In order to prevent the deleterious effects of genotoxic agents, cells have developed complex surveillance mechanisms and DNA repair pathways that allow them to maintain genome integrity. The ubiquitin-specific protease 9X (USP9X) contributes to genome stability during DNA replication and chromosome segregation. Depletion of USP9X leads to DNA double-strand breaks, some of which are triggered by replication fork collapse. Here, we identify USP9X as a novel regulator of homologous recombination (HR) DNA repair in human cells. By performing cellular HR reporter, irradiation-induced focus formation and colony formation assays, we show that USP9X is required for efficient HR. Mechanistically, we show USP9X is important to sustain the expression levels of key HR factors, namely BRCA1 and RAD51 through a non-canonical regulation of their mRNA abundance. Intriguingly, we find that the contribution of USP9X to BRCA1 and RAD51 expression is independent of its known catalytic activity. Thus, this work identifies USP9X as a regulator of HR, demonstrates a novel mechanism by which USP9X can regulate protein levels, and provides insights in to the regulation of BRCA1 and RAD51 mRNA.

This article has an associated First Person interview with the first author of the paper.

Cells are constantly exposed to agents that can damage our DNA. These can be from internal sources, such as products of metabolism and errors in DNA duplication, or from external sources, such as carcinogens and UV light (Jackson and Bartek, 2009). DNA damage left unresolved or inaccurately repaired results in genome instability, leading to mutations, chromosome breakages and translocations, events that can promote tumorigenesis (Khanna and Jackson, 2001). To counteract this constitutive DNA damage, cells have intricate mechanisms in place for surveillance and repair of DNA damage. DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and two main mechanisms exist to repair this type of lesion: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an homology- independent method of repair which relies on KU, DNA-PK and LIG IV to carry out minimal processing and ligation of broken DNA ends (Chiruvella et al., 2013). Such processes very often introduce mutations and it is therefore defined as an error-prone pathway. HR relies on DNA synthesis, using a homologous DNA sequence as the template to repair the break and is therefore a high-fidelity method of repair (Sancar et al., 2004).

The HR repair pathway is a complex hierarchical pathway in which numerous proteins and post-translational modifications (PTMs) are critical. A key step in initiating HR repair after DSB recognition is the recruitment of the E3 ligase BRCA1 and the nuclease CtIP (also known as RBBP8). This complex, together with additional nucleases resect the DNA DSB, revealing substantial single-stranded (ss)DNA committing the break to HR repair (Symington and Gautier, 2011). The revealed ssDNA is coated initially with the ssDNA-binding RPA heterotrimer. At later stages in the pathway, RPA is replaced by the recombinase RAD51 (Krejci et al., 2012; Sugiyama et al., 1997). The replacement of RPA with RAD51 is facilitated by BRCA1 through recruitment of a PALB2–BRCA2 complex, which binds and loads RAD51 on to the ssDNA (Davies et al., 2017; Zhang et al., 2009). The RAD51 nucleofilament carries out the strand invasion and homology search required for DNA synthesis and the repair of the DSB (Shinohara et al., 1992).

Protein ubiquitylation is one of the critical PTMs involved in HR. This PTM involves the addition of ubiquitin, an 8 kDa protein, to substrates via a three step enzymatic cascade consisting of a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase enzyme (E3). Substrates can be modified with a single moiety (monoubiquitylation) or polyubiquitin chains formed through a linkage using one of the seven lysine residues (K48, K63, K11, K27, K33, K6 or K29) or the N-terminal methionine (M1) present in ubiquitin (Komander and Rape, 2012). The presence of ubiquitin can affect substrate localisation, function and stability (Akutsu et al., 2016).

Several types of ubiquitin modifications are required to accomplish HR repair of DSB. The canonical K48 degradation ubiquitin signals have been shown to regulate the stability of key HR proteins CtIP, BRCA1 and the E3 ligase RNF168 (Gudjonsson et al., 2012; Lafranchi et al., 2014; Lu et al., 2012; Wu et al., 2010). In addition, the K63 ubiquitin chains placed on histones by RNF8 and RNF168 have been shown to be critical in generating a recruitment platform for various mediator proteins (Doil et al., 2009). Non-canonical roles of ubiquitin in HR have also been identified. The E3 complex, CRL3–KEAP1 has been shown to ubiquitylate PALB2 blocking its interaction with BRCA1 negatively regulating HR (Orthwein et al., 2015). In addition, monoubiquitylation of histones has been shown to indirectly facilitate chromatin remodelling enabling the recruitment of HR proteins (Nakamura et al., 2011).

