stability of heterochromatic DNA in mitotic and meiotic cells requires the siRNA and H3K9 methylation pathways. Increased spontaneous DNA damage in heterochromatin in Su(var)3-9 and dcr- 2 mutants results from reduced H3K9me2 levels in heterochroma- tin, due to loss of the major H3K9 methyltransferase or misregula- tion of H3K9me2 localization, respectively [34]. Most or all of the increased DNA damage observed in mutants are located in heterochromatin, suggesting that the observed chromosomal defects and rearrangements and activation of DNA damage checkpoints are consequences of heterochromatin damage. The lower phenotypic severity exhibited by dcr-2, compared to Su(var)3-9, is correlated with the higher heterochromatic H3K9me2 content in dcr-2 compared to Su(var)3-9 mutants [34]. This phenotypic difference may be due to the hypomorphic nature of the dcr-2 mutant [46]. A potential cause of increased damage to heterochromatic sequences in Su(var)3-9 and dcr-2 mutants is defective DNA replication. Replication of heterochromatin normally occurs in late S-phase [30], and analysis of PCNA staining revealed that the proportion of cells in S-phase in Su(var)3-9 mutants is significantly lower than that in wild type. In the absence of H3K9 methylation, regions of repetitive DNA may be incompletely replicated or defective in chromatin reassembly [49] due to a shortened S phase. Alternatively, repeated DNA in heterochromatin may undergo faster replication, resulting in more errors; this is supported by the demonstration that heterochromatic regions are more efficiently endoreplicated in Su(var)3-9 mutant polytene chromosomes [50]. Thus, H3K9 methylation may be required to delay replication in repetitive regions, allowing resolution of replication forks; DSBs would be produced by stalled forks and/or fork collapse in the absence of this control. The demonstration that gH2Av foci were detected in G1, S and G2 stages in Su(var)3-9 mutants suggests that defective DNA replication is not the only cause of the increased damage and repair foci in heterochromatin. Another explanation is that proper DNA damage detection and subsequent DNA repair response in heterochromatin may require H3K9me2. This model is supported by the recent demonstration that HP1b, whose localization requires H3K9me, is needed for efficient DNA damage detection in mammalian cells (Ayoub et al., 2008). This requirement for HP1 in the DNA damage response suggests that euchromatin (containing little H3K9me and HP1b) and heterochromatin likely exhibit different responses to DNA damaging agents. gH2A is not recruited to the silent HM region in S. cerevisiae when a DSB is generated in nearby euchromatin [51]. Studies of ionizing radiation followed by quantitation of DNA break frequencies over time indicated that the vast majority of DNA breaks are located outside the heterochromatin in interphase cells an hour after damage was induced [51,52]. The lower frequencies of repair foci observed in heterochromatin suggests that euchromatin may be more prone to damage by ionizing radiation. Alternatively, initial damage frequencies within euchromatin and heterochro- matin may be very similar, with faster repair of heterochromatic breaks. Experiments to differentiate between these two explana- tions are needed, such as comparing break frequencies within seconds/minutes of damage. In addition, analyses are required to determine if H3K9me chromatin at heterochromatic DNA is required for suppressing DSB formation by ensuring normal heterochromatic replication or protection from damaging agents, and/or proper function of the DNA damage response. Mutations in the DNA damage checkpoint (mei-4129D, grp06034, and lokP6) exhibited synthetic lethality when combined with the Su(var)3-9 mutations, further cementing the essential role of the DNA damage checkpoint for the viability of Su(var)3-9 mutant animals. The incomplete synthetic lethality of the double mutants Requirement for Heterochromatin Stability (,50%) likely reflects redundancy of the checkpoint proteins; this is supported by the observation that mutations in both Chk1 and Chk2 cause complete lethality of Su(var)3-9 mutants. Intriguingly, zw10 and rod mutations also exhibit synthetic lethality when combined with the Su(var)3-9 mutations, suggesting that the Spindle Assembly Checkpoint (SAC) is also important for viability when H3K9me2 levels are reduced. Partial depletion of heterochromatic cohesin [34] and mitotic delays (this study) were observed in Su(var)3-9 mutants, and smc1/+, Su(var)3-9 double mutant animals displayed synthetic lethality. These observations suggest that reduced cohesion in Su(var)3-9 mutants results in minor defects in bi-orientation and maintenance of spindle attachments, which normally do not affect cell or organismal viability due to SAC activation. However, when the SAC is abrogated by zw10 or rod mutations, there is no mitotic delay and attachment defects cannot be fixed, resulting in missegregation and lethality. Mutations in Chk1 (grp) or Chk2 (lok) also suppress the increased mitotic index in Su(var)3-9 cells. However, it is unclear at this time whether Chk1 and Chk2 are participating in an SAC response [25,26], or are involved in a different checkpoint that is activated by DNA damage that persists into mitosis in these mutants. The RNAi pathway directly impacts genome stability and the development of germlines in D. melanogaster, mammals, and C. elegans. Specifically, Piwi/Aubergine regulation of repeat associated small interfering RNAs (rasiRNAs) mediates silencing of retro- transposons and the repeated Stellate locus [53], and promotes normal embryonic axis specification and germline development [54]. Mutations in the rasiRNA pathway components, armitage and aubergine lead to disruption of embryonic axis specification and increased DNA breaks in meiotic cells [54]. In contrast to our observations, mei-W68 mutations did not suppress DSB formation in rasiRNA mutants. Furthermore, increased DNA damage in armitage and aubergine is specific to the germline [54], and we did not observe microtubule disorganization in Su(var)3-9 mutants (data not shown). Differences between the phenotypes in Su(var)3-9/dcr- 2 and rasiRNA mutants suggest that these pathways ensure genome integrity via different mechanisms. The majority of studies of chromatin modifications focus on transcriptional regulation. Current epigenomic characterizations of different cancer types also put great emphasis on epigenetic regulation of transcription, showing dramatic chromatin alter- ations of oncogenes or tumor suppressor genes whose transcrip- tional deregulation contribute to cancer progression [55,56]. Our findings contribute to the growing realization that local chromatin structure and epigenetic regulation also impact genome structural integrity (reviewed in [57]. We have shown that mutations affecting H3K9 methylation lead to increased spontaneous damage that is predominantly or exclusively located in hetero- chromatin. Consequences of this defect are chromosome structural defects and genome instability, events that are also correlated with uncontrolled cell growth and tumorigenesis. SUV39h double knockout mice exhibit partial embryonic lethality and genome instability [42], as well as global gene deregulation [58,59]. It is therefore unclear if the impact of SUV39h on mammalian genome stability is due to a direct effect on DNA damage and repair in heterochromatin, or misregulation of key developmental genes and cell cycle regulators. In contrast, H3K9 methylation by Su(var)3-9 in Drosophila is restricted to heterochromatin (Langley and Karpen, unpublished). Su(var)3-9null mutations in Drosophila therefore present a unique opportunity to study the role of H3K9me chromatin in heterochromatin stability, with minimal indirect effects from transcriptional deregulation. Drosophila heterochromatin, not euchromatin, resembles mam- malian euchromatic genomes in their complex DNA organization. PLoS Genetics | www.plosgenetics.org 10 March 2009 | Volume 5 | Issue 3 | e1000435