RNF4, a SUMO-Targeted Ubiquitin E3 Ligase, Promotes DNA Double-Strand Break Repair
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Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair Yaron Galanty, Rimma Belotserkovskaya, Julia Coates, and Stephen P. Jackson1 The Gurdon Institute, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (gH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and gH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems. [Keywords: RNF4; SUMO; ubiquitin–proteasome system (UPS); DNA double-strand breaks (DSBs); RPA; RAD51] Supplemental material is available for this article. Received January 26, 2012; revised version accepted April 30, 2012. DNA double-strand breaks (DSBs) are generated by ion- is normally restricted to S and G2 cells (Hartlerode and izing radiation (IR) and various DNA-damaging chem- Scully 2009; Pardo et al. 2009). HR but not NHEJ relies on icals. If they are not repaired or are repaired incorrectly, the presence of a sister chromatid and also requires cell DSBs result in cell death or genomic instability that can cycle-regulated 59-to-39 exonucleolytic processing (resec- lead to immune deficiencies, neurodegeneration, prema- tion) of DNA ends that generates stretches of ssDNA. ture aging, and cancer (Jackson and Bartek 2009; Ciccia This ssDNA is bound by replication protein A (RPA), and Elledge 2010). DSB formation triggers activation of which is then replaced by RAD51 to produce a RAD51- the DNA damage response (DDR) protein kinases ATM, ssDNA nucleoprotein filament, promoting DNA strand ATR, and DNA-PK, which phosphorylate many protein invasion and subsequent HR events. A key protein player targets, including the histone variant H2AX (H2AFX). in the essential step of replacing RPA with RAD51 is Phosphorylated H2AX (gH2AX) then mediates the accu- BRCA2, which directly binds RAD51 and promotes mulation of DDR proteins such as MDC1 (NFBD1), 53BP1 RAD51 loading onto RPA-coated ssDNA (Liu et al. (TP53BP1), and BRCA1 into IR-induced foci (IRIF) that are 2010; Thorslund et al. 2010; Holloman 2011). Notably, thought to amplify DSB signaling and promote DSB repair RPA-coated ssDNA also leads to recruitment and activa- (Downs et al. 2007; Bekker-Jensen and Mailand 2010; tion of the checkpoint kinase ATR, which phosphorylates Ciccia and Elledge 2010; Polo and Jackson 2011). various targets, including the downstream checkpoint DSBs are repaired by two principal pathways: non- kinase CHK1 (Cortez et al. 2001; Zou and Elledge 2003; homologous end-joining (NHEJ) that is active throughout Cimprich and Cortez 2008). Recent work has revealed the cell cycle, and homologous recombination (HR) that that resection is promoted by various factors, including human CtIP (RBBP8) (Sartori et al. 2007), and has in- dicated how resection is subject to cell cycle control 1Corresponding author. (Sartori et al. 2007; Gravel et al. 2008; Nimonkar et al. Email [email protected]. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad. 2008, 2011; Huertas 2010). However, it remains to be 188284.112. determined specifically how the activities of the various GENES & DEVELOPMENT 26:1179–1195 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org 1179 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Galanty et al. HR-promoting factors are controlled and precisely how 2007; Sun et al. 2007). These proteins act as SUMO- other HR events, such as the transition from RPA-ssDNA targeted ubiquitin E3 ligases (STUbLs), which recognize to RAD51-ssDNA, are mediated. sumoylated or SUMO-like domain-containing protein The reversible, covalent attachment of ubiquitin and targets. For example, sumoylation of the acute promye- small ubiquitin-like modifier (SUMO) proteins to DDR locytic leukemia (PML) protein triggers its recognition factors is critical for effective DSB repair and signaling in by RNF4, which then mediates PML ubiquitylation and eukaryotic cells (Bergink and Jentsch 2009; Al-Hakim proteasome-dependent PML degradation (Lallemand- et al. 2010; Bekker-Jensen and Mailand 2010; Morris Breitenbach et al. 2008; Tatham et al. 2008; Weisshaar 2010a,b). For example, ubiquitin conjugates and ubiquitin- et al. 2008). Since various DDR components are sumoylated conjugating enzymes such as UBC13 (UBE2N), BRCA1, and because RNF4 counterparts in yeast are implicated