# Cell Fate and Developmental Regulation Dynamics by Polycomb Proteins and 3D Genome Architecture

Vincent Loubiere, Anne-Marie Martinez, Giacomo Cavalli

► To cite this version:

Vincent Loubiere, Anne-Marie Martinez, Giacomo Cavalli. Cell Fate and Developmental Regulation Dynamics by Polycomb Proteins and 3D Genome Architecture. BioEssays, 2019, 41 (3), pp.e1800222. 10.1002/bies.201800222. hal-05529679

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# Cell Fate and Developmental Regulation Dynamics by Polycomb Proteins and 3D Genome Architecture

Vincent Loubiere, Anne-Marie Martinez,* and Giacomo Cavalli*

Targeted transitions in chromatin states at thousands of genes are essential drivers of eukaryotic development. Therefore, understanding the in vivo dynamics of epigenetic regulators is crucial for deciphering the mechanisms underpinning cell fate decisions. This review illustrates how, in addition to its cell memory function, the Polycomb group of transcriptional regulators orchestrates temporal, cell and tissue-specific expression of master genes during development. These highly sophisticated developmental transitions are dependent on the context- and tissue-specific assembly of the different types of Polycomb Group (PcG) complexes, which regulates their targeting and/or activities on chromatin. Here, an overview is provided of how PcG complexes function at multiple scales to regulate transcription, local chromatin environment, and higher order structures that support normal differentiation and are perturbed in tumorigenesis.

## 1. Introduction

Polycomb Group (PcG) proteins are transcriptional regulators that were first discovered in *Drosophila* for their ability to establish an epigenetic “cellular memory” system that ensures the stable repression of Hox genes throughout development.[1] However, PcG proteins can also dynamically regulate their target genes during differentiation processes in embryonic and adult lineages. In recent years, various experimental models have been used to study the dynamic role of PcG proteins in regulating cell fate decisions.[2] During differentiation, the activities of pluripotency-associated genes and differentiation-inducible genes are coordinately reversed. Thanks to proteomic, genomic and epigenomic analysis and to powerful methods for the molecular analysis of 3D genome architecture, the functional interplay between histone modifications, gene expression, and genome organization during lineage differentiation processes can now be elucidated. Here, we review the molecular features of

PcG protein complexes, the mechanisms of PcG-dependent transcriptional regulation and the current knowledge on PcG functions during neurogenesis as an example of dynamic genome regulation during cell differentiation. Finally, we recapitulate aberrant PcG function during tumorigenesis to highlight how this regulatory machinery, once thought to function in a monotonous way in order to maintain gene silencing during normal development can be perturbed in pathological processes.

## 2. PcG Proteins Assemble Into Two Main Classes of Multimeric Complexes

PcG proteins were originally discovered in *Drosophila*, where the *Polycomb* gene was shown to repress Hox gene expression.[3] To exert this repressive role, PcG proteins assemble into multimeric complexes that are highly conserved from flies to mammals.[11] The two main PcG complexes, termed Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), catalyze histone tail modifications and cooperate at multiple levels to establish a repressive chromatin environment. The core of the PRC2 consists of EZH2/1 (Enhancer of Zeste Homolog 2/1), EED (Embryonic Ectoderm Development), SUZ12 (Suppressor Of Zeste 12 Homolog Protein) and RBBP4/7 (Retinoblastoma Binding Protein 4/7), also known as NURF55, and catalyzes the tri-methylation of lysine 27 of histone H3 (H3K27me3) to mediate transcriptional repression (Figure 1A).[4] The deposition of this mark is mostly ensured by EZH2 in proliferating cells, whereas its EZH1 paralog, which has a weaker histone methyl-transferase (HMTase) activity, is more abundant in resting cells.[5] The PRC2 HMTase activity is enhanced by a positive feedback loop relying on the specific recognition of the H3K27me3 by EED, which in turn stimulates the complex via an allosteric mechanism.[6]

In addition to its core members, PRC2 interacts with several sub-stoichiometric components that modulate its enzymatic activity and/or its targeting to DNA. Proteomic analysis in human cell lines identified two sub-complexes called PRC2.1 and PRC2.2 (Figure 1A).[7] PRC2.1 contains one of the three PCL1-3 (Polycomb Like Protein 1-3) paralogs – also known as PHF1, MTF2, and PHF19, respectively – and either PALI1/2 (PRC2 Associated LCOR Isoform 1/2) or EPOP (Elongin B/C and PRC2-associated Protein).[8] PCL proteins specifically bind the elongation-associated H3K36me2/3 active marks through their TUDOR domain and stimulate H3K27me3 deposition at

Dr. V. Loubiere, Prof. A.-M. Martinez, Dr. G. Cavalli
Institute of Human Genetics
UMR 9002
CNRS and University of Montpellier
34396 Montpellier, France
E-mail: anne-marie.martinez@igh.cnrs.fr; giacomo.cavalli@igh.cnrs.fr

© 2019 The Authors. *BioEssays* Published by WILEY Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/bies.201800222
A

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<table><thead><tr><th>Complex</th><th>Proteins</th><th>Function</th></tr></thead><tbody><tr><td rowspan="4">core PRC2</td><td>EZH1/2</td><td>H3K27 methyltransferase</td></tr><tr><td>EED</td><td>H3K27me3 binding, allosteric stimulation of HMTase activity</td></tr><tr><td>SUZ12</td><td>protein-protein interactions</td></tr><tr><td>RBBP4/7</td><td>Histone binding</td></tr><tr><td rowspan="6">PRC2.1</td><td>PCL1/PHF1</td><td>H3K36me2/3 binding, stimulation of HMTase activity, DNA binding</td></tr><tr><td>PCL2/MTF2</td><td>H3K36me2/3 binding, stimulation of HMTase activity, DNA binding</td></tr><tr><td>PCL3/PHF19</td><td>H3K36me2/3 binding, stimulation of HMTase activity</td></tr><tr><td>PALI1/2</td><td>Stimulation of HMTase activity</td></tr><tr><td>EPOP</td><td>Interaction with Elongin B/C</td></tr><tr><td>JARID2</td><td>H2AK119Ub binding, stimulation of HMTase activity, DNA binding</td></tr><tr><td>PRC2.2</td><td>AEBP2</td><td>H2AK119Ub binding, stimulation of HMTase activity, DNA binding</td></tr></tbody></table>

