with respect to localization to DAPI-bright versus DAPI-weak regions. DAPI signals were not enhanced contrast. DAPI-bright regions were regions that contain contiguous (.5 pixels) bright DAPI signals; representative DAPI images are shown in Figure 1C. Statistical comparisons and p values were calculated using the Chi-square test or Student’s t test, assuming two-sample tails and unequal variance. Chromosome paints FISH chromosome paints were made by degenerate PCR. The PCR products were digested with 4-base restriction enzymes, AluI, HaeIII, MseI, MspI, RsaI, and Sau3AI. Digested DNAs were end- labeled with TdT using aminoallyl-dUTPs followed by dye conjugation. Templates for the chromosome 2 and 3 paints were provided by Aki Minoda and Roger Hoskins, and were composed of genome tiling-path BACS identified by the Berkeley Drosophila Genome Project as low in repeat content and spaced ,500 kb apart. Templates for the X chromosome paints were provided by Abby Dernburg, who micro-dissected polytene X chromosomes and amplified them via degenerate PCR [70]. FISH using chromosome paints were performed as follows. Acid-squashed preparations were treated with an ethanol series (incubation for 2 minutes each in 70%, 85%, and 95% ethanol at room temperature), incubated in 0.005% pepsin in 10 mM HCl for 1 minute, rinsed in PBS, and treated with an ethanol series to dry. The slides were treated with 26 SSCT (0.1% Tween-20) for 5 minutes, 50% formamide in 26 SSCT for 5 minutes, and 70% formamide in 26 SSCT for 5 minutes. Chromosomes on slides, incubated in 70% formamide and 26 SSCT, were denatured on the heat block of a PCR machine programmed to increase the temperature from 25 to 74uC within 1.5 minutes, stay at 74uC for 1.5 minutes, and decrease the temperature from 74 to 25uC within 1.5 minutes. The slides were dried with an ethanol series, and the denatured probes (in 50% formamide, 10% dextran sulfate, 26 SSCT, 1 ug Cot-1 DNA) were added to chromosomes and hybridized overnight. After the incubation, the coverslips were removed, and the slides were washed with 50% formamide, 26 SSCT at 37uC for 4 times, for 30 minutes each time. Mosaic eye and genome instability assay Described in the Figure 3D legend. Cell Cycle analysis Brain and imaginal discs were fixed in 4% paraformaldehyde and PBS for 5 minutes, then washed with PBS 4 times for 5 minutes each. The fixed tissues were incubated in Collagenase solution (0.04% Collagenase type IV, Sigma, in PBS) for 10 minutes, squashed onto slides using RainX-treated coverslips, and frozen in liquid nitrogen. After coverslips were removed, the slides were allowed to warm for less than 30 seconds, fixed in 4% paraformaldehyde and PBS for 5 minutes, and washed with PBS 4 times for 5 minutes each. IF with cell cycle markers were performed using methods described [71], except no TritonX100 was used for CycA IF. Images were captured using an Applied Precision Deltavision Workstation and converted to TIFF files. The multi-wavelength cell scoring application within Metamorph 7.0 software was used to score cells positive for cell cycle markers or TUNEL signals. For each genotype and each marker, .3000 cells from at least 3 animals were analyzed. Supporting Information Figure S1 Effects of Su(var)3-9 on chromosome segregation in female meiosis. Nonrecombinant chromosomes bypass the normal requirement for chiasma formation by using the ‘achiasmate Requirement for Heterochromatin Stability segregation system.’ 4th chromosomes are always nonrecombinant (achiasmate), and 5% of normal sequence X chromosomes (y/y) are nonrecombinant. The frequency of non-recombinant X chromosomes is increased to 100% in FM7 (balancer)/y heterozygotes. The following crosses were performed to monitor X and 4th chromosome segregation simultaneously in wild type and Su(var)3-9 females, with and without suppression of X recombination (FM7/y and y/y, respectively): FMY/y; Su(var)3- 9 null; spapol X y+/Y ; C(4)ci ey/0 (53 females, 2035 progeny). FMY/y; ry ; spapol X y+/Y ; C(4)ci ey/0 (89 females, 4226 progeny). y/y; Su(var)3-9 null; spapol X y+/Y; C(4)ci ey/0 (27 females, 1093 progeny). y/y; ry; spapol X y+/Y; C(4)ci ey/0 (49 females, 2391 progeny). Frequencies of total exceptions, which includes both loss and nondisjunction (ND) events, were all calculated using the methods described previously [72]. The frequencies of meiotic exceptions increased in Su(var)3-9 mutant females compared to wild type most dramatically for the X chromosome (17 fold for FM7/y, 25 fold for y/y), but also for the 4th chromosome (4.6 fold for FM7/y, 3.3 fold for y/y). 4th chromosome ND, rather than loss, was increased in the mutants, suggesting defects in achiasmate homolog pairing, as opposed to cohesion or spindle attachment. X chromosome loss increased dramatically in both FM7/y and y/y females, but X ND frequencies only increased when X recombination was suppressed. The observation that X exceptions increased even in the absence of an X chromosome balancer (y/y females) suggests that segregation of both recombinant and non- recombinant chromosomes are affected by reduced H3K9 methylation. Previous studies showed that chromosomes with very proximal recombination events are more prone to ND and loss [41]; thus, the observed increase in heterochromatic DSBs in Su(var)3-9 oocytes (Figure 2) is the most likely cause of the increased ND and loss. Found at: doi:10.1371/journal.pgen.1000435.s001 (1.04 MB EPS) Figure S2 dcr-2 mutant somatic cells also display increased DNA damage in heterochromatin. A) and B) cH2Av (red) and Rad51 (green) IF in whole-mount diploid tissues from wild type and dcr-2 mutants are shown. Each image is an optical section. cH2Av- and Rad51-positive cells in Su(var)3-9 are 2.1- and 3.5- 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. C) and D) show quantitative analysis of cH2Av and TUNEL signal localizations in wild type and dcr-2 cells. The distribution of cH2Av and TUNEL signals in DAPI-weak regions do not differ significantly between wild type and dcr-2 (p.0.05 by Chi-square test; n.40 for each genotype). Compared to wild type, cH2Av foci localized to DAPI- bright regions in dcr-2 is 24-fold higher and 29-fold for TUNEL (p,0.001 by Chi-square test; n.35 for each genotype). Found at: doi:10.1371/journal.pgen.1000435.s002 (2.74 MB EPS) Figure S3 dcr-2 mutant oocytes display increased DNA damage in heterochromatin. A) The images show cH2Av (white in top panel and red in bottom panel) and C(3)G (green) IF in whole-mount germaria from wild type and dcr-2 mutant. C(3)G is part of the synaptonemal complex and used to distinguish oocytes from nurse cells, both of which contain DSBs. Each image is an optical section; bar = 7 mm. B) and C) The graphs show the average numbers and volumes (relative to total nuclear volumes) of cH2Av foci in nurse cells and oocytes from wild type and dcr-2. Both quantitation methods showed that cH2Av foci in dcr-2 oocytes were significantly increased over wild type (p,0.01), while foci in mutant nurse cells do not. Error bars indicate standard deviations, p values were calculated by Student’s t test, and n.15 for each cell type. Found at: doi:10.1371/journal.pgen.1000435.s003 (7.17 MB EPS) PLoS Genetics | www.plosgenetics.org 12 March 2009 | Volume 5 | Issue 3 | e1000435