Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice

Radiotherapy and Oncology xxx (2016) xxx–xxx

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Original article

Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice Ali Bakr a,1, Sabrina Köcher a,1, Jennifer Volquardsen a, Rudolph Reimer b, Kerstin Borgmann a, Ekkehard Dikomey a, Kai Rothkamm a, Wael Y. Mansour a,c,⇑ a Laboratory of Radiobiology and Experimental Radio-oncology, University Medical Center Hamburg Eppendorf; b Heinrich-Pette-Institute Leibniz-Institute for Experimental Virology, Hamburg, Germany; and c Tumor Biology Department, National Cancer Institute, Cairo University, Egypt

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Article history: Received 3 July 2015 Received in revised form 28 October 2015 Accepted 1 November 2015 Available online xxxx Keywords: 53BP1 BRCA1 DSB-repair choice

a b s t r a c t Purpose: The aim of this study was to elucidate the impact of DNA damage response (DDR) proteins 53BP1 and BRCA1 on the double-strand break (DSB)-repair choice. This is important not only in order to understand the underlying mechanisms of DSB-repair pathway regulation but also to determine the therapeutic implications for BRCA1-associated tumors. Materials and methods: Human tumor cell lines A549 and HeLa were used. Non-homologous end-joining (NHEJ) and homologous recombination (HR) were assessed using NHEJ and HR reporter constructs. Colocalization of HR-proteins RPA and RAD51 with 53BP1 was evaluated by confocal microscopy and 3D-analysis. Results: We demonstrate a specific crosstalk between 53BP1 and BRCA1. While 53BP1 does not colocalize with RPA or RAD51 and prohibits the recruitment of BRCA1 to DSBs to stimulate NHEJ, BRCA1 promotes the 53BP1 displacement specifically in S/G2-phase to allow end-resection, initiating HR. HR-efficiency was restored in BRCA1-depleted cells upon additional 53BP1-knockdown. Further, we found that 53BP1-mediated end protection precedes BRCA1-dependent end-resection. Conclusion: These results demonstrate that the interplay between 53BP1/NHEJ and BRCA1/HR is of great relevance for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors. Ó 2015 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology xxx (2016) xxx–xxx

DSBs are highly toxic and, if un- or mis-repaired, can cause genomic rearrangements or cell death [1]. Therefore, a complex DDR network has evolved to avoid the deleterious consequences of these lesions. Upon DSB-induction, the histone variant H2AX is phosphorylated at serine-139 (cH2AX) in chromatin regions flanking the DSB, which then serves as a platform for the recruitment of other DSB-repair proteins to microscopically visible nuclear ‘‘foci”. These foci contain factors from the two main DSB-repair pathways: NHEJ and HR. While NHEJ is a ligation-based mechanism which can act throughout the cell cycle, HR is a resection-based process which requires a homologous DNA-template, thus being restricted to S- or G2-phase. The choice between these pathways is tightly regulated. When this regulation fails, translocations and other genomic alterations may result, increasing cancer risk [1,2]. Previously, we have described a functional hierarchy controlling ⇑ Corresponding author at: Lab. of Radiobiology & Experimental Radiooncology, Campus Science N27, Center of Oncology, University Medical Center HamburgEppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail address: [email protected] (W.Y. Mansour). 1 Equal contribution.

DSB-repair pathway choice [3]. This hierarchy is tightly regulated by multiple factors [4,5] to ensure appropriate recruitment of DSB-repair proteins. DSB end-resection, a process by which 50 –30 nucleolytic degradation generates single-stranded DNA (ssDNA) [6], has been suggested to be a main decisive step. Initiation of end-resection by CtIP/MRE11 removes the KU-heterodimer, the main player in NHEJ, from the DSB to generate ssDNA overhangs that commit the repair to HR. Deregulation of the DSB-repair hierarchy, due to mutations in DSB-repair genes (e.g. BRCA1), is associated with tumorigenesis. Defective HR is a major contributor to tumorigenesis in BRCA1defective tumors. Such tumors exhibit an extreme sensitivity to drugs that induce damage requiring HR for repair such as Mitomycin-C (MMC) or PARP1-inhibtors (e.g. olaparib) [7,8]. However, some BRCA1-mutated tumors are not hypersensitive to those agents [9]. It has previously been reported that BRCA1-deficient tumors show complex chromosomal rearrangements such as fusions which are apparently mediated by end-joining processes [10,11], confirming the deficient repair hierarchy in these tumors. For

http://dx.doi.org/10.1016/j.radonc.2015.11.001 0167-8140/Ó 2015 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Bakr A et al. Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice. Radiother Oncol (2016), http://dx.doi.org/10.1016/j.radonc.2015.11.001

