The role of Polycomb in stem cell genome architecture

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ScienceDirect The role of Polycomb in stem cell genome architecture Gloria Mas1,2 and Luciano Di Croce1,2,3 Polycomb-group proteins maintain embryonic stem cell identity by repressing genes that encode for developmental regulatory factors. Failure to properly control developmental transcription programs by Polycomb proteins is linked to disease and embryonic lethality. Recent technological advances have revealed that developmentally repressed genes tend to cluster in the three-dimensional space of the nucleus. Importantly, spatial clustering of developmental genes is fundamental for the correct regulation of gene expression during early development. Here, we outline novel insights and perspectives regarding the function of Polycomb complexes in shaping the stem cell genome architecture, and discuss how this function might be required to properly orchestrate transcriptional programs during differentiation. Addresses 1 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain 2 Universitat Pompeu Fabra (UPF), Barcelona, Spain 3 ICREA, Pg. Lluis Companys 23, 08010 Barcelona, Spain Corresponding authors: Mas, Gloria ([email protected]) and Di Croce, Luciano ([email protected])

Current Opinion in Cell Biology 2016, 43:87–95 This review comes from a themed issue on Differentiation and disease Edited by Tom Misteli and Graham Warren

http://dx.doi.org/10.1016/j.ceb.2016.09.006 0955-0674/# 2016 Elsevier Ltd. All rights reserved.

Introduction During development, pluripotent embryonic stem cells (ESCs) differentiate into all the specialized cell types of an embryo. At every stage of embryogenesis, a precise gene expression program determines the identity of each cell. The gene expression programs that dictate cell identity are orchestrated by a multilayered molecular system that includes transcription factors, epigenetic modifiers, and chromatin remodelers [1]. Recent advances in high-resolution microscopy and chromosome conformation capture (3C) technologies have revealed that the three-dimensional organization of the genome also plays a critical role in transcriptional regulation [2]. A growing body of research argues that chromosome folding www.sciencedirect.com

within the nucleus brings distant regions of the genome into close proximity, and that this physical interaction influences their function. On the large scale, chromosomes occupy discrete sections of the nuclear space known as chromosome territories (reviewed in [3]) (Figure 1). Within chromosome territories, the genome is folded into topologically associating domains (TADs). TADs are megabase-sized regions comprised of highly interacting genomic loci that are flanked by structural boundaries and stabilized by architectural proteins, such as CTCF and cohesin (Figure 1). Genomic regions within a TAD tend to share the same epigenetic and transcriptional features (reviewed in [2,3]). At increasing resolution, the physical contacts between distal regulatory sequences (‘enhancers’) and gene promoters form chromatin loops (Figure 1). Chromatin loops are mediated by protein effectors, non-coding RNAs, and histone posttranslational modifications (PTMs) [1,4,5]. Importantly, large-scale organization of the genomes in TAD structures is a highly conserved feature across mammalian cell types and species [6,7]. However, local dynamic topological changes can occur at the level of chromatin loops, and these have been proposed to influence gene transcription and ensure the proper orchestration of cellular differentiation [4,8,9]. Polycomb-group (PcG) proteins are key regulators of the transcriptional programs that maintain stem cell properties and dictate lineage specification [10–12]. In mouse ESCs, PcG proteins encompass two enzymatically distinct complexes, the Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). PRC1 comprises one of the E3 ligases Ring1A/B, which catalyze the mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub1), as well as one each of the Pcgf and Phc proteins and either a Cbx protein or RYBP. PRC2 contains the core subunits Ezh1/ 2, Suz12 and Eed, and catalyzes methylation of lysine 27 of histone H3 (H3K27me3). Association of PcG proteins at selected genomic loci maintains transcriptional gene repression. Gene silencing is achieved by direct inhibition of the transcription machinery and/or by preventing chromatin accessibility to remodeling complexes. Hence, in ESCs, Polycomb complexes maintain lineagespecific genes in a silenced state and prevent exit of cells from pluripotency. Here, we review the latest research on the consequence of Polycomb loss in genome topology at the level of a whole chromosome (for instance, the X chromosome), TADs, Polycomb domains and chromatin loops (promoter–promoter and promoter–enhancer interactions) (Figure 1) (Box 1). Current Opinion in Cell Biology 2016, 43:87–95

