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Home , DNA repair, Homologous recombination

© 2020. Published by The Company of Biologists Ltd | Journal of Science (2020) 133, jcs233437. doi:10.1242/jcs.233437

RESEARCH ARTICLE Non-canonical regulation of DNA repair by the USP9X deubiquitylase Rachel O’Dea and Corrado Santocanale*

ABSTRACT and it is therefore defined as an error-prone pathway. HR In order to prevent the deleterious effects of genotoxic agents, cells relies on DNA synthesis, using a homologous DNA sequence as the have developed complex surveillance mechanisms and DNA repair template to repair the break and is therefore a high-fidelity method pathways that allow them to maintain integrity. The - of repair (Sancar et al., 2004). specific 9X (USP9X) contributes to genome stability during The HR repair pathway is a complex hierarchical pathway in which DNA replication and segregation. Depletion of USP9X numerous and post-translational modifications (PTMs) are leads to DNA double-strand breaks, some of which are triggered by critical. A key step in initiating HR repair after DSB recognition is the replication fork collapse. Here, we identify USP9X as a novel regulator recruitment of the E3 ligase BRCA1 and the CtIP (also of homologous recombination (HR) DNA repair in human cells. By known as RBBP8). This complex, together with additional performing cellular HR reporter, irradiation-induced focus formation resect the DNA DSB, revealing substantial single-stranded (ss)DNA and colony formation assays, we show that USP9X is required for committing the break to HR repair (Symington and Gautier, 2011). efficient HR. Mechanistically, we show USP9X is important to sustain The revealed ssDNA is coated initially with the ssDNA-binding RPA the expression levels of key HR factors, namely BRCA1 and RAD51 heterotrimer. At later stages in the pathway, RPA is replaced by the through a non-canonical regulation of their mRNA abundance. RAD51 (Krejci et al., 2012; Sugiyama et al., 1997). The Intriguingly, we find that the contribution of USP9X to BRCA1 and replacement of RPA with RAD51 is facilitated by BRCA1 through – RAD51 expression is independent of its known catalytic activity. Thus, recruitment of a PALB2 BRCA2 complex, which binds and loads this work identifies USP9X as a regulator of HR, demonstrates a novel RAD51 on to the ssDNA (Davies et al., 2017; Zhang et al., 2009). mechanism by which USP9X can regulate levels, and provides The RAD51 nucleofilament carries out the strand invasion and insights in to the regulation of BRCA1 and RAD51 mRNA. search required for DNA synthesis and the repair of the DSB (Shinohara et al., 1992). This article has an associated First Person interview with the first Protein ubiquitylation is one of the critical PTMs involved in HR. author of the paper. This PTM involves the addition of ubiquitin, an 8 kDa protein, to substrates via a three step enzymatic cascade consisting of a KEY WORDS: USP9X, DNA repair, BRCA1, RAD51, Deubiquitylation, ubiquitin activating (E1), a ubiquitin conjugating enzyme Homologous recombination (E2) and a enzyme (E3). Substrates can be modified with a single moiety (monoubiquitylation) or polyubiquitin chains INTRODUCTION formed through a linkage using one of the seven lysine residues Cells are constantly exposed to agents that can damage our DNA. (K48, K63, K11, K27, K33, K6 or K29) or the N-terminal These can be from internal sources, such as products of methionine (M1) present in ubiquitin (Komander and Rape, 2012). and errors in DNA duplication, or from external sources, such as The presence of ubiquitin can affect substrate localisation, function carcinogens and UV light (Jackson and Bartek, 2009). DNA and stability (Akutsu et al., 2016). damage left unresolved or inaccurately repaired results in genome Several types of ubiquitin modifications are required to accomplish instability, leading to mutations, chromosome breakages and HR repair of DSB. The canonical K48 degradation ubiquitin signals translocations, events that can promote tumorigenesis (Khanna have been shown to regulate the stability of key HR proteins CtIP, and Jackson, 2001). To counteract this constitutive DNA damage, BRCA1 and the E3 ligase RNF168 (Gudjonsson et al., 2012; cells have intricate mechanisms in place for surveillance and repair Lafranchi et al., 2014; Lu et al., 2012; Wu et al., 2010). In addition, of DNA damage. DNA double-strand breaks (DSBs) are the most the K63 ubiquitin chains placed on by RNF8 and RNF168 deleterious type of DNA damage and two main mechanisms exist have been shown to be critical in generating a recruitment platform to repair this type of lesion: non-homologous end joining (NHEJ) for various mediator proteins (Doil et al., 2009). Non-canonical roles and homologous recombination (HR). NHEJ is an homology- of ubiquitin in HR have also been identified. The E3 complex, independent method of repair which relies on , DNA-PK and CRL3–KEAP1 has been shown to ubiquitylate PALB2 blocking its LIG IV to carry out minimal processing and ligation of broken DNA interaction with BRCA1 negatively regulating HR (Orthwein et al., ends (Chiruvella et al., 2013). Such processes very often introduce 2015). In addition, monoubiquitylation of histones has been shown to indirectly facilitate remodelling enabling the recruitment of HR proteins (Nakamura et al., 2011). Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway H91W2TY, Ireland. Protein ubiquitylation is a dynamic process and can be either fine- tuned or reversed by counteracting deubiquitylases (DUBs). The *Author for correspondence ([email protected]) negative regulation of ubiquitylation by DUBs has been shown to be R.O., 0000-0003-0487-5392; C.S., 0000-0003-1337-5656 critical for proper HR progression, as well as termination of the response to the DSB. The DUBs OTUB2, POH1 (also known as

