Diverse gene regulatory mechanisms mediated by Polycomb group proteins during neural development

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ScienceDirect Diverse gene regulatory mechanisms mediated by Polycomb group proteins during neural development Masafumi Tsuboi1, Yusuke Hirabayashi1 and Yukiko Gotoh2,3 While all the developmental genes are temporarily repressed for future activation in the pluripotent stem cells, non-neural genes become persistently repressed in the course of commitment to the neuronal lineage. Although Polycomb group proteins (PcG) are key factors for both temporary and persistent repression of the developmental genes, how the same group of proteins can differentially repress target genes remains unclarified. The identification of a variety of PcG complexes and activities sheds light on these issues. In this review, based on the recent findings including those with the use of interactome and Chromosome Conformation Capture (3C)-type analyses, we summarize the molecular mechanisms of PcG-mediated gene regulation and discuss how PcG regulates cell fate specification during neural development. Addresses 1 Graduate School of Engineering, The University of Tokyo, Tokyo 113-0033, Japan 2 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan 3 International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Tokyo 113-0033, Japan Corresponding author: Tsuboi, Masafumi ([email protected])

Current Opinion in Neurobiology 2019, 59:164–173 This review comes from a themed issue on Neural epigenetics Edited by Michael Greenberg and Stavros Lomvardas

https://doi.org/10.1016/j.conb.2019.07.003 0959-4388/ã 2019 Elsevier Ltd. All rights reserved.

Introduction Spatiotemporal control of gene expression is key to the determination of stem cell fate during development. Polycomb group (PcG) proteins regulate the expression of major developmental genes. PcG proteins usually repress gene expression by catalyzing repressive histone modification and altering the structure of chromatin in various cell types. The recent identification of noncanonical PcG complexes in addition to the canonical ones has revealed diverse mechanisms for the regulation of gene expression in different contexts. In this review, we first summarize the diverse mechanisms of PcG protein-mediated gene regulation and Current Opinion in Neurobiology 2019, 59:164–173

then address when and how these mechanisms contribute to the control of mammalian neural development.

Diverse gene regulatory mechanisms mediated by PcG proteins PcG proteins were first identified as transcriptional repressors of Hox genes in Drosophila melanogaster. They function in two main complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Whereas PRC2 includes either of the SET domain-containing methyltransferases Ezh1 or Ezh2, which catalyze the trimethylation of histone H3 at lysine-27 (H3K27me3), PRC1 contains the RING-finger proteins Ring1A and Ring1B, which are E3 ubiquitin ligases that catalyze the monoubiquitination of histone H2A at lysine-119 (H2Aub). Immunoprecipitation and mass spectrometric analysis performed to identify Ring1B-associated proteins revealed various forms of PRC1 [1,2]. In addition to canonical PRC1 containing Pcgf2 or Pcgf4 (PRC1.2 and PRC1.4, respectively) as well as Cbx and Phc family proteins [3], noncanonical PRC1 complexes that contain Pcgf1, 3, 5, or 6 (PRC1.1, 1.3, 1.5, and 1.6, respectively) thus also exist [1,2,4–7] and function in specific contexts [8] (Figure 1). PRC2 recruits canonical PRC1 to target gene loci through deposition of H3K27me3, to which Cbx proteins bind. Conversely, noncanonical PRC1 has recently been shown to recruit PRC2 to target genes as a result of the association of PRC2 with H2Aub, as described in the next section. In this review, we will focus on the role of PRC1 and its H2A ubiquitination-dependent and ubiquitination-independent functions.

H2A ubiquitination-dependent and H2A ubiquitination-independent functions of PRC1 Studies over the past two decades have suggested that H2A ubiquitination by PRC1 is essential for the repression of PcG target genes in various cell types including embryonic stem cells (ESCs) as well as in vitro [9–15]. For instance, a ubiquitination-defective mutant (I53A) of mouse Ring1B was not able to rescue the derepression of PcG target genes in Ring1 knockout ESCs [16]. H2Aub has been proposed to inhibit transcriptional initiation and elongation processes through various mechanisms including enhancement of the recruitment of the linker histone H1 and suppression of the histone chaperone FACT and the gene activation-related histone marks H3K4me2 and H3K4me3 [10–13,15] (Figure 2a). The precise biochemical functions of H2Aub have remained unclear, however. An attempt to identify ‘readers’ of H2Aub by the isolation of H2Aub binding proteins revealed that many of these www.sciencedirect.com