Protein ubiquitylation is a dynamic process and can be either fine-tuned or reversed by counteracting deubiquitylases (DUBs). The negative regulation of ubiquitylation by DUBs has been shown to be critical for proper HR progression, as well as termination of the response to the DSB. The DUBs OTUB2, POH1 (also known as PSMD14) and BRCC36 (also known as BRCC3) have been shown to directly antagonise histone RNF8 and RNF168 K63 chains (Butler et al., 2012; Kato et al., 2014; Shao et al., 2009), while USP3, USP16 and USP44 have been shown to remove histone monoubiquitin moieties (Mosbech et al., 2013; Nicassio et al., 2007; Shanbhag et al., 2010). This DUB activity is critical in preventing excessive modifications and removing the signal after the break has been repaired. There is also evidence of non-canonical DUB functions in HR. OTUB1 has been shown to regulate HR independent of its catalytic activity, through inhibitory binding of RNF168 E2 ligase, UBC13 (Nakada et al., 2010).

Ubiquitin-specific protease 9X (USP9X) is a member of the USP family of DUBs, a large family of DUBs that typically lack specificity for particular UB chains types (Faesen et al., 2011). USP9X in a cellular context has been shown to have the ability to remove K48, K63, K33 and K29 ubiquitin chains as well as monoubiquitin (Al-Hakim et al., 2008; Dupont et al., 2009; Marx et al., 2010; Mouchantaf et al., 2006; Vong et al., 2005). Predominantly by promoting K48 chain hydrolysis, USP9X has been implicated in a variety of different cellular events. Originally discovered for its role in deciding cell fate decisions during development in mice (Fischer-Vize et al., 1992), USP9X is now known to regulate the levels of proteins implicated in a variety of different cellular processes, including DNA replication, centrosome biogenesis and signalling pathways (Dupont et al., 2009; Izrailit et al., 2016; Li et al., 2017; McGarry et al., 2016; Wang et al., 2017; Wu et al., 2017).

USP9X is deregulated in a number of different cancers and has been shown to act as both a tumour suppressor and an oncogene, depending on the type and stage of cancer (Cox et al., 2014). Overexpression of USP9X has been identified in cervical, colorectal, kidney, breast, prostate, brain and sarcoma cancers (Murtaza et al., 2015). The oncogenic function of USP9X was initially explained by its ability to stabilise the anti-apoptotic factor MCL1. USP9X expression was shown to positively correlate with MCL1 in follicular lymphomas, colon adenocarcinoma, gastric cancer and small cell lung carcinomas (Fu et al., 2017b; Peddaboina et al., 2012; Schwickart et al., 2010; Yan et al., 2014). In breast cancer, USP9X has been shown to promote carcinogenesis by inducing centrosome amplification and stimulating proliferation and metastasis through activation of Notch and TGFβ signalling pathways (Li et al., 2017; Nanayakkara et al., 2016; Wu et al., 2017).

In contrast, USP9X has also been shown to behave as a tumour suppressor. In pancreatic cancer, low USP9X expression has been shown to correlate with poor survival and higher tumour burden (Pérez-Mancera et al., 2012). More recently, in an untransformed breast cell line, the depletion of USP9X was shown to induce epithelial to mesenchymal transition characteristics and enhance invasiveness, suggesting that USP9X may have tumour-suppressive properties in multiple cell types (Toloczko et al., 2017). In addition, loss of USP9X has been shown to disrupt normal DNA replication, and cause accumulation of the phosphorylated form of H2AX (γ-H2AX), a marker of DSBs, highlighting the importance of USP9X in maintaining genome stability (McGarry et al., 2016). A comprehensive understanding of the molecular mechanisms that facilitate the tumour-suppressive functions of USP9X and in what context it elicits these functions has not yet been achieved.

In this work, we show that USP9X contributes to efficient DNA repair. We find that depletion of USP9X decreases the ability of cells to carry out HR repair of DSB and sensitises cells to poly-ADP ribosylase (PARP, herein referring to PARP1 and PARP2) inhibition. Furthermore, we show that USP9X affects the functions and the levels of RAD51 and BRCA1 through a non-canonical regulation of their mRNA abundance. These findings offer novel insights into the regulation of BRCA1 and RAD51, demonstrate a novel role for USP9X in DNA repair, and potentially provide a novel mechanistic explanation for the tumour-suppressive properties of USP9X.

