Polycomb group-mediated gene silencing ... - Future Medicine

Review For reprint orders, please contact: [email protected]

Polycomb group-mediated gene silencing mechanisms: stability versus flexibility Polycomb group (PcG) proteins are highly conserved chromatin factors that repress transcription of particular target genes in animals and plants. PcG proteins form multimeric complexes that act on their target genes through the regulation of post-translational histone modifications, the modulation of chromatin structure and chromosome organization. PcG proteins have long been considered as a cellular memory system that stably locks regulatory chromatin states for the whole lifespan of the organism. However, recent work on the genome-wide distribution of PcG components and their associated chromatin marks in vertebrate cells and Drosophila have challenged this view, revealing that PcG proteins confer dynamic transcriptional control of key developmental genes during cell differentiation and development. KEYWORDS: chromatin marks n chromatin structure n development n differentiation n embryonic stem cells n epigenetic regulation n genome-wide mapping n nuclear organization n pluripotency n Polycomb group complexes

The conserved Polycomb group (PcG) proteins are central players in various epigenetic phenomena, such as the maintenance of homeotic (Hox) expression patterns from fruit flies to humans, X chromosome inactivation, genomic imprinting, stem cell plasticity and renewal, cell fate determination and cancer development [1–3] . Genes encoding PcG proteins were first identified in Drosophila melanogaster as factors required for maintaining the silent expression states of Hox genes during development. The Polycomb (Pc) gene was discovered by P. Lewis in 1947 after the isolation of a dominant mutation of Pc that produces a well-defined homeotic phenotype: ectopic sex combs on the second and third legs of adult male flies [4] . PcG-mediated repression is counteracted by trithorax group (trxG) proteins, which maintain the activation of the Hox gene in the appropriate spatial domains. In Drosophila, PcG components are recruited to specific DNA regions called PcG response elements (PREs). These proteins form conserved multimeric complexes, referred to as Polycomb repressor complexes (PRCs). Two major complexes, PRC1 and PRC2, were initially characterized. These complexes exert their functions on chromatin by the deposition of post-translational histone modifications, interference with the transcriptional machinery and the modulation of chromatin structure and chromosome organization. The paradigm of PcG activity is that they ‘freeze’ the chromatin of particular developmentally regulated target genes into a stable and

heritable repressive state [1,5] . However, this view has recently been challenged by a multitude of genome-wide mapping studies of PcG proteins, performed in a variety of tissues and cell types (reviewed in [6]). Models have now been proposed in which PcG proteins exert a dynamic regulation of chromatin states for stem cell maintenance and during cellular differentiation. Over the past few years, the PcG field has exploded and now covers a wide range of research topics. Many specific aspects of PcG regulation have recently been addressed in a number of focused reviews [7–10] . Here, we discuss the molecular mechanisms by which PcG protein complexes mediate and transmit stable chromatin silencing, as well as their roles in more dynamic regulatory processes, such as cellular plasticity and cell fate decisions.

10.2217/EPI.09.28 © 2009 Future Medicine Ltd

Epigenomics (2009) 1(2), 301–318

Virginie Roure & Frédéric Bantignies† Author for correspondence: Institut de Génétique Humaine, CNRS UPR 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France Tel.: +33 499 619 971 Fax: +33 499 619 901 [email protected] †

PcG complexes PRC1 and PRC2 were the first PcG complexes to be identified in Drosophila. These two complexes contain the key catalytic components of PcG-mediated repression. Other more recently described PcG complexes, such as PhoRC, Pcl-PRC2 and dRAF, may serve to recruit the core complexes or to participate in their repressive activities (for more details, see [7]). In mammals, PRC1 and PRC2 are well conserved. Duplication of many PcG genes, or alternative translation start site usage, allow variations in the composition of complexes which differs with cell type and developmental stage (reviewed in [10]).

part of

ISSN 1750-1911

301

Review

Roure & Bantignies

PRC2 complex: deposition of the repressive histone H3K27me3 mark The PRC2 complex biochemically purified from Drosophila contains four core components (Figure 1) : the histone methyltransferase Enhancer of zeste (E[z]), Extra sex combs (Esc), Suppressor of zeste 12 (Su[z]12) and nucleosome-remodeling factor 55 (Nurf-55). The catalytic subunit E(z), which contains a SET domain, deposits the characteristic repressive chromatin mark histone H3 trimethylated at lysine 27 (H3K27me3). Each of the four components contribute to the ability of PRC2 to bind and methylate nucleosomes,

as E(z) has no histone methyltransferase activity when alone [11–13] . Su(z)12 and Nurf-55 are essential for nucleosome binding, and Esc is important for boosting the enzymatic activity of E(z). A related mammalian complex containing homologous proteins has also been identified (EZH2, EED, SUZ12 and RbAp46/48) [14,15] . A distinct form of PRC2 containing the PcG protein Polycomblike (Pcl), the Pcl–PRC2 complex, has recently been isolated in Drosophila [16] . Similar mammalian PRC2 complexes that contain the Pcl homolog PHF1 have also been recently described [17,18] . Interestingly, Pcl

Drosophila Pcl PRC2 PhoRC Nurf55

dSfmbt Pho

Esc

+ Tri-methylation of H3K27

E(z) Su(z)12

PRE Ph PRC1 Ubiquitylation on K119 of H2A

dRing

Demethylation of H3K36me2

dRing

Pc Psc

Interaction with H3K27me3

Psc

dKdm2

dRAF

Mammals

SUZ12 PRC2

EED

BMI1

EZH2 RbAp46 RbAp48

PRC1

hPH1-3 hPC1-3

RING1B SCMH1–2

Epigenomics © Future Science Group 2009

Figure 1. PcG complexes and their biochemical activities. (A) In Drosophila, PRC1 (in dark green) and PRC2 (in red) complexes are recruited to PRE DNA sequences by a combination of DNA-binding proteins, including the PcG PhoRC complex (in blue). The subunit E(z) of PRC2 trimethylates H3K27 (represented by red balls). Association of Pcl with PRC2 allows high levels of H3K27 trimethylation. This repressive mark is recognized by the chromodomain of Pc. dRing is responsible for the deposition of another repressive mark, the histone H2A mono-ubiquitylated on lysine 119 (represented by green crosses). dRing, as well as Psc, can be present in either PRC1 or dRAF (in light green) complexes. dRAF notably contains the demethylase dKdm2 that possesses an H3K36me2 demethylase activity. This schematic representation has been adapted from [139] . (B) The principal PRC1 and PRC2 complexes in mammals.

302

Epigenomics (2009) 1(2)

future science group

Polycomb group-mediated gene silencing mechanisms: stability versus flexibility

appears to be specifically required to assist PRC2 at PREs to generate high levels of H3K27me3 needed for an effective PcG-mediated silencing. PRC1 complex: reading of repressive marks & transcriptional repression The Drosophila PRC1 complex contains a core quartet of PcG proteins (Figur e 1) : Polycomb (Pc), Polyhomeotic (Ph), Posterior sex combs (Psc), and dRing [19] . An orthologous vertebrate PRC1 complex has also been isolated [20] . PRC1 can interact with the PRC2-modified chromatin via the chromodomain of Pc, which specifically recognizes the H3K27me3 mark. PRC1 is responsible for the deposition of a second histone mark via the E3 ubiquitin ligase activity of dRing that mono-ubiquitylates lysine 119 of histone H2A (H2Aub) [21–23] . Very little is known about the role of this modification, but removal of dRing results in loss of H2A ubiquitylation and derepression of Hox target genes, suggesting that it is crucial for PcG-dependent silencing. Recently, a distinct Drosophila PcG complex harboring dRing and Psc was identified [24] . This complex, called dRING-associated factors (dRAF), additionally contains the demethylase dKdm2. On one hand, dKdm2 mediates demethylation of the active histone mark H3K36me2, thus counteracting trxG protein activity. On the other hand, dKdm2 is also required for efficient H2A ubiquitylation by dRing/Psc in cultured cells, suggesting that dRAF, rather than PRC1, is the major H2A ubiquitylase in Drosophila. This study reveals that dKdm2 plays a pivotal role in the ‘transhistone pathway’ by removing an active mark from H3 and adding a repressive one to H2A, thus mediating PcG-dependent silencing. PhoRC complex: PcG targeting Pleiohomeotic (Pho) and its closely related homolog PhoL (homologs of the mammalian factor YY1) are the only PcG proteins known to bind directly to DNA. The PhoRC complex, recently characterized in Drosophila, is composed of the PcG proteins Pho and dSfmbt (Scmrelated gene containing four MBT domains) (Figure 1) [25] . This complex is thought to play an important role in the recruitment of PRC2 and PRC1 to PREs (see following section).