Multiple modes of gene regulation by PcG in neural development Tsuboi, Hirabayashi and Gotoh 165

Figure 1

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Diversity of PRC1 complexes. PRC1 is classified into 6 subgroups (PRC1.1–6). Each of them contains one of Pcgf proteins (Pcgf1–6) and either Ring1B or Ring1A. Pcgf2/4 containing PRC1 complexes (PRC1.2 or 1.4, respectively) are called canonical PRC1 (cPRC1) and contain a Cbx protein that can bind to H3K27me3. Pcgf1/3/5/6 containing non-canonical PRC1 (ncPRC1) complexes (PRC1.1, 1.3, 1.5, 1.6, respectively) include RYBP or its homolog YAF2 instead of Cbx.

proteins were actually components of PRC2 [17]. Furthermore, several studies established that H2A ubiquitination by noncanonical PRC1 provides a binding platform for PRC2 [18–20]. In ESCs, a PRC1 complex containing Pcgf1 and Kdm2b was shown to recruit PRC2 [18]. In the case of X chromosome inactivation, H2A ubiquitination by PRC1 containing Pcgf3 or Pcgf5 is necessary for targeting of PRC2 to the inactive X chromosome [21,22]. Monitoring of temporal changes in histone modification revealed that H2A ubiquitination at the inactive X chromosome precedes H3K27me3 deposition [23]. Given that H3K27me3 formation by PRC2 can recruit PRC1, PRC1 and PRC2 may constitute a reciprocal (feedforward) loop that leads to the expansion of PcG domains at target loci and consequent repression of genes within these domains (Figure 2b). A recent study found that the expression of a certain set of PcG target genes in epidermal cells was upregulated by deletion of Ring1A and Ring1B genes or by knockin of a ubiquitination-defective mutant (I53A) at the Ring1B gene locus, but not by deletion of the gene for Eed, an essential component of PRC2 [24]. These findings suggest that H2A ubiquitination is able to repress target gene expression in both PRC2-dependent and PRC2-independent manners in the same cell type. What then might be the H2Aub readers responsible for the PRC2-independent functions of this modification? RSF1 (remodeling and spacing factor 1) was recently identified as a factor that associates with H2Aubcontaining nucleosomes (Figure 2c). RSF1 is a component of the RSF complex that remodels chromatin structure and generates regularly spaced nucleosome arrays. Despite the observation that 80% of genes enriched in H2Aub are www.sciencedirect.com

bound by RSF1 in mouse ESCs, RSF1 knockout resulted in derepression of only a subset of these genes and the release of linker histone H1 [25]. H2Aub-dependent gene repression thus appears to be executed by various mechanisms in a manner dependent on the genomic locus. Although all PRC1 complexes (PRC1.1 to 1.6) contain the E3 ubiquitin ligases Ring1A or Ring1B, ubiquitination activity does not account for all PRC1-dependent gene repression [16,26–28]. Indeed, a ubiquitination-defective mutant of Sce, the Drosophila ortholog of mammalian Ring1, was able to functionally replace the wild-type protein with regard to repression of target genes in the developing fly embryo [27]. In addition, ubiquitination-defective Ring1B(I53A) knockin mice were found to show milder phenotypes compared with Ring1B knockout mice [28], although this difference might be due in part to incomplete inactivation of H2A ubiquitination by the I53A mutation [29]. How then does PRC1 repress target gene expression independently of the ubiquitination activity of Ring1?

Chromatin looping and compaction as a PcGmediated mechanism of H2A ubiquitinationindependent gene regulation PRC1 is able to mediate the compaction of nucleosome arrays in vitro even those containing tailless histones such as a H2A mutant lacking the ubiquitination sites K118 and K119 [30,31] (Figure 3a). Fluorescence in situ hybridization (FISH) with closely apposed pairs of probes for quantitation of genomic separation revealed that Ring1B mediates chromatin looping or compaction at Hox loci in mouse ESCs [16,26,32,33]. Whereas this FISH-based Current Opinion in Neurobiology 2019, 59:164–173

