Figure S5 No preferential spacing of SU(HW) peaks in a range of 40kb. Histogram of the pair-wise distances between all SU(HW) peaks. X-axis depicts genomic distance between the peaks. Figure S6 Changes in NL interaction after altering SU(HW) expression levels are reproducible. Ave changes in NL interaction levels per LAD, for LADs without SU(HW) (grey), LADs with at least one SU(HW) peak (light blue or orange), the 25% of LADs with the highest SU(HW) peak density (dark blue or orange) after knockdown with amplicon 1 (blue, upper panles), knockdown with amplicon 2 (blue, middle panels) and overexpression of SU(HW) (orange, lower panels). First experi- ment (left panels), second experiment (right panels). Table S1 LAD positions. This file is a flat text file in GFF format (http://www.sanger.ac.uk/Software/formats/GFF) listing the positions of all 412 LADs (Drosophila melanogaster genome sequence release 4.3). Score (column 6) indicates the fraction of References 1. Prokocimer M, Davidovich M, Nissim-Rafinia M, Wiesel-Motiuk N, Bar DZ, et al. (2009) Nuclear lamins: key regulators of nuclear structure and activities. J Cell Mol Med 13: 1059–1085. 2. Belmont AS, Zhai Y, Thilenius A (1993) Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography. J Cell Biol 123: 1671–1685. 3. Schermelleh L, Carlton PM, Haase S, Shao L, Winoto L, et al. (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320: 1332–1336. 4. Takizawa T, Meaburn KJ, Misteli T (2008) The meaning of gene positioning. Cell 135: 9–13. 5. Fedorova E, Zink D (2009) Nuclear genome organization: common themes and individual patterns. Curr Opin Genet Dev 19: 166–171. 6. van Steensel B, Henikoff S (2000) Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18: 424–428. 7. Pickersgill H, Kalverda B, de Wit E, Talhout W, Fornerod M, et al. (2006) Characterization of the Drosophila melanogaster genome at the nuclear lamina. Nat Genet 38: 1005–1014. 8. Guelen L, Pagie L, Brasset E, Meuleman W, Faza MB, et al. (2008) Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453: 948–951. 9. Goldman RD, Shumaker DK, Erdos MR, Eriksson M, Goldman AE, et al. (2004) Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A 101: 8963–8968. 10. Malhas A, Lee CF, Sanders R, Saunders NJ, Vaux DJ (2007) Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression. J Cell Biol 176: 593–603. 11. Scaffidi P, Misteli T (2005) Reversal of the cellular phenotype in t0he premature aging disease Hutchinson-Gilford progeria syndrome. Nat Med 11: 440–445. 12. Columbaro M, Capanni C, Mattioli E, Novelli G, Parnaik VK, et al. (2005) Rescue of heterochromatin organization in Hutchinson-Gilford progeria by drug treatment. Cell Mol Life Sci 62: 2669–2678. 13. Shumaker DK, Dechat T, Kohlmaier A, Adam SA, Bozovsky MR, et al. (2006) Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging. Proc Natl Acad Sci U S A 103: 8703–8708. 14. Shevelyov YY, Lavrov SA, Mikhaylova LM, Nurminsky ID, Kulathinal RJ, et al. (2009) The B-type lamin is required for somatic repression of testis-specific gene clusters. Proc Natl Acad Sci U S A 106: 3282–3287. 15. Williams RR, Azuara V, Perry P, Sauer S, Dvorkina M, et al. (2006) Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus. J Cell Sci 119: 132–140. 16. Peric-Hupkes D, Meuleman W, Pagie L, Bruggeman SW, Solovei I, et al. (2010) Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol Cell 38: 603–613. 17. Yokochi T, Poduch K, Ryba T, Lu J, Hiratani I, et al. (2009) G9a selectively represses a class of late-replicating genes at the nuclear periphery. Proc Natl Acad Sci U S A 106: 19363–19368. 18. Zink D, Amaral MD, Englmann A, Lang S, Clarke LA, et al. (2004) Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei. J Cell Biol 166: 815–825. 19. Geyer PK, Corces VG (1992) DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev 6: 1865–1873. 20. Kellum R, Schedl P (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 12: 2424–2431. 21. Gaszner M, Felsenfeld G (2006) Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7: 703–713. 