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Figure 1

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Multiscale functionality of Polycomb in genome architecture. (a) At the genomic scale (2.8 Gbp for the mouse genome), chromosomes occupy discrete regions within the interphase nucleus, known as chromosome territories. Chromosomes are depicted in different colors. (b) Mammalian chromosomes are organized in topologically associating domains (TADs), megabase-sized structures that comprise frequently interacting genomic regions, which segregate from other TADs by sharp boundaries. In ESCs, the X chromosome (‘Active X’, depicted in purple) is organized into TADs. Induction of differentiation causes widespread dissociation of TADs and transcriptional silencing. The resulting inactive X chromosome is topologically organized into two megadomains. (c) At the sub-TAD scale, Polycomb and Super-enhancer domains are 3D structures averaging approximately 100 kb that contain one or two genes. Polycomb domains are densely occupied by Polycomb proteins and marked by H3K27me3. In mouse ESCs, in contrast, Super-enhancer domains are defined by the presence of concatenated enhancer regions with very high occupancy of pluripotency transcription factors (Oct4, Sox2, and Nanog), Mediator, RNA polymerase II and histone modifications that are associated with active transcription, such as H3K27ac. CTCF and cohesin sites demarcate the boundaries of both Polycomb and Super-enhancer domains. (d) Polycomb proteins can mediate the formation of chromatin loops between promoters and distal enhancer regions to regulate transcription in a cell type-specific manner.

Role of Polycomb in X-chromosome topology In mammalian female embryos, one copy of the X chromosome is silenced by several epigenetic mechanisms, in a process known as X-chromosome inactivation (XCI). Mouse female ESCs harbor two active X chromosomes, one of which undergoes XCI. XCI is initiated by the long non-coding RNA Xist, which is expressed from the X-inactivation center (Xic) locus on the X chromosome (Figure 2). The Xic locus also contains the non-coding RNA gene Tsix, which is transcribed antisense to Xist and Current Opinion in Cell Biology 2016, 43:87–95

represses its expression. Upon differentiation stimuli, Tsix is inhibited, thereby allowing Xist-mediated XCI (reviewed in [15]). In ESCs, active X chromosomes are topologically organized into 112 discrete TADs [16] (Figure 2). The Xic locus is divided into two TADs spanning around 750 kb [7,15–17]. The boundary of these two TADs is between the Xist and Tsix promoters (Figure 2). Preserving the Xic locus boundary is critical for the process of XCI, www.sciencedirect.com

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Box 1 Technologies for studying genome topology  Chromosome conformation capture (3C): Since the development of the first 3C assay over a decade ago [13], researchers have devised multiple derivatives of the 3C assay to study long-range genome interactions (reviewed in [14]). In a 3C assay, cells are crosslinked, their DNA is digested with a restriction enzyme and the resulting DNA fragments are ligated together. Crosslinking preserves the natural genome spatial organization, such that fragments are more likely to be ligated together if they are close in 3D space (within the A˚ range), irrespective of their location on the 2D genome. Longrange contacts between two defined regions of interest can be assessed by semi-quantitative PCR (3C-PCR). With the revolution of next-generation sequencing, other methods have been developed to capturing interactions between more than two selected loci. The choice of methodology for a specific study will depend on the biological question asked and on the resolution and throughput needed to answer it. In general, the resolution of 3C technologies relies on the restriction enzyme used to cut the genome (which are usually 6-bp or 4-bp cutters) and on sequencing depth.  4C-seq: 4C-seq can reveal contacts at a very high resolution with respect to the entire genome (i.e. at 200–400 bp, using a 4-bp cutter). Therefore, enhancer-promoter loops can be elucidated by 4C-seq. 4C-seq involves digesting the 3C template with a second restriction enzyme, usually a 4-bp cutter, to generate circular ligation fragments. A single biotinylated oligonucleotide specific to a ‘viewpoint’ region is then used to obtain a library of ligation fragments containing the specific viewpoint of interest.  Chromosome conformation capture carbon copy (5C): 5C is commonly utilized to comprehensively investigate higher-order chromosome structures within a region up to one megabase. In 5C, the 3C template is amplified with a set of forward and reverse primers that anneal at specific restriction sites of the genomic region of interest. These primers will amplify fragments linked by the selected restriction sites.  HiC-seq and in situ HiC: These methods identify contacts simultaneously occurring in cis and in trans throughout the whole genome. In HiC, restriction fragment ends are filled up with biotinylated nucleotides and then ligated. The output of the biotin pull-down is used to prepare a library of ligation fragments, which are sequenced. Subsequent analysis generates a map of the frequency of genome-wide intra-chromosomal and inter-chromosomal interactions. Initial HiC studies have identified TAD structures as fundamental higher-order organization units of the genome in multiple cell types and species (reviewed in [14]).  Capture C (CHI-C and HiCap): Capture C was devised to obtain high-resolution interaction maps of a large but specified fraction of the genome. Enrichment of the specific regions of interest is achieved by using a library of biotinylated RNA oligonucleotides that anneal to these regions, which are then selected with biotin pull-downs. Capture C provides high-resolution, genome-wide interaction maps of the selected regions and requires relatively lower sequencing depth as compared to the HiC method.  It is important to note that 3C-based assays average the interaction frequencies of a whole cell population. Single cell approaches, such as single-cell HiC and fluorescence in situ hybridization (FISH) using super-resolution microscopy (3D-SIM), have recently been developed to overcome cell-to-cell heterogeneity issues and also to provide information about the localization of the interacting loci within the nucleus. Nevertheless, neither of these methods currently provides sufficient resolution to detect interaction frequency within TADs (at a sub-megabase scale).

