Requirement for Heterochromatin Stability Figure 7. dcr-2 mutants display increased spontaneous DNA damage in heterochromatin. A) Analysis of the ratios of cH2Av foci to total cell numbers at different cell cycle stages are shown for wild type and dcr-2. cH2Av foci in dcr-2 cells only increased during S phase (p,0.05). P values were calculated by Chi-square test. B) The histograms show cell cycle stage analysis of wild type and dcr-2 cells. The percent of G1 cells in the two groups do not differ significantly (p.0.05). The percentage of S phase cells is not significantly lower in dcr-2 compared to wild type (p.0.05), but the percent of wild-type cells in G2 is significantly lower than in dcr-2 (p,0.05). The mitotic index in dcr-2 cells is 11-fold over wild type (p,0.001), and the percent of apoptotic cells (whole nuclei contain TUNEL signals, instead of foci) in dcr-2 is 18-fold over wild type (p,0.001). P values were calculated by Student’s t test, and n.1000 cells for each genotype. C) The chart lists the viability of double mutants of dcr-2 with mutations in the DNA damage checkpoint and mitotic checkpoint pathways. Viability was calculated relative to single homozygous checkpoint mutants, which are less viable than dcr-2 single mutants. Progeny counts are in Table S1. P values were calculated by the Chi-square test. doi:10.1371/journal.pgen.1000435.g007 damage or mitotic checkpoint proteins did not impact the viability of dcr-2 mutants (Figure 7C). Thus, in contrast to the effects of Su(var)3- 9, checkpoint activation is not necessary for the repair of endogenous DNA damage in dcr-2 diploid cells, or for mutant viability. It is possible that phenotypic differences in comparison to Su(var)3-9 mutants is due to the hypomorphic nature of the only available dcr-2 allele [46], and retention of more H3K9me2 at heterochromatic sequences (Peng and Karpen, 2007). Neverthe- less, these data demonstrated that compromising the siRNA pathway also leads to increased DNA damage in heterochromatin, reduced viability in embryogenesis, and activation of DNA damage checkpoints. We conclude that both the H3K9 methyl- ation and siRNA pathways safeguard the integrity of heterochro- matic DNA in meiotic and somatic cells, albeit with different levels of impact. Discussion Genomes with complex DNA organization and high repeat content present challenges for maintenance of genome stability during DNA replication, repair, and recombination. Heterochro- matin comprises approximately 30% of the Drosophila and human genomes, and ,30% of human euchromatin is composed of transposons and other repeats [2,47,48]. Persistence of heterochro- matin through evolution likely results from the many essential functions it encodes. We have demonstrated that maintaining the PLoS Genetics | www.plosgenetics.org 9 March 2009 | Volume 5 | Issue 3 | e1000435