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• W2005027557 abstract "Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC-fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation, and altered patterns of histone methylation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation. Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC-fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation, and altered patterns of histone methylation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation. PCL2 associates with the PRC2 complex in mouse ESCs PCL2 knockdown increases self-renewal and disrupts differentiation Reducing PLC2 decreases H3K27me3 at specific targets PCL2 represses transcription of self-renewal genes in ESCs Control over ESC-fate decisions is accomplished through a variety of molecular, genetic, and epigenetic events. Exogenous control of cell fate can be achieved by a limited number of factors. When grown in fetal bovine serum (FBS)-containing medium and in the presence of murine embryonic fibroblast feeder cells (Evans and Kaufman, 1981Evans M.J. Kaufman M.H. Establishment in culture of pluripotential cells from mouse embryos.Nature. 1981; 292: 154-156Crossref PubMed Scopus (6065) Google Scholar, Martin, 1981Martin G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc. Natl. Acad. Sci. USA. 1981; 78: 7634-7638Crossref PubMed Scopus (4064) Google Scholar) or the cytokine leukemia inhibitory factor (LIF) (Smith et al., 1988Smith A.G. Heath J.K. Donaldson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.Nature. 1988; 336: 688-690Crossref PubMed Scopus (1411) Google Scholar, Smith and Hooper, 1987Smith A.G. Hooper M.L. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.Dev. Biol. 1987; 121: 1-9Crossref PubMed Scopus (299) Google Scholar, Williams et al., 1988Williams R.L. Hilton D.J. Pease S. Willson T.A. Stewart C.L. Gearing D.P. Wagner E.F. Metcalf D. Nicola N.A. Gough N.M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.Nature. 1988; 336: 684-687Crossref PubMed Scopus (1475) Google Scholar), mouse ESCs remain undifferentiated. BMP4, provided by the serum, functions in the presence of LIF to maintain pluripotency by inducing phosphorylation and nuclear localization of Smad1, followed by upregulation of Id proteins that block neural differentiation (Ying et al., 2003aYing Q.L. Nichols J. Chambers I. Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1630) Google Scholar). Three transcription factors are known to be critical in the establishment and/or maintenance of ESC pluripotency. OCT4 (Pou5f1) has a highly conserved role in maintaining pluripotent cell populations (Morrison and Brickman, 2006Morrison G.M. Brickman J.M. Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development.Development. 2006; 133: 2011-2022Crossref PubMed Scopus (123) Google Scholar, Nichols et al., 1998Nichols J. Zevnik B. Anastassiadis K. Niwa H. Klewe-Nebenius D. Chambers I. Schöler H. Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.Cell. 1998; 95: 379-391Abstract Full Text Full Text PDF PubMed Scopus (2548) Google Scholar), and its expression level dictates ESC fate (Niwa et al., 2000Niwa H. Miyazaki J. Smith A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2785) Google Scholar). SOX2 forms a complex with OCT4 and is necessary to cooperatively activate target genes (Ambrosetti et al., 1997Ambrosetti D.C. Basilico C. Dailey L. Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites.Mol. Cell. Biol. 1997; 17: 6321-6329Crossref PubMed Scopus (298) Google Scholar, Yuan et al., 1995Yuan H. Corbi N. Basilico C. Dailey L. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3.Genes Dev. 1995; 9: 2635-2645Crossref PubMed Scopus (616) Google Scholar). NANOG is critical for initiating pluripotency and maintaining OCT4 levels, even in the absence of LIF (Chambers et al., 2003Chambers I. Colby D. Robertson M. Nichols J. Lee S. Tweedie S. Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.Cell. 2003; 113: 643-655Abstract Full Text Full Text PDF PubMed Scopus (2537) Google Scholar, Chambers et al., 2007Chambers I. Silva J. Colby D. Nichols J. Nijmeijer B. Robertson M. Vrana J. Jones K. Grotewold L. Smith A. Nanog safeguards pluripotency and mediates germline development.Nature. 2007; 450: 1230-1234Crossref PubMed Scopus (1089) Google Scholar, Mitsui et al., 2003Mitsui K. Tokuzawa Y. Itoh H. Segawa K. Murakami M. Takahashi K. Maruyama M. Maeda M. Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2453) Google Scholar), while it is itself regulated by OCT4 and SOX2 (Rodda et al., 2005Rodda D.J. Chew J.L. Lim L.H. Loh Y.H. Wang B. Ng H.H. Robson P. Transcriptional regulation of nanog by OCT4 and SOX2.J. Biol. Chem. 2005; 280: 24731-24737Crossref PubMed Scopus (787) Google Scholar). The polycomb group (PcG) proteins, first described in D. melanogaster, regulate epigenetic states and are required for proper repression of homeotic genes during development (Schuettengruber et al., 2007Schuettengruber B. Chourrout D. Vervoort M. Leblanc B. Cavalli G. Genome regulation by polycomb and trithorax proteins.Cell. 2007; 128: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar, 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 (683) Google Scholar). PcG proteins have been identified in either the PRC1 or the PRC2 multiprotein complex (Lund and van Lohuizen, 2004Lund A.H. van Lohuizen M. Polycomb complexes and silencing mechanisms.Curr. Opin. Cell Biol. 2004; 16: 239-246Crossref PubMed Scopus (245) Google Scholar). The core components of PRC2 in Drosophila are Extra Sex Combs, E(Z), and SU(Z)12. These proteins are found in distinct complexes with additional accessory proteins that undergo dynamic changes during development (Furuyama et al., 2003Furuyama T. Tie F. Harte P.J. Polycomb group proteins ESC and E(Z) are present in multiple distinct complexes that undergo dynamic changes during development.Genesis. 2003; 35: 114-124Crossref PubMed Scopus (32) Google Scholar, Kuzmichev et al., 2005Kuzmichev A. Margueron R. Vaquero A. Preissner T.S. Scher M. Kirmizis A. Ouyang X. Brockdorff N. Abate-Shen C. Farnham P. Reinberg D. Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation.Proc. Natl. Acad. Sci. USA. 2005; 102: 1859-1864Crossref PubMed Scopus (341) Google Scholar). Core PRC2 members are highly conserved between species (Schuettengruber et al., 2007Schuettengruber B. Chourrout D. Vervoort M. Leblanc B. Cavalli G. Genome regulation by polycomb and trithorax proteins.Cell. 2007; 128: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar), and their mouse orthologs are EED, EZH2, and SUZ12, respectively (Cao et al., 2002Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing.Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2574) Google Scholar, Czermin et al., 2002Czermin 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. 2002; 111: 185-196Abstract Full Text Full Text PDF PubMed Scopus (1176) Google Scholar). Through the methyltransferase activity of EZH2, PRC2 induces gene repression by trimethylating lysine 27 on histone 3 (3meH3K27) (Cao et al., 2002Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Role of histone H3 lysine 27 methylation in Polycomb-group silencing.Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2574) 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 (614) Google Scholar, Silva et al., 2003Silva J. Mak W. Zvetkova I. Appanah R. Nesterova T.B. Webster Z. Peters A.H. Jenuwein T. Otte A.P. Brockdorff N. Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes.Dev. Cell. 2003; 4: 481-495Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). In Drosophila, PCL (polycomb-like) is present in a subset of PRC2 complexes (O'Connell et al., 2001O'Connell S. Wang L. Robert S. Jones C.A. Saint R. Jones R.S. Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.J. Biol. Chem. 2001; 276: 43065-43073Crossref PubMed Scopus (92) Google Scholar, Tie et al., 2003Tie F. Prasad-Sinha J. Birve A. Rasmuson-Lestander A. Harte P.J. A 1-megadalton ESC/E(Z) complex from Drosophila that contains polycomblike and RPD3.Mol. Cell. Biol. 2003; 23: 3352-3362Crossref PubMed Scopus (110) Google Scholar). Evidence suggests that PCL is required to generate high levels of 3meH3K27 at some target genes. However, gene repression and 3meH3K27 of many known PRC2 targets is not abolished in the absence of PCL, suggesting that PRC2 can function independently of PCL at many target genes (Nekrasov et al., 2007Nekrasov M. Klymenko T. Fraterman S. Papp B. Oktaba K. Köcher T. Cohen A. Stunnenberg H.G. Wilm M. Müller J. Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes.EMBO J. 2007; 26: 4078-4088Crossref PubMed Scopus (198) Google Scholar). In contrast to other PRC2 members, the lack of PCL produces a mild homeotic phenotype, specifically affecting target gene expression in central nervous system and mesoderm tissues (Duncan, 1982Duncan I.M. Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia gene complexes of Drosophila melanogaster.Genetics. 1982; 102: 49-70Crossref PubMed Google Scholar, O'Connell et al., 2001O'Connell S. Wang L. Robert S. Jones C.A. Saint R. Jones R.S. Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.J. Biol. Chem. 2001; 276: 43065-43073Crossref PubMed Scopus (92) Google Scholar). PCL2 is a mammalian ortholog of PCL, and defects reported in animal models of Pcl2 deficiency are consistent with lack of PCL in Drosophila. In G. gallus (Wang et al., 2004Wang S. Yu X. Zhang T. Zhang X. Zhang Z. Chen Y. Chick Pcl2 regulates the left-right asymmetry by repressing Shh expression in Hensen's node.Development. 2004; 131: 4381-4391Crossref PubMed Scopus (32) Google Scholar), Pcl2 regulates left-right axis specification, and in X. laevis (Kitaguchi et al., 2001Kitaguchi T. Nakata K. Nagai T. Aruga J. Mikoshiba K. Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue.Dev. Genes Evol. 2001; 211: 309-314Crossref PubMed Scopus (14) Google Scholar), central nervous system gene expression is disrupted by the lack of Pcl2. Finally, hypomorphic Pcl2 gene trap mice have pleiotropic defects, including posterior transformation of axial skeleton, stunted growth, hydrocephaly, hunchback, and incisor abnormalities (Wang et al., 2007Wang S. He F. Xiong W. Gu S. Liu H. Zhang T. Yu X. Chen Y. Polycomblike-2-deficient mice exhibit normal left-right asymmetry.Dev. Dyn. 2007; 236: 853-861Crossref PubMed Scopus (27) Google Scholar). Here, we investigated the role of PCL2 in mouse ESCs. Reduction of PCL2 resulted in heightened self-renewal characteristics and inefficient differentiation to the three germ layers. PCL2 was found to associate with the core PRC2 complex, and using chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-seq), we found that PCL2 is highly enriched at many, but not all, locations of PRC2 enrichment. Loss of PCL2 did not abolish global levels of 3meH3K27 but did result in decreased 3meH3K27 at specific targets and altered patterns of 3meH3K27 during early commitment. Integrating PCL2-PRC2 targets with the ESC self-renewal circuitry revealed a key role for PCL2-PRC2 in limiting transcription of ESC self-renewal genes in undifferentiated ESCs, as well as controlling developmental regulators during commitment and early differentiation. In a screen for regulators of ESC fate, we hypothesized that networks critical for the stability of the ESC state would be differentially regulated at the initiation of commitment (Walker et al., 2007Walker E. Ohishi M. Davey R.E. Zhang W. Cassar P.A. Tanaka T.S. Der S.D. Morris Q. Hughes T.R. Zandstra P.W. et al.Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment.Cell Stem Cell. 2007; 1: 71-86Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Transcript levels of Mtf2 (metal response element binding transcription factor 2) decreased during differentiation (Figure 1A), and promoter occupancy data indicated that Mtf2 is bound by both OCT4 and NANOG (Loh et al., 2006Loh Y.H. Wu Q. Chew J.L. Vega V.B. Zhang W. Chen X. Bourque G. George J. Leong B. Liu J. et al.The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.Nat. Genet. 2006; 38: 431-440Crossref PubMed Scopus (1847) Google Scholar). Further inspection revealed that the protein product of Mtf2 is orthologous to Drosophila PCL. Both proteins contain a TUDOR domain and two PHD zinc finger domains (Figure 1B), and MTF2 shares 73% and 88% similarity to the PCL ortholog, PCL2, studied in X. laevis (Kitaguchi et al., 2001Kitaguchi T. Nakata K. Nagai T. Aruga J. Mikoshiba K. Xenopus Polycomblike 2 (XPcl2) controls anterior to posterior patterning of the neural tissue.Dev. Genes Evol. 2001; 211: 309-314Crossref PubMed Scopus (14) Google Scholar) and G. gallus (Wang et al., 2004Wang S. Yu X. Zhang T. Zhang X. Zhang Z. Chen Y. Chick Pcl2 regulates the left-right asymmetry by repressing Shh expression in Hensen's node.Development. 