Loss of USP9X leads to accumulation of DSBs

One of the earliest events in the response to DNA DSBs is the phosphorylation of the histone variant H2AX (encoded by H2AFX) on serine 139 on the chromatin surrounding the break. This modification, also known as γ-H2AX, is removed upon completion of DSB repair (Chowdhury et al., 2005), thus γ-H2AX is a widely accepted as a marker of DSBs. In previous work, we have reported that in the human embryonic kidney derived HEK-293 cells, USP9X depletion leads to an increase the number of γ-H2AX-positive cells (McGarry et al., 2016). In order to extend these observations, we used a different cell line and additional siRNA sequences. Osteosarcoma-derived U2OS cells were transfected with two different non-overlapping siRNAs [denoted siUSP9X (a) and siUSP9X (b)], and the number of γ-H2AX foci detectable after 48 h was quantified using high-throughput microscopy. Approximately 300 nuclei in three independent experiments were scored. We observed that the average number of γ-H2AX foci was increased when USP9X was depleted with both of the siRNAs (Fig. 1A,B). Decreased levels of USP9X upon siRNA transfections were confirmed by western blot analysis (Fig. 1C), suggesting that the depletion of USP9X leads to the accumulation of DSB.

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).

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).

The accumulation of DNA damage in USP9X-depleted proliferating cells may simply be due to the role of USP9X in protecting claspin levels and replication forks from collapsing during S-phase (McGarry et al., 2016); however, we showed that the overexpression of claspin in USP9X-depleted cells did not fully prevent the accumulation of DNA damage, suggesting there are multiple sources of DNA damage in the absence of USP9X. Owing to the emerging role of USP9X in regulating oxidative stress (Miotto et al., 2018; Nagai et al., 2009), we first hypothesised that USP9X depletion could disrupt cellular homeostasis leading to the accumulation of reactive oxygen species (ROS); however, our experiments with the carboxy-H2DCFDA ROS assay suggested that ROS homeostasis under these conditions was not grossly disrupted by USP9X depletion (Fig. S1).

Loss of USP9X leads to impaired HR-mediated repair of DNA DSBs

We then tested whether the DNA damage in USP9X-depleted cells was caused by defects in DNA repair pathways. First, we specifically investigated whether the loss of USP9X affected HR repair. To do this we directly assessed HR efficiency using a DR-GFP U2OS reporter cell line. This cell line contains two differentially mutated GFP transgenes, one of which contains an I-SceI restriction site. Upon transfection with the I-SceI endonuclease, a DSB is generated and, if HR repairs it using the downstream truncated gene, a functional gene expressing GFP is reconstituted. Thus, the percentage of GFP-positive cells is indicative of HR competency (Pierce et al., 1999). In this assay, the depletion of BRCA1 is used as a positive control as this is a major player in this pathway. Cells were first depleted of USP9X or BRCA1 and then transfected with expression plasmids for I-SceI and the fluorescent protein cerulean, which was used as an indicator of transfection efficiency. The percentage of GFP-positive cells in the transfected (cerulean positive) population was measured by flow cytometry 48 h later. In the cells transfected with a control siRNA, an average of 14.6% of the transfected cells were GFP positive. The percentage of GFP-positive cells in BRCA1 depleted cells was as expected, greatly reduced to 1.5%. The depletion of USP9X with siUSP9X (a) and siUSP9X (b) significantly reduced the percentage of GFP-positive cells to an intermediate level of 8.3% and 5.2%, respectively (Fig. 2A). Again, the efficiency of USP9X depletion upon siRNA transection in these experiments was confirmed by western blot analyses (Fig. S2A). These results suggest that USP9X loss results in defective HR. Cells deficient in HR repair are characteristically sensitive to several chemotherapeutic agents that directly or indirectly target the DNA, among which are PARP inhibitors (Bryant et al., 2005; Farmer et al., 2005). To assess the sensitivity of USP9X-depleted cells to PARP inhibition, U2OS cells were depleted of USP9X or BRCA1 and treated with increasing doses of the PARP inhibitor Olaparib. The percentage of cells that survived after Olaparib treatment was then assessed by a colony formation assay. We observed that compared to the control, BRCA1 depletion caused a strong reduction in the number of colonies formed at all doses. The depletion of USP9X, although to a lesser extent, also caused a significant reduction in the percentage of surviving cells after Olaparib treatment (Fig. 2B). USP9X depletion was confirmed by western blot analyses (Fig. S2B). The sensitivity of USP9X-depleted cells to Olaparib is a further indication that HR is impaired in these cells.

Fig. 2.

Loss of USP9X specifically decreases repair by HR and enhances sensitivity to Olaparib. (A) DR-GFP U2OS cells were transfected with siRNAs targeting USP9X, BRCA1 and a control siRNA. After 24 h, cells were transfected 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 normalised to transfection efficiency, which was indicated by cerulean fluorescence. Data shown is the mean±s.d. of three biologically independent experiments ****P<0.0001 (ordinary one-way ANOVA and Dunnett's test). (B) Colony formation assay in which U2OS cells were transfected with the indicated siRNAs and treated with increasing doses of Olaparib for three days. Graph depicts the number of colonies formed in three biologically independent experiments. (C) Colony formation assay in which U2OS cells transfected with the indicated siRNAs were treated with increasing concentrations of ICRF-193 for the duration of the assay. As a control, cells were treated with 10 µM of the DNA-PK inhibitor, NU 7026. Graph depicts the number of colonies formed in two biologically independent experiments. (D) U2OS cells transfected with siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy), 1 h later cells were stained with anti-53BP1 antibodies. (E) Quantification of the number of nuclear 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 siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy); 20 min later cells were stained with anti pS2056 DNA-PK antibodies. (G) Quantification of the nuclear intensity (arb, arbitrary units) of pS2056 DNA-PK. Data shown is the compilation of three biologically independent experiments, with the mean±s.e.m. highlighted.

