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PRC2 mediated H3K27 methylations in cellular identity and cancer
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ScienceDirect PRC2 mediated H3K27 methylations in cellular identity and cancer Eric Conway1, Evan Healy1 and Adrian P Bracken The Polycomb Repressive Complex 2 (PRC2) is a multiprotein chromatin modifying complex that is essential for vertebrate development and differentiation. It is composed of a trimeric core of SUZ12, EED and EZH1/2 and is responsible for catalysing both di-methylation and tri-methylation of Histone H3 at lysine 27 (H3K27me2/3). Both H3K27 methylations contribute to the role of PRC2 in maintaining cellular identity. In all cell types, the H3K27me3 modification is associated with repression of genes encoding regulators of alternative lineages. The less well-characterised H3K27me2 modification is ubiquitous throughout the genome and is thought to act like a protective blanket to maintain the repression of nonH3K27me3 associated genes and cell-type-specific enhancers of alternative lineages. Recent cancer genome sequencing studies highlighted that several genes encoding PRC2 components as well as Histone H3 are mutated in multiple cancer types. Intriguingly, these cancers have changes in the global levels of the H3K27me2 and H3K27me3 modifications as well as genome-wide redistributions. Exciting new studies suggest that these changes confer context dependent blocks in cellular differentiation and increased vulnerability to aberrant cancer signalling pathways. Address Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Corresponding author: Bracken, Adrian P ([email protected]) 1
These authors contributed equally.
Current Opinion in Cell Biology 2015, 37:42–48 This review comes from a themed issue on Differentiation and disease Edited by Michael Rape and Scott A Armstrong
http://dx.doi.org/10.1016/j.ceb.2015.10.003 0955-0674/# 2015 Elsevier Ltd. All rights reserved.
Introduction In recent years, the sequencing of cancer genomes has led to the remarkable realisation that a large proportion of cancers carry mutations in genes encoding chromatin regulators [1]. A seminal review by Vogelstein highlighted that out of 140 cancer ‘driver’ genes, 30 encode chromatin regulators, clearly illustrating that a better understanding of their function is a pressing need [2]. Current Opinion in Cell Biology 2015, 37:42–48
Our review is focused on a group of chromatin regulators called Polycomb group proteins, which includes the wellknown histone methyltransferase, EZH2 of the Polycomb Repressive Complex 2 (PRC2). We discuss the roles of PRC2 mediated H3K27me2 and H3K27me3 modifications in cell fate decisions and the maintenance of cellular identity, then further relate this to our burgeoning understanding of how the deregulation of PRC2 function and H3K27 methylation states contribute to cancer.
The normal role of Polycombs and H3K27 methylations in lineage specification Polycomb group proteins were first identified in Drosophila as repressors of HOX genes during embryonic development [3]. They are a family of evolutionarily conserved chromatin repressors, also present in mammals [4], that have essential roles in maintaining the correct identities of stem, progenitor and differentiated cells [5,6]. Biochemically, Polycombs form two main multiprotein complexes, the Polycomb Repressive Complex 1 (PRC1) and, the primary focus of this review, PRC2. PRC2 consists of three core components; EED, SUZ12 and one of the two histone H3K27 methyltransferases, EZH1 or EZH2 [7]. Together, these three core components are required for mediating both the H3K27me2 and H3K27me3 histone post-translational modifications [7,8]. The importance of the H3K27 residue for PRC2 function was elegantly confirmed by the demonstration that in Drosophila, cells with an H3K27R point mutation fail to maintain the repressed state of HOX genes [9]. The H3K27me3 modification has been linked with the repression of HOX genes due to its role in recruiting the PRC1 complex, which in turn mediates H2AK119ub (Figure 1). The importance of H2AK119ub for Polycomb function is disputed, however, it is clear that the PRC1 complex functions to repress transcription by additional mechanisms, such as chromatin compaction [10,11]. The first genome-wide mapping studies of components of the PRC1 and PRC2 complexes confirmed that they co-localise together with H3K27me3 on the promoters of 10–15% of all genes [12–15]. Importantly, these include HOX and other genes encoding proteins with key roles in development and cell fate determination in all types of cells, for example, BMP, WNT, NANOG and SOX, and the INK4A gene, which regulates proliferation [16]. A model emerged in which the PRC2 and PRC1 complexes function to preserve cellular identity of stem and differentiated cells by silencing genes of alternative cell fates [5] (Figure 1). www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 43
Figure 1
Cell Type A PRC1 PRC2
PRC1 PRC2 ON
OFF
CBX
CBX
OFF
Stem Cell PRC1 PRC2 ON
CBX
Stem Cell genes
PRC1 PRC2 OFF
CBX
Cell Type A genes Cell Type B genes
OFF
Cell Type B Stem Cell genes
Cell Type A genes Cell Type B genes PRC1 PRC2 CBX
PRC1 PRC2 OFF
Stem Cell genes
CBX
OFF
ON
Cell Type A genes Cell Type B genes
Current Opinion in Cell Biology
Polycombs and H3K27me3 during cellular differentiation. This model depicts a stem cell that has the potential to differentiate into two independent cell lineages: Cell type A and Cell type B. The stem cell expresses ‘stem cell genes’ that are required to maintain its proliferative and undifferentiated state. Before the signal to differentiate, the PRC2 and PRC1 complexes, co-bound at sites of H3K27me3 (represented by three red circles), function to repress the transcription of lineage-specific differentiation or ‘cell type’ genes. The CBX component of the PRC1 complex contains a chromodomain that specifically binds to the PRC2 mediated H3K27me3 modification. Upon stimulation to differentiate, both PRC2 and PRC1 complexes are re-located to the stem cell-specific gene promoters and displaced from the lineage-specific gene promoters. The mechanism by which the PRC2 complex is recruited to target genes is still not fully understood, although cessation of transcription and nucleosome compaction have been implicated in the process. In the differentiated cell types A and B, the Polycomb proteins silence not only the expression of stem cell genes, but also the expression of genes that encode regulators of alternative lineages. This mechanism of ‘locking’ cell fate is thought to be central to how cells maintain their identity through subsequent cell divisions. Importantly, these mechanisms and especially PRC2 and H3K27 methylations are deregulated in cancer.
Recent evidence suggests that the targeting or subsequent binding of the PRC2 complex to unmethylated CpG islands may be in part dependent on non-canonical PRC1 complexes containing CpG island binding proteins, such as KDM2B [17–22]. We also know that PRC2 is recruited to unmethylated CpG sites to mediate H3K27me3 upon cessation of transcription and nucleosome compaction [23–25]. Intriguingly, much less is known about the biological importance of H3K27me2. This modification is PRC2 complex dependent, is found ubiquitously throughout the genome and appears to be the ‘default setting’ for H3K27 [8,26]. Supporting this, in both mammals and Drosophila, H3K27me2 is localised to almost all euchromatin regions except the gene bodies of actively transcribed genes (instead marked by H3K27me1), active enhancers (instead marked by CBP/p300 mediated H3K27ac) and Polycomb repressed gene promoters (instead marked by H3K27me3), as depicted in Figure 2. Paradoxically, the PRC2 complex does not co-localise with these large H3K27me2 regions [8,26]. This has been rationalised by the fact that di-methylation of H3K27 is enzymatically easier for PRC2 and can be mediated by a transient interaction, unlike H3K27me3, www.sciencedirect.com
which requires a more stable association [8,27,28]. The H3K27me2 modification is likely mediated on nascent Histone H3 during or soon after DNA replication, while H3K27me3 accumulates more slowly [8,29]. The ubiquitous genome-wide profile and repressive nature of H3K27me2 suggests that its role is to act as a ‘repressive blanket’ to prevent the misfiring of cell-type-specific enhancers and gene promoters required for alternative lineages [8,26]. Consistent with this idea, the loss of PRC2 function in Drosophila cells leads to increases in the transcription of intragenic mRNAs coupled with increases in H3K27ac at sites previously protected by H3K27me2 [26]. A picture is emerging in which the PRC2 mediated H3K27me2 and H3K27me3 repressive modifications function at distinct genomic regions to maintain and protect cellular identity from trans-differentiation and de-differentiation (Figure 2). Further unravelling of the normal roles of H3K27me2 and H3K27me3 will be central to understanding the regulation of cell fate transitions and maintenance of cellular identity. Moreover, this will most likely provide important insights into how the frequent deregulation of PRC2 function and H3K27 methylation contributes to cancer (Table 1). Current Opinion in Cell Biology 2015, 37:42–48
44 Differentiation and disease
PRC1 CBX
Z1 2
EED
SU
SU
Z1 2
Figure 2
EED
EZH2/1
EZH2/1 H3K27me3 OFF
H3K27me2
Repressed Polycomb target gene
CBP/p300
H3K27me2
H3K27Ac
Active Enhancer
SU
Z1
2
H3K27me2
EED
EZH2/1 ?
