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RNF4 Is Required for DNA Double-Strand Break Repair in Vivo

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RNF4 Is Required for DNA Double-Strand Break Repair in Vivo Cell Death and Differentiation (2013) 20, 490–502 & 2013 Macmillan Publishers Limited All rights reserved 1350-9047/13 www.nature.com/cdd RNF4 is required for DNA double-strand break repair in vivo R Vyas1,2, R Kumar3, F Clermont1,2, A Helfricht3,4, P Kalev2,5, P Sotiropoulou6, IA Hendriks3, E Radaelli7, T Hochepied8, C Blanpain6, A Sablina2,5, H van Attikum4, JV Olsen9, AG Jochemsen3, ACO Vertegaal3 and J-C Marine*,1,2 Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation- induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events. Cell Death and Differentiation (2013) 20, 490–502; doi:10.1038/cdd.2012.145; published online 30 November 2012 DNA double-strand breaks (DSBs) represent the most constitute a barrier toward further DNA decay.5 Protein recruitment destructive type of chromosomal lesion that pose a major to such structures occurs in a highly dynamic and hierarchical threat to genome stability, if not correctly sensed and repaired. manner, and is controlled by post-translational modifications Cells counteract DNA damage via a sophisticated DNA within DSB-flanking chromatin.6 Phosphorylation of multiple damage signaling response, which impacts on diverse cellular DSB-signaling components by the ATM/ATR/DNA-PK processes, such as DNA repair itself, cell cycle progression, kinases have a central role in promoting this response; one DNA replication, and transcription.1,2 Most cellular DSBs are of such key events being ATM-mediated phosphorylation of repaired by homologous recombination (HR) or NEHJ,3 and H2AX on ser139, an epigenetic mark commonly referred to as defects in either pathway exhibit increased sensitivity to g-H2AX.7 Phosphorylation of g-H2AX provides a direct binding ionizing radiation (IR).4 platform for the MDC1 scaffold protein.8 Following the DSBs induction, multiple DNA damage Nonproteolytic ubiquitylation also has an important role in signaling and repair factors become concentrated at, or orchestrating protein interactions with DSB sites.9 ATM- excluded from, specific areas of the nucleus. In particular, a mediated phosphorylation of MDC1 provides a docking site protective and DNA repair-stimulating microenvironment is for the ubiquitin ligase RNF8.10,11 RNF8 promotes the formed around the DSB sites. These so-called IR-induced foci polyubiquitylation of core histones H2A and H2AX to trigger (IRIF) concentrate multiple DNA repair factors in the vicinity a second, ubiquitin-dependent round of protein recruitment to of the strand breaks, promote checkpoint signaling, and DSB-flanking chromatin. In addition to RNF8, other E3 ligases 1Center for the Biology of Disease, Laboratory for Molecular Cancer Biology, VIB, Leuven, Belgium; 2Center for Human Genetics, KU Leuven, Leuven, Belgium; 3Department of Molecular Cell Biology, University Medical Centre, Leiden, The Netherlands; 4Department of Toxicogenetics, University Medical Centre, Leiden, The Netherlands; 5Center for the Biology of Disease, Laboratory for Mechanisms of Cell Transformation, VIB, Leuven, Belgium; 6WELBIO, IRIBHM, Universite´ Libre de Bruxelles, Brussels, Belgium; 7Department of Veterinary Pathology, Hygiene, and Public Health, University of Milan, Faculty of Veterinary Medicine, Milan, Italy; 8Transgenic Core Facility, Department for Molecular Biomedical Research, VIB-Ghent University, Ghent, Belgium and 9Department of Proteomics, Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark *Corresponding author: J-C Marine, VIB-KULeuven, Laboratory For Molecular Cancer Biology, Campus Gasthuisberg O&N 4, 3000 Leuven, Belgium. Tel: þ 32 16 33 0368; Fax: þ 32 16 33 0145; E-mail: [email protected] Keywords: DNA damage; homologous recombination; RNF4; BRCA1; MDC1; Rad51; spermatogenesis Abbreviations: DSBs, double-strand breaks; HR, homologous recombination; NHEJ, non-homologous end joining; IR, ionizing radiation; UV, ultraviolet; IRIF, ionizing radiation-induced foci; DDR, DNA damage response; STUbl, SUMO-targeted