DNA repair is essential in maintaining genome integrity and defects in different steps of the process have been linked to cancer and aging. It is a long lasting question how DNA repair is spatially and temporarily organized in the highly compartmentalized nucleus and whether the diverse nuclear compartments regulate differently the efficiency of repair. Increasing evidence suggest the involvement of nuclear pore complexes in repair of double-strand breaks (DSBs) in yeast. Here, we show that the human nucleoporin 153 (NUP153) has a role in repair of DSBs and in the activation of DNA damage checkpoints. We explore the mechanism of action of NUP153 and we propose its potential as a novel therapeutic target in cancers.
DNA double-strand breaks (DSBs) are particularly dangerous as their inefficient or inaccurate repair can result in mutations and chromosomal translocations that may induce cancer.1 DSBs can be repaired by one of two major pathways: homology-based repair (homologous recombination (HR)) using the intact chromatid as a template present in proximity in S and G2 phases of the cell cycle, or direct joining across the break site (non-homologous end joining (NHEJ)).2
The coordination between cell cycle progression and DSB repair (DSBR) is regulated by the DNA damage response (DDR) signalling pathway, which activates the cell cycle checkpoints in the presence of DNA breaks.3 This pathway is initiated by the recruitment of the MRN (MRE11–RAD50–NBS1) sensor complex to sites of damage. The recruitment of MRN subsequently activates the ATM kinase, which associates with DSBs and phosphorylates the histone variant H2AX (γ-H2AX).2 MDC1 can then bind to γH2AX and recruit new MRN and ATM proteins, leading to spreading of the repair machinery along the chromosome. MDC1 also recruits ubiquitin ligases, such as RNF8 and RNF168, which facilitate the recruitment of the downstream factors 53BP1 and BRCA1.2 When the DNA is resected to single-stranded DNA, it is recognized by replication protein A, which results in the recruitment of ATR.2 Both the ATM and the ATR dependent branches of the pathway lead to the activation of the checkpoint kinases, CHK1 and CHK2, which stall damaged cells in their cell cycle until the lesions are resolved.3
DNA repair, like all DNA-dependent processes, occur in the highly compartmentalized nucleus. Most nuclear events do not occur ubiquitously, but are limited to defined sites.2 Several studies in yeast have shown that dedicated DNA repair centres exist as preferential sites of repair.2, 4 Furthermore, persistent DSBs in yeast migrate from their internal nuclear positions to the nuclear periphery, where they associate with nuclear pores.5, 6 This sequestration to the nuclear periphery was shown to require certain components of the yeast nuclear pore complex, like NUP84 and the nucleoporin NUP60, located in the basket of the pore.5, 6 Additional studies revealed that depletion of representative members of the NUP84 or NUP60 complex leads to synthetic lethality when combined with genes that are required for DSBR through HR.4 Moreover, mutants of the NUP84 complex are highly sensitive to DNA-damaging treatments.7 A more recent study has shown that key nucleoporins are phosphorylated upon DNA damage and act to neutralize the topological tension generated at nuclear pore tethered genes that is inhibitory to origin firing after replication stress.8
On the contrary, in mammalian cells, each DSB is repaired individually in the absence of nuclear repair centers.9 Furthermore, DSBs do not move towards the nuclear periphery, as their motion seems to be very limited in the mammalian nucleus.9 However, the evolutionary conserved role of nucleoporins in gene regulation raises the question whether nucleoporins have a conserved role in mammalian DSBR. We therefore explored the role of the Nucleoporin 153 (NUP153), a component of the nuclear basket of the mammalian nuclear pore in DSBR. We show that NUP153 is essential for proper activation of the DNA damage checkpoints and regulates the choice between NHEJ and HR. These functions can be partially explained by the role of NUP153 in promoting 53BP1 nuclear localization. Our results will set up the basis of investigation of the role of nuclear pore in DNA repair in mammals and can lead to potential therapeutic innovations.