B

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<table><caption>**Figure 1.** Schematic view of the two main mammalian PcG complexes. A) On the top panel, the mammalian core PRC2 complex is depicted with its accessory proteins belonging to the PRC2.1 (left) and PRC2.2 (right) sub-complexes. On the bottom part, a table recapitulating PRC2 components and related functions is shown. B) The mammalian core PRC1 complex is shown on the top. The cPRC1 (left) is characterized by the presence of one of the 5 CBX paralogs (in pink) and PCGF2/4 (in gray) while the various ncPRC1 (right) contain RYBP/YAF2 (in pink) together with one of the six PCGF paralogs (in gray). For each PRC1 complex, the specific components that are not part of the “core” are listed below their respective names. The sub-stoichiometric SCMH1/L2 of the cPRC1 is also shown. On the bottom part, a table recapitulating PRC1 components and related functions is shown.</caption><thead><tr><th>Complex</th><th>Proteins</th><th>Function</th></tr></thead><tbody><tr><td rowspan="2">Core PRC1</td><td>RING1A/B</td><td>H2AK119Ub deposition, protein-protein interactions</td></tr><tr><td>PCGF1-6</td><td>Stimulation of E3 ligase activity, protein-protein interactions</td></tr><tr><td rowspan="3">cPRC1</td><td>CBX2/4/6-8</td><td>H3K27me3/H3K9me3 binding, chromatin compaction (CBX2)</td></tr><tr><td>PHC1-3</td><td>Oligomerization, chromatin looping</td></tr><tr><td>SCMH1/L2</td><td>Oligomerization, protein-protein interactions</td></tr><tr><td>ncPRC1s</td><td>RYBP/YAF2</td><td>Stimulation of E3 ligase activity, DNA binding</td></tr><tr><td rowspan="4">PRC1.1</td><td>KDM2B</td><td>H3K36 demethylation, CG-rich DNA binding</td></tr><tr><td>USP7</td><td>Stimulation of E3 ligase activity</td></tr><tr><td>SKP1</td><td>Ubiquitin ligase</td></tr><tr><td>BCOR/BCORL1</td><td>Scaffold</td></tr><tr><td rowspan="3">PRC1.3/5</td><td>CKII</td><td>RING1B phosphorylation and inhibition of E3 ligase activity</td></tr><tr><td>AUTS2/FBRS/FBSL</td><td>Transcriptional activation</td></tr><tr><td>DCAF7</td><td>Transcriptional activation, scaffold</td></tr><tr><td rowspan="8">PRC1.6</td><td>HDAC1/2</td><td>Histone deacetylase</td></tr><tr><td>HP1γ</td><td>H3K9me3 binding</td></tr><tr><td>L3MBTL2</td><td>Histone methyl-lysine binding, chromatin compaction</td></tr><tr><td>DP-1</td><td>DNA binding</td></tr><tr><td>E2F6</td><td>DNA binding</td></tr><tr><td>MAX</td><td>DNA binding</td></tr><tr><td>MGA</td><td>DNA binding</td></tr><tr><td>WDR5</td><td>Scaffold</td></tr></tbody></table>
target sites.[9] Hence, it has been proposed that this mechanism
allows the intrusion of PRC2 at transcriptionally active sites to
induce de novo repression.[10] MTF2 and PHF1 have also been
shown to play a crucial role in recruiting PRC2 at unmethylated
CpG islands (CGIs).[11] EPOP mediates PRC2-Elongin B/C
association at a subset of sites in mouse Embryonic Stem Cells
(mESCs) and partially counteracts PRC2 occupancy and
transcriptional silencing in order to maintain low level
transcription at bivalent genes,[12] while PAL1/2 stimulates
PRC2 HMTase activity.[13] The PRC2.2 complex is characterized
by the presence of AEBP2 (Adipocyte Enhancer-Binding Protein
2) together with JARID2 (Jumonji, AT Rich Interactive Domain
2) (Figure 1A).[7,14] Both JARID2 and AEBP2 are thought to
recruit PRC2 on chromatin and to preferentially bind CG-rich
DNA, consistent with PRC2 occupying CGIs in vivo.[14b,15]
Notably, they also stimulate the HMTase activity of the complex
and specifically bind the H2AK119Ub (mono-ubiquitinated
Lysine 119 of histone H2A) mark deposited by the PRC1
complex, providing a functional bridge between the two main
PCG complexes.[16]

Compared to PRC2, PRC1 complexes are more diverse (Figure 1B). Their core components are RING1A or RING1B (Really Interesting New Gene 1 A and B, respectively, which are mutually exclusive components<sup>[17]</sup>), the E3 Ubiquitin ligases that catalyze H2AK119Ub deposition, together with one of the six PCGF1-6 (Polycomb Group Ring Finger 1-6) paralogs.<sup>[18]</sup> Five PRC1 subtypes have been identified, which are characterized by the incorporation of specific PCGF isoforms and other interacting partners and can be stratified into “canonical” and “non-canonical” PRC1 (cPRC1 and ncPRC1; Figure 1B).<sup>[18a]</sup>

The cPRC1 assembles around the RING1A/B-PCGF2/4 (PCGF2/4 are also known as MEL18 and BMI1, also giving rise to the two PRC1.2 and PRC1.4 non-canonical complexes, respectively) core and contains one of the CBX2/4/6-8 (Chromobox Homologs 2/4/6-8) proteins, one of the PHC1-3 subunits (Polyhomeotic Homologs 1-3) and SCMH1/L2 (Sex Combs on Midleg Homolog 1/Like 2) (Figure1B).[1,19] CBX proteins contain characteristic chromodomains with varying affinities for H3K27me3 and/or H3K9me3 repressive marks and are thought to play a key role in recruiting PRC1 at PcG-repressed sites (Figure 2A).[20] The PHC1-3 proteins contain a Sterile Alpha Motif (SAM) domain that allows the formation of homo- but also hetero-polymers with other PcG members, and is essential for PcG-mediated repression.[21] SCMH1/L2 also contain a SAM domain and are generally considered as sub-stoichiometric PRC1 components.[22] Their *Drosophila* homolog, SCM, forms homo- and hetero-polymers and has been shown to participate in the recruitment of PRC1 to chromatin.[23] In the future, it will be important to assess the implication of SCMH1/L2 polymerization in PcG-mediated repression in mammals and to understand whether the different variants of canonical PRC1 are fully or only partially redundant, depending on the presence of specific CBX and PHC subunits.