ON
H3K27me1
Active gene Current Opinion in Cell Biology
Diversity of Histone 3 modification states and their roles in the control of transcription. This model depicts the variable modifications of Histone 3 at lysine 27 in distinct genomic contexts and the role of the PRC2 complex in mediating different H3K27 methylations (represented by red circles). The PRC2 and PRC1 complexes co-bind at sites of H3K27me3 on Polycomb target genes. The PRC2 complex is also responsible for all H3K27me2 deposition genome wide, which represents the majority of the human genome and likely serves as the default modification on H3K27. H3K27me2 is a repressive modification and likely functions to suppress the aberrant activation of non-cell-type-specific enhancers and promoters. Importantly, H3K27me2 does not occupy the sites of active enhancers, which have CBP/p300 mediated H3K27ac (represented by one green circle), or the gene bodies of active genes, which have H3K27me1, which may also be mediated, at least in part, by PRC2 action.
PRC2 and H3K27 methylations are deregulated in cancer Since 2010, multiple cancer genome sequencing studies have revealed that the function of the PRC2 complex and H3K27 methylations are frequently disrupted on the genetic level [53,54] (Table 1). In 2003, EZH2 was reported to be highly expressed in multiple cancers as a result of being an E2F regulated gene, downstream of the pRB pathway [55]. However, this elevated expression is generally not considered to lead to deregulation of H3K27 methylations due to the stoichiometry of the PRC2 complex and the requirement for correspondingly high levels of Current Opinion in Cell Biology 2015, 37:42–48
EED and SUZ12. This contrasts with the remarkable nature of the recurrent ‘change-of-function’ mutations in EZH2, first identified in B-cell lymphomas, which confer an enhanced ability to convert H3K27me2 to H3K27me3 [27,28,30,31,32–35]. The consequence of these heterozygous mutations is aberrantly high global levels of H3K27me3 and reduced H3K27me2. The discovery that EZH2, EED and SUZ12 have deletions and inactivating mutations in T-cell leukemias (T-ALL) [38–40] and malignant peripheral nerve sheath tumours (MPNST) [44,47] was initially surprising, because PRC2 function was considered as being oncogenic. These ‘loss of function’ www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 45
Table 1 Mutations in PRC2 members and Histone H3 coding genes in cancer Aberration
Gene
EZH2 ‘change of function’ mutations EZH2 pTyr641X
pAla677Gly pAla687Val PRC2 loss of function mutations EZH2 Homozygous mutation Heterozygous mutation SUZ12 Mutation Heterozygous deletion Heterozygous deletion and mutation Homozygous deletion EED Heterozygous deletion and mutation Heterozygous deletion Homozygous deletion AEBP2 Mutation Histone H3 mutations H3F3A pLys27Met mutation HIST1H3B
pLys27Met mutation
Cancer type (frequency %)
References
Lymphoma (9–24%), parathyroid adenoma (1%), ALL (2%), melanoma (2%) Lymphoma (1–2%), Ewing sarcoma (5%) Lymphoma (1–2%)
Elevated H3K27me3 Reduced H3K27me2
[30,31,32–35]
Elevated H3K27me3 Reduced H3K27me2 Elevated H3K27me3 Reduced H3K27me2
[30,32,36]
Leukemia (4%), myeloid disorders (1–3%) Leukemia (1%), myeloid disorders (6%) MDS/MPN (1–3%), leukemia (2–3%) MPNST (4–25%) MPNST (15–22%)
Reduced H3K27me3
[38–40]
Reduced H3K27me3
[38–40]
Reduced H3K27me3
[41–46]
Reduced H3K27me3
[44,47]
MPNST (16–26%)
Reduced H3K27me3
[44,47]
MPNST (12%)
Reduced H3K27me3
[44,47]
MPNST (2–5%)
Reduced H3K27me3
[44,47]
MPNST (3–14%)
Reduced H3K27me3
[44,47]
MPNST (10%)
Reduced H3K27me3
[44,47]
Leukemia (1%), MPNST (7%)
Reduced H3K27me3
[32,41]
High grade glioma (18–71%), low grade glioma (1–2%), leukemia (1%) High grade glioma (3–18%)
Reduced Reduced Reduced Reduced
mutations feature reduced levels of both H3K27me2 and H3K27me3. Another example of apparent loss of PRC2 function in cancer was the discovery of paediatric gliomas with H3K27M mutations in two genes encoding histone H3, H3F3A (H3.3) or HIST3H1B (H3.1) [32,48–50,51,52]. These gliomas also have global reductions in H3K27me2 and H3K27me3, which is particularly remarkable when one considers that the H3F3A and HIST3H1B genes are just two of 15 different genes encoding Histone H3 and that the mutations are heterozygous. Subsequent work by Lewis and colleagues explained this phenomenon when they showed that transgenes of H3K27M act as dominant negatives to block the enzymatic activity of the PRC2 complex [56,57,58]. A key open question is how do all the various alterations in PRC2 and Histone H3 genes contribute to cancer progression.
Deregulation of H3K27 methylation confers a blockage of cellular differentiation in cancer cells Several studies have begun to address the genome-wide consequences of PRC2 deregulation in real tumours and model systems. In a mouse model of B-cell lymphoma, EZH2 ‘change of function’ mutants have been shown to www.sciencedirect.com
H3K27 methylation status
H3K27me3 H3K27me2 H3K27me3 H3K27me2
[30,36,37]
[32,48–50,51,52] [32,51]
increase global H3K27me3 levels in vivo and lead to a block in cellular differentiation [59]. Interestingly, in a mouse model of myelodysplastic syndrome (MDS) with a conditional biallelic deletion of EZH2, while the total levels of H3K27me3 were reduced, genome-wide profiling identified genes with H3K27me3 increases [60]. This was rationalised by the fact that the EZH1 methyltransferase is still available to methylate H3K27. Similarly, in an EZH2 knockout model of T-ALL, while H3K27me2 and H3K27me3 were both reduced on the global level, both modifications were never completely absent [61]. The implication of these studies is that key genes associated with differentiation gain elevated levels of H3K27me3 and become more potently repressed, thus conferring a differentiation block, while cohorts of genes associated with cancer become activated due to loss of H3K27me3. Similar redistributions of H3K27me3 are observed in gliomas with H3K27M mutations [57–62], which report global reductions, but not complete loss of H3K27me3, coupled with localised increases at particular genomic sites. This again suggests a form of epigenomic reprogramming. Therefore one link between the PRC2 ‘change of function’ mutations, the EZH2 loss of function mutants and the H3K27M gliomas is redistributions of Current Opinion in Cell Biology 2015, 37:42–48
46 Differentiation and disease
H3K27me3 contributing to a block of differentiation and aberrant activation of cancer-associated genes.
the importance of this less well characterised H3K27 modification and point to its potential importance as a global repressive mark whose function is to protect non-cell-type specific enhancers and promoters from aberrant activation, thus helping to preserve cellular identity.