Results and discussion
One of the hallmarks of defective DSBR and DDR is hypersensitivity to DNA damaging agents. To address whether the NUP153 has a role in repair of DSBs, we analysed the effect of its depletion in clonogenic survival of U2OS cells following exposure to genotoxic stress. RNAi-mediated downregulation of NUP153 led to an increased sensitivity to the radiomimetic drug phleomycin compared with control cells (Figure 1a). The efficiency of the NUP153 silencing was verified by RT–qPCR (Supplementary Figure 1) and western blot (Figure 1b). One possible explanation for the hypersensitivity to DNA damage upon depletion of NUP153 is the deregulation of cell cycle checkpoints resulting in mitotic progression with unrepaired DSBs. To test this hypothesis, we investigated whether the downregulation of NUP153 affects the activation of checkpoints after treatment with the radiomimetic drug Neocarzinostatin (NCS). Indeed, we observed compromised phosphorylation of ATM, CHK1 and CHK2 kinases and p53 (Figure 1b). In line with this observation, the NUP153 depleted cells didn’t properly activate the G2/M check point. Although, 31% of cells treated with si-scramble arrest in G2/M, 8 h after treatment with phleomycin, only 26% of siNUP153 cells exert similar arrest (Supplementary Figure 2). These results support the idea that NUP153 promotes DNA damage checkpoints.
To investigate the possibility that the hypersensitivity to DNA damaging agents stems also from persistent DSBs and defective DNA repair, we sought to directly test whether NUP153 facilitates DNA repair through a specific pathway. To this end, we utilized cell lines that contain stably integrated reporters to assess the rates of HR (DR-GFP10, 11) and NHEJ.12, 13, 14 For NHEJ, we used cell lines containing two types of NHEJ-reporter substrates; the pCOH-CD4 that permits analysis of the NHEJ of two distal ends (separated by 3.2 kb),12, 13 and a GFP-based substrate,14 to measure the NHEJ on closely adjacent ends, separated by only 34 bp.14 Interestingly, upon depletion of NUP153, we observed a significant drop in efficiency of NHEJ with both substrates compared with the scramble siRNA (2 fold for the CD4 and 5 fold for the GFP) (Figure 2a). Concomitantly, we observed a more than two fold increase in the rate of HR (Figure 2b). We used downregulation of RAD51 as a control and expectedly observed defective HR. Cell cycle analysis shows that the changes in NHEJ and HR rates cannot be explained by alterations in cell cycle profile upon depletion of NUP153 (Supplementary Figure 2). These results suggest that NUP153 has a role in the balance between NHEJ and HR.
To investigate whether the increase in HR was accompanied by increased resection, we visualized and quantified replication protein A and BRCA1 foci at a single DSB to mimic the break induced in the NHEJ and HR assays. We utilized U2OS cells with stably integrated I-SceI site harboring lacO operator repeats that can be visualized by GFP-lacR (lac repressor). As expected, we found that depletion of NUP153 leads to more than 2-fold increase in the number of replication protein A and BRCA1 foci in U2OS cells after expression of I-SceI (Figures 2c and d). DSB induction was verified by the phosphorylation of H2AX at the lacO array (Supplementary Figures 3A and B). Our results therefore suggest that NUP153 might inhibit HR by blocking resection of DSBs.