In ncPRC1 complexes, CBX proteins are replaced by RYBP (Ring and YY1 Binding Protein) or its YAF2 (YY1 Associated Factor 2) paralog, which compete for the very same binding pocket on the C-terminal domain of RING1B.[24] The ncPRC1s can be distinguished by the presence of PCGF2/4 (PRC1.2/4), PCGF1 (PRC1.1, also known as BCOR), PCGF3/5 (PRC1.3/5) or

PCGF6 (PRC1.6, also known as E2F6.com) (Figure 1B).[18a]
Importantly, PCGF4-RING1B dimers have a lower E3 ligase
activity compared to non-canonical PCGF-RING1B dimers in
vitro.[25] In vivo, most of the H2AK119Ub is thought to be the
product of ncPRC1 complexes, whose stronger basal activity is
further stimulated by the presence of RYBP.[26]

3. PcG Complexes Collaborate at Multiple
Levels to Repress Transcription

In the hierarchical model of PcG recruitment originally inferred from *Drosophila* studies, the recruitment of PRC2 was proposed to occur first and in turn deposit H3K27me3 to eventually recruit cPRC1 through its chromodomain-containing CBX homolog, PC (Polycomb).[20b] In mammals, the absence of CBX proteins within ncPRC1s implies the existence of alternative mechanisms to recruit RYBP-containing complexes independently of the H3K27me3 mark.[26a] Moreover, while PcG proteins are recruited at dedicated DNA sequences via a specific set of transcription factors in *Drosophila*, the most universal feature regarding PcG recruitment in mammals is their binding at unmethylated CGIs in vivo.[1,27] Research in this field has mostly focused on KDM2B, whose CxxC DNA-binding domain specifically binds non-methylated CG-rich DNA sequences and was shown to directly recruit PRC1.1 to chromatin in Embryonic Stem Cells (ESCs).[28] According to this reverse hierarchical model, the non-canonical PRC1.1 is thus tethered first, deposits the H2AK119Ub mark and this in turn recruits PRC2 via specific interactions with AEBP2 and JARID2 (Figure 2A).[16,28b] PRC2 recruitment at unmethylated CGIs is further supported by PCL1-3 and triggers the deposition of the H3K27me3 repressive mark, which is finally used as a docking site for the recruitment of the CBX-containing cPRC1 (Figure 2A).[11a,28b,29] Notably, this reverse hierarchical model has recently been extended to the initiation of PcG-dependent inactivation of the X chromosome via PRC1.3/5.[30] Almeida et al. also showed that the XN region of the *Xist* long non-coding RNA is crucial for the recruitment of the PRC1.3/5 and subsequent H2AK119Ub deposition. This observation contrasts with the previous model of *Xist*-mediated recruitment of PRC2,[31] and is consistent with recent reports showing that the *Xist* interactome contains several ncPRC1 members and recruiter proteins but no PRC2 members[32] (for a recent review on the role of PcG complexes in X inactivation, see ref. [33]). Therefore, there seem to exist several non-mutually exclusive crosstalks between PRC1 and PRC2 complexes that induce their co-recruitment on chromatin, where they collaborate at multiple levels to establish a repressive chromatin environment (Figure 2A,B).

PcG complexes are recruited at discrete foci around which their cognate repressive marks are spread bi-directionally to set up the repressive landscape of target genes (Figure 2B).[28b,34] Once deposited, H3K27me3 is propagated through DNA replication by equivalent segregation of marked nucleosomes on the two daughter chromatin molecules followed by PRC2-dependent replenishment of the mark on newly incorporated unmarked histones.[35] In this way, H3K27me3 persists through the cell-cycle, providing a memory of PcG-mediated repression.[36] Although H3K27me3 helps maintaining PRC2 on target, via EED-mediated recognition of the mark and allosteric
<figure><img src="image_4.png" /><figcaption>**Figure 2.** PcG recruitment model and transcriptional silencing mechanisms. A) The reverse hierarchical model of PcG recruitment is shown, in which ncPRC1 complex (in yellow) is first recruited to chromatin and deposits the H2AK119Ub mark (in red) that subsequently recruits the PRC2 complex (via JARID2 and AEBP2) that eventually deposits the repressive H3K27me3 mark (in blue). Subsequently, the H3K27me3 mark is used as a docking site to recruit the cPRC1 (in pink). B) Schematic view of PcG-dependent chromatin compaction and transcriptional repression. C) Cartoon depicting PcG-dependent chromatin loops. PRC1-bound sites contact each other in the 3D space of the nucleus through PHC-SAM polymerization. D) Top: the compartmentalization of the genome into active (green) and PcG-repressed (blue) is illustrated. Topologically Associating Domains (TADs) are represented by triangles. Dashed lines (in gray) correspond to intra-TAD contacts between PRC1 binding sites (in pink). In addition, PcG-repressed TADs contact each other at long distances in the 3D space of the nucleus (black arrow). Bottom: 3D view of long-range contacts (black arrow) between multi-looped PcG TADs 1 and 2 (in blue). Active TADs (in green) are segregated from PcG-repressed TADs.</figcaption></figure>

activation of PRC2, the mere presence of marked histones is not sufficient to propagate chromatin memory indefinitely. At least in *Drosophila*, H3K27me3 propagation to newly incorporated histones requires continuous sequence-specific tethering of PRC2 in order to avoid progressive dilution through multiple rounds of replication.[34b] Understanding the role of CpG island-dependent PcG tethering in the propagation of H3K27me3 through cell divisions in mammals will require further investigation.

Once recruited to their target loci, PcG complexes repress transcription through multiple mechanisms. One of the best characterized silencing mechanisms involves chromatin compaction, which makes it inaccessible to the ATP-dependent chromatin remodeling complexes of the SWI/SNF family (Figure 2B).[19] This function is ensured by a highly positively charged region found in CBX2, whose mutation leads to defects in PcG-mediated repression in vivo,[37] highlighting the importance of this non-catalytic role for proper repression. In addition, PcG proteins were proposed to block RNA PolII initiation and/or elongation[38] and CBX proteins were shown to directly interact with the major Histone Acetyl Transferase (HAT) CBP to inhibit its activity and prevent H3K27Ac deposition and transcriptional activation (Figure 2B).[39] Conversely, PRC2 binds nascent transcripts in vivo and this

interaction correlates with decreased H3K27me3.[15,40] Importantly, it has been shown that RNA and DNA compete for PRC2 binding[15,41] and, therefore, nascent RNAs might act as a “decoy” that prevents the stable binding of PRC2 to its nucleosome substrate and aberrant H3K27me3 deposition at active sites (Figure 2B).[15,42]