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Figure 2

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H2A ubiquitination-dependent functions of PRC1. (a) Ring1A and Ring1B are E3 ubiquitin ligases at Lysine 119 of histone H2A. H2Aub inhibits transcriptional initiation and elongation steps through the interaction with RNA polymerase II, the histone chaperone FACT, histone H1, or other histone modifiers. (b) H2Aub functions as a binding platform of PRC2 complex, which catalyzes H3K27me3. Given that PRC1 can bind to H3K27me3, PRC1 and PRC2 may constitute a reciprocal loop and lead to the expansion of PcG domains at their target loci. (c) H2Aub functions as a binding platform for reader proteins, which regulate transcription. In mouse ESCs, RSF1 protein has been found to bind H2Aub and repress a subset of PcG target genes.

approach is limited to the analysis of chromatin looping for a small number of genomic regions, Hi-C and 5C analyses can reveal genomic interactions at the genomewide level [34,35]. Such analyses have recently shown that PcG target gene loci (those associated with Ring1B or H3K27me3) interact with each other with high probabilities and generate PcG domains by chromatin looping in Current Opinion in Neurobiology 2019, 59:164–173

mouse ESCs [32,36,37,38] as well as in Drosophila embryos [39,40] (Figure 3b). Cbx2, a component of canonical PRC1, was found to mediate nucleosome compaction by bridging nucleosomes [31] and thereby to contribute to PcG-dependent axial patterning [41]. The Phc components of canonical PRC1 can also induce nucleosome compaction via tandem polymerization www.sciencedirect.com

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Figure 3

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Phc2-SAM PRC1 clustering Cbx

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H2A ubiquitination independent functions of PRC1. (a) PRC1 can create compacted chromatin structure independently of its ubiquitylase activity in vitro and in vivo (i.e. at Hox gene loci in mouse ESCs). Polymerization of Phc2, a component of cPRC1, via its sterile alpha motif (SAM) domain contributes to the clustering of PRC1 at Hox gene loci. (b) PcG target gene loci interact each other and form discrete domains (PcG domain) in mouse ESCs independently of E3 ubiquitin ligase activity of Ring1B. In contrast to the Ring1B KO, ubiquitylase-deficient Ring1B (I53A) knock-in did not reduce the frequency of interactions between PcG target gene loci in ESCs. (c) PRC2 forms enhancer–promoter chromatin looping at neural gene loci. PcG proteins generate permissive chromatin topology at the target gene loci while suppressing their premature activation. This looping might contribute to the future activation of those loci.

mediated by their SAM (sterile alpha motif) domain [33,42,43] (Figure 3a), and 5C analysis revealed that deletion of the Phc1 gene reduced the extent of PcG domains in mouse ESCs [32]. The I53A or I53S mutants www.sciencedirect.com

of Ring1B were able to rescue the loss of chromatin compaction at Hox loci in Ring1-deficient ESCs, despite defective H2A ubiquitination at these loci [16,26]. Moreover, Ring1B(I53A) knockin at the Ring1B gene locus did Current Opinion in Neurobiology 2019, 59:164–173

168 Neural epigenetics

not substantially affect the frequency of genome-wide chromatin interactions in ESCs, whereas Ring1B knockout reduced it [32] (Figure 3b). Together, these various observations suggest that chromatin compaction and formation of PcG domains are mediated by PRC1 independently of its E3 ubiquitin ligase activity. What are the roles of PcG-mediated chromatin looping and the formation of PcG domains? High-resolution Hi-C analysis of the Drosophila Kc cell line revealed that developmental genes located at the anchors of PRC1dependent loops are less likely to be expressed than those that are located elsewhere [39]. Inhibition of PcG domain formation by insertion of insulator elements between Polycomb response elements (PREs)—cis elements necessary for PcG protein recruitment in Drosophila— resulted in the destabilization of PcG-mediated gene repression [40]. Interactions between PRC1-repressed gene loci may therefore confer or maintain a more persistent repressive state compared with that achieved without such interactions. In addition to the repressive role of PcG-mediated chromatin looping, recent studies have revealed that PcG proteins may also contribute to gene activation via the establishment of enhancer–promoter interactions (Figure 3c). PRC1 was thus shown to mediate the association of a midbrain-specific enhancer and the promoter of the Meis2 gene during midbrain development, with the subsequent dissociation of PcG proteins resulting in the activation of Meis2 expression in the midbrain [44]. PcG proteins were found to play a similar role in the establishment of ‘poised’ enhancers—defined by the presence of the histone acetyltransferase p300 and H3K27me3 and the absence of the K27-acetylated form of histone H3 (H3K27ac) and H3K4me3—in ESCs. Importantly, poised enhancers physically contact their target genes in a PRC2-dependent manner, and the PRC2 components Suz12 and Eed are necessary for the induction of these genes in differentiated cells [45] (Figure 3c). These findings indicate the essential role of PcG proteins in the generation of permissive chromatin topology at target gene loci and concomitant suppression of their premature activation.

