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. 2005 Aug;37(8):906-10.
doi: 10.1038/ng1611. Epub 2005 Jul 17.

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

Angela H Ting  1 , Kornel E Schuebel, James G Herman, Stephen B Baylin
Affiliations

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

Angela H Ting et al. Nat Genet. 2005 Aug.

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). Whether a similar mechanism exists in mammalian systems is a vital and controversial issue. DNA methylation is an important component in mammalian gene silencing for normal processes such as gene imprinting and X-chromosome inactivation, and aberrant CpG island hypermethylation at tumor-suppressor promoters is associated with transcriptional silencing and loss of gene function in cancer. 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 cancers. 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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Figures

Figure 1
Figure 1
21-nucleotide long dsRNA species can induce transcriptional silencing in human colorectal cancer cells. (a) A schematic diagram showing the target sequence positions of dsCDH1s relative to the transcription start site (0). This region was analyzed for CpG methyaltion by MSP primers that amplify the regions indicated as I-2 (vertical line) and I-3 (diagonal line). The bisulfite sequenced fragment is indicated as a solid gray bar. ChIP analysis for H3 dimethyl-K4 and H3 dimethyl-K9 was performed with 2 sets of primers amplifying the 5′ (dotted black bar) and 3′ (dotted white bar) portions of this promoter. (b) Western blot analysis of HCT116 cells treated with dsCDH1s for 8 consecutive days. CDH1 protein levels were gradually depleted to barely detectable levels by day 7 when compared to the controls. The intensity of the CDH1 bands, stated below the pictures as normalized percentages, was quantified by densitometry and normalized to the β-actin bands. (c) qRT-PCR analysis of CDH1 expression. The dsCDH1s treated cells contained only 13±0.04% of CDH1 mRNA when compared to the mock treated HCT116 cells. (d) Western blot analysis on days 4 and 7 of HCT116 cells treated with dsCDH1-1 for 7 consecutive days. Moderate CDH1 protein depletions were observed when compared to the controls on each day. (e) Nuclear run-on assays of dsCtrl and dsCDH1s treated cells on day 7. CDH1-specific probe binding was reduced to 39% by densitometry quantification in the dsCDH1s treated cells when compared to the dsCtrl treated cells.
Figure 2
Figure 2
DNA methylation and ChIP assay analyses on the CDH1 promoter after dsCDH1s treatment in HCT116 cells. (a) MSP analysis of the CpG island within the CDH1 promoter at 2 regions specified in Fig. 1a. The CDH1 promoter in HCT116 is normally unmethylated. DNA methylation patterns showing the presence of only the unmethylated (U) alleles remain unchanged in the mock, dsCtrl, and dsCDH1s treated cells. (b) Bisulfite sequencing analysis of the CpG island in the CDH1 promoter in the mock, dsCtrl, and dsCDH1s treated cells. The CpG sites are shown by their positions relative to the transcription start site (0), the open circles (○) represent unmethylated CpG sites, and closed circles (•) represent methylated CpG sites. The promoter remains unmethylated for each treatment group. (c) Real time PCR analysis of H3 dimethyl-K4 modification at the CDH1 promoter by ChIP assay. Two sets of primers were used in the PCR analysis to span the promoter as indicated in Fig. 1a. H3 dimethyl-K4 residues were observed, as expected, at both regions at the CDH1 promoter in the mock, dsCtrl, and dsCDH1-1 treated cells. (d) Real time PCR analysis of the H3 dimethyl-K9 modification at the CDH1 promoter by ChIP assay. H3 dimethyl-K9 was enriched at the CDH1 promoter in the dsCDH1-1 treated cells when compared to either the mock or the dsCtrl treated cells.
Figure 3
Figure 3
MCF-7 human breast cancer cells treated with dsCDH1s also exhibit gene silencing in the absence of DNA methylation changes. (a) Western blot analysis of MCF-7 cells on day 7 after either the mock, dsCtrl, or dsCDH1s treatment for 7 days. A marked decrease in CDH1 protein level, 40% remaining by densitometry quantification, is evident in the dsCDH1s treated cells when compared to the controls. (b) qRT-PCR analysis of CDH1 expression in the mock, dsCtrl, and dsCDH1s treated MCF-7 cells. The dsCDH1s treated cells contained 50±0.03% of CDH1 mRNA when compared to the mock treated HCT116 cells. (c) MSP analysis of the CDH1 promoter in MCF-7 cells treated with the mock, dsCtrl, or dsCDH1s. The CDH1 promoter remains unmethylated at either the I-2 or the I-3 region as analyzed by MSP. (d) Bisulfite sequencing analysis of the CpG island in the CDH1 promoter in the mock, dsCtrl, and dsCDH1s treated cells on day 7. The CpG sites are shown by their positions relative to the transcription start site (0), the open circles (○) represent unmethylated CpG sites, and closed circles (•) represent methylated CpG sites. The promoter remains unmethylated for each treatment group.
Figure 4
Figure 4
RdTS is achieved in HCT116 cells genetically lacking both DNMT1 and DNMT3b (DKO) cells. (a) Western blot analysis of DKO cells on day 7 after the mock, dsCtrl, or dsCDH1s treatment for 7 days. CDH1 protein level was decreased to only 8% when compared to the mock treated cells, indicating RdTS was effectively achieved in the absence of two major DNA methyltransferases that are responsible of 95% of the total DNA methylation in these cells. (b) qRT-PCR analysis of CDH1 expression in the mock, dsCtrl, and dsCDH1s treated DKO cells. The dsCDH1s treated cells contained only 21±0.00% of CDH1 mRNA when compared to the mock treated HCT116 cells. (c) MSP analysis of the CDH1 promoter in DKO cells treated with the mock, dsCtrl, or dsCDH1s. The MSP pattern of only the U band amplification is preserved in all three treatment groups, indicating a lack of DNA methylation changes. (d) Bisulfite sequencing analysis of the CpG island in the CDH1 promoter in the mock, dsCtrl, and dsCDH1s treated cells on day 7. The CpG sites are shown by their positions relative to the transcription start site (0), the open circles (○) represent unmethylated CpG sites, and closed circles (•) represent methylated CpG sites. The promoter remains unmethylated for each treatment group.
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