Quantitative Mcm Perturbation in Mice by densitometry is shown in the center panel. The amounts relative to WT cells (after normalization to the controls) are plotted. Error bars represent SEM, derived from 4 replicate experiments. The rightmost panel graphs the results of flow cytometric analysis of unsynchronized MEF culture cell cycle profiles, based on DNA content. (B) Flow cytometric quantification of MCM2 content (fluorescence intensity of antibody staining) is plotted on the Y axis, vs DNA content on the X axis. Plotted at the right is the mean fluorescent intensity of the 3 gated (boxed) regions from the flow data. Se = early S phase; SL = late S phase. The labeled cell cycle stages are based on DNA content. However, because light scatter was inadequate to distinguish individual nuclei from clumped nuclei or artifactual structures, the 4c category (denoted G2/M*) contains events other than G2/M nuclei. *We drew a dashed line representing an arbitrary cutoff above which contains such undefined events. (C) qRT-PCR analysis of Mcm mRNAs (left panel) and control genes (right panel), in the three indicated genotypes of MEFs. Relative transcript levels were normalized to b-actin. Charted are the percent levels of the indicated RNAs in mutant compared to WT (considered to be 100%). At least 3 replicate cultures were analyzed for each genotype. Error bars are SEM. In all panels, the raw data shown are from MEFs established from littermates. Furthermore, the replicates involved MEFs from pairs of littermates. Chaos3 = Mcm4Chaos3/Chaos3; WT = +/+. doi:10.1371/journal.pgen.1001110.g001 decreased in Mcm2IRES-CreERT2/IRES-CreERT2 mice, although mRNA levels were not evaluated in that study [20]. After breeding the gene trap alleles into the C3HeB/FeJ genetic background for at least 2 generations (Mcm4Chaos3/Chaos3 females get mammary tumors in this background), blood MN levels were measured. Heterozygosity for each allele caused an increase in the fraction of cells with MN (Figure 2C). Compound heterozygosity further increased MN on average, as did heterozygosity for 3 or more gene traps (Figure 2C), indicating that genetically-based decreases in any of the MCMs precipitate GIN. Genetic reductions of Mcm2, Mcm6 or Mcm7 in an Mcm4Chaos3/Chaos3 background causes partial synthetic lethality, severe growth defects and (for Mcm2) dramatically accelerated cancer onset As outlined above, previous studies showed that reductions of particular MCMs in cells or mice reduces the levels of other MCMs, causing GIN, cancer, and developmental defects. However, the reduction in MCM levels required to precipitate these consequences, and whether there is a threshold effect, is unclear. To explore the consequences of incremental MCM reductions on viability and cancer in mice, we crossed the Mcm4Chaos3 and gene trap alleles into the same genome. In the case of Mcm2, there was a striking and highly significant shortfall of Mcm4Chaos3/Chaos3 Mcm2Gt/+ offspring at birth (Figure 3A; Figure S2). Heterozygosity for Mcm2Gt itself was not haploinsufficient, as indicated by Mendelian transmission of Mcm2Gt in crosses of heterozygotes to WT (119/250; x2 = 0.448). These results demonstrate that there is a synthetic lethal interaction between Mcm4Chaos3 and Mcm2Gt that is related to MCM2 levels. Additionally, the surviving Mcm4Chaos3/Chaos3 Mcm2Gt/+ offspring were severely growth retarded; males weighed ,50% less than Mcm4Chaos3/Chaos3 siblings (Figure 3B; this genotype causes disproportionate female lethality). Another indication of a quantitative MCM threshold effect is that C3H-Mcm4Chaos3/Chaos3 mice are developmentally normal, but Mcm4Chaos3/Gt animals die in utero or neonatally (Figure 3A) [23]. The synthetic interaction between Mcm4Chaos3 and Mcm2Gt might be specific, or it may reflect a general consequence of reduced replication licensing (and consequent elevated replication stress). We therefore tested whether hemizygosity for Mcm3, Mcm6 or Mcm7 would also cause