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• W2046804572 abstract "To prevent insertional mutagenesis arising from retroviral reactivation, cells of embryonic origin possess a unique capacity to silence retroviruses. Given the distinct modes of X chromosome inactivation between embryonic and extraembryonic lineages, we investigated paradigms of viral extinction. We show that trophectoderm stem cells do not silence retroviral transcription, whereas extraembryonic endoderm stem cells aggressively extinguish proviral transcription, even more rapidly than do embryonic stem cells. By using a short hairpin RNA library, we identified epigenetic modifiers of retroviral extinction in extraembryonic endoderm stem cells. Multiple chromatin remodeling and polycomb repressor complex proteins act to modulate integrated, as well as endogenous, retroviral element silencing, with a subset of factors displaying differential effects between stem cell types. Furthermore, our data suggest that small RNAs play a role in this process through interactions with the Argonaute family. Our results further the understanding of mechanisms regulating retroviral transcription in different stem cell lineages. To prevent insertional mutagenesis arising from retroviral reactivation, cells of embryonic origin possess a unique capacity to silence retroviruses. Given the distinct modes of X chromosome inactivation between embryonic and extraembryonic lineages, we investigated paradigms of viral extinction. We show that trophectoderm stem cells do not silence retroviral transcription, whereas extraembryonic endoderm stem cells aggressively extinguish proviral transcription, even more rapidly than do embryonic stem cells. By using a short hairpin RNA library, we identified epigenetic modifiers of retroviral extinction in extraembryonic endoderm stem cells. Multiple chromatin remodeling and polycomb repressor complex proteins act to modulate integrated, as well as endogenous, retroviral element silencing, with a subset of factors displaying differential effects between stem cell types. Furthermore, our data suggest that small RNAs play a role in this process through interactions with the Argonaute family. Our results further the understanding of mechanisms regulating retroviral transcription in different stem cell lineages. Mouse ESCs, TSCs, and XEN cells have different capacities to suppress retroviral activity RNAi screens identified epigenetic factors that mediate silencing of retroviruses Candidates modulate integrated, as well as endogenous, retroviral element silencing An Argonaute protein binds integrated provirus and may mediate retroviral extinction To contend with the constant threat of retroviral infection, both from exogenous as well as endogenous elements, mammalian genomes have evolved complex defense mechanisms to impede the life cycle of invading parasitic nucleic acids. Historically, these defensive strategies have been separated into two broad categories: viral restriction and viral extinction (Niwa et al., 1983Niwa O. Yokota Y. Ishida H. Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells.Cell. 1983; 32: 1105-1113Abstract Full Text PDF PubMed Scopus (127) Google Scholar, Cherry et al., 2000Cherry S.R. Biniszkiewicz D. van Parijs L. Baltimore D. Jaenisch R. Retroviral expression in embryonic stem cells and hematopoietic stem cells.Mol. Cell. Biol. 2000; 20: 7419-7426Crossref PubMed Scopus (235) Google Scholar). Restriction is a process whereby protein factors encoded by the host genome interact directly with viral elements to block some aspect of the invading virus's life strategy. Proteins such as Friend virus susceptibility 1 (FV1), tripartite interaction motif 5a (TRIM5a), zinc finger antiviral protein (ZAP), TRIM19/PML (promyelocytic leukemia), and the TRIM28-ZFP809 (zinc finger antiviral protein 809) complex all restrict retroviral tropism via direct biochemical interactions (Best et al., 1996Best S. Le Tissier P. Towers G. Stoye J.P. Positional cloning of the mouse retrovirus restriction gene Fv1.Nature. 