The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a(-/-) Dnmt3b(-/-) cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression.

Department of Cellular and Viral Genetics Institute of Molecular Genetics Academy of Sciences of the Czech Republic Vídeňská 1083 14220 Prague Czech Republic

Zobrazit více v PubMed

Jordan A, Defechereux P, Verdin E. The site of HIV-1 integration in the human genome determines basal transcriptional activity and response to Tat transactivation. EMBO J. 2001;20:1726–1738. PubMed PMC

Jordan A, Bisgrove D, Verdin E. HIV reproducibly establishes a latent infection after acute infection of T cells. EMBO J. 2003;22:1868–1877. PubMed PMC

Elleder D, Pavlicek A, Paces J, Hejnar J. Preferential integration of human immunodeficiency virus type 1 into genes, cytogenetic R bands and GC-rich regions: insight from the human genome sequence. FEBS Lett. 2001;517:285–286. PubMed

Schroder AR, Shinn P, Chen H, Berry C, Ecker JR, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002;110:521–529. PubMed

Mitchell RS, Beitzel BF, Schroder AR, Shinn P, Chen H, Berry CC, Ecker JR, Bushman FD. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol. 2004;2:e234. PubMed PMC

Ciuffi A, Llano M, Poeschla E, Hoffmann C, Leipzig J, Shinn P, Ecker JR, Bushman F. A role of LEDGF/p75 in targeting HIV DNA integration. Nat. Med. 2005;11:1287–1289. PubMed

Shun MC, Raghavendra NK, Vandegraaff N, Daigle JE, Hughes S, Kellam P, Cherepanov P, Engelman A. LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev. 2007;21:1767–1778. PubMed PMC

Meehan AM, Saenz DT, Morrison JH, Garcia-Rivera JA, Peretz M, Llano M, Poeschla EM. LEDGF/p75 proteins with alternative chromatin tethers are functional HIV-1 cofactors. PLoS Pathog. 2009;5:e1000522. PubMed PMC

Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for \MLV integration. Science. 2003;300:1749–1751. PubMed

Trobridge GD, Miller DG, Jacobs MA, Allen JM, Kiem HP, Kaul R, Russell DW. Foamy virus vector integration sites in normal human cells Proc. Natl Acad. Sci. USA. 2006;103:1498–1503. PubMed PMC

Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 2008;118:3132–3142. PubMed PMC

Studamire B, Goff SP. Host proteins interacting with the Moloney murine leukemia virus integrase: multiple transcriptional regulators and chromatin binding factors. Retrovirology. 2008;5:e48. PubMed PMC

Narezkina A, Taganov KD, Litwin S, Stoyanova R, Hayashi J, Seeger C, Skalka AM, Katz RA. Genome-wide analyses of avian sarcoma virus integration sites. J. Virol. 2004;78:11656–11663. PubMed PMC

Barr SD, Leipzig J, Shinn P, Ecker JR, Bushman FD. Integration targeting by avian sarcoma-leukosis virus and human immunodeficiency virus in the chicken genome. J. Virol. 2005;79:12035–12044. PubMed PMC

Faschinger A, Rouault F, Sollner J, Lukas A, Salmons B, Günzburg WH, Indik S. Mouse mammary tumor virus integration site selection in human and mouse genomes. J. Virol. 2008;82:1360–1367. PubMed PMC

Lewinski MK, Bisgrove D, Shinn P, Chen H, Hoffmann C, Hannenhalli S, Verdin E, Berry CC, Ecker JR, Bushman FD. Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J. Virol. 2005;79:6610–6619. PubMed PMC

Shan L, Yang H-C, Rabi A, Bravo HC, Shroff NS, Irizarry RA, Zhang H, Margolick JB, Siliciano JD, Siliciano RF. Influence of host cell transcriptional level and orientation on HIV-1 latency in a primary-cell model. J. Virol. 2011;85:5384–5393. PubMed PMC