In addition to their role in the regulation of local chromatin environment and structure, PcG proteins form repressive chromatin loops in the 3D space of the nucleus (Figure 2C). Hox clusters are canonical examples of PcG target sites and show a multi-looped structure.[43] Applying the Hi-C (High-throughput Chromosome Conformation Capture) method allowed the detection of PcG loops genome-wide and revealed that they are anchored at PRC1 binding sites and require the cPRC1 with a functional PHC SAM domain to be formed (Figure 2C).[44] Recent evidence from *Drosophila* suggests that these loops play an instructive role, given that the sole disruption of a loop is sufficient to destabilize PcG repression in this model organism.[45] At a larger scale, chromosomal regions are organized into Topologically Associating Domains (TADs) characterized by higher intra-contact frequencies relative to surrounding genomic regions, and in active and repressed chromosome compartments (Figure 2D).[46] Consistent with the role of PRC1 in compacting chromatin, Hi-C experiments and super-resolution microscopy revealed that PcG
domains fold into denser TADs compared to active ones and that
this compact packing relies on PRC1 function (Figure 2D).[44b,47]
Furthermore, very distant PcG sites contact each other in the 3D
space of the nucleus (Figure 2D).[46b,48] Such long-range contacts
also seem to rely on PRC1 proteins and *Drosophila* studies suggest
that they actively participate in PcG-mediated repression and
require the SAM domain of the PHC *Drosophila* homolog to be
established.[43b,48,49]

4. PcG Proteins Maintain the Identity of Embryonic Stem Cells and Prime Them for Differentiation

PcG complexes are thought to ensure the maintenance of ESC identity by collaboratively and stably repressing the transcription of key developmental and lineage-specific genes.[29] In agreement with this, *Eed* or *Ring1B* depletion leads to increased expression of differentiation markers in mESCs and *Ring1A/B* double KO impairs ESC self-renewal.[50] Interestingly, PCGF6 is the most abundant PCGF paralog in mESCs.[8b,51] The corresponding ncPRC1 complex, PRC1.6 (also known as E2F6.com), is composed of PCGF6/RYBP/RING1B, HP1γ (Heterochromatin protein 1 gamma), HDAC1/2 (Histone Deacetylases 1/2), the scaffold protein WDR5 (WD Repeat Domain 5) and a set of transcription factors that consists of E2F6 (E2F Transcription Factor 6), L3MBTL2 (Lethal(3) Malignant Brain Tumor-Like Protein 2), MAX (Myc-Associated Factor X), MGA (MAX Gene-Associated Protein), and DP1 (E2F Dimerization Partner 2) (Figure 3A).[8b,18a,52] The MAX-MGA heterodimer collaborates with E2F6 and L3MBTL2 to recruit the complex to chromatin,[52b,53] consistent with the extensive genome-wide co-localization between L3MBTL2, MGA, and E2F6 in mESCs.[53a,54] Importantly, PRC1.6 targets do not strictly overlap with cPRC1 and this complex was proposed to maintain ESCs identity via the repression of germ-cell related genes.[53a,c] Once recruited, the PRC1.6 represses its target genes via the deposition of the H2AK119Ub mark and subsequent deposition of the PRC2-mediated H3K27me3 mark[53a,c,54] and by L3MBTL2-mediated chromatin compaction.[54] PRC1.6 overlaps with the cPRC1, PRC1.1 and PRC2 at a small subset of sites,[53c] suggesting that its role in the maintenance of ESC identity could also partly rely on the transcriptional repression of lineage specific genes in conjunction with other PcG complexes.[51,53a,c,55] Interestingly, in addition to this repressive function, the PRC1.6 has been proposed to support the transcriptional activity of pluripotency genes.[51,56] Altogether, these data suggest that the PRC1.6 is a master regulator of mESC identity that acts in collaboration with but also independently of the canonical PcG axis, as further supported by the well documented premature differentiation phenotype observed in Loss Of Function (LOF) experiments affecting PRC1.6 members.[51,52b,53c,55,56]

ESCs are characterized by a prevalent co-localization between the H3K27me3 PcG repressive mark and the H3K4me3 active mark at a subset of promoters of lowly expressed key developmental genes (Figure 3B).[57] At these so-called bivalent promoters, the H3K4me3 active mark is deposited by MLL2 (Mixed Lineage Leukemia 2) from the TrxG (Trithorax Group),[58] which corresponds to the activating counterpart of PcG proteins (Figure 3B). Therefore, bivalent promoters are regulated via a tight PcG/TrxG balance, as further supported by the fact that *Mll2*

knock-out (KO) leads to an increase in PcG occupancy, reduced
accessibility and a redistribution of long-range interactions at
bivalent promoters.[57a] Although the molecular mechanisms
underlying this PcG/TrxG balance are still poorly characterized,
the UTX (Ubiquitously-Transcribed X Chromosome Tetratrico-
peptide Repeat Protein) H3K27-specific demethylase from the
TrxG family has been proposed to play a key role in this process
by facilitating the transition from a PcG repressed to a TrxG,
H3K27Ac marked active state (Figure 3B).[59]

Bivalent promoters are thought to provide a chromatin template that allows conjugating pluripotency while safeguarding cell differentiation potential.[60] Although they are poorly transcribed, bivalent promoters are often pre-loaded with poised RNA PolII, suggesting that they are ready for later activation (Figure 3B).[61] These primed promoters were originally proposed to convey rapid induction of underlying genes upon developmental cues, and their bivalent state was proposed to be resolved into a fully active or repressed state during differentiation (Figure 3B). Nevertheless, this hypothesis is disputed since there are no transcriptional induction defects after retinoic acid treatment of mESCs depleted for *Mll2*[58,62] and PcG-repressed promoters still bind poised RNA PolII in terminally differentiated neurons, suggesting that bivalent states could also play a role at later developmental stages.[63] Indeed, a study of PRC2 function in the epithelium of the adult mouse intestine identified hundreds of cell-type specific bivalent genes as the primary targets that become derepressed upon depletion of PRC2.[64] This suggests an alternative view in which the function of PRC2 in differentiated cells is primarily to repress tissue-specific bivalent genes. Perhaps these represent genes that were active in progenitor cells, prior to PRC2 recruitment, and acquired PRC2 in order to prevent them from inappropriate expression in differentiated progeny cells. In this respect, it will be important to decipher the mechanisms of PRC2 redeployment to these tissue and cell-type specific genes.