1996; 382: 826-829Crossref PubMed Scopus (389) Google Scholar, Kaiser et al., 2007Kaiser S.M. Malik H.S. Emerman M. Restriction of an extinct retrovirus by the human TRIM5alpha antiviral protein.Science. 2007; 316: 1756-1758Crossref PubMed Scopus (106) Google Scholar, Gao et al., 2002Gao G. Guo X. Goff S.P. Inhibition of retroviral RNA production by ZAP, a CCCH-type zinc finger protein.Science. 2002; 297: 1703-1706Crossref PubMed Scopus (302) Google Scholar, Turelli et al., 2001Turelli P. Doucas V. Craig E. Mangeat B. Klages N. Evans R. Kalpana G. Trono D. Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: Interference with early steps of viral replication.Mol. Cell. 2001; 7: 1245-1254Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, Wolf and Goff, 2009Wolf D. Goff S.P. Embryonic stem cells use ZFP809 to silence retroviral DNAs.Nature. 2009; 458: 1201-1204Crossref PubMed Scopus (240) Google Scholar, Rowe et al., 2010Rowe H.M. Jakobsson J. Mesnard D. Rougemont J. Reynard S. Aktas T. Maillard P.V. Layard-Liesching H. Verp S. Marquis J. et al.KAP1 controls endogenous retroviruses in embryonic stem cells.Nature. 2010; 463: 237-240Crossref PubMed Scopus (447) Google Scholar). Less well understood is the process of retroviral extinction, which is progressive silencing of proviral transcription that occurs over long-term cellular growth or differentiation (Cherry et al., 2000Cherry S.R. Biniszkiewicz D. van Parijs L. Baltimore D. Jaenisch R. Retroviral expression in embryonic stem cells and hematopoietic stem cells.Mol. Cell. Biol. 2000; 20: 7419-7426Crossref PubMed Scopus (235) Google Scholar, Laker et al., 1998Laker C. Meyer J. Schopen A. Friel J. Heberlein C. Ostertag W. Stocking C. Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation.J. Virol. 1998; 72: 339-348PubMed Google Scholar). This process is epigenetic in nature and has been associated with acquisition of DNA methylation and other transcriptionally repressive chromatin modifications at the viral integration site (Harbers et al., 1981Harbers K. Schnieke A. Stuhlmann H. Jähner D. Jaenisch R. DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning.Proc. Natl. Acad. Sci. USA. 1981; 78: 7609-7613Crossref PubMed Scopus (124) Google Scholar, Jähner et al., 1982Jähner D. Stuhlmann H. Stewart C.L. Harbers K. Löhler J. Simon I. Jaenisch R. De novo methylation and expression of retroviral genomes during mouse embryogenesis.Nature. 1982; 298: 623-628Crossref PubMed Scopus (348) Google Scholar, Pannell et al., 2000Pannell D. Osborne C.S. Yao S. Sukonnik T. Pasceri P. Karaiskakis A. Okano M. Li E. Lipshitz H.D. Ellis J. Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code.EMBO J. 2000; 19: 5884-5894Crossref PubMed Scopus (128) Google Scholar, Poleshko et al., 2008Poleshko A. Palagin I. Zhang R. Boimel P. Castagna C. Adams P.D. Skalka A.M. Katz R.A. Identification of cellular proteins that maintain retroviral epigenetic silencing: evidence for an antiviral response.J. Virol. 2008; 82: 2313-2323Crossref PubMed Scopus (39) Google Scholar, Matsui et al., 2010Matsui T. Leung D. Miyashita H. Maksakova I.A. Miyachi H. Kimura H. Tachibana M. Lorincz M.C. Shinkai Y. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET.Nature. 2010; (in press. Published online February 17, 2010)https://doi.org/10.1038/nature08858Crossref Scopus (454) Google Scholar). Cells of embryonic origin are unique in their capacity to silence retroviruses, which is understandable given the necessity of preventing insertional mutagenesis that could arise from retroviral reactivation. γ-retroviruses, like the mouse leukemia virus (MLV), can integrate into embryonic carcinoma and embryonic stem cells but are silenced by both restriction- and extinction-based mechanisms (Loh et al., 1990Loh T.P. Sievert L.L. Scott R.W. Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.Mol. Cell. Biol. 1990; 10: 4045-4057PubMed Google Scholar, Niwa et al., 1983Niwa O. Yokota Y. Ishida H. Sugahara T. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells.Cell. 1983; 32: 1105-1113Abstract Full Text PDF PubMed Scopus (127) Google Scholar, Teich et al., 1977Teich N.M. Weiss R.A. Martin G.R. Lowy D.R. Virus infection of murine teratocarcinoma stem cell lines.Cell. 1977; 12: 973-982Abstract Full Text PDF PubMed Scopus (119) Google Scholar). The early embryo possesses three stem cell lineages: embryonic stem (ES), trophectoderm stem (TS), and extraembryonic endoderm (XEN) stem cells that give rise to the embryo proper, placenta, and yolk sac, respectively. Although viral extinction has been well documented in cells of embryonic origin, the capacity of extraembryonic stem cells to epigenetically silence γ-retroviruses has not been examined. Given the distinct epigenetic mechanisms regulating X chromosome inactivation in embryonic and extraembryonic cells (Kunath et al., 2005Kunath T. Arnaud D. Uy G.D. Okamoto I. Chureau C. Yamanaka Y. Heard E. Gardner R.L. Avner P. Rossant J. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.Development. 2005; 132: 1649-1661Crossref PubMed Scopus (264) Google Scholar, Takagi and Sasaki, 1975Takagi N. Sasaki M. Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse.Nature. 1975; 256: 640-642Crossref PubMed Scopus (570) Google Scholar), we hypothesized that different modes of viral extinction may also exist between separate lineages of the preimplantation embryo. Specifically, we sought to determine whether stem cells derived from trophoblast and extraembryonic endoderm could epigenetically extinguish a MLV variant modified to escape retroviral restriction but which is susceptible to extinction (Cherry et al., 2000Cherry S.R. Biniszkiewicz D. van Parijs L. Baltimore D. Jaenisch R. Retroviral expression in embryonic stem cells and hematopoietic stem cells.Mol. Cell. Biol. 2000; 20: 7419-7426Crossref PubMed Scopus (235) Google Scholar, Grez et al., 1990Grez M. Akgün E. Hilberg F. Ostertag W. Embryonic stem cell virus, a recombinant murine retrovirus with expression in embryonic stem cells.Proc. Natl. Acad. Sci. USA. 1990; 87: 9202-9206Crossref PubMed Scopus (164) Google Scholar, Laker et al., 1998Laker C. Meyer J. Schopen A. Friel J. Heberlein C. Ostertag W. Stocking C. Host cis-mediated extinction of a retrovirus permissive for expression in embryonal stem cells during differentiation.J. Virol. 1998; 72: 339-348PubMed Google Scholar). To examine viral extinction in stem cells, we assayed transcriptional activity of genetically marked mouse γ-retroviruses in primary ESCs, TSCs, and XEN stem cells. Unlike ESCs, TSCs did not silence proviral transcription but rather maintained consistent and high expression levels over long-term culture. Surprisingly, integration of γ-retrovirus into XEN cells produced rapid transcriptional silencing, by comparison to ESCs. We present data that indicate XEN cell extinction is epigenetic in nature and mediated by multiple chromatin remodeling and polycomb repressor complexes. These complexes not only act to modulate the silent state of integrated retroviruses but also play significant roles in repressing endogenous retro-element transcription. Furthermore, our results suggest that these repressor complexes are recruited to sites of viral integration through interactions with the Argonaute family of proteins. For decades, scientists have intensively studied mechanisms of gene regulation in ESCs, including those involved in exogenous and endogenous retroviral silencing. However, only a few epigenetic factors employed in retroviral silencing have been identified. Furthermore, epigenetic mechanisms operating in cohabiting extraembryonic stem cells have been virtually ignored, the assumption being that all stem cells utilize similar mechanisms. To determine whether stem cells derived from trophoblast and extraembryonic endoderm possess similar or distinct capacities to silence retroviruses, we chose to examine transcriptional activity of a MLV variant that is closely related to the endogenous type C retroviral element. To achieve this, we constructed a series of genetically marked mouse embryonic stem cell viruses (MSCV) (Figures 1A and 2A) and assayed their transcriptional activity in infected primary ESCs, TSCs, and XEN cells. Within 24 hr of infection, expression