Fig. 2.

Loss of USP9X specifically decreases repair by HR and enhances sensitivity to Olaparib. (A) DR-GFP U2OS cells were transfected with siRNAs targeting USP9X, BRCA1 and a control siRNA. After 24 h, cells were transfected 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 normalised to transfection efficiency, which was indicated by cerulean fluorescence. Data shown is the mean±s.d. of three biologically independent experiments ****P<0.0001 (ordinary one-way ANOVA and Dunnett's test). (B) Colony formation assay in which U2OS cells were transfected with the indicated siRNAs and treated with increasing doses of Olaparib for three days. Graph depicts the number of colonies formed in three biologically independent experiments. (C) Colony formation assay in which U2OS cells transfected with the indicated siRNAs were treated with increasing concentrations of ICRF-193 for the duration of the assay. As a control, cells were treated with 10 µM of the DNA-PK inhibitor, NU 7026. Graph depicts the number of colonies formed in two biologically independent experiments. (D) U2OS cells transfected with siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy), 1 h later cells were stained with anti-53BP1 antibodies. (E) Quantification of the number of nuclear 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 siRNAs targeting USP9X for 48 h were subjected or not to IR (3 Gy); 20 min later cells were stained with anti pS2056 DNA-PK antibodies. (G) Quantification of the nuclear intensity (arb, arbitrary units) of pS2056 DNA-PK. Data shown is the compilation of three biologically independent experiments, with the mean±s.e.m. highlighted.

HR repair is, in general, limited to S and G2 phases of the cell cycle. To ensure that the observed deficiency of HR in USP9X-depleted cells was not being caused by confounding changes to the cell cycle the phase distribution was assessed by flow cytometry. No major changes to the cell cycle profile were observed in cells depleted of USP9X, suggesting the deficiency in HR was not due to changes in the cell cycle (Fig. S3).

We then investigated whether the NHEJ pathway was also affected. After DSB formation, multiple DNA repair proteins are immediately recruited at the DSB including 53BP1 (also known as TP53BP1), which plays a major role in promoting NHEJ (Chapman et al., 2013; Zimmermann et al., 2013). We tested whether USP9X depletion resulted in defective 53BP1 focal recruitment before or after γ-irradiation (IR). Intriguingly the loss of USP9X resulted in a significant increase in the number of 53BP1 foci in non-irradiated as well as in irradiated cells (Fig. 2D,E). In a similar setting, we investigated whether USP9X affected the autophosphorylation of DNA-dependent protein kinase (DNA-PK), an event which has been shown to occur in response to IR and be important for the role of DNA-PK in promoting end ligation in the later stages of NHEJ repair (Jiang et al., 2015; Uematsu et al., 2007). To assess autophosphorylation we used an antibody specific to DNA-PK phosphorylated at S2056 (p2056 DNA-PK). As previously reported, in wild-type (WT) cells, we observed an increase in the nuclear staining of pS2056 DNA-PK in response to IR (Fig. 2F,G; Fig. S2D). A similar increase was observed in USP9X-depleted cells, suggesting that the responses leading to NHEJ repair were not obviously affected.

Finally, in clonogenic assays we assessed the sensitivity of USP9X-depleted cells to ICRF-193, a topoisomerase II inhibitor which is particularly cytotoxic in NHEJ-deficient cells (Adachi et al., 2003). Importantly, unlike what was seen upon DNA-PK inhibition, USP9X depletion not did not increase cell death but in fact partially rescued the deleterious effects of the drug (Fig. 2C; Fig. S2E).

Loss of USP9X impairs BRCA1 and RAD51 IR induced foci formation

To understand how the HR pathway is affected in the absence of USP9X, we assessed the capability of several HR repair proteins to be recruited into foci at sites of DNA damage after IR. We first looked at RAD51 foci formation in three independent experiments. In cells transfected with the control siRNA, as expected, IR resulted in an increase in the average number of nuclear RAD51 foci. Cells depleted of USP9X with two distinct siRNAs had significantly less RAD51 foci formation after IR compared to the control (Fig. 3A,B). The decrease in RAD51 foci formation suggested that the loss of USP9X leads to HR being impaired at the stage of RAD51 recruitment or before.