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DSB repair pathway choice: a decision taken by 53BP1 and BRCA1

anti-cancer therapies the understanding of the DSB-repair pathway interplay is therefore of great importance. Interestingly, deletion of 53BP1 resulted in significantly fewer tumors in BRCA1-deficient mice [7]. Genetic studies have placed 53BP1 in the NHEJ pathway by demonstrating that 53BP1/ cells display G1-specific sensitivity to ionizing radiation (IR) that is epistatic to a KU-deletion [12]. The accumulation of both 53BP1 and BRCA1 at DSBs depends on the E3-ubiquitin ligases RNF8 and RNF168 [13]. It is not yet clear how a common pathway promotes the recruitment of two apparently antagonistic factors. Furthermore, how the accumulation of these proteins at DSBs may impact on the choice of a particular DSB-repair pathway is still not fully understood. Here we provide several lines of evidence for opposing activities of 53BP1 and BRCA1 in DSB-repair. Utilizing confocal microscopy and detailed 3D-analysis, we show that 53BP1 foci characteristics differ depending on cell cycle phase and repair pathway usage. We demonstrate for the first time that 53BP1-mediated endprotection and BRCA1-mediated end-resection are antagonistic processes and that end-protection precedes end-resection. Together, these results demonstrate the potential relevance of the 53BP1-status for BRCA1-associated cancers.

To directly address this question, we initially monitored the involvement of 53BP1 and BRCA1 in DSB-repair using GFP-based reporter assays for HR (pGC) and NHEJ (pEJ) [3] (Fig. S1A). Depletion of BRCA1 (Fig. S1B) reduced HR 2.5-fold (Fig. 1A), whereas 53BP1-knockdown (Fig. S1C) decreased NHEJ 2-fold compared to control-treated cells (Fig. 1B). For comparison, CtIP-depletion (Fig. S1D) abrogated HR similarly as BRCA1-knockdown and –as expected– had no effect on NHEJ. Next, we analyzed the recruitment kinetics of the HR intermediate factors RPA (end-resection) and RAD51 (recombination) to DSBs in CenpF-positive-S/G2 A549 cells after 2 Gy. Control-treated cells showed a steep increase and subsequent decline of RPA foci numbers, indicating efficient DSB end-resection and successful repair by HR (Fig. 1C). BRCA1- or CtIP-depleted cells displayed a greatly diminished number of RPA foci (3-fold at 2 h) (Fig. 1C, and Fig. S1C), reflecting impaired end-resection. Notably, 53BP1-knockdown did not change the

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Materials and methods Cell culture and siRNA knockdown Cells were cultured as previously described [14]. siRNA knockdown experiments were performed using smart-pool siRNA (Dharmacon) and Lipofectamine RNAiMAX (Invitrogen) according to manufacturers’ protocols. Knockdown was monitored by Western blot [5,15] using antiBRCA1 (Santa-Cruz, Sc-6954), anti-53BP1 (Novus, NB100-305), anti-CtIP (Active-Motif, 61141) and anti-b-actin antibodies (Sigma). Cells were irradiated as previously described [16]. Colony formation assay (CFA)

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CFA was performed as previously described [15]. MMC and olaparib were added for 24 h. Immunofluorescence analysis (IF) Cells were seeded on coverslips and treated for IF staining as previously described [15], using anti-cH2AX (Upstate, 05-636), anti-53BP1 (Novus, NB100-305), anti-RAD51 (Abcam, ab-14B4), anti-RPA (Santa-Cruz, sc-53496) and anti-CenpF antibodies (BD, 610768). Quantitative analysis of foci numbers and intensities was performed semi-automatically using ImageJ software. Bitplane Imaris 7.6.1 software (Bitplane AG, Zürich, Switzerland) was used for visualization of 3D-Super-Resolution images in addition to analysis of the single focus volume (surface rendering).

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Statistical analysis All statistical analyses were performed using Graphpad Prism version 6.02. Data are presented as mean ± SEM of at least three different experiments. Results Opposing activities of 53BP1 and BRCA1 in DSB-repair Despite having common regulators (RNF8/168), most studies indicate opposing activities for 53BP1 (stimulating NHEJ) and BRCA1 (promoting HR) [17]. How the accumulation of these proteins into IR-induced foci (IRIF) may impact on DSB repair choice is still not fully understood.

