USP4 Auto-Deubiquitylation Promotes Homologous Recombination.

Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A11224, CRUK Project Grant C6/A14831 and the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). R.N. was funded by the Daiichi Sankyo Foundation of Life Sciences fellowship. Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096 provided core infrastructure funding. S.P.J receives his salary from the University of Cambridge, supplemented by CRUK. The John Fell Fund 133/075 and the Wellcome Trust grant 097813/Z/11/Z funded research performed by B.M.K and R.K..This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.molcel.2015.09.01

The deubiquitylating enzyme UCHL3 regulates Ku80 retention at sites of DNA damage.

Non-homologous end-joining (NHEJ), which can promote genomic instability when dysfunctional, is a major DNA double-strand break (DSB) repair pathway. Although ubiquitylation of the core NHEJ factor, Ku (Ku70-Ku80), which senses broken DNA ends, is important for its removal from sites of damage upon completion of NHEJ, the mechanism regulating Ku ubiquitylation remains elusive. We provide evidence showing that the ubiquitin carboxyl-terminal hydrolase L3 (UCHL3) interacts with and directly deubiquitylates one of the Ku heterodimer subunits, Ku80. Additionally, depleting UCHL3 resulted in reduced Ku80 foci formation, Ku80 binding to chromatin after DSB induction, moderately sensitized cells to ionizing radiation and decreased NHEJ efficiencies. Mechanistically, we show that DNA damage induces UCHL3 phosphorylation, which is dependent on ATM, downstream NHEJ factors and UCHL3 catalytic activity. Furthermore, this phosphorylation destabilizes UCHL3, despite having no effect on its catalytic activity. Collectively, these data suggest that UCHL3 facilitates cellular viability after DSB induction by antagonizing Ku80 ubiquitylation to enhance Ku80 retention at sites of damage.This work was funded by Grant-in-Aid for Research Activity start-up 15H06738 (R.N.), Grant-in Aid for Young Scientists (A) 16H05888 (R.N.), Daiichi Sankyo Foundation of Life Science (R.N.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (R.N.), Cancer Research UK (CRUK) Grant C6/A11224 and C6/A18796 (P.W.), CRUK Project Grant C6/A14831 (R.N.). T.L.B. and Q.W. are funded by Wellcome Trust Investigator Award (200814_Z_16_Z). Research in the B.M.K. laboratory is supported by a John Fell Fund 133/075, the Wellcome Trust (097813/Z/11/Z) and the Engineering and Physical Sciences Research Council (EP/N034295/1). Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A18796, and Wellcome Trust Grant WT206388/Z/17/Z. Cancer Research UK Grant C6946/A24843 and Wellcome Trust Grant WT203144 provided core infrastructure funding

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.Research in S.P.J.’s laboratory is funded by Cancer Research UK (CRUK) program grant number C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement number HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. L.D.’s laboratory is funded by the Institut Pasteur as well as the European Research Council (ERC) under starting grant agreement number 310917. D.J.A.’s laboratory is supported by CRUK and the Wellcome Trust. A.N.B. is supported by a CRUK Career Development Fellowship (C29215/A20772).This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via https://doi.org/10.1101/gad.290510.11

MPHASYS: a mouse phenotype analysis system

<p>Abstract</p> <p>Background</p> <p>Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery.</p> <p>Results</p> <p>Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd.</p> <p>Conclusion</p> <p>MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Adaptive Stress Response in Segmental Progeria Resembles Long-Lived Dwarfism and Calorie Restriction in Mice

How congenital defects causing genome instability can result in the pleiotropic symptoms reminiscent of aging but in a segmental and accelerated fashion remains largely unknown. Most segmental progerias are associated with accelerated fibroblast senescence, suggesting that cellular senescence is a likely contributing mechanism. Contrary to expectations, neither accelerated senescence nor acute oxidative stress hypersensitivity was detected in primary fibroblast or erythroblast cultures from multiple progeroid mouse models for defects in the nucleotide excision DNA repair pathway, which share premature aging features including postnatal growth retardation, cerebellar ataxia, and death before weaning. Instead, we report a prominent phenotypic overlap with long-lived dwarfism and calorie restriction during postnatal development (2 wk of age), including reduced size, reduced body temperature, hypoglycemia, and perturbation of the growth hormone/insulin-like growth factor 1 neuroendocrine axis. These symptoms were also present at 2 wk of age in a novel progeroid nucleotide excision repair-deficient mouse model (XPD(G602D/R722W)/XPA(−/−)) that survived weaning with high penetrance. However, despite persistent cachectic dwarfism, blood glucose and serum insulin-like growth factor 1 levels returned to normal by 10 wk, with hypoglycemia reappearing near premature death at 5 mo of age. These data strongly suggest changes in energy metabolism as part of an adaptive response during the stressful period of postnatal growth. Interestingly, a similar perturbation of the postnatal growth axis was not detected in another progeroid mouse model, the double-strand DNA break repair deficient Ku80 (−/−) mouse. Specific (but not all) types of genome instability may thus engage a conserved response to stress that evolved to cope with environmental pressures such as food shortage

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

BACKGROUND & AIMS: Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barrett's and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. METHODS: We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. RESULTS: We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09-1.18; P = 1.8 × 10(-11)) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86-0.93; P = 7.5 × 10(-9)). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87-0.93; P = 3.72 × 10(-9)). CONCLUSIONS: We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response