Plachy J, Kotab J, Divina P, Reinisova M, Senigl F, Hejnar J. Proviruses selected for high and stable expression of transduced genes accumulate in broadly transcribed genome areas. J. Virol. 2010;84:4204–4211. PubMed PMC

Wolf D, Goff SP. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell. 2007;131:46–57. PubMed

Wolf D, Goff SP. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature. 2009;458:1201–1204. PubMed PMC

Rowe HM, Jakobsson J, Mesnard D, Rougemont J, Reynard S, Aktas T, Maillard PV, Layard-Lieschinget H, Verp S, Marquis J, et al. KAP1 controls endogenous retroviruses in embryonic stem cells. Nature. 2010;463:237–240. PubMed

Niwa 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;32:1105–1113. PubMed

Petersen R, Kempler G, Barklis E. A stem cell-specific silencer in the primer-binding site of a retrovirus. Mol. Cell. Biol. 1991;11:1214–1221. PubMed PMC

Grez M, Akgün E, Hilberg F, Ostertag W. Embryonic stem cell virus, a rekombinant murine retrovirus with expression in embryonic stem cells. Proc. Natl Acad. Sci. USA. 1990;87:9202–9206. PubMed PMC

Challita PM, Skelton D, el-Khoueiry A, Yu XJ, Weinberg K, Kohn DB. Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells. J. Virol. 1995;69:748–755. PubMed PMC

Stewart CL, Stuhlmann H, Jähner D, Jaenisch R. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc. Natl Acad. Sci. USA. 1982;79:4098–4102. PubMed PMC

Lorincz MC, Schübeler D, Goeke SC, Walters M, Groudine M, Martin DI. Dynamic analysis of proviral induction and de novo methylation: implications for a histone deacetalase-independent, methylation density-dependent mechanism of transcriptional repression. Mol. Cell. Biol. 2000;20:842–850. PubMed PMC

Lorincz MC, Schübeler D, Groudine M. Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation. Mol. Cell. Biol. 2001;21:7913–7922. PubMed PMC

Lorincz MC, Schübeler D, Hutchinson SR, Dickerson DR, Groudine M. DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. Mol. Cell. Biol. 2002;22:7572–7580. PubMed PMC

Hejnar J, Plachy J, Geryk J, Machon O, Trejbalova K, Guntaka RV, Svoboda J. Inhibition of the Rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells. Virology. 1999;255:171–181. PubMed

Bednarik DP, Cook JA, Pitha PM. Inactivation of the HIV LTR by DNA CpG methylation: evidence for a role in latency. EMBO J. 1990;9:1157–1164. PubMed PMC

Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, Verdin E, Olive D, Van Lint C, Hejnar J. CpG methylation controls reactivation of HIV from latency. PLoS Pathog. 2009;5:e1000554. PubMed PMC

Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog. 1999;5:1000495. PubMed PMC

Koiwa T, Hamano-Usami A, Ishida T, Okayama A, Yamaguchi K, Kamihira S, Watanabe T. 5′-long terminal repeat-selective CpG methylation of latent human T-cell leukemia virus type 1 provirus in vitro and in vivo. J. Virol. 2002;76:9389–9397. PubMed PMC

Taniguchi Y, Nosaka K, Yasunaga J, Maeda M, Mueller N, Okayama A, Matsuoka M. Silencing of human T-cell leukemia virus type 1 gene transcription by epigenetic mechanisms. Retrovirology. 2005;2:e64. PubMed PMC

Lavie L, Kitova M, Maldener E, Meese E, Mayer J. CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K (HML-2) J. Virol. 2005;79:876–883. PubMed PMC

Matouskova M, Blazkova J, Pajer P, Pavlicek A, Hejnar J. CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues. Exp. Cell. Res. 2006;312:1011–1020. PubMed

Gimenez J, Montgiraud C, Oriol G, Pichon JP, Ruel K, Tsatsaris V, Gerbaud P, Frendo JL, Evain-Brion D, Mallet F. Comparative methylation of ERVWE1/syncytin-1 and other human endogenous retrovirus LTRs in placenta tissues. DNA Res. 2009;16:195–211. PubMed PMC