In future studies, it will be important to consider that either loss or gain of PRC2 function not only affect H3K27me3, but also affect regions of H3K27me2 genome-wide. These reductions or complete losses of H3K27me2 have thus far been overlooked when examining epigenomic profiles of cancer cells. Although we do not yet fully understand the H3K27me2 modification or how it confers repression, one could speculate that its loss or redistribution could lead to a more open or ‘stem-like’ chromatin state, consistent with the more general idea of how the deregulation of chromatin regulators contribute to cancer [63]. Furthermore, the reductions in H3K27me2 and H3K27me3 methylation profiles possibly make the cells more amenable to the pathogenic signalling pathways associated with cancer [44]. In contrast, the increased global levels of H3K27me3 observed in lymphomas and melanomas could confer a more closed, refractory epigenomic state, non-responsive to differentiation signals.
9.
Future insights into the role of the H3K27 modifications in cellular identity will allow a better understanding of their contribution to cancer and potentially allow for improved treatments for cancers such as paediatric glioblastomas, melanomas and B-cell lymphomas.
15. Schwartz YB, Kahn TG, Nix DA, Li XY, Bourgon R, Biggin M, Pirrotta V: Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat Genet 2006, 38:700-705.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Dawson MA, Kouzarides T: Cancer epigenetics: from mechanism to therapy. Cell 2012, 150:12-27.
2.
Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW: Cancer genome landscapes. Science 2013, 339:1546-1558. This review provides an excellent overview of the key findings of the genome sequencing of human cancers and an objective list of all oncogenes and tumour suppressor genes.
Pengelly AR, Copur O, Jackle H, Herzig A, Muller J: A histone mutant reproduces the phenotype caused by loss of histonemodifying factor Polycomb. Science 2013, 339:698-699. This elegant study was the first to unambiguously confirm that the Lysine 27 residue on Histone H3 is essential for PRC2 complex mediated repression of HOX genes during development.
10. Pengelly AR, Kalb R, Finkl K, Muller J: Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation. Genes Dev 2015, 29:1487-1492. 11. Isono K, Endo TA, Ku M, Yamada D, Suzuki R, Sharif J, Ishikura T, Toyoda T, Bernstein BE, Koseki H: SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing. Dev Cell 2013, 26:565-577. 12. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK et al.: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006, 441:349-353. 13. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K: Genomewide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 2006, 20:1123-1136. 14. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K et al.: Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006, 125:301-313.
16. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, Theilgaard-Monch K, Minucci S, Porse BT, Marine JC et al.: The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev 2007, 21:525-530. 17. Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y et al.: Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell 2014, 157:1445-1459. 18. Cooper S, Dienstbier M, Hassan R, Schermelleh L, Sharif J, Blackledge NP, De Marco V, Elderkin S, Koseki H, Klose R et al.: Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment. Cell Rep 2014, 7:1456-1470. 19. Farcas AM, Blackledge NP, Sudbery I, Long HK, McGouran JF, Rose NR, Lee S, Sims D, Cerase A, Sheahan TW et al.: KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. Elife 2012, 1:e00205.
3.
Steffen PA, Ringrose L: What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory. Nat Rev Mol Cell Biol 2014, 15:340-356.
4.
Di Croce L, Helin K: Transcriptional regulation by Polycomb group proteins. Nat Struct Mol Biol 2013, 20:1147-1155.
5.
Bracken AP, Helin K: Polycomb group proteins: navigators of lineage pathways led astray in cancer. Nature Reviews Cancer 2009, 9:773-784.
21. Kalb R, Latwiel S, Baymaz HI, Jansen PW, Muller CW, Vermeulen M, Muller J: Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat Struct Mol Biol 2014, 21:569-571.
6.
Pietersen AM, van Lohuizen M: Stem cell regulation by polycomb repressors: postponing commitment. Curr Opin Cell Biol 2008, 20:201-207.
22. Wu X, Johansen JV, Helin K: Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation. Mol Cell 2013, 49:1134-1146.
7.
Margueron R, Reinberg D: The Polycomb complex PRC2 and its mark in life. Nature 2011, 469:343-349.
23. Yuan W, Wu T, Fu H, Dai C, Wu H, Liu N, Li X, Xu M, Zhang Z, Niu T et al.: Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 2012, 337:971-975.
8.
Ferrari KJ, Scelfo A, Jammula S, Cuomo A, Barozzi I, Stutzer A, Fischle W, Bonaldi T, Pasini D: Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. Mol Cell 2014, 53:49-62. This paper and Ref. [26] establish that the H3K27me2 modification is broadly located in both Drosophila and mammalian cells. They highlight
Current Opinion in Cell Biology 2015, 37:42–48
20. He J, Shen L, Wan M, Taranova O, Wu H, Zhang Y: Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat Cell Biol 2013, 15:373-384.
24. Jermann P, Hoerner L, Burger L, Schubeler D: Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation. Proc Natl Acad Sci U S A 2014, 111:E3415-E3421. www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 47
25. Riising EM, Comet I, Leblanc B, Wu X, Johansen JV, Helin K: Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol Cell 2014, 55:347-360. 26. Lee HG, Kahn TG, Simcox A, Schwartz YB, Pirrotta V: Genomewide activities of Polycomb complexes control pervasive transcription. Genome Res 2015, 25:1170-1181. 27. Sneeringer CJ, Scott MP, Kuntz KW, Knutson SK, Pollock RM, Richon VM, Copeland RA: Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci U S A 2010, 107:20980-20985. This paper helped to explain how EZH2 ‘change of function’ mutations in B-cell Lymphomas lead to deregulated H3K27 methylation. It established that the EZH2 mutants have unique substrate specificities allowing them to lead to elevated global levels of H3K27me3 and lower levels of H3K27me2. 28. McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, Smitheman KN, Ott HM, Pappalardi MB, Allen KE et al.: Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc Natl Acad Sci U S A 2012, 109:2989-2994. 29. Alabert C, Barth TK, Reveron-Gomez N, Sidoli S, Schmidt A, Jensen ON, Imhof A, Groth A: Two distinct modes for propagation of histone PTMs across the cell cycle. Genes Dev 2015, 29:585-590. 30. Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD, Johnson NA, Severson TM, Chiu R, Field M et al.: Frequent mutation of histone-modifying genes in nonHodgkin lymphoma. Nature 2011, 476:298-303.
