Polycomb Domains Interact in Nuclear Space Figure 3. PcG targets interact specifically with PcDs and K27Ds. (A) Proportion of DIDs (y-axis) that overlap with PcDs is shown (observed - grey bars) and compared to an overlap based after random permutation (expected – white bars). Random sampling was repeated 1000-times and the bars show the average random overlap, while the error-bars show the standard deviation. Significance levels were determined using Pearson’s chi- square (x2) goodness of fit test and are indicated. Bait names are given (x-axis). (B) Proportion of DIDs that overlap with K27Ds. Plot details are descirbed in A. (C) mRNA expression levels of all baits as detected in 3rd instar larval central brain cells are shown in violin plots. Violin plots are a combination of box plots and smoothed histograms. Median values are indicated by white dot, lower and upper quartiles by black rectangle, and sample minima/maxima by black line. Smoothed histograms of the four PcG-target baits are depicted in light grey, lowly expressed non-target genes in white and highly expressed genes in dark grey. Data represents four replicate microarray experiments. Genes were probed multiple times in each experiment, giving 48 (Antp), 32 (Abd-B), and 16 (all other genes) data points. Notably, Ptx1 is higher expressed than the three other PcG targets, because it is expressed in a subset of larval brain cells (Figure S6, Text S1). doi:10.1371/journal.pgen.1001343.g003 fraction of DIDs match with PcDs (Figure 3A and 3B, Figure 5D and 5E, and Table S2). Taken together, our data strongly indicates that long-range interactions of the four PcG-target genes are highly specific with a strong preference for other PcG target loci. Large PcDs interact more often When we further characterized interacting PcDs we observed two distinctive features. First, interacting PcDs encompass significantly larger genomic regions than non- interacting PcDs (Figure 5; Table S3). Second, we overlaid our PcD map with a computational prediction of PRE sequences [27] and observed that interacting PcDs contain significantly more predicted PRE sequences than non-inter- acting PcDs (Table S3). In contrast, we observed no differences for gene density, repetitive DNA sequences, and non-coding evolutionary conserved sequences (Table S3). These results suggest that, at least for our 4 PcG baits in larval brain, increased size and presence of PREs may contribute to the frequency of interaction between two PcDs. PLoS Genetics | www.plosgenetics.org 5 March 2011 | Volume 7 | Issue 3 | e1001343