Function of Polycomb repressive complexes in stem cells

Abstract

Stem cells are unique cell populations identified in a variety of normal tissues and some cancers. Maintenance of stem cell pools is essential for normal development, tissue homeostasis, and tumorigenesis. Recent studies have revealed that Polycomb repressive complexes (PRCs) play a central role in maintaining stem cells by repressing cellular senescence and differentiation. Here, we will review recent findings on dynamic composition of PRC complexes and sub-complexes, how PRCs are recruited to chromatin, and their functional roles in maintaining self-renewal of stem cells. Furthermore, we will discuss how PRCs, CpG islands (CGIs), the INK4A/ARF/INK4B locus, and developmental genes form a hierarchical regulatory axis that is utilized by a variety of stem cells to maintain their self-renewal and identities.

Keywords

Polycomb repressive complexes / gene silencing / CpG islands / stem cells / self-renewal

Introduction

Stem cells are unique cell populations that are capable of self-renewing and differentiating into multiple lineages of progeny cells. Maintenance of stem cells in the body is important for normal embryo development and tissue homeostasis. On the other hand, persistence of stem cells in some tumors results in cancer progression and relapse. Multiple lines of research evidence suggest that the “stemness” properties of stem cells are determined by the unique epigenetic landscape created by stem cell-specific transcription factors and epigenetic modulators. Among these factors, Polycomb repressive complexes (PRCs) are found to play a central role in stem cell maintenance by repressing both cellular senescence and lineage differentiation. In recent years, studies have further identified multiple new PRC variants, elucidated how PRCs are recruited to chromatin, and examined the function of PRCs in various stem cells. These findings provide new insight into the functional role of PRCs in stem cell maintenance and its underlying molecular mechanisms. In this review, we will discuss recent research advances on: (1) dynamic composition of PRCs and their biochemical properties; (2) mechanisms by which PRCs are recruited to chromatin; (3) PRC-mediated epigenetic regulation and its function in stem cell maintenance.

Polycomb repressive complexes

Polycomb-group (PcG) genes were initially discovered in Drosophila melanogaster ( Lewis, 1978). Phenotypic analysis of flies with various PcG gene mutations suggested that PcG proteins are essential for maintaining the proper body segmentation by controlling the expression of homeotic genes during fly embryogenesis ( Nusslein-Volhard et al., 1985; Gaytan de Ayala Alonso et al., 2007). Although the sequences of PcG genes are diverged significantly, the main function of PcG proteins in maintaining gene silencing remains conserved in fly and mammals.

In cells PcG proteins assemble into two major chromatin-modifying complexes, named Polycomb repressive complex 1 (PRC1) and 2 (PRC2), which have distinct components and biochemical functions. In mammalian cells, the core components of PRC1 include RING1A/1B and Polycomb group ring finger proteins (PCGFs), whereas PRC2 core components are composed of enhancer of zester homolog 2 (EZH2) or EZH1, embryonic ectoderm development (EED), and suppressor of zeste 12 (SUZ12). Recent studies revealed that PRC complexes form a variety of sub-complexes through association of different subunits or binding partners. For instances, several studies reported that PRC1 could be divided into canonical forms (cPRC1) that have CBX proteins associated with the core components, and non-canonical forms (ncPRC1) in which the catalytic components are associated with RYBP or YAF2 ( Gao et al., 2012; Luis et al., 2012; Tavares et al., 2012). Likewise, the core components of PRC2 associate with various proteins, such as JARID2, PCL1-3, and AEBP2, to form different PRC2 sub-complexes ( Kim et al., 2009; Pasini et al., 2010; Walker et al., 2010; Ballare et al., 2012; Cai et al., 2013) (Fig. 1).

Biochemically, PRC1 and PRC2 catalyze covalent modifications at different lysine residues of histones. Specifically, RING1B in PRC1 mediates H2AK119 mono-ubiquitylation (H2AK119u1) by its E3 ligase activity ( de Napoles et al., 2004; Wang et al., 2004a), whereas PRC2 catalyzes di-, and tri-methylation of H3K27 (H3K27me2/3) by the methyltransferase activity of EZH2 ( Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Muller et al., 2002). Recently an analysis of high-resolution crystal structure of PRC2 complex from the yeast Chaetomium thermophilum revealed that the catalytic activity of EZH2 is activated by its stimulation-responsive motif (SRM) bound with H2K27me3, thus facilitates the methylation of nearby unmethylated H3K27 substrates and propagates the H3K27me3 maker to neighboring regions ( Jiao and Liu, 2015).

Functionally, PRCs are involved in transcriptional silencing. Although H3K27me3 is a well-known histone marker associated with gene silencing, it remains unclear whether this modification causes transcriptional repression directly or through recruiting other repressive factors. Since H3K27me3 recruits cPRC1 through its interaction with the CBX proteins, it is highly possible that cPRC1 works with PRC2 synergistically to repress transcription at H3K27me3 sites ( Fischle et al., 2003; Min et al., 2003). Two mechanisms, catalytic activity dependent and independent, have been found to be involved in the PRC1-mediated transcriptional silencing ( Eskeland et al., 2010a; Endoh et al., 2012). In vitro assays showed that PRC1 induces the compaction of nucleosomal arrays, which is mediated by the charge interaction and independent on its E3 ligase activity ( Grau et al., 2010). The PRC1 catalytic activity and H2AK119u1 are also found to be dispensable for the target binding and chromatin compaction at Hox gene loci in mouse embryonic stem cells (ESCs), but indispensable for efficient repression of target genes ( Eskeland et al., 2010b; Endoh et al., 2012). Although the primary function of PRCs in transcriptional silencing is well established by numerous biochemical and genetic studies, recent studies revealed that PRC2 and a variant PRC1 activate gene expression in neural cells and cancers ( Xu et al., 2012; Gao et al., 2014). Therefore, it appears that PRCs could have dual functions in regulating gene expression under different cellular contexts.