since deleting the boundary element leads to the formation of ectopic long-range interactions over the TAD boundaries. These alterations in the topological landscape are accompanied by transcriptional changes, www.sciencedirect.com

including deregulation of Tsix and Xist, which result in impaired XCI [7]. Interestingly, there is a marked correlation between X chromosome TAD organization and the genomic distribution of H3K27me3. To examine the role of Polycomb in X chromosome TAD organization, Heard and colleagues compared 5C-seq profiles of wild-type and Eed / ESCs. Strikingly, the lack of PRC2 — and the associated H3K27me3 modification — altered neither TAD sizes nor the positions of the active X chromosome [7], indicating that PRC2 function is not required for the structural organization of active X chromosomes in TADs. During ESC differentiation, Xist expression is activated. Xist coats the X chromosome, inducing the recruitment of repressive epigenetic machineries including PRC1, PRC2 complexes, and DNA methyltransferases throughout the chromosome (reviewed in [15]). Xist spreading over the X chromosome is sufficient to induce a spatial reorganization and widespread transcriptional silencing. Recent reports indicate that chromosomal topology of the inactive X chromosome is rather disorganized: the TAD structure is dissolved and instead the topology is segregated at the DXZ4 satellite region into two megadomains [7,17,18] (Figure 2). Genes that escape X inactivation and remain transcriptionally active tend to be located at the periphery of the megadomains [17,18]. Notably, depletion of Xist reverts the megadomain conformation of the inactive X back to a TAD conformation that resembles the active X chromosome [16]. Thus, Xist function is essential in configuring the X chromosome topology and disrupting TAD structures, which is an important step for silencing the X-linked genes during X-inactivation. Is Polycomb involved in the genomic spatial reorganization during XCI? The inactive X chromosome (Xi) contains large regions covered in H3K27me3 and H2AK119ub (reviewed in [15]). Several lines of evidence indicate that Xist and Polycomb complexes interact and co-localize on Xi, and Xist seems to recruit PRC1 and PRC2 through direct or indirect mechanisms [16,19,20]. Thus, the presence of Xist RNA is necessary and sufficient to recruit PRC2 to Xi [21]. Additionally, direct physical interactions between Xist and PRC2 foci have been demonstrated in vivo and in vitro [19,20,22]. Recently, 3D-SIM super-resolution microscopy revealed that, during initial stages of differentiation, PRC2 genomic targets do not co-localize with regions that become transcriptionally silenced by Xist [23]. However, a second super-resolution microscopy study found significant colocalization of Xist and PRC2 signals on Xi [24]. They proposed a hit-and-run model whereby Xist recruits and co-associates with PRC2 at a relatively few loci, from which PRC2 spreads away, dissociating from the Xist RNA [24]. Nevertheless, the partial segregation of Current Opinion in Cell Biology 2016, 43:87–95

