Title: Polycomb Group Proteins: Multi-Faceted Regulators of Somatic Stem Cells and Cancer
Abstract: Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development. Polycomb Group (PcG) proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins play important roles in both stem cell self-renewal and in cancer development. Our understanding of their mechanism of action has greatly advanced over the past 10 years, but many unanswered questions remain. In this review, we present the currently available experimental data that connect PcG protein function with some of the key processes which govern somatic stem cell activity. We also highlight recent studies suggesting that a delicate balance in PcG gene dosage is crucial for proper stem cell homeostasis and prevention of cancer stem cell development. Throughout our lives, mature cells in adult tissues are continuously replenished through the activity of small populations of somatic stem cells. The defining feature of these cells is their capacity to undergo self-renewal division coupled with maintenance of multipotency. Somatic stem cells have been identified for most tissues (blood, brain, muscle, skin, gut, etc.), and harnessing their regenerative properties offers a great potential for future therapies. At the molecular level, much about self-renewal remains to be elucidated. However, we can envision this process as the fine orchestration of cell-cycle regulation with cell-fate decisions. To self-renew, stem cells must therefore enter the cell cycle and progress successfully through cell division. During this process, genome integrity has to be preserved through the coordinated regulation of cell-cycle checkpoints and DNA damage repair. While doing so, they must also ensure that at least one daughter cell restricts programs leading to differentiation, senescence, or apoptosis, thus retaining stemness. Accumulating evidence indicates that a subpopulation of cancer cells within tumors possess stem cell-like properties. Bonnet and Dick, 1997Bonnet D. Dick J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat. Med. 1997; 3: 730-737Crossref PubMed Scopus (3052) Google Scholar showed that most leukemic blasts are limited in their proliferative capacity and must be constantly replenished by a rare self-renewing population of “leukemic stem cells.” Similar findings have been reported for cancers of the breast, brain, colon, ovary, pancreas, and prostate (Al-Hajj et al., 2003Al-Hajj M. Wicha M.S. Benito-Hernandez A. Morrison S.J. Clarke M.F. Prospective identification of tumorigenic breast cancer cells.Proc. Natl. Acad. Sci. USA. 2003; 100: 3983-3988Crossref PubMed Scopus (4415) Google Scholar, Li et al., 2007Li C. Heidt D.G. Dalerba P. Burant C.F. Zhang L. Adsay V. Wicha M. Clarke M.F. Simeone D.M. Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (1464) Google Scholar, O'Brien et al., 2007O'Brien C.A. Pollett A. Gallinger S. 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In support of this model, cancer cells frequently exhibit stem cell-like gene expression and chromatin structure signatures (Ben-Porath et al., 2008Ben-Porath I. Thomson M.W. Carey V.J. Ge R. Bell G.W. Regev A. Weinberg R.A. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors.Nat. Genet. 2008; 40: 499-507Crossref PubMed Scopus (898) Google Scholar, Widschwendter et al., 2007Widschwendter M. Fiegl H. Egle D. Mueller-Holzner E. Spizzo G. Marth C. Weisenberger D.J. Campan M. Young J. Jacobs I. Laird P.W. Epigenetic stem cell signature in cancer.Nat. Genet. 2007; 39: 157-158Crossref PubMed Scopus (548) Google Scholar). This predicts similarities in the genes that determine self-renewal of normal and cancer stem cells and highlights the importance of identifying the key components regulating this function. As detailed below, the Polycomb Group (PcG) genes represent prime candidates for determining activity of normal and cancer stem cells. In this review, we discuss the proposed function of PcG proteins in stem cell activity with a particular focus on their role in cell-cycle regulation, differentiation, apoptosis, and senescence. We also briefly describe the growing importance of PcG genes in cancer development. PcG genes were identified in Drosophila more than 30 years ago as regulators of anterior-posterior body patterning through the repression of Hox genes. They have since been recognized as global epigenetic transcriptional repressors and key regulators of cell fate (reviewed in Schwartz and Pirrotta, 2007Schwartz Y.B. Pirrotta V. Polycomb silencing mechanisms and the management of genomic programmes.Nat. Rev. Genet. 