Received 18 April 2019; Accepted 30 December 2019 PSMD14) and BRCC36 (also known as BRCC3) have been shown Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs233437. doi:10.1242/jcs.233437 to directly antagonise RNF8 and RNF168 K63 chains their mRNA abundance. These findings offer novel insights into the (Butler et al., 2012; Kato et al., 2014; Shao et al., 2009), while regulation of BRCA1 and RAD51, demonstrate a novel role for USP3, USP16 and USP44 have been shown to remove histone USP9X in DNA repair, and potentially provide a novel mechanistic monoubiquitin moieties (Mosbech et al., 2013; Nicassio et al., explanation for the tumour-suppressive properties of USP9X. 2007; Shanbhag et al., 2010). This DUB activity is critical in preventing excessive modifications and removing the signal after RESULTS the break has been repaired. There is also evidence of non-canonical Loss of USP9X leads to accumulation of DSBs DUB functions in HR. OTUB1 has been shown to regulate HR One of the earliest events in the response to DNA DSBs is the independent of its catalytic activity, through inhibitory binding of of the histone variant H2AX (encoded by H2AFX) RNF168 E2 ligase, UBC13 (Nakada et al., 2010). on serine 139 on the chromatin surrounding the break. This Ubiquitin-specific protease 9X (USP9X) is a member of the USP modification, also known as γ-H2AX, is removed upon completion family of DUBs, a large family of DUBs that typically lack of DSB repair (Chowdhury et al., 2005), thus γ-H2AX is a widely specificity for particular UB chains types (Faesen et al., 2011). accepted as a marker of DSBs. In previous work, we have reported USP9X in a cellular context has been shown to have the ability to that in the human embryonic kidney derived HEK-293 cells, remove K48, K63, K33 and K29 ubiquitin chains as well as USP9X depletion leads to an increase the number of γ-H2AX- monoubiquitin (Al-Hakim et al., 2008; Dupont et al., 2009; Marx positive cells (McGarry et al., 2016). In order to extend these et al., 2010; Mouchantaf et al., 2006; Vong et al., 2005). observations, we used a different cell line and additional siRNA Predominantly by promoting K48 chain hydrolysis, USP9X has sequences. Osteosarcoma-derived U2OS cells were transfected with been implicated in a variety of different cellular events. Originally two different non-overlapping siRNAs [denoted siUSP9X (a) and discovered for its role in deciding cell fate decisions during siUSP9X (b)], and the number of γ-H2AX foci detectable after 48 h development in mice (Fischer-Vize et al., 1992), USP9X is now was quantified using high-throughput microscopy. Approximately known to regulate the levels of proteins implicated in a variety of 300 nuclei in three independent experiments were scored. We different cellular processes, including DNA replication, centrosome observed that the average number of γ-H2AX foci was increased biogenesis and signalling pathways (Dupont et al., 2009; Izrailit when USP9X was depleted with both of the siRNAs (Fig. 1A,B). et al., 2016; Li et al., 2017; McGarry et al., 2016; Wang et al., 2017; Decreased levels of USP9X upon siRNA were Wu et al., 2017). confirmed by western blot analysis (Fig. 1C), suggesting that the USP9X is deregulated in a number of different and has depletion of USP9X leads to the accumulation of DSB. been shown to act as both a tumour suppressor and an , The accumulation of DNA damage in USP9X-depleted depending on the type and stage of (Cox et al., 2014). proliferating cells may simply be due to the role of USP9X in Overexpression of USP9X has been identified in cervical, protecting claspin levels and replication forks from collapsing colorectal, kidney, breast, prostate, brain and sarcoma cancers during S-phase (McGarry et al., 2016); however, we showed that the (Murtaza et al., 2015). The oncogenic function of USP9X was overexpression of claspin in USP9X-depleted cells did not fully initially explained by its ability to stabilise the anti-apoptotic factor prevent the accumulation of DNA damage, suggesting there are MCL1. USP9X expression was shown to positively correlate with multiple sources of DNA damage in the absence of USP9X. Owing MCL1 in follicular , colon adenocarcinoma, gastric to the emerging role of USP9X in regulating (Miotto cancer and small cell lung (Fu et al., 2017b; Peddaboina et al., 2018; Nagai et al., 2009), we first hypothesised that USP9X et al., 2012; Schwickart et al., 2010; Yan et al., 2014). In breast depletion could disrupt cellular homeostasis leading to the cancer, USP9X has been shown to promote by accumulation of reactive oxygen (ROS); however, our inducing centrosome amplification and stimulating proliferation and experiments with the carboxy-H2DCFDA ROS assay suggested that through activation of Notch and TGFβ signalling pathways ROS homeostasis under these conditions was not grossly disrupted (Li et al., 2017; Nanayakkara et al., 2016; Wu et al., 2017). by USP9X depletion (Fig. S1). In contrast, USP9X has also been shown to behave as a tumour suppressor. In pancreatic cancer, low USP9X expression has been Loss of USP9X leads to impaired HR-mediated repair shown to correlate with poor survival and higher tumour burden of DNA DSBs (Pérez-Mancera et al., 2012). More recently, in an untransformed We then tested whether the DNA damage in USP9X-depleted cells breast cell line, the depletion of USP9X was shown to induce was caused by defects in DNA repair pathways. First, we specifically epithelial to mesenchymal transition characteristics and enhance investigated whether the loss of USP9X affected HR repair. To do invasiveness, suggesting that USP9X may have tumour-suppressive this we directly assessed HR efficiency using a DR-GFP U2OS properties in multiple cell types (Toloczko et al., 2017). In addition, reporter cell line. This cell line contains two differentially mutated loss of USP9X has been shown to disrupt normal DNA replication, GFP , one of which contains an I-SceI restriction site. and cause accumulation of the phosphorylated form of H2AX Upon with the I-SceI , a DSB is generated (γ-H2AX), a marker of DSBs, highlighting the importance of and, if HR repairs it using the downstream truncated , a USP9X in maintaining genome stability (McGarry et al., 2016). A functional gene expressing GFP is reconstituted. Thus, the percentage comprehensive understanding of the molecular mechanisms that of GFP-positive cells is indicative of HR competency (Pierce et al., facilitate the tumour-suppressive functions of USP9X and in what 1999). In this assay, the depletion of BRCA1 is used as a positive context it elicits these functions has not yet been achieved. control as this is a major player in this pathway. Cells were first In this work, we show that USP9X contributes to efficient DNA depleted of USP9X or BRCA1 and then transfected with expression repair. We find that depletion of USP9X decreases the ability of cells for I-SceI and the fluorescent protein cerulean, which was to carry out HR repair of DSB and sensitises cells to poly-ADP used as an indicator of transfection efficiency. The percentage of ribosylase (PARP, herein referring to PARP1 and PARP2) inhibition. GFP-positive cells in the transfected (cerulean positive) population Furthermore, we show that USP9X affects the functions and the was measured by 48 h later. In the cells transfected levels of RAD51 and BRCA1 through a non-canonical regulation of with a control siRNA, an average of 14.6% of the transfected cells Journal of Cell Science

