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PMID: 10629041 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression.

Molecular and cellular biology ·Vol. 20 ·No. 3 ·2000-02-00 ·Pages 842-50

Lorincz MC, Schübeler D, Goeke SC, Walters M, Groudine M, Martin DI

Abstract

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

MeSH Terms
Animals Azacitidine/pharmacology DNA Methylation DNA Primers Dinucleoside Phosphates/metabolism Gene Expression Regulation/drug effects Green Fluorescent Proteins Histone Deacetylases/metabolism Humans Luminescent Proteins/genetics Mice Moloney murine leukemia virus/genetics Proviruses/genetics Recombinant Proteins/biosynthesis Sulfites/pharmacology Transcription, Genetic Transfection Tumor Cells, Cultured
Chemicals
DNA Primers Dinucleoside Phosphates Luminescent Proteins Recombinant Proteins Sulfites Green Fluorescent Proteins cytidylyl-3'-5'-guanosine Histone Deacetylases Azacitidine sodium bisulfite
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Lorincz M C
Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, Washington, USA. [email protected]
Schübeler D
Goeke S C
Walters M
Groudine M
Martin D I
References (59)
59 references, click to expand
  1. Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.
    Virology. 1989 Aug;171(2):331-41 PMID: 2503932
  2. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs.
    Cell. 1989 Aug 11;58(3):499-507 PMID: 2758464
  3. Inactivation of the Moloney murine leukemia virus long terminal repeat in murine fibroblast cell lines is associated with methylation and dependent on its chromosomal position.
    J Virol. 1991 Feb;65(2):904-12 PMID: 1702844
  4. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein.
    Cell. 1991 Mar 22;64(6):1123-34 PMID: 2004419
  5. DNA methylation and gene expression.
    Microbiol Rev. 1991 Sep;55(3):451-8 PMID: 1943996
  6. Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.
    Mol Cell Biol. 1992 Jan;12(1):38-44 PMID: 1309593
  7. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein.
    EMBO J. 1992 Jan;11(1):327-33 PMID: 1310933
  8. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.
    Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827-31 PMID: 1542678
  9. Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.
    Cell. 1992 Jun 12;69(6):905-14 PMID: 1606614
  10. Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism.
    Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10119-23 PMID: 1332030
  11. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.
    Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3539-43 PMID: 8097319
  12. Histone H1-mediated inhibition of transcription initiation of methylated templates in vitro.
    J Biol Chem. 1993 Oct 15;268(29):21754-9 PMID: 8408030
  13. Number of CpG islands and genes in human and mouse.
    Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11995-9 PMID: 7505451
  14. B cell-specific demethylation: a novel role for the intronic kappa chain enhancer sequence.
    Cell. 1994 Mar 11;76(5):913-23 PMID: 8124725
  15. Dependence of transcriptional repression on CpG methylation density.
    Mol Cell Biol. 1994 Aug;14(8):5487-94 PMID: 7518564
  16. High sensitivity mapping of methylated cytosines.
    Nucleic Acids Res. 1994 Aug 11;22(15):2990-7 PMID: 8065911
  17. CpNpG methylation in mammalian cells.
    Nat Genet. 1995 May;10(1):20-7 PMID: 7647784
  18. Centrifugal enhancement of retroviral mediated gene transfer.
    J Virol Methods. 1995 Aug;54(2-3):131-43 PMID: 8530565
  19. Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation.
    EMBO J. 1996 Mar 1;15(5):1112-20 PMID: 8605881
  20. Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins.
    Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8508-11 PMID: 8710900
  21. Codon usage limitation in the expression of HIV-1 envelope glycoprotein.
    Curr Biol. 1996 Mar 1;6(3):315-24 PMID: 8805248
  22. Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.
    Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10741-6 PMID: 8855250
  23. Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3.
    Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12845-50 PMID: 8917507
  24. MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin.
    Cell. 1997 Feb 21;88(4):471-81 PMID: 9038338
  25. DNA methylation directs a time-dependent repression of transcription initiation.
    Curr Biol. 1997 Mar 1;7(3):157-65 PMID: 9395433
  26. Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression.
    Cell. 1997 May 2;89(3):349-56 PMID: 9150134
  27. Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase.
    Proc Natl Acad Sci U S A. 1997 May 27;94(11):5798-803 PMID: 9159154
  28. A component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins.