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).

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).

We next assessed the formation of foci of BRCA1 and RPA2, which are key HR proteins acting upstream of RAD51. Similar to what was seen for RAD51 foci, the depletion of USP9X also significantly reduced the average number BRCA1 and RPA2 foci after IR compared to the control (Fig. 3C–F). Interestingly, the levels of BRCA1 and RAD51 foci present in the absence of IR also seemed to be reduced by the depletion of USP9X.

The finding that RAD51 and BRCA1 foci were also partially reduced in the USP9X-depleted non-irradiated cells led us to hypothesise that USP9X might be regulating the overall levels of these proteins; thus western blot analysis was carried out on whole-cell extracts. A decrease in both RAD51 and BRCA1 protein levels was observed in cells depleted of USP9X in the presence and absence of IR (Fig. 4A,B). Quantification of three biologically independent experiments showed that, in U2OS cells, BRCA1 and RAD51 levels are decreased by ∼50% upon USP9X depletion (Fig. S4A,B). USP9X depletion was also shown to decrease BRCA1 levels in MDA-MB-231 cells, a breast cancer-derived cell line and MCF10A, a-non tumorigenic epithelial cell line (Fig. S4C). Importantly, the loss of USP9X did not affect the levels of BARD1, a BRCA1-stabilising binding partner, or RPA2 (Fig. 4C; Fig. S4D).

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.

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.

USP9X affects BRCA1 and RAD51 levels through regulation of mRNA abundance

USP9X has been shown to regulate the stability of numerous proteins by promoting their deubiquitylation and thus protecting them from proteasomal degradation. We therefore investigated whether USP9X regulates BRCA1 and RAD51 in a similar manner by assessing their levels in USP9X-depleted cells treated with MG132, a potent proteasome inhibitor (Lee and Goldberg, 1996; Rock et al., 1994). In USP9X-depleted cells, RAD51 protein levels increased slightly with proteasomal inhibition but remained lower than those observed in control cells (Fig. 5A). As previously reported proteasome inhibition resulted in the accumulation of a faster migrating BRCA1 band (Choudhury et al., 2004). This accumulation was also observed in USP9X-depleted cells; however, the overall levels of BRCA1 remained reduced (Fig. 5B). In addition to proteosomal degradation, ubiquitylation can also drive proteins for lysosomal-mediated degradation (Hurley and Emr, 2006). In our experiments, the inhibition of lysosome degradation by chloroquine also failed to rescue BRCA1 protein levels (Fig. S4E). The persisting reduction in BRCA1 and RAD51 proteins indicated that USP9X was not regulating their levels by influencing their degradation. To investigate whether USP9X was regulating BRCA1 or RAD51 at the transcriptional level, real-time quantitative PCR (RT-qPCR) was performed to measure their mRNA abundance in USP9X-depleted cells. The mRNA from three independent experiments was quantified. Analysis of the mRNA relative quantity demonstrated that the depletion of USP9X with distinct siRNAs decreased RAD51 and BRCA1 mRNA quantity by ∼50% (Fig. 5C,D; Fig. S2C), which was not due to an increase in their rate of degradation (Fig. S5).

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).

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).

To expand these observations, we profiled the expression of 90 DNA repair genes in WT and USP9X-depleted cells with two different siRNAs. We found that both siRNAs caused mild changes in the expression of several genes, with a good correlation between the two siRNAs.

With an arbitrary cut of >33% decrease caused by both siRNAs we observed that only four genes were downregulated, which included BRCA1, RAD51 as well as the immune-proteosome subunit PSMB9, and the TREX1 nuclease. Intriguingly, we also found that USP9X depletion increased the expression of DNA ligase IV by 2–3-fold, which is involved in NHEJ (Table S1).

These results indicate that USP9X regulates BRCA1 and RAD51 protein levels through regulation of their mRNA abundance.

USP9X regulation of BRCA1 and RAD51 is independent of its catalytic activity

To gain molecular insights into how USP9X may be regulating the mRNA abundance of BRCA1 and RAD51, we investigated whether this regulation was dependent on the protease activity of USP9X. In order to perform these experiments, we first generated a stable U2OS cell line conditionally expressing an shRNA targeting the 3′UTR of endogenous USP9X mRNA. Induction of this shRNA with doxycycline (dox), similar to the transfection with the siRNAs targeting the USP9X coding regions, deregulated BRCA1 and RAD51 proteins impairing their focal recruitment at sites of spontaneous or IR-induced DNA damage (Fig. 6A–D).

Fig. 6.

USP9X regulates BRCA1 and RAD51 independentlyof 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).

Fig. 6.

USP9X regulates BRCA1 and RAD51 independentlyof 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).

