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Repression of Retrotransposal Elements in Mouse Embryonic Stem Cells Is Primarily Mediated by a DNA Methylation-independent Mechanism*

  • Leah K. Hutnick
    Affiliations
    From the Department of Human Genetics, Los Angeles, California 90095

    Neuroscience Interdepartmental Program, David Geffen School of Medicine, UCLA, Los Angeles, California 90095
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  • Xinhua Huang
    Affiliations
    From the Department of Human Genetics, Los Angeles, California 90095
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  • Tao-Chuan Loo
    Affiliations
    From the Department of Human Genetics, Los Angeles, California 90095
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  • Zhicheng Ma
    Affiliations
    From the Department of Human Genetics, Los Angeles, California 90095
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  • Guoping Fan
    Correspondence
    A Carol Moss Spivak Scholar in Neuroscience. To whom correspondence should be addressed: Dept. of Human Genetics, David Geffen School of Medicine, UCLA, 695 Charles Young Dr. S., Los Angeles, CA 90095. Tel.: 310-267-0439; Fax: 310-794-5446;
    Affiliations
    From the Department of Human Genetics, Los Angeles, California 90095
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant RO1 NS051411 and P01 GM081621 (to G. F.). This work was also supported by California Institute of Regenerative Medicine Grant RC1-0111 (to G. F.) and National Research Service Award 5F31NS051 (to L. K. H.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
Open AccessPublished:July 01, 2010DOI:https://doi.org/10.1074/jbc.M110.125674
      In defense of deleterious retrotransposition of intracisternal A particle (IAP) elements, IAP loci are heavily methylated and silenced in mouse somatic cells. To determine whether IAP is also repressed in pluripotent stem cells by DNA methylation, we examined IAP expression in demethylated mouse embryonic stem cells (mESCs) and epiblast-derived stem cells. Surprisingly, in demethylated ESC cultures carrying mutations of DNA methyltransferase I (Dnmt1), no IAP transcripts and proteins are detectable in undifferentiated Oct4+ ESCs. In contrast, ∼3.6% of IAP-positive cells are detected in Oct4 Dnmt1−/− cells, suggesting that the previously observed increase in IAP transcripts in the population of Dnmt1−/− ESCs could be accounted for by this subset of Oct4 Dnmt1−/− ESCs undergoing spontaneous differentiation. Consistent with this possibility, a dramatic increase of IAP mRNA (>100-fold) and protein expression was observed in Dnmt1−/− ESC cultures upon induction of differentiation through the withdrawal of leukemia-inhibitory factor for 6 or more days. Interestingly, both mRNAs and proteins of IAP can be readily detected in demethylated Oct4+ epiblast-derived stem cells as well as differentiated mouse embryo fibroblasts, neurons, and glia upon conditional Dnmt1 gene deletion. These data suggest that mESCs are a unique stem cell type possessing a DNA methylation-independent IAP repression mechanism. This methylation-independent mechanism does not involve Dicer-mediated action of microRNAs or RNA interference because IAP expression remains repressed in Dnmt1−/−; Dicer−/− double mutant ESCs. We suggest that mESCs possess a unique DNA methylation-independent mechanism to silence retrotransposons to safeguard genome stability while undergoing rapid cell proliferation for self-renewal.

