Polycomb Domains Interact in Nuclear Space Figure 5. Large PcDs interact more often than small PcDs. (A) Strip charts show size differences between PcDs that interact with at least one of the 4 PcG baits (closed circles) and PcDs that do not interact (open circles). (B) Similar to a, but now for K27Ds. The x-axes indicate domain size in Kbp (log10 scale), and each dot represents a single domain on chromosome 3. Perpendicular bars are median values and p-values were calculated using a two-sided non-parametric Mann-Whitney U test. doi:10.1371/journal.pgen.1001343.g005 chromosome arm (Figure 6B). Thus, interactions within a chromosome arm are prevalent and contacts between sequences on different chromosome arms are rare. Interactions are constrained by chromosome architecture What mechanism limits interactions to a single chromosome arm? To date, it is well established that chromatin contacts can occur between cis-acting DNA elements [28]. It is possible that long-range chromatin interactions are driven by high-affinity interacting DNA elements, which are confined to a single chromosome arm. Alternatively, it is possible that interactions are topologically constrained by overall chromosome architecture, for example because each chromosome arm is a spatially confined unit. We tested these two models by analyzing PcD interactions using fly strain In(3LR)sep that carries a chromosomal inversion [29]. This inversion (Figure 7A) rearranges the centromere-proximal ,5.8 Mbp of chromosome 3R to the left arm [29]. This region contains 17 PcDs, including the ANT-C and grn. The other PcDs on 3R, including those annotated as hth, BX-C, lbl/lbe, and Ptx1, remain on the right arm. In addition, approximately 17 Mbp of chromosome 3L, including 43 PcDs, is moved to the right arm. For clarity, we will refer to the wild-type left and right arms as 3LWT and 3RWT, and to the inverted arms as 3LIN and 3RIN, respectively. We reasoned that if long-range contacts are only possible within the same arm, then on the inverted chromosome interactions across the inversion breakpoints, such as between BX- C and ANT-C, will be abolished and new interactions may be formed. On the other hand, if interactions depend solely on local cis-regulatory DNA sequences then they will not be affected by the inversion. To discriminate between these possibilities, we first mapped the long-range chromatin contacts in central brain nuclei of homozy- gous In(3LR)sep larvae using Abd-B as bait. For the domainograms (Figure 7B), we plotted the chromosomal position of probes identical to wild type, which facilitates direct comparison to wild type data (Figure 2B). Remarkably, all significantly enriched regions are located on 3RIN (Figure 7B, Figure 8A, and Table S4), demonstrating that long-range contacts are again limited to a single chromosome arm. Specifically, among PcDs that remained to the right of the centromere we observed contacts that we previously identified in wild type cells, including hth, lbl/lbe, and Ptx1 (Table S4, and Figure 7B). In contrast, all interactions on the inverted 5.8 Mbp, including ANT-C, were abolished. Instead, new contacts occur on the 17 Mbp that is inverted from chromosome 3LWT to 3RIN (Figure 7B), including PcG targets dorsocross (doc) genes and Iroquois-complex (IRO-C; araucan, caupolican, and mirror). We observed similar results when we mapped interactions using Antp as bait. Several interactions that occur on 3RWT, such as with BX-C, hth, and lbl/lbe are lost on 3RIN and many new contacts are found on 3LIN (Figure 7C, Table S4). Interactions are mostly limited to 3LIN, and only 2 out of 18 DIDs occur with sequences on chromosome 4 (Figure 8A, Table S4). Similar to wild type, we observed that ,95% of all Abd-B and Antp interactions (n = 37) occur within a single chromosome arm (Figure 8A). Next, we tested whether the dramatically changed interactions affect expression of genes on the In(3LR)sep chromosome. In a series of microarray expression profiling experiments we could not PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001343