Specific targeting of cytosine methylation to DNA sequences in vivo.
Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions.
Smith
Alexander E
AE
King's College London, Department of Haematological and Molecular Medicine, Guy's, King's and St Thomas' School of Medicine, The Rayne Institute 123 Cold Harbour Lane, London SE5 9NU, UK.
Ford
Kevin G
KG
D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D006801:Humans; D016296:Mutagenesis; D016335:Zinc Fingers
D003596:Cytosine; D004247:DNA; C037664:DNA modification methylase HhaI; C029641:DNA modification methylase HpaII; D015257:DNA-Cytosine Methylases