Cooperation of Blm and Mus81 in Development, Fertility, Genomic Integrity and Cancer Suppression

ORIGINAL ARTICLE Cooperation of Blm and Mus81 in development, fertility, genomic integrity and cancer suppression

S El Ghamrasni1,2, R Cardoso1, MJ Halaby1, D Zeegers3, S Harding1, R Kumareswaran1, T Yavorska4, N Chami2, A Jurisicova4, O Sanchez5, MP Hande3, R Bristow1, R Hakem1 and A Hakem1

BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81−/− and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.

Oncogene (2015) 34, 1780–1789; doi:10.1038/onc.2014.121; published online 26 May 2014

INTRODUCTION co-localizes with BLM at sites of stalled replication forks during 9,10 Defects in the DNA damage repair mechanisms promote genomic the S-phase arrest. Although MUS81 does not affect BLM instability and lead to increased risks for developmental defects helicase activity, its endonuclease activity on nicked Holliday and diseases including cancer and immunodeficiency.1 BLM junctions and 3’flap structures is stimulated by BLM because of its 10 protein, a member of the evolutionarily conserved RecQ helicase enhanced binding to these DNA substrates. MUS81 mutations – family, functions in unwinding various branched DNA structures2 promote genomic instability in murine and human cells,11 15 and Δ Δ and also facilitates the recruitment of a number of DNA repair Mus81 ex3-4/ ex3-4 mutant mice display increased cancer proteins (for example, EXO1, RAD51 and RAD51D) to DNA double- predisposition.11,13,15 In addition, decreased MUS81 expression strand breaks (DSBs).3–6 Importantly, mutations of BLM are has been observed in different human cancers.16,17 Using Bloom associated with the human Bloom’s syndrome, an autosomal syndrome cells, it was shown that deletion of MUS81 or SLX4 leads recessive disorder with clinical manifestations that include to decreased frequency of SCE, suggesting that in the absence of proportional dwarfism, immunodeficiency and infertility.2 Cells BLM nucleolytic-processing pathway, MUS81/SLX4 pathway is from Bloom’s syndrome patients display elevated frequency of activated to allow chromosomal segregation.18 It was also shown sister chromatid exchanges (SCEs). Bloom’s syndrome is also in Drosophila that dual loss of DmBlm and Mus81 leads to associated with elevated incidence of cancer and a high degree of increased apoptosis levels.19 genomic instability.2 With its partners RMI1/RMI2/TOP3α, BLM Given the importance of BLM and MUS81 in DNA damage forms a complex with FANCM at sites of DNA damage and defects repair, and in order to examine the in vivo effects of their in the formation of this complex result in higher SCE levels.7 BLM inactivation, we have generated a mouse model with dual mediates both anti-recombinogenic functions as well as pro- inactivation of these proteins. Using single and double mutant recombinogenic functions in the homologous recombination (HR) mice for Blm and Mus81, we examined the effects of the loss of repair pathway and its SUMOylation regulates the switch between these proteins on development, fertility, DNA damage repair its two functions.6 BLM responds to replication stress by pathways and cancer. We report that although Blmtm3Brd/tm3Brd and Δ Δ accumulating at stalled DNA replication forks where it contributes Mus81 ex3-4/ ex3-4 mice were viable, double mutants displayed to their restart.2 growth defects, infertility and impaired thymocyte homeostasis MUS81, with its partners EME1 or EME2, forms a DNA structure- compared with single mutant and wild-type (WT) littermates. specific endonuclease important for the restart of stalled Double mutant cells displayed remarkably reduced HR repair replication forks and the resolution of Holliday junctions, critical activity accompanied with elevated non-homologous-end-joining intermediates of HR repair.8 MUS81 physically interacts and (NHEJ) repair compared with single mutants. Irradiation (IR)-

1Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada; 2Laboratory of Biotechnology, Faculty of Sciences DHEM, University SMBA, Fez, Morocco; 3Department of Physiology, Yong Loo Lin School of Medicine and Tembusu College, National University of Singapore, Singapore, Singapore; 4Department of Obstetrics and Gynecology and Physiology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada and 5Department of Pathology, University of Ontario Institute of Technology, Oshawa, Ontario, Canada. Correspondence: Professor R Hakem or Dr A Hakem , Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, University Health Network, 610 University Avenue, 10th floor. Room 622, Toronto, Ontario, Canada M5G 2M9. E-mail: [email protected] or [email protected] Received 22 August 2013; revised 10 February 2014; accepted 26 March 2014; published online 26 May 2014 In vivo functional interaction of Blm and Mus81 S El Ghamrasni et al 1781 induced genomic instability was significantly higher in double Concomitant requirement for Blm and Mus81 for male and female mutants compared with single mutants. Although elevated SCE fertility was proposed to drive cancer development in Blm mutants, Defective DSB repair promotes sterility, and Bloom’s syndrome is double mutant mice were more cancer prone compared associated with male infertility and female subfertility.2 Therefore, with single mutant littermates despite their reduced SCE levels. we examined the fertility of Blm−/−Mus81−/− mice and their Collectively, our data highlight the importance of Blm and Mus81 control littermates. Previous studies of Blmtm3Brd/tm3Brd mice functions in DNA damage repair, fertility, development and indicated no fertility defects in these hypomorphic mutants.20 cancer. Similarly, Mus81Δex3-4/Δex3-4 mice display no significant changes in their reproductive capacity.11,15 Although interbreeding of Blm−/− or Mus81−/− mutants resulted in normal size litters compared RESULTS with WT littermates (Supplementary Table 1), crossing of double fi −/− −/− mutant males to WT females yielded signi cantly reduced number Blm Mus81 mice are viable but display growth defects of litters and the number of pups per litter was also smaller To generate mice with dual loss of function of Blm and Mus81, we compared with control littermates (Figure 2a). In addition, crossed mice that carry the Blmtm3Brd hypomorph allele20 to consistent with impaired fertility, testes of 6- to 8-week-old Δ − − Mus81 ex3-4 mutant mice11 (referred to herein as Blm / and Blm−/−Mus81−/− males were smaller in size compared with their Mus81−/− mice, respectively). Blm−/−Mus81−/− mice were obtained single mutant and WT littermates (Figure 2b). Hematoxylin and in the expected Mendelian ratio from intercrossing of eosin staining of testis sections indicated that in contrast to WT − − − − Blm+/-Mus81−/− mice (Supplementary Table 1). However, both (Figures 2c and d), Mus81 / (Figures 2f and g) and Blm / −/− −/− double mutant males and females were smaller in size and (Figures 2i and j) littermates, Blm Mus81 testes displayed the weighed significantly less than their WT and single mutant presence of tubules with disrupted spermiogenesis, occasional o - vacuolar degeneration and thinning of germinal epithelium littermates (P 0.0006; Figures 1a and b). Although 6- to 8-week −/− −/− old Blm−/−Mus81−/− mice showed no significant defects in their (Figures 2l and m). Some tubules of Blm Mus81 testes also total number of bone marrow cells and splenocytes (Figures 1c showed severe thinning of the germinal epithelium, with and d), their total number of thymocytes was significantly spermatogonia, spermatocytes and Sertoli cells still evident, but − − displayed a substantial decrease or total absence of more decreased (74.8 × 106 ± 11) compared with Mus81 / − − differentiated mature spermatids and sperm (Figures 2l and m). (120.9 × 106 ± 24.9; P = 0.005), Blm / (129 × 106 ± 25; P = 0.009) 6 These changes did not affect all seminiferous tubules, as some and WT (126.9 × 10 ± 2.7; P = 0.0004) littermates (Supplementary were histologically and functionally normal as evidenced by the Figure 1e). Flow cytometry analysis of bone marrow, spleen and presence of mature sperm cells in the corresponding epididymis thymocyte subpopulations indicated no significant difference −/− −/− (Figure 2n). No statistical difference was observed in the number between Blm Mus81 mice and their control littermates of PCNA-positive cells (for example, spermatogonia and pachy- (Supplementary Figure 1). The level of in vitro mitogen-induced tene spermatocytes) per tubule, indicating that disruption of Blm proliferation of B cells was also similar between double mutants and Mus81 does not compromise spermatogonial proliferation or and their controls (Supplementary Figure 2). These data indicate a entry to meiosis at young ages. Although cleaved caspase3 tissue-specific requirement for Blm and Mus81 during postnatal staining indicated no increased cell death in Blm−/−Mus81−/− development. testes compared with controls (Supplementary Figure 3),

Figure 1. Mice deﬁcient for Blm and Mus81 display growth defects. (a and b) Graphs showing body weight of 6- to 8-week-old WT, Mus81−/−, Blm−/− and Blm−/−Mus81−/− males and females (n = 4 to 6 for each genotype). *Po0.0007 compared with single mutants and WT controls. Graphs showing total cell number of bone marrow cells (c), splenocytes (d) and thymocytes (e) from 6- to 8-week-old mice from the indicated genotypes. *P = 0.0004 compared with single mutants and WT controls. Data are presented as the mean ± s.d. of four independent experiments.

