Polycomb Domains Interact in Nuclear Space Figure 2. Long-range interactions of PcG target genes. (A) Chromatin profile of third chromosome representing Polycomb binding (blue) and H3K27me3 levels (green) as detected in 3rd instar larval central brain cells. Below graphs, the blue and green rectangles show Pc (PcD) and H3K27me3 (K27D) domains, respectively. X-axes indicate the chromosomal position (in Mbp), while the fold-change (either PC-Dam over Dam or H3K27me3 over IgG) is shown on the y-axes. Positions of the 4 PcG target baits are indicated (red). (B–E) We mapped long-range chromatin interactions using four different PcG-targets as bait, namely: Abdominal-B (Abd-B) promoter (B), Antennapedia (Antp) P1 promoter (C), Ptx1 (D), and trachealess (trh - E). Plots are described in Figure 1B. Notably, the majority of all DIDs (red boxes) coincide with either PcDs (blue boxes) and/or K27Ds (green boxes), and a minority (grey boxes) contacts other sequences. doi:10.1371/journal.pgen.1001343.g002 Next, we determined interactions using bait sequences contain- ing lowly expressed genes, namely Wnt inhibitor of Dorsal (wntD) and CG5107. The mRNA expression levels of wntD and CG5107 are equally low as the Antp and Abd-B genes (Figure 3C), but they are not part of PcDs (Figure 4A). The interaction maps of neither the wntD nor the CG5107 gene show enrichment for PcDs (Figure 4B, 4C). Less than 15% of all DIDs coincide with PcDs (Figure 3A, 3B, Table S2), which is 1.5 to 2-fold less than an overlap based on random chance. These results indicate that interactions between PcG target genes cannot be attributed to general interactions between transcriptionally inactive loci. We also observed that actively transcribed genes do not favor interactions with PcDs. For this purpose, we used the highly expressed Calreticulin (Crc) and RNA polymerase II 140 kD subunit (RpII140) genes as bait (Figure 3C), which are not PcG-targets (Figure 4A). Again, we observed that only a small, non-significant PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001343