      Introduction

      DNA methylation, an epigenetic modification where a methyl group is covalently added to the cytosine of CpG dinucleotides, is catalyzed by a family of DNA methyltransferases (Dnmts)
      The abbreviations used are: Dnmt
      DNA methyltransferase
      IAP
      intracisternal A particle
      ESC
      embryonic stem cell
      mESC
      mouse ESC
      EpiSC
      epiblast-derived stem cell
      MEF
      mouse embryo fibroblast
      GST
      glutathione S-transferase
      LIF
      leukemia-inhibitory factor
      RT
      reverse transcription
      FISH
      fluorescent in situ hybridization
      PBS
      phosphate-buffered saline
      LTR
      long terminal repeat
      MSCV
      murine stem cell virus
      GFP
      green fluorescent protein
      Cre
      cre recombinase
      En
      embryonic day n.
      that include de novo (Dnmt3a and -3b) and maintenance methyltransferases (Dnmt1) (
      • Bestor T.H.
      ,
      • Goll M.G.
      • Bestor T.H.
      ). DNA methylation is known to regulate developmental gene expression, genomic imprinting, X-inactivation, and genomic stability (
      • Robertson K.D.
      • Wolffe A.P.
      ,
      • Jaenisch R.
      • Bird A.
      ,
      • Klose R.J.
      • Bird A.P.
      ,
      • Feng J.
      • Fouse S.
      • Fan G.
      ). Many retrotransposable repetitive elements are heavily methylated, and current evidence supports a causal relationship between DNA methylation and repression of retrotransposons (
      • Walsh C.P.
      • Chaillet J.R.
      • Bestor T.H.
      ,
      • Gaudet F.
      • Rideout 3rd, W.M.
      • Meissner A.
      • Dausman J.
      • Leonhardt H.
      • Jaenisch R.
      ,
      • Hutnick L.K.
      • Golshani P.
      • Namihira M.
      • Xue Z.
      • Matynia A.
      • Yang X.W.
      • Silva A.J.
      • Schweizer F.E.
      • Fan G.
      ). Intracisternal A particles (IAPs) are murine endogenous retroviral repetitive elements, with an estimation of over 1000 copies across the haploid murine genome (
      • Heidmann O.
      • Heidmann T.
      ,
      • Dupressoir A.
      • Heidmann T.
      ,
      • Dewannieux M.
      • Dupressoir A.
      • Harper F.
      • Pierron G.
      • Heidmann T.
      ). Germ line mutations due to IAP retrotransposition most often occur at intronic sites, disrupting gene expression through premature termination, aberrant splicing, or viral LTR-driven transcription (
      • Whitelaw E.
      • Martin D.I.
      ). Furthermore, insertional mutagenesis of IAP elements is noted in various murine cancer cell lines with subsequent activation of oncogenes or cytokine genes (
      • Ukai H.
      • Ishii-Oba H.
      • Ukai-Tadenuma M.
      • Ogiu T.
      • Tsuji H.
      ,
      • Lee J.S.
      • Haruna T.
      • Ishimoto A.
      • Honjo T.
      • Yanagawa S.
      ).
      When expressed, IAP proteins are non-infectious viral particles that are retained in the cisternae of the endoplasmic reticulum (
      • Kuff E.L.
      • Wivel N.A.
      • Lueders K.K.
      ). IAP proteins can be transiently detected in preimplantation embryos (
      • Calarco P.G.
      ,
      • Poznanski A.A.
      • Calarco P.G.
      ), although DNA methylation of IAP elements is largely maintained throughout embryogenesis (
      • Lane N.
      • Dean W.
      • Erhardt S.
      • Hajkova P.
      • Surani A.
      • Walter J.
      • Reik W.
      ,
      • Lees-Murdock D.J.
      • De Felici M.
      • Walsh C.P.
      ). In established lines of mouse embryonic stem cells (ESCs) derived from the inner cell mass, IAP elements are already heavily methylated with little detectable IAP expression (
      • Ramírez M.A.
      • Pericuesta E.
      • Fernandez-Gonzalez R.
      • Moreira P.
      • Pintado B.
      • Gutierrez-Adan A.
      ).
      Here, we examine expression of IAP proteins in control and demethylated somatic cells, EpiSCs, and mESCs. Using a previously generated antibody against IAP protein (p73) (
      • Kuff E.L.
      • Callahan R.
      • Howk R.S.
      ) as well as our polyclonal antibody raised against recombinant IAP gag protein (
      • Hutnick L.K.
      • Golshani P.
      • Namihira M.
      • Xue Z.
      • Matynia A.
      • Yang X.W.
      • Silva A.J.
      • Schweizer F.E.
      • Fan G.
      ,
      • Mietz J.A.
      • Grossman Z.
      • Lueders K.K.
      • Kuff E.L.
      ), we mapped changes in IAP protein levels after Dnmt1 gene deletion in floxed (Dnmt12lox/2lox) mouse embryonic fibroblasts (MEFs) using virus-mediated cre recombinase. Unexpectedly, we found that IAP mRNA and protein levels are not detected in Oct4+ Dnmt1−/− ESCs but are dramatically increased in demethylated mESC cultures upon in vitro differentiation, suggestive of the presence of DNA methylation-independent repression mechanism(s) in silencing IAP expression in undifferentiated mESCs.