Differential deployment of gene regulatory mechanisms by PcG proteins during neural development PcG proteins regulate gene expression through various mechanisms as summarized above, but how are these mechanisms actually deployed differentially during development? Given that many, if not all, development-related genes are PcG targets, the nature of PcG-mediated gene regulation would appear to be fundamental to the differentiation potential of a tissue stem cell. This differentiation potential is determined by the availability of developmental gene sets—with some developmental genes being Current Opinion in Neurobiology 2019, 59:164–173

temporarily repressed but ready for activation in response to differentiation-inducing cues (permissive or poised state), and others being persistently repressed and not expressed even in the presence of such cues. PcG proteins appear to contribute to both modes of repression. In ESCs, developmental genes, especially those subsequently expressed specifically in anterior brain regions, exist preferentially in a poised state dependent on PcG proteins [45,46,47] (Figure 4a). The differentiation of ESCs into neural progenitor cells (NPCs, which are tissue stem cells for the nervous system) is accompanied by the derepression and activation of neural genes such as the NPC markers Nestin and Sox1, whereas nonneural genes become persistently repressed. With regard to the mechanism of the derepression (activation) of the poised state of neural genes in response to ESC-to-NPC differentiation cues, the composition of PRC1 was found to change markedly during this differentiation step. Whereas noncanonical PRC1 variants such as PRC1.1 and PRC1.6 are predominant in ESCs, canonical PRC1 (PRC1.4) becomes relatively enriched in NPCs [48]. Among the components of PRC1, Pcgf5 was found to be necessary for induction of Nestin and Sox1 expression in NPCs. Given the necessity of Pcgf5 for the reduction in the amounts of H2Aub and H3K27me3 observed at these loci during their activation [49], Pcgf5 might compete with other PcG proteins that promote these modifications or play an unappreciated role in the activation of these genes (Figure 4a). In addition to the exchange of Pcgf proteins, a change in the levels of Cbx proteins has also been implicated in ESC-to-NPC differentiation [50] as well as in the differentiation of other tissue stem cells [51,52]. For example, overexpression of Pcgf4 and Cbx4 in ESCs results in downregulation of certain ESC-specific genes which are suppressed in NPCs, indicating an increase in the abundance of Pcgf4 and Cbx4 suppress these ESC-specific genes in NPCs [48]. While NPC-specific genes such as Nestin and Sox1 are expressed in NPCs during the neurogenic phase of development, genes related to terminal neuronal differentiation are maintained in the poised state in a PcG-dependent manner [29,53,54] (Figure 4b). Indeed, neuronal genes are the major PcG targets in NPCs at this time, and they are derepressed by deletion of either Ring1 or Ezh2 [29,53]. How is this PcG-mediated poised state maintained in NPCs and wiped out in response to neuronal differentiation signals? As is the case for developmental genes in ESCs, neuronal genes at the early (neurogenic) stage of NPC development are temporarily repressed by PRC1 in a ubiquitination-dependent manner, as revealed by the observation that this repression is lost on replacement of wild-type Ring1B with the ubiquitination-inactive mutant I53A/D56K [29] (Figure 4b). Given that activation of certain developmental genes during ESC differentiation depends on the histone deubiquitinase Usp16, deubiquitination may also be expected to occur at neuronal genes www.sciencedirect.com