Trejbalova K, Blazkova J, Matouskova M, Kucerova D, Pecnova L, Vernerova Z, Heracek J, Hirsch I, Hejnar J. Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin. Nucleic Acids Res. 2011;39:8728–8739. PubMed PMC

Swindle CS, Kim HG, Klug CA. Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells. J. Biol. Chem. 2004;279:34–41. PubMed

Hejnar J, Hajkova P, Plachy J, Elleder D, Stepanets V, Svoboda J. CpG island protects Rous sarcoma virus-derived vectors integrated into nonpermissive cells from DNA methylation and transcriptional suppression. Proc. Natl Acad. Sci. USA. 2001;98:565–569. PubMed PMC

Senigl F, Plachy J, Hejnar J. The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing. J. Virol. 2008;72:7818–7827. PubMed PMC

Pannell D, Osborne CS, Yao S, Sukonnik T, Pasceri P, Karaiskakis A, Okano M, Li E, Lipshitz HD, Ellis J. Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code. EMBO J. 2000;19:5884–5894. PubMed PMC

Yao S, Sukonnik T, Kean T, Bharadwaj RR, Pasceri P, Ellis J. Retrovirus silencing, variegation, extinction, and memory are controlled by a dynamic interplay of multiple epigenetic modifications. Mol. Therapy. 2004;10:27–36. PubMed

Mikkelsen TS, Kum M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. 2007;448:553–560. PubMed PMC

Dong KB, Maksakova IA, Mohn F, Leung D, Appanah R, Lee S, Yang HW, Lam LL, Mager DL, Schübeler D, et al. DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity. EMBO J. 2008;27:2691–2701. PubMed PMC

Matsui T, Leung D, Miyashita H, Maksakova IA, Miyachi H, Kimura H, Tachibana M, Lorincz CM, Shinkai Y. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature. 2010;464:927–931. PubMed

Leung DC, Dong KB, Maksakova IA, Goyal P, Appanah R, Lee S, Tachibana M, Shinkai Y, Lehnertz B, Mager DL, et al. Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing. Proc. Natl Acad. Sci. USA. 2011;108:5718–5723. PubMed PMC

Poleshko A, Einarson MB, Shalginskich N, Zhang R, Adams P, Skalka AM, Katz RA. Identification of a functional network of human epigenetic silencing factors. J. Biol. Chem. 2010;285:422–433. PubMed PMC

Federspiel MJ, Hughes SH. Retroviral gene delivery. Methods Cell. Biol. 1997;52:179–214. PubMed

Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW, Vogelstein B, Baylin SB, Schuebel KE. CpG methylation is maintained in human cancer cells lacking DNMT1. Nature. 2000;404:1003–1007. PubMed

Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, Cui H, Feinberg AP, Lengauer C, Kinzler KW, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature. 2002;416:552–556. PubMed

Jair KW, Bachman KE, Suzuki H, Ting AH, Rhee I, Yen RW, Baylin SB, Schuebel KE. De novo CpG island methylation in human cancer cells. Cancer Res. 2006;66:682–692. PubMed

Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat. Protoc. 2009;4:789–798. PubMed PMC

Shen L, Gao G, Zhang Y, Zhang H, Ye Z, Huang S, Huang J, Kang J. A single amino acid substitution confers enhanced methylation activity of mammalian Dnmt3b on chromatin DNA. Nucleic Acids Res. 2010;38:6054–6064. PubMed PMC

Holz-Schietinger C, Reich NO. The inherent processivity of the human DNA methyltransferase 3 a (DNMT3A) is enhanced by DNMT3L. J. Biol. Chem. 2010;285:29091–29100. PubMed PMC

Okitsu CY, Hsieh JC, Hsieh CL. Transcriptional activity affects the H3K4me3 level and distribution in the coding region. Mol. Cell. Biol. 2010;30:2933–2936. PubMed PMC

