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Activating gene expression in mammalian cells with promoter-targeted duplex RNAs

Nature Chemical Biology volume 3, pages 166–173 (2007)Cite this article

Abstract

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.

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Figure 1: Increased expression of PR protein or mRNA upon transfection of duplex RNAs into MCF7 or T47D breast cancer cells.
Figure 2: Probing the PR and MVP promoters with duplex RNAs.
Figure 3: QPCR analysis showing effects of adding activating RNA PR11 on mRNA levels of selected genes in varied media.
Figure 4: Time course of activation by PR11 or PR22 in MCF7 cells.
Figure 5: Effect of adding PR11 on histone modifications at the PR promoter in MCF7 cells.

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Acknowledgements

We thank N.-B. Nguyen for skilled assistance. This work was supported by the US National Institutes of Health (NIGMS 60642 and 73042 to D.R.C., CA 10151 to K.E.H. and HD011149 for D.B.H.), the Susan G. Komen Breast Cancer Foundation (PDF0600877 to D.B.H.) and the Robert A. Welch Foundation (I–1244 to D.R.C.). We thank J. Schwartz, C. Mendelson and D. Shames for their helpful comments.

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Authors and Affiliations

  1. Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    Bethany A Janowski, Scott T Younger, Rosalyn Ram, Kenneth E Huffman & David R Corey

  2. Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    Bethany A Janowski, Scott T Younger, Daniel B Hardy, Rosalyn Ram, Kenneth E Huffman & David R Corey

  3. Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    Daniel B Hardy

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Contributions

B.A.J., S.T.Y., D.B.H., R.R. and K.E.H. designed and performed experiments. B.A.J. and D.R.C. supervised experiments.

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Correspondence to Bethany A Janowski or David R Corey.

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Supplementary information

Supplementary Fig. 1

Increased expression of PR upon addition of selected RNAs in T47D breast cancer cells. (PDF 100 kb)

Supplementary Fig. 2

Reproducibility of probing the PR or MVP promoters with duplex RNAs. (PDF 162 kb)

Supplementary Fig. 3

Treatment of MCF7 cells with TSA yields increased COX-2 expression. (PDF 70 kb)

Supplementary Fig. 4

Contiguous and noncontiguous sequence similarity results (and common BLAST results) of PR11 and PR22. (PDF 125 kb)

Supplementary Methods (PDF 31 kb)

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Janowski, B., Younger, S., Hardy, D. et al. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Nat Chem Biol 3, 166–173 (2007). https://doi.org/10.1038/nchembio860

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