Figure 2. Infertility of Blm−/−Mus81−/− mice. (a) Graphs showing number of litters and pups per litter from breeding of double mutant males to WT females and interbreeding of single mutant and WT controls. *Po0.05. (b) Representative pictures of testis from 8-week-old double mutant male, single mutant and WT littermates. Image of hematoxylin and eosin (H&E)-stained sections of seminiferous tubules of 6-week-old WT (c and d), Mus81−/− (f and g), Blm−/− (i and j) and Blm−/−Mus81−/− (l and m) mice. Images of H&E-stained sections of epididymis of 6-week- old WT (e), Mus81−/− (h), Blm−/− (k) and Blm−/−Mus81−/− (n) mice. Image of anti-cleaved caspase3-stained epididymis sections from 6-week-old WT (o), Mus81−/− (p), Blm−/− (q) and Blm−/−Mus81−/− (r) mice. Graph showing the percentage of cleaved caspase3-positive cells in the epididymis (s). Cleaved caspase3-positive cells were counted from 10 different ﬁelds. Data are presented as the mean ± s.d. *Po0.0001 compared with single mutants and WT controls. Bar: 500 μm.

apoptosis was significantly higher in the epididymis basal cells of immortalized mouse embryonic fibroblasts (MEFs). Blm−/− and − − Blm−/−Mus81−/− males compared with single mutant and WT Mus81 / MEFs displayed no statistically significant increased littermates (Po0.0001; Figures 2o–s). sensitivity to 2 Gy compared with WT controls (Figure 3a). Blm−/−Mus81−/− females also displayed reduced number of However, in response to 4 Gy, radiosensitivity of these mutant litters and litter sizes compared with controls (Supplementary MEFs was elevated compared with WT controls (Po0.05; Figure 4a). Ovarian histology revealed comparable number and Figure 3a). Interestingly, dual loss of Blm and Mus81 highly distribution of follicles at various stages of folliculogenesis sensitized MEFs to both 2 and 4 Gy compared with single mutant (Supplementary Figure 4b). However, these ovaries displayed and WT controls (Po0.001; Figure 3a). cholesterol crystal deposits. The formation of these deposits may To examine the effect of loss of Mus81 endonuclease activity on be explained by accumulated cholesterol not converted to the hyper-radiosensitivity of double mutant cells, we have estrogen by the granulosa cells. Despite their partial infertility generated catalytically inactive murine Mus81 that carries substitution of Asparatic acids 338 and 339 to Alanine (A) as but consistent with their apparently normal ovarian reserve, 21–23 −/− −/− −/− induced ovulation of double mutant females showed normal previously described. Mus81 and Blm Mus81 MEFs ovarian response with comparable number of ovulated oocytes to were reconstituted with either MSCV-Flag-Mus81-WT or MSCV- fi Flag-Mus81-D338A-D339A (MSCV-Mus81-DD). Expression of exo- single mutants and WT littermates. Taken together, these ndings fi indicate the cooperation of Blm and Mus81 in male and female genous Mus81 was con rmed using anti-Mus81 antibodies fertility. (Supplementary Figure 5). Clonogenic assays performed using reconstituted cells indicated that in contrast to Mus81-WT, the catalytic inactive Mus81-DD failed to rescue increased radio- Combined disruption of Blm and Mus81 increases sensitivity to sensitivity associated with loss of Mus81 (Figure 3b). radiation and DNA interstrand crosslinks Next, cohorts of age-matched Blm−/−Mus81−/− mice, their single Blm and Mus81 have important roles in DNA damage repair.2,8 mutant and WT littermates were subjected to 8 Gy total body IR Using clonogenic assays, we first examined the effects of dual and their survival was monitored for 17 days. Kaplan–Meier mutation of Blm and Mus81 in reponses to IR of SV40- analysis indicated that although radiosensitivity level remained

