in mutational signatures obtained from various DNA-repair deficient genetic backgrounds. SIQ greatly facilitates the interpretation of complex sequence data by establishing mutational profiles at specific loci and is, to our knowledge, the first tool that can analyse Sanger sequence data as well as short and long-read NGS data (e.g. Illumina and PacBio).

Contains fulltext : 248862.pdf (Publisher’s version ) (Closed access) Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis.

Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems. Recent work points to fill-in synthesis at double-stranded DNA breaks with complementary sequences, regulated by end-joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain-of-function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy-induced activation of oncogenes and might expose novel vulnerabilities in cancer.

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells., Error-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA polymerase α primase plays part in tandem duplication formation at CRISPR/Cas9-induced complementary 3′ ssDNA protrusions.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance., Polθ has been recently identified as a therapeutic target in cancer but specific inhibitors are currently unavailable. Here, the authors identify small molecule inhibitors of Polθ’s polymerase activity which elicit BRCA1/2 synthetic lethality, enhance the effect of PARP inhibitors and target PARP inhibitor resistance caused by 53BP1/Shieldin pathway defects.

To target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining (TMEJ), without targeting Non-Homologous End Joining. Moreover, we show that exposure to ART558 can elicit DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumor cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which are a cause of PARP inhibitor resistance, result in in vitro and in vivo sensitivity to Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells. The inhibition of DNA nucleases that promote end-resection, such as Exo1 or Blm-Dna2 reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Citation Format: Diana Zatreanu, Helen Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luted Badder, Daniela Novo, Eleanor Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Chris Bot, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Andre Nussenzweig, Marcel Tijsterman, Andrew N. Tutt, Simon Boulton, Geoff Higgins, Stephen J. Pettitt, Graeme C. Smith, Christopher J. Lord. Targeting PARP inhibitor resistance with Polθ inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5697.

To target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight, allosteric inhibitors of the polymerase function of DNA polymerase Theta (Polθ), including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining (TMEJ), without targeting Non-Homologous End Joining. Moreover, we show that exposure to ART558 can elicit DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which are a cause of PARP inhibitor resistance, result in in vitro and in vivo sensitivity to Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells. The inhibition of DNA nucleases that promote end-resection reversed these effects, suggesting that resection via Exo1 or Blm-Dna2 being a cause, at least in part, of the ART558 sensitivity phenotype. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Citation Format: Diana A. Zatreanu, Helen M. R. Robinson, Omar Alkhatib, Marie Boursier, Harry Finch, Lerin Geo, Diego Grande, Vera Grinkevich, Robert Heald, Sophie Langdon, Jayesh Majithiya, Claire McWhirter, Niall M. B. Martin, Shaun Moore, Joana Neves, Eeson Rajendra, Marco Ranzani, Theresia Schaedler, Martin Stockley, Kimberley Wiggins, Rachel Brough, Sandhya Sridhar, Aditi Gulati, Nan Shao, Luned M Badder, Daniela Novo, Eleanor G. Knight, Rebecca Marlow, Syed Haider, Elsa Callen, Graeme Hewitt, Joost Schimmel, Remko Prevo, Christina Alli, Amanda Ferdinand, Cameron Bell, Peter Blencowe, Mathew Calder, Mark Charles, Jayne Curry, Tennyson Ekwuru, Katherine Ewings, Andre Nussenzweig, Marcel Tijsterman, Andrew Tutt, Simon J. Boulton, Geoff S. Higgins, Steve Pettitt, Graeme C. M. Smith, Christopher J. Lord. Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P056.