Fig. 1. Opposing activities of 53BP1 and BRCA1 in DSB-repair. Measurements of HR (A) and NHEJ (B) in HeLa cells harboring pGC or pEJ subjected to indicated siRNAs. RPA (C) or RAD51 (D) foci kinetics in 2 Gy-irradiated A549 cells subjected to the indicated siRNAs.

Please cite this article in press as: Bakr A et al. Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice. Radiother Oncol (2016), http://dx.doi.org/10.1016/j.radonc.2015.11.001

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kinetics of RPA foci. However, the total number of RPA foci even exceeded the number detected in controls (Fig. 1C, and Fig. S1C). Similar results were observed for RAD51 (Fig. 1D and Fig. S1D). Together, these results demonstrate BRCA1 promotes resection of DSB-ends, committing the repair to HR, whereas 53BP1 protects the ends from resection, directing the repair to NHEJ. Consequently, 53BP1 should be excluded from DSBs committed to HR. Indeed, no spatial overlap was found between 53BP1 and RPA or RAD51 foci in cells positive for both proteins (Fig. 2A/B and Fig. S2A and B). Detailed confocal microscopy and 3D-analysis also confirmed that 53BP1 and RPA or RAD51 foci are juxtaposed occupying different sub-nuclear compartments spanning the DSB but do not directly colocalize (Fig. 2C/D and Movies 1 and 2). In contrast, RPA/cH2AX or RAD51/cH2AX are perfectly colocalized (Fig. S2 C–F and Movies 3 and 4). Different patterns of 53BP1 foci account for different repair pathways The above data indicate that 53BP1 as a pro-NHEJ and anti-HR factor does not colocalize with HR factors. However, 53BP1 is considered a general DSB-marker which colocalizes with cH2AX. A paradox arises since the HR repair proteins RPA and RAD51 certainly colocalize with cH2AX (Fig. S2/D). We speculated that the 53BP1 foci patterns might differ depending on cell cycle phase and repair pathway usage. Therefore, we analyzed 53BP1 focus intensity and volume either in CenpF-negative G1- or CenpFpositive S/G2-cells. Strikingly, 2D- and 3D-analyses revealed the existence of two distinct patterns of 53BP1 foci in respect to the cell cycle phase. 53BP1 foci were more intense and occupied a larger volume within the IRIF in G1 (strong/big), while they were generally 50% weaker and 25% smaller in volume in S/G2 (weak/small) (Fig. 3A/B and Fig. S3). Together with data in Fig. 2, this suggests that the pattern of 53BP1 differs in respect to the repair pathway

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used, which is also supported by the finding that 53BP1 promotes NHEJ and inhibits HR (Fig. 1). Crosstalk between 53BP1 and BRCA1 for DSB-repair pathway choice Next, we sought to monitor BRCA1 foci in different cell cycle phases. To this end, A549 cells were irradiated with 2 Gy and foci enumerated in CenpF-negative G1- or -positive S/G2-cells (Fig. S4). BRCA1 foci formed only in S/G2- but not in G1-phase cells (Fig. 4A), confirming its pro-HR activity. Since the change in 53BP1 focal-intensity coincides with BRCA1 enrichment at DSB-ends, we speculated that 53BP1 and BRCA1 may cross-inhibit each other’s enrichment at the DSB. Therefore, we firstly investigated whether 53BP1 prevents BRCA1 foci formation. 53BP1 depletion resulted in an enhanced number of BRCA1 IRIF in S/G2-phase (2-fold) and in the appearance of BRCA1 foci also in G1-cells (Fig. 4A and Fig. S4), thus confirming that 53BP1 antagonizes BRCA1 accumulation at DSBs in both cell cycle phases. Further, we elucidated whether BRCA1-knockdown increases 53BP1 focal-intensity in S/G2-phase. Compared to control cells, the 53BP1 focal-intensity was greatly enhanced in the S/G2-phase in BRCA1-depleted cells and as intense as in G1-phase where no change was observed (Fig. 4B). Together, these results indicate a crosstalk between 53BP1 as an end-protection factor and BRCA1 as an end-resection factor for regulating DSB-repair choice. In G1, 53BP1 prevents the recruitment of BRCA1 to DSBs and protects the break ends from resection, thus stimulating NHEJ. In S/G2, BRCA1 excludes 53BP1 from IRIF and enables DSB end-resection, committing the repair to HR. Yet, what comes first: end-protection by 53BP1 or BRCA1mediated end-resection? To address this question, we analyzed the kinetics of 53BP1 focal-intensity in G1 and S/G2. Intense G1-patterns of 53BP1-foci were established as early as 15 min

