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

Subjects

Abstract

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.

Introduction

Defects in the DNA damage repair mechanisms promote genomic instability and lead to increased risks for developmental defects and diseases including cancer and immunodeficiency.1 BLM protein, a member of the evolutionarily conserved RecQ helicase family, functions in unwinding various branched DNA structures2 and also facilitates the recruitment of a number of DNA repair proteins (for example, EXO1, RAD51 and RAD51D) to DNA double-strand breaks (DSBs).3, 4, 5, 6 Importantly, mutations of BLM are associated with the human Bloom’s syndrome, an autosomal recessive disorder with clinical manifestations that include proportional dwarfism, immunodeficiency and infertility.2 Cells from Bloom’s syndrome patients display elevated frequency of sister chromatid exchanges (SCEs). Bloom’s syndrome is also associated with elevated incidence of cancer and a high degree of genomic instability.2 With its partners RMI1/RMI2/TOP3α, BLM forms a complex with FANCM at sites of DNA damage and defects in the formation of this complex result in higher SCE levels.7 BLM mediates both anti-recombinogenic functions as well as pro-recombinogenic functions in the homologous recombination (HR) repair pathway and its SUMOylation regulates the switch between its two functions.6 BLM responds to replication stress by accumulating at stalled DNA replication forks where it contributes to their restart.2

MUS81, with its partners EME1 or EME2, forms a DNA structure-specific endonuclease important for the restart of stalled replication forks and the resolution of Holliday junctions, critical intermediates of HR repair.8 MUS81 physically interacts and co-localizes with BLM at sites of stalled replication forks during the S-phase arrest.9,10 Although MUS81 does not affect BLM helicase activity, its endonuclease activity on nicked Holliday junctions and 3’flap structures is stimulated by BLM because of its enhanced binding to these DNA substrates.10 MUS81 mutations promote genomic instability in murine and human cells,11, 12, 13, 14, 15 and Mus81Δex3-4/Δex3-4 mutant mice display increased cancer predisposition.11,13,15 In addition, decreased MUS81 expression has been observed in different human cancers.16,17 Using Bloom syndrome cells, it was shown that deletion of MUS81 or SLX4 leads to decreased frequency of SCE, suggesting that in the absence of BLM nucleolytic-processing pathway, MUS81/SLX4 pathway is activated to allow chromosomal segregation.18 It was also shown in Drosophila that dual loss of DmBlm and Mus81 leads to increased apoptosis levels.19

Given the importance of BLM and MUS81 in DNA damage repair, and in order to examine the in vivo effects of their inactivation, we have generated a mouse model with dual inactivation of these proteins. Using single and double mutant mice for Blm and Mus81, we examined the effects of the loss of these proteins on development, fertility, DNA damage repair pathways and cancer. We report that although Blmtm3Brd/tm3Brd and Mus81Δex3-4/Δex3-4 mice were viable, double mutants displayed growth defects, infertility and impaired thymocyte homeostasis compared with single mutant and wild-type (WT) littermates. Double mutant cells displayed remarkably reduced HR repair activity accompanied with elevated non-homologous-end-joining (NHEJ) repair compared with single mutants. Irradiation (IR)-induced genomic instability was significantly higher in double mutants compared with single mutants. Although elevated SCE was proposed to drive cancer development in Blm mutants, double mutant mice were more cancer prone compared with single mutant littermates despite their reduced SCE levels. Collectively, our data highlight the importance of Blm and Mus81 functions in DNA damage repair, fertility, development and cancer.