Cockayne Syndrome (CS) is a severe neurodegenerative and premature aging autosomal-recessive disease, caused by inherited defects in the CSA and CSB genes, leading to defects in transcription-coupled nucleotide excision repair (TC-NER) and consequently hypersensitivity to ultraviolet (UV) irradiation. TC-NER is initiated by lesion-stalled RNA polymerase II, which stabilizes the interaction with the SNF2/SWI2 ATPase CSB to facilitate recruitment of the CSA E3 Cullin ubiquitin ligase complex. However, the precise biochemical connections between CSA and CSB are unknown. The small ubiquitin-like modifier SUMO is important in the DNA damage response. We found that CSB, among an extensive set of other target proteins, is the most dynamically SUMOylated substrate in response to UV irradiation. Inhibiting SUMOylation reduced the accumulation of CSB at local sites of UV irradiation and reduced recovery of RNA synthesis. Interestingly, CSA is required for the efficient clearance of SUMOylated CSB. However, subsequent proteomic analysis of CSA-dependent ubiquitinated substrates revealed that CSA does not ubiquitinate CSB in a UV-dependent manner. Surprisingly, we found that CSA is required for the ubiquitination of the largest subunit of RNA polymerase II, RPB1. Combined, our results indicate that the CSA, CSB, RNA polymerase II triad is coordinated by ubiquitin and SUMO in response to UV irradiation. Furthermore, our work provides a resource of SUMO targets regulated in response to UV or ionizing radiation.

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches., Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins. Of these acceptor lysines, 76 are situated in the SUMOylation consensus site [VILMFPC]KxE. Interestingly, eight sites. fit the inverted SUMOylation consensus motif [ED]xK[VILFP]. In addition, we found direct mass spectrometric evidence for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. In 16 proteins we identified a hydrophobic cluster SUMOylation motif (HCSM). SUMO conjugation of RanGAP1 and ZBTB1 via HCSMs is remarkably efficient.

The cell nucleus harbors a variety of different bodies that vary in number, composition, and size. Although these bodies coordinate important nuclear processes, little is known about how they are formed. Among the most intensively studied bodies in recent years is the PML body. These bodies have been implicated in gene regulation and other cellular processes and are disrupted in cells from patients suffering from acute promyelocytic leukemia. Using live cell imaging microscopy and immunofluorescence, we show in several cell types that PML bodies are formed at telomeric DNA during interphase. Recent studies revealed that both SUMO modification sites and SUMO interaction motifs in the promyelocytic leukemia (PML) protein are required for PML body formation. We show that SMC5, a component of the SUMO ligase MMS21-containing SMC5/6 complex, localizes temporarily at telomeric DNA during PML body formation, suggesting a possible role for SUMO in the formation of PML bodies at telomeric DNA. Our data identify a novel role of telomeric DNA during PML body formation.

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR.

Background— Hypertrophic growth, a risk factor for mortality in heart disease, is driven by reprogramming of cardiac gene expression. Although the transcription factor myocyte enhancer factor-2 (MEF2) is a common end point for several hypertrophic pathways, its precise cardiac gene targets and function in cardiac remodeling remain to be elucidated. Methods and Results— We report the existence of synergistic interactions between the nuclear factor of activated T cells and MEF2 transcription factors triggered by calcineurin signaling. To circumvent the embryonic lethality and mitochondrial deficiency associated with germ-line null mutations for MEF2C and MEF2A respectively, we used conditional transgenesis to express a dominant-negative form of MEF2 in the murine postnatal heart and combined this with magnetic resonance imaging to assess MEF2 transcriptional function in Ca 2+ /calcineurin-induced cardiac remodeling. Surprisingly, end-diastolic and end-systolic ventricular dimensions and contractility were normalized in the presence of severely hypertrophied left ventricular walls on MEF2 inhibition in calcineurin transgenic mice. In line, we generated lines of transgenic mice expressing MEF2A in the heart, which displayed primarily chamber dilation. Microarray profiling indicated that MEF2 promotes a gene profile functioning primarily to or at the nucleus, cytoskeletal and microtubular networks, and mitochondria. Conclusions— These findings assign a novel function to MEF2 transcription factors in the postnatal heart, where they activate a genetic program that minimally affects cardiac growth yet promotes chamber dilation, mechanical dysfunction, and dilated cardiomyopathy.

Loss of small ubiquitin-like modification (SUMOylation) in mice causes genomic instability due to the missegregation of chromosomes. Currently, little is known about the identity of relevant SUMO target proteins that are involved in this process and about global SUMOylation dynamics during cell-cycle progression. We performed a large-scale quantitative proteomics screen to address this and identified 593 proteins to be SUMO-2 modified, including the Forkhead box transcription factor M1 (FoxM1), a key regulator of cell-cycle progression and chromosome segregation. SUMOylation of FoxM1 peaks during G2 and M phase, when FoxM1 transcriptional activity is required. We found that a SUMOylation-deficient FoxM1 mutant was less active compared to wild-type FoxM1, implying that SUMOylation of the protein enhances its transcriptional activity. Mechanistically, SUMOylation blocks the dimerization of FoxM1, thereby relieving FoxM1 autorepression. Cells deficient for FoxM1 SUMOylation showed increased levels of polyploidy. Our findings contribute to understanding the role of SUMOylation during cell-cycle progression.