5. Changes in PcG Complexes Support Neural Differentiation

Somewhat paradoxically, besides their function as positive
regulators of ESC identity, PcG proteins play a crucial role in
differentiation.[29] Therefore, the initial view of these proteins as
setting up and maintaining cell memory of silent transcription
states from embryogenesis until the end of life is necessarily
limited. Indeed, recent evidence suggests that the pleiotropic
role of PcG complexes during mammalian development stems
from their highly dynamic interactome and composition, that
adjusts their targeting and functions to differentiation inputs.[29]
For example, CBX7 is replaced by CBX2/4 during mesoderm cell
fate-specification, and this swap triggers the displacement of the
cPRC1 from lineage-specific genes (via CBX7) to stem cell self-
renewal genes (via CBX2/4).[2b,65] Such an exchange has also
been reported for the PRC2 complex, whose catalytic subunit
EZH2 is progressively replaced by its close EZH1 paralog in
differentiated cells.[5] As an example of these dynamics, we will
discuss in more detail PcG function during differentiation of
stem cells toward the neural fate, but we invite the readers to
refer to recent reviews discussing the roles of these components
<figure><img src="image_5.png" /><figcaption>**Figure 3.** PcG function in maintaining ESCs identity and bivalent promoters. A) Several mutually-reinforcing molecular mechanisms govern the stable PcG binding and transcriptional repression of lineage-specific genes in ESCs. The non-canonical PRC1.6 complex is composed of core PRC1 members (in orange) and other repressive chromatin factors (in cyan), together with transcription factors (in purple) that trigger its recruitment on chromatin. The complex is assembled around a scaffold protein, WDR5, shown in white. PRC1.6 both collaborates with the canonical PcG axis at lineage-specific genes (depicted on the left side) and specifically represses germ-cell specific genes via H2AK119Ub deposition (in red) and subsequent H3K27 tri-methylation (in blue). B) Schematic view of bivalent promoters in ESCs (on the left) and their resolution into fully PcG-repressed (top right) or active (bottom right) state in the course of differentiation. The H3K27 UTX demethylase was proposed to facilitate the transition from a bivalent to active state.</figcaption></figure>

in cortical neurogenesis,[66] in the epithelial to mesenchymal transition[67] and in hematopoiesis[68] for further insights into dynamic PcG functions.

To understand the compositional changes of PcG complexes during neural differentiation, Kloet et al. performed a quantitative, MS-based characterization of the interactome of PcG proteins combined with genome-wide profiling of PRC1 and PRC2 in mESCs and Neural Progenitor Cells (NPCs) (Figure 4A).[8b] During differentiation, although the PRC2 core stoichiometry is stable, the overall levels of expression of PRC2 core subunits EED, EZH2, and SUZ12 decrease and PRC2 core binding also decreases while, interestingly, its associated H3K27me3 mark is maintained and PRC1 is retained (Figure 4A). However, cell-type specific dynamics exist in PRC2 sub-stoichiometric interactors. Indeed, FBXW11 (F-Box and WD-40 Domain Protein 11) and the uncharacterized AU022751 proteins only interact with PRC2 in mESCs, whereas PCL1/PHF1 and EHMT2 (Euchromatin Histone Lysine Methyltransferase 2) are NPC-specific PRC2 interactors. Moreover, the abundance of these sub-stoichiometric PRC2 interactors also varies during differentiation: EPOP, PCL2/MTF2, and Elongin B/C are tenfold more enriched in ESCs than in NPCs and, reciprocally, Gm340 and PCL3/PHF19 tenfold enriched in NPCs (Figure 4A). It remains to be determined whether all these

proteins assemble in a fraction of PRC2 complexes or whether they interact sub-stoichiometrically with all PRC2 variants. This second hypothesis is more plausible, since some of these interactors share a binding surface on EZH1 and EZH2.[8b] While the abundance of PRC2 components gradually decreases with differentiation, *Ezh2* is expressed in NPCs and its specific deletion triggers a premature activation of genes involved in neuronal differentiation such as *Insm1*, *Eomes*, *Neurog1/2*, *Neurod1/2*, and premature differentiation into neurons.[69] H3K4me3 and H3K27me3 marks have been shown to be highly dynamic at different stages of neural differentiation[69a] and these switches correlate with transcriptional changes, raising the question of how PcG targeting is dynamically regulated. Interestingly, the neuronal-specific TBR2 (T-box brain protein 2) transcription factor encoded by *Eomes* was shown to interact with the H3K27me3-specific demethylase JMJD3, leading to H3K27 demethylation at TBR2 target genes and suggesting a possible mechanism driving specific PRC2 inactivation for lineage gene induction.[69a]

PRC1 complexes also undergo profound compositional changes during NPC specification. A major change occurs in the abundance of PCGF and CBX subunits. Indeed, PCGF6 occupies 60% of RING1B complexes in ESCs. Differentiation to NPCs mainly correlates with an exchange of PCGF6 for PCGF4/
<figure><img src="image_6.png" /><figcaption>**Figure 4.** PCG function during neural lineage commitment. A) The stoichiometry of the core PRC2 members remains unchanged between ESCs (left) and NPCs (right) during neural differentiation, although the amount of core PRC2 members is greatly reduced. In contrast, neither the levels of H3K27me3 (in blue) nor PRC1 binding at target developmental genes are affected. In parallel, the changes in sub-stoichiometric PRC2 components between ESCs and NPCs are illustrated (in light blue). B) Top: The PRC1.6 is the most abundant PRC1 variant in ESCs where it represses lineage-and germ-cell specific genes and its components are strongly decreased in NPCs, where PCGF6 is mostly replaced by PCGF4. It remains to be determined what is the exact combination of PRC1 complexes existing in NPCs. Bottom: Concerning CBX members, NPC differentiation is accompanied by a swap between CBX7 (in ESCs), which allows the tethering of lineage-specific genes, to CBX6. In addition, CBX4 was proposed to promote the relocation of the cPRC1 complex from lineage-specific to ESCs-specific genes (*Orc2/Nodal*) during NPCs commitment. It remains to be addressed whether CBX exchanges more generally govern the relocation of the cPRC1 at pluripotency genes in NPCs, as previously proposed in other differentiation systems. Finally, PCGF5 expression is increased in NPCs where it represses genes from the SMAD2/TGFβ signaling pathway via H2AK119Ub deposition and subsequent H3K27me3. In parallel, PCGF5 was proposed to activate neural genes. This atypical role might rely on the previously described PRC1.5-AUTS2 complex, which collaborates with the p300 histone Acetyl Transferase to activate its target genes.</figcaption></figure>