2004; 131: 4381-4391Crossref PubMed Scopus (32) Google Scholar), respectively. The mouse and human MTF2 protein sequences share 96% identity. Thus, we will refer to the mammalian Mtf2 transcript as Pcl2. Mammals have two other PCL orthologs, PHF1 and PHF19, which have 63% homology with PCL2 among the conserved domains. Interestingly, expression analysis of Pcl2 in mice shows that it is uniquely expressed in undifferentiated ESCs and during early embryonic development (Figure S1A available online). To examine the function of Pcl2 in ESCs, endogenous Pcl2 expression was stably knocked down using short hairpin RNA (shRNA) (Kunath et al., 2003Kunath T. Gish G. Lickert H. Jones N. Pawson T. Rossant J. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype.Nat. Biotechnol. 2003; 21: 559-561Crossref PubMed Scopus (242) Google Scholar). We designed three shRNA sequences targeting different regions of the Pcl2 mRNA, as well as a mismatch control sequence to rule out indirect effects. One shRNA sequence resulted in the most significant reduction in Pcl2. Three of these clones, each exhibiting at least 70% knockdown in Pcl2 transcript compared to the mismatch control and wild-type R1 ESCs, were chosen for further analysis (Figure 1C) and used in all subsequent experiments. However, stable clones expressing the additional two shRNA sequences were analyzed in many of the assays and showed similar results (data not shown). We also confirmed that knockdown in Pcl2 mRNA levels resulted in reduced protein levels (Figures S2B and S2C). To test the effect of Pcl2 depletion on ESC self-renewal, we measured transcript levels of Oct4, Nanog, and Sox2 and found that they were increased in Pcl2 knockdowns (Figure 1D) over both wild-type and mismatch controls. Next, we performed a high-content fluorescence imaging assay to examine OCT4 protein levels in single cells (Davey and Zandstra, 2006Davey R.E. Zandstra P.W. Spatial organization of embryonic stem cell responsiveness to autocrine gp130 ligands reveals an autoregulatory stem cell niche.Stem Cells. 2006; 24: 2538-2548Crossref PubMed Scopus (50) Google Scholar, Walker et al., 2007Walker E. Ohishi M. Davey R.E. Zhang W. Cassar P.A. Tanaka T.S. Der S.D. Morris Q. Hughes T.R. Zandstra P.W. et al.Prediction and testing of novel transcriptional networks regulating embryonic stem cell self-renewal and commitment.Cell Stem Cell. 2007; 1: 71-86Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The OCT4 distribution is best estimated by a double Gaussian curve, and a threshold delineating OCT4 +ve and −ve cells was calculated as the intersection of these two curves (Davey and Zandstra, 2006Davey R.E. Zandstra P.W. Spatial organization of embryonic stem cell responsiveness to autocrine gp130 ligands reveals an autoregulatory stem cell niche.Stem Cells. 2006; 24: 2538-2548Crossref PubMed Scopus (50) Google Scholar). Results are reported as the percentage of cells above and below this threshold. At 3 hours after plating, the cells were viable and contained over 90% OCT4-positive cells (Figure 1E). Cells were cultured under multiple differentiative conditions to study the effects of known self-renewal mediators LIF and BMP4. In all cases, Pcl2 knockdowns maintained a larger OCT4 +ve population (Figures 1E and 1F, BMP4; data not shown). However, the intensity of the OCT4 +ve peak in knockdown cells was not increased compared to the control (Figure 1E). Thus, individual cells did not overexpress OCT4. Instead, a greater percentage of the population was OCT4 +ve. This resulted in the appearance of overexpression in bulk analyses (Figures 1D and 4A) and highlights the importance of single-cell analyses in distinguishing distinct cell states within a heterogeneous population. In clonogenic assays (Peerani et al., 2009Peerani R. Bauwens C. Kumacheva E. Zandstra P.W. Patterning mouse and human embryonic stem cells using micro-contact printing.Methods Mol. Biol. 2009; 482: 21-33Crossref PubMed Scopus (29) Google Scholar, Zhang et al., 2006Zhang W. Walker E. Tamplin O.J. Rossant J. Stanford W.L. Hughes T.R. Zfp206 regulates ES cell gene expression and differentiation.Nucleic Acids Res. 2006; 34: 4780-4790Crossref PubMed Scopus (40) Google Scholar), cultures were stained for alkaline phosphatase (ALP) activity and colonies were stringently scored, with only colonies resembling those in Figure 1G being counted as undifferentiated. Pcl2 knockdown cells formed undifferentiated colonies with much greater efficiency than controls (Figure 1H). Collectively, these results suggest that knockdown of Pcl2 promoted self-renewal and maintained the undifferentiated state of ESCs during the initial time points following the cue to differentiate. To test the effect of Pcl2 on differentiation capacity, we performed directed neuroectoderm differentiation (Ying et al., 2003bYing Q.L. Stavridis M. Griffiths D. Li M. Smith A. Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.Nat. Biotechnol. 2003; 21: 183-186Crossref PubMed Scopus (1105) Google Scholar) assays and found Pcl2 knockdown cells maintained high levels of OCT4 with no observable expression of Nestin within a 5 day period (data not shown). Next, we used embryoid body (EB) formation to determine how the cells would respond when stimulated to differentiate for a prolonged period. Compared to controls, knockdown EBs remained rounded, and in general, cells did not migrate outwards, nor did they acquire a differentiated morphology. Based on immunostaining, control EBs formed large areas of both smooth muscle actin (SMA, mesoderm) and Nestin (neuroectoderm)-positive cells while knockdown EBs formed very few differentiated cells (Figure 1I). Furthermore, knockdown EBs did not form α-fetoprotein (endoderm)-positive cells and maintained high levels of OCT4 expression throughout the cultures (Figure 1I). We also tested whether expression of lineage-specific genes was disrupted and found that although transcripts of some markers of mesoderm and ectoderm were eventually expressed, their expression was delayed (Figure S1B). We also observed that endoderm markers were expressed to different extents in knockdowns compared to controls (Figure S1B). To test whether knockdown EBs would eventually differentiate after extended culture, EBs were grown in suspension for 25 days (Kaji et al., 2006Kaji K. Caballero I.M. MacLeod R. Nichols J. Wilson V.A. Hendrich B. The NuRD component Mbd3 is required for pluripotency of embryonic stem cells.Nat. Cell Biol. 2006; 8: 285-292Crossref PubMed Scopus (289) Google Scholar). After 25 days, control cultures consisted of cystic EBs (Figure 1J, top left) that, when plated on gelatin, developed into cells of differentiated morphology that did not exhibit ALP activity (Figure 1J, top right). In contrast, knockdown EBs remained as small, tightly compacted groups of cells (Figure 1J, bottom left) and retained the capacity to form undifferentiated ALP +ve colonies (Figure 1J, bottom right). These data suggest that Pcl2 is critically involved in early commitment and differentiation. Knockdowns appeared unable to progress to terminal differentiation of some cell types and retained a population of self-renewing ESC-like cells. To verify that the phenotype observed was specific to the depletion of Pcl2, we performed rescue experiments by ectopically expressing Pcl2-IRES-βgeo with a doxycycline-inducible transposon expression system (Woltjen et al., 2009Woltjen K. Michael I.P. Mohseni P. Desai R. Mileikovsky M. Hämäläinen R. Cowling R. Wang W. Liu P. Gertsenstein M. et al.piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.Nature. 2009; 458: 766-770Crossref PubMed Scopus (1371) Google Scholar). Clones expressing the transgene encoding the predominate ESC isoforms (Figures S2A and S2B) were selected based on β-galactosidase expression (Figure 2A), PCL2 protein levels (Figure 2B), and Pcl2 transcript (Figure 2C) following 72 hr of treatment with doxycycline. In colony-forming assays, overexpression of PCL2 was not sufficient to return the normal ratio of differentiated to undifferentiated colonies in +LIF (Figures 2D and 2E). However, under –LIF conditions, rescue clones regained the ability to differentiate (Figure 2E) and contained a normal percentage of OCT4-negative cells (Figure 2F). Finally, after long-term culture in doxycycline, expression of ESC-specific genes Oct4, Sox2, Nanog, Esrrb, and Tcl1 were decreased (Figure 2G). These data demonstrate that following overexpression of Pcl2 in the knockdown clones, cells recover the ability to downregulate the pluripotency network and differentiate. In Drosophila, PCL associates with PRC2 (O'Connell et al., 2001O'Connell S. Wang L. Robert S. Jones C.A. Saint R. Jones R.S. Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.J. Biol. Chem. 2001; 276: 43065-43073Crossref PubMed Scopus (92) Google Scholar, Savla et al., 2008Savla U. Benes J. Zhang J. Jones R.S. Recruitment of Drosophila Polycomb-group proteins by Polycomblike, a component of a novel protein complex in larvae.Development. 