2007; 8: 9-22Crossref PubMed Scopus (473) Google Scholar). The Polycomb family comprises a structurally diverse set of proteins which assemble into chromatin-associated complexes. The composition of these complexes is variable and context-dependent (e.g., differentiation status). 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Wakao R. Tang Y.A. Endoh M. Appanah R. Nesterova T.B. Silva J. Otte A.P. Vidal M. et al.Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation.Dev. Cell. 2004; 7: 663-676Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, Wang et al., 2004Wang H. Wang L. Erdjument-Bromage H. Vidal M. Tempst P. Jones R.S. Zhang Y. Role of histone H2A ubiquitination in Polycomb silencing.Nature. 2004; 431: 873-878Crossref PubMed Scopus (622) Google Scholar). Although less well characterized, L3MBTL and SFMBT proteins are also found in PRC1 complexes, whereas ASXL1 was recently identified in the new Polycomb Repressive H2A Deubiquitinase complex (Grimm et al., 2009Grimm C. Matos R. Ly-Hartig N. Steuerwald U. Lindner D. Rybin V. Müller J. Müller C.W. 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Mechtler K. Spahn L. Koseki H. Jenuwein T. Wutz A. Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing.EMBO J. 2006; 25: 3110-3122Crossref PubMed Scopus (170) Google Scholar). The ASXL subunit has recently been found to be involved in a H2A deubiquitinase complex required for PcG-mediated repression, but its precise role remains unclear.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Following recruitment of the PRC2 complex to chromatin, the histone methyltransferase EZH1/2 catalyzes the trimethylation of the lysine 27 of histone H3 (H3K27me3). Subsequent recruitment of the PRC1 complex occurs in part through affinity binding of the chromodomain of the CBX subunit to the H3K27me3 covalent mark. The PRC1 RING1 E3 ligase then monoubiquitylates the lysine 119 of histone H2A (H2AK119ub1), which was proposed to consolidate transcriptional repression by preventing access to chromatin remodelers, inhibiting RNA polymerase II-dependent transcriptional elongation and facilitating chromatin compaction (Francis et al., 2004Francis N.J. Kingston R.E. Woodcock C.L. Chromatin compaction by a polycomb group protein complex.Science. 2004; 306: 1574-1577Crossref PubMed Scopus (306) Google Scholar, Zhou et al., 2008Zhou W. Zhu P. Wang J. Pascual G. Ohgi K.A. Lozach J. Glass C.K. Rosenfeld M.G. Histone H2A monoubiquitination represses transcription by inhibiting RNA polymerase II transcriptional elongation.Mol. Cell. 2008; 29: 69-80Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). PRC1 has also been reported to be targeted to chromatin independently of PRC2 (Boyer et al., 2006Boyer L.A. Plath K. Zeitlinger J. Brambrink T. Medeiros L.A. Lee T.I. Levine S.S. Wernig M. Tajonar A. Ray M.K. et al.Polycomb complexes repress developmental regulators in murine embryonic stem cells.Nature. 2006; 441: 349-353Crossref PubMed Scopus (1278) Google Scholar, Ku et al., 2008Ku M. Koche R.P. Rheinbay E. Mendenhall E.M. Endoh M. Mikkelsen T.S. Presser A. Nusbaum C. Xie X. Chi A.S. et al.Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains.PLoS Genet. 2008; 4: e1000242Crossref PubMed Scopus (403) Google Scholar, Schoeftner et al., 2006Schoeftner S. Sengupta A.K. Kubicek S. Mechtler K. Spahn L. Koseki H. Jenuwein T. Wutz A. Recruitment of PRC1 function at the initiation of X inactivation independent of PRC2 and silencing.EMBO J. 2006; 25: 3110-3122Crossref PubMed Scopus (170) Google Scholar). The ASXL subunit has recently been found to be involved in a H2A deubiquitinase complex required for PcG-mediated repression, but its precise role remains unclear. During the evolution from invertebrates to vertebrates the number of PcG genes underwent significant expansion, rising from approximately 15 in Drosophila to 37 in Mouse and Humans. The apparent gene duplications that occurred, most notably in mammals, resulted in alternative paralogous subunits that diverged in their structural domains (Kerppola, 2009Kerppola T.K. Polycomb group complexes—many combinations, many functions.Trends Cell Biol. 2009; 19: 692-704Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, Whitcomb et al., 2007Whitcomb S.J. Basu A. Allis C.D. Bernstein E. Polycomb Group proteins: an evolutionary perspective.Trends Genet. 2007; 23: 494-502Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In some situations, this structural diversification confers to paralogs the ability to execute novel activities. 