To understand the mechanism underlying the involvement of NUP153 in DDR, we first tested whether NUP153 is recruited to DSBs. We were unable to detect any accumulation of GFP-NUP153 at laser-induced breaks (Figure 3a). We then asked whether NUP153 regulates the localization and the ability of known DDR factors to form IRIF upon Neocarzinostatin treatment in U2OS cells. Whereas depletion of NUP153 did not affect γH2AX (Figure 3b) and MDC1 foci (Supplementary Figure 4), it significantly inhibited focal accumulation of 53BP1 quantified by high throughput imaging (Figure 3c). The results were validated by three independent siRNAs targeting distinct regions of NUP153 mRNA ranging from 30–50% decrease in the number of 53BP1 foci (Figure 3c). High-resolution imaging showed that NUP153 siRNA treated cells exerted massive and selective mislocalization of 53BP1 to the cytoplasm (Figure 3b, quantification in Figure 3d). The remaining nuclear 53BP1 either did not accumulate in foci or formed foci localized to the nuclear periphery (Figure 3b). Importantly, depletion of NUP153 did not affect the global levels of 53BP1 (Supplementary Figure 5). To test whether the 53BP1 cytoplasmic localization in the absence of NUP153 is because of an accelerated export or defective nuclear retention, we repeated our experiments in the presence of leptomycin B that selectively inhibits CRM1-dependent nuclear export.15 We observed no difference in the localization of 53BP1 or the foci formation (data not shown), suggesting that the presence of 53BP1 in the cytoplasm is due to an import defect or accelerated export through a CRM1-independent pathway. Indeed, previous work showed that depletion of NUP153 leads to import impairment of selective proteins.16 Moreover, while we were conducting this study, Moudry et al.17 confirmed the mislocalization of 53BP1 in NUP153 depleted cells and showed the requirement of NUP153 for 53BP1 nuclear import.
Recent studies have provided important mechanistic insights about how deficiency in 53BP1 restores HR levels in BRCA1-deficient cells by regulating the choice between HR and NHEJ.18, 19 These studies have placed 53BP1 as a top candidate for pharmacological targeting for future breast cancer therapies. Therefore, we sought to understand whether the impairment of 53BP1 is sufficient to explain the NUP153-deficient phenotype in our DNA repair assays, to suggest NUP153 as a potential candidate for targeted cancer therapy. To this end, we performed the survival assay in cells depleted for 53BP1 or for a combination of NUP153 and 53BP1. Indeed, 53BP1 knock down recapitulated the sensitivity to phleomycin treatment and the decreased survival (Figure 4a). Interestingly, combined 53BP1 and NUP153 depletion did not result in an additive survival defect suggesting that the radiosensitivity observed in NUP153 depleted cells is owing to the impairment in 53BP1 localization (Figure 4a). The efficiency of 53BP1 knock down was monitored by RT–qPCR and WB (Supplementary Figure 6). Moreover, 53BP1 was shown to promote ATM activity,20, 21, 22 and its depletion leads to checkpoint activation defects23 pointing to similar dependency on 53BP1 for activation of the checkpoints.
We then assessed whether the increase in HR at NUP153 depleted cells was mediated by the 53BP1 defect. We observed a moderate (1.3 fold) increase of HR upon depletion of 53BP1 using siRNA (Figure 4b). This effect was similar to that observed by Xie et al.14 in a previous study. However, this increase was smaller than the one observed in NUP153 depleted cells, and the combinatorial depletion of 53BP1 and NUP153 phenocopied the HR efficiency in cells depleted for NUP153 alone (Figure 4b).
Moreover, Guirouilh-Barbat et al. observed that 53BP1 silencing leads to a significant decrease in the frequency of end-joining, monitored with the GFP-based substrate, but has no impact on NHEJ frequencies, monitored with the CD4-based substrate (Bernard Lopez personal communication). This observation is different from our results that show that silencing of NUP153 leads to a decrease in NHEJ efficiency in both substrates (Figure 2a), suggesting that NUP153 promotes NHEJ through a pathway that does not involve only 53BP1. Additionally, Guirouilh-Barbat et al. showed a decrease in NHEJ accuracy upon 53BP1 depletion (Bernard Lopez personal communication). To test whether NUP153 depletion recapitulates these results, we analyzed repair junctions on the pCOH-CD4 substrate after the silencing of NUP153. Surprisingly, although NUP153 depletion affects the efficiency of NHEJ, it does not promote inaccurate repair (Figure 4c) further pointing to a role of NUP153 in NHEJ independent from 53BP1.