39. Jankowska AM, Makishima H, Tiu RV, Szpurka H, Huang Y, Traina F, Visconte V, Sugimoto Y, Prince C, O’Keefe C et al.: Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 2011, 118: 3932-3941. 40. Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, Waghorn K, Zoi K, Ross FM, Reiter A et al.: Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010, 42:722-726. 41. Zhang M, Wang Y, Jones S, Sausen M, McMahon K, Sharma R, Wang Q, Belzberg AJ, Chaichana K, Gallia GL et al.: Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors. Nat Genet 2014, 46:1170-1172. 42. Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, Easton J, Chen X, Wang J, Rusch M et al.: The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012, 481:157-163. 43. Score J, Hidalgo-Curtis C, Jones AV, Winkelmann N, Skinner A, Ward D, Zoi K, Ernst T, Stegelmann F, Dohner K et al.: Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms. Blood 2012, 119:1208-1213. 44. De Raedt T, Beert E, Pasmant E, Luscan A, Brems H, Ortonne N, Helin K, Hornick JL, Mautner V, Kehrer-Sawatzki H et al.: PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature 2014, 514:247-251. 45. Brecqueville M, Rey J, Bertucci F, Coppin E, Finetti P, Carbuccia N, Cervera N, Gelsi-Boyer V, Arnoulet C, Gisserot O et al.: Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes Cancer 2012, 51:743-755.
31. Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, Paul JE, Boyle M, Woolcock BW, Kuchenbauer F et al.: Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 2010, 42:181-185. This was the first paper to identify mutations in EZH2, which subsequently were shown to be ‘change of function’ mutations and led to the rapid advancement of EZH2 inhibitor drugs and their clinical trials for the personalised treatment of patients with these mutations in B-cell Lymphomas.
46. Beekman R, Valkhof MG, Sanders MA, van Strien PM, Haanstra JR, Broeders L, Geertsma-Kleinekoort WM, Veerman AJ, Valk PJ, Verhaak RG et al.: Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia. Blood 2012, 119:5071-5077.
32. Huether R, Dong L, Chen X, Wu G, Parker M, Wei L, Ma J, Edmonson MN, Hedlund EK, Rusch MC et al.: The landscape of somatic mutations in epigenetic regulators across 1,000 paediatric cancer genomes. Nat Commun 2014, 5:3630.
47. Lee W, Teckie S, Wiesner T, Ran L, Prieto Granada CN, Lin M, Zhu S, Cao Z, Liang Y, Sboner A et al.: PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nat Genet 2014, 46:1227-1232.
33. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, Nickerson E, Auclair D, Li L, Place C et al.: A landscape of driver mutations in melanoma. Cell 2012, 150:251-263.
48. Gielen GH, Gessi M, Hammes J, Kramm CM, Waha A, Pietsch T: H3F3A K27M mutation in pediatric CNS tumors: a marker for diffuse high-grade astrocytomas. Am J Clin Pathol 2013, 139:345-349.
34. Cromer MK, Starker LF, Choi M, Udelsman R, Nelson-Williams C, Lifton RP, Carling T: Identification of somatic mutations in parathyroid tumors using whole-exome sequencing. J Clin Endocrinol Metab 2012, 97:E1774-E1781.
49. Khuong-Quang DA, Buczkowicz P, Rakopoulos P, Liu XY, Fontebasso AM, Bouffet E, Bartels U, Albrecht S, Schwartzentruber J, Letourneau L et al.: K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol 2012, 124:439-447.
35. Barsotti AM, Ryskin M, Zhong W, Zhang WG, Giannakou A, Loreth C, Diesl V, Follettie M, Golas J, Lee M et al.: Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth. Oncotarget 2015, 6:2928-2938. 36. Bodor C, Grossmann V, Popov N, Okosun J, O’Riain C, Tan K, Marzec J, Araf S, Wang J, Lee AM et al.: EZH2 mutations are frequent and represent an early event in follicular lymphoma. Blood 2013, 122:3165-3168. 37. Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C, Cruz-Gordillo P, Knoechel B, Asmann YW, Slager SL et al.: Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci U S A 2012, 109:3879-3884. 38. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T et al.: Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010, 42:665-667. www.sciencedirect.com
50. Sturm D, Witt H, Hovestadt V, Khuong-Quang DA, Jones DT, Konermann C, Pfaff E, Tonjes M, Sill M, Bender S et al.: Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 2012, 22:425-437. 51. Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, Qu C, Ding L, Huether R, Parker M et al.: Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 2012, 44:251-253. This was the first paper to identify H3K27M mutations in gliomas. 52. Zhang J, Wu G, Miller CP, Tatevossian RG, Dalton JD, Tang B, Orisme W, Punchihewa C, Parker M, Qaddoumi I et al.: Wholegenome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat Genet 2013, 45:602-612. 53. Plass C, Pfister SM, Lindroth AM, Bogatyrova O, Claus R, Lichter P: Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nat Rev Genet 2013, 14:765-780. Current Opinion in Cell Biology 2015, 37:42–48
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54. Helin K, Dhanak D: Chromatin proteins and modifications as drug targets. Nature 2013, 502:480-488. 55. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K: EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. Embo J 2003, 22:5323-5335. 56. Lewis PW, Muller MM, Koletsky MS, Cordero F, Lin S, Banaszynski LA, Garcia BA, Muir TW, Becher OJ, Allis CD: Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 2013, 340:857-861. This paper, together with paper [58,59], established a dominant negative mechanism by which the H3K27M transgenes could cause global changes in H3K27 methylation. 57. Chan KM, Fang D, Gan H, Hashizume R, Yu C, Schroeder M, Gupta N, Mueller S, James CD, Jenkins R et al.: The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev 2013, 27:985-990. 58. Bender S, Tang Y, Lindroth AM, Hovestadt V, Jones DT, Kool M, Zapatka M, Northcott PA, Sturm D, Wang W et al.: Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas. Cancer Cell 2013, 24:660-672.
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59. Beguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, Shen H, Yang SN, Wang L, Ezponda T et al.: EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 2013, 23:677-692. 60. Muto T, Sashida G, Oshima M, Wendt GR, Mochizuki-Kashio M, Nagata Y, Sanada M, Miyagi S, Saraya A, Kamio A et al.: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders. J Exp Med 2013, 210:2627-2639. 61. Simon C, Chagraoui J, Krosl J, Gendron P, Wilhelm B, Lemieux S, Boucher G, Chagnon P, Drouin S, Lambert R et al.: A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia. Genes Dev 2012, 26:651-656. 62. Funato K, Major T, Lewis PW, Allis CD, Tabar V: Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation. Science 2014, 346: 1529-1533. 63. Suva ML, Riggi N, Bernstein BE: Epigenetic reprogramming in cancer. Science 2013, 339:1567-1570.
www.sciencedirect.com
ScienceDirect PRC2 mediated H3K27 methylations in cellular identity and cancer Eric Conway1, Evan Healy1 and Adrian P Bracken The Polycomb Repressive Complex 2 (PRC2) is a multiprotein chromatin modifying complex that is essential for vertebrate development and differentiation. It is composed of a trimeric core of SUZ12, EED and EZH1/2 and is responsible for catalysing both di-methylation and tri-methylation of Histone H3 at lysine 27 (H3K27me2/3). Both H3K27 methylations contribute to the role of PRC2 in maintaining cellular identity. In all cell types, the H3K27me3 modification is associated with repression of genes encoding regulators of alternative lineages. The less well-characterised H3K27me2 modification is ubiquitous throughout the genome and is thought to act like a protective blanket to maintain the repression of nonH3K27me3 associated genes and cell-type-specific enhancers of alternative lineages. Recent cancer genome sequencing studies highlighted that several genes encoding PRC2 components as well as Histone H3 are mutated in multiple cancer types. Intriguingly, these cancers have changes in the global levels of the H3K27me2 and H3K27me3 modifications as well as genome-wide redistributions. Exciting new studies suggest that these changes confer context dependent blocks in cellular differentiation and increased vulnerability to aberrant cancer signalling pathways. Address Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2, Ireland Corresponding author: Bracken, Adrian P ([email protected]) 1
These authors contributed equally.
