Author Summary DNA damage from the environment is very common in animal cells, yet most of the time they are repaired efficiently and the integrity of the hereditary material is maintained. The genomes of most eukaryotes, such as humans and fruitflies, contain repeated DNAs that pose major challenges to genome stability. For example, recombination between repeated DNAs can result in chromosome rearrangements, a hallmark of cancer and birth defects. Repeated DNAs are contained within the part of the genome known as heterochromatin, which is characterized by a special type of chromatin packaging not prevalent in the rest of the genome, known as euchroma- tin. We use cytological and genetic analyses of the fruitfly model organism and demonstrate that the chromatin structure of heterochromatin plays a key role in efficient DNA damage detection and/or repair of repeated DNAs. Loss of a key chromatin modification (methylation of histone H3 at lysine 9) leads to increased spontaneous DNA damage specific to heterochromatin and activation of DNA repair cell cycle checkpoints. If left unrepaired, these DNA breaks cause cellular and organismal death. Remark- ably, human euchromatin and fruitfly heterochromatin share similar physical features, such as repeated DNA content, intron lengths, and open reading frame sizes. Understanding heterochromatic DNA damage repair will yield critical information about how mammalian genome stability is maintained and how defects in these processes may contribute to human disease. Chromatin functions in the DNA damage response provokes questions about whether the distinct compositions of euchromatin and heterochromatin impact DNA integrity. Replication across repeated sequences can result in sequence alterations and replication fork stalling and collapse, which can lead to DSBs. In addition, recombination between homologous tandem and dispersed repeats alters repeat lengths and generates exchange between non-homologous chromosomes, which can cause trans- locations and aneuploidy [29]. These challenges suggest that heterochromatic DNAs may utilize different regulatory mecha- nisms during replication, repair, and recombination to ensure genome stability. This hypothesis is further supported by the observations that heterochromatin is consistently replicated later in S phase than euchromatin [30], and that reciprocal meiotic recombination does not occur in heterochromatin [31–33]. We previously showed that animals mutated for proteins that regulate heterochromatin structure (Su(var)s), including the H3K9 HMTase Su(var)3-9, the H3K9me binding protein Heterochro- matin Protein 1 (HP1), and components of the RNA interference (RNAi) pathway, contained significantly increased levels of extrachromosomal repeated DNAs [34]. This phenotype led us to hypothesize that these pathways may regulate additional aspects of genome stability in repeated DNAs and heterochromatin. Here we show that compromised heterochromatin composition, specif- ically due to mutations in Su(var)3-9 and the dcr-2 siRNA pathway component, results in increased spontaneous DNA damage in heterochromatin in somatic and meiotic cells. Detailed analyses of Su(var)3-9 mutants showed that diploid cells exhibit chromosomal defects such as translocations and aneuploidy. In addition, activation of DNA repair and mitotic checkpoints is required for cellular and organismal viability of mutant animals. We conclude that the H3K9 methylation and RNAi pathways are required to ensure the general stability of heterochromatic sequences. Requirement for Heterochromatin Stability Results Heterochromatic DNA damage is increased in Su(var)3-9 mutant somatic cells Spontaneous DNA damage in whole-mount (three dimensional) larval brain and imaginal disc tissues from wild type and Su(var)3- 9null mutants (generated from mothers and fathers) was examined by indirect immunofluorescence (IF) with antibodies specific to cH2Av and Rad51 (Figure 1A). cH2Av is the phosphorylated form of the histone variant H2Av (at serine 137) and is associated with DNA repair sites [35]. The Rad51 protein facilitates repair of double-stranded breaks via homologous recombination [36]. We observed that Su(var)3-9 somatic cells contained significantly increased frequencies of cH2Av and Rad51 foci in comparison to wild type (6.9-fold and 11-fold, respectively; p,0.001). Both cH2Av and Rad51 localization indicate sites of double-stranded breaks (DSBs), which we confirmed by the TUNEL (TdT-mediated dUTP nick end labeling) assay (Figure 1B; Materials and Methods). H3K9me2 modifications are highly enriched in heterochroma- tin of D. melanogaster, suggesting that DNA damage associated with loss or mislocalization of this modification would be enriched in heterochromatin. Additionally, we had previously demonstrated that repeated DNA integrity is severely compromised in Su(var)3-9 mutant cells, in terms of increased frequencies of extrachromo- somal repeated DNAs [34]. Comparing damage frequencies in heterochromatin and euchromatin is challenging in this mutant, due to loss of the standard heterochromatin markers H3K9me2 and HP1. However, heterochromatic regions are associated with intense DAPI staining, due to the AT-rich nature of many heterochromatic sequences, and perhaps a higher degree of condensation. Therefore, we measured the colocalization of markers for DNA damage and repair with ‘DAPI-bright’ and ‘DAPI-weak’ regions of interphase cells (Figure 1B). This method of quantitation is conservative; it underestimates the amount of damage in heterochromatin, because some heterochromatic sequences are present in DAPI-weak regions. cH2Av and TUNEL analysis showed that the numbers of foci localized in DAPI-weak regions do not significantly differ between wild type and Su(var)3-9 cells (Figure 1C). In sharp contrast, foci localized to DAPI-bright regions in Su(var)3-9 increased 96-fold over wild type for cH2Av and 88-fold for TUNEL (Figure 1C). IF analysis of Su(var)3-9 demonstrated that 70% (6s.d. 7.9%) of foci in DAPI-bright regions contained both cH2Av and Rad51, which identifies bona fide DSBs, as opposed to other kinds of damage. We conclude that loss of Su(var)3-9 leads to increased DNA damage in somatic cells, and that most or all of the additional damage is DSBs in heterochromatin. Reduced H3K9me in rDNA due to Su(var) mutations leads to formation of extrachromosomal ribosomal DNAs (rDNA) that can seed ectopic nucleolus formation [34]. Surprisingly, combined fluorescent in situ hybridization (FISH) and IF on whole-mount diploid tissues did not show significant colocalization between rDNA and cH2Av signals in the Su(var)3-9 diploid cells (data not shown). This suggests that DNA breaks within rDNA are repaired and form eccDNA more efficiently than DSBs in other regions of heterochromatin. Increased DNA breaks occur in Su(var)3-9 mutant oocytes and nurse cells Classical genetic studies have shown that reciprocal meiotic recombination (crossing-over) occurs on average once per euchromatic arm per nucleus, but does not occur in heterochro- matin [33,37]. Previous studies showed that heterozygous PLoS Genetics | www.plosgenetics.org 2 March 2009 | Volume 5 | Issue 3 | e1000435