Taken together, our results suggest that in assays where a large amount of DSBs is induced the depletion of NUP153 phenocopies the depletion of 53BP1. On the other hand, when a single DSB is induced, the NUP153 depletion has a stronger and/or divergent phenotype. A possible explanation for this phenomenon is that the amount of protein remaining in the nucleus upon depletion of NUP153, is limited and there is active competition between the breaks for focal accumulation of 53BP1. On the other hand, when one or limited breaks are induced as it is the case with the I-SceI break at the HR and NHEJ assays, there is enough 53BP1 protein to form a repair focus. To test this hypothesis, we used the LacO-I-SceI cell line, where a break is induced at a single locus in the nucleus. Interestingly, we detected a normal recruitment of 53BP1 to I-SceI breaks upon depletion of NUP153 (Figures 4d and e). This finding is in agreement with the observation that the recruitment of 53BP1 at endogenous foci is not impaired upon depletion of NUP153 (Figure 3c–Neocarzinostatin condition).
The stronger effect on HR efficiency observed upon depletion of NUP153 could be explained by the loss of a potential 53BP1 modification and that the unmodified 53BP1 accumulates at the single DSB, acting as dominant negative. An alternative explanation could be that NUP153 has a role in addition to the regulation of nuclear import of 53BP1. It could promote the nuclear accumulation of a NHEJ factor and/or its recruitment to DSBs. However, silencing of classical NHEJ factors that affect the efficiency of end ligation affect the fidelity of repair as well.13 An alternative scenario could be that NUP153 negatively regulates a protein that promotes HR. Furthermore, we can imagine that the nuclear soluble fraction of 53BP1 has a role in the regulation of the repair pathways, by sequestering certain factors away from the break. Therefore, we can speculate that upon depletion of NUP153, even if 53BP1 has still the ability to bind to DSBs, the absence of its soluble pool can impair DSBR.
Here, we describe a novel role of NUP153 in DDR and DNA repair. TPR, the binding partner of NUP153 at the nuclear basket, is phosphorylated upon DNA damage by ATM/ATR and is involved in the proper activation of G2/M and intra S check point.24 One interesting aspect for further investigation is whether these factors have distinct or overlapping roles in DDR and whether the overlapping roles are mediated through their interaction. In yeast, the nucleoporin complexes NUP84 (hNUP107) and NUP60 (hNUP153) protect against genomic instability through maintenance of proper levels of the sumo protease Ulp1 at NPCs, and through appropriate sumoylation of several proteins, including yKu.25 It is tempting to speculate that a similar mechanism is conserved in mammals. Furthermore, it would be very interesting to investigate whether the role of NUP153 in DDR is unique or if other mammalian nucleoporins have similar role.
We show here that NUP153 regulates the choice between NHEJ and HR. This observation positions NUP153 as a candidate gene whose reduced expression could promote synthetic lethality in tumor cells that bear mutations in HR factors, like BRCA1 and BRCA2. However, as NUP153 promotes 53BP1 IRIF foci formation after DNA damage, impairment of NUP153 in BRCA1 cancer cells could mimic the phenotype of 53BP1 depletion, rescuing lethality and conferring resistance to PARP inhibition.18, 19 It will be consequently very interesting to exploit in the future the potential of NUP153 as a therapeutic target in certain cancers.
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We thank Amélie Weiss, Laure Froidevaux and Audrey Furst for excellent technical assistance. The GFP-NUP153 vector was provided by Ian Ellenberg (EMBL, Heidelberg). We are grateful to Zita Nagy and Tibor Pankotai for critical reading of the manuscript. C.L. is supported by the Région Alsace. The research in E.S. lab is funded by CNRS, ANR, INCA and HFSP. We thank the imaging center of IGBMC and particularly Marc Koch for the help with the multi photon laser experiments.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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