of virally delivered GFP could be detected within each of the three stem cell lineages (Figure 1B; Table S1 available online). To assay for viral extinction, cells were propagated for 6–15 weeks, with the percentage of GFP-positive cells monitored via flow cytometry at each passage (Figure 1C). Consistent with previous observations (Cherry et al., 2000Cherry S.R. Biniszkiewicz D. van Parijs L. Baltimore D. Jaenisch R. Retroviral expression in embryonic stem cells and hematopoietic stem cells.Mol. Cell. Biol. 2000; 20: 7419-7426Crossref PubMed Scopus (235) Google Scholar), ESCs exhibited a progressive decline in the number of cells expressing GFP with near complete viral extinction occurring after 15 weeks in culture (passage 40) (Figure S1). Surprisingly, TSCs did not inactivate the γ-retroviral reporter but rather maintained a constant level of GFP-expressing cells. This trend was maintained through 12 weeks of observation (30 passages) and correlated with continued expression of TSC-specific markers (data not shown). By comparison, XEN cells exhibited aggressive silencing of viral transcription such that within 3 weeks (6–7 passages), GFP expression was virtually undetectable. Given the surprising speed with which the γ-retroviral reporter was silenced in the XEN cell lineage, we examined viral extinction in this relatively uncharacterized cell type. To ensure that the observed extinction was an epigenetic phenomenon, and not an artifact of cellular differentiation or GFP toxicity, three experiments were conducted. First, to determine whether loss of viral expression was a result of cellular differentiation, RNA was extracted from MSCV-GFP-infected XEN cells at passages 2, 7, and 10 and assayed for expression of XEN cell markers. Abundant expression of transcripts encoding GATA binding protein 4 (Gata4), Forkhead box protein A2 (Foxa2), Hepatocyte nuclear factor 4 (Hnf4), and Sex determining region Y-box 7 (Sox7) was observed via reverse transcription polymerase chain reaction (RT-PCR) (Figure S1; Kunath et al., 2005Kunath T. Arnaud D. Uy G.D. Okamoto I. Chureau C. Yamanaka Y. Heard E. Gardner R.L. Avner P. Rossant J. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.Development. 2005; 132: 1649-1661Crossref PubMed Scopus (264) Google Scholar). Maintenance of XEN cell marker expression, together with consistent XEN cell morphology throughout the culture period, indicated that silencing of virally delivered GFP was not associated with loss of stemness. Second, to ensure that the observed extinction was not due to loss of integrated virus, nor to GFP toxicity, infected XEN cell DNA was examined for MSCV-GFP provirus at passage 2 through 10 by quantitative PCR (qPCR). No difference in the ratio of GFP to Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) promoter amplification was detected throughout the experiment, indicating that the number of cells containing the integrated provirus was relatively unchanged (Figure S1). Finally, to determine whether viral reporter silencing was epigenetic in nature, pharmacological inhibition of DNA methylation and histone deacetylation was employed. To this end, XEN cells were again infected with MSCV-GFP, cultured for 10 passages to allow γ-retroviral reporter silencing, then treated with 5-Aza-2′-deoxycytidine (5-Aza) or trichostatin-A (TSA) either alone or in combination. Treatment with 5-Aza or TSA alone induced a 5- to 15-fold increase in reactivation of proviral transcription compared to vehicle alone. Treatment with both drugs produced a synergistic response leading to transcriptional activation (Figure 1D). Thus, integration of γ-retrovirus into the XEN cell genome rapidly initiated an aggressive epigenetic-based antiviral response, resulting in complete silencing of proviral transcription. To identify specific biochemical factors that mediate this epigenetic response, we conducted a loss-of-function, RNA interference (RNAi), positive selection screen. By sing a microRNA-based short hairpin RNA library (shRNAmir) (Silva et al., 2005Silva J.M. Li M.Z. Chang K. Ge W. Golding M.C. Rickles R.J. Siolas D. Hu G. Paddison P.J. Schlabach M.R. et al.Second-generation shRNA libraries covering the mouse and human genomes.Nat. Genet. 