Figure 3. Dual loss of Blm and Mus81 leads to higher sensitivity to ionizing radiation and MMC. (a) Graph showing percent survival in a clonogenic assay of SV40-immortalized MEFs irradiated with 2 or 4 Gy γ-rays. (b) Graph showing clonogenic survival 10 days post IR (2-4 Gy) of SV40-immortalized MEFs reconstituted with MSCV-Mus81-WT or MSCV-Mus81-DD (D338A and D339A). (c) Kaplan–Meier survival analysis of cohorts of age-matched WT (n = 12), Mus81−/− (n = 9), Blm−/− (n = 12) and double mutant (n = 12) mice subjected to whole-body IR (8 Gy). Mice were monitored for survival for 17 days post IR. Log-rank test analysis indicated increased radiosensitivity of double mutant mice compared with controls (P = 0.0007 vs Mus81−/−, P = 0.027 vs Blm−/− and P = 0.0025 vs WT). (d) Graph showing percent survival in a clonogenic assay of SV40-immortalized MEFs treated with MMC (1–4 ng/ml). (e) Graph showing percent survival in a clonogenic assay of SV40-immortalized MEFs reconstituted with either MSCV-Mus81-WT or MSCV-Mus81-DD (D338A and D339A) and treated with MMC (1–4 ng/ml). (f) Kaplan–Meier survival analysis of cohorts of age-matched WT (n = 27), Mus81−/− (n = 24), Blm−/− (n = 23) and double mutant (n = 24) mice injected with 10 mg MMC/kg of body mass. Mice were monitored for survival for 28 days post MMC injection. Log-rank test analysis: P = 0.014 double mutant vs Mus81−/−; P = 0.014 double mutant vs Blm−/− mice; P = 0.03 Mus81−/− vs Blm−/−; P = 0.006 Mus81−/− vs WT mice. (a, b, d and e) Data are presented as the mean ± s.d. of three independent experiments. *Po0.05 double mutant vs single mutants. similar between single mutant and WT littermates, it was compared with WT controls (Po0.007; Figure 3f). Interestingly, − − − − significantly elevated in Blm / Mus81 / mice (Po0.03 compared sensitivity of Blm−/−Mus81−/− mice to MMC was significantly with single mutants; Figure 3c). exacerbated compared with single mutants (Po0.01; Figure 3f). The effect of dual loss of Blm and Mus81 on the response to Collectively, these data indicate a strong cooperation between DNA interstrand crosslinking agent mitomycin C (MMC) was also Blm and Mus81 in the response to IR- and interstrand crosslinking- examined. Clonogenic assays using SV40-immortalized MEFs induced DNA damage. They also demonstrate that the endonu- −/− −/− indicated that although Blm and Mus81 MEFs displayed clease activity of Mus81 is required for this cooperation. higher sensitivity to different MMC concentrations (1–4 ng) compared with WT controls, this sensitivity was further increased Mus81 deletion rescues the exacerbated SCE associated with Blm for double mutants (Po0.001, compared with single mutants; deficiency Figure 3d). Our data also indicated the importance of the loss of −/− Elevated level of SCE is a hallmark feature for Bloom’s syndrome Mus81 catalytic activity in the higher MMC sensitivity of Mus81 2,24 20 and double mutant cells (Figure 3e). cells, and was also observed in Blm mutant mice. To examine We also examined in vivo response to MMC and monitored the in vivo effect of Mus81 loss on elevated SCE associated with −/− −/− Blm mutation, we examined SCE levels in B cells from Blm−/− survival of cohorts of age-matched Blm Mus81 mice and their − − Blm−/−, Mus81−/− and WT littermates injected with MMC (10 mg/ Mus81 / mice, single mutants and WT littermates. kg of body mass). Blm−/− mice displayed no increased sensitivity Lipopolysaccharide-activated B cells were either left untreated or to MMC compared with WT littermates; however, the survival of treated with hydroxyurea (HU) or MMC. In accordance with Mus81−/− mice to MMC treatment was significantly reduced previous studies,11 Mus81−/− and WT B cells presented similar

Figure 4. Loss of Mus81 restrains elevated SCE and promotes IR-induced genomic instability of Blm−/− B cells. (a) Graphs showing the incidence of SCEs. Cells were either left untreated or treated with HU (80 μM) or MMC (50 ng/ml). A minimum of 25 metaphase spreads were analyzed for SCEs for each genotype and treatment in three independent experiments. Data are presented as the mean ± s.d. *Po0.0001. (b) Representative metaphases of B lymphocytes from Blm−/−Mus81−/−, Blm−/−, Mus81−/− and WT mice. Cells were treated with MMC (40 ng/ml) or IR (2 Gy) and metaphase spreads were prepared 24 h later. A minimum of 60 metaphase spreads were analyzed for each genotype and treatment in three independent experiments. b, chromosome/chromatid break; d, dicentric chromosome; f, chromosome fragment; rlc, Robertsonian fusion-like conﬁgurations; tce, triradial-like/chromatid-type exchange-like structures.