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No role has been ascribed yet to PcGs in G0/G1 transition. However, expression levels of the PRC2 proteins EZH2 and SUZ12 are greatly reduced in serum-starved nondividing fibroblasts and rapidly increase as they enter cell cycle, whereas EZH1 levels are not affected (Bracken et al., 2003Bracken A.P. 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: 5323-5335Crossref PubMed Scopus (516) Google Scholar, Pasini et al., 2004Pasini D. Bracken A.P. Jensen M.R. Lazzerini Denchi E. Helin K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity.EMBO J. 2004; 23: 4061-4071Crossref PubMed Scopus (339) Google Scholar). It will be important to determine whether this differential regulation of PcG protein levels also characterizes somatic stem cells and what role these changes play in their G0/G1 transition. Regulation of the G1/S transition occurs mainly through the INK4A-pRB/E2F pathway. The PcG-mediated repression of the p16Ink4a/p19Arf locus is essential for G1/S progression (Figure 3A). The PRC1 proteins BMI1, PCGF1, PCGF2/MEL18, CBX2, CBX7, CBX8, RING1B, and the PRC2 proteins EED, SUZ12, and EZH2 have been shown in multiple cell types to bind the p16Ink4a/p19Arf promoter directly and repress this locus (Gil and Peters, 2006Gil J. Peters G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all.Nat. Rev. Mol. Cell Biol. 2006; 7: 667-677Crossref PubMed Scopus (349) Google Scholar, Maertens et al., 2009Maertens G.N. El Messaoudi-Aubert S. Racek T. Stock J.K. Nicholls J. Rodriguez-Niedenführ M. Gil J. Peters G. Several distinct polycomb complexes regulate and co-localize on the INK4a tumor suppressor locus.PLoS ONE. 2009; 4: e6380Crossref PubMed Scopus (50) Google Scholar). 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In fly and mammals, physical interaction between L3MBTL1 and pRB-E2F appears to be required for repression of target genes, notably cyclin E, an E2F2-pRB target gene crucial for S phase initiation (Lu et al., 2007Lu J. Ruhf M.L. Perrimon N. Leder P. A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression.Proc. Natl. Acad. Sci. USA. 2007; 104: 9381-9386Crossref PubMed Scopus (38) Google Scholar, Trojer et al., 2007Trojer P. Li G. Sims 3rd, R.J. Vaquero A. Kalakonda N. Boccuni P. Lee D. Erdjument-Bromage H. Tempst P. Nimer S.D. et al.L3MBTL1, a histone-methylation-dependent chromatin lock.Cell. 2007; 129: 915-928Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Moreover, EZH2 and HDAC1 compete for interaction with pRB2/p130, indicating that PRC2 participates in the regulation of pRB/E2F activity (Tonini et al., 2004Tonini T. Bagella L. D'Andrilli G. Claudio P.P. Giordano A. Ezh2 reduces the ability of HDAC1-dependent pRb2/p130 transcriptional repression of cyclin A.Oncogene. 2004; 23: 4930-4937Crossref PubMed Scopus (45) Google Scholar). Interestingly, E2Fs bind to and activate the Ezh2 and Eed promoters, indicating the presence of a regulatory feedback loop between PcG and pRB-E2F proteins (Bracken et al., 2003Bracken A.P. 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: 5323-5335Crossref PubMed Scopus (516) Google Scholar). Prohibitin (PHB), an antiproliferative protein that interacts with E2F-pRB and p53, represses E2F target genes by recruiting corepressors and RING1B/PRC1 complexes. This latter interaction is essential for the transcriptional repressive function of PHB, and knockdown of PHB results in reduction of RING1B levels and increase in p16Ink4a expression (Choi et al., 2008Choi D. Lee S.J. Hong S. Kim I.H. Kang S. Prohibitin interacts with RNF2 and regulates E2F1 function via dual pathways.Oncogene. 2008; 27: 1716-1725Crossref PubMed Scopus (29) Google Scholar). Thus, RING1B and PHB directly interact with and inhibit E2F1's transcriptional activity. Curiously, they also indirectly promote E2F1 activity by repressing p16Ink4a expression, which allows Cyclin D-CDK4/6-mediated phosphorylation of pRB and E2F1 release. This dual function could serve as a fine-tuning system for G1/S transition in response to mitogenic signals. The PRC1 complex has been implicated in regulation of S phase initiation. Within the PRC1 complex, SCMH1 directly interacts with Geminin, an inhibitor of the DNA replication licensing factor CDT1 (Figure 3B). The PRC1 complex, comprising PHC1, SCMH1, RING1B, and BMI1, exerts E3 ubiquitin ligase activity on Geminin. PRC1-induced ubiquitination of Geminin leads to its protea