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Figure 2

Active X Chromosome DXZ4 satellite 0 Mb

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Genome architecture dynamics during X-chromosome inactivation. The active X chromosome is topologically organized into TADs. The X-inactivation center (Xic) spans around 750 kb and is arranged into two adjacent TADs (enlarged cartoon). The promoters of the non-coding RNAs Tsix and Xist (in blue and orange, respectively) lie in separate TADs. In the active X chromosome, Tsix is transcribed and the expression of Xist is inhibited. Upon differentiation stimuli, Xist becomes activated and its non-coding RNA spreads along the X chromosome. This results in the loss of the TAD organization and widespread gene silencing. Xist induces the recruitment of Polycomb complexes to the X chromosome, and this contributes to the maintenance of gene silencing. The inactive X chromosome is structured in two large megadomains separated by the DXZ4 macrosatellite.

PRC2 and Xist challenges the notion that PRC2 is required for X-linked gene silencing and might explain why PRC2 loss does not lead to increased X-linked transcription as well as why PRC2 null embryos show mild XCI phenotypes [22,25–27]. In agreement with this, analyses of Eed / mice embryos suggest that Polycomb might play a relevant role in maintenance of Xi silencing in extraembryonic tissues, but not in embryonic tissues of the blastocyst [25,28]. Similar to PRC2, PRC1 is recruited to Xi via Xist but independently from PRC2, and it is not essential for initiating transcriptional silencing during XCI [29,30]. In sum, these studies point to independent functions of Xist and Polycomb complexes at early stages of XCI, with Xist as a major player in the topological rearrangements associated with X-chromosome transcriptional silencing. The Polycomb domains seem to be involved in maintaining XCI, but not in establishing the initial transcriptional silencing on Xi in ESCs (Figure 2) [31]. Further studies utilizing high-resolution 3C and microscopy technologies Current Opinion in Cell Biology 2016, 43:87–95

on PRC2 and PRC1 mutants should clarify whether Polycomb complexes regulate X chromosome topology, both in ESCs and differentiating cells.

Role of Polycomb in TAD organization of ESC genomes As stem cells differentiate, gene expression patterns change and correlate with widespread modifications of the epigenomic landscape [4,11]. Remarkably, chromosome conformation assays performed in ESCs and differentiated cells show that the size and position of TADs is largely conserved between cell types and stages of differentiation [4,6,7]. More recently, high-resolution studies have highlighted the existence of genomic regions that switch from repressed to active TADs correlating with gene expression (i.e. facultative TADs) [2]. In ESCs, facultative TADs include regions densely marked by Polycomb proteins, also known as Polycomb domains (Figure 1). Polycomb domains and super-enhancer domains describe two distinct types of chromatin www.sciencedirect.com

