Ubiquitin regulates dissociation of DNA repair factors from chromatin.

This is the final version of the article. It first appeared from Impact Journals via http://www.impactjournals.com/oncotarget/misc/linkedout.php?pii=441

Neddylation promotes ubiquitylation and release of Ku from DNA-damage sites.

The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.We thank Thimo Kurz (University of Dundee, UK) for providing MLN4924 and Kate Dry, Rimma Berlotserkovskaya (S.P.J.’s laboratory), and Eric Lightcap (Takeda Pharmaceuticals) for critical reading of the manuscript. We thank Sylvie Urbe and Michael Clague (University of Liverpool, UK) for providing the GFP-CSN5 plasmid, the Division of Signal Transduction Therapy (University of Dundee, UK) for providing UBE2M and UBE2F plasmids, Matthew Petroski (Sanford-Burnham Medical Research Institute, US) for providing FLAG-UBA3 wild-type (WT) and FLAG-UBA3-A171T constructs, and Nico Dantuma (Karolinska Institute, Sweden) and Changshun Shao (Rutgers University) for providing CUL4A and CUL4B plasmids, respectively. We also thank Nicola Lawrence, Alex Sossick, and Richard Butler (Gurdon Institute, Cambridge, UK) for help with microscopy, Volocity, and Fiji. Research in the S.P.J.’s laboratory is funded by Cancer Research UK programme grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement no. 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. N.L. is funded by CRUK programme grant C6/A11224, J.S.B. is funded by a Wellcome Trust Clinical Fellowship (WT083416), and Y.G. and M.S.-C. are funded by European Research Council grant DDREAM. S.B. was funded by an EMBO long-term fellowship ALTF 93-2010, Cancer Research UK, and a post-doctoral grant from Ligue Nationale Contre le Cancer. P.B. is supported by the Emmy Noether Programme of the German Research Foundation (DFG, BE 5342/1-1).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S2211124715003496

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

UV-light-induced DNA damage affects RNA metabolism but the underlying signalling pathways are largely unexplored. Here, the authors show that UV light triggers p38-MK2-mediated phosphorylation of the NELF complex, promoting its release from chromatin and concurrent transcriptional elongation

Chronic irradiation of human cells reduces histone levels and deregulates gene expression

Abstract: Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6–20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology

Chronic irradiation of human cells reduces histone levels and deregulates gene expression

Abstract: Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6–20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology

PARP1 proximity proteomics reveals interaction partners at stressed replication forks

PARP1 mediates poly-ADP-ribosylation of proteins on chromatin in response to different types of DNA lesions. PARP inhibitors are used for the treatment of BRCA1/2-deficient breast, ovarian, and prostate cancer. Loss of DNA replication fork protection is proposed as one mechanism that contributes to the vulnerability of BRCA1/2-deficient cells to PARP inhibitors. However, the mechanisms that regulate PARP1 activity at stressed replication forks remain poorly understood. Here, we performed proximity proteomics of PARP1 and isolation of proteins on stressed replication forks to map putative PARP1 regulators. We identified TPX2 as a direct PARP1-binding protein that regulates the auto-ADP-ribosylation activity of PARP1. TPX2 interacts with DNA damage response proteins and promotes homology-directed repair of DNA double-strand breaks. Moreover, TPX2 mRNA levels are increased in BRCA1/2-mutated breast and prostate cancers, and high TPX2 expression levels correlate with the sensitivity of cancer cells to PARP-trapping inhibitors. We propose that TPX2 confers a mitosis-independent function in the cellular response to replication stress by interacting with PARP1

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex

IKKα controls ATG16L1 degradation to prevent ER stress during inflammation

Inhibition of the IκB kinase complex (IKK) has been implicated in the therapy of several chronic inflammatory diseases including inflammatory bowel diseases. In this study, using mice with an inactivatable IKKα kinase (IkkαAA/AA), we show that loss of IKKα function markedly impairs epithelial regeneration in a model of acute colitis. Mechanistically, this is caused by compromised secretion of cytoprotective IL-18 from IKKα-mutant intestinal epithelial cells because of elevated caspase 12 activation during an enhanced unfolded protein response (UPR). Induction of the UPR is linked to decreased ATG16L1 stabilization in IkkαAA/AA mice. We demonstrate that both TNF-R and nucleotide-binding oligomerization domain stimulation promote ATG16L1 stabilization via IKKα-dependent phosphorylation of ATG16L1 at Ser278. Thus, we propose IKKα as a central mediator sensing both cytokine and microbial stimulation to suppress endoplasmic reticulum stress, thereby assuring antiinflammatory function during acute intestinal inflammation

Ubiquitin regulates dissociation of DNA repair factors from chromatin

This is the final version of the article. It first appeared from Impact Journals via http://www.impactjournals.com/oncotarget/misc/linkedout.php?pii=441