Results

Blm−/−Mus81−/− mice are viable but display growth defects

To generate mice with dual loss of function of Blm and Mus81, we crossed mice that carry the Blmtm3Brd hypomorph allele20 to Mus81Δex3-4 mutant mice11 (referred to herein as Blm−/− and Mus81−/− mice, respectively). Blm−/−Mus81−/− mice were obtained in the expected Mendelian ratio from intercrossing of Blm+/-Mus81−/− mice (Supplementary Table 1). However, both double mutant males and females were smaller in size and weighed significantly less than their WT and single mutant littermates (P<0.0006; Figures 1a and b). Although 6- to 8-week-old Blm−/−Mus81−/− mice showed no significant defects in their total number of bone marrow cells and splenocytes (Figures 1c and d), their total number of thymocytes was significantly decreased (74.8 × 106±11) compared with Mus81−/− (120.9 × 106±24.9; P=0.005), Blm−/− (129 × 106±25; P=0.009) and WT (126.9 × 106±2.7; P=0.0004) littermates (Supplementary Figure 1e). Flow cytometry analysis of bone marrow, spleen and thymocyte subpopulations indicated no significant difference between Blm−/−Mus81−/− mice and their control littermates (Supplementary Figure 1). The level of in vitro mitogen-induced proliferation of B cells was also similar between double mutants and their controls (Supplementary Figure 2). These data indicate a tissue-specific requirement for Blm and Mus81 during postnatal development.

Figure 1
figure1

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). *P<0.0007 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.

Concomitant requirement for Blm and Mus81 for male and female fertility

Defective DSB repair promotes sterility, and Bloom’s syndrome is associated with male infertility and female subfertility.2 Therefore, we examined the fertility of Blm−/−Mus81−/− mice and their control littermates. Previous studies of Blmtm3Brd/tm3Brd mice indicated no fertility defects in these hypomorphic mutants.20 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 with WT littermates (Supplementary Table 1), crossing of double mutant males to WT females yielded significantly reduced number of litters and the number of pups per litter was also smaller compared with control littermates (Figure 2a). In addition, consistent with impaired fertility, testes of 6- to 8-week-old Blm−/−Mus81−/− males were smaller in size compared with their single mutant and WT littermates (Figure 2b). Hematoxylin and eosin staining of testis sections indicated that in contrast to WT (Figures 2c and d), Mus81−/− (Figures 2f and g) and Blm−/− (Figures 2i and j) littermates, Blm−/−Mus81−/− testes displayed the presence of tubules with disrupted spermiogenesis, occasional vacuolar degeneration and thinning of germinal epithelium (Figures 2l and m). Some tubules of Blm−/−Mus81−/− testes also showed severe thinning of the germinal epithelium, with spermatogonia, spermatocytes and Sertoli cells still evident, but displayed a substantial decrease or total absence of more differentiated mature spermatids and sperm (Figures 2l and m). These changes did not affect all seminiferous tubules, as some were histologically and functionally normal as evidenced by the presence of mature sperm cells in the corresponding epididymis (Figure 2n). No statistical difference was observed in the number of PCNA-positive cells (for example, spermatogonia and pachytene spermatocytes) per tubule, indicating that disruption of Blm and Mus81 does not compromise spermatogonial proliferation or entry to meiosis at young ages. Although cleaved caspase3 staining indicated no increased cell death in Blm−/−Mus81−/− testes compared with controls (Supplementary Figure 3), apoptosis was significantly higher in the epididymis basal cells of Blm−/−Mus81−/− males compared with single mutant and WT littermates (P<0.0001; Figures 2o–s).

Figure 2
figure2

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. *P<0.05. (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. *P<0.0001 compared with single mutants and WT controls. Bar: 500 μm.

Blm−/−Mus81−/− females also displayed reduced number of litters and litter sizes compared with controls (Supplementary Figure 4a). Ovarian histology revealed comparable number and distribution of follicles at various stages of folliculogenesis (Supplementary Figure 4b). However, these ovaries displayed cholesterol crystal deposits. The formation of these deposits may be explained by accumulated cholesterol not converted to estrogen by the granulosa cells. Despite their partial infertility but consistent with their apparently normal ovarian reserve, induced ovulation of double mutant females showed normal ovarian response with comparable number of ovulated oocytes to single mutants and WT littermates. Taken together, these findings indicate the cooperation of Blm and Mus81 in male and female fertility.

Combined disruption of Blm and Mus81 increases sensitivity to radiation and DNA interstrand crosslinks

Blm and Mus81 have important roles in DNA damage repair.2,8 Using clonogenic assays, we first examined the effects of dual mutation of Blm and Mus81 in reponses to IR of SV40-immortalized mouse embryonic fibroblasts (MEFs). Blm−/− and Mus81−/− MEFs displayed no statistically significant increased sensitivity to 2 Gy compared with WT controls (Figure 3a). However, in response to 4 Gy, radiosensitivity of these mutant MEFs was elevated compared with WT controls (P<0.05; Figure 3a). Interestingly, dual loss of Blm and Mus81 highly sensitized MEFs to both 2 and 4 Gy compared with single mutant and WT controls (P<0.001; Figure 3a).