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domains that organize stem cell identity genes [1,32]. Super-enhancer domains encompass actively transcribed pluripotency genes and enhancers, and are defined by the high occupancy of pluripotent transcription factors, histone modifications associated with active transcription and the transcriptional coactivator complex Mediator (Figure 1) [32,33]. In contrast, Polycomb domains encompass lineage-specification genes that are transcriptionally repressed in ESCs [33]. There are around 350 Polycomb domains in ESCs, averaging 112 kb in size and containing at least one or two developmental genes. Polycomb domains are typically multi-looped structures highly enriched in H3K27me3 and Polycomb proteins, limited by sites of CTCF and cohesin (Figure 1) [33,34]. In ESCs, Polycomb domains include the Hox gene clusters and imprinted regions. The topology of Hox gene clusters during stem cell differentiation is particularly well studied. Hox genes are localized within four clusters: HoxA, HoxB, HoxC and HoxD in chromosomes 7, 17, 12 and 2, respectively. In ESCs, Hox clusters form large TADs decorated by the repressive H3K27me3 and active H3K4me3 marks, and exhibit low levels of transcription [11,35,36,37]. The coexistence of active and repressive histone modifications at specific genomic regions defines the so-called ‘bivalent’ domains and characterizes developmental genes [38]. Hox clusters seem to preferably aggregate with other H3K27me3-marked loci. Although they are not actively transcribed, clusters of Polycomb-decorated loci tend to occupy active regions of the nuclear space, and segregate away from inactive lamina-associated domains of the nuclear periphery [37]. Strikingly, 4C-seq and FISH analyses at the Hox clusters have shown that depletion of H3K27me3 in Eed / ESCs results in minimal changes in overall TAD configuration, with only specific interactions between densely H3K27me3-marked regions lost or reduced [39]. Eed / ESCs retain functional pluripotency features, but differentiation to the germ layers of the embryo is impaired [40]. Further, roughly 300 genes become upregulated in Eed / ESCs as compared to wild-type ESCs [41]. Whether the misregulated genes correspond to those involved in the interactions lost in Eed / ESCs remains to be determined. The causal relationship between Polycomb binding, transcription and genome topology in mouse ESCs was recently investigated [42]. The authors tethered the PRC2 subunit Ezh2 using a lacO/lacR recruitment system and assessed the topology by 4C-seq experiments. Ezh2 targeting induced the formation of long-range contacts with neighboring Polycomb-occupied regions, and led to local deposition of H3K27me3 at contacting regions. However, the establishment of new contacts was uncoupled from transcriptional repression. Together, these results suggest that Polycomb occupancy drives spatial crowding of H3K27me3-rich regions, but raise the question of www.sciencedirect.com

whether this 3D organization has a direct role in gene expression. Upon differentiation, the sequential activation of the Hox genes causes a progressive switch of the Polycomb domain to an active TAD [35,43]. Such topological reorganization is concomitant with the epigenetic and transcriptional changes of the Hox genes [37,44]. For example, even though the entire HoxA cluster is repressed by Polycomb in ESCs, induction of neuronal differentiation activates the portion of the cluster corresponding to Hoxa1–6 genes but not the part containing Hoxa7–13, which remains silenced. The sequential activation of Hoxa1–6 correlates with changes in topology and epigenetic marks [45]. The presence of CTCF binding sites between Hoxa5 and Hoxa6 is essential to properly switch the Hoxa1–6 portion from a repressed to an active state. Indeed, disrupting the CTCF binding sites between Hoxa5 and Hoxa6 in neurons leads to aberrant spreading of the H3K4me3 mark over the boundary, indicating that CTCF sites restrict Trithorax activity [45]. Future work will be needed to specifically address whether the presence and enzymatic functions of Polycomb and/or Trithorax complexes are required for the proper switch of Hoxa1–6 from a repressed to an active TAD when ESC differentiation is induced. Taken together, these studies indicate that neither Polycomb nor H3K27me3 distribution determine overall size or position of TAD boundaries in undifferentiated ESCs. Rather, the preservation of intact Polycomb domain boundaries seems to be the critical factor that restricts chromatin states and maintains transcriptional repression [33,44,45]. Nevertheless, the increased resolution of newly developed chromosome conformation technologies has started to reveal that Polycomb recruitment may control genome organization within TADs — at the sub-megabase scale — during ESCs differentiation [46]. The latest literature investigating the role of Polycomb complexes in shaping intra-TAD organization in stem cells, and its correlation with developmental gene expression programs, is discussed below.

Role of PRC1 in chromatin loop formation Multiple lines of evidence establish a role of the PRC1 complex in the compaction of chromatin arrays in vitro [47,48]. In Drosophila cells, the Polyhomeotic (Ph) subunit of PRC1 has been recently shown to be required for clustering Polycomb-occupied regions and for establishing chromatin interactions, resulting in transcriptional repression [49]. An intriguing hypothesis that remains to be tested is that the mammalian homologues Phc1/2/3 also shape genome architecture in mammals. Several reports further establish that PRC1 maintains chromatin compaction to repress Hox loci transcription in ESCs (reviewed in [50]). The chromatin compaction activity of PRC1 does not depend on either PRC2 or the histone Current Opinion in Cell Biology 2016, 43:87–95