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Fig. 1. Loss of USP9X leads to accumulation of γ-H2AX. (A) U2OS cells were transfected with a control siRNA and siRNAs targeting USP9X for 48 h, and cells were stained with an anti-γ-H2AX antibody. (B) Quantification of the number of γ-H2AX foci in individual nuclei. Data shown are a compilation of three biologically independent experiments, with the mean±s.e.m. highlighted. (C) Western blot analysis of whole-cell lysates. Total protein staining (TPS) of membrane is used as loading control. Scale bars: 20 µm. ****P<0.0001 (ordinary one-way ANOVA and Dunnett’s test). were GFP positive. The percentage of GFP-positive cells in BRCA1 after γ-irradiation (IR). Intriguingly the loss of USP9X resulted in a depleted cells was as expected, greatly reduced to 1.5%. The significant increase in the number of 53BP1 foci in non-irradiated as depletion of USP9X with siUSP9X (a) and siUSP9X (b) significantly well as in irradiated cells (Fig. 2D,E). In a similar setting, we reduced the percentage of GFP-positive cells to an intermediate level investigated whether USP9X affected the autophosphorylation of of 8.3% and 5.2%, respectively (Fig. 2A). Again, the efficiency of DNA-dependent protein (DNA-PK), an event which has USP9X depletion upon siRNA transection in these experiments was been shown to occur in response to IR and be important for the role confirmed by western blot analyses (Fig. S2A). These results suggest of DNA-PK in promoting end ligation in the later stages of NHEJ that USP9X loss results in defective HR. Cells deficient in HR repair repair (Jiang et al., 2015; Uematsu et al., 2007). To assess are characteristically sensitive to several chemotherapeutic agents autophosphorylation we used an antibody specific to DNA-PK that directly or indirectly target the DNA, among which are PARP phosphorylated at S2056 (p2056 DNA-PK). As previously inhibitors (Bryant et al., 2005; Farmer et al., 2005). To assess the reported, in wild-type (WT) cells, we observed an increase in the sensitivity of USP9X-depleted cells to PARP inhibition, U2OS cells nuclear staining of pS2056 DNA-PK in response to IR (Fig. 2F,G; were depleted of USP9X or BRCA1 and treated with increasing doses Fig. S2D). A similar increase was observed in USP9X-depleted of the PARP inhibitor . The percentage of cells that survived cells, suggesting that the responses leading to NHEJ repair were not after Olaparib treatment was then assessed by a colony formation obviously affected. assay. We observed that compared to the control, BRCA1 depletion Finally, in clonogenic assays we assessed the sensitivity of caused a strong reduction in the number of colonies formed at all USP9X-depleted cells to ICRF-193, a II inhibitor doses. The depletion of USP9X, although to a lesser extent, also which is particularly cytotoxic in NHEJ-deficient cells (Adachi caused a significant reduction in the percentage of surviving cells et al., 2003). Importantly, unlike what was seen upon DNA-PK after Olaparib treatment (Fig. 2B). USP9X depletion was confirmed inhibition, USP9X depletion not did not increase cell death but in by western blot analyses (Fig. S2B). The sensitivity of USP9X- fact partially rescued the deleterious effects of the drug (Fig. 2C; depleted cells to Olaparib is a further indication that HR is impaired in Fig. S2E). these cells. HR repair is, in general, limited to S and G2 phases of the cell Loss of USP9X impairs BRCA1 and RAD51 IR induced foci cycle. To ensure that the observed deficiency of HR in USP9X- formation depleted cells was not being caused by confounding changes to the To understand how the HR pathway is affected in the absence of the phase distribution was assessed by flow cytometry. No USP9X, we assessed the capability of several HR repair proteins to major changes to the cell cycle profile were observed in cells be recruited into foci at sites of DNA damage after IR. We first depleted of USP9X, suggesting the deficiency in HR was not due to looked at RAD51 foci formation in three independent experiments. changes in the cell cycle (Fig. S3). In cells transfected with the control siRNA, as expected, IR resulted We then investigated whether the NHEJ pathway was also in an increase in the average number of nuclear RAD51 foci. Cells affected. After DSB formation, multiple DNA repair proteins are depleted of USP9X with two distinct siRNAs had significantly less immediately recruited at the DSB including 53BP1 (also known as RAD51 foci formation after IR compared to the control (Fig. 3A,B). TP53BP1), which plays a major role in promoting NHEJ (Chapman The decrease in RAD51 foci formation suggested that the loss of et al., 2013; Zimmermann et al., 2013). We tested whether USP9X USP9X leads to HR being impaired at the stage of RAD51 depletion resulted in defective 53BP1 focal recruitment before or recruitment or before. Journal of Cell Science