2005; 37: 1281-1288Crossref PubMed Scopus (512) Google Scholar) that was designed to target 250 known protein-coding genes involved in epigenetic gene regulation (∼3 shRNAmir/gene; Table S2), we addressed the question of how γ-retroviruses are silenced in mouse XEN cells. To conduct our screen, we designed two separate γ-retroviral reporters with distinct fluorescent and drug-selectable markers (Figure 2A ). These constructs were packaged into infectious retroviral particles and delivered into early-passage XEN cells. Cells were drug selected, withdrawn from drug selection, and passaged to allow for retroviral extinction. As previously observed, integrated virus was rapidly silenced (Figure S2). Additionally, cultures were verified for maintenance of stemness via XEN cell markers as described above (Figure S1). At passage 11, replication-deficient lentiviral particles (Figure 2B) were used to deliver the shRNAmir epigenetic library into XEN cells containing a silenced γ-retroviral reporter. These constructs were specifically designed to facilitate delivery into ESCs, TSCs, and XEN cells (unpublished data). shRNAmir constructs were matched to contain distinct fluorescent and drug selection markers from the γ-retroviral reporters (Figure 2C). As a control, a shRNAmir targeting the firefly luciferase (Luc) gene was stably transduced into XEN cells, and experiments were conducted in parallel. Upon delivery of the shRNAmir library, cells were passaged for 1 week, then drug selected with either neomycin or puromycin to enrich for XEN cells with reactivated MSCV ChIN or MSCV PIG reporters, respectively. Surviving colonies were picked and DNA isolated, and perspective shRNAmirs conferring resistance to silencing were amplified and sequenced (425 colonies in total). Results of the screen based on a comprehensive scoring system (Figure S3) are summarized in Table 1. Three of the top 25 identified factors have known interactions with viral elements, which validates our experimental design: SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 (SMARCA5); DNA methyltransferase 1 (DNMT1); and Helicase, lymphoid-specific (HELLS) (Chong et al., 2007Chong S. Vickaryous N. Ashe A. Zamudio N. Youngson N. Hemley S. Stopka T. Skoultchi A. Matthews J. Scott H.S. et al.Modifiers of epigenetic reprogramming show paternal effects in the mouse.Nat. Genet. 2007; 39: 614-622Crossref PubMed Scopus (137) Google Scholar, De La Fuente et al., 2006De La Fuente R. Baumann C. Fan T. Schmidtmann A. Dobrinski I. Muegge K. Lsh is required for meiotic chromosome synapsis and retrotransposon silencing in female germ cells.Nat. Cell Biol. 2006; 8: 1448-1454Crossref PubMed Scopus (99) Google Scholar). In contrast, the majority of the candidates identified in this screen are novel regulators of retroviral silencing.Table 1Top 25 Candidate Genes Mediating γ-Retroviral Silencing in XEN CellsGene NameOther NamesScoreComplex AssociationSmarca1Snf2L, Nurf140637NURFSmarca5Snf2h, MommeD4349PRC1, SIN3a-HDAC, SNF2h-COHESIN-NuRD CENHdac7a201SIN3-HDACHdac2Rpd3, Yaf1, Yyibp180PRC2, SIN3a, NcORHdac9Hdac7b, Hdrp, Mitr135?Pcgf6Mblr, Rnf134108PRC1Smarcc2Baf17099BRG, BAFDnmt3a90PRC2, PRC3Eif2c1Ago180RISCEed77PRC2, PRC3, PRC4Myst4Morf, Moz2, Kat6b, Qkf63?Smarcd1Baf60a36BRG, BAFSmarcad1Etl131?Sfmbt224PHO-RCPhc2Mph2, Edr224PRC1SirT1Sir2, Sir2alpha24PRC4Myst2Hbo1, Hboa, Kat721?Smyd3Zmynd121?Dnmt1MommeD219PRC2, PRC3HellsLsh, Smarca6, Yfk8, Pasg18DNMTs / HMTsSuv39h1Kmt1a16PRC1, PRC2Myst1Mof, Kat816PRC1?Ezh2Enx-1, Kmt614PRC2, PRC3, PRC4Phc3Hph3, Edr39PRC1Actl6bActl6, Baf53b, ArpNa8BAF Open table in a new tab To validate the most promising candidates that emerged from our screen, shRNAmirs targeting the top ten candidates based on our scoring system were reintroduced into XEN cells. Target mRNA depletion was verified with a combination of quantitative RT-PCR (qRT-PCR) and western blot analysis (Figures S4 and S5). As controls, candidate shRNAmir-targeted XEN cells were