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Figure 4

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Usage of different gene regulatory mechanisms by PcG during neural development. (a) In ESCs, neural genes such as Nestin and Sox1 are temporarily repressed and poised for activation by PcG. Those gene loci are organized into the structure called A compartment (enriched with permissive or active gene loci). During differentiation from ESCs into NPCs, poised state of neural genes is resolved for activation in response to the differentiation signal. A large fraction of H3K27me3-associated gene loci coding non-neuronal genes moves from A compartment to B compartment (enriched with inactive gene loci) upon terminal differentiation into neurons. Those gene loci are persistently repressed through PcG independent repression such as H3K9me2/3 mediated heterochromatin. (b) In the early stage (neurogenic) NPCs, gene loci coding genes associated with neuronal differentiation (neuronal genes) are temporarily repressed and maintained in a poised state by the H2Aub-dependent mechanism. The neurogenic cues can induce the expression of these genes at this stage. By contrast, in the late-stage (gliogenic) NPCs, those loci become persistently repressed by the H2Aub independent clustering of PRC1 and no longer respond to neurogenic cues.

during NPC differentiation [55]. Dissolution of the poised state may also be accompanied by displacement of PRC1 (as well as PRC2) from chromatin mediated by competing factors such as the transcription factor ZRF1 or the chromatin-remodeling BAF complex [56,57]. Forced recruitment of Brg1, a central component of the BAF complex, was shown to trigger fast (within 1–5 min) eviction of www.sciencedirect.com

Ring1B (PRC1) and Suz12 (PRC2) from a target locus and a subsequent reduction in the amounts of H2Aub (within 5 min) and H3K27me3 (within 10 min) at this locus [58]. Indeed, ablation of the BAF complex components Baf155 and Baf170 was found to increase the global level of H3K27me3 and to reduce the expression of neuronal genes such as Neurog2, Tcfap2c, and Tbr1 in NPCs [57]. Current Opinion in Neurobiology 2019, 59:164–173

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Interestingly, Baf155 and Baf170 interact with and enhance the activity of the histone demethylases Jmjd3 and UTX. Given that Jmjd3 activates neuronal gene expression in various contexts [59], the BAF complex may cooperate with histone demethylases (and perhaps also histone deubiquitinases) to compete with PcG proteins and activate neuronal genes in differentiating NPCs. The poised neuronal genes in NPCs are not derepressed simultaneously during neuronal differentiation, suggesting that the eviction of PcG proteins from and the derepression of these genes may be regulated in a target-specific manner, although what might serve as the cue for such target-specific derepression is unclear. In contrast to early stage (neurogenic) NPCs, late-stage (gliogenic) NPCs have lost neurogenic potential and produce only glial cells such as astrocytes [60–62]. This loss of neuronal differentiation potential has also been shown to involve PcG proteins [63–65]. A key question regarding the differentiation potential of NPCs concerns how PcG proteins are able to mediate both the temporary repression of neural genes during the neurogenic phase and the persistent repression of these same genes during the gliogenic phase. A recent study revealed that ubiquitination-dependent and ubiquitination-independent functions of Ring1B contribute to the repression of neural genes during the neurogenic and gliogenic phases, respectively [29] (Figure 4b). The ubiquitination-defective I53A/D56K mutant of Ring1B was thus able to suppress the expression of neurogenic genes such as Neurog1, Fezf2, and Tcfap2c during the gliogenic phase but not during the neurogenic phase. Of note, Phc2, a polymerization-inducing subunit of PRC1, was found to accumulate at neurogenic gene loci in NPCs during the gliogenic phase. Furthermore, disruption of PRC1 clustering by expression of a dominant negative mutant of Phc2 resulted in derepression of neurogenic genes in late-stage NPCs, indicating that PRC1 clustering is responsible for the H2A ubiquitination-independent mode of PcG-mediated persistent gene repression (Figure 4b). A developmental stage-dependent switch in the mode of gene repression by Ring1B (PRC1) has also been demonstrated for Hox genes, with their temporary repression in ESCs and persistent repression in NPCs being ubiquitination-dependent and ubiquitination-independent, respectively. Together, these observations support the notion that certain PcG targets become more resistant to activation cues during development as a result of a change in repression mode related to chromatin structure. Hi-C-based chromatin interaction studies have revealed that a large proportion of H3K27me3-associated gene loci moves from the A compartment (enriched in permissive or active gene loci) to the B compartment (enriched in inactive loci) during terminal differentiation of NPCs into neurons [38], perhaps reflecting persistent repression of nonneural PcGassociated genes. Indeed, most H3K27me3-associated genes, including nonneural genes, are not derepressed by deletion of Current Opinion in Neurobiology 2019, 59:164–173