ubiquitin ligase; SA, splice acceptor; PCR, polymerase chain reaction; MEFs, mouse embryonic fibroblasts; RT-qPCR, reverse transcription quantitative PCR; ROS, reactive oxygen species; PFGE, pulsed-field gel electrophoresis; IHC, immunohistochemistry; RING, really interesting new gene; H&E, hematoxylin and eosin; (E)GFP, (enhanced) green fluorescent protein; Hypo, hypomorphic; SIM, SUMO interaction motif Received 30.5.12; revised 17.9.12; accepted 05.10.12; Edited by G Melino; published online 30.11.12 RNF4 and DNA double-strand break repair R Vyas et al 491 such as HERC2 and RNF168 are also recruited to these sites, correspond to a null mutation. The Rnf4-trapped allele is to assist RNF8 in promoting the formation of nonproteolytic, thereafter referred to as Rnf4 À . K63-linked polyubiquitin chains on histones and possibly on Rnf4 þ / À mice are viable and fertile. However, none of the other targets at sites of DNA damage. These modifications 123 offspring from heterozygous intercrosses were Rnf4 À generate permissive conditions for recruitment of downstream homozygous (supplementary Table S1). Although expected, DNA repair factors such as 53BP1 and BRCA1-containing Mendelian ratios were observed in embryos isolated at E9.5, protein complexes.12 Although several other E3 ligases are the proportion of homozygous embryos declined at later recruited to IRIF,12 including RAD18, BRCA1, BMI1, the stages such that no live homozygous embryos were identified identity of their substrates within these structures, remains among 42 embryos at E12.5 (Supplementary Table S1 and largely unknown. Figure 1b). At E10.5, all Rnf4 À / À embryos were developmen- The small ubiquitin-like modifier (SUMO) has also been tally delayed (Figure 1d). implicated in genome stability and repair responses.13 The We crossed Rnf4 þ / À mice with a Cre-deleter strain in an SUMO E3 ligases PIAS1 and PIAS4 are required for the attempt to generate a hypomorphic allele. We reasoned that recruitment of BRCA1 and 53BP1 to IRIF, and both SUMO1 low levels of Rnf4 activity may rescue the recessive lethality and SUMO2/3 accumulate in the DSB-associated chromatin associated with the null allele. We chose the Sox2-Cre areas.14,15 Interestingly, these studies uncovered a more transgenic line as it promotes high-efficiency recombination in direct function of SUMO in facilitating DSB-induced histone all epiblast-derived tissues.23 A PCR-based genotyping ubiquitylation upstream of 53BP1 and BRCA1 recruitment. strategy confirmed efficient Cre-mediated excision of the SA However, the molecular mechanisms that govern the relation- site; the non-recombined allele could not be detected by PCR ship between SUMOylation and ubiquitylation remain unknown. in E9.5 Cre-positive homozygous embryos for the trap allele RNF4 is currently the only known mammalian member of (Figure 1b). Viable mice heterozygous for the converted, the SUMO-targeted ubiquitin ligase family.16 Given that these putative hypomorphic allele (Rnf4hypo/ þ ) were obtained and proteins selectively ubiquitylate SUMOylated proteins they intercrossed. Importantly, viable Rnf4hypo/hypo mice were are well suited to have an important role in the cross talk found, but at a reduced Mendelian distribution at P4 between SUMO and ubiquitin pathways.17 RNF4 possesses (Supplementary Table S2). Both male and female Rnf4hypo/ four amino-terminal SUMO-interacting motifs (SIMs) and a hypo viable mice weighted B20% less than wild-type mice both C-terminal RING-finger domain that together facilitate the at P20 and P120 (Figure 1e). specific ubiquitylation of poly-SUMOylated substrates.18 The Timed harvests revealed that, although growth retarded, yeast and Drosophila orthologues of RNF4 have been several Rnf4hypo/hypoembryos are viable at E12.5 implicated in the maintenance of genome stability and DNA (Supplementary Table S2 and Figure 1c). In order to confirm repair.19,20 Because of the early embryonic lethality of Rnf4- hypomorphic expression of RNF4 from the Cre-recombined null mice, it has not been possible to study the role of Rnf4 in allele, we harvested E9.5 embryos of various
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