Figure 3
figure3

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. *P<0.05 double mutant vs single mutants.

To examine the effect of loss of Mus81 endonuclease activity on the hyper-radiosensitivity of double mutant cells, we have generated catalytically inactive murine Mus81 that carries substitution of Asparatic acids 338 and 339 to Alanine (A) as previously described.21, 22, 23 Mus81−/− and Blm−/−Mus81−/− MEFs were reconstituted with either MSCV-Flag-Mus81-WT or MSCV-Flag-Mus81-D338A-D339A (MSCV-Mus81-DD). Expression of exogenous Mus81 was confirmed using anti-Mus81 antibodies (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 radiosensitivity associated with loss of Mus81 (Figure 3b).

Next, cohorts of age-matched Blm−/−Mus81−/− mice, their single mutant and WT littermates were subjected to 8 Gy total body IR and their survival was monitored for 17 days. Kaplan–Meier analysis indicated that although radiosensitivity level remained similar between single mutant and WT littermates, it was significantly elevated in Blm−/−Mus81−/− mice (P<0.03 compared with single mutants; Figure 3c).

The effect of dual loss of Blm and Mus81 on the response to DNA interstrand crosslinking agent mitomycin C (MMC) was also examined. Clonogenic assays using SV40-immortalized MEFs indicated that although Blm−/− and Mus81−/− MEFs displayed higher sensitivity to different MMC concentrations (1–4 ng) compared with WT controls, this sensitivity was further increased for double mutants (P<0.001, compared with single mutants; Figure 3d). Our data also indicated the importance of the loss of Mus81 catalytic activity in the higher MMC sensitivity of Mus81−/− and double mutant cells (Figure 3e).

We also examined in vivo response to MMC and monitored survival of cohorts of age-matched Blm−/−Mus81−/− mice and their Blm−/−, Mus81−/− and WT littermates injected with MMC (10 mg/kg of body mass). Blm−/− mice displayed no increased sensitivity to MMC compared with WT littermates; however, the survival of Mus81−/− mice to MMC treatment was significantly reduced compared with WT controls (P<0.007; Figure 3f). Interestingly, sensitivity of Blm−/−Mus81−/− mice to MMC was significantly exacerbated compared with single mutants (P<0.01; Figure 3f).

Collectively, these data indicate a strong cooperation between Blm and Mus81 in the response to IR- and interstrand crosslinking-induced DNA damage. They also demonstrate that the endonuclease activity of Mus81 is required for this cooperation.

Mus81 deletion rescues the exacerbated SCE associated with Blm deficiency

Elevated level of SCE is a hallmark feature for Bloom’s syndrome cells,2,24 and was also observed in Blm mutant mice.20 To examine the in vivo effect of Mus81 loss on elevated SCE associated with Blm mutation, we examined SCE levels in B cells from Blm−/− Mus81−/− mice, single mutants and WT littermates. Lipopolysaccharide-activated B cells were either left untreated or treated with hydroxyurea (HU) or MMC. In accordance with previous studies,11 Mus81−/− and WT B cells presented similar levels of spontaneous SCE, whereas Blm−/− cells displayed elevated level of spontaneous as well as HU- or MMC-induced SCE (P<0.05; Figure 4a, Supplementary Figure 6). Remarkably, loss of Mus81 in Blm−/− B cells fully restored their spontaneous and induced SCE levels similar to WT controls (P>0.05; Figure 4a,Supplementary Figure 6).

Figure 4
figure4

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. *P<0.0001. (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.

To verify that restrained SCE level in Blm−/−Mus81−/− cells was not due to cell cycle defects, we examined replication index of activated B cells from the different genotypes. Under untreated conditions, as well as post-treatment with MMC or HU, the ratios between metaphases at the first, second and third cycle of replication remained similar between the four genotypes (P>0.05; Supplementary Figure 7). These data indicate that in the absence of Mus81, restrained levels of SCE of Blm−/− cells was not due to replication defects. Collectively, these data suggest that elevated level of SCE in Blm-deficient cells is Mus81 dependent.

