Author Summary The folding of chromosomes inside the cell nucleus is a fascinating yet poorly understood topological problem. It is thought that certain genomic loci that are distant in the linear genome may come together in nuclear space by folding of the chromosome fiber. Previously, such a long- range interaction was found in Drosophila for two genomic loci that are known to be bound by the Polycomb Repressive Complex. Because hundreds of genes are known to be bound by Polycomb proteins, we asked whether such long-range contacts are more common. To address this, we optimized the Chromosome Conforma- tion Capture on Chip (4C) technology for use in small tissue samples. Using this technique in dissected larval brains, we found that indeed Polycomb target genes interact frequently with each other, even when they are separated by megabases of sequence. However, these long-range interactions occur almost exclusively on the same chromosome arm. By using a rearranged chromo- some in which segments are swapped between two arms, we demonstrate that not DNA sequence but chromosome architecture imposes this restriction. Taken together, our data demonstrate that Polycomb target genes extensively interact in nuclear space, but only when they are located on the same chromosome arm. Results Experimental design We applied Chromosome Conformation Capture on Chip (4C) to map long-range chromatin interactions among PcDs on a genome-wide scale [20]. We initially focused on the Abdominal-B (Abd-B) and Antennapedia (Antp) genes, because they are part of the Bithorax (BX-C) and Antennapedia (ANT-C) complexes, which were found to interact in nuclear space by microscopy [13,14]. Importantly, these interactions occur more frequently when these Homeotic genes are silenced [14]. We therefore used central brain tissue from 3rd instar larvae, in which both Antp and Abd-B are repressed by PcG proteins (Figure S1) [21]. In addition, when we mapped Polycomb (PC) binding and H3K27me3 levels in central brain tissue (Text S1 and Table S1), we observed that both genes reside in chromatin domains with high levels of PC and H3K27me3 (Figure S2A, S2B). Hence, we expected Abd-B and Antp to interact in nuclear space of central brain cells. The available 4C assay requires large numbers of cells due to a relatively low efficiency in detecting long-range interactions [22]. To accommodate this requirement, we dissected and pooled central brain hemispheres from 40 larvae. However, this still did not yield enough material for subsequent microarray analyses (data not shown). To overcome this, we implemented a modification of the 4C protocol in which the 4C PCR products are further amplified in a linear fashion using a T7 RNA amplification procedure [23]. Finally, we fluorescently labeled the amplified products with dye and hybridized them to a specially designed microarray, which covers approximately 92% of the non- repetitive fly genome. In this way, we could identify all fragments that are in close contact with a chosen locus in larval brain cells. Detection and visualization of long-range chromatin interactions We used recently developed [24] and novel computational tools to detect and visualize long-range chromatin interactions. Here, we briefly describe the technical aspects of these tools using 4C Polycomb Domains Interact in Nuclear Space assay results obtained with the Abd-B promoter region (hereafter: Abd-B) as bait. In a typical 4C experiment, reliable interactions can only be detected as genomic clusters of multiple restriction fragments with increased signals [22]. Such clusters can be identified using a sliding window filter that improves the signal/noise ratio by integrating signals from neighboring probes [20]. Indeed, after applying a running median filter (window size = 51 probes) we observed genomic clusters with increased ratios on the right arm of chromosome 3 (3R, Figure 1A). Disadvantages of this method are that it is difficult to address the statistical significance of these interactions and the choice of window size is arbitrary, limiting the identification of interacting regions to a certain size range. We used instead a multi-scale algorithm that evaluates statistical significance at all possible window sizes [24]. The results are visualized in so- called domainograms (Figure 1B) in which statistically significantly interacting regions are depicted in red, while non-interacting regions are black. This approach implements both unbiased statistics and detection of interactions at multiple scales. While domainograms are a powerful means to visualize statistically significant interactions at all scales, they do not identify the exact boundaries of interacting regions. For this purpose we used an adapted version (see Materials and Methods) of a dynamic programming algorithm [24], which identifies the statistically most probable discrete interaction domains (DIDs). DIDs represent the center of gravity of a long-range chromatin interaction (Figure 1B). We note that DIDs are an oversimplification of the 4C interaction maps, but are nevertheless useful for subsequent computational analyses. Finally, in 4C assays the strongest signals are found centered around the chromosomal position of the bait, because interaction frequencies are inversely proportional to the distance (in base pairs) between physically linked DNA sequences [20,25]. Indeed, we observed a broad region of interacting sequences surrounding Abd- B, which spans several megabases and includes a large proportion of all DIDs (Figure 1B). It seems likely that many of these interactions are caused by linear proximity rather than by spatial proximity as a result of higher-order chromatin folding. Because our aim was to identify interactions caused by higher-order chromatin folding, we modeled the linear proximity effect by fitting a monotonously declining smoothing curve to the data (Figure 1A). After subtraction of this background model from the interaction data, the number of interactions close to the bait has been dramatically reduced, while long-range interactions are still detectable (Figure 1C). Homeotic genes prefer to interact with other PcDs To capture long-range contacts using Abd-B as bait, we hybridized two biological replicates of amplified PCR products to our custom microarray. The domainogram of the combined data revealed that neither chromosome 3L nor any other chromosome showed significant enrichments, whereas chromosome 3R holds all 17 DIDs (Figure 2B, and Table S2). Importantly, three centromere proximal DIDs overlapped with ANT-C, which is in line with the previously reported microscopic data [13,14]. Next, we wanted to know how many DIDs overlap with Polycomb domains (PcD) and H3K27me3 domains (K27D). For this purpose, we overlaid the Abd-B DIDs with either the PcD or K27D maps from central brain tissue (Figure 2B, and Table S1). We observed that 88 and 94% of the DIDs overlap with PcDs and K27Ds, respectively, which is significantly more than may be expected by random chance (Figure 3A, 3B, and Table S2). Thus, our data provides evidence that Abd-B has a preference to interact with other PcDs. In total, we identified 25 PcDs that are in contact with the Abd-B promoter, including ANT-C, grain (grn), homothorax (hth), lbl/lbe, and Ptx1 (Table S1). PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001343