levels of spontaneous SCE, whereas Blm−/− cells displayed (Figure 4b, Table 1). Under untreated conditions, Blm−/−Mus81−/− elevated level of spontaneous as well as HU- or MMC-induced and Mus81−/− B cells displayed significantly higher levels of SCE (Po0.05; Figure 4a, Supplementary Figure 6). Remarkably, genomic instability compared with Blm−/− and WT controls loss of Mus81 in Blm−/− B cells fully restored their spontaneous (Po0.0003). In response to MMC (40 ng/ml) treatment, the level and induced SCE levels similar to WT controls (P>0.05; Figure 4a, of chromosomal aberrations was similarly elevated in Blm−/− Supplementary Figure 6). Mus81−/− and single mutant B-cell controls (P>0.05). However, To verify that restrained SCE level in Blm−/−Mus81−/− cells was post-IR (2 Gy), the level of total chromosomal aberrations was not due to cell cycle defects, we examined replication index of significantly higher in Blm−/−Mus81−/− B cells compared with single activated B cells from the different genotypes. Under untreated mutants and WT controls (Po0.00001). We also examined conditions, as well as post-treatment with MMC or HU, the ratios lipopolysaccharide-activated B cells for their number of γH2ax foci, between metaphases at the first, second and third cycle of a marker for DSBs.1 In response to IR (2 Gy) Blm−/−Mus81−/− Bcells replication remained similar between the four genotypes (P>0.05; showed significantly higher retention of γH2ax foci 24 h post- Supplementary Figure 7). These data indicate that in the absence treatment compared with Mus81−/−,Blm−/− and WT controls of Mus81, restrained levels of SCE of Blm−/− cells was not due to (Supplementary Figures 6a and b; Po0.001). Therefore, although replication defects. Collectively, these data suggest that elevated dual loss of Blm and Mus81 did not significantly increase level of SCE in Blm-deficient cells is Mus81 dependent. spontaneous and MMC-induced genomic instability compared with their individual loss, it resulted in far more pronounced IR- Increased genomic instability in the presence of dual mutations of induced genomic instability. Blm and Mus81 Loss of either Blm or Mus81 proteins has been associated with Dual loss of Blm and Mus81 restrains HR and restores NHEJ repair increased levels of genomic instability.2,11–15,20,25 To analyze the Cell cycle checkpoints are activated in response to DSBs and are effect of concomitant loss of Blm and Mus81 on genomic critical to the overall cellular response to DNA damage. To instability, we performed metaphase spread analysis of examine the effect of dual loss of Blm and Mus81 on the activation lipopolysaccharide-activated B cells from 6- to 8-week old Blm−/− of cell cycle checkpoints, we performed G2/M checkpoint analysis Mus81−/− mice and their single mutants and WT littermates in Blm−/−Mus81−/− MEFs and their single mutants and WT controls.

Table 1. Effect of dual loss of Blm and Mus81 on genomic stability

Sample ID Metaphases Aberrant Fragments/ Fusions Triradial-like Total scored cells breaks structures aberrations

WT UT 113 0 0 0 0 0 0.00 0.00 0.00 0.00 WT IR 75 8 5 9 1 15 10.67 6.67 12.00 1.33 20.00 WT MMC 102 15 8 10 4 22 14.71 7.84 9.80 21.57 Mus81−/− UT 75 4 6 1 2 9 5.33 8.00 1.33 2.67 12.00 Mus81−/− IR 91 17 18 10 3 31 18.68 19.78 10.99 3.30 34.07 Mus81−/− MMC 108 32 12 22 6 40 29.63 11.11 20.37 5.56 37.04 Blm−/− UT 91 3 3 0 0 3 3.30 3.30 0.00 3.30 Blm−/− IR 115 27 25 19 0 44 23.48 21.74 16.52 0.00 38.26 Blm−/− MMC 125 36 14 14 21 49 28.80 11.20 11.20 16.80 39.20 Blm−/−Mus81−/− UT 69 5 5 3 0 8 7.25 7.25 4.35 0.00 11.59 Blm−/−Mus81−/− IR 81 32 37 24 3 64 39.51 45.68 29.63 3.70 79.01 Blm−/−Mus81−/− MMC 120 35 13 22 14 49 29.17 10.83 18.33 11.67 40.83 Abbreviations: IR, irradiation; MMC, mitomycin C; UT, untreated; WT, wild type. Second row – percentage data shown. Fusions involve dicentrics, sister chromatid fusion, ring chromosomes and Robertsonian fusion-like conﬁgurations. Triradial-like structures include quadriradials and multicentric chromosomes.