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Fig. 2. See next page for legend.

We next assessed the formation of foci of BRCA1 and RPA2, the absence of IR also seemed to be reduced by the depletion which are key HR proteins acting upstream of RAD51. of USP9X. SimilartowhatwasseenforRAD51 foci, the depletion of The finding that RAD51 and BRCA1 foci were also partially USP9X also significantly reduced the average number BRCA1 reduced in the USP9X-depleted non-irradiated cells led us to and RPA2 foci after IR compared to the control (Fig. 3C–F). hypothesise that USP9X might be regulating the overall levels

Interestingly, the levels of BRCA1 and RAD51 foci present in of these proteins; thus western blot analysis was carried out on Journal of Cell Science

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Fig. 2. Loss of USP9X specifically decreases repair by HR and enhances distinct siRNAs decreased RAD51 and BRCA1 mRNA quantity by sensitivity to Olaparib. (A) DR-GFP U2OS cells were transfected with siRNAs ∼50% (Fig. 5C,D; Fig. S2C), which was not due to an increase in targeting USP9X, BRCA1 and a control siRNA. After 24 h, cells were transfected their rate of degradation (Fig. S5). with pCBASce and cerulean, and 48 h later the percentage of GFP-positive cells was assessed by flow cytometry. The percentage of GFP-positive cells was To expand these observations, we profiled the expression of 90 normalised to transfection efficiency, which was indicated by cerulean DNA repair in WT and USP9X-depleted cells with two fluorescence. Data shown is the mean±s.d. of three biologically independent different siRNAs. We found that both siRNAs caused mild changes experiments ****P<0.0001 (ordinary one-way ANOVA and Dunnett’s test). in the expression of several genes, with a good correlation between (B) Colony formation assay in which U2OS cells were transfected with the the two siRNAs. indicated siRNAs and treated with increasing doses of Olaparib for three days. With an arbitrary cut of >33% decrease caused by both siRNAs Graph depicts the number of colonies formed in three biologically independent experiments. (C) Colony formation assay in which U2OS cells transfected with we observed that only four genes were downregulated, which the indicated siRNAs were treated with increasing concentrations of ICRF-193 included BRCA1, RAD51 as well as the immune-proteosome for the duration of the assay. As a control, cells were treated with 10 µM of the subunit PSMB9, and the TREX1 nuclease. Intriguingly, we also DNA-PK inhibitor, NU 7026. Graph depicts the number of colonies formed in two found that USP9X depletion increased the expression of DNA biologically independent experiments. (D) U2OS cells transfected with siRNAs ligase IV by 2–3-fold, which is involved in NHEJ (Table S1). targeting USP9X for 48 h were subjected or not to IR (3 Gy), 1 h later cells were These results indicate that USP9X regulates BRCA1 and RAD51 stained with anti-53BP1 antibodies. (E) Quantification of the number of nuclear protein levels through regulation of their mRNA abundance. 53BP1 foci. Data shown is the compilation of three biologically independent experiments, with the mean±s.e.m. highlighted. ***P<0.001, ****P<0.0001 (ordinary one-way ANOVA and Sidak’s test). (F) U2OS cells transfected with USP9X regulation of BRCA1 and RAD51 is independent of its siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy); 20 min later catalytic activity cells were stained with anti pS2056 DNA-PK antibodies. (G) Quantification of the To gain molecular insights into how USP9X may be regulating the nuclear intensity (arb, arbitrary units) of pS2056 DNA-PK. Data shown is the mRNA abundance of BRCA1 and RAD51, we investigated whether compilation of three biologically independent experiments, with the mean this regulation was dependent on the protease activity of USP9X. In ±s.e.m. highlighted. order to perform these experiments, we first generated a stable U2OS cell line conditionally expressing an shRNA targeting the whole-cell extracts. A decrease in both RAD51 and BRCA1 protein 3′UTR of endogenous USP9X mRNA. Induction of this shRNA levels was observed in cells depleted of USP9X in the presence and with doxycycline (dox), similar to the transfection with the siRNAs absence of IR (Fig. 4A,B). Quantification of three biologically targeting the USP9X coding regions, deregulated BRCA1 and independent experiments showed that, in U2OS cells, BRCA1 and RAD51 proteins impairing their focal recruitment at sites of RAD51 levels are decreased by ∼50% upon USP9X depletion spontaneous or IR-induced DNA damage (Fig. 6A–D). (Fig. S4A,B). USP9X depletion was also shown to decrease We then transfected these cells with expression plasmids for BRCA1 levels in MDA-MB-231 