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Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation

Nature Genetics volume 37, pages 906–910 (2005)Cite this article

Abstract

Double-stranded RNA molecules targeted to gene promoter regions can induce transcriptional gene silencing in a DNA cytosine methylation–dependent manner in plants (RNA-dependent DNA methylation)1,2,3. Whether a similar mechanism exists in mammalian systems is a vital and controversial issue4,5,6. DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation7,8,9, and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer10. Hence, we investigated whether RNA-dependent DNA methylation might operate in human cancers to mediate epigenetic silencing using the endogenous gene CDH1 as a potential target. The loss of this cell-cell adhesion factor facilitates the metastatic process, and its promoter is frequently hypermethylated in breast and other cancers11,12,13. We found that, although small double-stranded RNAs targeted exclusively to the CDH1 promoter could effectively induce transcriptional repression with chromatin changes characteristic of inactive promoters, this was entirely independent of DNA methylation. Moreover, we could accomplish such silencing in a cancer cell line genetically modified to lack virtually any capacity to methylate DNA.

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Figure 1: 21-nucleotide-long dsRNA species can induce transcriptional silencing in human colorectal cancer cells.
Figure 2: DNA methylation and ChIP assay analyses on the CDH1 promoter after treatment with dsCDH1s in HCT116 cells.
Figure 3: MCF-7 human breast cancer cells treated with dsCDH1s also show gene silencing in the absence of DNA methylation changes.
Figure 4: RdTS is achieved in HCT116 cells genetically lacking both DNMT1 and DNMT3b (DKO cells).

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Acknowledgements

We thank B. Lee for suggestions, support and critical reading of the manuscript and T. Jenuwein for providing the antibodies to H3K9me2 used in the ChIP assay. This work was supported by a grant from the US National Institutes of Health.

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Authors and Affiliations

  1. Graduate Program in Cellular and Molecular Medicine, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, 21231, Maryland, USA

    Angela H Ting, James G Herman & Stephen B Baylin

  2. Cancer Biology Division, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, 21231, Maryland, USA

    Angela H Ting, Kornel E Schuebel, James G Herman & Stephen B Baylin

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  1. Angela H Ting
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  2. Kornel E Schuebel
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  3. James G Herman
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Correspondence to Stephen B Baylin.

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Supplementary information

Supplementary Fig. 1

Real time ChIP analyses of hMLH1 promoter in HCT116 cells. (PDF 73 kb)

Supplementary Table 1

Sequences of dsRNA oligonucleotides and PCR primers. (PDF 18 kb)

Supplementary Note (PDF 14 kb)

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Ting, A., Schuebel, K., Herman, J. et al. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 37, 906–910 (2005). https://doi.org/10.1038/ng1611

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