DNA. Nevertheless, cH2Av IF and satellite FISH signals overlapped in 21% (s.d. 9.6%, n = 151) of Su(var)3-9 oocytes and nurse cells, and was never observed in wild type cells (n = 55, Figure 2D; p,0.001). These data show that a significant proportion of the elevated levels of DNA breaks in Su(var)3-9 mutant oocytes and nurse cells occur in heterochromatin. Elevated frequencies of DNA breaks in Su(var)3-9 mutant ovaries could arise from increased access of heterochromatin to normal meiotic recombination pathways, which are mediated by Spo11, or by an independent mechanism, such as replication errors. We therefore examined the impact of meiW68 loss on repair foci formation in Su(var)3-9 mutants. The number and volume of cH2Av foci in mei-W68; Su(var)3-9 double mutant nurse cells and oocytes were significantly reduced in comparison to single Su(var)3-9 mutant cells, and were not significantly different from levels observed in wild type cells (Figure 2A, B, and C). Therefore, Spo11/mei-W68 mediated events cause the majority of the increased DSBs in Su(var)3-9 germ-line cells. The residual foci observed in the double mutants likely represent Spo11-indepen- dent breaks in response to eliminating Su(var)3-9. Previous studies demonstrated that non-recombinant (achias- mate) chromosomes in Drosophila females are paired in the heterochromatin [40] and that heterochromatic homology is required for normal segregation [4]. We observed significant increases in the frequencies of 4th and X chromosome exceptions (nondisjunction or loss) in Su(var)3-9 mutant females compared to wild type females (Figure S1). These results demonstrate that Su(var)3-9 and H3K9 methylation are required for normal homolog segregation in female meiosis. Depletion of H3K9me2 could cause loss and nondisjunction due to defective heterochro- Requirement for Heterochromatin Stability matin-mediated pairing, sister chromatid cohesion, and/or high levels of recombination in heterochromatin [41]. We conclude that H3K9 methylation is important for maintaining the structural integrity of heterochromatin in meiotic as well as mitotic cells, and that most of the increased DSBs in Su(var)3-9 meiotic cells arise through the canonical recombination pathway mediated by Spo11/mei-W68. Su(var)3-9 mutant exhibits defective chromosome structures and increased loss of heterozygosity Persistent DNA damage could lead to chromosomal structural defects, rearrangements and aneuploidy. To test this hypothesis, we first examined wild type and Su(var)3-9 mitotic chromosomes by DAPI-staining. All wild-type mitotic chromosomes exhibited banding patterns characteristic of heterochromatin (Figure 3A, first panel). In contrast, Su(var)3-9 mitotic chromosomes exhibited a variety of phenotypes such as hypo-condensation (Figure 3A, second panel) and extra DAPI-bright bands (Figure 3A, third panel; complete list of phenotypic analyses is in Figure 3B). We used FISH paints that hybridize to the euchromatic regions of three Drosophila chromosomes (X, 2 and 3; Materials and Methods) to determine if Su(var)3-9 cells contain increased frequencies of rearranged chromosomes compared to wild type (Figure 3C). Quantitative analysis of the ‘painted’ chromosomes showed that 1.1% of Su(var)3-9 mitotic chromosomes exhibit structural defects such as deletions, duplications, and transloca- tions (Figure 3D), which were never observed in wild type. We also determined if Su(var)3-9 mutant animals exhibit increased loss of heterozygosity (LOH) compared to wild type. A genetic assay was performed to monitor the loss of w+ from wild type and Su(var)3- Figure 3. Su(var)3-9 somatic cells exhibit genome instability phenotypes. A) DAPI staining of mitotic chromosomes from wild type and Su(var)3-9 mutant diploid cells. Structural defects in Su(var)3-9 mitotic chromosomes are indicated by white arrows. Each image is an optical section; bar = 2 mm. B) The chart shows quantitation of defective Su(var)3-9 mitotic chromosomes. Some mitotic chromosomes exhibited more than one defect. C) Chromosome painting of mitotic chromosomes from wild type and Su(var)3-9 mutant. Red = 3rd chromosomes, green = 2nd chromosomes, and blue = X chromosomes. Fourth and Y chromosomes are only stained with DAPI. Structural defects, such as deletions and translocations, are indicated by white arrows. Each image is an optical section; bar = 2 mm. D) Quantitation showed that 1.1% of the Su(var)3-9 mitotic chromosomes exhibited structural defects, compared to 0% for wild type (p,0.05 by Chi-square test). E) The diagram illustrates the genetic assay used to quantitate genome instability (loss of heterozygosity) in wild type and Su(var)3-9 animals. Wild type virgin flies were mated with males hemizygous for w2 (recessive white1118 mutation) to produce females heterozygous for w2 and w+. A clone of w2 adult eye cells (pictured) arises during larval development when the w+ allele is lost due to mitotic recombination, deletion, or chromosome loss events. f) Quantitative analysis of w2 clone frequencies in wild type and Su(var)3-9 animals. The p values were calculated using the Chi-square test. doi:10.1371/journal.pgen.1000435.g003 PLoS Genetics | www.plosgenetics.org 5 March 2009 | Volume 5 | Issue 3 | e1000435