We observed no difference in the activation of G2/M cell cycle reconstituted with Mus81-WT or the catalytic inactive Mus81-DD. checkpoint between double mutant and single mutant cells As expected Mus81−/− and Blm−/−Mus81−/− MEFs reconstituted (Figures 5a and b). with Mus81-WT displayed similar HR and NHEJ repair efficiency BLM and MUS81 have important roles in HR repair of DSBs.8,26,27 compared with WT and Blm−/− MEFs, respectively (Figures 5c and In addition, BLM has been also reported to interact with proteins d,). However, Mus81−/− and Blm−/−Mus81−/− MEFs reconstituted involved in NHEJ repair pathway (for example, 53BP1 and with Mus81-DD showed no differences in their efficiency of HR – LIGIV).28 30 To examine the effect of individual or combined loss and NHEJ repair compared with non-reconstituted controls. These of Blm and Mus81 on the repair pathways of DSBs, we used data demonstrate the importance of Mus81 endonuclease activity 31 reporter assays for HR and NHEJ and SV40-immortalized MEFs. for DSB repair pathways. 27 Consistent with a previous study, we observed reduced HR Collectively, these data indicated that although dual loss of Blm −/− repair activity in Blm MEFs compared with WT controls and Mus81 had no effect on G2/M checkpoint activation, it −/− (Po0.02; Figure 5c). Mus81 MEFs also displayed significantly exacerbated HR defects and significantly rescued impaired NHEJ reduced HR repair compared with WT controls (Po0.007; associated with individual loss of function of these proteins. Figure 5c). Interestingly, dual loss of Blm and Mus81 resulted in even higher HR defects compared with single loss of these Effect of dual loss of Mus81 and Blm on cancer development and proteins (Po0.0005; Figure 5c). In order to determine whether the the response of tumors to radiation observed HR defects were due directly to the loss of both Mus81 ’ 2 and Blm or whether they resulted from a defect in the recruitment Bloom s syndrome patients develop various types of cancer of more upstream molecules important for HR repair pathway, we and MUS81 has been reported to be downregulated in −/− −/− −/− human colorectal cancer, hepatocellular carcinoma and gastric have examined IR-induced Rad51 foci in Blm Mus81 , Blm , 16,17,33 −/− cancer. A MUS81 variant allele (rs545500) was also Mus81 and WT MEFs. No defect in Rad51 recruitment to DSBs 34 was observed in double and single mutant cells 6 h post IR associated with high risk of human breast cancer. Studies of (Supplementary Figures 8a and b). mouse models have also indicated that loss of either Blm or 2,13,15,20,35 Our analysis of NHEJ repair in Blm−/− and Mus81−/− cells Mus81 functions promotes tumorigenesis. To examine indicated its significant decrease compared with WT controls the effect of dual loss of Blm and Mus81 on cancer development, −/− −/− −/− (Po0.0001; Figure 5d). However, although the level of NHEJ repair we monitored cohorts of Blm Mus81 (n = 15), Blm (n = 16), −/− in double mutant MEFs remained lower compared with WT Mus81 (n = 20) and WT (n = 20) littermates for a period of controls, it was significantly rescued compared with single 19 months and examined their survival and incidence of mutants (Po0.0001; Figure 5d). These data are consistent with spontaneous tumors. Log-rank statistical analysis indicated that − − the mounting evidence that suggests crosstalk between HR and although the survival of Mus81 / mice was significantly reduced − − NHEJ repair pathways and compensatory activation of the NHEJ compared with WT littermates (P = 0.024), lifespan of Blm / mice pathway in cases of defective HR repair.32 was not statistically different from WT littermates (P>0.05; − − To examine the role the endonuclease activity of Mus81 has Figure 6a). Interestingly, additional loss of Blm in Mus81 / mice − − − − in DSB repair pathways, HR and NHEJ reporter assays further reduced their survival (Po0.05, Blm / Mus81 / mice vs were performed using Mus81−/− and Blm−/−Mus81−/− MEFs Mus81−/− mice; Figure 6a). Examination of moribund mice

Figure 5. Dual disruption of Blm and Mus81 has no effect on G2/M checkpoint but it exacerbates HR defects associated with single mutants. (a) Representative G2/M fluorescence-activated cell sorting (FACS) analysis of SV40-immortalized MEFs either untreated (UT) or 1 h post IR (2 Gy). (b) Graphs showing percentages of mitotic cells (positive for p-Ser10 Histone H3). At least five independent experiments using one mouse per group were performed. (c) Graphs showing the percentage of HR repair efficiency in SV40-immortalized Blm−/−Mus81−/−, Mus81−/−, Blm−/− and WT MEFs and in Blm−/−Mus81−/− and Mus81−/− MEFs reconstituted with either MSCV-Mus81-WT or MSCV-Mus81-DD. Immortalized MEFs were co-transfected with linearized pHR reporter and undigested red fluorescent protein (RFP) plasmid and FACS analysis was performed 48 h post transfection. (d) Graphs showing the percentage of NHEJ repair efficiency in Blm−/−Mus81−/−, Mus81−/−, Blm−/− and WT SV40-immortalized MEFs and in Blm−/−Mus81−/−, Mus81−/− counterparts reconstituted with MSCV-Mus81-WT or MSCV-Mus81-DD. MEFs were co-transfected with linearized pNHEJ reporter and undigested RFP plasmid and FACS analysis was performed as in c. Data are presented as the mean ± s.d. *Po0.02; ** Po0.0005.

