Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation Jamy C. Peng1,2¤, Gary H. Karpen1,2* 1 Lawrence Berkeley National Laboratory, Department of Genome and Computational Biology, Berkeley, California, United States of America, 2 Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, United States of America Abstract Heterochromatin contains many repetitive DNA elements and few protein-encoding genes, yet it is essential for chromosome organization and inheritance. Here, we show that Drosophila that lack the Su(var)3-9 H3K9 methyltransferase display significantly elevated frequencies of spontaneous DNA damage in heterochromatin, in both somatic and germ-line cells. Accumulated DNA damage in these mutants correlates with chromosomal defects, such as translocations and loss of heterozygosity. DNA repair and mitotic checkpoints are also activated in mutant animals and are required for their viability. Similar effects of lower magnitude were observed in animals that lack the RNA interference pathway component Dcr2. These results suggest that the H3K9 methylation and RNAi pathways ensure heterochromatin stability. Citation: Peng JC, Karpen GH (2009) Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation. PLoS Genet 5(3): e1000435. doi:10.1371/journal.pgen.1000435 Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received May 2, 2008; Accepted February 23, 2009; Published March 27, 2009 Copyright: 2009 Peng, Karpen. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No specific funding was received for this work. Competing Interests: The authors have declared that no competing interests exist. * E-mail: karpen@fruitfly.org ¤ Current address: Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, United States of America Introduction The eukaryotic genome is partitioned into cytologically and functionally distinct heterochromatin and euchromatin. Hetero- chromatin was initially defined as the part of the genome that remains compacted throughout the cell cycle and stains intensively with DNA dyes [1]. Heterochromatin is rich in tandemly repeated sequences and transposable elements [2], and as a result was mistakenly assumed to be genomic ‘junk’ with no function. In fact, heterochromatin contains essential protein coding genes [3], and encodes indispensable chromosomal functions such as centromeres, telomeres, nuclear organization, and meiotic homolog pairing [4–7]. Different chromatin states have been correlated with patterns of post-translational histone modifications. Chromatin associated with actively expressed genes contains methylated lysine 4 of histone H3 (H3K4me) and hyper-acetylated histones. In contrast, H3K9me2 and me3 modifications in ‘silent’ chromatin have become a standard characteristic of heterochromatin [8]. Recent studies have shown that RNA interference (RNAi) pathways are required for the initial recruitment of H3K9 methyltransferases (HMTases), such as clr4 in S. pombe and Su(var)3-9 in Drosophila, and for the establishment and maintenance of heterochromatin [9,10]. In addition to regulating functions such as transcription and chromosome organization, chromatin is involved in the cellular response to DNA damage, especially double-stranded breaks (DSBs) [11]. The chromatin structure around DSBs assists recruitment and retention of DNA repair components and cell cycle checkpoint proteins. For example, phosphorylated H2A variants—serine 139 of H2Ax in mammals, serine 129 of H2A in yeast (cH2AX in mammals), and serine 137 of H2Av (cH2Av) in Drosophila—are important for recruitment of cohesins [12], ATP-dependent chromatin remodelers and various DNA repair factors [13,14]. Dephosphorylation of H2A variants serves as a signal for cell cycle checkpoint recovery [15]. Other histone modifications implicated in the DNA damage response are phosphorylation, acetylation, and methylation of histone H4 residues, H3K79 methylation, H2BK123 ubiquitination, and H2AS129 phosphorylation [16]. The two main repair responses to DSBs are homologous recombination (HR) and non-homologous end joining (NHEJ). Comparative studies in S. cerevisiae, mammals and Drosophila showed many similarities while highlighting significant differences [17]. For example, a mutation in Rad51, a critical component of the HR pathway, causes lethality in mouse but does not impact viability in S. cerevisiae or Drosophila [18]. Compared to other systems, DNA repair factors in Drosophila appear to be highly redundant, and repair pathways can functionally compensate for each other [19]. Differences between organisms also exist in the DNA damage checkpoint pathways, which delay cell cycle progression to facilitate efficient DNA repair. The main regulators are the phosphoinositol kinases ATM and ATR in mammalian and yeast systems, and ATR/mei-41 in Drosophila [20–22]. ATM and ATR recruitment to DSBs results in H2Av phosphorylation and activation of checkpoint kinase 1 (Chk1, grp in Drosophila) and/or checkpoint kinase 2 (Chk2, lok in Drosophila), which then delay cell cycle progression until the damage is repaired [20,23,24]. Chk1- and/or Chk2- mediated phosphorylation in response to DNA damage regulates the G1-S transition, S phase progression, G2-M transitions, and the metaphase-anaphase transition [25–27]. ATM (tefu) in Drosophila functions in telomere protection in addition to apoptotic signaling via the p53 pathway (Oikemus et al., 2004). In contrast to mammalian systems, p53 in Drosophila does not directly participate in the DNA damage checkpoint response, and instead activates the apoptosis pathway in response to persistent, unrepaired DNA damage [28]. PLoS Genetics | www.plosgenetics.org 1 March 2009 | Volume 5 | Issue 3 | e1000435