BMI1 containing complexes that represent 54% of the PcG-NPC complexes (Figure 4B). Consistently, PCGF6-specific interactors show a radical change between ESCs and NPCs, due to the loss of PCGF6 from the complex during neural lineage differentiation.[8b] This observation is in line with the overexpression

phenotype of PCGF6, which was proposed to reduce ESC differentiation.[56] Conversely, PCGF4 interactors are more abundant in NPCs (Figure 3B). A similar switch is observed for CBX subunits, with a notable increase of CBX association to RING1B-containing complexes during differentiation. As
expected, CBX7 is associated with RING1B only in ESCs,[65,70]
but is replaced by CBX6 in NPCs (Figure 3B).[88] Overexpression
of the NPC-CBX4 and PCGF4 subunits in ESCs is sufficient to
drive PRC1 to NPC-specific target genes such as *Nodal* and *Orc2*
and trigger their transcriptional repression. The MBD5 subunit
of the Polycomb Repressive DeUBiquitinase (PR-DUB) is also
enriched in NPCs, which is consistent with its brain
function.[1,71] This complex also contains BAP1 (BRCA1-
Associated Protein 1) together with ASXL1/2 (Additional Sex
Combs Like 1/2) and is responsible for the removal of the
H2AK119Ub mark, although its precise role in PcG repression
remains unclear.[1,72] Finally, PHC1, which interacts with the
PCGF2 and PCGF4 cPRC1 complexes, is more abundant in
ESCs than in NPCs.

Another example of a PRC1 subunit that plays a differentiation-
specific role is the PCGF5 paralog, which belongs to the PRC1.5
complex and is dispensable for the self-renewal of mESCs, whereas
it is necessary for neural differentiation.[73] PCGF5 engages
mESCs toward neural specification by repressing the SMAD2/
TGFß signaling pathway target genes *Nodal*, *Lefty1* and *Lefty2* and,
intriguingly, by concomitantly activating neural specific genes
(Figure 3B).[73a] How PCGF5 might activate transcription in NPCs
was not addressed in this study, but PCGF5 had previously been
associated with transcriptional activation when associated with the
non-canonical PRC1-AUTS2 complex that can recruit the p300
histone acetyl transferase at neural genes in the mouse central
nervous system (Figure 3B).[74] AUTS2 and RING1B co-localize at
promoters of neuronal genes in the mouse brain and brain-specific
loss of *Auts2* leads to developmental phenotypes compatible with
*Auts2* disruption in humans.[74] Recently, Wang et al. have shown
that DCAF7 (also known as WDR68), a bona fide member of the
PRC1.5 complex (Figure 1B) is required for PRC1-AUTS2-
mediated transcription activation and proper neuronal differ-
entiation.[73b] Whether this is the mechanism by which PRC1.5
activates genes in NPCs remains to be determined. Another
possible case of PRC1-dependent gene activation involves
RING1B. Its deletion in the mouse developing neocortex during
the neurogenic phase leads to an altered neuronal subtype
specification.[75] Strikingly, in NPCs RING1B is enriched on
thousands of active promoters and enhancers of genes lacking
H3K27me3 and involved almost exclusively in neural function,
although whether this binding can be explained by a role for PRC1
complexes in transcriptional activation remains to be seen.[8b]

Altogether, these results strongly suggest that the redistribu-
tion of PcG complexes during development is correlated with
profound changes in their composition, stoichiometry, and/or
the acquisition of new partners.[8b] What remains to be
understood is how these subunit swaps are able to redirect
PRC1 and PRC2 complexes from differentiation to pluripotency
genes, since the structural and molecular data available today do
not inform about DNA sequence specificity of variant PcG
complexes.

6. PcG Proteins Form Dynamic Higher Order Structures

One way in which PcG components regulate expression of their
target genes is via orchestration of their nuclear architecture.

This can involve both silencing as well as predisposition of these
genes for transcriptional activation. The role of 3D genome
regulation in PcG-dependent silencing has been documented in
both flies and mammals, where PcG proteins condense their
target chromatin in a way that seems to prevent its tran-
scription.[44b,47c,76] This condensation might depend on the
formation of extensive long-range chromatin contacts
(Figure 2D).[43b,44c,48,77] These contacts depend at least in part
on the PH protein and its ortholog, PHC1, and are mediated by
oligomerization of their SAM domains (Figure 2C).[44c,49a]
Indeed, super-resolution microscopy analysis of chromatin
interactions identified by the 5C method in mESCs revealed
PRC1-occupied regions that were transcriptionally silenced and
formed nuclear nano-compartments including compact
domains defined by PRC1 binding.[44b] In order to investigate
the dynamics of genome architecture during neural lineage,
Kundu et al.[44b] focused on Hox clusters (Figure 5A), but also on
neural transcription factor *loci*, such as *Nkx2.4*, *Nkx2.2*, *Pax6*,
and *Pax1*. These neural transcription factors are PRC1-bound
and -repressed in ESCs but expressed in NPCs, where they lose
PcG occupancy (Figure 5B). Conversely, the *lgf2bp3* and *Stk31
loci* are expressed in ESCs and are silenced upon neural
differentiation when they gain PcG binding (Figure 5C). As
previously described, the PcG-multi-looped structures are
dynamic and newly active *HOX* genes are progressively released
from these repressive hubs to active ones in tissues where they
are expressed (Figure 5A).[49b,78] While in ESCs long-distance
loops are formed either between the *Nkx2.2* and *Pax1* loci, or
between *Nkx2.2* and *Nkx2.4*, these loops are lost in NPCs,
suggesting that the loss of PRC1 disrupts repressive loops at
neural-specific genes (Figure 5B). Intriguingly, neither the
compact Hox domain formation nor the neural-specific loops
require the catalytic activity of RING1B, whereas the catalytic
activity of EZH2 promotes both types of chromatin compac-
tion.[44b] Therefore, the role of H2AK119Ub in this regulatory
process remains unknown.

Although 5C is powerful when studying individual *loci*, this
approach cannot deliver a genome-wide view. Using high-
resolution Hi-C maps, Bonev et al. have been able to bypass the
technical limits to analyze 3D organization at the smallest scale
and have linked 3D nuclear architecture to normal in vitro and
in vivo physiological function during neural differentiation
genome-wide[46b] in a homogeneous population of synchro-
nized cells. Importantly, major changes are detected between
PcG-regions where many PcG-mediated repressive interac-
tions, that occur between RING1B enriched regions, decrease
sharply during neural differentiation following a decrease in
RING1B targeted sites (Figure 5D), whereas some new PcG-
dependent contacts are detected during differentiation.
Whether these contacts have a regulatory function *per se*, or
whether they are rather a consequence of gene expression
changes remains to be studied, but work in *Drosophila* suggests
that PcG-dependent contacts may play a regulatory role, at least
in some cases.[43b,45]

Importantly, PcG-dependent regulation of chromatin archi-
tecture is not necessarily linked to gene silencing. During mouse
brain development, the *Meis2* gene is regulated in a spatiotem-
poral manner, with early repression followed by activation
during mid embryogenesis. In addition to the expected loss of
<figure><img src="image_7.png" /><figcaption>**Figure 5.** Dynamic PcG regulatory functions in 3D genome organization. A) While the *HoxA* cluster forms a unique homogenous structure (or TAD) in ESCs (top left, in blue), newly activated genes are progressively released from this PcG-dependent repressive hub in NPCs (top right, in green). Their activation correlates with the acquisition of active histone marks (H3K4me3, in green), decreased H3K27me3 (in blue), and decompaction of the locus (Top right). B) The *Nkx2.4*, *Nkx2.2*, *Pax6*, and *Pax1* neural genes lose PRC1 binding at the NPC stage, which correlates with a decompaction of the locus and transcriptional activation. C) Conversely, the *Igf2bp3* and *Stk31* genes acquire new PRC1 binding sites in the course of differentiation, become compacted and transcriptionally repressed. D) Finally, long-range PcG contacts are decreased in NPCs compared to ESCs, an observation that correlates with a decreased binding of RING1B.</figcaption></figure>

silencing, *Ring1B* mutant embryos displayed loss of Meis2 in the normal expression domains. This was shown to depend on failure of a specific enhancer to set up the appropriate promoter contacts upon loss of *Ring1B*. [79] It would be important in future work to study what other genes participate in this phenomenon in order to identify the specific complex involved in this regulation. An additional case of PcG function assisting enhancer activation involves PRC2. In pluripotent ESCs,

thousands of poised enhancers characterized by the presence of H3K4me1, histone acetyltransferase P300 binding and the repressive H3K27me3 mark have been found to establish contacts with their target promoter genes, such as *Lhx5*, *Six3*, *Sox1*, and *Wnt8b* that will be mainly activated later in brain tissues.[80] These enhancer-promoter contacts have been shown to be mediated by PRC2 binding, which provides a permissive topology that facilitates the robust induction of major neural
genes upon differentiation. Whether PRC1 components are involved in this regulation is unknown, but RING1B was shown to induce an extensive network of chromatin contacts in ESCs, including many promoter-enhancer contacts at developmental transcription factor genes that are prevented from inappropriate activation, suggesting that the role of PRC1 complexes in this case might rather be to maintain developmental genes in a poised but repressed state prior to cell differentiation.[48] Together, these data suggest that PcG protein-mediated gene regulation involves a tight regulation of 3D genome architecture. Future work should dissect the molecular mechanisms of the different regulatory responses established by different PcG complexes and disentangle silencing from activating functions.

## 7. PcG Proteins Play a Pleiotropic Role in Tumorigenesis

PcG integrity is crucial to maintain embryonic and adult stem cell homeostasis and identity. It is also important to emphasize that depleting PcG-mediated silencing promotes trans- or de-differentiation in both *Drosophila* and mammals.[81] It is therefore not surprising that PRC2, canonical but also non-canonical PRC1 members are frequently deregulated in cancer.[1,82] They can notably promote tumor progression by supporting cellular self-renewal and proliferation, while blocking differentiation.[83] In this section, we will describe how molecular mechanisms which normally regulate the targeting and/or the function of PcG on chromatin might also underpin their aberrant function in cancer.

The oncogenic role of PcG proteins has been further confirmed by the observation of frequent over-expression of PcG members in human tumors[82c] as well as high levels of H3K27me3 and EZH2 levels that correlate with poor prognosis in prostate tumors.[84] Moreover, the heterozygous substitution of the K27 residue of H3.3 into a non-methylatable methionine has been identified as a driver mutation in pediatric glioblastomas.[85] This mutation has a dominant negative effect and leads to a global decrease of H3K27me3, which nevertheless intensifies on other genes implicated in glioma progression including the *Cdkn2A* locus coding for cell cycle inhibitors (Figure 6A,B).[86] Therefore, redistribution of H3K27me3 could play a dual role both in repressing tumor-suppressor genes and in de-repressing oncogenes (Figure 6B). Nevertheless, the role of PcG proteins in hematological cancers[68,87] has become controversial as inactivating mutations have been identified in PRC2 gene *loci* in tumors, including *Ezh2*, *Suz12*, *Eed*, and *Jarid2*.[82c,88] Thus, the implication of PRC2 activity in tumor growth in vivo is still debated and it has been proposed that the frequent over-expression of PRC2 components is, at least in some cases, a consequence of excessive proliferation and the subsequent requirement for continuous replication.[89] When normalized to the proliferation rate, low EZH2 levels and “hypomorphic” deposition of H3K27me3 are associated with transcriptional instability and to a worse prognosis of breast cancer cases.[89]

On the PRC1 side, BMI1/PCGF4 was first identified as a cooperative oncoprotein in a mouse model of Myc-induced lymphomagenesis.[90] Subsequent studies have suggested that

this oncogenic role relies on PcG-dependent repression of the *Cdkn2a* locus, involved in cell-cycle arrest and senescence (Figure 6A).[82c] However, PcG transformation can be independent of this *Cdkn2a* cell-cycle checkpoint and be triggered by the mis-regulation of different signaling pathways.[91] *Pcgf4* also regulates the epithelial-to-mesenchymal transition via several mechanisms and thus promotes metastasis.[92] *Mel-18/PCgf2* is a close homolog of *Bmi1/PCgf4* that exerts partially redundant functions during embryonic development[93] but is often down-regulated in tumors and was proposed to act as a tumor-suppressor gene.[82c,94] It is interesting to note that PCGF2-PRC1 has lower E3 ligase activity compared to PCGF4-PRC1 in vitro[95] and that, while PCGF4 plays a crucial role in the maintenance of Hematopoietic Stem Cells (HSCs), PCGF2 takes over during the differentiation of the hematopoietic lineage.[96] Finally, the role of *Cbx7* in tumorigenesis is also controversial. This is the main CBX paralog in HSCs, where its over-expression promotes self-renewal and causes leukemia development via the aberrant repression of the *Cdkn2a* locus (Figure 6B).[97] However, independent studies have suggested that *Cbx7* KO mice, which are viable, have increased susceptibility to liver and lung neoplasia.[98]

These discrepancies are now thought to derive from the extreme versatility of PcG proteins, which act in a tissue-stage- and complex-specific manner to regulate a plethora of biological processes.[99] In addition, PcG members may acquire new interactors and functions that are specific and/or exacerbated in cancer, adding another level of complexity. For instance, it has recently been shown that the cPRC1 functionally associates with the Estrogen Receptor and the FOXA1 pioneer factor in breast cancer cells and is recruited at a set of active enhancers decorated with the enhancer-specific H3K4me1 mark, the active H3K27Ac mark and in the absence of the H3K27me3 mark (Figure 6C).[100] Unexpectedly, more genes are downregulated than upregulated in *Ring1B* depleted breast cancer cells – suggesting that *RING1B* could facilitate gene activation – and mis-regulated genes include key genes involved in breast cancer progression and metastasis.[100] In parallel, the authors show that the cPRC1 regulates chromatin accessibility at cancer-specific enhancer sites and suggest that *RING1B* could play a dual role in the repression but also the activation of cognate genes (Figure 6C).[100] Hence, the pleiotropy of PcG function in cancer is likely reflecting its multi-faceted role during normal development.[96b,97a] In the future, it will be crucial to systematically address the dynamics of the PcG interactome during tumorigenesis and its impact on gene regulation and on 3D genome organization.