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Fig. 2. 53BP1 does not colocalize with HR proteins. Quantification of RPA (A) or RAD51 (B) foci individually or colocalized with 53BP1 (colo.) in irradiated A549 cells. Representative projections of 3D-images of 53BP1 foci juxtaposed to but not colocalized with RPA (C) or RAD51 (D).

Please cite this article in press as: Bakr A et al. Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice. Radiother Oncol (2016), http://dx.doi.org/10.1016/j.radonc.2015.11.001

DSB repair pathway choice: a decision taken by 53BP1 and BRCA1

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Fig. 3. Different patterns of 53BP1 foci in G1- and S/G2-cells. Automated quantification of 53BP1 focal-intensity (A) and volume (B) in A549 cells 2 h post 2 Gy. AntiCenpF staining was used to distinguish between G1 and G2 phases.

following IR and persisted for up to 8 h (Fig. 4C). Intriguingly, S/G2cells also showed a 53BP1 focal-intensity comparable to that of G1cells at 15 min post IR, which declined thereafter to form the previously described weak S/G2 foci-pattern of 53BP1, suggesting a progression from end-protection to end-resection in S/G2-phase. Altogether, these data reveal that all DSB-ends are initially protected by 53BP1. In S/G2, BRCA1 enrichment at the DSB then displaces 53BP1 from the DSB to facilitate end-resection.

Knockdown of 53BP1 rescues the HR-deficiency in BRCA1-depleted cells BRCA1-deficient cells are sensitive to drugs which induce lesions requiring HR for repair (e.g. olaparib, MMC). We have shown that in BRCA1-depleted cells 53BP1 promotes endprotection guiding the repair toward NHEJ, a fatal repair mechanism for DSBs requiring HR [18]. These cells could potentially be rescued by additional knockdown of 53BP1, thus freeing up the DSB-ends for BRCA1-independent resection processes. To verify this, we enumerated RPA and RAD51 foci in BRCA1-depleted cells after additional knockdown of 53BP1. As anticipated, BRCA1knockdown showed decreased foci numbers of both RPA and RAD51 (Fig. 5A/B). Remarkably, foci numbers were significantly restored after additional knockdown of 53BP1 (Fig. 5A/B), confirming that removal of 53BP1 from the DSB-ends rescues HR in BRCA1deficient cells. Next, we used pGC to directly monitor HR in doubledepleted backgrounds. Importantly, additional knockdown of 53BP1 significantly increased HR-efficiency in BRCA1-depleted cells. Thus, loss of 53BP1 rescues HR-impairment in BRCA1deficient cells by restoring the ability to resect DSB-ends, hence initiating HR. Therefore, we tested whether restoring endresection and recombination processes also suppresses the profound sensitivity of BRCA1-deficient cells to MMC or olaparib. Depletion of BRCA1 alone led to an increased sensitivity to MMC or olaparib, whereas 53BP1-knockdown alone had no effect. Notably, however, additional depletion of 53BP1 in BRCA1-knockdown cells rescued their MMC or olaparib sensitivity (Fig. 5D). In conclusion, our data provide mechanistic insights into the repair pathway choice which is governed by a crosstalk of

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Fig. 4. Crosstalk between 53BP1 and BRCA1 for DSB-repair pathway choice. Quantification of BRCA1 foci (A) and 53BP1 focal-intensity (B) at 2 h post 2 Gy in A549 cells treated with indicated siRNAs. Anti-CenpF staining was used to distinguish between G1 and G2 phases. (C) Quantification of 53BP1 focal-intensity at indicated time points post 2 Gy.

