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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Braasch, D.A. & Corey, D.R. Novel antisense strategies for controlling gene expression. Biochemistry 41, 4503–4510 (2002).

    CAS  Article  Google Scholar 

  2. 2

    Eckstein, F. Small noncoding RNAs as magic bullets. Trends Biochem. Sci. 30, 445–452 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Dykxhoorn, D.M., Palliser, D. & Lieberman, J. The silent treatment: siRNAs as small molecule drugs. Gene Ther. 13, 541–552 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Arora, P.S., Ansari, A.Z., Best, T.P., Ptashne, M. & Dervan, P.B. Design of artificial transcriptional activators with rigid poly-L-proline linkers. J. Am. Chem. Soc. 124, 13067–13071 (2002).

    CAS  Article  Google Scholar 

  5. 5

    Kwon, Y. et al. Small molecule transcription factor mimic. J. Am. Chem. Soc. 126, 15940–15941 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Liu, B., Han, Y., Ferdous, A., Corey, D.R. & Kodadek, T. Transcription activation by a PNA–peptide chimera in a mammalian cell extract. Chem. Biol. 10, 909–916 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Majmudar, C.Y. & Mapp, A.K. Chemical approaches to transcriptional regulation. Curr. Opin. Chem. Biol. 9, 467–474 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Janowski, B.A. et al. Inhibition of gene expression at transcription start sites using antigene RNAs (agRNAs). Nat. Chem. Biol. 1, 216–222 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Janowski, B.A. et al. Involvement of Ago1 and Ago2 in mammalian transcriptional silencing. Nat. Struct. Mol. Biol. 13, 787–792 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Janowski, B.A., Hu, J. & Corey, D.R. Antigene inhibition by peptide nucleic acids and duplex RNAs. Nat. Protoc. 1, 436–443 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Morris, K.V., Chan, S.W., Jacobsen, S.E. & Looney, D.J. Small interfering RNA–induced transcriptional silencing in human cells. Science 305, 1289–1292 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Ting, A.H., Schuebel, K.E., Herman, J.G. & Baylin, S.B. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat. Genet. 37, 906–910 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Suzuki, K. et al. Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. J. RNAi Gene Silencing 1, 66–78 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Zhang, M.-X. et al. Regulation of endothelial nitric oxide synthase by small RNA. Proc. Natl. Acad. Sci. USA 102, 16967–16972 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Kim, D.H. et al. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 13, 793–797 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Corey, D.R. Regulating mammalian transcription with RNA. Trends Biochem. Sci. 30, 655–658 (2005).

    CAS  Article  Google Scholar 

  17. 17

    Morris, K.V. siRNA-mediated transcriptional gene silencing: the potential mechanism and a possible role in the histone code. Cell. Mol. Life Sci. 62, 3057–3066 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Kastner, P. et al. Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor isoforms A and B. EMBO J. 9, 1603–1614 (1990).

    CAS  Article  Google Scholar 

  19. 19

    Misrahi, M. et al. Structure of the human progesterone receptor gene. Biochim. Biophys. Acta 1216, 289–292 (1993).

    CAS  Article  Google Scholar 

  20. 20

    Jenster, G. et al. Steroid receptor induction of gene transcription: a two-step model. Proc. Natl. Acad. Sci. USA 94, 7879–7884 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Hurd, C. et al. Hormonal regulation of the p53 tumor suppressor protein in T47D human breast carcinoma cell line. J. Biol. Chem. 270, 28507–28510 (1995).

    CAS  Article  Google Scholar 

  22. 22

    Janowski, B.A. et al. Inhibiting transcription of chromosomal DNA using antigene peptide nucleic acids. Nat. Chem. Biol. 1, 210–215 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Conneely, O.M., Jericevic, B.M. & Lydon, J.P. Progesterone receptors in mammary gland development and tumorigenesis. J. Mammary Gland Biol. Neoplasia 8, 205–214 (2003).

    Article  Google Scholar 

  24. 24

    Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    CAS  Article  Google Scholar 

  25. 25

    Huffman, K.E. & Corey, D.R. Inhibition of expression of major vault protein does not alter chemoresistance or drug localization in cancer cells. Biochemistry 44, 2253–2261 (2005).

