Polycomb Domains Interact in Nuclear Space Figure 1. Detection and visualization of long-range chromatin interactions. (A) Genomic clusters of multiple restriction fragments with increased 4C signals were identified using a sliding window filter on the normalized data (running median, window = 51 probes; grey bars) along chromosome 3R. We applied a ‘‘background filter’’ to correct for interactions that are a consequence of short nucleotide distance to the bait sequence (green curve). After filtering the normalized data, clusters of restriction fragments that exceed the curve (running median, window = 51 probes; red bars) represent long-range chromatin interactions. X-axis indicates the chromosomal position (in Mbp), while log2-transformed 4C signals are shown on the y-axes. Position of Abd-B is blue dashed line.(B) Domainograms visualize long-range chromatin contacts detected using normalized data obtained with the Abd-B promoter as bait (red arrowhead). X-axis indicates the chromosomal position (in Mbp), while the window size of the domainogram algorithm is shown on the y-axis. Interactions are plotted using a color scale with black for non-significant and red for highly significant (scale bar). Only window size #200 probes (corresponding to 840 Kbp on average) are shown. We used a dynamic programming algorithm to identify non-overlapping Discrete Interaction Domains (DIDs, black rectangles bottom). (C) Domainogram of Abd-B data after applying the ‘‘background filter.’’ Plotted as in B. Note, centromeric sequences (CEN) are not drawn to scale. doi:10.1371/journal.pgen.1001343.g001 We performed a reciprocal experiment, using the Antp P1 promoter as bait (hereafter: Antp; Figure 2A, Figure S2). We aimed to: (1) confirm the interaction between BX-C and ANT-C; and (2) assess whether this bait also prefers to interact with other PcDs. Indeed, a highly significant proportion of all DIDs overlap with PcDs and K27Ds (Figure 3A, 3B, Table S2), including an interaction with the BX-C (,12.5–13.0 Mbp, Figure 2C). Finally, we identified 26 PcDs that are in close contact with Antp, of which 16 also interact with Abd-B (Table S1). As for Abd-B, most interactions take place within chromosome 3R, which contains the Antp bait sequence. However, we found one interaction with a PcD located on chromosome 4, demon- strating that interactions between two different chromosomes can occur, albeit with low frequency. Thus, we confirmed the previously reported long-range interaction between the Homeotic gene clusters [13,14], validating the specificity of our 4C assay. Importantly, we observed that the majority of other interacting regions overlap with PcDs, indicating that Abd-B and Antp both interact extensively with other PcDs. Non-Homeotic PcG targets prefer to interact with other PcDs Next, we asked whether non-Homeotic PcG target genes also prefer to contact other PcDs. We performed experiments using Ptx1 and trachealess (trh) as baits. The results are comparable to those obtained with Abd-B and Antp. First, most interactions are again mostly restricted to the chromosome arm that contains the bait (Figure 2D, 2E; Figure S2). Second, the observed overlap between DIDs and PcDs/K27Ds is highly signficant (Figure 3A, 3B). Altogether, we conclude that Ptx1 and trh, like the Homeotic genes, preferentially interact with other PcDs. Non-PcG target loci lack preference for PcDs While the vast majority of all DIDs of the four PcG target genes coincide with PcDs, a small subset lacks PcDs, suggesting that PcG-target genes can sometimes interact with other chromatin types. In mammalian cells, it has been found that the genome spatially segregates into open/active and closed/ inactive compartments [20,26]. Therefore, we tested the possibility that our largely inactive PcDs can interact with other inactive genome regions and together make an inactive nuclear compartment. We first generated microarray expression profiles of brain tissue to assess the transcriptional activity of genes in DIDs. We did not observe any gene expression differences when we compared genes located inside DIDs to genes from other parts of the chromosome arm (Figure S3). This suggests that low transcriptional activity in DIDs cannot simply account for the observed long-range interactions. We note however that the resolution of 4C may be rather low, because most DIDs are large genomic regions (average size = 170 Kbp), and contain many genes (on average 22) that vary greatly in expression level. PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1001343