RNF8, HERC2, RNF168, RAD18, and the Fanconi ane- in suppressing HR via an as-yet-undefined mechanism mia (FA) protein complex accumulate at DSB sites in (Burgess et al. 2007; Kosoy et al. 2007; Prudden et al. mammalian cells, and defects in these factors result in 2007), we investigated whether human RNF4 functions impaired DSB repair and signaling associated with hy- in the DDR. Indeed, as described below, we show that persensitivity toward DNA-damaging agents (Huen et al. RNF4 accumulates at DSBs, regulates protein turnover/ 2007; Kolas et al. 2007; Mailand et al. 2007; Wang and exchange at these sites, and is required for effective DSB Elledge 2007; Zhao et al. 2007; Bekker-Jensen et al. 2009; repair. Doil et al. 2009; Huang et al. 2009; Stewart et al. 2009; Watanabe et al. 2009; Kee and D’Andrea 2010; Yang et al. 2010). While the ubiquitin–proteasome system (UPS) Results was originally described as the main protein turnover/ SUMO-interacting motif (SIM)-dependent degradation machinery of eukaryotic cells, it is now accumulation of RNF4 at DSB sites evident that it is also a major nondegradative regulator of various cellular processes, including the DDR (Kirkin To investigate potential DDR functions for RNF4, we and Dikic 2007; Bergink and Jentsch 2009; Motegi et al. first determined whether it accumulates at DNA damage 2009; Al-Hakim et al. 2010). Furthermore, it has been sites. Although we could not detect RNF4 recruitment to shown that proteasomes accumulate on damaged chro- IRIF, by using both live-cell imaging and fixed-cell stain- matin, suggesting that they may promote DDR signaling ing, we found that endogenous RNF4 (Fig. 1A,B) and as well as DDR-dependent protein turnover and HR recombinant RNF4 (Fig. 1C,D; Supplemental Fig. S1A,B) (Ustrell et al. 2002; Blickwedehl et al. 2007; Jacquemont accumulated at DNA damage sites induced by laser and Taniguchi 2007; Murakawa et al. 2007; Shi et al. micro-irradiation. RNF4 accrual was weakly detectable 2008; Motegi et al. 2009; Al-Hakim et al. 2010; Ben- within 15 min after irradiation (data not shown) but Aroya et al. 2010; Levy-Barda et al. 2011). The kinetics became readily apparent after 30 min (Fig. 1D) and then and mechanisms governing proteasome accumulation persisted for up to several hours (note that Fig. 1A–C at DNA damage regions and the functions and targets displays cells 2 h after laser micro-irradiation; Supple- of proteasomes at such sites, however, remain largely mental Fig. S1B; data not shown). This persistence of obscure. RNF4 recruitment within laser-micro-irradiated regions It was recently established that modification of the suggested that RNF4 might function at DSBs, which in largest RPA subunit (RPA1) by SUMO2/3 promotes the many cases take several hours to repair. Consistent with interaction of RPA with RAD51 and HR-mediated DSB this idea, RNF4 depletion by either of two distinct repair (Dou et al. 2010). Furthermore, we and others siRNAs enhanced the persistence of SUMO proteins have established that SUMO conjugates and the SUMO- and the well-defined DSB marker gH2AX at micro- conjugating enzymes UBC9 (UBE2I), PIAS1 (protein inhib- irradiated sites (Fig. 1A,B). Furthermore, effective RNF4 itor of activated STAT 1), and PIAS4 (PIASy) accumulate accrual at DNA damage sites required the DSB-responsive at DSB sites and are required for effective DSB signaling proteins MDC1, RNF8, 53BP1, and BRCA1 (Fig. 1C; Sup- and repair (Galanty et al. 2009; Morris et al. 2009). This plemental Fig. S1A, numbers represent the proportion of work also revealed that PIAS1- and PIAS4-mediated cells displaying gH2AX recruitment that also displayed sumoylation promotes accumulation of 53BP1 and ef- RNF4 recruitment), with RNF4 recruitment being most fective RNF8-, RNF168-, and BRCA1-dependent accu- markedly diminished when several of these factors were mulation of ubiquitin conjugates at DSB sites and that codepleted (see Supplemental Fig. S1A for effects of PIAS1/4-mediated sumoylation directly promotes BRCA1 individual factor depletions). These data therefore sug- E3 ubiquitin ligase activity (Galanty et al. 2009; Morris gested that RNF4 might be targeted to DNA damage et al. 2009). Thus, rather than operating separately, DDR– sites, most likely DSBs, by interactions with multiple protein sumoylation and ubiquitylation are intimately DDR proteins.