References 1. Kornberg RD (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184: 868–871. 2. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389: 251– 260. 3. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21: 381–395. 4. Leung W, Shaffer CD, Cordonnier T, Wong J, Itano MS, et al. (2010) Evolution of a distinct genomic domain in Drosophila: comparative analysis of the dot chromosome in Drosophila melanogaster and Drosophila virilis. Genetics 185: 1519– 1534. 5. Locke J, McDermid HE (1993) Analysis of Drosophila chromosome 4 using pulsed field gel electrophoresis. Chromosoma 102: 718–723. 6. Barigozzi C, Dolfini S, Fraccaro M, Raimondi GR, Tiepolo L (1966) In vitro study of the DNA replication patterns of somatic chromosomes of Drosophila melanogaster. Exp Cell Res 43: 231–234. 7. Arguello JR, Zhang Y, Kado T, Fan C, Zhao R, et al. (2010) Recombination yet inefficient selection along the Drosophila melanogaster subgroup’s fourth chromo- some. Mol Biol Evol 27: 848–861. 8. Bridges CB (1935) The mutants and linkage data of chromosome four of Drosophila melanogaster. Biol Zh 4: 401–420. 9. Riddle NC, Elgin SC (2008) A role for RNAi in heterochromatin formation in Drosophila. Curr Top Microbiol Immunol 320: 185–209. 10. Sun FL, Haynes K, Simpson CL, Lee SD, Collins L, et al. (2004) cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromo- some four. Mol Cell Biol 24: 8210–8220. 11. Wallrath LL, Elgin SC (1995) Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev 9: 1263–1277. 12. James TC, Eissenberg JC, Craig C, Dietrich V, Hobson A, et al. (1989) Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur J Cell Biol 50: 170–180. 13. 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. 14. Kharchenko PV, Alekseyenko AA, Schwartz YB, Minoda A, Riddle NC, et al. (2011) Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471: 480–485. 15. Riddle NC, Minoda A, Kharchenko PV, Alekseyenko AA, Schwartz YB, et al. (2011) Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin. Genome Res 21: 147–163. 16. Larsson J, Chen JD, Rasheva V, Rasmuson-Lestander A, Pirrotta V (2001) Painting of fourth, a chromosome-specific protein in Drosophila. Proc Natl Acad Sci U S A 98: 6273–6278. 17. Johansson AM, Stenberg P, Pettersson F, Larsson J (2007) POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation. PLoS Genet 3: e209. doi:10.1371/journal.pgen.0030209 18. Stenberg P, Lundberg LE, Johansson AM, Ryden P, Svensson MJ, et al. (2009) Buffering of segmental and chromosomal aneuploidies in Drosophila melanogaster. PLoS Genet 5: e1000465. doi:10.1371/journal.pgen.1000465 19. Johansson AM, Stenberg P, Allgardsson A, Larsson J (2012) POF Regulates the Expression of Genes on the Fourth Chromosome in Drosophila melanogaster by Binding to Nascent RNA. Mol Cell Biol 32: 2121–2134. 20. Brower-Toland B, Riddle NC, Jiang H, Huisinga KL, Elgin SC (2009) Multiple SET methyltransferases are required to maintain normal heterochromatin domains in the genome of Drosophila melanogaster. Genetics 181: 1303–1319. 21. Seum C, Reo E, Peng H, Rauscher FJ, 3rd, Spierer P, et al. (2007) Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet 3: e76. doi:10.1371/journal.pgen.0030076 22. Tzeng TY, Lee CH, Chan LW, Shen CK (2007) Epigenetic regulation of the Drosophila chromosome 4 by the histone H3K9 methyltransferase dSETDB1. Proc Natl Acad Sci U S A 104: 12691–12696. 23. Roy S, Ernst J, Kharchenko PV, Kheradpour P, Negre N, et al. (2010) Identification of functional elements and regulatory circuits by Drosophila modENCODE. Science 330: 1787–1797. 24. Riddle NC, Leung W, Haynes KA, Granok H, Wuller J, et al. (2008) An investigation of heterochromatin domains on the fourth chromosome of Drosophila melanogaster. Genetics 178: 1177–1191. 25. Schwartz YB, Kahn TG, Stenberg P, Ohno K, Bourgon R, et al. (2010) Alternative epigenetic chromatin states of polycomb target genes. PLoS Genet 6: e1000805. doi:10.1371/journal.pgen.1000805 26. Larschan E, Bishop EP, Kharchenko PV, Core LJ, Lis JT, et al. (2011) X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila. Nature 471: 115–118. 27. Core LJ, Waterfall JJ, Lis JT (2008) Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322: 1845–1848. 28. Slawson EE, Shaffer CD, Malone CD, Leung W, Kellmann E, et al. (2006) Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains. Genome Biol 7: R15. Drosophila Chromosome 4 Chromatin Structure 29. Hendrix DA, Hong JW, Zeitlinger J, Rokhsar DS, Levine MS (2008) Promoter elements associated with RNA Pol II stalling in the Drosophila embryo. Proc Natl Acad Sci U S A 105: 7762–7767. 