Table S1 Progeny counts for genetic crosses used to calculate the viability of single and double mutants. Found at: doi:10.1371/journal.pgen.1000435.s004 (0.02 MB XLS) Acknowledgements The authors thank Michael Brodsky, Richard Carthew, Kent Golic, Michael Goldberg, Scott Hawley, Daryl Henderson, Jim Kadonaga, Kim McKim, Gunter Reuter, and Tin Tin Su for fly mutants, Abby Dernburg for the X chromosome templates, and Aki Minoda and Roger Hoskins for the 2nd and 3rd chromosome templates. We thank David Acevedo and References 1. Heitz E (1928) Das heterochromatin der moose. I Jb wiss Bot 69: 762–818. 2. Hoskins RA, Carlson JW, Kennedy C, Acevedo D, Evans-Holm M, et al. (2007) Sequence Finishing and Mapping of Drosophila melanogaster Heterochromatin. Science 316: 1625–1628. 3. Smith CD, Shu S, Mungall CJ, Karpen GH (2007) The Release 5.1 annotation of Drosophila melanogaster heterochromatin. Science 316: 1586–1591. 4. Karpen GH, Le MH, Le H (1996) Centric heterochromatin and the efficiency of achiasmate disjunction in Drosophila female meiosis. Science 273: 118–122. 5. Dernburg AF, Sedat JW, Hawley RS (1996) Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell 86: 135–146. 6. de Lange T (2005) Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19: 2100–2110. 7. Yasuhara JC, Wakimoto BT (2006) Oxymoron no more: the expanding world of heterochromatic genes. Trends in Genetics 22: 330–338. 8. Jenuwein T, Allis CD (2001) Translating the histone code. Science 293: 1074–1080. 9. Grewal SI, Moazed D (2003) Heterochromatin and epigenetic control of gene expression. Science 301: 798–802. 10. Moazed D, Buhler M, Buker SM, Colmenares SU, Gerace EL, et al. (2006) Studies on the Mechanism of RNAi-dependent Heterochromatin Assembly. Cold Spring Harb Symp Quant Biol 71: 461–471. 11. Downs JA, Nussenzweig MC, Nussenzweig A (2007) Chromatin dynamics and the preservation of genetic information. Nature 447: 951–958. 12. Unal E, Arbel-Eden A, Sattler U, Shroff R, Lichten M, et al. (2004) DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16: 991–1002. 13. van Attikum H, Fritsch O, Hohn B, Gasser SM (2004) Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119: 777–788. 14. Morrison AJ, Highland J, Krogan NJ, Arbel-Eden A, Greenblatt JF, et al. (2004) INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119: 767–775. 15. Keogh M-C, Kim J-A, Downey M, Fillingham J, Chowdhury D, et al. (2006) A phosphatase complex that dephosphorylates [gamma]H2AX regulates DNA damage checkpoint recovery. Nature 439: 497–501. 16. Karagiannis TC, El-Osta A (2006) Chromatin modifications and DNA double- strand breaks: the current state of play. Leukemia 21: 195–200. 17. Su TT (2006) Cellular Responses to DNA Damage: One Signal, Multiple Choices. Annual Review of Genetics 40: 187–208. 18. Staeva-Vieira E, Yoo S, Lehmann R (2003) An essential role of DmRad51/ SpnA in DNA repair and meiotic checkpoint control. Embo J 22: 5863–5874. 19. Johnson-Schlitz DM, Flores C, Engels WR (2007) Multiple-Pathway Analysis of Double-Strand Break Repair Mutations in Drosophila. PLoS Genetics 3: e50. 20. Jaklevic BR, Su TT (2004) Relative contribution of DNA repair, cell cycle checkpoints, and cell death to survival after DNA damage in Drosophila larvae. Curr Biol 14: 23–32. 21. Brodsky MH, Sekelsky JJ, Tsang G, Hawley RS, Rubin GM (2000) mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development. Genes Dev 14: 666–678. 22. Hari KL, Santerre A, Sekelsky JJ, McKim KS, Boyd JB, et al. (1995) The mei-41 gene of D. melanogaster is a structural and functional homolog of the human ataxia telangiectasia gene. Cell 82: 815–821. 23. Brodsky MH, Weinert BT, Tsang G, Rong YS, McGinnis NM, et al. (2004) Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol Cell Biol 24: 1219–1231. 24. Xu J, Xin S, Du W (2001) Drosophila Chk2 is required for DNA damage- mediated cell cycle arrest and apoptosis. FEBS Lett 508: 394–398. 25. Royou A, Macias H, Sullivan W (2005) The Drosophila Grp/Chk1 DNA Damage Checkpoint Controls Entry into Anaphase. Current Biology 15: 334–339. 26. Xu J, Du W (2003) Drosophila chk2 plays an important role in a mitotic checkpoint in syncytial embryos. FEBS Letters 545: 209–212. 27. Zachos G, Black EJ, Walker M, Scott MT, Vagnarelli P, et al. (2007) Chk1 is required for spindle checkpoint function. Dev Cell 12: 247–260. 