cells, a -derived cell either WT USP9X or USP9X containing a C1566A , line and MCF10A, a-non tumorigenic epithelial cell line (Fig. S4C). which abrogates its catalytic activity (Li et al., 2018). Under these Importantly, the loss of USP9X did not affect the levels of BARD1, experimental conditions we could rescue the overall levels of a BRCA1-stabilising binding partner, or RPA2 (Fig. 4C; Fig. S4D). USP9X with both constructs (Fig. 6A,B). Interestingly, BRCA1 and RAD51 protein levels were rescued by the expression of both the USP9X affects BRCA1 and RAD51 levels through regulation WT and C1566A USP9X. Most importantly the expression of both of mRNA abundance WT and the C1566A USP9X was sufficient to fully rescue USP9X has been shown to regulate the stability of numerous formation of RAD51 and BRCA1 foci with or without IR proteins by promoting their deubiquitylation and thus protecting (Fig. 6C,D; Fig. S6). them from proteasomal degradation. We therefore investigated The ability of the WT USP9X to rescue the BRCA1 and RAD51 whether USP9X regulates BRCA1 and RAD51 in a similar manner protein levels and formation of foci demonstrates that this by assessing their levels in USP9X-depleted cells treated with phenotype is a direct result of the loss of USP9X and not an off MG132, a potent proteasome inhibitor (Lee and Goldberg, 1996; target effect of RNAi. Furthermore, the rescue of this phenotype by Rock et al., 1994). In USP9X-depleted cells, RAD51 protein levels a catalytically inactive USP9X expression suggests that the increased slightly with proteasomal inhibition but remained lower regulation of BRCA1 and RAD51 is not dependent on the than those observed in control cells (Fig. 5A). As previously catalytic activity of USP9X. reported proteasome inhibition resulted in the accumulation of a faster migrating BRCA1 band (Choudhury et al., 2004). This DISCUSSION accumulation was also observed in USP9X-depleted cells; however, USP9X has been shown to regulate genome stability, and low levels the overall levels of BRCA1 remained reduced (Fig. 5B). In addition of USP9X have been associated with enhanced DNA damage to proteosomal degradation, ubiquitylation can also drive proteins (McGarry et al., 2016). In an effort to elucidate the mechanisms by for lysosomal-mediated degradation (Hurley and Emr, 2006). In our which USP9X may be involved in preventing DNA damage, we experiments, the inhibition of lysosome degradation by chloroquine investigated the role of USP9X in DNA repair. In this work, we also failed to rescue BRCA1 protein levels (Fig. S4E). The provide evidence that the depletion of USP9X impairs HR-mediated persisting reduction in BRCA1 and RAD51 proteins indicated that repair of DSB; such impairment occurs at very early stages in the USP9X was not regulating their levels by influencing their process, most likely affecting ssDNA generation and pathway degradation. To investigate whether USP9X was regulating choice as IR-induced RPA2 recruitment to foci is impaired in BRCA1 or RAD51 at the transcriptional level, real-time USP9X-depleted cells. We find that USP9X is required to sustain quantitative PCR (RT-qPCR) was performed to measure their the expression of the HR proteins BRCA1 and RAD51. The mRNA abundance in USP9X-depleted cells. The mRNA from three importance of USP9X is likely due to this regulation; however, independent experiments was quantified. Analysis of the mRNA additional roles for USP9X in facilitating this process cannot be relative quantity demonstrated that the depletion of USP9X with excluded. Journal of Cell Science

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Fig. 3. Loss of USP9X decreases RAD51, BRCA1 and RPA2 IR induced focus formation. (A) U2OS cells transfected with siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy); 4 h later cells were stained with anti-RAD51 antibodies. (B) Quantification of the number of nuclear RAD51 foci. Data shown is the compilation of three biologically independent experiments, with the mean±s.e.m. highlighted. (C) U2OS cells transfected with siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy); 4 h later cells were stained with anti-BRCA1 antibodies. (D) Quantification of the number of nuclear BRCA1 foci. Data shown is the compilation of three biologically independent experiments, with the mean±s.e.m. highlighted. (E) U2OS cells transfected with siRNAs targeting USP9X for 48 h were treated with IR (3 Gy), 1 h later cells were stained with anti-RPA2 antibodies. (F) Quantification of the number of nuclear RPA2 foci. Data shown is the compilation of 200 cells in each of two biologically independent experiments, with the mean±s.e.m. highlighted. Scale bars: 20 µm. **P<0.01, ****P<0.0001 (ordinary one-way ANOVA and Sidak’s test).