Genome-wide PcG complex recruitment to chromatin PcG proteins act on their target genes via binding to specific cis-regulatory DNA sequences, the PREs. PREs are sequences functionally defined future science group

Review

by silencing assays. Recently, the identification of the PcG binding sites in both fly and mammalian genomes clearly indicates that PREs exist in both organisms; however, PREs have been functionally validated mostly in flies [26] . Drosophila PREs do not share sequence homology, and no consensus PRE sequence has been identified so far. Genome-wide mapping studies in flies show that approximately 50% of putative PREs are located at promoters, but they can also be found upstream or downstream of the genes [27] . Given that neither PRC1 nor PRC2 complexes contain DNA-binding proteins, the recruitment of PcG complexes to PREs is thought to be effected by DNA-binding proteins that directly interact with them. The initial analysis of Drosophila PREs revealed the presence of binding sites for several DNA-binding proteins, such as Pho/PhoL, GAGA factor (GAF)/Pipsqueak (Psq), Zeste, DSP-1, SP1/KLF and Grainyhead (GRH) (reviewed in [1,28]). However, the number and the distribution of their binding sites are quite variable, and no factor on its own is sufficient for PcG recruitment. Candidate PREs could be identified in the genome on the basis of PcG protein binding. Three genome-wide distribution studies in Drosophila have shone light on the pathways of PcG protein recruitment [27,29,30] (for a focused review, see [10]). These studies revealed that Pho or PhoRC share many genomic locations with PRC1 and PRC2 components, suggesting that PhoRC is a crucial determinant for anchoring PRC1 and PRC2 at their target genes [27,29,30] . However, a large proportion of Pho binding sites are not associated with PcG-bound regions. The same observation was made for the other DNAbinding proteins PhoL, Dsp1, GAF and Zeste, and confirms previous genetic analysis suggesting that clusters of binding sites for these proteins are insufficient to define a PRE [31,32] . It is likely that PREs are still hiding some genetic information for the targeting of PcG proteins. In agreement with this idea, an extended Pho-binding motif, as well as a novel motif for an unknown factor, were recently proposed as new signatures of PREs [27,30] . Additionally, the relative equilibrium between recruiter proteins at PREs could be important to predict a PRE, notably a high ratio between Pho and its homolog PhoL [27] . Finally, the genomic context and the chromatin conformation (see below) of these sites could also play a role in the effective recruitment of PcG components. To date, two mammalian PREs have been characterized: one from the HoxD locus [33] and, more recently, one named PRE-kr that regulates www.futuremedicine.com

303

Review

Roure & Bantignies

expression of the mouse MafB/Kreisler gene [34] . Interestingly, these mammalian PREs are sensitive to PcG mutations in transgenic assays in Drosophila, and act as PRC1-binding sites in this species. Genome-wide mapping studies show that PcG proteins are mainly associated with transcriptional start sites [35–37] . A major difference between Drosophila and vertebrates is that the mammalian PcG sites are characterized by a high density of CpG dinucleotides [38,39] . Moreover, the recruitment of PcG proteins on chromatin appears quite different. Among the Drosophila recruiters described above, only Pho and Dsp1 have homologs in vertebrates, named YY1 and HMGB2, respectively. Biochemical evidence suggests that these factors could participate in the recruitment of vertebrate PcG complexes [40,41] , and consensus Pho/YY1-binding sites are found in the PRE-kr [34] . Other DNAbinding proteins, such as AEBP2, SNAIL1 and PLZF, might also contribute to this function [42–45] . Strikingly, in embryonic stem (ES) cells, PRC2 binding sites are characterized by an over-representation of repressor motifs and strong depletion of transcriptional activator motifs [46] . One of the most represented motifs corresponds to the binding motif for the neuronrestrictive silencing factor (NRSF), which is a potent transcriptional repressor essential for ES cell pluripotency. Otherwise, many of the developmental PcG target genes identified in murine and human ES cells have been shown to be bound by three key pluripotency transcription factors, OCT4, SOX2 and NANOG [35,36,47] . Therefore, this raises the possibility that PcG proteins could be recruited by this specific set of transcription factors in ES cells, but this issue needs to be tested. Finally, RNA species could contribute to PcG recruitment [48,49] . Remarkably, a very recent work reports the association of the PRC2 complex with more than 600 long noncoding RNAs (ncRNAs) in human cells [50] .

Mechanisms of stable transcriptional repression Several, non-exclusive modes of action might contribute to the silencing of PcG target genes. The time-honored view for the mechanism of transcriptional repression by PcG proteins was that their complexes establish a highly compact chromatin structure that interferes with the access of the transcriptional and remodeling machineries. Much evidence supports the chromatin-compaction model. In vitro experiments show that PRC1 can repress ATP-dependent 304


remodeling by the SWI/SNF complex [19] . The PRC1 core components were also shown in vitro to cause compaction of a nucleosome array in a Psc-dependent manner [51,52] . Moreover, the mammalian PRC2–EZH1 (an alternative PRC2 complex containing an EZH2 paralog) was recently reported to mediate repression through its capacity to compact chromatin in vitro and in vivo by the decreased nuclease accessibility [53] , in agreement with precedent works [54,55] . Interestingly, different EZH2-containing complexes exhibit H1K26 methyltransferase activity [56] , and this activity is supposed to reinforce the chromatin compaction. Nevertheless, some findings argue against this model. Drosophila PREs are depleted in nucleosomes and are nuclease hypersensitive [57–59] . PcG-binding sites also represent peaks of histone replacement [60] . Finally, a stable condensed chromatin model is difficult to accommodate with the finding that, in Drosophila, PcG proteins have residence times of only a few minutes on chromatin [61] . This dynamic behavior implies that PcG repressed genes are not stably locked in an impervious state, but rather suggests that PcGmediated repression could be more dynamic than previously expected. Consistent with this idea, several studies in Drosophila indicate that PcG protein binding does not necessarily prevent the access of the transcriptional machinery. Indeed, basal transcription factors, elongation factors and RNA Polymerase II (Pol II) can be found at PREs and PcG-repressed promoters [58,62–65] , whereas these factors are absent along the coding region of PcG-repressed genes, indicative of a transcriptional blocking [58,64] . In agreement with these studies, a recent work in murine ES cells shows that PRC1-mediated ubiquitylation of H2A at specific repressed loci does not prevent recruitment of Pol II, but rather prevents elongation by inhibiting the release of the initiative form (Ser5-phosphorylated) of Pol II bound to promoters [66] . Therefore, in particular cases, rather than altering chromatin accessibility, PcG silencing may directly target the activity of the transcriptional machinery at the promoter. However, larger scale analysis needs to be performed to generalize this model. Finally, ncRNAs could also play a role in regulating PcG silencing (reviewed in [9]). Initially, transcription of untranslated RNA through the regulatory elements of the Drosophila Hox genes has been proposed to counteract PcGdependent silencing [67–70] . Another study also supports an activating role for the ncRNAs, future science group


showing that transcripts of the regulatory region bxd can recruit the trxG protein Ash1 to this region to induce transcription of its Hox target gene Ubx in larval tissues [71] . However, more recent evidence indicates that these untranslated RNAs instead silence the Hox gene Ubx in early embryos, and the authors proposed a mechanism of transcriptional interference [72] . Finally, the PRC2 complex was found to be associated with the Kcnq1ot1 ncRNA for the silencing of the Kcnq1 imprinted cluster in mammals [73] . Although ncRNAs are clearly implicated in PcG-dependent gene regulation, more studies are needed to clarify their exact function and mechanisms of action during development.

Reinforcement of PcG-mediated silencing implies large-scale genome organization In addition to their direct action on chromatin, increasing evidence in various organisms has

Review

suggested that PcG proteins play a critical role in large-scale chromatin organization (Figure 2) . Chromatin loops The looping model for control of gene expression postulates that various regulatory elements communicate through direct interactions of chromatin constituents. Several reports have described the existence of ‘chromatin hubs’, which appear important for efficient transcriptional regulation [74] . Long-range chromatin interactions by chromatin looping have been thought to be one of the potential mechanisms to explain PcG action over long distances in cis [75] . Genomewide studies in Drosophila have confirmed that the position of PREs relative to their target genes is variable, sometimes being positioned at very large distances. One possible explanation for the silencing of distant promoters is that PcG proteins bound to a PRE might contact the promoter via looping. Contact between PREs and promoters by looping was first demonstrated

Repressive compartment

cis-interactions Repressive compartment PcG

Interacting region 2

Interacting region 1 cis-interactions or trans-interactions Epigenomics © Future Science Group 2009

Figure 2. PcG-mediated silencing implies large-scale genome organization. (A) A linear representation of a regulatory element (in red) and its target gene promoter (in blue). (B) The first hierarchical level of chromatin folding corresponds to the interaction between the regulatory element and its target promoter, which leads to a chromatin loop conformation. (C) A second hierarchical level involves multiple interactions in cis between various regulatory elements and promoters inside a repressive PcG nuclear compartment or PcG bodies to form a complex topological domain also called ‘chromatin hub’. (D) Finally, a further level of complexity involves long-range chromosomal interactions in between two distinct domains into the repressive PcG nuclear compartment. These domains can be located on the same chromosome (cis-interactions) or on different chromosomes (trans-interactions). These long-range chromosomal interactions could reinforce gene repression mediated by the PcG system. PcG: Polycomb group.


www.futuremedicine.com

305

Review

Roure & Bantignies

by the tethering of Dam methyltransferase to the Drosophila Fab-7 PRE [76] . Another study in Drosophila using a transgenic system also indicated that PRE looping can drive promoter silencing [77] . More recently, Lanzuolo et al. investigated the physical and spatial interactions between regulatory regions of the Hox multigenic bithorax complex (BX-C) by fluorescent in situ hybridization and chromosome conformation capture [78] . This work provided the first direct evidence in Drosophila of a PcGdependent hub-like structure. All major PcGbound elements at the bithorax locus, including PREs and core promoters, physically associate at a distance upon PcG-dependent silencing. Importantly, active genes are specifically out of the hub, indicating that this higher-order structure could play an important role in maintaining the stable repression of the locus. In mammals, some examples of PcG proteins mediating higher order chromatin conformation have also been recently described. In the human GATA-4 gene locus, PcG-bound regions physically interact to form chromatin loops in order to maintain the gene in a silent but poised state in undifferentiated ES-like cells [79] . Upon cell differentiation, GATA-4 expression is accompanied by a complete loss of these chromatin loops and loss of PcG protein binding. However, in adult cancer cells, the chromatin loop forms a tighter structure, dependent on DNA methylation, resulting in strong transcriptional repression. In mammals, methyl-DNA-binding protein at CpG islands may thus reinforce the PcG chromatin hub [79] . Terranova et al. studied the mouse Kcnq1 imprinted gene cluster and reported that the paternal Kcnq1 cluster becomes progressively competent for silencing through the formation of a higher-order repressive compartment and PcG-mediated genomic reorganization [80] . Moreover, in comparison with the active maternal cluster, the paternally repressed cluster exists in a PcG-mediated contracted state in embryos. In the absence of EZH2 expression, paternal genomic contraction is abrogated, and several genes of the cluster failed to be repressed. Genomic contraction could reflect a condensed spatial configuration formed either through looping and/or local chromatin compaction of specific genomic regions. Importantly, these PcG chromatin hubs might serve to create a nuclear compartment devoid of Pol II for highly stable chromatin states [79,80] . Finally, in the Caenorhabditis elegans embryo, the mes-2/E(z) PcG protein influences the chromatin compaction of endogenous loci, 306


