,2-fold increased inside K27Ds (green area). (H). Violin plots show distribution of PcD (blue, top) and K27D (green, bottom) sizes (x-axis). Median sizes are indicated. (I). Distribution of gene content in PcDs. Number of genes inside a PcD are given on the x- axis and the total fraction of PcDs with a specific number of genes is on the y-axis. More than 60% of all PcDs contains more than 2 genes. (J). Violin plots show distributions of mRNA signal intensities (y-axis) of PC (blue), and H3K27me3 (green) targets, and nontarget genes (grey). mRNA signal intensities of PC and H3K27me3 targets are slightly reduced. P-values were calculated using a two-sided non-parametric Mann-Whitney U test. Found at: doi:10.1371/journal.pgen.1001343.s002 (7.73 MB TIF) Figure S3 mRNA expression levels of interacting genes on chromosome 3. Violin plots show distributions of mRNA signal intensities (y-axis) of genes that interact with PcG baits (Abd-B, Antp, Ptx1, and/or trh; blue), low expressed genes (wntD and/or CG5107; green), and high expressed genes (Crc and/or RpII140; red). The remainder genes that do not interact are shown in grey. Median values and population sizes (N) are indicated. Found at: doi:10.1371/journal.pgen.1001343.s003 (0.28 MB JPG) Figure S4 Gene expression is not detectably changed on In(3LR)sep chromosome. (A) Violin plots show distribution of gene expression changes (log2-transformed ratios of In(3LR)sep over wild type) of genes annotated to 3LIN (red), 3RIN (blue), or other chromosome arms (grey). Median values and population sizes (N) are indicated. (B) Histograms show frequency distributions of number of sex combs per 1st thoracic leg (x-axes) for crosses between wild type and mutant Sex combs reduced alleles (Scr; green) and crosses between In(3LR)sep and mutant Scr alleles (red). We examined Scr1 (left panel) and Scr4 (right panel) alleles. Total number of counted legs per group is indicated (n). P-values were calculated using a two- sided non-parametric Mann-Whitney U test. Found at: doi:10.1371/journal.pgen.1001343.s004 (0.43 MB JPG) Figure S5 Background filter. (A) Background filter applied to chromosome 3R using illustrative data, Abd-B (left) and Ptx1 (right). Bait sequence is positioned to the left of each graph (blue dashed line). All other sequences are plotted according to their physical distance to the bait sequence (x-axis). Data is plotted as log2- transformed 4C signals (y-axis) after applying a running median with a window of 50 probes (grey bars). The background filters (green lines) are highest closest to the bait sequence and gradually decrease as linear distance increases. As such, the filters represent a hypothetical linear conformation of the chromosome without any long-range chromatin interactions. For this filters a smoothening parameter (a) of 2 was used. (B) The smoothening parameter (a) affects the shape of the background filter. Abd-B 4C signal intensities (y-axis) after applying a running median with a window of 50 probes (grey bars) are plotted against chromosomal probe positions (x-axis). The position of the bait sequence is indicated (blue dashed line). Colored lines are background filters with different a values (red: a = 0.25; blue: a = 0.5; yellow: a = 1; and green: a = 1). The lower the a value the more tightly the line follows the original data. Higher a values show a more gradual slope. Found at: doi:10.1371/journal.pgen.1001343.s005 (1.11 MB JPG) References Polycomb Domains Interact in Nuclear Space Figure S6 Expression of Ptx1 in larval central nervous system. Ptx1 positive cells are found both in the central brain and ventral nerve cord (VNC). In the VNC Ptx1 is expressed in a segmental manner (right panel) that was also observed at embryonic stages [52]. In central brain (middle panel), a subset of all cells express Ptx1, while the majority does not. Found at: doi:10.1371/journal.pgen.1001343.s006 (1.88 MB TIF) Table S1 Genomic coordinates of PcDs and K27Ds in larval brain tissue, including gene annotation and long-range chromatin interactions. Found at: doi:10.1371/journal.pgen.1001343.s007 (0.19 MB XLS) Table S2 Genomic coordinates of DIDs in wild type larval brain tissue, including gene annotation and PcD/K27D specific interactions. Found at: doi:10.1371/journal.pgen.1001343.s008 (0.07 MB XLS) Table S3 Comparison of genomic features between interacting and non-interacting PcDs. Found at: doi:10.1371/journal.pgen.1001343.s009 (0.02 MB XLS) Table S4 Genomic coordinates of DIDs in In(3LR)sep larval brain tissue, including gene annotation and PcD/K27D specific interactions. Found at: doi:10.1371/journal.pgen.1001343.s010 (0.03 MB XLS) Table S5 Description of bait sequences used in this study, including 4C PCR primer sequences. Found at: doi:10.1371/journal.pgen.1001343.s011 (0.01 MB XLS) Table S6 Comparison of background filters with various levels of smoothening parameter (a) to test effects on overlap between DIDs and PcDs/K27Ds. Found at: doi:10.1371/journal.pgen.1001343.s012 (0.03 MB XLS) Text S1 Additional information on PC and H3K27me3 maps in larval brain tissue. Found at: doi:10.1371/journal.pgen.1001343.s013 (0.08 MB DOC) Acknowledgments We thank K. Basler, W. Brugman, U. Braunschweig, G. Filion, J. Kind, J. Vissers, A. Sparmann, G. Gargiulo, M. Vogel, B. van Veen, J. Noordermeer, and L. Fradkin for technical assistance and/or critically reading the manuscript. Author Contributions Conceived and designed the experiments: BT MvL BvS. Performed the experiments: BT MB HT. Analyzed the data: BT. Contributed reagents/ materials/analysis tools: RMK LP HT MN MS WdL. Wrote the paper: BT MvL BvS. 1. Schwartz YB, Pirrotta V (2007) Polycomb silencing mechanisms and the management of genomic programmes. Nat Rev Genet 8: 9–22. 2. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, et al. (2006) Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441: 349–353. 3. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K (2006) Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 20: 1123–1136. 4. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, et al. (2006) Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125: 301–313. 5. Schwartz YB, Kahn TG, Nix DA, Li XY, Bourgon R, et al. (2006) Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat Genet 38: 700–705. 6. 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. PLoS Genetics | www.plosgenetics.org 13 March 2011 | Volume 7 | Issue 3 | e1001343