Our observation that USP9X is affecting BRCA1 and RAD51 substrates through traditional regulation of their proteasomal protein level by regulating mRNA abundance is somewhat degradation. Furthermore, while this manuscript was in revision, surprising as USP9X typically regulates the protein levels of it has been reported that USP9X may regulate BRCA1 protein, but Journal of Cell Science

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Fig. 4. Loss of USP9X results in a decrease in BRCA1 and RAD51 protein levels. (A,B) Western blot analyses of RAD51 (A) and BRCA1 (B) in whole-cell lysates from USP9X-depleted cells 4 h post IR (3 Gy). (C) Western blot analyses of whole-cell lysates in USP9X-depleted cells, assessing the levels of BARD1. TPS, total protein staining. not its RNA, through direct deubiquitylation and protection from of function mutations in USP9X may be particularly susceptible to degradation, a mechanism, which initially we investigated treatments such as Olaparib. Interestingly, although no link has thoroughly but which in our experiments did not appear to be previously been made between USP9X and DSB repair, USP9X relevant (Lu et al., 2019). This identification of USP9X as regulator disruption has been shown to sensitise cancer cells to the DNA- of mRNA abundance increases our understanding of the roles of damaging agents Fluorouracil, and (Fu et al., USP9X and elucidating the exact mechanism behind this regulation 2017a; Harris et al., 2012; Liu et al., 2015). will no doubt reveal further intriguing facets of this protein. This USP9X has been shown to have tumour-suppressive properties in finding also offers insight into the regulation of RAD51 and certain contexts. Here, we show that USP9X is required for BRCA1 mRNA, which is important, as BRCA1 and RAD51 maintaining BRCA1 and RAD51 protein levels. BRCA1 and mRNA levels are decreased in a number of breast and ovarian RAD51 are well-known tumour suppressors. In breast and ovarian cancers (Hedenfalk et al., 2003; Yang et al., 2001; Yoshikawa et al., cancers, mutations in the BRCA1 gene, as well decreased BRCA1 2000) and the mechanism behind this deregulation remains to be mRNA, are frequently observed (Rizzolo et al., 2011). RAD51 is fully understood. also often deregulated in cancer cells. In one study 30% of breast We demonstrate that USP9X regulates the levels of BRCA1 and carcinomas analysed were shown to have decreased levels of RAD51 independently of its catalytic activity. While USP9X has not RAD51 protein (Yoshikawa et al., 2000). It is likely that sustaining previously been shown to function in this manner, there are a small the levels of these HR factors is an additional mechanism by which number of DUBs that have been demonstrated to have functions USP9X can supress tumorigenesis. irrespective of their protease activity. As an example, the DUB In conclusion, we have identified USP9X as a novel regulator of OTUB1 has been shown to function in a catalytically independent BRCA1 and RAD51, and we have revealed a novel, protease- manner during HR by binding to the E2 enzyme UBC13 and thus independent mechanism by which USP9X can regulate protein preventing RNF168 ubiquitylation (Nakada et al., 2010). levels. Further investigations in other cancer cell lines and primary Further investigation is required to determine how USP9X samples are required to understand the implications of this regulates BRCA1 and RAD51 . However, it is regulation in cancer development and its potential for exploitation interesting to note that the association of transcription factors in treatments targeting DNA integrity. with the of both BRCA1 and RAD51 is regulated by the histone acetyl transferase CBP (Ogiwara and Kohno, 2012) and that MATERIALS AND METHODS USP9X has been described to interact with the Cell culture HDAC6 (Joshi et al., 2013). It is appealing to speculate that USP9X Osteosarcoma-derived U2OS cells were purchased from American Type could act in a manner similar to OTUB1 through physical Culture Collection at the beginning of the project and routinely tested for interaction with one or more proteins to regulate this pathway. contamination. DR-GFP U2OS cells were a gift from the group of Noel Cells that are deficient in DNA repair are particularly sensitive to Lowndes (Centre for Chromosome Biology, National University of Ireland DNA-damaging agents. This is a property that can be exploited Galway). Cells were cultured at 37°C and 5% CO2 in DMEM supplemented when treating tumours deficient in DNA repair pathways. Treatment with 1% penicillin-streptomycin and heat-inactivated 10% fetal bovine serum (Sigma-Aldrich). To generate an inducible USP9X shRNA cell line, with the DNA-damaging PARP inhibitor Olaparib has been shown shRNAs targeting the 3′ UTR and coding sequence of USP9X were cloned to specifically kill cells deficient in HR repair. Here, we demonstrate into pRSITEP-U6Tet-sh-Ef1-TetRep-2A-Puro, a lentiviral transfer that the depletion of USP9X decreases the long-term survival of (Cellecta). HEK-293 cells were used to package this vector into viral U2OS cells following treatment with Olaparib. The sensitivity of particles, which were then transduced into U2OS cells. Single clones were

USP9X-depleted cells to Olaparib suggests that tumours with loss isolated using puromycin selection. USP9X depletion was induced in these Journal of Cell Science