    Nat Genet. 1997 Jul;16(3):256-9 PMID: 9207790
  29. Transcriptional regulation of mammalian genes in vivo. A tale of two templates.
    J Biol Chem. 1997 Oct 31;272(44):27493-6 PMID: 9346875
  30. Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells.
    Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13724-9 PMID: 9391093
  31. Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin.
    Cell. 1997 Dec 12;91(6):845-54 PMID: 9413993
  32. An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA.
    EMBO J. 1998 Mar 2;17(5):1446-53 PMID: 9482741
  33. Histone acetylation and transcriptional regulatory mechanisms.
    Genes Dev. 1998 Mar 1;12(5):599-606 PMID: 9499396
  34. Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells.
    Immunity. 1998 Apr;8(4):461-71 PMID: 9586636
  35. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.
    Nat Genet. 1998 Jun;19(2):187-91 PMID: 9620779
  36. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
    Nature. 1998 May 28;393(6683):386-9 PMID: 9620804
  37. Consistent, persistent expression from modified retroviral vectors in murine hematopoietic stem cells.
    Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):10182-7 PMID: 9707621
  38. Methyl-CpG-binding protein MeCP2 represses Sp1-activated transcription of the human leukosialin gene when the promoter is methylated.
    Mol Cell Biol. 1998 Sep;18(9):5492-9 PMID: 9710633
  39. DNA methylation models histone acetylation.
    Nature. 1998 Aug 27;394(6696):842 PMID: 9732866
  40. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators.
    Genes Dev. 1998 Sep 15;12(18):2852-62 PMID: 9744862
  41. In vivo footprinting of the enhancer sequences in the upstream long terminal repeat of Moloney murine leukemia virus: differential binding of nuclear factors in different cell types.
    J Virol. 1998 Nov;72(11):8961-70 PMID: 9765441
  42. Identification and characterization of a family of mammalian methyl-CpG binding proteins.
    Mol Cell Biol. 1998 Nov;18(11):6538-47 PMID: 9774669
  43. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.
    Nat Genet. 1999 Jan;21(1):103-7 PMID: 9916800
  44. Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells.
    Nat Genet. 1999 May;22(1):98-101 PMID: 10319871
  45. n-Butyrate causes histone modification in HeLa and Friend erythroleukaemia cells.
    Nature. 1977 Aug 4;268(5619):462-4 PMID: 268489
  46. Chromatin structure of endogenous retroviral genes and activation by an inhibitor of DNA methylation.
    Nature. 1981 Jul 23;292(5821):311-7 PMID: 6166864
  47. Clonal analysis of the integration and expression of endogenous avian retroviral DNA acquired by exogenous viral infection.
    J Virol. 1981 Aug;39(2):584-96 PMID: 6268844
  48. 5-Azacytidine induction of mouse endogenous type C virus and suppression of DNA methylation.
    Proc Natl Acad Sci U S A. 1981 Oct;78(10):6290-4 PMID: 6171816
  49. Infectivity and methylation of retroviral genomes is correlated with expression in the animal.
    Cell. 1981 Oct;26(2 Pt 2):221-32 PMID: 6277495
  50. De novo methylation and expression of retroviral genomes during mouse embryogenesis.
    Nature. 1982 Aug 12;298(5875):623-8 PMID: 6285203
  51. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells.
    Proc Natl Acad Sci U S A. 1982 Jul;79(13):4098-102 PMID: 6955793
  52. Delayed de novo methylation in teratocarcinoma suggests additional tissue-specific mechanisms for controlling gene expression.
    Nature. 1983 Jan 6;301(5895):32-7 PMID: 6296681
  53. Independent mechanisms involved in suppression of the Moloney leukemia virus genome during differentiation of murine teratocarcinoma cells.
    Cell. 1983 Apr;32(4):1105-13 PMID: 6188535
  54. DNA methylation and the regulation of globin gene expression.
    Cell. 1983 Aug;34(1):197-206 PMID: 6883509
  55. Effect of regional DNA methylation on gene expression.
    Proc Natl Acad Sci U S A. 1985 May;82(9):2560-4 PMID: 3857599
  56. Assembly and propagation of repressed and depressed chromosomal states.
    Cell. 1985 Oct;42(3):705-11 PMID: 2996773
  57. DNA methylation affects the formation of active chromatin.
    Cell. 1986 Feb 28;44(4):535-43 PMID: 3456276
  58. Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene.
    Proc Natl Acad Sci U S A. 1987 Mar;84(5):1177-81 PMID: 3029768
  59. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A.
    J Biol Chem. 1990 Oct 5;265(28):17174-9 PMID: 2211619
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.20.3.842-850.2000
Email
[email protected]
Published
2000-02-00
Pages
842-50
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC85201
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.20.3.842-850.2000 PMC Full Text View journal details
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