Quantitative Mcm Perturbation in Mice Mcm2Gt/+ mice. Error bars represent SEM. (C) Mcm4Chaos3/Chaos3 Mcm2Gt/+ MEF proliferation defects are partially rescued by Mcm3 hemizygosity. The effect is significant after 6 days in culture (P,0.05, Student’s t-test; Error bars represent SEM). (D) Micronucleus levels in Mcm4Chaos3/Chaos3 mice bearing additional gene trap alleles. At least 5 males were analyzed for each genotype. Error bars represent SEM. Asterisk indicates P,0.05 (student’s t-test.) compared to Mcm4Chaos3/Chaos3 alone. (E) CD71+ reticulocyte ratios in mutant male mice. At least 5 animals were analyzed from each class. The samples are identical to those in ‘‘D’’. All scored cells were anucleate peripheral blood cells Error bars represent SEM. Asterisks and ‘‘#’’ indicate P,0.05 (Student’s t-test) when compared to Mcm4Chaos3/Chaos3 and Mcm4Chaos3/Chaos3 Mcm2Gt/+ cohorts, respectively. (F) Mcm2 hemizygosity decreases efficiency of reprogramming Mcm4Chaos3/Chaos3 MEFs into iPS cells, and Mcm3 hemizygosity significantly increases reprogramming efficiency. Two methods of quanitifying reprogramming were used as described in Materials and Methods. ‘‘Cell number’’ refers flow cytometric quantification of LIN28/SSEA1 double positive cells from primary cultures of reprogrammed MEFs. Relative reprogramming efficiencies were normalized to Mcm4Chaos3/Chaos3 MEFs (considered to be 100%). Error bars represent SEM. All samples within quantification class are significantly different from one another (P,0.05, Student’s t-test). C3 = Mcm4Chaos3; M = Mcm. doi:10.1371/journal.pgen.1001110.g005 we measured MN levels in Mcm4Chaos3/Chaos3 mice with different combinations of other Mcm mutations. As shown in Figure 5D, hemizygosity for Mcm2 and Mcm7 caused a significant elevation in MN levels, unlike Mcm3. However, the increased MN in Mcm4Chaos3/Chaos3 Mcm2Gt/+ was not rescued by Mcm3 hemizygosity. This suggests that the synthetic lethality and mouse/cell growth defects are not related to GIN per se. However, in the course of measuring MN in enucleated peripheral blood cells, we noticed that the ratio of CD71+ cells was significantly higher in both Mcm4Chaos3/Chaos3 Mcm2Gt/+ and Mcm4Chaos3/Chaos3 Mcm7Gt/+ mice (3.3 and 6.2 fold, respectively; Figure 5E). This increase in the ratio of reticulocytes (erythrocyte precursors; immature RBCs) to total RBCs is character- istic of anemia. Hemizygosity for Mcm3, which alone had no effect on CD71 ratios of Chaos3 mice, corrected completely this abnormal phenotype in Mcm4Chaos3/Chaos3 Mcm2Gt/+animals (Figure 5E). Because MCM2-depleted mice were reported to have stem cell defects [20], and Mcm4Chaos3/Chaos3 Mcm#Gt/+ mice had clear developmental abnormalities, we examined the efficiency of reprogramming mutant MEFs into induced pluripotent stem cells (iPS). The efficiency was quantified using either : 1) iPS-like colony formation, or 2) cells counts of SSEA1 and LIN28 positive cells by flow cytometry. Both gave similar results. Mcm4Chaos3/Chaos3 Mcm2Gt/+ cells were severely compromised in the ability to form iPS cells compared to Mcm4Chaos3/Chaos3 (,200 fold less efficient; Figure 5F). However, additionally reducing Mcm3 by 50% increased iPS formation from both Mcm4Chaos3/Chaos3and Mcm4Chaos3/Chaos3 Mcm2Gt/+ MEFs by ,2.5 and 10 fold, respectively. Finally, we found that reduced MCM3 levels could rescue the cancer susceptibility of two different Chaos3 models. As shown earlier (Figure 4), Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice were highly cancer-prone with an average latency of ,4 months. When a dose of Mcm3 was removed from mice of this genotype, lifespan was extended dramatically in both sexes as a consequence of delayed cancer onset, and the cancer spectrum shifted from lymphoma/thymoma towards mammary tumors (Figure 6A). Additionally, hemizygosity of Mcm3 delayed (or eliminated) the onset of mammary tumorigenesis in Mcm4Chaos3/Chaos3 females by ,4 or more months (Figure 6B). However, although Mcm3 