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ubiquitination activity of the catalytic subunit Ring1B [51,52]. Taken together, these studies advocate a model whereby PRC1-mediated local chromatin compaction pre-exists before deposition of H2AK119ub1 and PRC2 binding. More recently, Fraser and colleagues performed promoter Capture HiC to examine, at very high resolution, whether PRC1 regulates the formation of chromatin loops in mouse ESCs [8,53]. Analysis of the long-range interactions established by the 22 000 annotated ESC promoters uncovered a strong spatial network involving promoters of the Hox genes and of genes encoding mostly developmental transcription factors. PRC1-bound promoters interacted preferentially with poised enhancers (marked by both H3K4me1 and H3K27me3) as opposed to active enhancers (marked by H3K4me1 and H3K27ac). The authors found that PRC1 plays a major role in stabilizing the network of long-range interactions between Hox gene promoters and other PRC1-occupied promoters of lineage-specification genes [8]. Intriguingly, PRC1 is not necessary to maintain contacts between promoters and poised enhancers in the Hox network, implying distinctive requirements in promoter-promoter versus promoter–enhancer interactions. In addition, changes in topology correlate with transcriptional output. In PRC1-depleted cells, the Ring1b-target promoters that lose contact between each other become upregulated as they form new contacts with actively transcribed promoters. In parallel, poised enhancers transition to an active state and establish new interactions with genes, which are then upregulated [8]. These data indicate that PRC1 is required to maintain a spatial network of developmental genes to keep them transcriptionally poised for activation before developmental stimuli. It will be interesting to address whether these interaction networks depend on the catalytic activity of Ring1B and/or the Phc subunit of PRC1. The role of Polycomb in establishing chromatin loops has been also studied in the context of transcriptional activation. The Meis2 gene encodes for a homeobox protein involved in transcriptional regulation of developmental programs. Analogously to Hox genes, Meis2 is transcriptionally silent in ESCs and is activated in neuronal development. Transcriptional activation correlates with the formation of a chromatin loop between the Meis2 promoter and a midbrain-specific enhancer (MBE). Ring1B stabilizes this promoter-enhancer loop to allow the activation of Meis2 gene expression [54]. In sum, these studies clearly indicate that the spatial configuration of developmental gene networks largely relies on the presence of PRC1. Future work will be needed to further delineate the molecular mechanisms underlying the topological functions of PRC1, and how these functions relate to transcription regulation. Current Opinion in Cell Biology 2016, 43:87–95

Role of PRC2 in chromatin loop formation The two main subtypes of PRC2 complexes are defined by which catalytic subunit they contain: Ezh1 or Ezh2. The two PRC2 subtypes seem to be mutually exclusive depending on cell type: PRC2-Ezh2 is expressed mostly in proliferating tissues and stem cells, while PRC2-Ezh1 is found in non-dividing adult tissues [55]. Although PRC1-Ezh2 seems to methylate H3K27me3 more robustly than PRC1-Ezh1, the latter was shown in vitro to compact chromatin arrays more efficiently [55]. These and complementary observations imply that the chromatin compaction activity of PRC2 complexes is independent from their histone methylation activity. The role of PRC2 in stabilizing chromatin loops in cells was recently studied. The imprinted Kcnq1 locus in the paternal allele is spatially organized in a repressed nuclear cluster, in contrast to the more relaxed conformation of the maternal non-imprinted locus. The genomic compaction of the imprinted Kcnq1 locus depends on PRC1 and PRC2 and is required for full transcriptional repression of these genes in vivo, as shown by 3D RNA/DNA FISH experiments [56]. By performing chromosome conformation assays, Baylin and colleagues further confirmed that PRC2 contributes to maintain a transcriptionally repressed and multi-looped structure of the bivalently modified GATA4 locus in embryonic carcinoma cells [57]. PRC2 was also shown to mediate inter-chromosomal and intrachromosomal interactions throughout the genome in these cells [58]. More recently, promoter Capture HiC performed in mouse ESCs provided additional evidence that Eed depletion leads to a reduced frequency in long-range interactions involving Hox clusters [8]. Further, extremely long-range promoter–promoter interactions (ELRIs) were found to be established in the transition from naı¨ve to primed ESCs [59]. Naı¨ve ESCs are characterized by low levels of H3K27me3 as well as by an epigenetic and transcriptional landscape reminiscent of ESCs at the blastocyst stage. In primed ESCs, H3K27me3-marked loci increase and cells become more restricted in their capability of generating all lineages of the embryo. ELRIs are formed during this transition, and these interactions involve genes that are decorated with H3K27me3. Importantly, PRC2 activity, together with the presence of PRC1, is required to mediate the formation of ELRIs [59]. To summarize, several lines of research support that both PRC1 and PRC2 are important players in securing local interactions to maintain repressive gene expression. Notably, the interdependency between PRC1 and PRC2 for the recruitment at chromatin is still under debate, implying that future work should clarify whether the two protein complexes exert independent roles in stem cell chromatin topology. Along these lines, it is interesting to point out that both PRC1 and PRC2 have been shown to interact with long non-coding RNAs (lncRNAs) to regulate gene expression [19,26,60–63]. lncRNAs are often www.sciencedirect.com

