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Molecular and Cellular Biology, February 2000, p. 842-850, Vol. 20, No. 3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression

Matthew C. Lorincz,1,* Dirk Schübeler,1 Scott C. Goeke,1 Mark Walters,1 Mark Groudine,1,2 and David I. K. Martin1,dagger

Fred Hutchinson Cancer Research Center1 and Department of Radiation Oncology, University of Washington School of Medicine,2 Seattle, Washington

Received 31 August 1999/Returned for modification 22 October 1999/Accepted 28 October 1999

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.

* Corresponding author. Mailing address: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. North, Mailstop A3-025, Seattle, WA 98109-1024. Phone: (206) 667-4492. Fax: (206) 667-5894. E-mail: mlorincz{at}fhcrc.org.

dagger Present address: Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW 2010, Australia.

Molecular and Cellular Biology, February 2000, p. 842-850, Vol. 20, No. 3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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