TGF-beta members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-beta family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-beta signaling. Depletion of RNF12 dramatically reduced TGF-beta/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-beta family signaling.

Covalent attachment of Small Ubiquitin-like MOdifiers (SUMOs) to the epsilon-amino group of lysine residues in target proteins regulates many cellular processes. Previously, we have identified the 110 kDa U4/U6.U5 tri-snRNP component SART1 as a target protein for SUMO-1 and SUMO-2. SART1 contains lysines on positions 94, 141, 709 and 742 that are situated in tetrameric sumoylation consensus sites. Recombinant SART1 was produced in E. coli, conjugated to SUMO-2 in vitro, digested by trypsin and analysed by MALDI-ToF, MALDI-FT-ICR or nanoLC-iontrap MS/MS. We found that Lys(94) and Lys(141) of SART1 were preferentially conjugated to SUMO-2 monomers and multimers in vitro. In agreement with these results, mutation of Lys(94) and Lys(141), but not Lys(709) and Lys(742), resulted in a reduced sumoylation of SART1 in HeLa cells. A detailed characterization of the four sumoylation sites of SART1 using full-length recombinant SART1 and a peptide sumoylation approach indicated that positively charged amino acids adjacent to the tetrameric sumoylation consensus site enhance the sumoylation of Lys(94). These results show that amino acids surrounding the classic tetrameric SUMO consensus site can regulate sumoylation efficiency and validate the use of an in vitro sumoylation-mass spectrometry approach for the identification of sumoylation sites. (C) 2010 Elsevier B.V. All rights reserved.

Many proteins are regulated by a variety of post-translational modifications, and orchestration of these modifications is frequently required for full control of activity. Currently little is known about the combinatorial activity of different post-translational modifications. Here we show that extensive cross-talk exists between sumoylation and ubiquitination. We found that a subset of SUMO-2-conjugated proteins is subsequently ubiquitinated and degraded by the proteasome. In a screen for preferential SUMO-1 or SUMO-2 target proteins, we found that ubiquitin accumulated in purified SUMO-2 conjugates but not in SUMO-1 conjugates. Upon inhibition of the proteasome, the amount of ubiquitin in purified SUMO-2 conjugates increased. In addition, we found that endogenous SUMO-2/3 conjugates, but not endogenous SUMO-1 conjugates, accumulated in response to proteasome inhibitors. Quantitative proteomics experiments enabled the identification of 73 SUMO-2-conjugated proteins that accumulated in cells treated with proteasome inhibitors. Cross-talk between SUMO-2/3 and the ubiquitin-proteasome system controls many target proteins that regulate all aspects of nucleic acid metabolism. Surprisingly the relative abundance of 40 SUMO-2-conjugated proteins was reduced by proteasome inhibitors possibly because of a lack of recycled SUMO-2. We conclude that SUMO-2/3 conjugation and the ubiquitin-proteasome system are tightly integrated and act in a cooperative manner.

The length and precise linkage of polyubiquitin chains is important for their biological activity. Although other ubiquitin-like proteins have the potential to form polymeric chains their identification in vivo is challenging and their functional role is unclear. Vertebrates express three small ubiquitin-like modifiers, SUMO-1, SUMO-2, and SUMO-3. Mature SUMO-2 and SUMO-3 are nearly identical and contain an internal consensus site for sumoylation that is missing in SUMO-1. Combining state-of-the-art mass spectrometry with an "in vitro to in vivo" strategy for post-translational modifications, we provide direct evidence that SUMO-1, SUMO-2, and SUMO-3 form mixed chains in cells via the internal consensus sites for sumoylation in SUMO-2 and SUMO-3. In vitro, the chain length of SUMO polymers could be influenced by changing the relative amounts of SUMO-1 and SUMO-2. The developed methodology is generic and can be adapted for the identification of other sumoylation sites in complex samples.