Current Opinion in Cell Biology 2015, 37:42–48 This review comes from a themed issue on Differentiation and disease Edited by Michael Rape and Scott A Armstrong
http://dx.doi.org/10.1016/j.ceb.2015.10.003 0955-0674/# 2015 Elsevier Ltd. All rights reserved.
Introduction In recent years, the sequencing of cancer genomes has led to the remarkable realisation that a large proportion of cancers carry mutations in genes encoding chromatin regulators [1]. A seminal review by Vogelstein highlighted that out of 140 cancer ‘driver’ genes, 30 encode chromatin regulators, clearly illustrating that a better understanding of their function is a pressing need [2]. Current Opinion in Cell Biology 2015, 37:42–48
Our review is focused on a group of chromatin regulators called Polycomb group proteins, which includes the wellknown histone methyltransferase, EZH2 of the Polycomb Repressive Complex 2 (PRC2). We discuss the roles of PRC2 mediated H3K27me2 and H3K27me3 modifications in cell fate decisions and the maintenance of cellular identity, then further relate this to our burgeoning understanding of how the deregulation of PRC2 function and H3K27 methylation states contribute to cancer.
The normal role of Polycombs and H3K27 methylations in lineage specification Polycomb group proteins were first identified in Drosophila as repressors of HOX genes during embryonic development [3]. They are a family of evolutionarily conserved chromatin repressors, also present in mammals [4], that have essential roles in maintaining the correct identities of stem, progenitor and differentiated cells [5,6]. Biochemically, Polycombs form two main multiprotein complexes, the Polycomb Repressive Complex 1 (PRC1) and, the primary focus of this review, PRC2. PRC2 consists of three core components; EED, SUZ12 and one of the two histone H3K27 methyltransferases, EZH1 or EZH2 [7]. Together, these three core components are required for mediating both the H3K27me2 and H3K27me3 histone post-translational modifications [7,8]. The importance of the H3K27 residue for PRC2 function was elegantly confirmed by the demonstration that in Drosophila, cells with an H3K27R point mutation fail to maintain the repressed state of HOX genes [9]. The H3K27me3 modification has been linked with the repression of HOX genes due to its role in recruiting the PRC1 complex, which in turn mediates H2AK119ub (Figure 1). The importance of H2AK119ub for Polycomb function is disputed, however, it is clear that the PRC1 complex functions to repress transcription by additional mechanisms, such as chromatin compaction [10,11]. The first genome-wide mapping studies of components of the PRC1 and PRC2 complexes confirmed that they co-localise together with H3K27me3 on the promoters of 10–15% of all genes [12–15]. Importantly, these include HOX and other genes encoding proteins with key roles in development and cell fate determination in all types of cells, for example, BMP, WNT, NANOG and SOX, and the INK4A gene, which regulates proliferation [16]. A model emerged in which the PRC2 and PRC1 complexes function to preserve cellular identity of stem and differentiated cells by silencing genes of alternative cell fates [5] (Figure 1). www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 43
Figure 1
Cell Type A PRC1 PRC2
PRC1 PRC2 ON
OFF
CBX
CBX
OFF
Stem Cell PRC1 PRC2 ON
CBX
Stem Cell genes
PRC1 PRC2 OFF
CBX
Cell Type A genes Cell Type B genes
OFF
Cell Type B Stem Cell genes
Cell Type A genes Cell Type B genes PRC1 PRC2 CBX
PRC1 PRC2 OFF
Stem Cell genes
CBX
OFF
ON
Cell Type A genes Cell Type B genes
Current Opinion in Cell Biology
Polycombs and H3K27me3 during cellular differentiation. This model depicts a stem cell that has the potential to differentiate into two independent cell lineages: Cell type A and Cell type B. The stem cell expresses ‘stem cell genes’ that are required to maintain its proliferative and undifferentiated state. Before the signal to differentiate, the PRC2 and PRC1 complexes, co-bound at sites of H3K27me3 (represented by three red circles), function to repress the transcription of lineage-specific differentiation or ‘cell type’ genes. The CBX component of the PRC1 complex contains a chromodomain that specifically binds to the PRC2 mediated H3K27me3 modification. Upon stimulation to differentiate, both PRC2 and PRC1 complexes are re-located to the stem cell-specific gene promoters and displaced from the lineage-specific gene promoters. The mechanism by which the PRC2 complex is recruited to target genes is still not fully understood, although cessation of transcription and nucleosome compaction have been implicated in the process. In the differentiated cell types A and B, the Polycomb proteins silence not only the expression of stem cell genes, but also the expression of genes that encode regulators of alternative lineages. This mechanism of ‘locking’ cell fate is thought to be central to how cells maintain their identity through subsequent cell divisions. Importantly, these mechanisms and especially PRC2 and H3K27 methylations are deregulated in cancer.
Recent evidence suggests that the targeting or subsequent binding of the PRC2 complex to unmethylated CpG islands may be in part dependent on non-canonical PRC1 complexes containing CpG island binding proteins, such as KDM2B [17–22]. We also know that PRC2 is recruited to unmethylated CpG sites to mediate H3K27me3 upon cessation of transcription and nucleosome compaction [23–25]. Intriguingly, much less is known about the biological importance of H3K27me2. This modification is PRC2 complex dependent, is found ubiquitously throughout the genome and appears to be the ‘default setting’ for H3K27 [8,26]. Supporting this, in both mammals and Drosophila, H3K27me2 is localised to almost all euchromatin regions except the gene bodies of actively transcribed genes (instead marked by H3K27me1), active enhancers (instead marked by CBP/p300 mediated H3K27ac) and Polycomb repressed gene promoters (instead marked by H3K27me3), as depicted in Figure 2. Paradoxically, the PRC2 complex does not co-localise with these large H3K27me2 regions [8,26]. This has been rationalised by the fact that di-methylation of H3K27 is enzymatically easier for PRC2 and can be mediated by a transient interaction, unlike H3K27me3, www.sciencedirect.com
which requires a more stable association [8,27,28]. The H3K27me2 modification is likely mediated on nascent Histone H3 during or soon after DNA replication, while H3K27me3 accumulates more slowly [8,29]. The ubiquitous genome-wide profile and repressive nature of H3K27me2 suggests that its role is to act as a ‘repressive blanket’ to prevent the misfiring of cell-type-specific enhancers and gene promoters required for alternative lineages [8,26]. Consistent with this idea, the loss of PRC2 function in Drosophila cells leads to increases in the transcription of intragenic mRNAs coupled with increases in H3K27ac at sites previously protected by H3K27me2 [26]. A picture is emerging in which the PRC2 mediated H3K27me2 and H3K27me3 repressive modifications function at distinct genomic regions to maintain and protect cellular identity from trans-differentiation and de-differentiation (Figure 2). Further unravelling of the normal roles of H3K27me2 and H3K27me3 will be central to understanding the regulation of cell fate transitions and maintenance of cellular identity. Moreover, this will most likely provide important insights into how the frequent deregulation of PRC2 function and H3K27 methylation contributes to cancer (Table 1). Current Opinion in Cell Biology 2015, 37:42–48
44 Differentiation and disease
PRC1 CBX
Z1 2
EED
SU
SU
Z1 2
Figure 2
EED
EZH2/1
EZH2/1 H3K27me3 OFF
H3K27me2
Repressed Polycomb target gene
CBP/p300
H3K27me2
H3K27Ac
Active Enhancer
SU
Z1
2
H3K27me2
EED
EZH2/1 ?