Zhou VW, Goren A, Bernstein BE. Charting histone modifications and the functional organization of mammalian genomes Nat. Rev. Genet. 2011;12:7–18. PubMed

Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin SP, Allis CD, et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007;448:714–717. PubMed PMC

Wu H, Coskun V, Tao J, Xie W, Ge W, Yoshikawa K, Li E, Zhang Y, Sun YE. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neuronic genes. Science. 2010;329:444–448. PubMed PMC

Dhayalan H, Rajavelu A, Rathert P, Tamas R, Jurkowska RZ, Ragozin S, Jeltsch A. The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation. J. Biol. Chem. 2010;285:26114–26120. PubMed PMC

Macleod D, Charlton J, Mullins J, Bird AP. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 1994;8:2282–2292. PubMed

Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schübeler D. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat. Genet. 2007;39:457–466. PubMed

Hodges E, Smith AD, Kendall J, Xuan Z, Ravi K, Rooks M, Zhang MQ, Ye K, Bhattacharjee A, Brizuela L, et al. High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing. Genome Res. 2009;19:1593–1605. PubMed PMC

Jacinto FV, Ballestar E, Esteller M. Impaired recruitment of the histone methyltransferase DOT1L contributes to the incomplete reactivation of tumor suppressor genes upon DNA demethylation. Oncogene. 2009;28:4212–4224. PubMed

Yang PK, Kuroda MI. Noncoding RNAs and intracellular positioning in monoallelic gene expression. Cell. 2007;128:777–786. PubMed

Carrozza MJ, Li B, Florens L, Suganuma T, Swanson SK, Lee KK, Shia WJ, Anderson S, Yates J, Washburn MP, et al. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell. 2005;123:581–592. PubMed

Yoh SM, Lucas JS, Jones KA. The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev. 2008;22:3422–3434. PubMed PMC

Lickwar CR, Rao B, Shabalin AA, Nobel AB, Strahl BD, Lieb JD. The Set2/Rpd3S pathway suppresses cryptic transcription without regard to gene length or transcription frequency. PloS One. 2009;4:e4886. PubMed PMC

Searle S, Gillespie DAF, Chiswell DJ, Wyke JA. Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells. Nucleic Acids Res. 1984;12:5193–5210. PubMed PMC

Hejnar J, Svoboda J, Geryk J, Fincham VJ, Hak R. High rate of morphological reversion in tumor cell line H-19 associated with permanent transcriptional suppression of the LTR, v-src, LTR provirus. Cell Growth Differ. 1994;5:277–285. PubMed

Poleshko A, Palagin I, Zhang R, Boimel P, Castagna C, Adams PD, Skalka AM, Katz RA. Identification of cellular proteins that maintain retroviral epigenetic silencing: evidence for an antiviral response. J. Virol. 2008;82:2313–2323. PubMed PMC

He J, Yang Q, Chang L-J. Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells. J. Virol. 2005;79:13497–13508. PubMed PMC

Ellis J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Therapy. 2005;16:1241–1246. PubMed

Trono D, Van Lint C, Rouzioux C, Verdin E, Barré-Sinoussi F, Chun TW, Chomont N. HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science. 2010;329:174–180. PubMed

Keedy KS, Margolis DM. Therapy for persistent HIV. Trends Pharmacol. Sci. 2010;31:206–211. PubMed PMC

Mok HP, Javed S, Lever A. Stable gene expression occurs from a minority of integrated HIV-1-based vectors: transcriptional silencing is present in the majority. Gene Therapy. 2007;14:741–751. PubMed

Long Terminal Repeats of Gammaretroviruses Retain Stable Expression after Integration Retargeting

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

Accumulation of long-term transcriptionally active integrated retroviral vectors in active promoters and enhancers

Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals

Role of DNA methylation in expression and transmission of porcine endogenous retroviruses

Nonconserved tryptophan 38 of the cell surface receptor for subgroup J avian leukosis virus discriminates sensitive from resistant avian species