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expressed in a tissue-specific manner and control the recruitment of Polycomb complexes at chromatin [64], and they have recently emerged as critical mediators of long-range interactions [65]. For example, the lncRNA HOTAIR acts in trans (e.g. across different chromosomes) to regulate the Hox cluster gene expression [63,66–68]. As discussed above, the lncRNA Xist also regulates PRC2 occupancy and is involved in chromatin organization of the inactive X chromosome. We predict that the role of long non-coding transcripts as docking sites for Polycomb proteins is a driving force in helping shape genome architecture, and that this will be an exciting new avenue of research in the near future.

Concluding remarks In the last few years, the expansion of high-resolution genome-wide 3C technologies has revealed the existence of contacts between promoters and distant genomic regions. During early stages of animal development, many of the contacts involving promoters of developmental genes are formed. Polycomb complexes and a transcriptionally paused RNA polymerase II occupy developmental gene promoters and maintain them in a transcriptionally repressed state before cell differentiation. The latest research indicates that Polycomb binding contributes to stabilizing such long-range interactions between transcriptionally inactive regions of the stem cell genome. Whether binding and stabilization of chromatin loops by Polycomb dictates a specific transcriptional output is still under debate. While a number of reports suggest that Polycomb-dependent genome topology poises genes for activation upon developmental stimuli, other studies question a direct causal relationship between chromatin topology and transcriptional regulation. As discussed above, reorganization of specific promoter– enhancer interactions can be uncoupled from transcriptional changes, suggesting that clustering of particular Polycomb-occupied loci is a consequence of the affinity between proteins and/or histone modifications. The most recent studies point to an integrative model whereby the formation of long-range interactions precedes gene activation, and does not cause major changes in transcriptional output. Instead, the contact network stabilized by Polycomb may be required for fine-tuning of gene expression programs upon developmental stimuli, ensuring proper space-temporal activation during embryonic development. In the context of disease, the integrity of TAD boundaries appears to be essential to prevent ectopic formation of long-range contacts that would cause aberrant activation of genes during embryogenesis (reviewed in [69]). Although depletion of Polycomb proteins and their associated histone modifications does not seem affect overall TAD boundary distribution or size, further studies should assess a direct role of Polycomb in TAD reorganization. www.sciencedirect.com

New analyses utilizing advanced microscopy and 3C technologies, coupled to next-generation ChIP-sequencing and RNA-sequencing, will be required to directly determine the consequences of Polycomb occupancy in the spatial organization of developmental loci and transcriptional regulation, both in health and disease states. We anticipate that future research will also clarify whether Polycomb mechanistically cooperates with pluripotent transcription factors, architectural proteins and lncRNAs in mediating long-range chromosomal interactions.

Acknowledgements We apologize to all authors whose related work could not be included due to space constraints. We thank L. Morey, B. Payer, S. Aranda and the members of the Di Croce Laboratory for critical reading of the manuscript and insightful discussions, and V.A. Raker for scientific editing. G.M. received the support of ‘La Convocatoria de Ayudas Fundacio´n BBVA a Investigadores, Innovadores y Creadores Culturales’. The Di Croce Laboratory is supported by grants from the Spanish ‘Ministerio de Educacio´n y Ciencia’ (SAF2013-48926-P), from AGAUR, from Fundacio´ ‘La Marato´ de TV3’, from the European Commission’s 7th Framework Program 4DCellFate (277899).

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