22. Sun FL, Elgin SC (1999) Putting boundaries on silence. Cell 99: 459–462. SU(HW) Fine-Tunes Nuclear Lamina Interactions array probes inside the LAD with a positive LAM DamID logratio, after applying a running median filter with window size 5. Acknowledgments We thank Marja Nieuwland for mRNA isolation, labeling and microarray hybridization; members of our laboratory, Paul Koppen, Maarten Fornerod and Bernike Kalverda for helpful discussions; and Pam Geyer for providing SU(HW) antibody. Author Contributions Conceived and designed the experiments: JvB BvS. Performed the experiments: JvB. Analyzed the data: JvB LP. Wrote the manuscript: JvB BvS. Performed microarray hybridizations: WB RMK. Generated the Lamin DamID profile: UB. Performed sequence motif searches: WM. 23. Hagstrom K, Muller M, Schedl P (1996) Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev 10: 3202–3215. 24. Mihaly J, Hogga I, Gausz J, Gyurkovics H, Karch F (1997) In situ dissection of the Fab-7 region of the bithorax complex into a chromatin domain boundary and a Polycomb-response element. Development 124: 1809–1820. 25. Zhao K, Hart CM, Laemmli UK (1995) Visualization of chromosomal domains with boundary element-associated factor BEAF-32. Cell 81: 879–889. 26. Gaszner M, Vazquez J, Schedl P (1999) The Zw5 protein, a component of the scs chromatin domain boundary, is able to block enhancer-promoter interaction. Genes Dev 13: 2098–2107. 27. Ohtsuki S, Levine M (1998) GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo. Genes Dev 12: 3325–3330. 28. Bushey AM, Dorman ER, Corces VG (2008) Chromatin insulators: regulatory mechanisms and epigenetic inheritance. Mol Cell 32: 1–9. 29. Capelson M, Corces VG (2005) The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol Cell 20: 105–116. 30. Braunschweig U, Hogan GJ, Pagie L, van Steensel B (2009) Histone H1 binding is inhibited by histone variant H3.3. Embo J 28: 3635–3645. 31. Bushey AM, Ramos E, Corces VG (2009) Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev 23: 1338–1350. 32. de Wit E, Braunschweig U, Greil F, Bussemaker HJ, van Steensel B (2008) Global chromatin domain organization of the Drosophila genome. PLoS Genet 4: e1000045. 33. van Steensel B, Braunschweig U, Filion GJ, Chen M, van Bemmel JG, et al. (2010) Bayesian network analysis of targeting interactions in chromatin. Genome Res 20: 190–200. 34. Moorman C, Sun LV, Wang J, de Wit E, Talhout W, et al. (2006) Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster. Proc Natl Acad Sci U S A 103: 12027–12032. 35. Blanton J, Gaszner M, Schedl P (2003) Protein:protein interactions and the pairing of boundary elements in vivo. Genes Dev 17: 664–675. 36. Gauhar Z, Sun LV, Hua S, Mason CE, Fuchs F, et al. (2009) Genomic mapping of binding regions for the Ecdysone receptor protein complex. Genome Res 19: 1006–1013. 37. Tolhuis B, de Wit E, Muijrers I, Teunissen H, Talhout W, et al. (2006) Genome- wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster. Nat Genet 38: 694–699. 38. Adryan B, Woerfel G, Birch-Machin I, Gao S, Quick M, et al. (2007) Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila. Genome Biol 8: R167. 39. Pai CY, Lei EP, Ghosh D, Corces VG (2004) The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol Cell 16: 737–748. 40. Gerasimova TI, Lei EP, Bushey AM, Corces VG (2007) Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila. Mol Cell 28: 761–772. 41. Mohan M, Bartkuhn M, Herold M, Philippen A, Heinl N, et al. (2007) The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning. Embo J 26: 4203–4214. 42. Gerasimova TI, Corces VG (1998) Polycomb and trithorax group proteins mediate the function of a chromatin insulator. Cell 92: 511–521. 43. Gerasimova TI, Byrd K, Corces VG (2000) A chromatin insulator determines the nuclear localization of DNA. Mol Cell 6: 1025–1035. 44. Comet I, Savitskaya E, Schuettengruber B, Negre N, Lavrov S, et al. (2006) PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter. Dev Cell 11: 117–124. 45. Kyrchanova O, Chetverina D, Maksimenko O, Kullyev A, Georgiev P (2008) Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements. Nucleic Acids Res 36: 7019–7028. PLoS ONE | www.plosone.org 12 November 2010 | Volume 5 | Issue 11 | e15013