compared to wild-type XEN cells, XEN cells infected with MSCV ChIN or MSCV PIG, as well as Luc shRNAmir-targeted XEN cells. For analysis, results were normalized to wild-type expression levels. All tested shRNAmirs produced target mRNA depletion. Our scoring system gives strong preference to genes for which multiple independent shRNAs emerged. Although arguably we may be bypassing strong candidates, it minimized the potential for off-target shRNAs to be included. The observation that shRNAmirs eliciting potent depletion scored higher in the screen then weaker ones validates the scoring system. To determine whether differences in proviral silencing between ESCs, TSCs, and XEN cells could be explained by a lineage-specific absence of candidate factors or differences in expression levels, relative transcript abundance was investigated via qRT-PCR analysis. To ensure accurate quantitation of candidate transcript levels between cell types, measurements were normalized against the geometric mean of β-actin, 7SK, and Hexokinase transcript levels. Very little SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 1 (Smarca1) expression was detected in TSCs; both ESCs and XEN cells possessed greater than 1000-fold higher transcript abundance than did TSCs (Figure 3). The remaining factors showed a 2- to 10-fold increase in transcript abundance in ESCs compared to TSCs and XEN cells, including SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 (Smarca5), Histone deactylases Hdac2, Hdac7, and Hdac9, Polycomb group ring finger 6 (Pcgf6), DNA methyltransferase 3a (Dnmt3a), Argonaute1 (Ago1), and Embryonic ectoderm development (Eed). The lone exception was SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 2 (Smarcc2), which was 3 times more abundantly expressed in XEN cells than in ESCs or TSCs. Given that SMARCA5 was identified in a previous genetic screen for modifiers of retroviral gene silencing (Chong et al., 2007Chong S. Vickaryous N. Ashe A. Zamudio N. Youngson N. Hemley S. Stopka T. Skoultchi A. Matthews J. Scott H.S. et al.Modifiers of epigenetic reprogramming show paternal effects in the mouse.Nat. Genet. 2007; 39: 614-622Crossref PubMed Scopus (137) Google Scholar), it is not surprising that other gene family members (SMARCA1 and SMARCC2) may be involved in proviral suppression. Chromatin remodeling is associated with numerous heterogeneous protein complexes and diverse changes in chromatin structure, including histone deacetylation (Xue et al., 1998Xue Y. Wong J. Moreno G.T. Young M.K. Côté J. Wang W. NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities.Mol. Cell. 1998; 2: 851-861Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). To characterize histone acetylation changes associated with proviral silencing, we conducted quantitative chromatin immunoprecipitation (ChIP) analysis of the MSCV 5′ long terminal repeat (LTR) and proximal packaging region (Psi) (Figure 4A ) over the course of XEN cell viral extinction. A 1000-fold reduction in signal between passages 2 and 6 was observed when lysates were immunoprecipitated with antibodies recognizing histone 3 acetylation (H3Ac) (Figure 4B). This dramatic drop in acetylated histones strongly supports the involvement of histone deacetylases (HDAC2, HDAC7, and/or HDAC9) in proviral extinction. In further support of this data, Poleshko et al., 2008Poleshko A. Palagin I. Zhang R. Boimel P. Castagna C. Adams P.D. Skalka A.M. Katz R.A. Identification of cellular proteins that maintain retroviral epigenetic silencing: evidence for an antiviral response.J. Virol. 2008; 82: 2313-2323Crossref PubMed Scopus (39) Google Scholar and Keedy et al., 2009Keedy K.S. Archin N.M. Gates A.T. Espeseth A. Hazuda D.J. Margolis D.M. A limited group of class I histone deacetylases acts to repress human immunodeficiency virus type 1 expression.J. Virol. 