synthetic phenotypes in the Mcm4Chaos3/Chaos3 background. The Mcm4Chaos3/Chaos3 Mcm6Gt/+ genotype caused highly penetrant embryonic lethality; only 10% of the expected number of such animals survived to birth (Figure 3A; Figure S2). The Mcm4Chaos3/Chaos3 Mcm7Gt/+ genotype caused both embryonic and postnatal lethality. The number of liveborns was ,50% of the expected value, and only 8% of those (5/62) survived to weaning (Figure 3A; Figure S2). Additionally, as with Mcm2, hemizygosity for Mcm6Gt and Mcm7Gt in the Mcm4Chaos3/Chaos3 background caused growth retardation (Figure 3B). The decrease in male weight was ,20% and ,80% respectively, compared to Mcm4Chaos3/Chaos3 siblings at the oldest age measured (Mcm4Chaos3/Chaos3 Mcm7Gt/+ animals died before wean, so the oldest weights were taken at 10 dpp). In contrast to the synthetic phenotypes with Mcm2, 4, 6 and 7, there was no significant decrease in viability (Figure 3A) or weight (not shown) in Mcm4Chaos3/Chaos3 Mcm3Gt/+ mice. This seeming inconsistency is addressed in the following section. As mentioned earlier, mice with ,35% of WT MCM2 protein, but not 62%, showed early latency (10–12 week) lymphoma susceptibility [20]. To identify if there is a critical MCM threshold for cancer susceptibility, we aged a cohort of Mcm2Gt/+ mice, representing approximately intermediate MCM2 levels. As shown in Figure 4A, these animals did not show a dramatic cancer- related mortality in the first 12 months of life. However, we did find that ,3/4 of these animals had tumors at death or necropsy by 18 months of age (data not shown). These combined data are suggestive of a potential gradient of susceptibility, but that there is a critical minimum threshold of MCM levels, between ,35 and 50% in the case of MCM2, required to avoid early cancer and other developmental defects. To further resolve this phenomenon, surviving Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice were aged and monitored. They began dying at 2 months of age, and all were dead (or sacrificed when they appeared moribund) by 7 months (Figure 4A). Gross necropsy and histopathological analyses revealed or suggested lymphomas/ leukemias in 20 of these animals (summarized in Table S1 with histological examples in Figure S3; detailed histopathology analysis of a T cell leukemic lymphoma is presented in Figure 4B). Six of these had chest tumors that were likely thymic lymphomas. The cause of death for the remaining 7 animals was undetermined. Consistent with previous studies [17], most Mcm4Chaos3/Chaos3 mice hadn’t yet succumbed from tumors or other causes by 12 months of age. Additional animals of these genotypes are incorporated in Figure 6, but histopathological analyses weren’t conducted. These data show clearly that removing a half dose of MCM2 from Mcm4Chaos3/Chaos3 cells is sufficient to produce greatly elevated cancer predisposition to the already-underrepresented survivors at wean. Mcm4Chaos3/Chaos3 Mcm2Gt/+ MEFs had 45% the amount of Mcm2 mRNA as Mcm4Chaos3/Chaos3 cells (Figure 7C), which already had a 38% reduction compared to WT (Figure 1). Thus, Mcm2 RNA was reduced to ,17% of WT. To determine if elevated GIN might be responsible for the cancer susceptibility phenotype, we measured erythrocyte MN. Whereas the percentage of micro- nucleated RBCs in Mcm4Chaos3/Chaos3 mice was 4.1860.26 (mean6SEM, N = 12), Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice averaged 5.8560.47 (N = 16), indicating a synergistic increase (P,0.01). Overall, the data support the notion that in whole animals, reduction of MCMs to under 50% of WT causes severe developmental and physiological problems. Rescue of phenotypic defects in Mcm4Chaos3/Chaos3 and Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice by reducing Mcm3 genetic dosage The data reported here and elsewhere [17,20] support a model where phenotypic severity is proportionally related to MCM PLoS Genetics | www.plosgenetics.org 4 September 2010 | Volume 6 | Issue 9 | e1001110