9 animals; loss of w+ in this assay could result from either chromosome loss or mitotic recombination (Figure 3E). We observed that Su(var)3-9 animals spontaneously lose w+ at a 2.5-fold higher frequency than in wild type (Figure 3F; p,0.01). Thus, Su(var)3-9 animals exhibit hallmark characteristics of genome instability that can be observed cytologically and genetically. Su(var)3-9 mutants are predominantly viable and fertile Previous analysis showed that mice deleted for both Suv3-9 genes exhibit genome instability and partial prenatal lethality [42]. Surprisingly, despite elevated frequencies of DNA damage, chromosome rearrangements and LOH shown here, Su(var)3-9 mutant animals derived from mothers are homozygous viable and fertile [43]. However, our analysis of survival at various developmental stages showed significant differences between wild type and mutant animals (Figure 4A). Su(var)3-9 mutant parents produced 93% fertilized eggs, 72% of which hatch to embryos, compared to 94% hatching for wild type (Figure 4A). Defective development during embryogenesis is the likely cause of the lower hatch rates. Once they hatched into larvae, developmental timing and eclosion rates of Su(var)3-9 mutants were comparable to wild type. Thus, Su(var)3-9 mutant animals are mostly viable and fertile; larval and pupal development are normal, but they have elevated levels of unfertilized eggs and embryonic lethality compared to wild type. In addition, Su(var)3-9 females exhibit significantly shorter adult lifespans compared to wild type (Figure 4B, p,0.01). DNA damage checkpoints are activated in Su(var)3-9 cells We hypothesized that Su(var)3-9 mutant animals are mostly viable and fertile [43] due to activation of checkpoints that delay Figure 4. Developmental progression and lifespan of wild type and Su(var)3-9 animals. A) Analysis of developmental progression for wild type and Su(var)3-9 animals. Animals in the two groups laid comparable numbers of eggs. In all three assays, the p values comparing Su(var)3-9 to wild type are ,0.001 by Student’s t test. n.150 for each genotype. B) The graph shows lifespan analysis of wild type and Su(var)3-9 adult female flies. Su(var)3-9 females displayed significantly shorter lifespan than wild type (p,0.01 by Wilcoxon signed rank test). n.50 female flies. doi:10.1371/journal.pgen.1000435.g004 Requirement for Heterochromatin Stability cell cycle progression until DNA damage is repaired. To test this hypothesis, we compared the proportions of cells in different cell cycle stages in wild type and Su(var)3-9 cells. IF was performed on squashed diploid cells using antibodies to PCNA (S phase), Cyclin A (CycA, S phase, G2 and mitosis), and PH3 (H3 phosphorylated at serine 10, mitosis); the TUNEL assay was performed to identify apoptotic cells, which contain labeling throughout the nucleus, as opposed to foci observed in response to DNA damage (Materials and Methods). Cells in G2 were identified by staining for CycA, but not PCNA or PH3, replicating cells were stained by PCNA, and G1 cells were not stained with CycA, PCNA, or PH3. Comparative analysis showed that the percentage of Su(var)3-9 cells in S phase was lower than in wild type, and that the percentages of Su(var)3-9 cells in G2, mitosis, and apoptosis increased relative to wild type (Figure 5A). These data suggest that G2 and mitotic checkpoints are activated in these mutant cells. Increased apoptosis in the mutant animals is likely caused by unrepaired DNA damage, which is mediated by the p53 pathway [28]. It is important to note that although the fold increases for mitotic and apoptotic cells in Su(var)3-9 are large, the actual percent of cells are low (1 to 3%), in comparison to the 24% of cells in G2 (4.5 fold over wild type). We next determined which cell cycle stages displayed spontaneous DNA damage in Su(var)3-9 mutants. Comparisons of gH2Av foci with the cell cycle markers PCNA and CycA showed that DNA breaks are present in all interphase stages of the cell cycle in Su(var)3-9 (Figure 5B and C). This assay cannot determine when DSBs occur, since they can persist from one cell cycle stage into another if they are not repaired. Nevertheless, the finding of DSBs in G1 strongly suggests that increased DNA breaks in heterochromatin of Su(var)3-9 cells are not specific to DNA replication in S phase. DNA damage checkpoint pathways are critical for the viability of Su(var)3-9 adults Increased proportions of G2 and mitotic cells in Su(var)3-9 animals suggests that the G2 and mitotic cell cycle checkpoints may be activated by the increased frequencies of DSBs in heterochro- matin. We thereby hypothesized that compromising the DNA damage checkpoint, using mutations in the checkpoint compo- nents, may result in lethality of Su(var)3-9 animals. To test this hypothesis, we analyzed flies homozygous for Su(var)3-9 and homozygous for mutations in DNA damage checkpoint activation (ATR/mei-41, Checkpoint kinase 1 (Chk1)/grp, and Checkpoint kinase 2 (Chk2)/lok). Animals double mutant for Su(var)3-9 and cell cycle checkpoint mutations showed sub-viability ranging from 50% to 64.6% (Figure 6A; viability of double mutants were compared to single checkpoint mutants, which exhibit lower viability than Su(var)3-9 mutants). Su(var)3-9 mutant animals containing both grp and lok mutations are 100% lethal (Figure 6A). This demonstrates that DNA damage checkpoints are essential to the survival of Su(var)3-9 animals. Cell cycle analysis showed that the percentage of grp; Su(var)3-9 or lok; Su(var)3-9 cells in S phase and G2 were lower than in Su(var)3-9 alone, with a corresponding increase in cells in G1 (Figure 6B). Cell cycle characterization combined with the observed genetic interactions between Su(var)3-9 and DNA damage checkpoint mutations demonstrate that the DNA damage checkpoint is activated in Su(var)3-9 mutant animals, and is required for mutant viability. We previously demonstrated significantly reduced levels of cohesin in heterochromatin in Su(var)3-9 cells [34], which is likely to cause defects in sister chromatid cohesin and biorientation that result in an increased mitotic index. Indeed, reduction of cohesin levels by half (smc1 heterozygotes) in Su(var)3-9 animals reduced PLoS Genetics | www.plosgenetics.org 6 March 2009 | Volume 5 | Issue 3 | e1000435