2008; 135: 813-817Crossref PubMed Scopus (57) Google Scholar); therefore, we examined whether murine PCL2 associated with the PRC2 complex. Gel filtration analysis indicated that PCL2 is found in two chromatographically distinct peaks: a large molecular mass peak that coeluted with SUZ12, EZH2, and EED and a smaller peak, which may represent the monomeric form of PCL2 or smaller subcomplexes. SUZ12 and EED were also detected in fractions corresponding to the smaller PCL2 peak (Figure 3A). These associations were confirmed by coimmunoprecipitation experiments (Figure 3B). Previous reports have suggested that PCL2 does not directly interact with EZH2 (O'Connell et al., 2001O'Connell S. Wang L. Robert S. Jones C.A. Saint R. Jones R.S. Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.J. Biol. Chem. 2001; 276: 43065-43073Crossref PubMed Scopus (92) Google Scholar), which prompted us to further substantiate its association with SUZ12 using a nonbiased epitope-tagged SUZ12 system coupled to mass spectrometry. We purified SUZ12-associated proteins from a mouse ESC line containing a 6xHis-3xFLAG tag (HF) targeted to the C terminus of one allele of the Suz12 gene, which resulted in expression of a full-length tagged SUZ12 protein (Singla et al., 2010Singla V. Hunkapiller J. Santos N. Seol A.D. Norman A.R. Wakenight P. Skarnes W.C. Reiter J.F. Floxin, a resource for genetically engineering mouse ESCs.Nat. Methods. 2010; 7: 50-52Crossref PubMed Scopus (22) Google Scholar). SUZ12-associated proteins were then separated by SDS-PAGE and stained with Coomassie blue to identify interacting proteins (Figure 3C). Mass spectrometric analysis indicated that the two prominent bands at approximately 100 and 55 kDa contained proteins SUZ12, EZH1, and EZH2 and RBBP4, RBBP7, AEBP2, EED, and PCL2, respectively (Figure 3D). In addition to a number of previously known interactors of the PRC2 complex including RBBP4, RBBP7, and AEBP2 (Figure 3D) (Kim et al., 2009Kim H. Kang K. Kim J. AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2.Nucleic Acids Res. 2009; 37: 2940-2950Crossref PubMed Scopus (137) Google Scholar, Kuzmichev et al., 2002Kuzmichev A. Nishioka K. Erdjument-Bromage H. Tempst P. Reinberg D. Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein.Genes Dev. 2002; 16: 2893-2905Crossref PubMed Scopus (1170) Google Scholar), we identified 15 peptides of PCL2 (Table S1). Taken together, these data demonstrate that PCL2 associates with the PRC2 complex in mouse ESCs. The PRC2 complex is responsible for mediating gene repression by trimethylating H3K27. Loss of the core members of PRC2 results in disruption of the complex and decreased global 3meH3K27. Reduction of PCL2 using shRNA led to moderately increased levels of EZH2 (Figures 4A and 4C) and global 3meH3K27 (Figures 4A and 4D) in the undifferentiated state, while levels of 1meH3K27 were unchanged (Figure 4B). Following LIF withdrawal, EZH2 initially declined, and by day 6, the control cells comprised three distinct cell populations expressing different levels of EZH2 (Figure 4C). In the knockdown cells, EZH2 levels did not decrease during the first 4 days of LIF withdrawal, and a small portion of cells displayed decreased EZH2 at day 6 (Figure 4C). 3meH3K27 levels decreased in both control and knockdown cells immediately following differentiation. Consistent with their undifferentiated state, the knockdown cells had slightly more 3meH3K27 during the first day of differentiation but contained similar levels to the control cells by day 4 of differentiation. At day 6, control cells consisted of two different populations of cells displaying two low but distinct levels of 3meH3K27, whereas knockdown cells comprised a single population (Figure 4D). These data reveal Pcl2 is dispensable for EZH2 expression and global 3meH3K27 in undifferentiated cells. However, upon the withdrawal of LIF, levels of both EZH2 and 3meH3K27 decline below the levels of the control. To dete" @default.
• W2005027557 created "2016-06-24" @default.
• W2005027557 creator A5019223167 @default.
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• W2005027557 date "2010-02-01" @default.
• W2005027557 modified "2023-10-16" @default.
• W2005027557 title "Polycomb-like 2 Associates with PRC2 and Regulates Transcriptional Networks during Mouse Embryonic Stem Cell Self-Renewal and Differentiation" @default.
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