Genomic targeting of methylated DNA: Influence of methylation on transcription, replication, chromatin structure, and histone acetylation

We have developed a strategy to introduce in vitro-methylated DNA into defi ned chromosomal locations. Using this system, we examined the effects of me thylation on transcription, chromatin structure, histone acetylation, and r eplication timing by targeting methylated and unmethylated constructs to ma rked genomic sites, At two sites, which support stable expression from an u nmethylated enhancer-reporter construct, Introduction of an in vitro-methyl ated but otherwise identical construct results in specific changes in trans gene conformation and activity, including loss of the promoter DNase I-hype rsensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of met hylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences, M ethylation at the promoter and gene is stable over time, as is the repressi on of transcription. Surprisingly, sequences within the enhancer are demeth ylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoac etylated reporter. Our findings suggest that CpG methylation represses tran scription by interfering with RNA polymerase initiation via a mechanism tha t involves localized histone deacetylation. This repression is dominant ove r a remodeled enhancer but neither results in nor requires region-wide chan ges in DNA replication or chromatin structure.