    CAS  Article  Google Scholar 

  26. 26

    Hardy, D.B., Janowski, B.A., Corey, D.R. & Mendelson, C.R. Progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-κB activation of cyclooxygenase 2 expression. Mol. Endocrinol. 20, 2724–2733 (2006).

    CAS  Article  Google Scholar 

  27. 27

    Read, L.D., Snider, C.E., Miller, J.S., Greene, G.L. & Katzenellenbogen, B.S. Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines. Mol. Endocrinol. 2, 263–271 (1988).

    CAS  Article  Google Scholar 

  28. 28

    Cho, H., Aronica, S.M. & Katzenellenbogen, B. Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: a comparison of the effects of cyclic adenosine 3′,5′-monophhosphate, estradiol, insulin-like growth factor-I and serum factors. Endocrinology 134, 658–664 (1994).

    CAS  Article  Google Scholar 

  29. 29

    Alexander, I.E., Clarke, C.L., Shine, J. & Sutherland, R.L. Progestin inhibition of progesterone receptor gene expression in human breast cancer cells. Mol. Endocrinol. 3, 1377–1386 (1989).

    CAS  Article  Google Scholar 

  30. 30

    Margueron, R., Trojer, P. & Reinberg, D. The key to development: interpreting the histone code. Curr. Opin. Genet. Dev. 15, 163–176 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Miao, F., Gonzalo, I.G., Lanting, L. & Natarajan, R. In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J. Biol. Chem. 279, 18091–18097 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Ruh, M.F., Tian, S., Cox, L.K. & Ruh, T.S. The effect of histone acetylation on estrogen responsiveness in MCF-7 cells. Endocrine 11, 157–164 (1999).

    CAS  Article  Google Scholar 

  33. 33

    Song, M.-R. & Ghosh, A. FGF2-induced chromatin remodelling regulates CNTF-mediated gene expression and astrocyte differentiation. Nat. Neurosci. 7, 229–235 (2004).

    Article  Google Scholar 

  34. 34

    Williams-Ashman, H.G., Seidenfeld, J. & Galletti, P. Trends in the biochemical pharmacology of 5′-deoxy-5′-methylthioadenosine. Biochem. Pharmacol. 31, 277–288 (1982).

    CAS  Article  Google Scholar 

  35. 35

    Chau, C.M. & Lieberman, P.M. Dynamic chromatin boundaries delineate a latency control region of Epstein Barr virus. J. Virol. 78, 12308–12319 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Santos-Rosa, H. et al. Active genes are trimethylated at K4 of histone H3. Nature 419, 407–411 (2002).

    CAS  Article  Google Scholar 

  37. 37

    Strahl, B.D., Ohba, R., Cook, R.G. & Allis, C.D. Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc. Natl. Acad. Sci. USA 96, 14967–14972 (1999).

    CAS  Article  Google Scholar 

  38. 38

    Schneider, R. et al. Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat. Cell Biol. 6, 73–77 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Roh, T.Y., Cuddapah, S., Cui, K. & Zhao, K. The genomic landscape of histone modifications in human T cells. Proc. Natl. Acad. Sci. USA 103, 15782–15787 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Birmingham, A. et al. 3′ UTR see matches, but not overall identity, are associated with RNAi off targets. Nat. Methods 3, 199–204 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Wassenegger, M. et al. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567–576 (1994).

    CAS  Article  Google Scholar 

  42. 42

    Badia, E. et al. Long-term hydroxytamoxifen treatment of an MCF-7–derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodelling. Cancer Res. 60, 4130–4138 (2000).

    CAS  PubMed  Google Scholar 

  43. 43

    Kuwabara, T. et al. A small modulatory dsRNA specifies the fate of adult neural stem cells. Cell 116, 779–793 (2004).

    CAS  Article  Google Scholar 

  44. 44

    Li, L.C. et al. Small dsRNAs induce transcriptional activation in human cells. Proc. Natl. Acad. Sci. USA 103, 17337–17342 (2006).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding authors

Correspondence to Bethany A Janowski or David R Corey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing