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

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 thi...

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Hlavní autoři: Lorincz, Matthew C., Schübeler, Dirk, Goeke, Scott C., Walters, Mark, Groudine, Mark, Martin, David I. K.
Médium: Článek
Jazyk:en
Vydáno: American Society for Microbiology 2000
Témata:
Transcriptional Regulation
On-line přístup:https://ncbi.nlm.nih.gov/pmc/articles/PMC85201/
https://ncbi.nlm.nih.gov/pubmed/10629041
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spelling pubmed-852012002-09-13 Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression Lorincz, Matthew C. Schübeler, Dirk Goeke, Scott C. Walters, Mark Groudine, Mark Martin, David I. K. Mol Cell Biol Transcriptional Regulation 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. American Society for Microbiology 2000-02 /pmc/articles/PMC85201/ /pubmed/10629041 Text en Copyright © 2000, American Society for Microbiology
institution US National Library of Medicine
collection PubMed Central
language en
format Article
topic Transcriptional Regulation
spellingShingle Transcriptional Regulation
Lorincz, Matthew C.
Schübeler, Dirk
Goeke, Scott C.
Walters, Mark
Groudine, Mark
Martin, David I. K.
Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
description 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.
author Lorincz, Matthew C.
Schübeler, Dirk
Goeke, Scott C.
Walters, Mark
Groudine, Mark
Martin, David I. K.
author_facet Lorincz, Matthew C.
Schübeler, Dirk
Goeke, Scott C.
Walters, Mark
Groudine, Mark
Martin, David I. K.
author_sort Lorincz, Matthew C.
title Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
title_short Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
title_full Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
title_fullStr Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
title_full_unstemmed Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression
title_sort dynamic analysis of proviral induction and de novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression
publisher American Society for Microbiology
publisher_facet American Society for Microbiology
publishDate 2000
url https://ncbi.nlm.nih.gov/pmc/articles/PMC85201/
https://ncbi.nlm.nih.gov/pubmed/10629041
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