We then transfected these cells with expression plasmids for either WT USP9X or USP9X containing a C1566A mutation, which abrogates its catalytic activity (Li et al., 2018). Under these experimental conditions we could rescue the overall levels of USP9X with both constructs (Fig. 6A,B). Interestingly, BRCA1 and RAD51 protein levels were rescued by the expression of both the WT and C1566A USP9X. Most importantly the expression of both WT and the C1566A USP9X was sufficient to fully rescue formation of RAD51 and BRCA1 foci with or without IR (Fig. 6C,D; Fig. S6).

The ability of the WT USP9X to rescue the BRCA1 and RAD51 protein levels and formation of foci demonstrates that this phenotype is a direct result of the loss of USP9X and not an off target effect of RNAi. Furthermore, the rescue of this phenotype by a catalytically inactive USP9X expression suggests that the regulation of BRCA1 and RAD51 is not dependent on the catalytic activity of USP9X.

USP9X has been shown to regulate genome stability, and low levels of USP9X have been associated with enhanced DNA damage (McGarry et al., 2016). In an effort to elucidate the mechanisms by which USP9X may be involved in preventing DNA damage, we investigated the role of USP9X in DNA repair. In this work, we provide evidence that the depletion of USP9X impairs HR-mediated repair of DSB; such impairment occurs at very early stages in the process, most likely affecting ssDNA generation and pathway choice as IR-induced RPA2 recruitment to foci is impaired in USP9X-depleted cells. We find that USP9X is required to sustain the expression of the HR proteins BRCA1 and RAD51. The importance of USP9X is likely due to this regulation; however, additional roles for USP9X in facilitating this process cannot be excluded.

Our observation that USP9X is affecting BRCA1 and RAD51 protein level by regulating mRNA abundance is somewhat surprising as USP9X typically regulates the protein levels of substrates through traditional regulation of their proteasomal degradation. Furthermore, while this manuscript was in revision, it has been reported that USP9X may regulate BRCA1 protein, but not its RNA, through direct deubiquitylation and protection from degradation, a mechanism, which initially we investigated thoroughly but which in our experiments did not appear to be relevant (Lu et al., 2019). This identification of USP9X as regulator of mRNA abundance increases our understanding of the roles of USP9X and elucidating the exact mechanism behind this regulation will no doubt reveal further intriguing facets of this protein. This finding also offers insight into the regulation of RAD51 and BRCA1 mRNA, which is important, as BRCA1 and RAD51 mRNA levels are decreased in a number of breast and ovarian cancers (Hedenfalk et al., 2003; Yang et al., 2001; Yoshikawa et al., 2000) and the mechanism behind this deregulation remains to be fully understood.

We demonstrate that USP9X regulates the levels of BRCA1 and RAD51 independently of its catalytic activity. While USP9X has not previously been shown to function in this manner, there are a small number of DUBs that have been demonstrated to have functions irrespective of their protease activity. As an example, the DUB OTUB1 has been shown to function in a catalytically independent manner during HR by binding to the E2 enzyme UBC13 and thus preventing RNF168 ubiquitylation (Nakada et al., 2010).

Further investigation is required to determine how USP9X regulates BRCA1 and RAD51 transcription. However, it is interesting to note that the association of E2F transcription factors with the promoter of both BRCA1 and RAD51 is regulated by the histone acetyl transferase CBP (Ogiwara and Kohno, 2012) and that USP9X has been described to interact with the histone deacetylase HDAC6 (Joshi et al., 2013). It is appealing to speculate that USP9X could act in a manner similar to OTUB1 through physical interaction with one or more proteins to regulate this pathway.

Cells that are deficient in DNA repair are particularly sensitive to DNA-damaging agents. This is a property that can be exploited when treating tumours deficient in DNA repair pathways. Treatment with the DNA-damaging PARP inhibitor Olaparib has been shown to specifically kill cells deficient in HR repair. Here, we demonstrate that the depletion of USP9X decreases the long-term survival of U2OS cells following treatment with Olaparib. The sensitivity of USP9X-depleted cells to Olaparib suggests that tumours with loss of function mutations in USP9X may be particularly susceptible to treatments such as Olaparib. Interestingly, although no link has previously been made between USP9X and DSB repair, USP9X disruption has been shown to sensitise cancer cells to the DNA-damaging agents Fluorouracil, Doxorubicin and Cisplatin (Fu et al., 2017a; Harris et al., 2012; Liu et al., 2015).