Increased genomic instability in the presence of dual mutations of Blm and Mus81

Loss of either Blm or Mus81 proteins has been associated with increased levels of genomic instability.2,11, 12, 13, 14, 15,20,25 To analyze the effect of concomitant loss of Blm and Mus81 on genomic instability, we performed metaphase spread analysis of lipopolysaccharide-activated B cells from 6- to 8-week old Blm−/− Mus81−/− mice and their single mutants and WT littermates (Figure 4b, Table 1). Under untreated conditions, Blm−/−Mus81−/− and Mus81−/− B cells displayed significantly higher levels of genomic instability compared with Blm−/− and WT controls (P<0.0003). In response to MMC (40 ng/ml) treatment, the level of chromosomal aberrations was similarly elevated in Blm−/−Mus81−/− and single mutant B-cell controls (P>0.05). However, post-IR (2 Gy), the level of total chromosomal aberrations was significantly higher in Blm−/−Mus81−/− B cells compared with single mutants and WT controls (P<0.00001). We also examined lipopolysaccharide-activated B cells for their number of γH2ax foci, a marker for DSBs.1 In response to IR (2 Gy) Blm−/−Mus81−/− B cells showed significantly higher retention of γH2ax foci 24 h post-treatment compared with Mus81−/−, Blm−/− and WT controls (Supplementary Figures 6a and b; P<0.001). Therefore, although dual loss of Blm and Mus81 did not significantly increase spontaneous and MMC-induced genomic instability compared with their individual loss, it resulted in far more pronounced IR-induced genomic instability.

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

Dual loss of Blm and Mus81 restrains HR and restores NHEJ repair

Cell cycle checkpoints are activated in response to DSBs and are critical to the overall cellular response to DNA damage. To examine the effect of dual loss of Blm and Mus81 on the activation of cell cycle checkpoints, we performed G2/M checkpoint analysis in Blm−/−Mus81−/− MEFs and their single mutants and WT controls. We observed no difference in the activation of G2/M cell cycle checkpoint between double mutant and single mutant cells (Figures 5a and b).

Figure 5
figure5

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. *P<0.02; ** P<0.0005.

BLM and MUS81 have important roles in HR repair of DSBs.8,26,27 In addition, BLM has been also reported to interact with proteins involved in NHEJ repair pathway (for example, 53BP1 and LIGIV).28, 29, 30 To examine the effect of individual or combined loss of Blm and Mus81 on the repair pathways of DSBs, we used reporter assays for HR and NHEJ31 and SV40-immortalized MEFs. Consistent with a previous study,27 we observed reduced HR repair activity in Blm−/− MEFs compared with WT controls (P<0.02; Figure 5c). Mus81−/− MEFs also displayed significantly reduced HR repair compared with WT controls (P<0.007; Figure 5c). Interestingly, dual loss of Blm and Mus81 resulted in even higher HR defects compared with single loss of these proteins (P<0.0005; Figure 5c). In order to determine whether the observed HR defects were due directly to the loss of both Mus81 and Blm or whether they resulted from a defect in the recruitment of more upstream molecules important for HR repair pathway, we have examined IR-induced Rad51 foci in Blm−/−Mus81−/−, Blm−/−, Mus81−/− and WT MEFs. No defect in Rad51 recruitment to DSBs was observed in double and single mutant cells 6 h post IR (Supplementary Figures 8a and b).

Our analysis of NHEJ repair in Blm−/− and Mus81−/− cells indicated its significant decrease compared with WT controls (P<0.0001; Figure 5d). However, although the level of NHEJ repair in double mutant MEFs remained lower compared with WT controls, it was significantly rescued compared with single mutants (P<0.0001; Figure 5d). These data are consistent with the mounting evidence that suggests crosstalk between HR and NHEJ repair pathways and compensatory activation of the NHEJ pathway in cases of defective HR repair.32

To examine the role the endonuclease activity of Mus81 has in DSB repair pathways, HR and NHEJ reporter assays were performed using Mus81−/− and Blm−/−Mus81−/− MEFs reconstituted with Mus81-WT or the catalytic inactive Mus81-DD. As expected Mus81−/−and Blm−/−Mus81−/− MEFs reconstituted with Mus81-WT displayed similar HR and NHEJ repair efficiency compared with WT and Blm−/− MEFs, respectively (Figures 5c and d). However, Mus81−/− and Blm−/−Mus81−/− MEFs reconstituted with Mus81-DD showed no differences in their efficiency of HR and NHEJ repair compared with non-reconstituted controls. These data demonstrate the importance of Mus81 endonuclease activity for DSB repair pathways.

