Discussion We successfully adapted the 4C method to systematically map long-range chromatin contacts with limited material from a single fly tissue. With this method, we detect interactions between the ANT-C and BX-C in central brain. This observation is in good agreement with earlier microscopic reports [13,14], underscoring the strength of our method. Importantly, we further demonstrate that not only the two Homeotic gene clusters, but also many other PcG target genes interact, suggesting that long-range chromatin contacts between PcDs are common in central brain tissue. The control fragments (wntD, CG5107, Crc, and RpII140), which do not reside in PcDs, have interactions that are distinct from PcDs, emphasizing the specificity of our findings. PcG targets show a strong preference for interaction with other PcG targets, suggesting that PcG proteins help to establish these interactions. This is in line with earlier in vivo and in vitro results that indicated that PcG proteins can keep certain DNA sequences together [10,11,14,15,32]. However, interactions among PcG target genes are constrained by overall chromosome architecture, because our data demonstrate that loci need to be on the same chromosome arm for efficient interaction Interpretation of the 4C data DIDs range in size from 6 to 600 kb, with an average of ,170 kb. Thus, we find highly local strong enrichments as well as moderate enrichments over larger regions, which may reflect different types of long-range interactions. We emphasize that interactions as detected by 4C technically represent events of molecular proximity of DNA sequences, and not necessarily physical binding. Based on the current data it is therefore not possible to identify within the DIDs sequence elements that may mediate direct contact with other DIDs. It is conceivable that contacts between PcDs may occur at any position within the PcDs; if PcG protein complexes have an intrinsic propensity to aggregate, as has been observed in vitro [33], then large PcDs may have a higher chance of interacting with each other due to their larger ‘sticky’ surface area. The 4C method is a cell population based assay that only detects the most frequent interactions in the population. Previous 4C studies in mammalian cells suggested an extensive network of long- range interactions [20]. Our data also suggests an extensive network among interacting PcDs. However, microscopic studies in mam- malian cells revealed that specific long-range interactions occur only in a small proportion of the cells [20]. Likewise, only a proportion of D. melanogaster cells show contacts between the two Homeotic clusters [13,14]. Therefore, 4C data have to be carefully interpreted, because the identified interactions are in part stochastic and do not all occur simultaneously [20]. As a consequence, we do not know how many PcDs interact in a single cell, but we do know the most common interactions in the population of larval brain cells. Chromosome arm territories Interphase chromosomes in most eukaryotes occupy distinct ‘‘territories’’ inside the nucleus, with only a limited degree of intermingling [34,35]. Although some studies have reported interactions between some loci that are on two different chromosomes (interchromosomal) [reviewed in: [28]], unbiased 4C mapping in mouse tissues has indicated that interactions within the same chromosome (intrachromosomal) occur much more frequently than between different chromosomes [20]. In addition, a recent genome-wide map of chromatin interactions in human cells showed that intrachromosomal interactions occur with higher frequency than interchromosomal contacts [26]. Our data are in Polycomb Domains Interact in Nuclear Space agreement with these observations, and show that most interac- tions are even limited to single chromosome arms, at least in D. melanogaster larval brain. Since our experiments indicate that a topological mechanism prevents interactions between the two arms of a chromosome, we propose that each arm (rather than the chromosome as a whole) forms a distinct territory. This is consistent with early microscopy studies of chromosome architec- ture in D. melanogaster, which suggested that chromosome arms are units of spatial organization [36–38]. What topological mechanism may limit contacts between the two chromosome arms? About ,16 Mbp of pericentric hetero- chromatin are located in between the two euchromatic arms of chromosome 3 [39]. This heterochromatin region could act as a long spacer and prevent efficient interactions between DNA fragments that are located on either chromosome arm. However, our data show that interactions within one arm can span even longer distances, such as between Ptx1 and grn (,22.7 Mbp). Another explanation may be that the pericentric regions of all chromosomes assemble into a nuclear compartment, called chromocenter [40]. This large structure could physically obstruct interactions between chromosome arms. Previous reports have demonstrated that certain PcG-bound PREs can pair in trans (i.e. when they are located on different chromosomes). First, a Fab7 PRE-element integrated on the X chromosome (Fab-X) was often found in close spatial proximity to the endogenous Fab-7 in the BX-C (chromosome 3R) [41], although this phenomenon appears to be tissue-specific and dependent on the transgene integration site [13]. Second, a microscopy assay based on Lac repressor/operator recognition showed that the Mcp PRE-element is able to pair with copies of that same element inserted at remote sites in the genome either in cis (i.e. when they are located on the same chromosome) or in trans [42]. Our 4C experiments also identified cases of trans interactions between endogenous loci, although they occur with low frequency (approximately 5% occurs in trans; Figure 6B). Although rare, such interactions between loci on different chromosome arms are of interest, because they indicate that the topological constraints imposed by chromosome architecture can in principle be overcome. In mammalian cells, there is evidence that the relative position of a gene locus within its chromosome territory (CT) influences its ability to form either cis- or trans- interactions. Peripheral regions of mammalian CTs intermingle their chromatin [43], which may allow for interactions between chromosomes. Indeed, more trans contacts are identified by 4C using a bait that often resides in the CT periphery compared to a bait located in the interior of a CT [20]. Likewise, activation of the HoxB gene cluster during differentiation coincides with relocation away from its CT interior [34], and the active HoxB1 gene more frequently contacts sequences on other chromosomes compared to the inactive gene [44]. In line with this, we observed a varying degree of trans interactions among eight bait sequences, suggesting distinct capacities to contact chromatin on other chromosomes. The trh gene has the strongest capacity to contact other chromosome arms. Interestingly, trh is located within 500 Kbp of the telomere of chromosome 3L, and 5 out of 6 contacts in trans occur within 500 Kbp of other telomeres (Figure 6A, and Table S2). Thus, interactions between chromosome arms may be possible if loci are favorably positioned on the edge of chromosome (arm) territories, which could be the case for telomeric sequences in larval brain cells. Relevance for gene regulation The experiments with strain In(3LR)sep showed dramatic changes in interactions, such as loss of contacts between the PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001343