Interactions among Polycomb Domains Are Guided by Chromosome Architecture Bas Tolhuis1*, Marleen Blom1, Ron M. Kerkhoven2, Ludo Pagie3, Hans Teunissen1, Marja Nieuwland 2 , Marieke Simonis4, Wouter de Laat4, Maarten van Lohuizen1*, Bas van Steensel3* 1 Division of Molecular Genetics and the Centre for Biomedical Genetics, Netherlands Cancer Institute, Amsterdam, The Netherlands, 2 Central Microarray Facility, Netherlands Cancer Institute, Amsterdam, The Netherlands, 3 Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, The Netherlands, 4 Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Abstract Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture. Citation: Tolhuis B, Blom M, Kerkhoven RM, Pagie L, Teunissen H, et al. (2011) Interactions among Polycomb Domains Are Guided by Chromosome Architecture. PLoS Genet 7(3): e1001343. doi:10.1371/journal.pgen.1001343 Editor: Wendy A. Bickmore, Medical Research Council Human Genetics Unit, United Kingdom Received August 13, 2010; Accepted February 16, 2011; Published March 24, 2011 Copyright: 2011 Tolhuis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the NWO-ALW ,http://www.nwo.nl., the Netherlands Genomics Initiative ,http://www.genomics.nl., and the Centre for Biomedical Genetics ,http://www.biomedicalgenetics.nl/.. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: b.v.steensel@nki.nl (BvS); m.v.lohuizen@nki.nl (MvL); b.tolhuis@nki.nl (BT) Introduction Polycomb group (PcG) proteins are evolutionary conserved and important epigenetic regulators that bind and modify chromatin. They repress transcription of Homeotic genes [1] and many other key regulators of development [2–6]. As such, PcG proteins control morphogenesis, cellular differentiation, proliferation, and cancer development [7]. PcG proteins are thought to mediate gene silencing by changing the structure of chromatin, but the exact molecular mechanisms are not yet fully understood. In D. melanogaster, biochemical studies have indicated that PcG proteins exist in at least three multiprotein complexes [reviewed in: [1]]. First, the Pleiohomeotic Repressive Complex binds directly to DNA sequences. In addition, there are two Polycomb Repressive Complexes (PRC1 and PRC2) that bind indirectly to DNA via protein-protein interactions. PRC2 includes the histone methyl transferase E(Z), which deposits the characteristic lysine 27 trimethyl mark at histone H3 (H3K27me3). In turn, the chromodomain of Polycomb (PC), one of the components of PRC1, binds to this mark. Recent genome-wide mapping studies revealed that PC and H3K27me3 tend to form large contiguous chromatin domains, which sometimes span more than 100 kb in size covering multiple genes [5,6,8,9]. These domains have been named Polycomb domains (PcDs). Other PRC1 components (PSC, and PH) and E(Z) bind as specific local enrichments within PcDs [5,9]. Some of these local enrichments coincide with well-known cis-regulatory sequences, called Polycomb Response Elements (PREs). PREs are essential for PcG-mediated gene silencing and thought to be initial recruitment sites for PcG proteins [1]. There are some indications that PcG proteins can be involved in long-range chromatin interactions in nuclear space. First, in D. melanogaster, some PREs interact over long distances with gene promoter and termination sequences, which is essential for PcG- mediated silencing [10,11]. Likewise, PcG proteins mediate the formation of a complex multi-loop chromatin structure around the silenced mammalian GATA4 locus [12]. Second, two PcDs, the two fly Homeotic gene clusters (Antennapedia complex – ANT-C and Bithorax complex – BX-C) were found to co-localize in nuclei of certain tissues [13,14]. This interaction is intriguing, because the Homeotic genes are separated by 10 Mbp. Similarly, in mammalian cells, long-range chromatin interactions were shown to be mediated by EZH2 protein (an E(z) homolog) [15]. Third, PcG proteins accumulate as microscopically visible foci in the nuclear space [14,16–19], suggesting that PcG-bound chromatin is concentrated in nuclear compartments. These observations raise the question whether long-range chromatin interactions among PcDs are a general phenomenon. However, a systematic survey to address this question is still lacking. Therefore, we decided to map the genome-wide interactions of 4 established PcG target genes. We adapted the Chromosome Conformation Capture on Chip (4C) assay [20] to generate genome-wide maps of PcD interactions in D. melanogaster larval brain tissue. Our results indicate that PcDs extensively and specifically interact with each other, yet these interactions are topologically constrained by the overall chromosome architecture. PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001343