Collectively, these data indicated that although dual loss of Blm and Mus81 had no effect on G2/M checkpoint activation, it exacerbated HR defects and significantly rescued impaired NHEJ associated with individual loss of function of these proteins.

Effect of dual loss of Mus81 and Blm on cancer development and the response of tumors to radiation

Bloom’s syndrome patients develop various types of cancer2 and MUS81 has been reported to be downregulated in human colorectal cancer, hepatocellular carcinoma and gastric cancer.16,17,33 A MUS81 variant allele (rs545500) was also associated with high risk of human breast cancer.34 Studies of mouse models have also indicated that loss of either Blm or Mus81 functions promotes tumorigenesis.2,13,15,20,35 To examine the effect of dual loss of Blm and Mus81 on cancer development, we monitored cohorts of Blm−/−Mus81−/−(n=15), Blm−/−(n=16), Mus81−/−(n=20) and WT (n=20) littermates for a period of 19 months and examined their survival and incidence of spontaneous tumors. Log-rank statistical analysis indicated that although the survival of Mus81−/− mice was significantly reduced compared with WT littermates (P=0.024), lifespan of Blm−/− mice was not statistically different from WT littermates (P>0.05; Figure 6a). Interestingly, additional loss of Blm in Mus81−/− mice further reduced their survival (P<0.05, Blm−/−Mus81−/− mice vs Mus81−/− mice; Figure 6a). Examination of moribund mice indicated increased frequency of tumors in Blm−/−Mus81−/− mice (12 out of 15; 80%) compared with Mus81−/− mice (6 out of 20; 30%) and Blm−/− mice (4 out of 16; 25%). Histological analysis of tumors indicated no differences in the spectrum of tumors between double and single mutants, with lymphomas being the most frequently observed tumors (Figures 6b–i).

Figure 6
figure6

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−/−; P<0.0001, Blm−/−Mus81−/− vs WT; P<0.02, Mus81−/− vs WT. (bi) 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) of Blm−/−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. *P<0.05. Bar: 500 μm.

The effect of dual loss of Blm and Mus81 on the response of tumors to radiation was also examined. B-cell lymphomas from Blm−/−Mus81−/− mice and single mutant littermates were either left untreated or subjected to ex vivo IR (1 and 6 Gy) and their survival level was determined 24 h later using Annexin V/propidium iodide assay and flow cytometry. Although no significant death was observed with the various lymphomas following 1 Gy of IR, Blm−/−Mus81−/− tumors displayed higher radiosensitivity to 6 Gy compared with Blm−/− tumors (P=0.04) and Mus81−/− tumors (P=0.009; Figure 6j). These data indicate the cooperation of Blm and Mus81 in suppressing cancer and in tumor radiosensitivity.

Discussion

The helicase BLM and the DNA structure-specific endonuclease MUS81 are important for the restart of stalled replication forks and for HR.8,9,26,36,37 Mutations of BLM in human are associated with Bloom’s syndrome.36 Patients with this syndrome and Blm mutant mice display elevated level of SCE and cancer risk.2,20,38

Despite the interaction of BLM with MUS81,10,37 and their involvement in DSB repair and the restart of stalled replication, there is currently no evidence for in vivo interplay between these mammalian proteins. To examine such interplay, we have targeted Mus81 mutation to homozygous Blmtm3Brd mice and assessed the effect of these dual mutations on development, fertility, DNA damage repair, genomic integrity and cancer.

Studies of lower organisms, including Saccharomyces cerevisiae and Drosophila, indicated synthetic lethality of mutations of the orthologs of BLM and MUS81.19,39 Our study indicated that Blmtm3Br/tm3BrdMus81Δex3-4/Δex3-4 mice were viable; however, in contrast to their single mutant littermates, they displayed developmental defects including growth retardation. The synthetic lethality of double mutants in S. cerevisiae and Drosophila, but not in mice, supports the existence of a more complex and overlapping repair mechanisms in mammalian cells.