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Fig. 5. USP9X regulates the mRNA abundance of BRCA1 and RAD51. (A,B) Western blot analyses of whole cell lysates RAD51 (A) and BRCA1 (B) of USP9X-depleted cells treated with 10 μM MG132 for 3 h. (C,D) RT-qPCR was performed on cDNA derived from USP9X-depleted U2OS cells to determine the relative quantity (RQ) of RAD51 (C) and BRCA1 mRNA (D). 18 s RNA was used as endogenous control. Data shown is the mean±s.d. RQ of three biologically independent experiments. **P<0.01, ****P<0.0001 (ordinary one-way ANOVA and Dunnett’s test). cell lines by addition of Doxycycline (1 µg/ml) to cell culture medium, Scientific). Protein concentration was determined using Bradford reagent alleviating TET repression. (Sigma-Aldrich). Proteins were resolved by SDS-PAGE (6% or 10%) and transferred onto nitrocellulose membrane prior to overnight incubation siRNA and drugs at 4°C with primary antibodies and infrared-labelled secondary antibodies The following siRNA sequences were used in this study; siUSP9X (a) (5′- (see antibodies). For immunoblotting of BRCA1, overnight transfers (16 h AGAAATCGCTGGTATAAATTT-3′) (Schwickart et al., 2010), siUSP9X at 35 V) were performed. To ensure equal loading of protein between lanes, (b) (5′-AGTGTATAGTGTTTTGTAATA-3′), siUSP9X (c) (5′-GCAGU- the Revert™ Total Protein Stain (TPS) (Li-Cor Biosciences) was used. TPS GAGUGGCUGGAAGU-3′) (Dupont et al., 2009), siBRCA1 (5′-GGAA- and immunoreactive bands were visualised and quantified using Odyssey CCUGUCUCCACAAAG-3′) (Lou et al., 2003) and siControl (5′- Infrared Imaging Systems (Li-Cor Biosciences). GCAUAUCGUCGUAUACUAU-3′). All siRNAs were purchased from Sigma-Aldrich. Unless otherwise stated, cells were transfected with 100 nM Immunofluorescence microscopy siRNAs for 48 h using JetPrime transfection reagent (Polyplus) according to U2OS cells were grown on coverslips. Cells were fixed with 4% ’ the manufacturer s instructions. Depletion of protein was confirmed by paraformaldehyde for 10 min at room temperature. After three washes western blot analyses. Olaparib was purchased from MedChem express (cat. with PBS, cells were permeabilised with PBS with 0.1% Triton X-100 for no. HY-10162). ICRF-193 from Enzo lifesciences (cat. no. GR-332) and 2 min at room temperature. The coverslips were rinsed three times with PBS NU7026 from Tocris (cat. no. 2828). and blocked for 1 h in 1% BSA in PBS at 37°C. After blocking, the coverslips were sequentially incubated with the indicated primary and Protein manipulation secondary antibody (see antibodies) for 1 h at 37°C. Coverslips were then Whole-cell extracts from cells were prepared in buffer A (50 mM Tris-HCl mounted using SlowFade (Thermo Fisher Scientific) and cells were imaged pH 7.4, 300 mM NaCl, 1 mM EDTA and 1% Triton X-100) containing and quantified using the high-throughput operetta imaging system (Perkin- protease and phosphatase inhibitors (Sigma-Aldrich), the DUB inhibitor Elmer). The operetta analysis programme quantified nuclear foci using the

N-ethylmaleimide (Sigma-Aldrich) and universal nuclease (Thermo Fisher following analysis sequence: (1) identify nuclei based on DAPI intensity, Journal of Cell Science

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Fig. 6. USP9X regulates BRCA1 and RAD51 independently of its catalytic activity. U2OS cells containing a dox-inducible shRNA targeting USP9X were treated with dox for 24 h and then transfected with HA–USP9X-WT and HA–USP9X-C1566A expression plasmids. After 44 h, cells were subjected or not to IR (3 Gy) and harvested 4 h later. (A,B) Western blot analyses of whole-cell lysate for BRCA1 (A) and RAD51 (B). (C,D) Quantification of the number of BRCA1 (C) and RAD51 (D) nuclear foci in individual cells. Data shown a compilation of three biologically independent experiments, with the mean±s.e.m. highlighted. *P<0.05**P<0.01, ***P<0.001, ****P<0.0001 (ordinary one-way ANOVA and Sidak’s test).

(2) exclude border cells, (3) exclude doublet cells, (4) detect foci based on PBS. Cells were then resuspended in DAPI (1 µg/ml) in 1% BSA in PBS to signal to background ratio, (5) output foci per nucleus counts. stain the DNA. Data was acquired on a BD FACS Canto II and analysed using FlowJo vX software. Flow cytometry To label nascent DNA, cells were incubated with EdU (10 μM) for 30 min Colony formation assay prior to harvest. Cells were fixed overnight (70% ethanol in PBS) and Following transfection with siControl, siUSP9X (a), (b) and (c) or siBRCA1 stained for EdU and DAPI analysis. Incorporated EdU was labelled with for 48 h, cells were trypsinised, counted and replated into Olaparib- CLICK chemistry [10 μM 6-carboxyfluorescine-TEG-azide, 10 mM containing medium for 3 days. The medium was replaced and, 10 days later, sodium-L-ascorbate, 2 mM copper (II) sulphate] for 30 min. Cells were colonies of surviving cells were fixed with methanol, stained with Crystal washed twice, once in 1% BSA PBST (PBS with 5% Tween-20) and once in Violet (Sigma-Aldrich) and colonies greater than >50 cells were counted. Journal of Cell Science

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The surviving fraction was calculated by normalising to plating efficiency. Supplementary information Statistical analysis of three independent experiments was performed using Supplementary information available online at Prism (GraphPad Software). http://jcs.biologists.org/lookup/doi/10.1242/jcs.233437.supplemental

