Requirement for Heterochromatin Stability Figure 5. G2, mitotic, and apoptotic cell percentages are increased in Su(var)3-9. A) The histograms show cell cycle stage analysis of wild type and Su(var)3-9 cells. The percent of G1 cells do not differ significantly (p.0.05). The percentage of S phase cells in Su(var)3-9 is significantly lower than wild type (p,0.05). The percent of wild-type cells in G2 is significantly lower than in Su(var)3-9. Mitotic indicex in Su(var)3-9 is 4-fold over wild type (p,0.001). The percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in Su(var)3-9 is 9-fold over wild type (p,0.001). P values were calculated by Student’s t test, and n.1000 cells for each genotype. B) The chart shows cH2Av foci numbers in G1, S, and G2 cells of wild type and Su(var)3-9. Analysis of the ratios of cH2Av foci to total cell numbers during the cell cycle in wild type and Su(var)3-9. cH2Av foci in Su(var)3-9 cells increased over wild type in G1, S, and G2 (p,0.01). P values were calculated by Chi-square test, and n.40 cells for each genotype. doi:10.1371/journal.pgen.1000435.g005 viability to 76.4% (Figure 6A). Chk1/grp and Chk2/lok have been shown to also regulate the metaphase-anaphase transition during mitosis [25,26]. Our analysis showed that grp and lok mutations almost entirely suppress the mitotic index increase in Su(var)3-9 mutants (Figure 6C). Mitotic checkpoint proteins Rod and ZW10 form a complex in the outer kinetochore, monitor microtubule attachments, and regulate the metaphase to anaphase transition [44]. Su(var)3-9 and rod or zw10 double mutants displayed synthetic lethality (51.7% and 0%, respectively, Figure 6A). The only available rodEY04576 mutation is hypomorphic, and the zw10 mutations are nulls, which could account for the viability differences in the double mutants. Regardless, these synthetic lethality data demonstrate that the mitotic checkpoint is also essential for Su(var)3-9 survival. p5311-1B-1, Su(var)3-9 double mutants exhibited 100% viability compared to Su(var)3-9 single mutants (Figure 6A), suggesting that the apoptosis pathway, regulated by p53 in flies, does not impact the viability of Su(var)3-9 animals. Even though apoptotic cells increase by 10-fold in Su(var)3-9 mutants, they only account for 0.86% of cells (Figure 5A; s.d. 0.29%). We reasoned that the apoptotic pathway is only activated upon persistent DNA damage, which could produce the observed mitotic chromosomal defects (Figure 3). Indeed, triple mutant mei-41; p53, Su(var)3-9 animals were less viable than mei-41; Su(var)3-9 double mutants (Figure 6A). Thus, even though p53 mutation (hence apoptosis) alone does not have significant impact, p53 does help ensure the viability of Su(var)3-9 adults when the DNA damage checkpoint is compro- mised by mei-41/ATR mutations. Are DNA repair factors also critical for the viability of Su(var)3-9 animals? We observed that lig4; Su(var)3-9 double mutants exhibited 100% viability (data not shown). However, the NHEJ pathway does not entirely depend on Ligase IV in Drosophila [45]. Such redundancy, in addition to functional compensations among different DNA repair pathways in Drosophila [19] make this viability analysis inconclusive. dcr-2 mutant animals also exhibit increased DNA damage in heterochromatin Heterochromatin formation and maintenance also requires the RNAi pathway, which can be subdivided into siRNA- and miRNA- based mechanisms. Dcr-2 is a critical component of the small-interfering RNA (siRNA) pathway in D. melanogaster [46], which regulates H3K9me2 localization to heterochromatin. We previously showed that H3K9me2 is mislocalized to a broader region of the nucleus in dcr2L811 fsx mutant cells, different from the strong reductions observed by IF in Su(var)3-9 cells. However. dcr2 mutants do contain significantly reduced levels of H3K9me2 at repeated DNAs, and significant increases in extrachromosomal repeated DNAs and ectopic nucleolus formation [34]. We have investigated whether patterns of DNA damage and repair are affected by loss of Dcr2, as reported here for Su(var)3-9 mutants. Quantitative analyses of gH2Av and Rad51 foci in dcr-2 mutant cells showed significant increases in spontaneous DNA damage and repair (2.1- and 3.5- fold over wild type, respectively; PLoS Genetics | www.plosgenetics.org 7 March 2009 | Volume 5 | Issue 3 | e1000435