ON
H3K27me1
Active gene Current Opinion in Cell Biology
Diversity of Histone 3 modification states and their roles in the control of transcription. This model depicts the variable modifications of Histone 3 at lysine 27 in distinct genomic contexts and the role of the PRC2 complex in mediating different H3K27 methylations (represented by red circles). The PRC2 and PRC1 complexes co-bind at sites of H3K27me3 on Polycomb target genes. The PRC2 complex is also responsible for all H3K27me2 deposition genome wide, which represents the majority of the human genome and likely serves as the default modification on H3K27. H3K27me2 is a repressive modification and likely functions to suppress the aberrant activation of non-cell-type-specific enhancers and promoters. Importantly, H3K27me2 does not occupy the sites of active enhancers, which have CBP/p300 mediated H3K27ac (represented by one green circle), or the gene bodies of active genes, which have H3K27me1, which may also be mediated, at least in part, by PRC2 action.
PRC2 and H3K27 methylations are deregulated in cancer Since 2010, multiple cancer genome sequencing studies have revealed that the function of the PRC2 complex and H3K27 methylations are frequently disrupted on the genetic level [53,54] (Table 1). In 2003, EZH2 was reported to be highly expressed in multiple cancers as a result of being an E2F regulated gene, downstream of the pRB pathway [55]. However, this elevated expression is generally not considered to lead to deregulation of H3K27 methylations due to the stoichiometry of the PRC2 complex and the requirement for correspondingly high levels of Current Opinion in Cell Biology 2015, 37:42–48
EED and SUZ12. This contrasts with the remarkable nature of the recurrent ‘change-of-function’ mutations in EZH2, first identified in B-cell lymphomas, which confer an enhanced ability to convert H3K27me2 to H3K27me3 [27,28,30,31,32–35]. The consequence of these heterozygous mutations is aberrantly high global levels of H3K27me3 and reduced H3K27me2. The discovery that EZH2, EED and SUZ12 have deletions and inactivating mutations in T-cell leukemias (T-ALL) [38–40] and malignant peripheral nerve sheath tumours (MPNST) [44,47] was initially surprising, because PRC2 function was considered as being oncogenic. These ‘loss of function’ www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 45
Table 1 Mutations in PRC2 members and Histone H3 coding genes in cancer Aberration
Gene
EZH2 ‘change of function’ mutations EZH2 pTyr641X
pAla677Gly pAla687Val PRC2 loss of function mutations EZH2 Homozygous mutation Heterozygous mutation SUZ12 Mutation Heterozygous deletion Heterozygous deletion and mutation Homozygous deletion EED Heterozygous deletion and mutation Heterozygous deletion Homozygous deletion AEBP2 Mutation Histone H3 mutations H3F3A pLys27Met mutation HIST1H3B
pLys27Met mutation
Cancer type (frequency %)
References
Lymphoma (9–24%), parathyroid adenoma (1%), ALL (2%), melanoma (2%) Lymphoma (1–2%), Ewing sarcoma (5%) Lymphoma (1–2%)
Elevated H3K27me3 Reduced H3K27me2
[30,31,32–35]
Elevated H3K27me3 Reduced H3K27me2 Elevated H3K27me3 Reduced H3K27me2
[30,32,36]
Leukemia (4%), myeloid disorders (1–3%) Leukemia (1%), myeloid disorders (6%) MDS/MPN (1–3%), leukemia (2–3%) MPNST (4–25%) MPNST (15–22%)
Reduced H3K27me3
[38–40]
Reduced H3K27me3
[38–40]
Reduced H3K27me3
[41–46]
Reduced H3K27me3
[44,47]
MPNST (16–26%)
Reduced H3K27me3
[44,47]
MPNST (12%)
Reduced H3K27me3
[44,47]
MPNST (2–5%)
Reduced H3K27me3
[44,47]
MPNST (3–14%)
Reduced H3K27me3
[44,47]
MPNST (10%)
Reduced H3K27me3
[44,47]
Leukemia (1%), MPNST (7%)
Reduced H3K27me3
[32,41]
High grade glioma (18–71%), low grade glioma (1–2%), leukemia (1%) High grade glioma (3–18%)
Reduced Reduced Reduced Reduced
mutations feature reduced levels of both H3K27me2 and H3K27me3. Another example of apparent loss of PRC2 function in cancer was the discovery of paediatric gliomas with H3K27M mutations in two genes encoding histone H3, H3F3A (H3.3) or HIST3H1B (H3.1) [32,48–50,51,52]. These gliomas also have global reductions in H3K27me2 and H3K27me3, which is particularly remarkable when one considers that the H3F3A and HIST3H1B genes are just two of 15 different genes encoding Histone H3 and that the mutations are heterozygous. Subsequent work by Lewis and colleagues explained this phenomenon when they showed that transgenes of H3K27M act as dominant negatives to block the enzymatic activity of the PRC2 complex [56,57,58]. A key open question is how do all the various alterations in PRC2 and Histone H3 genes contribute to cancer progression.
Deregulation of H3K27 methylation confers a blockage of cellular differentiation in cancer cells Several studies have begun to address the genome-wide consequences of PRC2 deregulation in real tumours and model systems. In a mouse model of B-cell lymphoma, EZH2 ‘change of function’ mutants have been shown to www.sciencedirect.com
H3K27 methylation status
H3K27me3 H3K27me2 H3K27me3 H3K27me2
[30,36,37]
[32,48–50,51,52] [32,51]
increase global H3K27me3 levels in vivo and lead to a block in cellular differentiation [59]. Interestingly, in a mouse model of myelodysplastic syndrome (MDS) with a conditional biallelic deletion of EZH2, while the total levels of H3K27me3 were reduced, genome-wide profiling identified genes with H3K27me3 increases [60]. This was rationalised by the fact that the EZH1 methyltransferase is still available to methylate H3K27. Similarly, in an EZH2 knockout model of T-ALL, while H3K27me2 and H3K27me3 were both reduced on the global level, both modifications were never completely absent [61]. The implication of these studies is that key genes associated with differentiation gain elevated levels of H3K27me3 and become more potently repressed, thus conferring a differentiation block, while cohorts of genes associated with cancer become activated due to loss of H3K27me3. Similar redistributions of H3K27me3 are observed in gliomas with H3K27M mutations [57–62], which report global reductions, but not complete loss of H3K27me3, coupled with localised increases at particular genomic sites. This again suggests a form of epigenomic reprogramming. Therefore one link between the PRC2 ‘change of function’ mutations, the EZH2 loss of function mutants and the H3K27M gliomas is redistributions of Current Opinion in Cell Biology 2015, 37:42–48
46 Differentiation and disease
H3K27me3 contributing to a block of differentiation and aberrant activation of cancer-associated genes.
the importance of this less well characterised H3K27 modification and point to its potential importance as a global repressive mark whose function is to protect non-cell-type specific enhancers and promoters from aberrant activation, thus helping to preserve cellular identity.
In future studies, it will be important to consider that either loss or gain of PRC2 function not only affect H3K27me3, but also affect regions of H3K27me2 genome-wide. These reductions or complete losses of H3K27me2 have thus far been overlooked when examining epigenomic profiles of cancer cells. Although we do not yet fully understand the H3K27me2 modification or how it confers repression, one could speculate that its loss or redistribution could lead to a more open or ‘stem-like’ chromatin state, consistent with the more general idea of how the deregulation of chromatin regulators contribute to cancer [63]. Furthermore, the reductions in H3K27me2 and H3K27me3 methylation profiles possibly make the cells more amenable to the pathogenic signalling pathways associated with cancer [44]. In contrast, the increased global levels of H3K27me3 observed in lymphomas and melanomas could confer a more closed, refractory epigenomic state, non-responsive to differentiation signals.