2 days versus 5 days of postoperative antibiotics for complex appendicitis:a pragmatic, open-label, multicentre, non-inferiority randomised trial

Background: The appropriate duration of postoperative antibiotics for complex appendicitis is unclear. The increasing global threat of antimicrobial resistance warrants restrictive antibiotic use, which could also reduce side-effects, length of hospital stay, and costs. Methods: In this pragmatic, open-label, non-inferiority trial in 15 hospitals in the Netherlands, patients with complex appendicitis (aged ≥8 years) were randomly assigned (1:1) to receive 2 days or 5 days of intravenous antibiotics after appendicectomy. Randomisation was stratified by centre, and treating physicians and patients were not masked to treatment allocation. The primary endpoint was a composite endpoint of infectious complications and mortality within 90 days. The main outcome was the absolute risk difference (95% CI) in the primary endpoint, adjusted for age and severity of appendicitis, with a non-inferiority margin of 7·5%. Outcome assessment was based on electronic patient records and a telephone consultation 90 days after appendicectomy. Efficacy was analysed in the intention-to-treat and per-protocol populations. Safety outcomes were analysed in the intention-to-treat population. This trial was registered with the Netherlands Trial Register, NL5946. Findings: Between April 12, 2017, and June 3, 2021, 13 267 patients were screened and 1066 were randomly assigned, 533 to each group. 31 were excluded from intention-to-treat analysis of the 2-day group and 30 from the 5-day group owing to errors in recruitment or consent. Appendicectomy was done laparoscopically in 955 (95%) of 1005 patients. The telephone follow-up was completed in 664 (66%) of 1005 patients. The primary endpoint occurred in 51 (10%) of 502 patients analysed in the 2-day group and 41 (8%) of 503 patients analysed in the 5-day group (adjusted absolute risk difference 2·0%, 95% CI −1·6 to 5·6). Rates of complications and re-interventions were similar between trial groups. Fewer patients had adverse effects of antibiotics in the 2-day group (45 [9%] of 502 patients) than in the 5-day group (112 [22%] of 503 patients; odds ratio [OR] 0·344, 95% CI 0·237 to 0·498). Re-admission to hospital was more frequent in the 2-day group (58 [12%] of 502 patients) than in the 5-day group (29 [6%] of 503 patients; OR 2·135, 1·342 to 3·396). There were no treatment-related deaths. Interpretation: 2 days of postoperative intravenous antibiotics for complex appendicitis is non-inferior to 5 days in terms of infectious complications and mortality within 90 days, based on a non-inferiority margin of 7·5%. These findings apply to laparoscopic appendicectomy conducted in a well resourced health-care setting. Adopting this strategy will reduce adverse effects of antibiotics and length of hospital stay. Funding: The Netherlands Organization for Health Research and Development.</p

Neoadjuvant chemoradiotherapy plus surgery versus active surveillance for oesophageal cancer: A stepped-wedge cluster randomised trial

Background: Neoadjuvant chemoradiotherapy (nCRT) plus surgery is a standard treatment for locally advanced oesophageal cancer. With this treatment, 29% of patients have a pathologically complete response in the resection specimen. This provides the rationale for investigating an active surveillance approach. The aim of this study is to assess the (cost-)effectiveness of active surveillance vs. standard oesophagectomy after nCRT for oesophageal cancer. Methods: This is a phase-III multi-centre, stepped-wedge cluster randomised controlled trial. A total of 300 patients with clinically complete response (cCR, i.e. no local or disseminated disease proven by histology) after nCRT will be randomised to show non-inferiority of active surveillance to standard oesophagectomy (non-inferiority margin 15%, intra-correlation coefficient 0.02, power 80%, 2-sided α 0.05, 12% drop-out). Patients will undergo a first clinical response evaluation (CRE-I) 4-6 weeks after nCRT, consisting of endoscopy with bite-on-bite biopsies of the primary tumour site and other suspected lesions. Clinically complete responders will undergo a second CRE (CRE-II), 6-8 weeks after CRE-I. CRE-II will include 18F-FDG-PET-CT, followed by endoscopy with bite-on-bite biopsies and ultra-endosonography plus fine needle aspiration of suspected lymph nodes and/or PET- positive lesions. Patients with cCR at CRE-II will be assigned to oesophagectomy (first phase) or active surveillance (second phase of the study). The duration of the first phase is determined randomly over the 12 centres, i.e., stepped-wedge cluster design. Patients in the active surveillance arm will undergo diagnostic evaluations similar to CRE-II at 6/9/12/16/20/24/30/36/48 and 60 months after nCRT. In this arm, oesophagectomy will be offered only to patients in whom locoregional regrowth is highly suspected or proven, without distant dissemination. The main study parameter is overall survival; secondary endpoints include percentage of patients who do not undergo surgery, quality of life, clinical irresectability (cT4b) rate, radical resection rate, postoperative complications, progression-free survival, distant dissemination rate, and cost-effectiveness. We hypothesise that active surveillance leads to non-inferior survival, improved quality of life and a reduction in costs, compared to standard oesophagectomy. Discussion: If active surveillance and surgery as needed after nCRT leads to non-inferior survival compared to standard oesophagectomy, this organ-sparing approach can be implemented as a standard of care