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Induction of DNA methylation and gene silencing by short interfering RNAs in human cells

Nature volume 431, pages 211–217 (2004)Cite this article

A Retraction to this article was published on 29 June 2006

A Corrigendum to this article was published on 14 October 2004

Abstract

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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Figure 1: DNA methylation of the E-cadherin promoter by siRNAs in MCF-7 and normal breast epithelial cells.
Figure 2: Effects of E-cadherin-siRNAs targeted to the E-cadherin promoter on E-cadherin mRNA expression.
Figure 3: Effects of E-cadherin-siRNAs targeted to the E-cadherin promoter in DNMT1-, DNMT2- and DNMT3B-knockdown cells.
Figure 4: Induction of DNA methylation of the erbB2 promoter by tRNA-shRNAs.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo, 113-8656, Japan

    Hiroaki Kawasaki & Kazunari Taira

  2. Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City, 305-8562, Tokyo, Japan

    Hiroaki Kawasaki & Kazunari Taira

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  1. Hiroaki Kawasaki
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Correspondence to Hiroaki Kawasaki or Kazunari Taira.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

DNA methylation of the E-cadherin promoter by the mixture of siRNAs (sites 1-10) in MCF-7 cells. (PDF 47 kb)

Supplementary Figure 2

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)

Supplementary Figure 3

Induction of DNA methylation of the erbB2 promoter by the mixture of tRNA-shRNAs (site 1-5). (PDF 37 kb)

Supplementary Methods

This file contains additional Methods (preparation of synthetic siRNAs, construction of tRNA-shRNA expression plasmids, chromatin immunoprecipitaion assay). (RTF 48 kb)

Supplementary Legends

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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