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. 2006 Nov 14;103(46):17337-42.
doi: 10.1073/pnas.0607015103. Epub 2006 Nov 3.

Small dsRNAs induce transcriptional activation in human cells

Long-Cheng Li  1 , Steven T Okino, Hong Zhao, Deepa Pookot, Robert F Place, Shinji Urakami, Hideki Enokida, Rajvir Dahiya
Affiliations

Small dsRNAs induce transcriptional activation in human cells

Long-Cheng Li et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5' end of the antisense strand, or "seed" sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
dsRNAs targeting the E-cadherin gene promoter induce E-cadherin mRNA and protein expression. (A) A schematic representation of the E-cadherin promoter with its CpG island, Alu repeat element, transcription start site, and dsRNA targets. (B) PC-3 cells were transfected with 50 nM dsRNA for 72 h. mRNA expression of E-cadherin and GAPDH were analyzed by RT-PCR. Samples for each treatment are shown in duplicate. (C) E-cadherin, β-actin, and GAPDH protein levels were detected by Western blot analysis in PC-3 cells treated as in B. (D) PC-3 cells were treated with dsEcad-215 at the indicated concentrations for 72 h. E-cadherin and GAPDH expression was detected by Western blotting. (E) PC-3 cells were transfected with 50 nM dsEcad-215 for the indicated lengths of time. E-cadherin and β-actin expression was detected by Western blotting. (F) PC-3 cells were transfected with 50 nM dsCon-1 or dsEcad-215 for the indicated periods of time. Mock samples were transfected in the absence of dsRNA. Western blot analysis shows E-cadherin protein levels in PC-3 cells on days 10 or 13 after single transfections.
Fig. 2.
Fig. 2.
dsRNAs induce p21 expression in different human cell lines. Cells were transfected with 50 nM dsRNA for 72 h. mRNA and protein levels were analyzed by RT-PCR and Western blotting, respectively. (A) A schematic representation of the p21 promoter with its CpG island, SP1 sites, TATA signal, transcription start site, and the dsRNA target. (B) p21 and GAPDH mRNA expression levels in PC-3, MCF-7, and HeLa cells after mock, dsCon-2, or dsP21–322 transfections. (C) p21 mRNA expression levels were normalized to GAPDH. The results are presented as the mean ± SEM of two independent experiments (two sample repeats within each experiment). (D) Induction of p21 protein expression was confirmed by Western blot analysis in PC-3, MCF-7, and HeLa cells. GAPDH levels were also detected and served as a loading control. (E) Western blot analysis of p21 and GAPDH after mock, dsCon-2, or dsP21–322 transfections in HEK293, J82, LNCaP, and T24 cells.
Fig. 3.
Fig. 3.
dsRNAs induce VEGF expression in HeLa cells. Cells were transfected with 50 nM dsRNA for 72 h. mRNA levels were analyzed by RT-PCR. (A) A schematic representation of the VEGF promoter and the location of the dsRNA target. (B) mRNA expression of two VEGF isoforms (VEGF-189 and -165) and GAPDH in HeLa cells after mock, dsCon-2, or dsVEGF-706 transfections. (C) VEGF mRNA expression levels were normalized to GAPDH. The results are presented as the mean ± SEM of two independent experiments with each sample repeated twice.
Fig. 4.
Fig. 4.
Sequence specificity for RNAa. (A) Sequence for E-cadherin dsRNA dsEcad-215. The antisense (guide) strand is shown in blue. Mutation of the first and last 5 bp of the dsEcad-215 duplex resulted in dsEcad-215-G3′ and dsEcad-215-G5′, respectively. The mutated bases are in red. (B) PC-3 cells were transfected with 50 nM of the indicated dsRNA molecules for 72 h. Expression of E-cadherin and β-actin was assessed by Western blot analysis. (C) E-cadherin levels from corresponding Western blots were normalized to β-actin and are presented as the mean ± SEM of two independent experiments. (D) The sequence of dsP21–322 and its corresponding mutated dsRNAs. (E) PC-3 and HeLa cells were transfected with 50 nM of the indicated dsRNA molecules for 72 h. Expression levels of p21 and GAPDH were assessed by Western blot analysis.
Fig. 5.
Fig. 5.
RNAa requires the Ago2 protein. (A) PC-3 cells were transfected with 50 nM of the indicated dsRNA molecules for 72 h. mRNA expression of Ago1–4 was assessed by RT-PCR. Each Ago family member was knocked down by its corresponding Ago siRNA (siAgo1, 2, 3, or 4). (B) RT-PCR results were normalized to GAPDH expression levels and are presented as the mean ± SEM from two independent experiments. (C and D) PC-3 cells were transfected with 50 nM of each indicated dsRNA for 72 h. Both p21 and GAPDH mRNA (C) and protein (D) levels were determined by RT-PCR and Western blot analysis, respectively. (E) p21 mRNA levels were normalized to GAPDH and are presented as the mean ± SEM from at least two independent experiments (containing two sample repeats per experiment).
Fig. 6.
Fig. 6.
dsRNA-induced transcriptional activation is associated with loss of histone 3 methylation at lysine-9. PC-3 cells were transfected with 50 nM of the indicated dsRNA molecules for 72 h. ChIP assays were performed by using antibodies against H3m2K9, H3m2K4, and H3m3K9 to pull down associated DNA. The precipitated DNA was amplified by PCR using two primer sets specific for regions 1 and 2. Input DNA was amplified as a control. (A) A schematic representation of the E-cadherin promoter and the location of ChIP PCR regions 1 and 2 (double-arrowed lines). (B and C) PCR amplification of DNA precipitated with H3m2K9 and H3m2K4 antibodies. (D and E) PCR amplification of DNA precipitated with H3m3K9 antibody.
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