indicated increased frequency of tumors in Blm−/−Mus81−/− mice Despite the interaction of BLM with MUS81,10,37 and their (12 out of 15; 80%) compared with Mus81−/− mice (6 out of 20; involvement in DSB repair and the restart of stalled replication, 30%) and Blm−/− mice (4 out of 16; 25%). Histological analysis of there is currently no evidence for in vivo interplay between these tumors indicated no differences in the spectrum of tumors mammalian proteins. To examine such interplay, we have targeted between double and single mutants, with lymphomas being the Mus81 mutation to homozygous Blmtm3Brd mice and assessed the most frequently observed tumors (Figures 6b–i). effect of these dual mutations on development, fertility, DNA The effect of dual loss of Blm and Mus81 on the response of damage repair, genomic integrity and cancer. tumors to radiation was also examined. B-cell lymphomas from Studies of lower organisms, including Saccharomyces cerevisiae Blm−/−Mus81−/− mice and single mutant littermates were either and Drosophila, indicated synthetic lethality of mutations of the left untreated or subjected to ex vivo IR (1 and 6 Gy) and their orthologs of BLM and MUS81.19,39 Our study indicated that survival level was determined 24 h later using Annexin Blmtm3Br/tm3BrdMus81Δex3-4/Δex3-4 mice were viable; however, in V/propidium iodide assay and flow cytometry. Although no contrast to their single mutant littermates, they displayed significant death was observed with the various lymphomas developmental defects including growth retardation. The syn- following 1 Gy of IR, Blm−/−Mus81−/− tumors displayed higher thetic lethality of double mutants in S. cerevisiae and Drosophila, radiosensitivity to 6 Gy compared with Blm−/− tumors (P = 0.04) but not in mice, supports the existence of a more complex and and Mus81−/− tumors (P = 0.009; Figure 6j). These data indicate the overlapping repair mechanisms in mammalian cells. cooperation of Blm and Mus81 in suppressing cancer and in tumor In fission yeast, mus81 mutations drastically reduce meiotic radiosensitivity. crossovers and spore viability,24,40,41 whereas in budding yeast, mus81 mutations only mildly affect meiosis.42–45 In contrast, Mus81 mutations in arabidopsis and Drosophila result in no significant DISCUSSION meiotic defects.19,46 The BLM ortholog Sgs1 in budding yeast The helicase BLM and the DNA structure-specific endonuclease collaborates with mus81/mms4 to promote meiotic recombina- MUS81 are important for the restart of stalled replication forks and tion, resolve replication forks generated during DNA damage for HR.8,9,26,36,37 Mutations of BLM in human are associated with response and has a major role in the resolution of unregulated Bloom’s syndrome.36 Patients with this syndrome and Blm mutant joint molecules.47–50 In the absence of Sgs1, these joint molecules mice display elevated level of SCE and cancer risk.2,20,38 are cleaved by Cdc5-dependent nucleases, including mus81-

Figure 6. Cooperation of Blm and Mus81 in tumor suppression. (a) Kaplan–Meier analysis representing the percent survival for Blm−/−Mus81−/− (n = 18), Blm−/− (n = 23), Mus81−/− (n = 18) and WT (n = 19) cohort mice. Log-rank test: P = 0.04, Blm−/−Mus81−/− vs Mus81−/−; Po0.0001, Blm−/− Mus81−/− vs WT; Po0.02, Mus81−/− vs WT. (b–i) Representative histology pictures of the observed tumors. Blm−/− (b and c) and Blm−/− Mus81−/− (d and e) lymph nodes stained with hematoxylin and eosin (b and d). Inset in b shows higher magnification of plasma cells. Inset in d, shows higher magnification of anti-B220 (brown) and anti-CD3 (purple) stained tumors. (c and e) A large area of lymph nodes invaded by B220-CD3- cells. Representative images of sections from thymus (f and g) and lung (h and I)ofBlm−/−Mus81−/− mice showing infiltration by a B220+ cell population. (j) Graphs showing the percentage of survival of Blm−/−Mus81−/−, Blm−/− and Mus81−/− tumor cells post-IR (1 and 6 Gy). Data are presented as the mean ± s.d. *Po0.05. Bar: 500 μm. mms4.47–49 In human, male patients with Bloom’s syndrome are targeted disruption of Rad54 in Blmtm3Brd/tm3Brd ES cells, rescued infertile, whereas females display subfertility.2 Studies of mutant elevated SCE associated with mutations of Blm.18,26 mice indicated that although the hypomorph Blmtm3Brd/tm3Brd It is possible that the case of Blm deficiency, DNA structures that mutants displayed no fertility defects, males carrying homozygous are preferentially repaired by a Blm-dependent pathway are left Blm null mutation in spermatocytes were sterile, indicating a unresolved and may be processed by Rad54-dependent or other requirement for the full function of this helicase for meiotic repair pathways. If mammalian Mus81 has a Blm-independent recombination.51 Mus81−/− mice (Mus81Δex3-4 and Mus81Δex9-12 function in DNA damage repair, its disruption should exacerbate strains) displayed no significant fertility defects,11,12,15 although the sensitivity to clastogenic agents. Indeed, double mutant MEFs Mus81 has been suggested to be involved in a minor alternative and mice displayed increased sensitivity to IR and MMC compared pathway that regulates crossover frequency during mammalian with single mutants. These data suggest the involvement of Blm spermatogenesis.51 Interestingly, in contrast to the fertility of and Mus81 in independent DNA damage repair pathways or that Blmtm3Brd and Mus81Δex3-4 homozygous mutants, both double they partially compensate for the loss of each other. Defective repair of DNA damage is a major cause for loss of homozygous males and females displayed infertility with reduced 1 number of litters and litter sizes and increased apoptosis of double genomic integrity. Although spontaneous genomic instability was observed in Blm and Mus81 mutant cells, its level in double mutants epididymis basal cells. These data support the impor- mutants was similar to Mus81 mutants. The levels of genomic tance of the collaboration of Mus81 and Blm in maintaining male instability in double mutant cells in response to MMC remained and female fertility. similar to single mutants, whereas it was significantly higher in Elevated genomic instability and SCE are hallmarks of Bloom’s response to IR. To examine mechanisms that may contribute to syndrome.2 Although the precise mechanism for increased fi increased IR-induced genomic instability in double mutants, we genomic instability in BLM-de cient cells remains unknown, it examined DSB repair pathways using reporter assays. Although likely results from collapsed replication forks induced DNA breaks loss of either Blm or Mus81 abrogated HR, impairment of this and their defective repair. Increased SCE in the Bloom's syndrome repair pathway was greater when both genes were defective and is likely the result of these DSBs seeking templates for repair. was not associated with impaired Rad51 recruitment to DSB sites. Several studies demonstrated the implication of Mus81-Ercc1 and Therefore, both Blm and Mus81 are required for efficient HR repair. Mus81-Rad54 pathways in promoting separation of sister chro- Remarkably, although NHEJ activity was reduced in Blm and matids, maintaining common fragile site integrity during replica- Mus81 mutant cells, we observed that concomitant to their 52–55 tion and resolving recombination. If Blm loss of function leads pronounced HR defects, double mutant cells displayed enhanced to a higher activation of these two pathways, disruption of Mus81 NHEJ-repair compared with single mutants. This finding suggests in Blm mutant cells should decrease their elevated level of SCE. that increased NHEJ activity in double mutant cells is likely a Indeed, our data indicate that Mus81 disruption suppresses compensatory mechanism for their reduced HR activity and is in − − elevated SCE of Blm / B cells. In accordance with these data, accordance with the reported interplay between these two knockdown of human MUS81 in Bloom’s syndrome cells and pathways.32