Recruitment of Polycomb repressive complexes

Although the biochemical properties of PRCs are well characterized, the underlying mechanisms by which PRCs are recruited to their targets in cells remain to be fully elucidated. In recent years the advances of next-generation sequencing technologies enables us to examine the genome-wide PRC occupancy, histone modifications, and gene expression, which has shed new light in our understanding on PRC recruitment and their function in regulating gene expression in cells.

Since no distinct DNA binding motifs have been identified in the components of PRCs, sequence-specific transcriptional factors (TFs) such as YY1, REST, and RUNX1 have been postulated to mediate the recruitment of PRCs through direct interaction in different cells ( Woo et al., 2010; Ren and Kerppola, 2011; Dietrich et al., 2012; Yu et al., 2012). In addition to TFs, non-coding RNAs (ncRNAs) are also found to mediate the PRC recruitment. For example, HOX transcript antisense RNA (HOTAIR) is reported to recruit PRC2 to the HOXD loci in human cells ( Rinn et al., 2007). The most extensively studied ncRNA is XIST whose expression is required for PRC2 targeting to the inactive X chromosome ( Zhao et al., 2008). However, although TF- and ncRNA-mediated PRC recruitment accounts for the PRC occupancy at individual genomic locations or in specific cells, it is difficult to explain the genome-wide colocalization of PRCs with CpG islands (CGIs), a common PRC binding pattern observed in a variety of cells ( Mikkelsen et al., 2007; Ku et al., 2008).

In Drosophila, PRCs binds to the cis-elements termed Polycomb response elements (PREs) ( Chan et al., 1994; Poux et al., 2001; Mohd-Sarip et al., 2002; Mohd-Sarip et al., 2005). However, a definitive PRE in mammalian cells remains elusive. Instead, genome-wide chromatin immuno-precipitation coupled with sequencing (ChIP-Seq) analyses revealed that PRC binding sites are highly overlapped with CGIs in mammalian cells ( Ku et al., 2008). Additionally, a piece of CpG-rich DNA inserted into the moue ESC genome is sufficient to recruit PRC2 to the exogenous DNA site, further suggesting that CGIs function as surrogate PREs and are sufficient to initiate the PRC recruitment in mammalian cells ( Mendenhall et al., 2010). CGIs are identified as short stretches of DNA sequences with rich GC content and higher frequency of CpG dinucleotides in vertebrate genomes. Typically, CGIs are resistant to CpG DNA methylation and form local unmethylated regions that are embedded in a highly methylated genome background in vertebrates ( Deaton and Bird, 2011). The unique sequence feature and DNA methylation status at CGIs are recognized and bound specifically by a family of proteins containing CxxC zinc finger (CxxC-ZF) domains ( Long et al., 2013). Notably, majority of CxxC-ZF domain-containing proteins, such as mix lineage leukemia protein 1/2 (MLL1/2), histone H3 lysine 36 demethylase 2A/2B (KDM2A/2B), DNA methyltransferase 1 (DNTM1) and methylcytosine dioxygenase TET1, contain known chromatin modifying activities, suggesting that the initial chromatin structure at CGIs could be set up without the involvement of sequence-specific TFs or ncRNAs but by the CGI binding proteins only.

In line with this concept, recently several studies demonstrated that one of CxxC-ZF containing proteins, histone lysine demethylase 2B (KDM2B), associates with a ncPRC1 variant (PRC1.1) and recruits the complex to most CGIs in mammalian cells. Depletion of KDM2B largely reduces the RING1B occupancy at CGIs, further suggesting the KDM2B plays a major role in recruiting PRC1 to CGIs ( Farcas et al., 2012; He et al., 2013; Wu et al., 2013). Similarly, it has been reported that PRC2 component JARID2 preferentially binds to CG-rich sequences ( Li et al., 2010). Interestingly, Robert Kloseʼ’s laboratory recently reported that targeting KDM2B to chromatin de novo recruits both the variant PRC1 complex and the PRC2 complex, suggesting the binding of KDM2B to DNA could be the first step to initiate the recruitment for both PRC1 and PRC2 ( Blackledge et al., 2014). The finding of PRC1-dependent PRC2 recruitment is surprising since it is different from the long-term held view that the PRC1 recruitment depends on initial PRC2 targeting to chromatin and depositing H3K27me3 for the binding of CBX components in PRC1( Min et al., 2003; Wang et al., 2004b). Since PRC2 occupancy at CGIs is not completely lost in the KDM2B-depleted cells ( Farcas et al., 2012; He et al., 2013; Wu et al., 2013), it is likely that both KDM2B-dependent and independent mechanisms are involved in the recruitment of PRC2 to CGIs in cells (Fig. 2).