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Discussion In the present study, we describe the interplay between 53BP1 and BRCA1 to establish an appropriate NHEJ-HR equilibrium in a cell cycle specific manner (Fig. S5). In G1-phase, 53BP1 prevents BRCA1 loading and hence protects the ends from nuclease processing. In S/G2-phase, the inhibitory effect of 53BP1 on end-resection is antagonized by BRCA1 which is recruited to DSBs to initiate endresection (Fig. 1C) committing the repair to HR (Fig. 1A). Depletion of 53BP1 rescues the BRCA1-deficiency phenotype as evidenced by (i) restored RPA and RAD51 focus formation upon IR (Fig. 5A/B), (ii) recovered HR-efficiency (Fig. 5C), and (iii) reduced sensitivity to MMC or olaparib (Fig. 5D). In agreement with these results, it was shown that loss of BRCA1 results in persistence of 53BP1 at DSB-ends, switching the repair to NHEJ in S/G2-phase resulting in chromosomal abnormalities and tumorigenesis, which were relieved by further 53BP1-deletion [7]. 3D-analysis identified different patterns of 53BP1 foci, which appear to mark the repair pathway used (Fig. 3). While strong/ big foci are found mostly in the G1-phase, small/weak foci appear

Please cite this article in press as: Bakr A et al. Functional crosstalk between DNA damage response proteins 53BP1 and BRCA1 regulates double strand break repair choice. Radiother Oncol (2016), http://dx.doi.org/10.1016/j.radonc.2015.11.001

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Fig. 5. Knockdown of 53BP1 rescues the HR-deficiency in BRCA1-depleted cells. Quantification of RPA (A) or RAD51 (B) foci in 2 Gy-irradiated A549 cells treated with indicated siRNAs. (C) HR-efficiency measured as in Fig. 1. (D) Sensitivity of A549 cells subjected to indicated siRNAs and olaparib (left) or MMC (right) measured by CFA.

in the G2-phase, as already reported by several labs [7,9,19,20]. Most importantly, two data-sets are presented for the first time in the current study. First, we demonstrate that the HR-proteins RPA and RAD51 appear only juxtaposed to 53BP1 in S/G2 phase where it forms small/weak foci (Fig. 2, Fig. S2 and Fig. 3). Second, we found that 53BP1-mediated end-protection precedes BRCA1dependent end-resection in S/G2-phase (Fig. 4C). This crosstalk is specific to 53BP1 and BRCA1 because loss of classical NHEJ proteins such as KU does not overcome the HR-defects associated with BRCA1 deficiency [7,21]. Also disrupting 53BP1 does not reverse the DNA repair defects associated with other HR proteins such as CtIP (Fig. 5A/B), XRCC2, BRCA2 or PALB2 [7,19]. How exactly 53BP1 and BRCA1 counteract each other remains a subject for future investigations. Since it has been shown that BRCA1 is an ubiquitin ligase, and 53BP1 oligomers are recruited to DSBs by recognizing the H4K20me2-residue in an ubiquitindependent manner [22], BRCA1 may then influence the abundance or the exposure of the H4K20me2 for 53BP1-binding. The proposed model for the 53BP1/NHEJ-BRCA1/HR interplay is of great relevance for clinical implications, since a subset of BRCAmutated tumors exists which develop resistance against chemotherapeutics such as PARPi and carboplatin [23]. Based on the data presented here, 53BP1 deficiency could explain this resistance. In line with this assumption, low 53BP1-expression levels have been reported in triple-negative BRCA1-associated breast cancers [19]. Furthermore, 53BP1 was lost in a fraction of BRCA1deficient mouse mammary tumors that acquired PARPi resistance in vivo [9]. Conflict of interest None declared. Acknowledgments This work is supported by DAAD, Germany and BMBF, Germany grant # 02NUK035B.

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[16] Kasten-Pisula U et al. The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair. Radiother Oncol 2009;90:257–64. [17] Schlegel BP, Jodelka FM, Nunez R. BRCA1 promotes induction of ssDNA by ionizing radiation. Cancer Res 2006;66:5181–9. [18] Patel AG, Sarkaria JN, Kaufmann SH. Nonhomologous end joining drives poly (ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells. Proc Natl Acad Sci USA 2011;108:3406–11. [19] Bouwman P et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol 2010;17:688–95.

[20] Chapman JR et al. BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. J Cell Sci 2012;125:3529–34. [21] Bunting SF et al. BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair. Mol Cell 2012;46:125–35. [22] Zimmermann M, de Lange T. 53BP1: pro choice in DNA repair. Trends Cell Biol 2014;24:108–17. [23] Norquist B et al. Secondary somatic mutations restoring BRCA1/2 predict chemotherapy resistance in hereditary ovarian carcinomas. J Clin Oncol 2011;29:3008–15.

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