In fission yeast, mus81 mutations drastically reduce meiotic crossovers and spore viability,24,40,41 whereas in budding yeast, mus81 mutations only mildly affect meiosis.42, 43, 44, 45 In contrast, Mus81 mutations in arabidopsis and Drosophila result in no significant meiotic defects.19,46 The BLM ortholog Sgs1 in budding yeast collaborates with mus81/mms4 to promote meiotic recombination, resolve replication forks generated during DNA damage response and has a major role in the resolution of unregulated joint molecules.47, 48, 49, 50 In the absence of Sgs1, these joint molecules are cleaved by Cdc5-dependent nucleases, including mus81-mms4.47, 48, 49 In human, male patients with Bloom’s syndrome are infertile, whereas females display subfertility.2 Studies of mutant mice indicated that although the hypomorph Blmtm3Brd/tm3Brd mutants displayed no fertility defects, males carrying homozygous Blm null mutation in spermatocytes were sterile, indicating a requirement for the full function of this helicase for meiotic recombination.51 Mus81−/− mice (Mus81Δex3-4 and Mus81Δex9-12 strains) displayed no significant fertility defects,11,12,15 although Mus81 has been suggested to be involved in a minor alternative pathway that regulates crossover frequency during mammalian spermatogenesis.51 Interestingly, in contrast to the fertility of Blmtm3Brd and Mus81Δex3-4 homozygous mutants, both double homozygous males and females displayed infertility with reduced number of litters and litter sizes and increased apoptosis of double mutants epididymis basal cells. These data support the importance of the collaboration of Mus81 and Blm in maintaining male and female fertility.

Elevated genomic instability and SCE are hallmarks of Bloom’s syndrome.2 Although the precise mechanism for increased genomic instability in BLM-deficient cells remains unknown, it likely results from collapsed replication forks induced DNA breaks and their defective repair. Increased SCE in the Bloom's syndrome is likely the result of these DSBs seeking templates for repair. Several studies demonstrated the implication of Mus81-Ercc1 and Mus81-Rad54 pathways in promoting separation of sister chromatids, maintaining common fragile site integrity during replication and resolving recombination.52, 53, 54, 55 If Blm loss of function leads to a higher activation of these two pathways, disruption of Mus81 in Blm mutant cells should decrease their elevated level of SCE. Indeed, our data indicate that Mus81 disruption suppresses elevated SCE of Blm−/− B cells. In accordance with these data, knockdown of human MUS81 in Bloom’s syndrome cells and targeted disruption of Rad54 in Blmtm3Brd/tm3Brd ES cells, rescued elevated SCE associated with mutations of Blm.18,26

It is possible that the case of Blm deficiency, DNA structures that are preferentially repaired by a Blm-dependent pathway are left unresolved and may be processed by Rad54-dependent or other repair pathways. If mammalian Mus81 has a Blm-independent function in DNA damage repair, its disruption should exacerbate the sensitivity to clastogenic agents. Indeed, double mutant MEFs and mice displayed increased sensitivity to IR and MMC compared with single mutants. These data suggest the involvement of Blm and Mus81 in independent DNA damage repair pathways or that they partially compensate for the loss of each other.

Defective repair of DNA damage is a major cause for loss of genomic integrity.1 Although spontaneous genomic instability was observed in Blm and Mus81 mutant cells, its level in double mutants was similar to Mus81 mutants. The levels of genomic instability in double mutant cells in response to MMC remained similar to single mutants, whereas it was significantly higher in response to IR. To examine mechanisms that may contribute to increased IR-induced genomic instability in double mutants, we examined DSB repair pathways using reporter assays. Although loss of either Blm or Mus81 abrogated HR, impairment of this repair pathway was greater when both genes were defective and was not associated with impaired Rad51 recruitment to DSB sites. Therefore, both Blm and Mus81 are required for efficient HR repair. Remarkably, although NHEJ activity was reduced in Blm and Mus81 mutant cells, we observed that concomitant to their pronounced HR defects, double mutant cells displayed enhanced NHEJ-repair compared with single mutants. This finding suggests that increased NHEJ activity in double mutant cells is likely a compensatory mechanism for their reduced HR activity and is in accordance with the reported interplay between these two pathways.32