concomitant with the loss of developmental plasticity [81] , indicating that this mechanism of action may be conserved. Long-range chromosomal interactions The formation of subnuclear silencing compartments might also contribute to the stable repression of transcription. The Drosophila and mammalian PcG proteins are organized into nuclear foci called PcG bodies [61,82–84] , which form distinct repressive nuclear compartments [78,80,85] . In Drosophila, recent results suggest that PcG bodies may be able to silence multiple genes simultaneously, allowing coordinated control of developmental pathways. Indeed, the number of PcG bodies is fewer than the number of genomic binding sites [27,86,87] , suggesting that endogenous target genes may share PcG foci. Evidence from studies in flies revealed that PcG target elements located on different chromosomes can associate in the nucleus to strengthen PcG-mediated silencing [85,88,89] . This was first demonstrated by colocalization of the endo genous Fab-7 PRE with a transgenic Fab-7 element inserted at a heterologous site, and was subsequently confirmed by similar experiments with the Mcp PRE element [88,89] . Furthermore, these contacts have been shown to occur at PcG bodies [85] . Clustering of PcG target genes into PcG bodies probably corresponds to a higher concentration of PcG activities for a more efficient repression, and may thus facilitate silencing by partial exclusion of Pol II bodies. Interestingly, the association of PcG target elements requires nuclear components of the RNA interference machinery that partially colocalize with PcG proteins [85] . More recently, Tiwari et al. used a novel combined chromosome conformation capture–chromatin immunoprecipitation-cloning assay to reveal specific proteins implicated in long-range chromatin interactions and provide evidence that the PcG protein EZH2 is able to bring distant chromatin regions, both intra- and interchromosomally, together in the three-dimensional space of the mammalian nucleus [90] . siRNA-mediated knockdown of EZH2 leads to the loss of pairing between genomic regions, concomitant with the upregulation of multiple flanking genes around each of the interacting chromatin regions. Therefore, PcG-mediated regulation involves specific gene nuclear organization. How widespread these chromosomal interactions are and how they arise still remain important questions to be resolved. future science group


Dynamic regulation Beyond their role in maintaining stable repressive chromatin states during development, it has become clear that the PcG system also confers dynamic control of a variety of biological processes. The recent genome-wide mapping studies of PcG proteins in Drosophila and vertebrates have identified a large set of target genes and opened new avenues to understand the functional significance of PcG protein regulation. In both systems, PcG components are mainly bound to genes encoding transcription factors involved in diverse cellular functions and developmental pathways (reviewed in [5,6]). Moreover, some of these genes are not silent while associated with PcG complexes [58,66,87,91] , and some PcG target genes change their transcriptional status during cell differentiation. These findings challenge the view whereby PcG proteins permanently shut down target genes. Bivalent chromatin domains One of the most important areas of interest is the finding that PcG proteins are implicated in the control of cell differentiation (Figure 3) . Indeed, genome-wide mapping in mouse and human ES cells, as well as in human embryonic fibroblasts (EF), showed that PcG proteins bind genes encoding key developmental regulators, such as HOX, GATA, PAX, SOX, RUNX, NEUROD and TBX transcription factors [35–37] . All these developmental factors are repressed in ES cells. However, upon differentiation, specific sets of these factors become expressed according to cell lineage commitment, indicating that not all genes bound by PcG are predetermined to be silenced permanently. Unexpectedly, the majority of the repressed PcG targets in ES cells are covered not only by the H3K27me3 repressive mark, but also by the activating H3K4me3 mark, and were termed ‘bivalent domains’ [92,93] . It was initially proposed that these bivalent chromatin marks silence key developmental genes in ES cells, while maintaining them poised for lineagespecific activation [92] . The coexistence of these histone marks led three groups to investigate the genome-wide distribution of these bivalent domains in human and mouse ES cells [38,94,95] . These studies revealed three major chromatin states in ES cells, which correspond to functionally distinct groups: genes marked solely by H3K4me3 are highly expressed and represent mainly pluri potency and housekeeping genes; genes marked by bivalent domains have no or low expression future science group

Review

and are characterized by developmental regulators. Finally, genes lacking either mark are generally not expressed and are enriched for genes involved in the response of transient external stimuli related to tissue-specific functions [94,95] . The analysis of histone modifications in more committed mouse neural progenitor cells and EF indicates that a large proportion of PcG target genes resolve to a monovalent status, although some remain bivalent [38] . Strikingly, the resolution of bivalent domains in lineage-committed cells appears to be closely related to their developmental potential – that is, genes that have a function in unrelated lineages tend to lose their active mark, whereas genes that have a function in the specific lineage become monovalent for H3K4me3 and become activated, or remain bivalent, poised for further activation [38] . Therefore, bivalency predisposes not only for gene activation, but also for inactivation, and the resolution of the bivalent domains into either an active or a repressed chromatin state constitutes an important feature for cell-fate decisions. Bivalent domains are not unique to pluripotent cells; they also exist in more committed cells [38,96,97] . Mounting evidence suggests that these domains may be more dynamic during differentiation than initially thought, perhaps reflecting a general regulatory mechanism. Mohn et al., using a well-defined murine cellular differentiation model, monitored various chromatin states from ES cells to lineage-committed progenitors, and then to terminally differentiated neurons [39] . This study revealed that some lineage-specific genes (neuron-specific genes), which are not initially repressed by the PcG system in ES cells (i.e., not marked by H3K27me3), become bivalent in progenitor cells, poised for the activation necessary in the final steps of differentiation. More globally, the authors observed a weak fluctuation in the total number of bivalent domains in any differentiation step, suggesting that their loss in ES cells is largely compensated by the formation of new ones at progenitor and terminal states [39] . Therefore, many de novo bivalent domains are formed during the differentiation process, suggesting that the chromatin state of some lineage-specific PcG target genes does not necessarily need to be predetermined in stem cells. Although bivalent domains seem to play important functions during cell-fate progression in mammalian cells, there is no evidence of such chromatin signatures in Drosophila. In fly embryos, H3K4me3 and H3K27me3 are generally mutually exclusive [27] . In vertebrate ES www.futuremedicine.com

307

Review

Roure & Bantignies

ES cells

Pluripotency genes

K4me3

ON

Terminally differentiated cells

Progenitor cells

DNA met

OFF

DNA met

OFF

Non PcG target genes CpG-poor Pm

Early differentiation genes

ON (cell lineage specific)

K4me3

OFF

K27me3

OFF (unrelated cell lineage)

PcG target genes CpG-rich Pm

Late differentiation genes

K27me3 K27me3 DNA met

K4me3

K4me3

OFF

DNA met

OFF

K4me3

OFF

OFF

DNA met

ON (cell fate specific)

K4me3

OFF (unrelated cell fate)

K27me3

K27me3

PcG target genes CpG-rich Pm


Figure 3. The PcG system dynamically regulates developmental factors during cell differentiation. Most of the pluripotency genes are CpG-poor promoters and are not PcG target genes, while differentiation genes are PcG targets and CpG-rich promoters. In ES cells, PcG complexes repress early differentiation genes to maintain ES cell identity, whereas the pluripotency genes are highly expressed and methylated at H3K4me3. The majority of PcG target genes correspond to ‘bivalent domains’, and carry the repressive H3K27me3 mark (in red) together with the active H3K4me3 mark (in green). Late differentiation genes are not expressed but can be methylated at H3K4. Upon cell fate commitment, pluripotency genes are stably silenced by a PcG independent mechanism like DNA methylation (in gray). In parallel, early differentiation genes specific for the cell lineage resolve their bivalent domains – that is, lose their H3K27me3 repressive marks and become activated – while early differentiation genes not involved in the cell lineage lose their active H3K4me3 mark and remain stably repressed by the DNA methylation. Importantly, at this progenitor stage, late differentiation genes become de novo methylated at H3K27, resulting in the formation of new bivalent domains. Upon terminal differentiation, early differentiation genes specific for the cell lineage become stably repressed by DNA methylation, while late differentiation genes specific for a particular cell fate resolve their bivalent domains. Therefore, bivalent domains poise the genes in a permissive active or repressed chromatin state able to be resolved throughout cell fate commitment. It is interesting to note that the DNA methylation occurs mainly during lineage commitment, while it is less dynamic during the late stage of differentiation. ES: Embryonic stem.

cells, bivalent domains are virtually all associated with CpG-rich promoters [38,39,94] , a situation that does not exist in flies. However, further characterization awaits studies in more specific cell types in flies and other organisms. Dynamics of histone methylation In agreement with the dynamic nature of PcG-mediated regulation, a flurry of recent 308


studies identified two Jumonji C domain (JmjC)-containing proteins, JMJD3 and UTX, as demethylases that specifically remove the repressive H3K27me3 mark [98,99] (for review, see [100]). Moreover, these studies revealed that this repressive mark, associated with PcG silencing and long thought to be stable and irreversible, is able in fact to rapidly turn over. Interestingly, the UTX demethylase has been found in complex future science group


with the mixed-lineage leukaemia (MLL) 2/3 protein, a histone H3K4 methyltransferase, indicating that both activities can be associated in cells to reinforce their activating function [98,101] . In Drosophila, dUtx associates with elongating Pol II to remove the repressive H3K27me3 mark during transcriptional elongation [102] . However, there is no evidence to date showing that UTX counteracts PcG silencing. Several other studies have shown that UTX occupies the promoters of Hox gene clusters [98,99,103] , and that UTX inhibition causes some Hox gene mis-expression [99,103] , but these results do not exclude the possibility that UTX could have a more general role in transcription. All PcG repressive chromatin marks appear reversible: deubiquitylating enzymes that remove the ubiquitin from H2A have also been recently described [104–107] . Conversely, members of the JARID1 family specifically catalyze the demethylation of the H3K4me3 active mark (for an extensive review, see [100]). Recently, it has been shown that the histone demethylase Rbp2/Jarid1 colocalizes with PRC2 at many target sites in mouse ES cells and, importantly, Rbp2 is required for the maintenance of PcG target gene repression [8] . Therefore, histone demethylase activities may work cooperatively with PcG complexes to assist the PcG silencing function. Collectively, these studies suggest that multi protein complexes made of different enzyme activities act together or antagonistically, forming an equilibrium to ‘fine-tune’ the level of histone modifications at specific loci during cell fate progression. Importantly, changes in this equilibrium might serve to resolve the bivalent chromatin domains, as illustrated when JMJD3 H3K27 demethylase is specifically induced during macrophage activation [108] , or the default of bivalency resolution in cells where the MLL1 H3K4 methyltransferase is mutated [109] . Additional work is now required to better understand how the bivalent domains, which appear to be the target of a cohort of antagonistic enzyme activities, are formed and resolved during cellular differentiation by external stimuli. Role of DNA methylation in cell fate commitment Bracken et al. observed that only a small percentage of genes that were bound by PcG in human EF cells become re-expressed after the knockdown of PcG components [37] . Similarly, in the precursor basal cells of the epidermal lineage in mouse, genes controlling pluripotency or other differentiation pathways do not future science group

Review

become derepressed upon conditional removal of EZH2 [110] . These results indicate that the genes that remain silent are probably subject to additional regulation, such as DNA methylation, which is considered as a stable repressive epigenetic modification (Figure 3) [111] . In ES cells, the majority of CpG-rich promoters are unmethylated [39,112] . These CpG islands are either associated with H3K4me3 (mono valent) or with both H3K4me3 and H3K27me3 (bivalent) [38] . The large majority of PcG target genes are associated with the bivalently marked and CpG-rich promoters [38,39] . Throughout ES cell differentiation, the majority of CpG island promoters remain unmethylated [39,112] , suggesting that, in ES cells, PcG proteins represent the main repressive system at CpG-rich promoters, independently of DNA methylation. However, some of the bivalent genes gain DNA methylation during differentiation of ES to neural precursor cells, in correlation with resolution of the bivalent state to the repressive state [39,112] . Thus, upon cellular commitment, de novo DNA methylation could lock some developmental genes in a silent state, which were poised in a more flexible bivalent state in ES cells. Moreover, on these particular sites, the PcG complexes could play a part in mediating the DNA methylation reaction. Indeed, in vitro studies indicate that the PcG proteins EZH2 and BMI1 might recruit DNA methyltransferase to PcG target genes [113,114] . Importantly, the cross-talk between PcG-dependent histone modification and DNA methylation occurs during normal somatic differentiation, and misregulation of this process could contribute to premature or aberrant cancer-specific methylation profiles [115–118] . In summary, PcG proteins seem to be part of a flexible silencing system that allows the postponement of lineage choices until the appropriate signals are received. Upon cellular commitment, the PcG system co-operates with other silencing mechanisms, such as DNA methylation, to lead to a more permanent cell fate. PcG dosage during differentiation & reprogramming In flies, many mutations in PcG genes produce haplo-insufficiency phenotypes, indicating that the dosage of PcG might be an important para meter for their epigenetic function [26] . In mammals, during the mouse epidermal lineage, the precursor basal cells are rich in PcG components, whereas several PcG genes are downregulated upon terminal differentiation [110] . Importantly, this global change leads to a selective derepression www.futuremedicine.com

309

Review

Roure & Bantignies

of PcG target genes involved in epidermal lineage determination, indicating that regulation of PcG levels could play an important role during development and cell fate commitment. The PcG dosage also plays an important role in controlling cell reprogramming. During tissue regeneration after injury of Drosophila imaginal discs, cell identity can occasionally switch fates, a process known as transdetermination, during which the cells that change their identity are subject to intense signaling by powerful morphogens [119,120] . Two independent reports have linked transdetermination to PcG gene regulation [119,121] . Notably, Lee et al. found that the downregulation of PcG in cells undergoing regeneration is controlled by the Jun amino-terminal kinase (JNK) signaling pathway [121] . Similar regulatory pathways may also exist in mammals since PcG gene downregulation has been recently associated with wound healing in mice [122] . Interestingly, the authors observed in parallel a marked upregulation of the newly described demethylases JMJD3 and UTX, which may counteract PcG function and lead to the induction of repair genes, such as Egfr and Myc. Therefore, PcG downregulation induces shifts of chromatin states at particular target sites to render the cells compatible with reprogramming for wound healing. During senescence or under exposition to cellular stress, EZH2, but not other PcG genes, is downregulated, which leads to the transcriptional activation of the Ink4a/Arf locus, a signal for proliferation arrest [123,124] . In parallel, the specific downregulation of EZH2 was concomitant with an upregulation of the JMJD3 H3K27 demethylase [124–126] . Therefore, the simultaneous downregulation of PcG components and the upregulation of antagonist activities is a common theme used by the cell to achieve very different biological processes, such as wound healing or senescence. All these results reveal the functional importance of controlling the cellular pool of PcG components during differentiation and reprogramming. However, an important question is how the cellular level of PcG components is regulated. Some studies indicate that, in addition to transcriptional regulation, sequestration or posttranslational modifications might be other methods by which PcG protein levels can be regulated (Figure 4) . During Drosophila spermatogenesis, testis-specific transcription factors counteract the action of PcG proteins on specific target genes by inducing their relocalization to the nucleolus in precursor cells [127] . Moreover, in vertebrate 310


cells, the PRC2 complex can be exported from the nucleus to the cytoplasm in response to the activation of signaling pathways [128,129] . This partial depletion of PRC2 from the nucleus might thus cause the derepression of some target genes. Other examples suggest the role of posttranscriptional modification in the regulation of PcG dosage. Indeed, the phosphorylation of PcG proteins could induce their dissociation from chromatin under various signaling pathways [130– 132] . Additional work needs to be carried out to identify new post-translational modifications and to measure their influence on specific target genes.

How are epigenetic marks stably maintained? An important remaining question is how chromatin marks can be maintained after several rounds of DNA replication? Indeed, during DNA replication new core histones are deposited after the replication fork to form the daughter chromatin fiber, and need to be post-translationally modified to conserve the chromatin state. The H3K27me3 domains associated with PcG components are rather large regions in both vertebrates and Drosophila [27,35,36,38,92,97] . These large regions may provide a robust epigenetic memory of their target genes. Notably, it is tempting to speculate that their large size would ensure that a substantial proportion of the modified histones would be distributed on each daughter chromosome, which could then promote similar modifications of new histones in the immediate vicinity [133] . This hypothesis could also account for the maintenance of the H3K4me3 domains or bivalent domains in vertebrates. Furthermore, the DNA methylation, which has an autonomous and faithful mechanism of maintenance, might also play a role in chromatin regions containing both repressive histone marks and DNA methylation [111] . Recently, two groups directly addressed the role of PcG proteins in the reproduction of epigenetic states during replication [134,135] . On the one hand, Hansen et al. found that EZH2, but not the PRC1 components, colocalizes well to replication foci during S phase, and suggest that this might depend on a direct interaction of the PRC2 complex with its own H3K27me3 site [134] . Using a heterologous reporter system, they also demonstrated that the transient recruitment of PRC2 is sufficient to induce a repressed chromatin state that is then maintained by the endogenous PRC2 through many cell generations. On the other hand, Francis et al. observed that the PRC1 complex remains associated to future science group


Review

R Transcriptional regulation

PcG gene PcG proteins

Promoter

PcG Post-translational regulation

Differentiation or reprogramming under specific signals

Target gene PRE Promoter

Plasma membrane

PcG target genes

Nucleolus PcG

Sequestration PcG

ne

t ge

ge Tar


Figure 4. Regulation of PcG cellular levels upon differentiation or cell reprogramming. Cell differentiation or cell reprogramming, which occur during wound healing, senescence or stress, are induced under specific signaling pathways, and can be accompanied by a decrease of the PcG protein functional cellular pool. This PcG dosage can be regulated at different levels: (A) Transcriptionally, to directly prevent mRNA and protein production. (B) Post-translationally, such as phosphorylation, to counteract the repressive action of PcG proteins (putative post-translational modifications are illustrated by a pink star). (C) ‘Physically’, either by sequestrating PcG proteins to a specific nuclear compartment, for example the nucleolus, or by recruiting them to the plasma membrane (the brown circle demarcates the cell nucleus). PcG: Polycomb group; PRE: PcG response elements; R: Transcriptional repressor.

chromatin or DNA during passage of the DNA replication fork in vitro [135] , suggesting a role of PRC1 in the maintenance of chromatin states during DNA replication. These reports provide possible PcG-dependent mechanisms for stable epigenetic inheritance through cell divisions. Other mechanisms might also exist to perturb this epigenetic memory system in order to permit specific changes of chromatin states occurring during cell fate commitment. However, it remains to be determined whether PcG proteins interact with the replication machinery, either directly or through intermediary factors, and how these interactions are regulated throughout the cell cycle and differentiation switches. Interestingly, a recent paper identified an essential regulator of DNA replication, CDC6, as a new partner of the PcG member BMI1 [124] .

Future perspective Over the past few years, our understanding of the PcG system has considerably improved. The extensive amount of work around this family future science group

of repressive factors has led to the discovery of new epigenetic regulatory phenomena such as bivalent domains, the formation of stable chromatin repressive loops or the cross-talk between PcG proteins and the DNA methylation or the replication machinery. The ongoing characterization of new PcG complexes, as well as their counteracting activities such as demethylases and deubiquitinylases, will certainly enrich the panoply of epigenetic mechanisms. The genome-wide mapping studies of PcG proteins and their associated chromatin marks have provided valuable insights onto the paradoxical function of this epigenetic system. On the one hand, the PcG system is required for the stable memory of epigenetic states, such as Hox gene regulation, X chromosome inactivation or gene imprinting. On the other hand, the PcG system dynamically controls many other targets for cell fate maintenance and determination. Much work has to be carried out to understand how this paradoxical situation can be achieved; notably how cell signaling and cell communication together www.futuremedicine.com

311

Review

Roure & Bantignies

with some nuclear organization features can affect this conserved and complex regulatory system. To add even more complexity, recent data indicate extranuclear functions for the PcG machinery, including regulation of actin polymerization [129] and mitochondrial function [136] . Therefore, the PcG machinery should no longer be seen solely as a stable cellular memory repressive system, but more as a flexible, reversible system. This makes the PcG mechanisms all the more important to obtain insight into the epigenetic reprogramming occurring during cancer, tissue regeneration and the production of induced pluripotent stem (iPS) cells from differentiated tissues [137,138] . This field of research will have great implications for cancer and regenerative medicine.

Author note In a recent paper, Margueron et al. add an important insight into the mechanism of inheritance of the PcGdependent chromatin marks [140] . They show that EED, a PRC2 component, specifically binds to histone tails carrying trimethyl-lysine residues such as H3K27me3, leading to the stimulation of the methyltransferase activity of PRC2. This work suggests a model for the propagation of silent chromatin states through DNA replication.

Acknowledgements We thank Bernd Schuettengruber, Tom Sexton and Thierry Cheutin for helpful discussions and for critical readings of the manuscript. We apologize to those whose recent publications we were unable to quote due to space limitations.

Executive summary PcG complexes In Drosophila, three major Polycomb group (PcG) complexes have been identified: – The PRC2 complex deposits the characteristic repressive chromatin mark H3K27me3 via its catalytic subunit E(z). – The PRC1 complex can interact with H3K27me3 and deposits a second histone mark, the mono-ubiquitylated lysine 119 of histone H2A, which might be crucial for PcG-dependent silencing. – The PhoRC complex is thought to play an important role in the recruitment of PRC2 and PRC1 to chromatin. – PRC1 and PRC2 are well conserved in mammals. Genome-wide PcG complex recruitment to chromatin PcG proteins act on their target genes via binding to specific cis-regulatory DNA sequences called Polycomb response elements (PREs). The recruitment of PcG complexes to PREs is thought to be effected by DNA-binding proteins that directly interact with them. No consensus PRE sequence has been identified so far. Mechanisms of stable transcriptional repression Several non-exclusive modes of action have been proposed to contribute to the PcG-dependent silencing of target genes, such as chromatin compaction, inhibition of transcriptional elongation and the implication of noncoding RNAs. Reinforcement of PcG-mediated silencing implies large-scale genome organization PcG-bound regions physically interact at a distance to form chromatin loops in order to maintain the gene silenced. Longer range chromosomal interactions between distant PcG-target loci intra- and inter-chromosomally could reinforce the gene repression. These phenomena of long-distance chromosomal interactions are conserved in Drosophila and mammals. Dynamic regulation The concomitant presence of the H3K27me3 repressive mark and the H3K4me3 active mark, named the ‘bivalent domain’, is observed at PcG target genes in embryonic stem cells. The resolution of these bivalent domains into either an active or a repressed chromatin state constitutes an important feature for cell-fate decisions. All PcG repressive chromatin marks appear reversible by demethylase and deubiquitylating enzymes. Upon cellular commitment, the PcG system cooperates with other silencing mechanisms, such as DNA methylation, to lead to a more permanent cell fate. The simultaneous downregulation of PcG components and the upregulation of antagonist enzyme activities is a common theme used by the cell to achieve very different biological processes, such as wound healing or senescence. How are epigenetic marks stably maintained? PRC2 components bind to replication foci during S phase, and functionally maintained the H3K27me3 repressive mark through cell division. The PRC1 complex remains associated to chromatin or DNA during the passage of the DNA replication fork in vitro. Conclusion The PcG machinery can no longer be seen solely as a stable cellular memory repressive system, but more as a flexible, reversible system. The implication of the PcG mechanism in epigenetic reprogramming occurring during tumorigenesis and tissue regeneration made this field of research all the more important for cancer and regenerative medicine.

312




Financial & competing interests disclosure The authors are members of the Giacomo Cavalli laboratory and are supported by the CNRS, the Agence Nationale de la Recherche, and the Association pour la Recherche sur le Cancer. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

11

1

Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111, 197–208 (2002).

Sparmann A, Van Lohuizen M: Polycomb silencers control cell fate, development and cancer. Nat. Rev. Cancer 6(11), 846–856 (2006).

3

Martinez AM, Schuettengruber B, Sakr S, Janic A, Gonzalez C, Cavalli G: Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling. Nat. Genet. 41(10), 1076–1082 (2009).

4

Lewis EB: A gene complex controlling segmentation in drosophila. Nature 276(5688), 565–570 (1978).

5

Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G: Genome regulation by Polycomb and trithorax proteins. Cell 128(4), 735–745 (2007).

6

Ringrose L: Polycomb comes of age: genome-wide profiling of target sites. Curr. Opin. Cell Biol. 19(3), 290–297 (2007).

7

Muller J, Verrijzer P: Biochemical mechanisms of gene regulation by Polycomb group protein complexes. Curr. Opin. Genet. Dev. 19(2), 150–158 (2009).

8

Pasini D, Bracken AP, Agger K et al.: Regulation of stem cell differentiation by histone methyltransferases and demethylases. Cold Spring Harb. Symp. Quant. Biol. 73, 253–263 (2008).

9

10


23 Cao R, Tsukada Y, Zhang Y: Role of Bmi-1

and Ring1a in H2A ubiquitylation and hox gene silencing. Mol. Cell 20, 845–854 (2005). 24 Lagarou A, Mohd-Sarip A, Moshkin YM

et al.: dkdm2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22(20), 2799–2810 (2008).

binding and histone methyltransferase activity of Drosophila PRC2. EMBO Rep. 6(4), 348–353 (2005). 14

Cao R, Wang L, Wang H et al.: Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039–1043 (2002).

15

Kuzmichev A, Nishioka K, ErdjumentBromage H, Tempst P, Reinberg D: Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein. Genes Dev. 16, 2893–2905 (2002).

16

n

Nekrasov M, Klymenko T, Fraterman S et al.: Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes. EMBO J. 26(18), 4078–4088 (2007). Reports a distinct form of Drosophila Polycomb repressor complex (PRC) 2 that contains the Polycomb group (PcG) protein Polycomb-like (Pcl) and provides biochemical and genetic evidence that Pcl is required to generate high levels of H3K27me3 at PcG target genes that are needed to maintain repression.

17

Cao R, Wang H, He J, Erdjument-Bromage H, Tempst P, Zhang Y: Role of hPHF1 in H3K27 methylation and hox gene silencing. Mol. Cell. Biol. 28(5), 1862–1872 (2008).

18

Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D: EZH2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol. Cell. Biol. 28(8), 2718–2731 (2008).

19

Shao Z, Raible F, Mollaaghababa R et al.: Stabilization of chromatin structure by PRC1, a Polycomb complex. Cell 98(1), 37–46 (1999).

20 Levine SS, Weiss A, Erdjument-Bromage H,

Shao Z, Tempst P, Kingston RE: The core of the Polycomb repressive complex is compositionally and functionally conserved in flies and humans. Mol. Cell. Biol. 22(17), 6070–6078 (2002).

Hekimoglu B, Ringrose L: Non-coding RNAs in Polycomb/trithorax regulation. RNA Biol. 6(2), (2009). Schuettengruber B, Cavalli G: Recruitment of polycomb group complexes and their role in the dynamic regulation of cell fate choice. Development 136(21), 3531–3542 (2009).

al.: Polycomb group proteins ring1a/b link ubiquitylation of histone h2A to heritable gene silencing and x inactivation. Dev. Cell 7, 663–676 (2004).

13 Nekrasov M, Wild B, Muller J: Nucleosome

Bantignies F, Cavalli G: Cellular memory and dynamic regulation of Polycomb group proteins. Curr. Opin. Cell Biol. 18(3), 275–283 (2006).

2

22 De Napoles M, Mermoud JE, Wakao R et

12 Müller J, Hart CM, Francis NJ et al.:

Bibliography Papers of special note have been highlighted as: n of interest nn of considerable interest

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

21

Wang H, Wang L, Erdjument-Bromage H et al.: Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873–878 (2004).


Review

n

Reveals a novel mode of histone cross-talk during gene silencing mediated by a new PcG complex called dRAF. dRAF harbors dRing, Posterior sex combs (Psc) and the demethylase dKdm2, and coordinates histone regulation by removing the active H3K36me2 mark and adding the repressive H2Aub mark.

25 Klymenko T, Papp B, Fischle W et al.:

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20(9), 1110–1122 (2006). 26 Grimaud C, Negre N, Cavalli G: From

genetics to epigenetics: the tale of Polycomb group and trithorax group genes. Chromosome Res. 14(4), 363–375. (2006). 27 Schuettengruber B, Ganapathi M, Leblanc B

et al.: Functional anatomy of Polycomb and trithorax chromatin landscapes in Drosophila embryos. PLoS Biol. 7(1), E13 (2009). nn

Reports the genome-wide binding profile of PcG proteins in Drosophila together with [29,30] . The authors mapped the chromosomal distribution in embryos for the PRC1 components PC and PH, the Trithorax protein and the H3K27me3 and H3K4me3 marks, as well as four candidate DNA-binding factors for PcG recruitment: Pleiohomeotic (PHO), PHOL, DSP1 and GAF. PcG proteins strongly colocalize and form large domain-containing multiple binding sites. However, the majority of PcG recruiter binding sites are associated with H3K4me3 and not with PcG binding. Interestingly, the PHO protein is strongly bound to silenced regions, whereas PHOL binds preferentially to active promoters.

28 Muller J, Kassis JA: Polycomb response

elements and targeting of Polycomb group proteins in Drosophila. Curr. Opin. Genet. Dev. 16, 476–484 (2006). 29 Kwong C, Adryan B, Bell I et al.: Stability

and dynamics of Polycomb target sites in Drosophila development. PLoS Genet. 4(9), E1000178 (2008).

313

Review n

Roure & Bantignies

Reports the genome-wide binding profile of PcG proteins in Drosophila. The authors analyzed the binging profile of Pc and Pho in embryos and larvae. The majority of the Pc-bound regions are also associated with Pho, but a large number of regions are only bound by Pho. Pc and Pho binding does not prevent transcription, but levels of Pc/Pho binding can change for a substantial set of genes during development, suggesting a dynamic relationship between Pc/Pho binding and gene transcription.

30 Oktaba K, Gutierrez L, Gagneur J et al.:

Dynamic regulation by Polycomb group protein complexes controls pattern formation and the cell cycle in Drosophila. Dev. Cell 15(6), 877–889 (2008). nn

31

Reports the genome-wide binding profile of PcG proteins in Drosophila. The binding profile of PhoRC is analyzed in embryos and larvae. PhoRC constitutively occupies short PcG response elements (PREs) of a large set of developmental regulators in both embryos and larvae. Many of the PhoRC binding sites are also co-occupied by PRC1 and PRC2. Functional analysis reveals that PcG proteins dynamically modulate the expression of several key developmental and cell-cycle regulators. Brown JL, Fritsch C, Mueller J, Kassis JA: The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130, 285–294 (2003).

nn

35

33 Mishra RK, Yamagishi T, Vasanthi D,

Ohtsuka C, Kondo T: Involvement of Polycomb-group genes in establishing hoxd temporal colinearity. Genesis 45(9), 570–576 (2007). n

Together with [34] , this study firstly identifies and functionally validates a vertebrate PRE. The authors investigate the region in the 5´ end of the mouse HoxD complex genes, which is required for their repression. They identify a 5-kb DNA fragment from this region that represses transgenes across species, from mouse to fly and in a PcG-dependent manner in flies. Moreover, PcG proteins directly associate with this repressive fragment in both fly and mouse.

34 Sing A, Pannell D, Karaiskakis A et al.: A

vertebrate Polycomb response element governs segmentation of the posterior hindbrain. Cell 138(5), 885–897 (2009).

314

42 Kim H, Kang K, Kim J: AEBP2 as a potential

Boyer LA, Plath K, Zeitlinger J et al.: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441(7091), 349–353 (2006).

44 Boukarabila H, Saurin AJ, Batsche E et al.:

targeting protein for Polycomb repression complex PRC2. Nucleic Acids Res. 37(9), 2940–2950 (2009). 43 Herranz N, Pasini D, Diaz VM et al.:

Polycomb complex 2 is required for e-cadherin repression by the snail1 transcription factor. Mol. Cell. Biol. 28(15), 4772–4781 (2008). The PRC1 Polycomb group complex interacts with PLZF/RARA to mediate leukemic transformation. Genes Dev. 23(10), 1195–1206 (2009).

36 Lee TI, Jenner RG, Boyer LA et al.: Control of

developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313 (2006).

45

37 Bracken AP, Dietrich N, Pasini D, Hansen

KH, Helin K: Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136 (2006). wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448(7153), 553–560 (2007). nn

Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4(10), E1000242 (2008). n

Provides genome-wide map histone modifications in pluripotent and lineage-committed mouse cells using ChIP-Seq technology. They report that H3K4me3 and H3K27me3 discriminate genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential.

39 Mohn F, Weber M, Rebhan M et al.:

Lineage-specific Polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30(6), 755–766 (2008). nn

Presents genome-wide DNA methylation and H3K27me3 mark during cellular differentiation from stem cells to lineagecommitted progenitors to terminally differentiated neurons in mouse. It reveals gain of DNA methylation during lineage commitment to restrict pluripotency, and a parallel acquisition of PcG repression to new target genes poised to be activated in subsequent terminal differentiation. This work challenges the view that PcG targets are mostly predetermined in stem cells.

transcriptional regulatory circuitry in human embryonic stem cells. Cell 122(6), 947–956 (2005). 48 Rinn JL, Kertesz M, Wang JK et al.:

Functional demarcation of active and silent chromatin domains in human hox loci by noncoding RNAs. Cell 129(7), 1311–1323 (2007). 49 Zhao J, Sun BK, Erwin JA, Song JJ, Lee JT:

Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science 322(5902), 750–756 (2008). 50 Khalil AM, Guttman M, Huarte M et al.:

Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl Acad. Sci. USA 106(28), 11667–11672 (2009).

Sartorelli V: The Polycomb EZH2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation. Genes Dev. 18(21), 2627–2638 (2004). Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110(3), 339–348 (2002).


Uses the ChIP-Seq method to map the genome-wide distribution of key histone modifications, as well as the subunits of PRC1 and PRC2 in human and mouse embryonic stem (ES) cells. Two classes of bivalent domains can be distinguished – the first occupied by both PRC1 and PRC2, and the second exclusively bound by PRC2 – that appear to be functionally distinct. Their analysis suggests that the genome-wide localizations of PRC2 and PRC1 in ES cells can be predicted from the locations, sizes and underlying motif contents of CpG islands.

47 Boyer LA, Lee TI, Cole MF et al.: Core

40 Caretti G, Di Padova M, Micales B, Lyons GE,

41 Gabellini D, Green MR, Tupler R:

Barna M, Merghoub T, Costoya JA et al.: Plzf mediates transcriptional repression of hoxD gene expression through chromatin remodeling. Dev. Cell 3(4), 499–510 (2002).

46 Ku M, Koche RP, Rheinbay E et al.:

38 Mikkelsen TS, Ku M, Jaffe DB et al.: Genome-

32 Déjardin J, Rappailles A, Cuvier O et al.:

Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1. Nature 434, 533–538 (2005).

Together with [33] , this study firstly identifies and functionally validates a vertebrate PRE. This PRE is called PRE-kr and it regulates expression of the mouse MafB/Kreisler gene. PRE-kr can recruit PcG proteins in flies and mouse and represses gene expression in a PcG/trxG-dependent manner in transgenic assays.

n

Reports that approximately 3300 functional large intergenic noncoding (linc) RNAs are encoded in the human genome, and that a significant proportion of these lincRNAs are physically associated with PRC2



(approximately 20%) or other chromatinmodifying complexes. They suggest a role for lincRNAs as a guide for chromatinmodifying complexes to specific genomic loci, in order to regulate gene expression. 51

Francis NJ, Kingston RE, Woodcock CL: Chromatin compaction by a Polycomb group protein complex. Science 306, 1574–1577 (2004).

52

Lo SM, Ahuja NK, Francis NJ: Polycomb group protein suppressor 2 of zeste is a functional homolog of posterior sex combs. Mol. Cell. Biol. 29(2), 515–525 (2009).

53 Margueron R, Li G, Sarma K et al.: EZH1

64 Chopra VS, Hong JW, Levine M: Regulation

Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells. Nat. Cell Biol. 9(12), 1428–1435 (2007). nn

54 Boivin A, Dura JM: In vivo chromatin

accessibility correlates with gene silencing in Drosophila. Genetics 150, 1539–1549 (1998). Fitzgerald DP, Bender W: Polycomb group repression reduces DNA accessibility. Mol. Cell. Biol. 21, 6585–6597 (2001).

transcription through a Polycomb group response element counteracts silencing. Genes Dev. 19, 697–708 (2005). Drewell RA: Characterization of the intergenic RNA profile at abdominal-a and abdominal-b in the Drosophila bithorax complex. Proc. Natl Acad. Sci. USA 99(26), 16847–16852 (2002).

Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell 32(2), 232–246 (2008).

V: General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412(6847), 651–655 (2001).


PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 6(12), 2911–2927 (2008). nn

72 Petruk S, Sedkov Y, Riley KM et al.:

73 Pandey RR, Mondal T, Mohammad F et al.:

74

Sexton T, Bantignies F, Cavalli G: Genomic interactions: chromatin loops and gene meeting points in transcriptional regulation. Semin. Cell Dev. Biol. 20(7), 849–855 (2009).


Analyses in vivo the three-dimensional structure of the bithorax complex locus in Drosophila by chromosome conformation capture (3C) and fluorescent in situ hybridization (FISH) immunostaining analysis and reveals that, in the repressed state, PcG-bound PREs and core promoters can interact at a distance to form a topologically complex structure. Pc depletion or gene activation disturbs these long-range interactions, suggesting that this chromatin loop structure is important for epigenetic silencing of the bithorax locus.

79 Tiwari VK, Mcgarvey KM, Licchesi JD et al.:

F: Noncoding RNAs of trithorax response elements recruit Drosophila ash1 to ultrabithorax. Science 311, 1118–1123 (2006).

62 Breiling A, Turner BM, Bianchi ME, Orlando

BM, Orlando V: Epigenome changes in active and inactive Polycomb-groupcontrolled regions. EMBO Rep. 5(10), 976–982 (2004).

nn

71 Sanchez-Elsner T, Gou D, Kremmer E, Sauer

Transcription of bxd noncoding RNAs promoted by trithorax represses ubx in cis by transcriptional interference. Cell 127(6), 1209–1221 (2006).

63 Breiling A, O’Neill LP, D’Eliseo D, Turner

Orlando V: Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex. Nat. Cell Biol. 9(10), 1167–1174 (2007).

70 Bae E, Calhoun VC, Levine M, Lewis EB,

Ficz G, Heintzmann R, Arndt-Jovin DJ: Polycomb group protein complexes exchange rapidly in living Drosophila. Development 132, 3963–3976 (2005).

61

78 Lanzuolo C, Roure V, Dekker J, Bantignies F,

69 Schmitt S, Prestel M, Paro R: Intergenic

60 Mito Y, Henikoff JG, Henikoff S: Histone

replacement marks the boundaries of cis-regulatory domains. Science 315(5817), 1408–1411 (2007).

et al.: Pre-mediated bypass of two su(hw) insulators targets PcG proteins to a downstream promoter. Dev. Cell 11(1), 117–124 (2006).

through intergenic chromosomal memory elements of the Drosophila bithorax complex correlates with an epigenetic switch. Mol. Cell. Biol. 22, 8026–8034 (2002).

59 Mishra RK, Mihaly J, Barges S et al.: The

iab-7 Polycomb response element maps to a nucleosome-free region of chromatin and requires both GAGA and pleiohomeotic for silencing activity. Mol. Cell. Biol. 21(4), 1311–1318 (2001).

Provides evidences that an important subset of bivalent genes in ES cells assembles the poised RNA Pol II complexes phosphorylated on Ser 5 and are weakly transcribed. Interestingly, this poised Pol II may have an unusual conformation that is enforced by PRC1 via H2Aub, indicative of a direct role of PcG-mediated ubiquitination in limiting RNA Pol II processivity at bivalent genes.

77 Comet I, Savitskaya E, Schuettengruber B

68 Rank G, Prestel M, Paro R: Transcription

58 Papp B, Muller J: Histone trimethylation and

the maintenance of transcriptional on and off states by trxG and PcG proteins. Genes Dev. 20, 2041–2054 (2006).

Probing long-distance regulatory interactions in the Drosophila melanogaster bithorax complex using dam identification. Nat. Genet. 38(8), 931–935 (2006).

iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing. Development 129, 4915–4922 (2002).

57 Mohd-Sarip A, Van Der Knaap JA,

Wyman C, Kanaar R, Schedl P, Verrijzer CP: Architecture of a Polycomb nucleoprotein complex. Mol. Cell 24(1), 91–100 (2006).

76 Cleard F, Moshkin Y, Karch F, Maeda RK:

67 Hogga I, Karch F: Transcription through the

56 Kuzmichev A, Jenuwein T, Tempst P,

Reinberg D: Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3. Mol. Cell 14, 183–193 (2004).

Dellino GI, Schwartz YB, Farkas G, McCabe D, Elgin SC, Pirrotta V: Polycomb silencing blocks transcription initiation. Mol. Cell 13, 887–893 (2004).

mechanisms and the management of genomic programmes. Nat. Rev. Genet. 8(1), 9–22 (2007).

66 Stock JK, Giadrossi S, Casanova M et al.:

and EZH2 maintain repressive chromatin through different mechanisms. Mol. Cell 32(4), 503–518 (2008).

55

75 Schwartz YB, Pirrotta V: Polycomb silencing

of hox gene activity by transcriptional elongation in Drosophila. Curr. Biol. 19(8), 688–693 (2009). 65

Review

PcG-bound regions physically interact around the GATA-4 locus to form chromatin loops in mammalian cells. The loop formation is associated with a poised, low transcription state in undifferentiated carcinoma cells, whereas upon cell differentiation, the chromatin loops completely dissolve accompanied by an increase of GATA-4 transcription. Interestingly, in colon cancer cells, abnormally DNA hypermethylated CpG islands are concomitant with the increase of long-range interaction frequency, leading to a complete repression.

80 Terranova R, Yokobayashi S, Stadler MB

et al.: Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev. Cell 15(5), 668–679 (2008). n

The authors combine RNA-FISH with immunostaining and DNA-FISH to characterize the higher-order chromatin and genomic organization of the mouse Kcnq1 imprinted cluster during extraembryonic

315

Review

Roure & Bantignies

development. They reveal that, contrary to the active maternal cluster, the paternal repressed cluster exists in a threedimensionally contracted state and organizes a repressive subnuclear compartment associated with PRC1/PRC2 but excluding RNA Pol II. 81

Yuzyuk T, Fakhouri TH, Kiefer J, Mango SE: The Polycomb complex protein mes-2/e(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos. Dev. Cell 16(5), 699–710 (2009).

82 Messmer S, Franke A, Paro R: Analysis of the

They focus their assay on EZH2 and identify EZH2-mediated long-range intra- and inter-chromosomal interactions that can regulate transcriptional downregulation of several genes by facilitating physical proximities between distant chromatin regions. 91

nn

84 Saurin AJ, Shiels C, Williamson J et al.: The

85 Grimaud C, Bantignies F, Pal-Bhadra M,

Ghana P, Bhadra U, Cavalli G: RNAi components are required for nuclear clustering of Polycomb group response elements. Cell 124(5), 957–971 (2006). 86 Tolhuis B, De Wit E, Muijrers I et al.:

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster. Nat. Genet. 38(6), 694–699 (2006). 87 Schwartz YB, Kahn TG, Nix DA et al.:

Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nature Genet. 38, 700–705 (2006).

89 Vazquez J, Muller M, Pirrotta V, Sedat JW:

The mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila. Mol. Biol. Cell 17, 2158–2165 (2006). 90 Tiwari VK, Cope L, Mcgarvey KM, Ohm JE,

Baylin SB: A novel 6c assay uncovers Polycomb-mediated higher order chromatin conformations. Genome Res. 18(7), 1171–1179 (2008). n

The authors develop a novel combined 3C–ChIP-cloning assay to address the question of long-range chromatin interactions mediated by specific proteins.

316

Demethylation of H3K27 regulates Polycomb recruitment and H2A ubiquitination. Science 318(5849), 447–450 (2007). nn

Reports for the first time the coexistence of both H3K27me3 repressive and H3K4me3 active chromatin marks at important developmental transcription factor genes in mouse ES cells. This specific modification pattern was termed ‘bivalent domain’. The authors initially proposed that these bivalent domains serve to silence developmental genes in ES cells, while keeping them poised for activation.

and JMJD3 are histone H3K27 demethylases involved in hox gene regulation and development. Nature 449(7163), 731–734 (2007). nn

Demonstrates the existence of the ‘bivalent domains’ at some silent lineage-specific genes in pluripotent murine ES cells.

Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes Dev. 22(9), 1115–1140 (2008). 101 Issaeva I, Zonis Y, Rozovskaia T et al.:

Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol. Cell. Biol. 27(5), 1889–1903 (2007).

94 Pan G, Tian S, Nie J et al.: Whole-genome

analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 1(3), 299–312 (2007). nn

genome mapping of histone H3 lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell Stem Cell 1(3), 286–298 (2007). nn

102 Smith ER, Lee MG, Winter B et al.:

Drosophila UTX is a histone H3 lys27 demethylase that colocalizes with the elongating form of RNA polymerase II. Mol. Cell. Biol. 28(3), 1041–1046 (2008).

One of the three studies, with [38] and [95] , of genome-wide mapping of the bivalent domains, in this case in human ES cells.

95 Zhao XD, Han X, Chew JL et al.: Whole-

Employed the ChIP coupled with a genome-wide paired-end-ditag (ChIP-PET) sequencing approach to map at high resolution the bivalent domains in human ES cells. In addition, they provide a comparison of their dataset with other ChIP-seq datasets from murine ES cells [38] and murine and human differentiated cells [38,97] . Bivalent domains are well conserved between the two species and reduced in more committed cells.

96 Roh TY, Cuddapah S, Cui K, Zhao K:

The genomic landscape of histone modifications in human T cells. Proc. Natl Acad. Sci. USA 103(43), 15782–15787 (2006).


Describes the human UTX, as well as JMJD3, as H3K27me3 demethylases.

100 Cloos PA, Christensen J, Agger K, Helin K:

signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538 (2006). nn

Describes UTX, a member of JmjC-family proteins, as a specific H3K27me3/me2 demethylase. Moreover, UTX associates with mixed-lineage leukemia (MLL) 2/3 complexes, a H3K4 methyltransferase. Therefore, this work uncovers a concerted mechanism for transcriptional activation in which cycles of H3K4 methylation by MLL2/3 are linked with demethylation of H3K27 by UTX.

99 Agger K, Cloos PA, Christensen J et al.: UTX

93 Azuara V, Perry P, Sauer S et al.: Chromatin

88 Bantignies F, Grimaud C, Lavrov S, Gabut

M, Cavalli G: Inheritance of Polycombdependent chromosomal interactions in Drosophila. Genes Dev. 17, 2406–2420 (2003).

98 Lee MG, Villa R, Trojer P et al.:

A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006).

Jovin DJ: The distribution of Polycombgroup proteins during cell division and development in drosophila embryos: impact on models for silencing. J. Cell Biol. 141, 469–481 (1998). human Polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain. J. Cell Biol. 142(4), 887–898 (1998).

High-resolution profiling of histone methylations in the human genome. Cell 129(4), 823–837 (2007).

92 Bernstein BE, Mikkelsen TS, Xie X et al.:

functional role of the Polycomb chromo domain in Drosophila melanogaster. Genes Dev. 6(7), 1241–1254 (1992). 83 Buchenau P, Hodgson J, Strutt H, Arndt-

Beisel C, Buness A, Roustan-Espinosa IM et al.: Comparing active and repressed expression states of genes controlled by the Polycomb/trithorax group proteins. Proc. Natl Acad. Sci. USA 104(42), 16615–16620 (2007).

97 Barski A, Cuddapah S, Cui K et al.:

n

As in mammals, the Drosophila dUtx specifically demethylates di- and trimethylated H3K27. Moreover, dUtx colocalizes with the elongating form of PolII (Ser2-phosphorylated) on active loci in larval polytene chromosomes.

103 Lan F, Bayliss PE, Rinn JL et al.: A histone

H3 lysine 27 demethylase regulates animal posterior development. Nature 449(7163), 689–694 (2007). 104 Nicassio F, Corrado N, Vissers JH et al.:

Human usp3 is a chromatin modifier required for S phase progression and genome stability. Curr. Biol. 17(22), 1972–1977 (2007). 105 Joo HY, Zhai L, Yang C et al.: Regulation of

cell cycle progression and gene expression by H2A deubiquitination. Nature 449(7165), 1068–1072 (2007).



most CpG islands remain umethylated upon differentiation. However, some of them completely lose H3K4 and H3K27 methylation concomitant with DNA hypermethylation. At low-CpG-density promoters, the loss or gain of H3K4 methylation during differentiation is a strong predictor of inverse changes in CpG methylation levels at these promoters.

106 Nakagawa T, Kajitani T, Togo S et al.:

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation. Genes Dev. 22(1), 37–49 (2008). 107 Zhu P, Zhou W, Wang J et al.: A histone H2A

deubiquitinase complex coordinating histone acetylation and H1 dissociation in transcriptional regulation. Mol. Cell 27(4), 609–621 (2007). 108 De Santa F, Totaro MG, Prosperini E,

Notarbartolo S, Testa G, Natoli G: The histone H3 lysine-27 demethylase jmjd3 links inflammation to inhibition of Polycombmediated gene silencing. Cell 130(6), 1083–1094 (2007). nn

This paper illustrates well that the H3K27me3 histone mark is involved in the control of differentiation and in the maintenance of cell identity, and that this mark can be rapidly erased by an inducible demethylase such as JMJD3 to permit cellular plasticity.

Frequent switching of Polycomb repressive marks and DNA hypermethylation in the pc3 prostate cancer cell line. Proc. Natl Acad. Sci. USA 105(35), 12979–12984 (2008). Polycomb-mediated methylation on lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat. Genet. 39(2), 232–236 (2007).

nn

As in [39] , this genome-wide DNA methylation study sheds light on the link between DNA methylation and histone methylation patterns in mouse pluripotent and differentiated cells. At high-CpGdensity promoters corresponding to ‘housekeeping genes’ and ‘key developmental genes’ (PcG target genes),


124 Agherbi H, Gaussmann-Wenger A, Verthuy C,

Chasson L, Serrano M, Djabali M: Polycomb mediated epigenetic silencing and replication timing at the ink4a/arf locus during senescence. PLoS ONE 4(5), E5622 (2009). nn

118 Widschwendter M, Fiegl H, Egle D et al.:

Epigenetic stem cell signature in cancer. Nat. Genet. 39(2), 157–158 (2007). 119 Klebes A, Sustar A, Kechris K, Li H, Schubiger

G, Kornberg TB: Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes. Development 132, 3753–3765 (2005). n

Using a DNA microarray approach, the authors analyze changes in gene expression during leg to wing transdetermination in Drosophila imaginal disc cells. Among the genes undergoing changes in expression levels, they find members of the PcG and the trxG. transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling. Development 125, 115–124 (1998).

The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4AARF locus in response to oncogene- and stress-induced senescence. Genes Dev. 23(10), 1171–1176 (2009). n

121 Lee N, Maurange C, Ringrose L, Paro R:

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs. Nature 438, 234–237 (2005). nn

The downregulation of PcG function, as monitored by the reactivation of a silent PcG-regulated reporter gene, is observed in transdetermined Drosophila imaginal disc


Demonstrates the downregulation of EZH2, but not BMI1, concomitant with an upregulation of the antagonist JMJD3 H3K27 demethylase during senescence. This leads to the transcriptional activation of the Ink4a/Arf locus. Moreover, the authors report that the PcG protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. This adds important information for the comprehensive role of PcG proteins during replication.

125 Agger K, Cloos PA, Rudkjaer L et al.:

120 Maves L, Schubiger G: A molecular basis for

112 Meissner A, Mikkelsen TS, Gu H et al.:

Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature 454(7205), 766–770 (2008).

N et al.: The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev. 21(5), 525–530 (2007).

117 Ohm JE, Mcgarvey KM, Yu X et al.: A stem

cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing. Nat. Genet. 39(2), 237–242 (2007).

Similar to the phenomenon observed during tissue regeneration in Drosophila, the PRC2 components Eed, Ezh2 and Suz12 are significantly downregulated during murine skin repair. Conversely, the demethylases JMJD3 and UTX that specifically remove the PcG-mediated histone mark, are coordinately upregulated during the repair process.

123 Bracken AP, Kleine-Kohlbrecher D, Dietrich

116 Schlesinger Y, Straussman R, Keshet I et al.:

111 Cedar H, Bergman Y: Linking DNA

methylation and histone modification: patterns and paradigms. Nat. Rev. Genet. 10(5), 295–304 (2009).

n

115 Gal-Yam EN, Egger G, Iniguez L et al.:

EZH2 orchestrates gene expression for the stepwise differentiation of tissue-specific stem cells. Cell 136(6), 1122–1135 (2009). The authors study the role of PcG-mediated chromatin repression in epidermal lineage differentiation in mouse embryo. They unveil EZH2 as a critical mediator of chromatin repression in progenitor basal cells, with its expression that diminishes concomitant with differentiation. Importantly, loss of Ezh2 function in these basal cells selectively derepresses genes associated with terminal epidermal differentiation, accelerating the normal maturation process of epidermal development.

during wound healing: loss of Polycombmediated silencing may enable upregulation of repair genes. EMBO Rep. 10(8), 881–886 (2009).

cooperates with DNMT1-associated protein 1 in gene silencing. Biochem. Biophys. Res. Commun. 353(4), 992–998 (2007).

110 Ezhkova E, Pasolli HA, Parker JS et al.:

nn

122 Shaw T, Martin P: Epigenetic reprogramming

114 Negishi M, Saraya A, Miyagi S et al.: BMI1

109 Lim DA, Huang YC, Swigut T et al.:

Chromatin remodelling factor MLL1 is essential for neurogenesis from postnatal neural stem cells. Nature 458(7237), 529–533 (2009).

cells. This downregulation is dependent on the JNK signaling pathway, which is activated in cells undergoing regeneration. The role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting chromatin to a reprogrammable state.

113 Vire E, Brenner C, Deplus R et al.: The

Polycomb group protein EZH2 directly controls DNA methylation. Nature 439(7078), 871–874 (2006).

Review

Using an oncogene-induced senescence (OIS) cell system, they report the concomitant downregulation of EZH2 (as well as SUZ12) with the specific upregulation of the antagonist JMJD3 demethylase, which affects the transcription of the Ink4a/Arf locus.

126 Barradas M, Anderton E, Acosta JC et al.:

Histone demethylase JMJD3 contributes to epigenetic control of INK4A/ARF by oncogenic ras. Genes Dev. 23(10), 1177–1182 (2009). n

Similar observations as in [125] , also using an OIS cell system.

317

Review

Roure & Bantignies

127 Chen X, Hiller M, Sancak Y, Fuller MT:

133 Henikoff S, Furuyama T, Ahmad K: Histone

Tissue-specific tafs counteract Polycomb to turn on terminal differentiation. Science 310(5749), 869–872 (2005). 128 Witte V, Laffert B, Rosorius O et al.: HIV-1

nef mimics an integrin receptor signal that recruits the Polycomb group protein eed to the plasma membrane. Mol. Cell 13(2), 179–190 (2004). 129 Su IH, Dobenecker MW, Dickinson E et al.:

Polycomb group protein EZH2 controls actin polymerization and cell signaling. Cell 121(3), 425–436 (2005). 130 Voncken JW, Schweizer D, Aagaard L, Sattler

L, Jantsch MF, Van Lohuizen M: Chromatinassociation of the Polycomb group protein bmi1 is cell cycle-regulated and correlates with its phosphorylation status. J. Cell Sci. 112 (Pt 24), 4627–4639 (1999). 131 Voncken JW, Niessen H, Neufeld B et al.:

MAPKAP kinase 3pK phosphorylates and regulates chromatin association of the Polycomb group protein BMI1. J. Biol. Chem. 280(7), 5178–5187 (2005). 132 Cha TL, Zhou BP, Xia W et al.: Akt-mediated

phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3. Science 310(5746), 306–310 (2005).

318

variants, nucleosome assembly and epigenetic inheritance. Trends Genet. 20(7), 320–326 (2004). 134 Hansen KH, Bracken AP, Pasini D et al.: A

model for transmission of the H3K27me3 epigenetic mark. Nat. Cell Biol. 10(11), 1291–1300 (2008). nn

Authors use three distinct approaches to propose a model for maintenance of the H3K27me3 epigenetic mark during cell division. Firstly, they show that EZH2 colocalizes with the H3K27me3 mark in G1 and at replication foci along with BrdU, CAF1 and PCNA throughout S phase. Secondly, using an in vitro histone peptide binding experiment, they report that EZH2 can bind directly with its own H3K27me3 site only as an EZH2–EED–SUZ12 complex. Third, using a heterologous reporter system, they show that the transient recruitment of the PRC2 to chromatin is sufficient to maintain the memory of the repressed chromatin mark through several cell divisions.

135 Francis NJ, Follmer NE, Simon MD, Aghia G,

Butler JD: Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro. Cell 137(1), 110–122 (2009).


n

Authors perform extensive in vitro studies with a Drosophila PRC1 core complex (PCC) containing PC, PSC and dRING. They show that PCC, once bound to chromatin or naked DNA, remains bound to it after replication, and that H3K27me3 is not required for this maintenance. They also report that PcG proteins are bound to chromatin during S phase and to newly replicated DNA in Drosophila S2 cells.

136 Liu J, Cao L, Chen J et al.: Bmi1 regulates

mitochondrial function and the DNA damage response pathway. Nature 459(7245), 387–392 (2009). 137 Takahashi K, Yamanaka S: Induction of

pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006). 138 Yamanaka S: A fresh look at iPS cells. Cell

137(1), 13–17 (2009). 139 Schwartz YB, Pirrotta V: Polycomb complexes

and epigenetic states. Curr. Opin. Cell Biol. 20(3), 266–273 (2008). 140 Margueron R, Justin N, Ohno K et al.: Role

of the polycomb protein EED in the propagation of repressive histone marks. Nature 461(7265), 762–767 (2009).