28. Song YH (2005) Drosophila melanogaster: a model for the study of DNA damage checkpoint response. Mol Cells 19: 167–179. 29. Pearson CE, Edamura KN, Cleary JD (2005) Repeat Instability: Mechanisms of dynamic mutations. Nature Reviews Genetics 6: 729–742. Requirement for Heterochromatin Stability Jessica Trang for assistance with genetic crosses. We thank Michael Brodsky, Irene Chiolo, Abby Dernburg, Sylvia Erhardt, Barbara Mellone and Weiguo Zhang for helpful discussions and critical comments on the manuscript. Author Contributions Conceived and designed the experiments: JCP GHK. Performed the experiments: JCP. Analyzed the data: JCP GHK. Wrote the paper: JCP GHK. 30. Sullivan B, Karpen G (2001) Centromere identity in Drosophila is not determined in vivo by replication timing. J Cell Biol 154: 683–690. 31. Stack SM (1984) Heterochromatin, the synaptonemal complex and crossing over. J Cell Sci 71: 159–176. 32. Carpenter AT (1975) Electron microscopy of meiosis in Drosophila melanoga- ster females: II. The recombination nodule–a recombination-associated structure at pachytene? Proc Natl Acad Sci U S A 72: 3186–3189. 33. Mehrotra S, McKim KS (2006) Temporal analysis of meiotic DNA double- strand break formation and repair in Drosophila females. PLoS Genet 2: e200. 34. Peng JC, Karpen GH (2007) H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 9: 25–35. 35. Madigan JP, Chotkowski HL, Glaser RL (2002) DNA double-strand break- induced phosphorylation of Drosophila histone variant H2Av helps prevent radiation-induced apoptosis. Nucl Acids Res 30: 3698–3705. 36. Shinohara A, Ogawa H, Matsuda Y, Ushio N, Ikeo K, et al. (1993) Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA. Nat Genet 4: 239–243. 37. Stern C (1936) Somatic Crossing over and Segregation in Drosophila Melanogaster. Genetics 21: 625–730. 38. Westphal T, Reuter G (2002) Recombinogenic Effects of Suppressors of Position-Effect Variegation in Drosophila. Genetics 160: 609–621. 39. McKim KS, Hayashi-Hagihara A (1998) mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved. Genes Dev 12: 2932–2942. 40. Dernburg AF, Broman KW, Fung JC, Marshall WF, Philips J, et al. (1996) Perturbation of nuclear architecture by long-distance chromosome interactions. Cell 85: 745–759. 41. Koehler KE, Boulton CL, Collins HE, French RL, Herman KC, et al. (1996) Spontaneous X chromosome MI and MII nondisjunction events in Drosophila melanogaster oocytes have different recombinational histories. Nat Genet 14: 406–414. 42. Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, et al. (2001) Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107: 323–337. 43. Schotta G, Ebert A, Krauss V, Fischer A, Hoffmann J, et al. (2002) Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. Embo J 21: 1121–1131. 44. Chan GKT, Jablonski SA, Starr DA, Goldberg ML, Yen TJ (2000) Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores. Nat Cell Biol 2: 944–947. 45. McVey M, Radut D, Sekelsky JJ (2004) End-Joining Repair of Double-Strand Breaks in Drosophila melanogaster Is Largely DNA Ligase IV Independent. Genetics 168: 2067–2076. 46. Lee YS, Nakahara K, Pham JW, Kim K, He Z, et al. (2004) Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117: 69–81. 47. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, et al. (2001) Initial sequencing and analysis of the human genome. Nature 409: 860–921. 48. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al. (2001) The Sequence of the Human Genome. Science 291: 1304–1351. 49. Houlard M, Berlivet S, Probst AV, Quivy J-P, ry P, et al. (2006) CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells. PLoS Genetics 2: e181. 50. Andreyeva EN, Kolesnikova TD, Demakova OV, Mendez-Lago M, Pokholkova GV, et al. (2007) High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants. PNAS 104: 12819–12824. 51. Kim J-A, Kruhlak M, Dotiwala F, Nussenzweig A, Haber JE (2007) Heterochromatin is refractory to {gamma}-H2AX modification in yeast and mammals. J Cell Biol 178: 209–218. 52. Cowell IG, Sunter NJ, Singh PB, Austin CA, Durkacz BW, et al. (2007) gammaH2AX Foci Form Preferentially in Euchromatin after Ionising- Radiation. PLoS ONE 2: e1057. 53. Theurkauf WE, Klattenhoff C, Bratu DP, McGinnis-Schultz N, Koppetsch BS, et al. (2006) rasiRNAs, DNA Damage, and Embryonic Axis Specification. Cold Spring Harbor Symposia on Quantitative Biology 71: 171–180. PLoS Genetics | www.plosgenetics.org 13 March 2009 | Volume 5 | Issue 3 | e1000435