NBS1 is required for SPO11-linked DNA double-strand break repair in male meiosis

Abstract

DNA double-strand breaks (DSBs) pose a serious threat to genomic stability. Paradoxically, hundreds of programed DSBs are generated by SPO11 in meiotic prophase, which are exclusively repaired by homologous recombination (HR) to promote obligate crossover between homologous chromosomes. In somatic cells, MRE11-RAD50-NBS1 (MRN) complex-dependent DNA end resection is a prerequisite for HR repair, especially for DSBs that are covalently linked with proteins or chemicals. Interestingly, all meiotic DSBs are linked with SPO11 after being generated. Although MRN complex's function in meiotic DSB repair has been established in lower organisms, the role of MRN complex in mammalian meiotic DSB repair is not clear. Here, we show that MRN complex is essential for repairing meiotic SPO11-linked DSBs in male mice. In male germ cells, conditional inactivation of NBS1, a key component of MRN complex, causes dramatic reduction of DNA end resection and defective HR repair in meiotic prophase. NBS1 loss severely disrupts chromosome synapsis, generates abnormal chromosome structures, and eventually leads to meiotic arrest and male infertility in mice. Unlike in somatic cells, the recruitment of NBS1 to SPO11-linked DSB sites is MDC1-independent but requires other phosphorylated proteins. Collectively, our study not only reveals the significance of MRN complex in repairing meiotic DSBs but also discovers a unique mechanism that recruits MRN complex to SPO11-linked DSB sites.

Fig. 1. NBS1 deficiency leads to etoposide sensitivity.

a Western blotting analyses of HeLa cells after NBS1 depletion by siRNA. α-tubulin was used as loading control. b Cell survival assays of NBS1-depleted HeLa cells with indicated doses of etoposide. c Western blotting analyses of TDP2 KO HeLa cells. α-tubulin was used as loading control. d Cell survival assays of TDP2 KO HeLa cells with indicated doses of etoposide. e Western blotting analyses of NBS1 protein level in TDP2 KO HeLa cells transfected with NBS1 siRNAs. α-tubulin was used as loading control. f Cell survival assays of TDP2 KO HeLa cells after NBS1 depletion with indicated doses of etoposide. g Summary of IC₅₀ values (half maximal inhibitory concentration) of etoposide in the indicated HeLa cell lines. Error bars represent standard deviation, n = 3.

Fig. 2. NBS1 deficiency leads to male infertility.

a Schematic illustrations of mating strategies for obtaining Nbs1 vKO mice. b Western blotting analyses of NBS1 in germ cells of Nbs1 vKO male mice at postnatal day 21 (P21). α-tubulin was used as loading control. c Cumulative numbers of pups per male obtained by mating control or Nbs1 vKO male mice with WT female mice. Three males of each genotype were used for mating. d H/E staining of paraffin sections of epididymis from control and Nbs1 vKO male mice (P42). Scale bar, 200 µm. e H/E staining of paraffin sections of ovaries from control and Nbs1 vKO female mice (P21). Scale bar, 200 µm. f Testes image of adult control and Nbs1 vKO male mice. g Testis to body weight ratios of control and Nbs1 vKO male mice (P7, P21, and P42). Error bars represent standard error of mean (SEM), n = 6. h H/E staining of paraffin sections of testes from control and Nbs1 vKO male mice (P21). Scale bar, 200 µm. i TUNEL staining of testis frozen sections from control and Nbs1 vKO mice (P21). The DNA was stained with DAPI. Scale bar, 20 µm. j Immunofluorescence staining analyses of PLZF in testis frozen sections from control and Nbs1 vKO mice (P21). Scale bar, 40 µm. The DNA was stained with DAPI.

Fig. 3. NBS1 is required for chromosome synapsis and meiotic progression.

a Immunofluorescence staining of SYCP3 and γH2AX was used for analyses of stages of meiotic prophase in spermatocytes from control and Nbs1 vKO mice. Scale bar, 5 µm. b Percentage of spermatocytes at each stage of meiotic prophase from control and Nbs1 vKO mice. Error bars represent standard deviation, n = 3. L leptotene, Z zygotene, P pachytene, D diplotene. c Typical abnormal chromosome structures in Nbs1 vKO zygotene spermatocytes. White boxes indicate the sites of abnormal chromosomes, and are magnified on the right. Scale bar, 5 µm. d Number of abnormal chromosome structures per zygotene spermatocyte from control and Nbs1 vKO mice. Error bars represent SEM, n = 30. e Immunofluorescence staining of SYCP1 and SYCP3 were used for analyses of chromosome synapsis at late leptotene and zygotene stages of meiotic prophase from control and Nbs1 vKO mice. Scale bar, 5 µm. Defective chromosome synapsis was observed in all Nbs1 vKO spermatocytes (100%) but none of the control spermatocytes (0%).