Finally, another level of PcG functional perturbation by the SSX family of testis-specific proteins has been described in multiple types of human cancers, including melanoma.[101] When ectopically expressed, SSX2 affects the formation of PcG bodies and derepresses PcG targets.[102] In sarcoma, SSX1/2 have been found in fusion with the SYT oncogene, and the SYT-SSX fusion protein co-localizes with PcG nuclear foci and derepresses underlying PcG genes.[103] In synovial carcinoma, the SS18-SSX fusion protein interacts with the PRC1.1, associates with the SWI/SNF chromatin remodeling complex and is targeted by KDM2D to aberrantly activate PcG targets.[104]
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Context-dependent role of PcG proteins in cancer. A) PcG represses the *Cdkn2a* locus via canonical H3K27me3-dependent mechanism. This locus encodes for two cell-cycle inhibitors and plays a key role in controlling cell-cycle arrest and senescence. Under physiological conditions, the *Cdkn2a* locus controls the self-renewal of adult stem cells and plays a key role in maintaining the homeostasis of the tissues. B) Therefore, PcG Gain Of Function (GOF) and increased H3K27me3 deposition triggers aberrant repression and promotes tumorigenesis. Noteworthy, H3.3K27Me mutation also increases H3K27me3 deposition at the *Cdkn2a* locus. Conversely, PcG loss of function (LOF) can also promote tumorigenesis by facilitating aberrant derepression of oncogenes. Of note, these two mechanisms are not mutually exclusive. C) PcG proteins can also acquire new functions in cancer cells, as exemplified by the functional association between cPRC1, the pioneer factor FOXA and Estrogen Receptors alpha in breast cancer cell lines. In this pathological context, the cPRC1 is redeployed at active enhancers decorated with the H3K4me1 and H3K27Ac active marks and controls their openness. Eventually, these new PRC1 sites affect the expression of cognate promoters both positively and negatively.</figcaption></figure>

## 8. Conclusion and Outlook

The diverse functions of ncPRC1 complexes are just starting to be addressed[105] and could revolutionize our understanding of PcG biology. Future research is likely to answer several critical open questions:

1. How are the different PcG complexes targeted? The transition from “closed” to “open” chromatin, and vice versa, is determined primarily by a class of transcription factors called pioneer factors.[106] During neural differentiation, pioneer factors have been proposed to remodel the repertoire of pro-neural factor binding sites at the NPC stage by modifying the epigenetic landscape at their respective target sites.[107] It will be interesting to study whether these or other factors contribute to the relocation of PcG complexes, either by altering chromatin accessibility or via direct protein-protein interactions.

2. What is the role of PcG proteins at active sites? While cPRC1 and ncPRC1s often co-localize at canonical target genes enriched for H3K27me3, ncPRC1 complexes are also found

in the absence of this mark in mESCs.[18a,26a,53c] This suggests that, while H3K27me3 has a strong predictive power for silencing, binding of PRC1 proteins is not restricted to repressed sites and might either dampen transcription or even play context-dependent activating roles. Whether these seemingly contrasting functions depend on the binding of differential complexes, the targeting of histone demethylases or other post-translational modifications, either on histones or on complex components, remains to be determined.

3. What regulatory outputs depend on PcG-mediated chromatin looping? PcG proteins have a conserved function in generating long-range chromatin contacts. In many cases, these correspond to repressed genes, but in other cases 3D architecture can be essential for the establishment of the appropriate gene regulatory patterns, for instance by sustaining enhancer-promoter contacts of poised enhancer before activation in the next stage of the lineage.[79] A detailed investigation of the consequence of stoichiometric changes or switches in PcG subunits on 3D genome organization and gene regulation would likely broaden our
understanding of PcG function in normal physiology and in diseases.

(4) What are the targets of sub-stoichiometric PRC1 complexes? Changes in the composition of PcG complexes seem to regulate their activities during normal development and rearrangements could also participate in the development of diseases. In the future, systematic proteomics and genomic approaches will be crucial to further clarify the repertoire of existing PRC1 complexes and their specific targeting on chromatin during lineage commitment.

(5) What is the precise function of the PRC1-mediated H2AK119Ub mark in gene regulation and in cell differentiation? Indeed, this mark seems dispensable in some processes and essential for others and it remains unclear whether, in addition to the recruitment of the PRC2, there are other roles for this histone modification. The contribution of this PRC1-dependent epigenetic mark might be cell-tissue-context- or timing-dependent and future work should address its role in different differentiation models.

## Acknowledgements

The authors would like to thank Maximilian Fitz-James for reading and correcting this manuscript. Research in the laboratory of G.C. was supported by the CNRS, the University of Montpellier and by grants from the European Research Council (ERC-2008-AdG No 232947), the FP7 European Network of Excellence EpiGeneSys, the European Union's Horizon 2020 research and innovation programme under grant agreement No 676556 (MuG), the Agence Nationale de la Recherche (N. ANR-15-CE12-006-01), the Fondation pour la Recherche Médicale (Grant N. DEI20151234396), the French National Cancer Institute (INCa, Project N. PLBIO16-222), and the Laboratory of Excellence EpiGenMed (N. ANR-10-LABX-12). A.M.M. is supported by a grant of the Fondation ARC pour la Recherche contre le Cancer. V.L. is supported by a doctoral fellowship from the Laboratory of Excellence EpiGenMed and the Fondation ARC pour la Recherche contre le Cancer.

## Conflict of Interest

The authors declare no conflict of interest.

## Keywords

3D genome, chromatin, Polycomb

Received: November 9, 2018
Revised: December 22, 2018
Published online: February 22, 2019

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