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 wi th transcriptional repression in mammalian cells, and recent findings impli cate histone deacetylation in methylation-mediated repression, Analyses of histone acetylation in in vitro-methylated transfected plasmids support thi s 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 th at permits the isolation and expansion of cells harboring expressing or sil ent retroviruses. MEL cells were infected with a Moloney murine leukemia vi rus encoding the green fluorescent protein (GFP), and single-copy, silent p roviral clones were treated weekly with the histone deacetylase inhibitor t richostatin A or the DNA methylation inhibitor 5-azacytidine, Expression wa s monitored concurrently by flow cytometry, allowing for repeated phenotypi c analysis over time, and proviral methylation was determined by Southern b lotting and bisulfite methylation mapping. Shortly after infection, provira l expression was inducible and the reporter gene and proviral enhancer show ed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the prov irus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the prov irus. Repression could be overcome in late-passage clones only by pretreatm ent with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experim ents reveal the presence of a silencing mechanism which acts on densely met hylated DNA and appears to function independently of histone deacetylase ac tivity.