Volume 12, Issue 6, Pages 1511-1523 (December 2003) Checkpoint Failure and Chromosomal Instability without Lymphomagenesis in Mre11ATLD1/ATLD1 Mice  Jan-Willem F Theunissen, Mark I Kaplan, Patricia A Hunt, Bret R Williams, David O Ferguson, Frederick W Alt, John H.J Petrini  Molecular Cell  Volume 12, Issue 6, Pages 1511-1523 (December 2003) DOI: 10.1016/S1097-2765(03)00455-6

Figure 1 Generation of Mre11ATLD1/ATLD1 Mice (A) MRE11 targeting vector. Bold vertical lines depict exons. The XbaI site (indicated by Xb*) was lost in the targeting vector due to the A→T change at nucleotide 1894 of the coding sequence. Targeted clones were identified with a 5′ and a 3′ external probe. (B) Southern blot of genomic DNA using a 3′ external probe from wild-type (WT) (1), heterozygous (2), and homozygous (3) mutant littermates digested with SacI and XhoI. MRE11 indicates the WT locus (8.8 kb) and Mre11ATLD1 the targeted locus (8.1 kb). (C) Immunoprecipitations from WT (lanes 1 and 2) and Mre11ATLD1/ATLD1 (3, 4, and 5) MEF cell extracts (100 μg for 1 and 2; 500 μg for 3 and 4, and 1500 μg for 5) were carried out with preimmune (1 and 3) or Nbs1 antiserum (2, 4, and 5), and immunoblotted sequentially with Mre11 and Nbs1 antisera. As a control for cell extract concentration, 100 μg of Mre11ATLD1/ATLD1 (6) and WT (7) cell extract was immunoblotted with SMC1 antiserum. SMC1, Nbs1, Mre11, and Mre11ATLD1 proteins are indicated. (D) Radiosensitivity. IR sensitivity was determined by colony formation assay in Mre11ATLD1/ATLD1 (black diamonds) and WT (gray diamonds) SV40 transformed MEFs. The surviving fractions (normalized to mock-irradiated cultures) were plotted in a dose-dependent manner. Error bars indicate standard deviations of triplicate samples for each dose in two independent experiments. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 2 DNA Damage Checkpoints in Mre11ATLD1/ATLD1 MEFs (A) Intra-S phase checkpoint. The DNA synthesis ratios in WT (gray diamonds), Mre11ATLD1/ATLD1 (black diamonds), and Atm−/− (black circles) γ-irradiated SV40 transformed MEFs, normalized to untreated cultures, are presented. This plot is a compilation of three independent experiments segregated from left to right, in which each experiment counted five plates at each dose for each genotype. (B) G2/M checkpoint. Exponentially growing WT (gray bars), Mre11ATLD1/ATLD1 (black bars), and Atm−/− (white bars) primary MEF cultures were harvested at the indicated times after 10 Gy of IR; mock treated cultures designated T = 0. The percentage of mitotic cells (based on phospho-H3 staining) is plotted. Error bars indicate standard deviations of triplicate plates for each treatment (C) G1/S checkpoint. The S phase ratios, obtained by dividing the S phase percentages after 0 and 10 Gy of IR by the average S phase percentages after 0 Gy, are plotted. Symbols as in Figure 2A; p53−/− (gray circles). Five different WT and Mre11ATLD1/ATLD1 primary MEF cultures, segregated in columns, were assessed. P[WT versus Mre11ATLD1/ATLD1] = 3.7 × 10−4; P[Mre11ATLD1/ATLD1 versus Atm−/−] = 3.1 × 10−4. (D) Analysis of ATM targets. For Chk2 activation, extracts from WT (I), Mre11ATLD1/ATLD1 (II), and Atm−/− (III) primary MEF cultures were prepared after mock treatment (0), and 1 hr after the indicated IR doses in Gy. For p53 stabilization and p21 induction, extracts from WT (I) and Mre11ATLD1/ATLD1 (II) primary MEF cultures were prepared after mock treatment (0), and 1 or 2 hr after 10 Gy. β-Actin is included as a loading control. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 3 Karyotypic Analysis in Mre11ATLD1/ATLD1 MEFs (A) Representative metaphases from p7 (left) and irradiated p2 (right) Mre11ATLD1/ATLD1 MEFs. Open arrowheads indicate chromatid breaks. (B) The percentage of normal (0) and aberrant metaphases (subdivided in three categories of 1 to 2, 3 to 4, or >4 aberrations/metaphase) in p7 WT (gray bars) and Mre11ATLD1/ATLD1 (black bars) MEFs. (C) The percentage of normal (0) and aberrant metaphases (subdivided in four categories of 1 to 2, 3 to 4, 5 to 6, or 7–11 aberrations/ metaphase) in p2 WT (gray bars) and Mre11ATLD1/ATLD1 (black bars) MEFs irradiated with 1 Gy of IR. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 4 Survival of Mre11ATLD1/ATLD1 and Nbs1ΔB/ΔB Mice (A and B) Each data point represents the fraction of surviving mice at a given age in months. The genotype legend is depicted to the right of the survival curves. N, total number of mice for each genotype. The average age in death in months (mo) and the percentage of tumors in each relevant cohort (%) are shown. Portions of the data regarding Nbs1ΔB/ΔB mice have been published previously (Williams et al., 2002). Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 5 Mre11ATLD1/ATLD1 Lymphocytes (A) Transrearrangements in Mre11ATLD1/ATLD1 thymocytes. Amplicons arising from normal rearrangements at the TCRß (β) and TCRγ (γ) loci, and transrearrangements between the TCRß and TCRγ loci (β-γ and γ-β) are shown. Lanes 3, 6, 9, and 12 are water controls; lanes 1, 4, 7, 10, 13, 15, 17, and 19, WT; lanes 2, 5, 8, and 11 Mre11ATLD1/ATLD1; lanes 14 and 16, Nbs1ΔB/ΔB; lanes 18 and 20, Atm−/−. The β and β-γ products are +/− 200 bp in size, and the γ and γ-β products are +/− 250 bp in size. (B) IR-dependent thymus apoptosis. Represented is the viability (= AnnexinV/PI double negative %) after mock treatment and 20 hr after 5 Gy, divided by the average percentage of viability after mock treatment, of thymocytes from 5 WT, 4 Mre11ATLD1/ATLD1, 4 Atm−/−, and 3 p53−/− mice. For every genotype, each column of symbols represents a single mouse, and the symbols in each column represent duplicate or triplicate cultures. Genotypes indicated as in Figure 2C. P[WT versus Mre11ATLD1/ATLD1] = 1.7 × 10−3; P[Mre11ATLD1/ATLD1 versus Atm−/−] = 3.4 × 10−5. (C) IR-induced effects on p53 and Chk2. Immunoblotting for p53, Chk2, and SMC1 (loading control). WT, Mre11ATLD1/ATLD1, Nbs1ΔB/ΔB and Atm−/− thymocytes were prepared after mock treatment (lanes 1, 3, 5, and 7) and 1 hr after 5 Gy of IR (lanes 2, 4, 6, and 8). WT, lanes 1 and 2; Mre11ATLD1/ATLD1, lanes 3 and 4; Nbs1ΔB/ΔB, lanes 5 and 6; Atm−/−, lanes 7 and 8. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 6 Subfertility in Mre11ATLD1/ATLD1 Females (A) Littersizes from Mre11ATLD1/ATLD1 females. The bars are colored according to the male genotype: WT, gray; Mre11ATLD1/+(Het), white; Mre11ATLD1/ATLD1 (Mut), black, and grouped according to the female genotype. The numbers in parentheses indicate the number of litters scored for each mating and error bars indicate the standard deviations. (B) Blastocyst outgrowth. Blastocysts were monitored for 5 days after harvest (0, 1, 2, 3, 4, and 5). (C) Hatching. The percentage of in vitro hatching is plotted according to the indicated matings (genotype symbols as in [A]), and the numbers in parentheses represent the average number of blastocysts harvested per mating. At least 70 embryos were analyzed for each mating configuration. (D) Cavitation failure in embryos from Mre11ATLD1/ATLD1 mothers. WT (I and II) and Mre11ATLD1/ATLD1 (III and IV) blastocysts are depicted. (E) Representative diakinesis preparations of oocytes from Mre11ATLD1/+ (I) and Mre11ATLD1/ATLD1 (II) females, illustrating the presence of chiasmata and absence of univalents. (F) Qualitative analysis of MII arrested oocyte morphology. Representative MII oocyte chromosome preparation from heterozygous (I) and Mre11ATLD1/ATLD1 (II) females. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)

Figure 7 Cellular Inviability as a Result of Chromosome Instability p53-dependent apoptosis (red bar) is the primary mechanism of suppressing murine lymphomagenesis (Schmitt et al., 2002). p53 surveillance is not operative in S and G2 cells (green bar) when Mre11 complex-dependent checkpoints (intra-S and G2/M) are active, and mitotic recombination events that could lead to loss of heterozygosity would occur. In Mre11ATLD1/ATLD1 cells, failure of the intra-S and G2/M checkpoints would lead to cell death during the execution of the mitotic program or as a result of loss of genetic information upon cell division (Zheng et al., 2000). In the context of an initiating lesion such as p53 heterozygosity, this aspect of the Mre11ATLD1/ATLD1 or Nbs1ΔB/ΔB phenotype would promote malignancy. p53 does not appear to control apoptosis in embryonic cells (Aladjem et al., 1998); thus embryonic death in Mre11ATLD1/ATLD1 mice would ensue from the same general mechanism. Molecular Cell 2003 12, 1511-1523DOI: (10.1016/S1097-2765(03)00455-6)