References DR-GFP HR assay Adachi, N., Suzuki, H., Iiizumi, S. and Koyama, H. (2003). Hypersensitivity of To measure HR competency, DR-GFP U2OS cells were transfected with nonhomologous DNA end-joining to VP-16 and ICRF-193: implications siControl, siUSP9X (a), (b) and (c) or siBRCA1 24 h after plating. The for the repair of topoisomerase II-mediated DNA damage. J. Biol. Chem. 278, following day cells were transfected with pCBASceI (Addgene plasmid 35897-35902. doi:10.1074/jbc.M306500200 #26477) and cerulean-n1 (Addgene plasmid #54742). After 48 h the number Akutsu, M., Dikic, I. and Bremm, A. (2016). Ubiquitin chain diversity at a glance. J. Cell Sci. 129, 875-880. doi:10.1242/jcs.183954 of GFP-positive cells was assessed and normalised to the transfection Al-Hakim, A. K., Zagorska, A., Chapman, L., Deak, M., Peggie, M. and Alessi, efficiency indicated by cerulean positivity. Data was acquired on a BD FACS D. R. (2008). 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The secondary antibodies used were obtained molcel.2005.10.003 from Li-COR Biosciences (800CW anti-rabbit-IgG, cat. no. 926-32211, Cox, J. L., Wilder, P. J., Wuebben, E. L., Ouellette, M. M., Hollingsworth, M. A. 800CW anti-mouse-IgG, cat. no. 926-32210). For immunofluorescence, the and Rizzino, A. (2014). Context-dependent function of the deubiquitinating following primary antibodies were used: BRCA1 (1:500, Santa Cruz enzyme USP9X in pancreatic ductal adenocarcinoma. Cancer Biol. Ther. 15, Biotechnology, cat. no. sc6954), RAD51 (1:1000, Calbiochem, cat. no. 1042-1052. doi:10.4161/cbt.29182 PC130), 53BP1 (1:200, Novus Biologicals, cat. no. NB100-304) and γ-H2AX Davies, A. A., Masson, J.-Y., McIlwraith, M. J., Stasiak, A. Z., Stasiak, A., (1:500, Merck, cat. no. 05-636), pS2056 DNA-PK (1:200, Abcam, cat. no. Venkitaraman, A. R. and West, S. C. (2017). Role of BRCA2 in control of the ab18192). 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FAM/ The authors thank Sandra Healy, Aisling Quinlan and Mark McDermott for critical USP9x, a deubiquitinating enzyme essential for TGFβ signaling, controls Smad4 reading of manuscript, Michael Rainey and Janna Luessing for technical assistance monoubiquitination. Cell 136, 123-135. doi:10.1016/j.cell.2008.10.051 and critical reading of the manuscript, Noel Lowndes for the kind gift of the DR-GFP Faesen, A. C., Luna-Vargas, M. P. A., Geurink, P. P., Clerici, M., Merkx, R., van U2OS cell line and all the members of the Santocanale laboratory for discussion and Dijk, W. J., Hameed, D. S., El Oualid, F., Ovaa, H. and Sixma, T. K. (2011). The support. The authors also acknowledge the facilities and technical assistance of differential modulation of USP activity by internal regulatory domains, interactors Enda O Connell, Shirley Hanley and the Flow Cytometry Core Facility at NUI Galway and eight ubiquitin chain types. Chem. Biol. 18, 1550-1561. doi:10.1016/j. that is funded by NUI Galway, Science Foundation Ireland, the Irish Government’s chembiol.2011.10.017 Programme for Research in Third Level Institutions, Cycle 5 and the European Farmer, H., McCabe, N., Lord, C. J., Tutt, A. N. J., Johnson, D. A., Richardson, Regional Development Fund. T. B., Santarosa, M., Dillon, K. J., Hickson, I., Knights, C. et al. (2005). Targeting the DNA repair defect in BRCA cells as a therapeutic strategy. Competing interests Nature 434, 917-921. doi:10.1038/nature03445 The authors declare no competing or financial interests. Fischer-Vize, J. A., Rubin, G. M. and Lehmann, R. (1992). The fat facets gene is required for eye and embryo development. Development 116, 985-1000. Author contributions Fu, P., Du, F., Liu, Y., Yao, M., Zhang, S., Zheng, X. and Zheng, S. (2017a). Conceptualization: R.O., C.S.; Methodology: R.O., C.S.; Validation: R.O.; Formal WP1130 increases cisplatin sensitivity through inhibition of usp9x in analysis: R.O.; Investigation: R.O.; Data curation: R.O.; Writing - original draft: R.O.; receptor-negative breast cancer cells. Am. J. Transl. Res. 9, 1783-1791. Writing - review & editing: C.S.; Visualization: R.O.; Supervision: C.S.; Project Fu, X., Xie, W., Song, X., Wu, K., Xiao, L., Liu, Y. and Zhang, L. (2017b). Aberrant administration: C.S.; Funding acquisition: R.O., C.S. expression of deubiquitylating enzyme USP9X predicts poor prognosis in gastric cancer. Clin. Res. Hepatol. Gastroenterol. 41, 687-692. doi:10.1016/j.clinre.2017. Funding 01.008 The Santocanale lab is mostly funded by the Science Foundation Ireland (SFI) with Gudjonsson, T., Altmeyer, M., Savic, V., Toledo, L., Dinant, C., Grøfte, M., grant 16/IA/4476. R.O. was funded by a National University of Ireland, Galway Bartkova, J., Poulsen, M., Oka, Y., Bekker-Jensen, S. et al. (2012). TRIP12 and College of Science Fellowship and received support from NUI Galway Thomas UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

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