Reduced usage of the error-free HR pathway combined with the higher activity of the error-prone NHEJ repair pathway in double mutant cells suggested the possibility of higher cancer risks for double mutant mice. On the other hand, elevated SCE is expected to promote cancer through LOH of tumor suppressors.20 Therefore, decreased SCE levels in double mutants would suggest that it may reduce their cancer predisposition. However, higher frequency 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 similar to WT does not protect them from cancer and that reduced HR levels together with increased activity of the error-prone NHEJ repair pathway are likely to contribute to increased tumorigenesis of double mutants.

In summary, our study highlights the in vivo importance of Mus81 and Blm in postnatal development and fertility. It also further supports the importance of these proteins in DNA damage 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, infertility and increased cancer risks were observed with their additional loss of Mus81.

Thus, fully functional Blm and Mus81 proteins are both required to prevent developmental abnormalities and the pathogenesis of diseases including cancer.

Materials and methods

Mice

In order to obtain double heterozygote mice, Mus81Δex3-4 mice11 were crossed with Blmtm3Brd mice20 obtained from the MMHCC repository at the NIH (strain B6; 129S-Blmtm3Brd). Double heterozygote mice were then crossed to obtain Blmtm3Brd/tm3BrdMus81Δex3-4/Δex3-4, single mutants and WT littermates. All mice were in a mixed 129/J x C57BL/6 genetic background and were genotyped by Southern blot or PCR as previously described.11,20 All experiments were performed in agreement with the guidelines of the animal care committee of the Ontario Cancer Institute/University Health Network.

Site-directed mutagenesis

Two point mutations of Mus81 (D338A and D339A) were generated using Phusion Site-Directed Mutagenesis Kit (Thermo Science, Burlington, ON, Canada). Mus81-WT and Mus81-DD were cloned into MSCV-Puro 3’-Flag retrovirus vector. MEFs were infected with these retroviruses and selected with Puromycin.

Chromosomal aberration analysis

Lipopolysaccharide (10 μg/ml)-activated splenocytes were cultured for 48 h in the presence or absence of MMC (40 ng/ml) or treated with 2 Gy of IR for 24 h and metaphase spreads were prepared as previously described.13,15 Chromosomal aberrations were determined for a minimum of 60 metaphase spreads per cell type. Statistical analysis was done using Fisher Exact test as reported previously.56

In vivo response to clastogenic agents

Mice from each genotype (n=12–15) were injected intraperitoneally with 10 mg MMC/Kg of body mass or treated with 8 Gy of IR. Mice were killed when they became moribund and the day of killing was counted as day of death.

Clonogenic assay

Immortalized SV40 MEFs were seeded at 500 cells per 60 mm dish. MEFs were either left untreated or treated with IR or MMC. Ten days later, colonies were fixed with ice-cold methanol and stained with 25% methanol/crystal violet and were counted.

HR and NHEJ reporter assays

pNHEJ and pHR reporter plasmids31 were linearized using I-Sce1 (NEB, Ipswich, MA, USA). Immortalized SV40 MEFs of each genotype were transfected with either linearized pNHEJ or pHR reporters together with ref fluorescent protein plasmid using GenJet system (Frogga-Bio, North york, ON, Canada). Cells were analyzed by flow cytometry 48 h post-transfection and the efficiency of repair was determined as the ratio of green fluorescent protein-positive cells to red fluorescent protein-positive cells.

Tumor response to IR

Tumors were cultured in vitro and were either left untreated or irradiated (1 and 6 Gy). Cell death was assessed using 7 aminoactinomycin D (7AAD) or Annexin V-propidium iodide staining and fluorescence-activated cell sorting analysis.

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Acknowledgements

We thank G Brown, P McPherson, R Hill and A Koch for reviewing the manuscript and for their valuable comments and suggestions. This study was supported by the Canadian Institute of Health Research (R Hakem).

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El Ghamrasni, S., Cardoso, R., Halaby, M. et al. Cooperation of Blm and Mus81 in development, fertility, genomic integrity and cancer suppression. Oncogene 34, 1780–1789 (2015). https://doi.org/10.1038/onc.2014.121

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