Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2?-deoxycytidine 5?-triphosphate

Abstract

DNA methylation is an important process contributing to transcriptional regulation in animal and plant cells. We observed that electroporation of Chinese hamster V-79 cells in the presence of millimolar concentrations of 5-methyl-2′-deoxycytidine 5′-triphosphate (5mdCTP) led to high-level direct incorporation of this nucleotide into DNA polymer. Under optimum conditions, approximately 2×10⁸ molecules of 5mdCTP were calculated to have been incorporated into each unit genome (6 pg of DNA). Since a diploid mammalian genome contains approximately 1.2–1.5×10⁹ cytosines, this level of 5mdCTP incorporation corresponds to substitution of up to 16.6% of total cytosines by 5-methylcytosine, or about 100–150 new methylated cytosines per average gene. The pattern of genomic methylation produced under these conditions differed from that produced physiologically in that 5mdCTP was substituted into DNA at random cytosines, superimposing a novel methylation pattern upon that derived from the normal enzyme-driven postreplicational process. This novel pattern of methylation showed no preference for CpG dinucleotides, but was nevertheless found capable of silencing HPRT gene expression and producing a condition of resistance to 6-thioguanine. The epigenetic nature of this gene silencing event was shown by the very high level of reexpression of HPRT mRNA following exposure of HPRT⁻ cells to the demethylating agent 5-azadeoxycytidine.