USP9X has been shown to have tumour-suppressive properties in certain contexts. Here, we show that USP9X is required for maintaining BRCA1 and RAD51 protein levels. BRCA1 and RAD51 are well-known tumour suppressors. In breast and ovarian cancers, mutations in the BRCA1 gene, as well decreased BRCA1 mRNA, are frequently observed (Rizzolo et al., 2011). RAD51 is also often deregulated in cancer cells. In one study 30% of breast carcinomas analysed were shown to have decreased levels of RAD51 protein (Yoshikawa et al., 2000). It is likely that sustaining the levels of these HR factors is an additional mechanism by which USP9X can supress tumorigenesis.

In conclusion, we have identified USP9X as a novel regulator of BRCA1 and RAD51, and we have revealed a novel, protease-independent mechanism by which USP9X can regulate protein levels. Further investigations in other cancer cell lines and primary samples are required to understand the implications of this regulation in cancer development and its potential for exploitation in treatments targeting DNA integrity.

Cell culture

Osteosarcoma-derived U2OS cells were purchased from American Type Culture Collection at the beginning of the project and routinely tested for contamination. DR-GFP U2OS cells were a gift from the group of Noel Lowndes (Centre for Chromosome Biology, National University of Ireland Galway). Cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 1% penicillin-streptomycin and heat-inactivated 10% fetal bovine serum (Sigma-Aldrich). To generate an inducible USP9X shRNA cell line, shRNAs targeting the 3′ UTR and coding sequence of USP9X were cloned into pRSITEP-U6Tet-sh-Ef1-TetRep-2A-Puro, a lentiviral transfer plasmid (Cellecta). HEK-293 cells were used to package this vector into viral particles, which were then transduced into U2OS cells. Single clones were isolated using puromycin selection. USP9X depletion was induced in these cell lines by addition of Doxycycline (1 µg/ml) to cell culture medium, alleviating TET repression.

siRNA and drugs

The following siRNA sequences were used in this study; siUSP9X (a) (5′-AGAAATCGCTGGTATAAATTT-3′) (Schwickart et al., 2010), siUSP9X (b) (5′-AGTGTATAGTGTTTTGTAATA-3′), siUSP9X (c) (5′-GCAGUGAGUGGCUGGAAGU-3′) (Dupont et al., 2009), siBRCA1 (5′-GGAACCUGUCUCCACAAAG-3′) (Lou et al., 2003) and siControl (5′-GCAUAUCGUCGUAUACUAU-3′). All siRNAs were purchased from Sigma-Aldrich. Unless otherwise stated, cells were transfected with 100 nM siRNAs for 48 h using JetPrime transfection reagent (Polyplus) according to the manufacturer's instructions. Depletion of protein was confirmed by western blot analyses. Olaparib was purchased from MedChem express (cat. no. HY-10162). ICRF-193 from Enzo lifesciences (cat. no. GR-332) and NU7026 from Tocris (cat. no. 2828).

Protein manipulation

Whole-cell extracts from cells were prepared in buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM EDTA and 1% Triton X-100) containing protease and phosphatase inhibitors (Sigma-Aldrich), the DUB inhibitor N-ethylmaleimide (Sigma-Aldrich) and universal nuclease (Thermo Fisher Scientific). Protein concentration was determined using Bradford reagent (Sigma-Aldrich). Proteins were resolved by SDS-PAGE (6% or 10%) and transferred onto nitrocellulose membrane prior to overnight incubation at 4°C with primary antibodies and infrared-labelled secondary antibodies (see antibodies). For immunoblotting of BRCA1, overnight transfers (16 h at 35 V) were performed. To ensure equal loading of protein between lanes, the Revert™ Total Protein Stain (TPS) (Li-Cor Biosciences) was used. TPS and immunoreactive bands were visualised and quantified using Odyssey Infrared Imaging Systems (Li-Cor Biosciences).

Immunofluorescence microscopy

U2OS cells were grown on coverslips. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After three washes with PBS, cells were permeabilised with PBS with 0.1% Triton X-100 for 2 min at room temperature. The coverslips were rinsed three times with PBS 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 secondary antibody (see antibodies) for 1 h at 37°C. Coverslips were then mounted using SlowFade (Thermo Fisher Scientific) and cells were imaged and quantified using the high-throughput operetta imaging system (Perkin-Elmer). The operetta analysis programme quantified nuclear foci using the following analysis sequence: (1) identify nuclei based on DAPI intensity, (2) exclude border cells, (3) exclude doublet cells, (4) detect foci based on signal to background ratio, (5) output foci per nucleus counts.

Flow cytometry cell cycle analysis

To label nascent DNA, cells were incubated with EdU (10 μM) for 30 min prior to harvest. Cells were fixed overnight (70% ethanol in PBS) and stained for EdU and DAPI analysis. Incorporated EdU was labelled with CLICK chemistry [10 μM 6-carboxyfluorescine-TEG-azide, 10 mM sodium-L-ascorbate, 2 mM copper (II) sulphate] for 30 min. Cells were washed twice, once in 1% BSA PBST (PBS with 5% Tween-20) and once in PBS. Cells were then resuspended in DAPI (1 µg/ml) in 1% BSA in PBS to stain the DNA. Data was acquired on a BD FACS Canto II and analysed using FlowJo vX software.