      DISCUSSION

      Host cell defensive strategies have evolved to counteract the deleterious consequences of IAP element reactivation. The best documented mechanism, DNA methylation of LTR promoters, can directly impede access of transcription factors or lead to an inactive form of chromatin at target loci (
      • Li E.
      ). As the time course of Dnmt1 conditional deletion in MEF cells indicates, IAP protein expression is tightly correlated with genomic DNA methylation levels, making IAP protein reactivation a reliable indicator of global changes in DNA methylation levels in individual cells. Previous tools for examining global DNA hypomethylation levels relied on evaluating DNA or RNA extracted from a population of cells. Although these techniques are sensitive, no evaluation could be made for global DNA methylation changes in individual cells within tissue. As shown in Fig. 3, IAP immunohistochemistry allows for recognition of DNA demethylation at a cellular level in different tissue types. In cultured N2a mouse neuroblastoma cells, a robust reactivation of IAP protein is present. Similarly, in hypomethylated neural lineage cells, IAP protein can be used to trace the demethylation status at the individual cell level. Thus, IAP antibodies are valuable reagents to detect demethylated cells in the murine system.
      Hypomethylated mESCs appear to possess an alternative mechanism for IAP silencing beyond the DNA methylation-mediated pathway. Interestingly, EpiSCs, which are also pluripotent yet differ from mESCs in both transcriptional networks and epigenetic modifications (
      • Tesar P.J.
      • Chenoweth J.G.
      • Brook F.A.
      • Davies T.J.
      • Evans E.P.
      • Mack D.L.
      • Gardner R.L.
      • McKay R.D.
      ,
      • Brons I.G.
      • Smithers L.E.
      • Trotter M.W.
      • Rugg-Gunn P.
      • Sun B.
      • Chuva de Sousa Lopes S.M.
      • Howlett S.K.
      • Clarkson A.
      • Ahrlund-Richter L.
      • Pedersen R.A.
      • Vallier L.
      ), show strong IAP protein reactivation after Dnmt1 deletion. Thus, a methylation-independent repressional mechanism is unique to mESCs. Further analysis of transcriptional and epigenetic profiles may highlight this unique repressional mechanism in mESCs.
      It can be potentially argued that DNA methylation directed by de novo methyltransferases in Dnmt1−/− mESCs silences IAP elements. We evaluated IAP protein expression levels in Dnmt1/Dnmt3a/Dnmt3b-deficient mESCs (TKO cells) (
      • Meissner A.
      • Gnirke A.
      • Bell G.W.
      • Ramsahoye B.
      • Lander E.S.
      • Jaenisch R.
      ). Under standard mESC culture conditions, the IAP-positive TKO cell population was not Oct4-positive (supplemental Fig. 1). Because TKO cells have less than 2% of the normal methylation levels (
      • Meissner A.
      • Gnirke A.
      • Bell G.W.
      • Ramsahoye B.
      • Lander E.S.
      • Jaenisch R.
      ), we argue that mESCs possess an alternative mechanism in the absence of DNA methylation that mediates IAP gene silencing.
      We have considered the involvement of both transcriptional and translational mechanisms controlling IAP expression in demethylated mESCs. It is possible that redundant repressive mechanisms are present in mESCs that include DNA methylation and histone modifications, yet HDAC inhibitors did not induce IAP expression in demethylated Dnmt1−/− mESCs (Fig. 6). Furthermore, preliminary results showed that deficiency of either PRC2 (eed−/− mESCs) or PRC1 (Bmi−/− brain tissues) alone did not relieve IAP protein repression (data not shown). To definitively ascertain PcG complex involvement, deficiency of both polycomb complexes and DNA methylation in mESCs may be needed to trigger IAP protein expression.
      Small RNA-mediated silencing pathways have been reported to regulate retrotransposons. Presently, contradictory reports regarding Dicer involvement in IAP silencing appear in the literature. One group observed increased transcription from centromeric repeats, L1s, and IAPs (
      • Kanellopoulou C.
      • Muljo S.A.
      • Kung A.L.
      • Ganesan S.
      • Drapkin R.
      • Jenuwein T.
      • Livingston D.M.