30. Muse GW, Gilchrist DA, Nechaev S, Shah R, Parker JS, et al. (2007) RNA polymerase is poised for activation across the genome. Nat Genet 39: 1507– 1511. 31. Zeitlinger J, Stark A, Kellis M, Hong JW, Nechaev S, et al. (2007) RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat Genet 39: 1512–1516. 32. Zhang Y, Malone JH, Powell SK, Periwal V, Spana E, et al. (2010) Expression in aneuploid Drosophila S2 cells. PLoS Biol 8: e1000320. doi:10.1371/ journal.pbio.1000320 33. Nechaev S, Fargo DC, dos Santos G, Liu L, Gao Y, et al. (2010) Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327: 335–338. 34. Wakimoto BT, Hearn MG (1990) The effects of chromosome rearrangements on the expression of heterochromatic genes in chromosome 2L of Drosophila melanogaster. Genetics 125: 141–154. 35. Piacentini L, Fanti L, Negri R, Del Vescovo V, Fatica A, et al. (2009) Heterochromatin protein 1 (HP1a) positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila. PLoS Genet 5: e1000670. doi:10.1371/journal.pgen.1000670 36. Johansson AM, Stenberg P, Bernhardsson C, Larsson J (2007) Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster. Embo J 26: 2307–2316. 37. Phalke S, Nickel O, Walluscheck D, Hortig F, Onorati MC, et al. (2009) Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2. Nat Genet 41: 696–702. 38. Cryderman DE, Tang H, Bell C, Gilmour DS, Wallrath LL (1999) Heterochromatic silencing of Drosophila heat shock genes acts at the level of promoter potentiation. Nucleic Acids Res 27: 3364–3370. 39. Nechaev S, Adelman K (2011) Pol II waiting in the starting gates: Regulating the transition from transcription initiation into productive elongation. Biochim Biophys Acta 1809: 34–45. 40. Min IM, Waterfall JJ, Core LJ, Munroe RJ, Schimenti J, et al. (2011) Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells. Genes Dev 25: 742–754. 41. Bao X, Deng H, Johansen J, Girton J, Johansen KM (2007) Loss-of-function alleles of the JIL-1 histone H3S10 kinase enhance position-effect variegation at pericentric sites in Drosophila heterochromatin. Genetics 176: 1355–1358. 42. Ebert A, Schotta G, Lein S, Kubicek S, Krauss V, et al. (2004) Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev 18: 2973–2983. 43. Lerach S, Zhang W, Bao X, Deng H, Girton J, et al. (2006) Loss-of-function alleles of the JIL-1 kinase are strong suppressors of position effect variegation of the wm4 allele in Drosophila. Genetics 173: 2403–2406. 44. Zhang W, Deng H, Bao X, Lerach S, Girton J, et al. (2006) The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila. Development 133: 229–235. 45. Regnard C, Straub T, Mitterweger A, Dahlsveen IK, Fabian V, et al. (2011) Global analysis of the relationship between JIL-1 kinase and transcription. PLoS Genet 7: e1001327. doi:10.1371/journal.pgen.1001327 46. Deng H, Bao X, Zhang W, Girton J, Johansen J, et al. (2007) Reduced levels of Su(var)3-9 but not Su(var)2-5 (HP1) counteract the effects on chromatin structure and viability in loss-of-function mutants of the JIL-1 histone H3S10 kinase. Genetics 177: 79–87. 47. Cryderman DE, Grade SK, Li Y, Fanti L, Pimpinelli S, et al. (2005) Role of Drosophila HP1 in euchromatic gene expression. Dev Dyn 232: 767–774. 48. de Wit E, Greil F, van Steensel B (2007) High-resolution mapping reveals links of HP1 with active and inactive chromatin components. PLoS Genet 3: e38. doi:10.1371/journal.pgen.0030038 49. Liu LP, Ni JQ, Shi YD, Oakeley EJ, Sun FL (2005) Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription. Nat Genet 37: 1361–1366. 50. Lu BY, Emtage PC, Duyf BJ, Hilliker AJ, Eissenberg JC (2000) Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics 155: 699–708. 51. Yasuhara JC, Wakimoto BT (2008) Molecular landscape of modified histones in Drosophila heterochromatic genes and euchromatin-heterochromatin transition zones. PLoS Genet 4: e16. doi:10.1371/journal.pgen.0040016 52. Cryderman DE, Vitalini MW, Wallrath LL (2011) Heterochromatin protein 1a is required for an open chromatin structure. Transcription 2: 95–99. 53. Lin CH, Li B, Swanson S, Zhang Y, Florens L, et al. (2008) Heterochromatin protein 1a stimulates histone H3 lysine 36 demethylation by the Drosophila KDM4A demethylase. Mol Cell 32: 696–706. 54. Kim T, Buratowski S (2007) Two Saccharomyces cerevisiae JmjC domain proteins demethylate histone H3 Lys36 in transcribed regions to promote elongation. J Biol Chem 282: 20827–20835. 55. Larsson J, Svensson MJ, Stenberg P, Makitalo M (2004) Painting of fourth in genus Drosophila suggests autosome-specific gene regulation. Proc Natl Acad Sci U S A 101: 9728–9733. PLOS Genetics | www.plosgenetics.org 19 September 2012 | Volume 8 | Issue 9 | e1002954