Fig. 4. NBS1 is required for DNA end resection and HR repair at meiotic prophase.

Surface spreads of control and Nbs1 vKO mice were incubated with antibodies against RAD51 (a), DMC1 (b), RPA2 (c), and MEIOB (d). Representative surface spreads were shown on the left and the statistical charts were shown on the right. Scale bar, 5 µm. Error bars represent SEM, n ≥ 60.

Fig. 5. MDC1 is dispensable for NBS1’s function at meiotic prophase.

a Immunofluorescence staining analyses of MDC1 and NBS1 in Mdc1^+/+ and Mdc1^−/− MEFs after treatments of 10 µM bleomycin for 6 h or 5 µM etoposide for 2 h. γH2AX marks DNA damage sites. Scale bar, 10 µm. b Immunofluorescence staining of SYCP3 and γH2AX were used for analyses of stages of meiotic prophase in spermatocytes from Mdc1^−/− mice. Scale bar, 5 µm. Surface spreads of zygotene spermatocytes from Mdc1^+/+ and Mdc1^−/− mice were incubated with antibodies against RAD51 (c), DMC1 (d), RPA2 (e), and MEIOB (f). Scale bar, 5 µm. g Localization of MDC1 in surface spreads of pachytene spermatocytes from Mdc1^+/+ and Mdc1^−/− mice. Scale bar, 5 µm. h Localization of endogenous NBS1 in surface spreads of pachytene spermatocytes from Mdc1^+/+ and Mdc1^−/− mice. Scale bar, 5 µm. Cartoons depicting proteins (red) localized on chromosome loops and axes (green) are shown on the right. Arrows mark chromosome axes and arrowheads mark chromosome loops. i Localization of endogenous NBS1 in surface spreads of pachytene spermatocytes from H2ax^+/+ and H2ax^−/− mice. Scale bar, 5 µm. Cartoons depicting proteins (red) localized on chromosome loops and axes (green) are shown on the right. Arrows mark chromosome axes and arrowheads mark chromosome loops.

Fig. 6. NBS1 is recruited to SPO11-linked DSBs by a phosphorylated protein.

a Schematic representation of in vitro binding of recombinant proteins to surface spread of spermatocytes and visualization. b Localization of GST-tagged NBS1 FHA + BRCT domain protein at pachytene stage in surface spreads of spermatocytes from Mdc1^+/+ and Mdc1^−/− mice. Scale bar, 5 µm. Cartoons depicting proteins (red) localized on chromosome loops and axes (green) are shown on the right. Arrows mark chromosome axes and arrowheads mark chromosome loops. c Localization of GST-tagged NBS1 FHA + BRCT domain protein at zygotene stage in surface spreads of spermatocytes from Mdc1^+/+ and Mdc1^−/− mice. Scale bar, 5 µm. Localization of GST-tagged NBS1 FHA + BRCT domain protein at pachytene (d) and zygotene (e) stages in surface spreads of spermatocytes from wild-type mice with or without λ-phosphatase treatment. Scale bar, 5 µm. Localizations of wild type, FHA domain mutant (R28A/R43A), and BRCT domain mutant (K160A) forms of GST-tagged NBS1 FHA + BRCT domain proteins at pachytene (f) and zygotene (g) stages in surface spreads of spermatocytes from wild-type mice. Scale bar, 5 µm.

Fig. 7. Working model: functions of MRN complex in protein-linked DSB repair in somatic cells and meiotic prophase.

In somatic cells, TDP2-dependent NHEJ pathway and MRN complex-dependent HR pathway function in parallel to repair TOP2-linked DSBs. MRN complex is recruited to DSB sites by phosphorylated MDC1. In meiotic prophase, TDP2-dependent NHEJ pathway is inactivated by unknown mechanisms and MRN complex-dependent HR pathway is essential for repairing SPO11-linked DSBs. The recruitment of NBS1 to SPO11-linked DSB sites on meiotic chromosome axes is independent of MDC1 but requires another unknown phosphorylated protein. MDC1 is required for spreading NBS1 signals from DSB sites to chromosome loops in XY body to mediate meiotic sex chromosome silencing (MSCI).