© 2015 Macmillan Publishers Limited Oncogene (2015) 1780 – 1789 In vivo functional interaction of Blm and Mus81 S El Ghamrasni et al 1788 Reduced usage of the error-free HR pathway combined with the HR and NHEJ reporter assays higher activity of the error-prone NHEJ repair pathway in double pNHEJ and pHR reporter plasmids31 were linearized using I-Sce1 (NEB, mutant cells suggested the possibility of higher cancer risks for Ipswich, MA, USA). Immortalized SV40 MEFs of each genotype were double mutant mice. On the other hand, elevated SCE is expected transfected with either linearized pNHEJ or pHR reporters together with ref to promote cancer through LOH of tumor suppressors.20 There- fluorescent protein plasmid using GenJet system (Frogga-Bio, North york, fl fore, decreased SCE levels in double mutants would suggest that it ON, Canada). Cells were analyzed by ow cytometry 48 h post-transfection fi may reduce their cancer predisposition. However, higher fre- and the ef ciency of repair was determined as the ratio of green fluorescent protein-positive cells to red fluorescent protein-positive cells. quency and earlier onset of tumors were observed in double mutants compared with their single mutant littermates. These data support that decreased SCE in double mutant mice to a level Tumor response to IR similar to WT does not protect them from cancer and that reduced Tumors were cultured in vitro and were either left untreated or irradiated HR levels together with increased activity of the error-prone NHEJ (1 and 6 Gy). Cell death was assessed using 7 aminoactinomycin D (7AAD) or Annexin V-propidium iodide staining and fluorescence-activated cell repair pathway are likely to contribute to increased tumorigenesis sorting analysis. of double mutants. In summary, our study highlights the in vivo importance of Mus81 and Blm in postnatal development and fertility. It also CONFLICT OF INTEREST further supports the importance of these proteins in DNA damage The authors declare no conflict of interest. repair and genome integrity. Although Blmtm3Br/tm3Brdd hypomorphic mutants failed to recapitulate many features of Bloom’s syndrome, some of these features, including growth defects, ACKNOWLEDGEMENTS infertility and increased cancer risks were observed with their We thank G Brown, P McPherson, R Hill and A Koch for reviewing the manuscript and additional loss of Mus81. for their valuable comments and suggestions. This study was supported by the Thus, fully functional Blm and Mus81 proteins are both required Canadian Institute of Health Research (R Hakem). to prevent developmental abnormalities and the pathogenesis of diseases including cancer. REFERENCES 1 Bohgaki T, Bohgaki M, Hakem R. DNA double-strand break signaling and human 1 MATERIALS AND METHODS disorders. Genome Integr 2010; : 15. 2 Chu WK, Hickson ID. 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