2009; 83: 4749-4756Crossref PubMed Scopus (155) Google Scholar identified a role for the class 1 histone deacetylases HDAC1-3 in suppressing retroviral transcription. Here, we have potentially expanded the family of HDACs involved in retroviral extinction to include both class 1 (HDAC2) and class 2 (HDAC7 and HDAC9) histone deacetylases. One interesting candidate to emerge from our screen is PCGF6. This protein, which was recently identified as a key component of the self-renewal process in ESCs (Hu et al., 2009Hu G. Kim J. Xu Q. Leng Y. Orkin S.H. Elledge S.J. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal.Genes Dev. 2009; 23: 837-848Crossref PubMed Scopus (301) Google Scholar), is a putative subunit of the Polycomb Repressive Complex 1 (PRC1) (Lee et al., 2007Lee M.G. Norman J. Shilatifard A. Shiekhattar R. Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein.Cell. 2007; 128: 877-887Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). PRC1 is a multimeric protein complex known to possess histone demethylating activity. Specifically, PCGF6 directly associates with members of the Jumonji AT-rich interactive domain (JARID) family of proteins, and thus the enzymatic removal of histone 3 lysine 4 trimethylation (H3K4me3) (Lee et al., 2007Lee M.G. Norman J. Shilatifard A. Shiekhattar R. Physical and functional association of a trimethyl H3K4 demethylase and Ring6a/MBLR, a polycomb-like protein.Cell. 2007; 128: 877-887Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). When lysates were precipitated with an antibody recognizing H3K4me3, a 60-fold reduction in signal was observed between passages 2 and 6 (Figure 4B). The third protein complex identified in our screen was PRC2, a repressive complex composed minimally of EED, Enhancer of zeste homolog 2 (EZH2), and Suppressor of zeste 12 homolog (SUZ12) (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, Sparmann and van Lohuizen, 2006Sparmann A. van Lohuizen M. Polycomb silencers control cell fate, development and cancer.Nat. Rev. Cancer. 2006; 6: 846-856Crossref PubMed Scopus (1021) Google Scholar). Two of the three core components (EED and EZH2) were identified in our screen, strongly implicating this complex in proviral silencing. Furthermore, PRC2 interacts with suppressor of variegation 3-9 homolog 1 (SUV39H1), HDAC2, and the DNMT family of proteins, all of which were identified in our screen. Through these interactions, PRC2 mediates transcriptional repression via histone 3 lysine 9 (H3K9) and lysine 27 (H3K27) methylation, as well as by directing DNA methylation (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, 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, Sewalt et al., 2002Sewalt R.G. Lachner M. Vargas M. Hamer K.M. den Blaauwen J.L. Hendrix T. Melcher M. Schweizer D. Jenuwein T. Otte A.P. Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins.Mol. Cell. Biol. 2002; 22: 5539-5553Crossref PubMed Scopus (80) Google Scholar, Viré et al., 2006Viré E. Brenner C. Deplus R. Blanchon L. Fraga M. Didelot C. Morey L. Van Eynde A. Bernard D. Vanderwinden J.M. et al.The Polycomb group protein EZH2 directly controls DNA methylation.Nature. 2006; 439: 871-874Crossref PubMed Scopus (1589) Google Scholar). To examine the role of PRC2 in viral extinction, we examined changes in H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3) via ChIP. When lysates were immunoprecipitated with antibodies recognizing H3K9me3, a 300-fold increase in signal was detected between passages 2 and 6 (Figure 4B). By comparison, a slower, modest increase in H3K27me3 signal was observed over the experimental time course. PRC2 associates with all three members of the DNA methyltransferase family and directs DNA methylation of EZH2 target promoters (Viré et al., 2006Viré E. Brenner C. Deplus R. Blanchon L. Fraga M. Didelot C. Morey L. Van Eynde A. Bernard D. Vanderwinden J.M. e" @default.
• W2046804572 created "2016-06-24" @default.
• W2046804572 creator A5067398286 @default.
• W2046804572 creator A5074863991 @default.
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• W2046804572 date "2010-05-01" @default.
• W2046804572 modified "2023-10-12" @default.
• W2046804572 title "Multiple Epigenetic Modifiers Induce Aggressive Viral Extinction in Extraembryonic Endoderm Stem Cells" @default.
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