      • Rajewsky K.
      ); however, other groups state that mammalian DICER and Eif2c2 (Ago2) have no roles in maintaining genomic methylation in mESCs, with direct evidence that the RISC complex does not repress IAP expression post-transcriptionally (
      • Murchison E.P.
      • Partridge J.F.
      • Tam O.H.
      • Cheloufi S.
      • Hannon G.J.
      ,
      • Morita S.
      • Horii T.
      • Kimura M.
      • Goto Y.
      • Ochiya T.
      • Hatada I.
      ). Our double deletion model in mESCs reveals no large induction of IAP protein reactivation, arguing that Dicer-mediated transcriptional silencing is not strongly targeting IAP repression. Although we observed weak IAP immunostaining in a few Dnmt1−/−; Dicer−/− cells, this could be due to the fact that Dicer mutation dramatically increases Oct3/4 levels in mESCs (
      • Kanellopoulou C.
      • Muljo S.A.
      • Kung A.L.
      • Ganesan S.
      • Drapkin R.
      • Jenuwein T.
      • Livingston D.M.
      • Rajewsky K.
      ,
      • Murchison E.P.
      • Partridge J.F.
      • Tam O.H.
      • Cheloufi S.
      • Hannon G.J.
      ). The appearance of a few Oct3/4 immunopositive cells weakly co-stained for IAP could be explained by longer turnover of Oct3/4 protein; thus, cells undergoing early stages of spontaneous differentiation would retain residual Oct3/4 protein.
      While this manuscript was in preparation, two recent publications demonstrated that KAP1/Trim28 repressor protein coupled with histone lysine 9 methyltransferase KMT1E (ESET/SETDB1) is involved in repressed IAP gene transcription in mESCs (
      • Rowe H.M.
      • Jakobsson J.
      • Mesnard D.
      • Rougemont J.
      • Reynard S.
      • Aktas T.
      • Maillard P.V.
      • Layard-Liesching H.
      • Verp S.
      • Marquis J.
      • Spitz F.
      • Constam D.B.
      • Trono D.
      ,
      • Matsui T.
      • Leung D.
      • Miyashita H.
      • Maksakova I.A.
      • Miyachi H.
      • Kimura H.
      • Tachibana M.
      • Lorincz M.C.
      • Shinkai Y.
      ). These studies revealed a novel role of repressive histone modifications as an alternative transcriptional repressive mechanism independent of DNA methylation. Interestingly, in the absence of all three Dnmts in mESCs, Matsui et al. (
      • Matsui T.
      • Leung D.
      • Miyashita H.
      • Maksakova I.A.
      • Miyachi H.
      • Kimura H.
      • Tachibana M.
      • Lorincz M.C.
      • Shinkai Y.
      ) reported that the KAP-repressive complex remains associated with IAP retrotransposon elements. Furthermore, KAP1/Trim28 acts synergistically with DNA methylation to repress IAP transcription (
      • Rowe H.M.
      • Jakobsson J.
      • Mesnard D.
      • Rougemont J.
      • Reynard S.
      • Aktas T.
      • Maillard P.V.
      • Layard-Liesching H.
      • Verp S.
      • Marquis J.
      • Spitz F.
      • Constam D.B.
      • Trono D.
      ). However, neither of the two groups have examined whether IAP proteins are present in KAP1−/− mESCs. In our study, we found that IAP gene transcription is only moderately increased when compared with differentiated Dnmt1−/− cells. Furthermore, IAP protein is not detected in demethylated mESCs, consistent with an additional inhibitory mechanism that blocks IAP expression in undifferentiated mESCs.
      Based on the findings of this current study, we conclude that IAP protein expression can be used to detect DNA hypomethylation at the cellular level in murine cells ranging from epiblast-derived stem cells to adult neurons. Both IAP transcription and protein translation can be detected upon DNA hypomethylation in lineage-committed cells. However, in undifferentiated mESCs, there exists a compensatory mechanism(s) to repress retrotransposon elements independent of DNA methylation. The need for an alternative mechanism further highlights the importance of maintaining genomic stability to prevent insertional mutations in fast replicating mESCs.

      Acknowledgments

      We thank Dr. Kira Lueders for the gifts of the p73 antibody and the MIA14 plasmid and Thuc Le for editing of the manuscript.

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