9.
Future insights into the role of the H3K27 modifications in cellular identity will allow a better understanding of their contribution to cancer and potentially allow for improved treatments for cancers such as paediatric glioblastomas, melanomas and B-cell lymphomas.
15. Schwartz YB, Kahn TG, Nix DA, Li XY, Bourgon R, Biggin M, Pirrotta V: Genome-wide analysis of Polycomb targets in Drosophila melanogaster. Nat Genet 2006, 38:700-705.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Dawson MA, Kouzarides T: Cancer epigenetics: from mechanism to therapy. Cell 2012, 150:12-27.
2.
Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzler KW: Cancer genome landscapes. Science 2013, 339:1546-1558. This review provides an excellent overview of the key findings of the genome sequencing of human cancers and an objective list of all oncogenes and tumour suppressor genes.
Pengelly AR, Copur O, Jackle H, Herzig A, Muller J: A histone mutant reproduces the phenotype caused by loss of histonemodifying factor Polycomb. Science 2013, 339:698-699. This elegant study was the first to unambiguously confirm that the Lysine 27 residue on Histone H3 is essential for PRC2 complex mediated repression of HOX genes during development.
10. Pengelly AR, Kalb R, Finkl K, Muller J: Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation. Genes Dev 2015, 29:1487-1492. 11. Isono K, Endo TA, Ku M, Yamada D, Suzuki R, Sharif J, Ishikura T, Toyoda T, Bernstein BE, Koseki H: SAM domain polymerization links subnuclear clustering of PRC1 to gene silencing. Dev Cell 2013, 26:565-577. 12. Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig M, Tajonar A, Ray MK et al.: Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 2006, 441:349-353. 13. Bracken AP, Dietrich N, Pasini D, Hansen KH, Helin K: Genomewide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev 2006, 20:1123-1136. 14. Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K et al.: Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 2006, 125:301-313.
16. Bracken AP, Kleine-Kohlbrecher D, Dietrich N, Pasini D, Gargiulo G, Beekman C, Theilgaard-Monch K, Minucci S, Porse BT, Marine JC et al.: The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells. Genes Dev 2007, 21:525-530. 17. Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y et al.: Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation. Cell 2014, 157:1445-1459. 18. Cooper S, Dienstbier M, Hassan R, Schermelleh L, Sharif J, Blackledge NP, De Marco V, Elderkin S, Koseki H, Klose R et al.: Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment. Cell Rep 2014, 7:1456-1470. 19. Farcas AM, Blackledge NP, Sudbery I, Long HK, McGouran JF, Rose NR, Lee S, Sims D, Cerase A, Sheahan TW et al.: KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands. Elife 2012, 1:e00205.
3.
Steffen PA, Ringrose L: What are memories made of? How Polycomb and Trithorax proteins mediate epigenetic memory. Nat Rev Mol Cell Biol 2014, 15:340-356.
4.
Di Croce L, Helin K: Transcriptional regulation by Polycomb group proteins. Nat Struct Mol Biol 2013, 20:1147-1155.
5.
Bracken AP, Helin K: Polycomb group proteins: navigators of lineage pathways led astray in cancer. Nature Reviews Cancer 2009, 9:773-784.
21. Kalb R, Latwiel S, Baymaz HI, Jansen PW, Muller CW, Vermeulen M, Muller J: Histone H2A monoubiquitination promotes histone H3 methylation in Polycomb repression. Nat Struct Mol Biol 2014, 21:569-571.
6.
Pietersen AM, van Lohuizen M: Stem cell regulation by polycomb repressors: postponing commitment. Curr Opin Cell Biol 2008, 20:201-207.
22. Wu X, Johansen JV, Helin K: Fbxl10/Kdm2b recruits polycomb repressive complex 1 to CpG islands and regulates H2A ubiquitylation. Mol Cell 2013, 49:1134-1146.
7.
Margueron R, Reinberg D: The Polycomb complex PRC2 and its mark in life. Nature 2011, 469:343-349.
23. Yuan W, Wu T, Fu H, Dai C, Wu H, Liu N, Li X, Xu M, Zhang Z, Niu T et al.: Dense chromatin activates Polycomb repressive complex 2 to regulate H3 lysine 27 methylation. Science 2012, 337:971-975.
8.
Ferrari KJ, Scelfo A, Jammula S, Cuomo A, Barozzi I, Stutzer A, Fischle W, Bonaldi T, Pasini D: Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. Mol Cell 2014, 53:49-62. This paper and Ref. [26] establish that the H3K27me2 modification is broadly located in both Drosophila and mammalian cells. They highlight
Current Opinion in Cell Biology 2015, 37:42–48
20. He J, Shen L, Wan M, Taranova O, Wu H, Zhang Y: Kdm2b maintains murine embryonic stem cell status by recruiting PRC1 complex to CpG islands of developmental genes. Nat Cell Biol 2013, 15:373-384.
24. Jermann P, Hoerner L, Burger L, Schubeler D: Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation. Proc Natl Acad Sci U S A 2014, 111:E3415-E3421. www.sciencedirect.com
H3K27 methylations in cell identity and cancer Conway, Healy and Bracken 47
25. Riising EM, Comet I, Leblanc B, Wu X, Johansen JV, Helin K: Gene silencing triggers polycomb repressive complex 2 recruitment to CpG islands genome wide. Mol Cell 2014, 55:347-360. 26. Lee HG, Kahn TG, Simcox A, Schwartz YB, Pirrotta V: Genomewide activities of Polycomb complexes control pervasive transcription. Genome Res 2015, 25:1170-1181. 27. Sneeringer CJ, Scott MP, Kuntz KW, Knutson SK, Pollock RM, Richon VM, Copeland RA: Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas. Proc Natl Acad Sci U S A 2010, 107:20980-20985. This paper helped to explain how EZH2 ‘change of function’ mutations in B-cell Lymphomas lead to deregulated H3K27 methylation. It established that the EZH2 mutants have unique substrate specificities allowing them to lead to elevated global levels of H3K27me3 and lower levels of H3K27me2. 28. McCabe MT, Graves AP, Ganji G, Diaz E, Halsey WS, Jiang Y, Smitheman KN, Ott HM, Pappalardi MB, Allen KE et al.: Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27). Proc Natl Acad Sci U S A 2012, 109:2989-2994. 29. Alabert C, Barth TK, Reveron-Gomez N, Sidoli S, Schmidt A, Jensen ON, Imhof A, Groth A: Two distinct modes for propagation of histone PTMs across the cell cycle. Genes Dev 2015, 29:585-590. 30. Morin RD, Mendez-Lago M, Mungall AJ, Goya R, Mungall KL, Corbett RD, Johnson NA, Severson TM, Chiu R, Field M et al.: Frequent mutation of histone-modifying genes in nonHodgkin lymphoma. Nature 2011, 476:298-303.
