Double-stranded RNAs (dsRNAs) induce post-transcriptional gene silencing in several species of animal and plant1,2. In plants, dsRNAs targeted to CpG islands within a promoter can also induce RNA-directed DNA methylation3,4,5,6,7,8; however, it remains unclear whether gene silencing mediated by DNA methylation can be induced by dsRNAs in mammalian cells. Here, we demonstrate that short interfering RNAs (siRNAs; 21–25-nucleotide RNA molecules) induce DNA methylation and histone H3 methylation in human cells. Synthetic siRNAs targeted to CpG islands of an E-cadherin promoter induced significant DNA methylation and histone H3 lysine 9 methylation in both MCF-7 and normal mammary epithelial cells. As a result, these siRNAs repressed expression of the E-cadherin gene at the transcriptional level. In addition, disrupting the expression of either one of two DNA methyltransferases (DNMT1 or DNMT3B) by specific siRNAs abolished the siRNA-mediated methylation of DNA. Moreover, vector-based siRNAs targeted to the erbB2 (also known as HER2) promoter also induced DNA methylation in MCF-7 cells. Thus, siRNAs targeted to CpG islands within the promoter of a specific gene can induce transcriptional gene silencing by means of DNA-methyltransferase-dependent methylation of DNA in human cells, and might have potential as a new type of gene therapeutic agent.
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This research was supported by grants from AIST and by a Grant-in-Aid for Scientific Research and for the 21st Century COE programmes, Center for Integrated Brain Medical Science and Human-Friendly Materials based on Chemistry, from the Ministry of Education, Culture, Sports, Science and Culture (MEXT) of Japan.
The authors declare that they have no competing financial interests.
DNA methylation of the E-cadherin promoter by the mixture of siRNAs (sites 1-10) in MCF-7 cells. (PDF 47 kb)
Sites of DNA methylation induced by siRNAs targeted to site 6 and site 10 of the E-cadherin promoter in normal human mammary epithelial cells, as determined by bisulfite sequencing. (PDF 16 kb)
Induction of DNA methylation of the erbB2 promoter by the mixture of tRNA-shRNAs (site 1-5). (PDF 37 kb)
This file contains additional Methods (preparation of synthetic siRNAs, construction of tRNA-shRNA expression plasmids, chromatin immunoprecipitaion assay). (RTF 48 kb)
This file contains legends for Supplementary Figs 1–3. (RTF 8 kb)
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Kawasaki, H., Taira, K. Induction of DNA methylation and gene silencing by short interfering RNAs in human cells. Nature 431, 211–217 (2004). https://doi.org/10.1038/nature02889
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