Requirement for Heterochromatin Stability Figure 1. Su(var)3-9 mutant somatic cells display increased DNA damage in heterochromatin. A) cH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and Su(var)3-9 mutants are shown. Each image is an optical section. cH2Av- and Rad51-positive cells in Su(var)3-9 are 96- and 11- fold increase over wild type. The p values were ,0.01 by the Student’s t test, and n.800 cells for each group. The scale bars = 25 mm in cH2Av IF images and 8 um in Rad51 images. B) An optical section shows a wild type diploid cell stained with DAPI; dashed lines encircle the DAPI-bright regions. Bar = 0.8 mm. Right panel are optical sections of Su(var)3-9 mutant diploid cells stained by the TUNEL assay (red foci). The foci are double-stranded breaks recognized by TdT. Enlarged images showed examples of TUNEL foci in DAPI-weak and DAPI-bright regions. Bar = 4 mm. C) shows quantitative analysis of cH2Av and TUNEL signal localizations in wild type and Su(var)3-9 cells. The distribution of cH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and Su(var)3-9 (p.0.05 by Chi-square test; n.40 for each genotype). Compared to wild type, cH2Av foci localized to DAPI-bright regions in Su(var)3-9 is 96-fold higher and 88-fold for TUNEL (p,0.001 by Chi-square test; n.35 for each genotype). doi:10.1371/journal.pgen.1000435.g001 combinations of Su(var) mutants results in reciprocal exchange in heterochromatin [38], suggesting that heterochromatin compo- nents repress recombination during meiosis. We determined whether increased heterochromatic DNA damage and DSBs occur in Su(var)3-9 mutant meiotic cells, as observed in somatic cells. The germarium is the part of Drosophila ovary that contains developing oocytes and nurse cells, which share the same cytoplasm. Although only the oocyte will progress through meiosis, both cell types express Spo11 (mei-W68 in Drosophila), which is required for generating DSBs during meiosis [33,39]. IF analysis showed a dramatic increase in cH2Av signals in Su(var)3-9 mutant germaria compared to wild type (Figure 2A). We performed two kinds of quantitative analyses because a high PLoS Genetics | www.plosgenetics.org 3 March 2009 | Volume 5 | Issue 3 | e1000435