both Ezh1 and Ezh2 in mature neurons such as striatal medial spiny neurons [66], suggesting that PcG target genes become persistently repressed by a PRC2-independent mechanism after terminal differentiation (Figure 4a). Similar phenomena have been described for other cell types including adult intestinal, blood, and skin cells, in which H3K27me3associated developmental genes related to other lineages were not derepressed by Eed deletion [67]. In fibroblasts, genes related to nonfibroblast lineages were found to be localized to sonication-resistant (physically compacted) heterochromatin domains and to be more refractory to activation signals compared with genes localized to euchromatin domains [68]. In the context of trophoblast lineage commitment, Ring1B-associated embryonic genes become persistently repressed by a mechanism dependent on the H3K9 methyltransferase Suv39h1 and DNA methylation [69]. It is therefore possible that poised genes suppressed by PcG proteins in tissue stem cells become persistently repressed by PcG-independent mechanisms including those mediated by changes in chromatin structure or in H3K9 or DNA methylation in order to ensure the loss of differentiation potential for certain lineages (Figure 4a).

Conclusions We have here summarized recent advances in the identification of various PcG protein complexes and their different functions in the regulation of gene expression. However, key questions remain to be answered with regard to the deployment of these diverse mechanisms of PcG function. Firstly, how are they implemented differentially at the right time in the right cell type? In other words, how are transitions in PcG function—such as those associated with the shift from poised to activated or from poised to persistently repressed states—achieved? A clue to this question has been provided by studies of the developmental switch from neurogenic NPCs (in which neurogenic genes are poised for activation by differentiation-inducing cues such as Wnt signaling and Notch inhibition) to gliogenic NPCs (in which neurogenic genes are more persistently repressed and resistant to differentiation-inducing signals). As described above, the mode of PcG-mediated repression of neurogenic genes switches from being ubiquitination-dependent to ubiquitinationindependent during this transition. Moreover, the NuRD chromatin-remodeling complex with its MBD3 and HDAC1 components has been implicated in this switch. The amount of MBD3, as well as that of Phc2, thus increases at neurogenic gene loci in association with the switch in repression mode, suggesting that these factors may function as a developmental timer in NPCs. In ESCs, PcG proteins are preferentially associated with unmethylated CpG islands such as those present at gene promoters [70], but these promoters are differentially regulated in response to distinct differentiation cues. The mechanisms that determine the locus-specific regulation of PcG targets have been an enigma in mammalian www.sciencedirect.com

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cells, which do not appear to harbor-specific cis elements for PcG targeting equivalent to PRE in Drosophila. It is possible that transcription or chromatin-remodeling factors cooperate with PcG proteins to determine target specificity. It is also possible that individual PcG domains may be separately regulated by differentiation cues. It would thus be of interest to determine whether genes located in the same PcG domain are coregulated during differentiation. Information on spatial organization and chromatin interactions obtained with detailed threedimensional analyses at the single-cell level (such as Hi-C, 5C, Genome Architecture Mapping (GAM), ChIA-PET, HiChIP, and PLAC-seq [71–73] combined with information on histone modifications and transcription and remodeling factors) should shed light on the nature of PcG domains and selective regulation of PcG target genes. Finally, given the pivotal role of PcG proteins in stem cell regulation, dysfunction of PcG-mediated mechanisms likely contributes to developmental disorders such as psychiatric diseases. Indeed, mutations of PcG-related genes have been associated with diseases such as autism spectrum disorder. Mutation of the gene for AUTS2, a component of PRC1 that activates neuronal genes in the mouse cortex, has thus been identified as a risk factor for autism spectrum disorder and other neurological conditions [74,75]. An understanding of the diverse mechanisms of PcG-mediated gene regulation may provide new insights into the pathogenesis of neurodevelopmental disorders as well as a basis for the development of new therapeutic strategies.

Conflict of interest statement Nothing declared.

Acknowledgements We thank the Drs Haruhiko Koseki and Shinsuke Ito for helpful suggestions. We apologize to all researchers whose work could not be cited owing to space limitation. The work in the authors’ laboratory is supported by MEXT/JSPS KAKENHI (JP15H05773, JP16H06481, and JP16H06479 for Y.G.), JST AMED-CREST (JP18gm0610013 for Y.G.), JST/PRESTO (JPMJPR16F7 for Y.H.) and the International Research Center for Neurointelligence (WPI-IRCN) at The University of Tokyo Institutes for Advanced Study.

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