39. Jankowska AM, Makishima H, Tiu RV, Szpurka H, Huang Y, Traina F, Visconte V, Sugimoto Y, Prince C, O’Keefe C et al.: Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 2011, 118: 3932-3941. 40. Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, Waghorn K, Zoi K, Ross FM, Reiter A et al.: Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010, 42:722-726. 41. Zhang M, Wang Y, Jones S, Sausen M, McMahon K, Sharma R, Wang Q, Belzberg AJ, Chaichana K, Gallia GL et al.: Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors. Nat Genet 2014, 46:1170-1172. 42. Zhang J, Ding L, Holmfeldt L, Wu G, Heatley SL, Payne-Turner D, Easton J, Chen X, Wang J, Rusch M et al.: The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature 2012, 481:157-163. 43. Score J, Hidalgo-Curtis C, Jones AV, Winkelmann N, Skinner A, Ward D, Zoi K, Ernst T, Stegelmann F, Dohner K et al.: Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms. Blood 2012, 119:1208-1213. 44. De Raedt T, Beert E, Pasmant E, Luscan A, Brems H, Ortonne N, Helin K, Hornick JL, Mautner V, Kehrer-Sawatzki H et al.: PRC2 loss amplifies Ras-driven transcription and confers sensitivity to BRD4-based therapies. Nature 2014, 514:247-251. 45. Brecqueville M, Rey J, Bertucci F, Coppin E, Finetti P, Carbuccia N, Cervera N, Gelsi-Boyer V, Arnoulet C, Gisserot O et al.: Mutation analysis of ASXL1, CBL, DNMT3A, IDH1, IDH2, JAK2, MPL, NF1, SF3B1, SUZ12, and TET2 in myeloproliferative neoplasms. Genes Chromosomes Cancer 2012, 51:743-755.
31. Morin RD, Johnson NA, Severson TM, Mungall AJ, An J, Goya R, Paul JE, Boyle M, Woolcock BW, Kuchenbauer F et al.: Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nat Genet 2010, 42:181-185. This was the first paper to identify mutations in EZH2, which subsequently were shown to be ‘change of function’ mutations and led to the rapid advancement of EZH2 inhibitor drugs and their clinical trials for the personalised treatment of patients with these mutations in B-cell Lymphomas.
46. Beekman R, Valkhof MG, Sanders MA, van Strien PM, Haanstra JR, Broeders L, Geertsma-Kleinekoort WM, Veerman AJ, Valk PJ, Verhaak RG et al.: Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia. Blood 2012, 119:5071-5077.
32. Huether R, Dong L, Chen X, Wu G, Parker M, Wei L, Ma J, Edmonson MN, Hedlund EK, Rusch MC et al.: The landscape of somatic mutations in epigenetic regulators across 1,000 paediatric cancer genomes. Nat Commun 2014, 5:3630.
47. Lee W, Teckie S, Wiesner T, Ran L, Prieto Granada CN, Lin M, Zhu S, Cao Z, Liang Y, Sboner A et al.: PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors. Nat Genet 2014, 46:1227-1232.
33. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, Nickerson E, Auclair D, Li L, Place C et al.: A landscape of driver mutations in melanoma. Cell 2012, 150:251-263.
48. Gielen GH, Gessi M, Hammes J, Kramm CM, Waha A, Pietsch T: H3F3A K27M mutation in pediatric CNS tumors: a marker for diffuse high-grade astrocytomas. Am J Clin Pathol 2013, 139:345-349.
34. Cromer MK, Starker LF, Choi M, Udelsman R, Nelson-Williams C, Lifton RP, Carling T: Identification of somatic mutations in parathyroid tumors using whole-exome sequencing. J Clin Endocrinol Metab 2012, 97:E1774-E1781.
49. Khuong-Quang DA, Buczkowicz P, Rakopoulos P, Liu XY, Fontebasso AM, Bouffet E, Bartels U, Albrecht S, Schwartzentruber J, Letourneau L et al.: K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta Neuropathol 2012, 124:439-447.
35. Barsotti AM, Ryskin M, Zhong W, Zhang WG, Giannakou A, Loreth C, Diesl V, Follettie M, Golas J, Lee M et al.: Epigenetic reprogramming by tumor-derived EZH2 gain-of-function mutations promotes aggressive 3D cell morphologies and enhances melanoma tumor growth. Oncotarget 2015, 6:2928-2938. 36. Bodor C, Grossmann V, Popov N, Okosun J, O’Riain C, Tan K, Marzec J, Araf S, Wang J, Lee AM et al.: EZH2 mutations are frequent and represent an early event in follicular lymphoma. Blood 2013, 122:3165-3168. 37. Lohr JG, Stojanov P, Lawrence MS, Auclair D, Chapuy B, Sougnez C, Cruz-Gordillo P, Knoechel B, Asmann YW, Slager SL et al.: Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing. Proc Natl Acad Sci U S A 2012, 109:3879-3884. 38. Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, van der Heijden A, Scheele TN, Vandenberghe P, de Witte T et al.: Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010, 42:665-667. www.sciencedirect.com
50. Sturm D, Witt H, Hovestadt V, Khuong-Quang DA, Jones DT, Konermann C, Pfaff E, Tonjes M, Sill M, Bender S et al.: Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer Cell 2012, 22:425-437. 51. Wu G, Broniscer A, McEachron TA, Lu C, Paugh BS, Becksfort J, Qu C, Ding L, Huether R, Parker M et al.: Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet 2012, 44:251-253. This was the first paper to identify H3K27M mutations in gliomas. 52. Zhang J, Wu G, Miller CP, Tatevossian RG, Dalton JD, Tang B, Orisme W, Punchihewa C, Parker M, Qaddoumi I et al.: Wholegenome sequencing identifies genetic alterations in pediatric low-grade gliomas. Nat Genet 2013, 45:602-612. 53. Plass C, Pfister SM, Lindroth AM, Bogatyrova O, Claus R, Lichter P: Mutations in regulators of the epigenome and their connections to global chromatin patterns in cancer. Nat Rev Genet 2013, 14:765-780. Current Opinion in Cell Biology 2015, 37:42–48
48 Differentiation and disease
54. Helin K, Dhanak D: Chromatin proteins and modifications as drug targets. Nature 2013, 502:480-488. 55. Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K: EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. Embo J 2003, 22:5323-5335. 56. Lewis PW, Muller MM, Koletsky MS, Cordero F, Lin S, Banaszynski LA, Garcia BA, Muir TW, Becher OJ, Allis CD: Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma. Science 2013, 340:857-861. This paper, together with paper [58,59], established a dominant negative mechanism by which the H3K27M transgenes could cause global changes in H3K27 methylation. 57. Chan KM, Fang D, Gan H, Hashizume R, Yu C, Schroeder M, Gupta N, Mueller S, James CD, Jenkins R et al.: The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes Dev 2013, 27:985-990. 58. Bender S, Tang Y, Lindroth AM, Hovestadt V, Jones DT, Kool M, Zapatka M, Northcott PA, Sturm D, Wang W et al.: Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas. Cancer Cell 2013, 24:660-672.
Current Opinion in Cell Biology 2015, 37:42–48
59. Beguelin W, Popovic R, Teater M, Jiang Y, Bunting KL, Rosen M, Shen H, Yang SN, Wang L, Ezponda T et al.: EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation. Cancer Cell 2013, 23:677-692. 60. Muto T, Sashida G, Oshima M, Wendt GR, Mochizuki-Kashio M, Nagata Y, Sanada M, Miyagi S, Saraya A, Kamio A et al.: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders. J Exp Med 2013, 210:2627-2639. 61. Simon C, Chagraoui J, Krosl J, Gendron P, Wilhelm B, Lemieux S, Boucher G, Chagnon P, Drouin S, Lambert R et al.: A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia. Genes Dev 2012, 26:651-656. 62. Funato K, Major T, Lewis PW, Allis CD, Tabar V: Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation. Science 2014, 346: 1529-1533. 63. Suva ML, Riggi N, Bernstein BE: Epigenetic reprogramming in cancer. Science 2013, 339:1567-1570.
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