Colony formation assay

Following transfection with siControl, siUSP9X (a), (b) and (c) or siBRCA1 for 48 h, cells were trypsinised, counted and replated into Olaparib-containing medium for 3 days. The medium was replaced and, 10 days later, colonies of surviving cells were fixed with methanol, stained with Crystal Violet (Sigma-Aldrich) and colonies greater than >50 cells were counted. The surviving fraction was calculated by normalising to plating efficiency. Statistical analysis of three independent experiments was performed using Prism (GraphPad Software).

DR-GFP HR assay

To measure HR competency, DR-GFP U2OS cells were transfected with siControl, siUSP9X (a), (b) and (c) or siBRCA1 24 h after plating. The following day cells were transfected with pCBASceI (Addgene plasmid #26477) and cerulean-n1 (Addgene plasmid #54742). After 48 h the number of GFP-positive cells was assessed and normalised to the transfection efficiency indicated by cerulean positivity. Data was acquired on a BD FACS Canto II and analysed using FlowJo software. Statistical analysis of three independent experiments was performed using Prism (GraphPad Software).

Real-time qPCR

Total RNA was extracted using NucleoSpin RNA columns (Machery Nagel). cDNA was generated from 1 µg of RNA with random hexamer primers using SuperScript First Strand Synthesis System (Thermo Fisher Scientific). The resulting cDNA was diluted 1:10 and used for RT-qPCR using the following TaqMan gene expression assays: Hs00947967_m1 (RAD51), Hs01556193_m1(BRCA1), Hs99999901_s1 (18 s), and TaqMan DNA repair assay (441 8773). The relative mRNA levels were normalised to the 18 s rRNA and calculated using the comparative Ct method.

Antibodies

The following primary antibodies were used for immunoblotting: BRCA1 (1:500, Merck, cat. no. MS110), USP9X (1:1000, Bethyl, cat. no. A301-351A), 53BP1 (1:1000, Novus Biologicals, cat. no. NB100-304), BARD1 (1:500, Santa Cruz Biotechnology, cat. no. Sc74559), ubiquitin (1:1000, Santa Cruz Biotechnology, cat. no. sc8017) and RAD51 (1:1000, Santa Cruz Biotechnology, cat. no. sc8349). The secondary antibodies used were obtained from Li-COR Biosciences (800CW anti-rabbit-IgG, cat. no. 926-32211, 800CW anti-mouse-IgG, cat. no. 926-32210). For immunofluorescence, the following primary antibodies were used: BRCA1 (1:500, Santa Cruz Biotechnology, cat. no. sc6954), RAD51 (1:1000, Calbiochem, cat. no. PC130), 53BP1 (1:200, Novus Biologicals, cat. no. NB100-304) and γ-H2AX (1:500, Merck, cat. no. 05-636), pS2056 DNA-PK (1:200, Abcam, cat. no. ab18192). The following secondary antibodies used were obtained from Life Technologies: Alexa Fluor 488-conjugated goat anti-mouse-IgG (A11001), Alexa Fluor 647-conjugated goat anti-mouse-IgG (A21235), Alexa Fluor 647-conjugated goat anti-rabbit-IgG (A31573).

The authors thank Sandra Healy, Aisling Quinlan and Mark McDermott for critical reading of manuscript, Michael Rainey and Janna Luessing for technical assistance and critical reading of the manuscript, Noel Lowndes for the kind gift of the DR-GFP U2OS cell line and all the members of the Santocanale laboratory for discussion and support. The authors also acknowledge the facilities and technical assistance of Enda O Connell, Shirley Hanley and the Flow Cytometry Core Facility at NUI Galway that is funded by NUI Galway, Science Foundation Ireland, the Irish Government's Programme for Research in Third Level Institutions, Cycle 5 and the European Regional Development Fund.

Author contributions

Conceptualization: R.O., C.S.; Methodology: R.O., C.S.; Validation: R.O.; Formal analysis: R.O.; Investigation: R.O.; Data curation: R.O.; Writing - original draft: R.O.; Writing - review & editing: C.S.; Visualization: R.O.; Supervision: C.S.; Project administration: C.S.; Funding acquisition: R.O., C.S.

Funding

The Santocanale lab is mostly funded by the Science Foundation Ireland (SFI) with grant 16/IA/4476. R.O. was funded by a National University of Ireland, Galway College of Science Fellowship and received support from NUI Galway Thomas Crawford Hayes Research Fund and a Beckman Research Fund.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information