hemizygosity rescued viability of Mcm4Chaos3/Gt mice (Figure 5A), these animals were cancer prone with a shorter latency (by ,6 months) and different spectrum (primarily lymphomas) than Mcm4Chaos3 homozygotes. Decreased MCM3 increases chromatin-bound levels of other MCMs We considered two possibilities to explain the surprising phenotypic rescues of reduced MCM genotypes (Mcm4Chaos3/Chaos3 ; Mcm4Chaos3/Chaos3 Mcm2/6Gt/+ ; Mcm4Chaos3/Gt) by additional MCM3 reduction (Mcm3Gt/+). One is that the phenotypes are related to altered stoichiometry of MCM monomers, and that disproportionally high amounts of MCM3 relative to MCM4 and MCM2/6/7 have a dominant negative effect. However, as demonstrated above, levels of MCM3 are proportionally reduced in Mcm4Chaos3/Chaos3 cells (Figure 1). The second possibility is that decreased levels of MCM3 leads to a favorable change in the amounts or subcellular localization of MCMs. Various experiments have indicated that MCM2-7 hexamers or subcomplexes must be assembled in the cytoplasm before nuclear import in yeast [4], and in mice, nuclear import appears to require MCM2 and MCM3 [24]. MCMs shuttle between the nucleus and cytoplasm during the cell cycle in S. cerevisiae. Although in most other organisms MCMs are reported to be predominantly and constitutively nuclear localized throughout the cell cycle, dynamic redistribution between the nucleus and cytoplasm has been observed in hormonally-treated mouse uterine cells [25]. In budding yeast, nuclear export is dependent upon Mcm3, which has a nuclear export signal (NES) that is recognized by Cdc28 to promote export of MCM2-7 [22]. Analysis of mouse and human MCM3 using NES prediction software (www.cbs.dtu.dk/services/NetNES/) [26] revealed the presence of homologously-positioned, leucine-rich potential NESs (Figure 7A). Therefore, we hypothesized that the rescue of phenotypes by Mcm3 hemizygosity is due to decreased MCM protein export from the nucleus, or alternatively, increased nuclear import or stabilization that allows greater access of all MCMs for licensing chromatin. To explore this hypothesis, we performed Western blot analysis of MCM levels in Mcm4Chaos3/Chaos3 MEFs with or without the Mcm3Gt and/or Mcm2Gt alleles, and examined the effects of Mcm3 dosage on the levels of nuclear and chromatin-bound MCM2 and MCM4. The results are presented in Figure 7B. In all cases, the genetic reductions of Mcm2 and Mcm3 led to corresponding decreases in the cognate mRNA levels (Figure 7C), with only minor additional decreases of other MCM mRNAs (beyond that already caused by homozygosity for Mcm4Chaos3) occuring in the context of Mcm2 hemizygosity (similar to Mcm2Gt/+ MEFs in Figure 2B). The overall levels of total, nuclear, and chromatin- bound MCM2 and MCM4 were unaffected by hemizygosity of Mcm3 in Mcm4Chaos3/Chaos3 cells (Figure 7B). When Mcm2 levels were genetically reduced by half, a condition causing the severe phenotypic effects described earlier, this caused a marked decrease in the level of chromatin-bound MCM3 and MCM4 (in addition to MCM2 itself), although total and nuclear MCM3/ 4 levels were affected to a lower degree or not at all. Strikingly, the decreased levels of chromatin-bound MCM2/3/4 in Mcm4Chaos3/Chaos3 Mcm2Gt/+ MEFs were reversed by Mcm3 heterozygosity, but levels of total MCM2 and MCM4 were not restored. The increase of chromatin-bound MCMs occured despite the presence of less MCM3, suggesting that MCM3 is present at levels in excess of that needed to bind chromatin, presumably for pre-RC formation in the context of the MCM2-7 hexamer. In conclusion, a 50% reduction in total MCM3 increases MCM2/4 loading onto chromatin when MCM2 is otherwise limiting, and this rescue is associated with amelioration of several phenotypes. We found that elevation of nuclear MCMs in the Mcm3Gt/+ MEFs was often (as shown in Figure 7B), but not consistently elevated across PLoS Genetics | www.plosgenetics.org 9 September 2010 | Volume 6 | Issue 9 | e1001110