The occupancy of PRCs at CGIs is not static but dynamically changes in response to local transcriptional activities. Although KDM2B binds to CGIs associated with both active and inactive promoters, PRC1 largely occupies at transcriptionally inactive promoters, suggesting local strong transcriptional activity is sufficient to disrupt the binding of PRC1 with KDM2B and removes PRC1 from chromatin ( He et al., 2013). This is consistent with the observation that inhibition of transcription in mouse ESCs is sufficient to induce genome-wide ectopic PRC2 recruitment to the CGIs associated with transcriptionally repressed promoters ( Riising et al., 2014). All these observations favor a CGI-based PRC recruitment model proposing that the chromatin configuration at transcriptionally inactive CGIs is initially set up by the CGI binding proteins and their associated PRCs. Typically, it is marked by H3K4me3 and H3K27me3, a bivalent domain modified by MLL1/2 and PRC2 complexes. Once local transcription is activated by sequence-specific transcriptional factors, PRCs are removed from the CGIs and bivalent domains resolve to monovalent domains marked by H3K4me3 only (Fig. 2). This model also implicates that gene silencing is more likely to be resulted from absence of transcription factors or weak transcriptional activity but not from the direct repression by PRCs, while PRCs are important for maintaining gene silencing by increasing the transcriptional threshold. Overall, transcription factors and transcriptional activity are the dominant forces to determine PRC binding, gene expression, and cell fate.

Function of Polycomb repressive complexes in stem cell maintenance

PRCs were originally regarded as master epigenetic regulators in establishing cell fates and locking cell identity in multi-cellular organisms. In recent years numerous studies have demonstrated PRCs also play crucial roles in stem cell maintenance, lineage specification, and caner development. In this review, we will primarily focus on the function of PRCs in maintaining normal and cancer stem cells through examining the regulatory axis formed by PRCs, CGIs, and Polycomb target genes.

Embryonic stem cells (ESCs) are derived from epiblasts of preimplantation embryos. Under the LIF- or FGF2-dependent culture conditions, mouse or human ESCs self-renew indefinitely and maintain their pluripotency in vitro. After re-introduced into blastocytes, pluripotent mouse ESCs are able to develop into all cell lineages ( Evans and Kaufman, 1981; Martin, 1981). Tissue stem cells (TSCs) are more developmentally committed cells identified in various developed organs and serve as the cell sources to maintain tissue homeostasis after birth. Cancer stem cells (CSCs) are isolated from some leukemias and solid tumors in which a normal developmental hierarchy is still or partially preserved. Similar to the function of TSCs in normal tissue regeneration, CSCs promote tumor growth through self-renewal and generation of massive nontumorigenic cancer cells ( Kreso and Dick, 2014). Although ESCs, TSCs, and CSCs are very different in terms of cellular origins, developmental status, and functions, they all acquire a self-renewing capability to maintain the stem cell pool. Self-renewal is an unique process for stem cells to reproduce themselves, in which not only mother cells continuously divide to generate daughter cells, but also daughter cells maintain the same stem cell identity by repressing lineage differentiation. At the molecular level, PRCs are involved in regulating both critical cell cycle regulatory genes and lineage-specific genes. As such, PRC-mediated gene silencing emerges as a key epigenetic mechanism in maintaining stem cell self-renewal.

Function of PRCs in stem cell proliferation

The INK4A/ARF/INK4B locus encodes three tumor suppressors including p16^INK4A, p14^ARF (p19^Arf in mouse), and p15^INK4B in human cells. p16^INK4A and p15^INK4B inhibit the phosphorylation of Rb family proteins by blocking the cyclin D-dependent kinase 4/6. Overexpression of p16^INK4A and p15^INK4B blocks the cell cycle at G1-S phase transition. p14^ARF is found to induce cell cycle arrest and apoptosis by activating the p53-p21^CIP1 pathway ( Kim and Sharpless, 2006). Therefore, the INK4A/ARF/INK4B are located on the top of both pRb and p53 regulatory pathways and play a central role in regulating cell proliferation, cellular senescence, and cancer development.

The INK4A/ARF/INK4B genes have the typical CGI-associated promoters that are targeted by PRCs in normal TSCs. Deletion of Bmi1, a core component of PRC1, impairs the self-renewal of adult hematopoietic stem cells (HSCs) and causes a postnatal bone marrow failure in a mouse model. At the molecular level, both p16^Ink4a and p19^Arf are de-repressed in the Bmi1-depeleted bone marrow ( Park et al., 2003). Similarly, Bmi1is required for maintaining the self-renewal of adult neural stem cells (NSCs). Knockout of Bmi1 de-represses p16^Ink4a and leads to a progressive depletion of NSCs ( Molofsky et al., 2003; Molofsky et al., 2005). Consistent with the function of KDM2B in recruiting PRC1 to CGIs, deletion of Kdm2b in mouse embryonic fibroblasts induces the expression of Ink4-Arf genes and causes premature cellular senescence ( He et al., 2008; Pfau et al., 2008). Therefore, PRC1, KDM2B, INK4A/ARF/INK4B genes and their promoter-associated CGIs form a conserved pathway to regulate cell proliferation in a variety of TSCs. Of note, individual PRC sub-complex has been found to play distinct roles in maintaining TSC self-renewal under different developmental or species-specific contexts. For instance, Cbx7 is found to play a dominant role in maintaining murine HSC self-renewal, whereas in human HSCs it is regulated by the CBX2-containg PRC1( Klauke et al., 2013; van den Boom et al., 2013). Similarly, the self-renewal of murine fetal and adult HSCs are found to be regulated by the Ezh2-or Ezh1-containing PRC2 complex respectively ( Mochizuki-Kashio et al., 2011; Hidalgo et al., 2012).

The cell cycle of ESCs is not subjected to the INK4-mediated regulation since the RB family proteins are constitutively phosphorylated in G1 phase and the pRB pathway is functionally inactive in ESCs. Therefore, repression of INK4/ARF locus by PRCs is not essential for the proliferation of ESCs. This is consistent with the observation that PRC2-depleted mouse ESCs proliferate normally as wild-type cells ( Riising et al., 2014). However, the INK4/ARF-induced cellular senescence and apoptosis are found to be a major roadblock for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) ( Utikal et al., 2009). Consistently, depletion of core components of either PRC1 or PRC2 largely reduces the somatic cell reprogramming efficiency, while overexpression of PRC2 components facilitates the reprogramming process ( Zhang et al., 2011; Onder et al., 2012). Interestingly, overexpression of the PRC1 recruiting factor KDM2B also promote somatic cell reprogramming although its underlying mechanism is independent of its role in antagonizing cellular senescence ( Liang et al., 2012).

Both PRC1 and PRC2 complexes are found to be important in maintaining the self-renewal of cancer stem cells. For instances, loss of Bmi1 or Kdm2b in the Hoxa9/MeisI-induced acute myeloid leukemia cells leads to the aberrant expression of Ink4/Arf genes and impairs the self-renewal of leukemia stem cells (LSCs) ( Lessard and Sauvageau, 2003; He et al., 2011). Deletion of Bmi1 in murine bronchiolalveolar stem cells (BASCs) compromises their self-renewing capability and abrogates the K-ras-initiated lung cancer development, which is partially due to the de-repression of p19^Arf in BASCs ( Ueda et al., 2014). Similarly, either deletion of PRC2 core components or inhibition of PRC2 enzymatic activity by small molecules blocks the CSC self-renewal in a variety of MLL rearranged leukemias and solid tumors ( Ueda et al., 2014; Xu et al., 2015). Although these results are encouraging for the development of therapeutic approaches to block CSC self-renewal by targeting Polycomb complexes, it is worth to note that in some mouse cancer models the critical PRC downstream effectors, INK4A/INK4B-pRB and ARF-p53 pathways, remain intact and are de-repressed upon PRC loss. However, these pathways are frequently mutated, silenced by DNA methylation, or deleted in human cancers. Therefore, human cancers with various genetic background could have very different responses to PRC deletion or inhibition.

Function of PRCs in repressing lineage differentiation

In mammalian cells CpG islands are normally co-localized with the promoters of virtually all constitutively expressed genes as well as 40% tissue-specific and developmental genes ( Deaton and Bird, 2011). Therefore, the chromatin configuration at developmental gene promoters is dynamically modified by PcG-group proteins, trithorax-group proteins, and transcriptional factors during differentiation. The primary function of PRCs in stem cells is to maintain the silencing of lineage-specific genes by increasing the gene activation threshold, which is crucial for both stem cell maintenance and normal lineage specification.

As mentioned earlier, MLL1/2 and PRC2 are recruited and deposit H3K4me3 and H3K27me3 markers to form bivalent domains at transcriptionally inactive CGI promoters. In mouse ESCs bivalent domains were initially identified to locate at approximate 22% of high CpG promoters, some of which regulate the expression of key developmental genes ( Mikkelsen et al., 2007). During lineage differentiation, the activation of developmental genes is associated with PRC removal from promoters as well as resolution of bivalent domains to H3K4me3-marked monovalent domains ( Bernstein et al., 2006). These results suggest the bivalent domains at developmental gene promoters create a local chromatin environment to maintain gene silencing in mouse ESCs but also allow a rapid gene activation upon differentiation stimuli. Although PRCs were originally regarded to repress transcription directly, recently Kristian Helinʼ’s laboratory reported that loss of Suz12 in mouse ESCs does not de-repress the developmental genes in the “2i” culture medium containing GSK3 and MEK inhibitors ( Riising et al., 2014). Of note, “2i” medium maintains mouse ESCs in a primitive state by blocking non-specific differentiation signals from extracellular environments ( Ying et al., 2008). These results suggest that the silencing of developmental genes in mouse ESCs is caused by absence of differentiation signals but not by the Polycomb-mediated transcriptional repression. On the other hand, PRCs is import for maintaining the differentiation gene silencing by increasing gene activation threshold. In the absence of PRCs, the transcriptional threshold becomes shallow and differentiation genes are easily activated by non-specific and weak transcriptional signals received from the environment. This is consistent with the observations that Suz12- and Ring1b-null mouse ESCs incline to express differentiation genes under the serum-containing ESC culture conditions ( Pasini et al., 2007; van der Stoop et al., 2008). Similarly, depletion of Kdm2b in mouse ESCs causes a leaky expression of multiple primitive endodermal genes ( He et al., 2013). The high transcriptional threshold imposed by PRCs becomes more critical for lineage specification since this provides an epigenetic barrier to prevent non-specific gene activation during lineage differentiation. Consistent with this concept, it was found that although various PRC2 mutant mouse ESC lines are still able to express proper neural lineage-specific genes after directed differentiation into spinal motor neurons, they also aberrantly express promiscuous genes specific for other lineages ( Thornton et al., 2014).

Bivalent domains are also identified in hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) ( Cui et al., 2009; Abraham et al., 2013). Similar to ESCs, the activation of lineage-specific genes in committed hematopoietic lineages is associated with the resolution of bivalent domains to H3K4me3-marked monovalent domains, suggesting HSCs and ESCs share a common PRC-mediated mechanism in silencing lineage-specific genes at the molecular level. Although de-repressed INK4A-induced HSC senescence and bone marrow failure are the dominant phenotypes, upregulated differentiation genes and impaired B cell development are also observed in the Ezh1-knockout mice ( Hidalgo et al., 2012). Similarly, Eed-null murine HSCs are defective in both self-renewal and lineage differentiation. Deletion of Ink4a in the Eed-null HSCs enhances the HSC survival but fails to restore normal HSC functions in vivo, suggesting that Ink4a-independent pathways are also involved in the PRC2-mediated HSC maintenance ( Xie et al., 2014). In addition to HSCs, PRCs are also involved in repressing lineage differentiation of other TSCs. For instances, deletion of Ring1b in neural progenitors cells results in both defective self-renewal and premature neuronal differentiation in vitro ( Roman-Trufero et al., 2009). Consistently, in vivo deletion of Ezh2 in cortical neural progenitor cells before the early neurogenic stage accelerates neuronal differentiation and exhausts the neural progenitor pools, suggesting PRC2 is critical for maintaining neural progenitor cells in vivo by keeping the balance of self-renewal and differentiation ( Pereira et al., 2010).

Block of differentiation is a hallmark of CSCs. Similar to their function in normal TSCs, PRCs are also found to repress lineage differentiation of CSCs. Loss of Bmi1 in the MLL-AF9-induced acute myeloid leukemia largely reduces the leukmogenic capability of LSCs, concomitantly de-represses Ink4a/Arf genes and lineage differentiation genes. Overexpression of myeloid lineage-specific transcriptional factor TBX15 reduces the self-renewal of Ink4a/Arf-null LSCs, suggesting that repression of both Ink4a/Arf and lineage differentiation genes by PRC1 is required for the self-renewal of LSCs ( Yuan et al., 2011). Consistently, inhibition of EZH2 and EZH1 methyltransferase activities by small molecules in a variety of human MLL rearranged leukemia cells induces the de-repression of lineage differentiation genes and reduces their tumorigenecity in vitro ( Xu et al., 2015).

Concluding remarks

The function of PRCs in maintaining stem cells relies on a regulatory axis formed by PRCs, CGI-associated promoters, the INK4A/ARF/INK4B locus, and key lineage developmental genes in cells. At the individual CGI level, the local chromatin structure is dynamically modified by the CGI binding proteins, their associated PRCs, and transcriptional activities. The primary function of PRCs at CGI-promoters is to increase the local transcriptional threshold, which is important for preventing aberrant gene activation during lineage differentiation. At the genome-wide level, the promoters of key cell cycle regulatory genes and lineage developmental genes are associated with CGIs and their expression is subject to the general PRC-mediated transcriptional regulation. Therefore, proliferative defect and aberrant differentiation become the major phenotypes observed in PRC-depleted stem cells (Fig. 3). Although this regulatory mechanism plays a dominant role in stem cell maintenance, recent studies also identified multiple PRC variants that have distinct genomic location and regulate different sets of genes in cells. Further investigation on these PRC variants under different cellular and developmental contexts will largely broaden our knowledge on the function of PRCs in regulating gene expression during development and other biological processes.

References

[1] Abraham B J, Cui K, Tang Q, Zhao K (2013). Dynamic regulation of epigenomic landscapes during hematopoiesis. BMC Genomics, 14(1): 193

[2] Ballare C, Lange M, Lapinaite A, Martin G M, Morey L, Pascual G, Liefke R, Simon B, Shi Y, Gozani O, Carlomagno T, Benitah S A, Di Croce L (2012). Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity. Nat Struct Mol Biol, 19(12): 1257–1265

[3] Bernstein B E, Mikkelsen T S, Xie X, Kamal M, Huebert D J, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber S L, Lander E S (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125(2): 315–326

[4] Blackledge N P, Farcas A M, Kondo T, King H W, McGouran J F, Hanssen L L, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long H K, Sheahan T W, Brockdorff N, Kessler B M, Koseki H, Klose R J (2014). Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell, 157(6): 1445–1459

[5] Cai L, Rothbart S B, Lu R, Xu B, Chen W Y, Tripathy A, Rockowitz S, Zheng D, Patel D J, Allis C D, Strahl B D, Song J, Wang G G (2013). An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting. Mol Cell, 49(3): 571–582

[6] Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones R S, Zhang Y (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science, 298(5595): 1039–1043

[7] Chan C S, Rastelli L, Pirrotta V (1994). A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. EMBO J, 13: 2553–2564

[8] Cui K, Zang C, Roh T Y, Schones D E, Childs R W, Peng W, Zhao K (2009). Chromatin signatures inmultipotent humanhematopoietic stem cells indicatethefate of bivalent genes during differentiation. Cell Stem Cell, 4(1): 80–93

[9] Czermin B, Melfi R, McCabe D, Seitz V, Imhof A, Pirrotta V (2002). Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell, 111(2): 185–196

[10] de Napoles M, Mermoud J E, Wakao R, Tang Y A, Endoh M, Appanah R, Nesterova T B, Silva J, Otte A P, Vidal M, Koseki H, Brockdorff N (2004). Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation. Dev Cell, 7(5): 663–676

[11] Deaton A M, Bird A (2011). CpG islands and the regulation of transcription. Genes Dev, 25(10): 1010–1022

[12] Dietrich N, Lerdrup M, Landt E, Agrawal-Singh S, Bak M, Tommerup N, Rappsilber J, Sodersten E, Hansen K (2012). REST-mediated recruitment of polycomb repressor complexes in mammalian cells. PLoS Genet, 8(3): e1002494

[13] Endoh M, Endo T A, Endoh T, Isono K, Sharif J, Ohara O, Toyoda T, Ito T, Eskeland R, Bickmore W A, Vidal M, Bernstein B E, Koseki H (2012). Histone H2A mono-ubiquitination is a crucial step to mediate PRC1-dependent repression of developmental genes to maintain ES cell identity. PLoS Genet, 8(7): e1002774

[14] Eskeland R, Freyer E, Leeb M, Wutz A, Bickmore W A (2010a). Histone acetylation and the maintenance of chromatin compaction by Polycomb repressive complexes. Cold Spring Harb Symp Quant Biol, 75(0): 71–78

[15] Eskeland R, Leeb M, Grimes G R, Kress C, Boyle S, Sproul D, Gilbert N, Fan Y, Skoultchi A I, Wutz A, Bickmore W A (2010b). Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination. Mol Cell, 38(3): 452–464

[16] Evans M J, Kaufman M H (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature, 292(5819): 154–156

[17] Farcas A M, Blackledge N P, Sudbery I, Long H K, McGouran J F, Rose N R, Lee S, Sims D, Cerase A, Sheahan T W, Koseki H, Brockdorff N, Ponting C P, Kessler B M, Klose R J (2012). KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. eLife, 1: e00205

[18] Fischle W, Wang Y, Jacobs S A, Kim Y, Allis C D, Khorasanizadeh S (2003). Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev, 17(15): 1870–1881

[19] Gao Z, Lee P, Stafford J M, von Schimmelmann M, Schaefer A, Reinberg D (2014). An AUTS2-Polycomb complex activates gene expression in the CNS. Nature, 516(7531): 349–354

[20] Gao Z, Zhang J, Bonasio R, Strino F, Sawai A, Parisi F, Kluger Y, Reinberg D (2012). PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes. Mol Cell, 45(3): 344–356

[21] Gaytan de Ayala Alonso A, Gutierrez L, Fritsch C, Papp B, Beuchle D, Muller J (2007). A genetic screen identifies novel polycomb group genes in <?Pub Caret?>Drosophila. Genetics, 176(4): 2099–2108

[22] Grau D J, Antao J M, Kingston R E (2010). Functional dissection of Polycomb repressive complex 1 reveals the importance of a charged domain. Cold Spring Harb Symp Quant Biol, 75(0): 61–70

[23] He J, Kallin E M, Tsukada Y, Zhang Y (2008). The H3K36 demethylase Jhdm1b/Kdm2b regulates cell proliferation and senescence through p15(Ink4b). Nat Struct Mol Biol, 15(11): 1169–1175

[24] He J, Nguyen A T, Zhang Y (2011). KDM2b/JHDM1b, an H3K36me2-specific demethylase, is required for initiation and maintenance of acute myeloid leukemia. Blood, 117(14): 3869–3880

[25] He J, Shen L, Wan M, Taranova O, Wu H, Zhang Y (2013). Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat Cell Biol, 15(4): 373–384

[26] Hidalgo I, Herrera-Merchan A, Ligos J M, Carramolino L, Nunez J, Martinez F, Dominguez O, Torres M, Gonzalez S (2012). Ezh1 is required for hematopoietic stem cell maintenance and prevents senescence-like cell cycle arrest. Cell Stem Cell, 11(5): 649–662

[27] Jiao L, Liu X (2015). Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2. Science, 350(6258): aac4383

[28] Kim H, Kang K, Kim J (2009). AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2. Nucleic Acids Res, 37(9): 2940–2950

[29] Kim W Y, Sharpless N E (2006). The regulation of INK4/ARF in cancer and aging. Cell, 127(2): 265–275

[30] Klauke K, Radulovic V, Broekhuis M, Weersing E, Zwart E, Olthof S, Ritsema M, Bruggeman S, Wu X, Helin K, Bystrykh L, de Haan G (2013). Polycomb Cbx family members mediate the balance between haematopoietic stem cell self-renewal and differentiation. Nat Cell Biol, 15(4): 353–362

[31] Kreso A, Dick J E (2014). Evolution of the cancer stem cell model. Cell Stem Cell, 14(3): 275–291

[32] Ku M, Koche R P, Rheinbay E, Mendenhall E M, Endoh M, Mikkelsen T S, Presser A, Nusbaum C, Xie X, Chi A S, Adli M, Kasif S, Ptaszek L M, Cowan C A, Lander E S, Koseki H, Bernstein B E (2008). Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet, 4(10): e1000242

[33] Kuzmichev A, Nishioka K, Erdjument-Bromage H, Tempst P, Reinberg D (2002). Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein. Genes Dev, 16(22): 2893–2905

[34] Lessard J, Sauvageau G (2003). Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature, 423(6937): 255–260

[35] Lewis E B (1978). A gene complex controlling segmentation in Drosophila. Nature, 276(5688): 565–570

[36] Li G, Margueron R, Ku M, Chambon P, Bernstein B E, Reinberg D (2010). Jarid2 and PRC2, partners in regulating gene expression. Genes Dev, 24(4): 368–380

[37] Liang G, He J, Zhang Y (2012). Kdm2b promotes induced pluripotent stem cell generation by facilitating gene activation early in reprogramming. Nat Cell Biol, 14(5): 457–466

[38] Long H K, Blackledge N P, Klose R J (2013). ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection. Biochem Soc Trans, 41(3): 727–740

[39] Luis N M, Morey L, Di Croce L, Benitah S A (2012). Polycomb in stem cells: PRC1 branches out. Cell Stem Cell, 11(1): 16–21

[40] Martin G R (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA, 78(12): 7634–7638

[41] Mendenhall E M, Koche R P, Truong T, Zhou V W, Issac B, Chi A S, Ku M, Bernstein B E (2010). GC-rich sequence elements recruit PRC2 in mammalian ES cells. PLoS Genet, 6(12): e1001244

[42] Mikkelsen T S, Ku M, Jaffe D B, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim T K, Koche R P, Lee W, Mendenhall E, O’Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander E S, Bernstein B E (2007). Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature, 448(7153): 553–560

[43] Min J, Zhang Y, Xu R M (2003). Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev, 17(15): 1823–1828

[44] Mochizuki-Kashio M, Mishima Y, Miyagi S, Negishi M, Saraya A, Konuma T, Shinga J, Koseki H, Iwama A (2011). Dependency on the polycomb gene Ezh2 distinguishes fetal from adult hematopoietic stem cells. Blood, 118(25): 6553–6561

[45] Mohd-Sarip A, Cleard F, Mishra R K, Karch F, Verrijzer C P (2005). Synergistic recognition of an epigenetic DNA element by Pleiohomeotic and a Polycomb core complex. Genes Dev, 19(15): 1755–1760

[46] Mohd-Sarip A, Venturini F, Chalkley G E, Verrijzer C P (2002). Pleiohomeotic can link polycomb to DNA and mediate transcriptional repression. Mol Cell Biol, 22(21): 7473–7483

[47] Molofsky A V, He S, Bydon M, Morrison S J, Pardal R (2005). Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev, 19(12): 1432–1437

[48] Molofsky A V, Pardal R, Iwashita T, Park I K, Clarke M F, Morrison S J (2003). Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature, 425(6961): 962–967

[49] Muller J, Hart C M, Francis N J, Vargas M L, Sengupta A, Wild B, Miller E L, O'Connor M B, Kingston R E, Simon J A (2002). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell, 111(2): 197–208

[50] Nusslein-Volhard C, Kluding H, Jurgens G (1985). Genes affecting the segmental subdivision of the Drosophila embryo. Cold Spring Harb Symp Quant Biol, 50(0): 145–154

[51] Onder T T, Kara N, Cherry A, Sinha A U, Zhu N, Bernt K M, Cahan P, Marcarci B O, Unternaehrer J, Gupta P B, Lander E S, Armstrong S A, Daley G Q (2012). Chromatin-modifying enzymes as modulators of reprogramming. Nature, 483(7391): 598–602

[52] Park I K, Qian D, Kiel M, Becker M W, Pihalja M, Weissman I L, Morrison S J, Clarke M F (2003). Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature, 423(6937): 302–305

[53] Pasini D, Bracken A P, Hansen J B, Capillo M, Helin K (2007). The polycomb group protein Suz12 is required for embryonic stemcell differentiation. Mol Cell Biol, 27(10): 3769–3779

[54] Pasini D, Cloos P A, Walfridsson J, Olsson L, Bukowski J P, Johansen J V, Bak M, Tommerup N, Rappsilber J, Helin K (2010). JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells. Nature, 464(7286): 306–310

[55] Pereira J D, Sansom S N, Smith J, Dobenecker M W, Tarakhovsky A, Livesey F J (2010). Ezh2, the histone methyltransferase of PRC2, regulates the balance between self-renewal and differentiation in the cerebral cortex. Proc Natl Acad Sci USA, 107(36): 15957–15962

[56] Pfau R, Tzatsos A, Kampranis S C, Serebrennikova O B, Bear S E, Tsichlis P N (2008). Members of a family of JmjC domain-containing oncoproteins immortalize embryonic fibroblasts via aJmjC domain-dependent process. Proc Natl Acad Sci USA, 105(6): 1907–1912

[57] Poux S, Melfi R, Pirrotta V (2001). Establishment of Polycomb silencing requires a transient interaction between PC and ESC. Genes Dev, 15(19): 2509–2514

[58] Ren X, Kerppola T K (2011). REST interacts with Cbx proteins and regulates polycomb repressive complex 1 occupancy at RE1 elements. Mol Cell Biol, 31(10): 2100–2110

[59] Riising E M, Comet I, Leblanc B, Wu X, Johansen J V, Helin K (2014). Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol Cell, 55(3): 347–360

[60] Rinn J L, Kertesz M, Wang J K, Squazzo S L, Xu X, Brugmann S A, Goodnough L H, Helms J A, Farnham P J, Segal E, Chang H Y (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell, 129(7): 1311–1323

[61] Roman-Trufero M, Mendez-Gomez H R, Perez C, Hijikata A, Fujimura Y, Endo T, Koseki H, Vicario-Abejon C, Vidal M (2009). Maintenance of undifferentiated state and self-renewal of embryonic neural stem cells by Polycomb protein Ring1B. Stem Cells, 27(7): 15591570

[62] Tavares L, Dimitrova E, Oxley D, Webster J, Poot R, Demmers J, Bezstarosti K, Taylor S, Ura H, Koide H, Wutz A, Vidal M, Elderkin S, Brockdorff N (2012). RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3. Cell, 148(4): 664–678

[63] Thornton S R, Butty V L, Levine S S, Boyer L A (2014). Polycomb Repressive Complex 2 regulates lineage fidelity during embryonic stem cell differentiation. PLoS ONE, 9(10): e110498

[64] Ueda K, Yoshimi A, Kagoya Y, Nishikawa S, Marquez V E, Nakagawa M, Kurokawa M (2014). Inhibition of histone methyltransferase EZH2 depletes leukemia stem cell of mixed lineage leukemia fusion leukemia through upregulation of p16. Cancer Sci, 105: 512–519

[65] Utikal J, Polo J M, Stadtfeld M, Maherali N, Kulalert W, Walsh R M, Khalil A, Rheinwald J G, Hochedlinger K (2009). Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature, 460(7259): 1145–1148

[66] van den Boom V, Rozenveld-Geugien M, Bonardi F, Malanga D, van Gosliga D, Heijink A M, Viglietto G, Morrone G, Fusetti F, Vellenga E, Schuringa J J (2013). Nonredundant and locus-specific gene repression functions of PRC1 paralog family members in human hematopoietic stem/progenitor cells. Blood, 121(13): 2452–2461

[67] van der Stoop P, Boutsma E A, Hulsman D, Noback S, Heimerikx M, Kerkhoven R M, Voncken J W, Wessels L F, van Lohuizen M (2008). Ubiquitin E3 ligase Ring1b/Rnf2 of polycomb repressive complex 1 contributes to stable maintenance of mouse embryonic stem cells. PLoS ONE, 3(5): e2235

[68] Walker E, Chang W Y, Hunkapiller J, Cagney G, Garcha K, Torchia J, Krogan N J, Reiter J F, Stanford W L (2010). Polycomb-like 2 associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell self-renewal and differentiation. Cell Stem Cell, 6(2): 153–166

[69] Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones R S, Zhang Y (2004a). Role of histone H2A ubiquitination in Polycomb silencing. Nature, 431(7010): 873–878

[70] Wang L, Brown J L, Cao R, Zhang Y, Kassis J A, Jones R S (2004b). Hierarchical recruitment of polycomb group silencing complexes. Mol Cell, 14(5): 637–646

[71] Woo C J, Kharchenko P V, Daheron L, Park P J, Kingston R E (2010). A region of the human HOXD cluster that confers polycomb-group responsiveness. Cell, 140(1): 99–110

[72] Wu X, Johansen J V, Helin K (2013). Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation. Mol Cell, 49(6): 1134–1146

[73] Xie H, Xu J, Hsu J H, Nguyen M, Fujiwara Y, Peng C, Orkin S H (2014). Polycomb repressive complex 2 regulates normal hematopoietic stem cell function in a developmental-stage-specific manner. Cell Stem Cell, 14(1): 68–80

[74] Xu B, On D M, Ma A, Parton T, Konze K D, Pattenden S G, Allison D F, Cai L, Rockowitz S, Liu S, Liu Y, Li F, Vedadi M, Frye S V, Garcia B A, Zheng D, Jin J, Wang G G (2015). Selective inhibition of EZH2 and EZH1 enzymatic activity by a small molecule suppresses MLL-rearranged leukemia. Blood, 125(2): 346–357

[75] Xu K, Wu Z J, Groner A C, He H H, Cai C, Lis R T, Wu X, Stack E C, Loda M, Liu T, Xu H, Cato L, Thornton J E, Gregory R I, Morrissey C, Vessella R L, Montironi R, Magi-Galluzzi C, Kantoff P W, Balk S P, Liu X S, Brown M (2012). EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science, 338(6113): 1465–1469

[76] Ying Q L, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008). The ground state of embryonic stem cell self-renewal. Nature, 453(7194): 519–523

[77] Yu M, Mazor T, Huang H, Huang H T, Kathrein K L, Woo A J, Chouinard C R, Labadorf A, Akie T E, Moran T B, Xie H, Zacharek S, Taniuchi I, Roeder R G, Kim C F, Zon L I, Fraenkel E, Cantor A B (2012). Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors. Mol Cell, 45(3): 330–343

[78] Yuan J, Takeuchi M, Negishi M, Oguro H, Ichikawa H, Iwama A (2011). Bmi1 is essential for leukemic reprogramming of myeloid progenitor cells. Leukemia, 25(8): 1335–1343

[79] Zhang Z, Jones A, Sun C W, Li C, Chang C W, Joo H Y, Dai Q, Mysliwiec M R, Wu L C, Guo Y, Yang W, Liu K, Pawlik K M, Erdjument-Bromage H, Tempst P, Lee Y, Min J, Townes T M, Wang H (2011). PRC2 complexes with JARID2, MTF2, and esPRC2p48 in ES cells to modulate ES cell pluripotency and somatic cell reprogramming. Stem Cells, 29(2): 229–240

[80] Zhao J, Sun B K, Erwin J A, Song J J, Lee J T (2008). Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science, 322(5902): 750–756

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