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Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs

Nature Chemical Biology volume 1, pages 216–222 (2005)Cite this article

Abstract

Transcription start sites are critical switches for converting recognition of chromosomal DNA into active synthesis of RNA. Their functional importance suggests that they may be ideal targets for regulating gene expression. Here, we report potent inhibition of gene expression by antigene RNAs (agRNAs) complementary to transcription start sites within human chromosomal DNA. Silencing does not require methylation of DNA and differs from all known mechanisms for inhibiting transcription. agRNAs overlap DNA sequences within the open complex formed by RNA polymerase, and silencing is acutely sensitive to single base shifts. agRNAs effectively silence both TATA-less and TATA-box-containing promoters. Transcription start sites occur within every gene, providing predictable targets for agRNAs. Potent inhibition of multiple genes suggests that agRNAs may represent a natural mechanism for controlling transcription, may complement siRNAs and miRNAs that target mRNA, and will be valuable agents for silencing gene expression.

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Figure 1: Inhibition of gene expression by agRNAs complementary to the transcription start site of hPR-B.
Figure 2: Inhibition of gene expression by agRNAs is potent and selective and is observed at the level of RNA.
Figure 3
Figure 4: Inhibition of MVP, AR and COX-2 expression.
Figure 5: Gene silencing by agRNAs is independent of methylation.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIGMS 60642 and 73042 to D.R.C. and Lung Cancer SPORE P50CA70907 to J.D.M.) and the Robert A. Welch Foundation (I-1244 to D.R.C.). We thank M. McPhaul for providing antibody to the androgen receptor.

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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, Kenneth E Huffman, Jacob C Schwartz, Rosalyn Ram, David S Shames, John D Minna & David R Corey

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

    Bethany A Janowski, Kenneth E Huffman, Jacob C Schwartz, Rosalyn Ram, Daniel Hardy & David R Corey

  3. Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    David S Shames & John D Minna

  4. The Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390, Texas, USA

    David S Shames & John D Minna

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  1. Bethany A Janowski
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Correspondence to David R Corey.

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Supplementary information

Supplementary Fig. 1

Effect of using moderately active agRNAs in combination. agRNAs were used at 25 nM and were added to cells singly or in combination. (PDF 542 kb)

Supplementary Fig. 2

Dose response profiles for inhibition of hPR by agRNA PR9 compared to other knock-down strategies. (PDF 1077 kb)

Supplementary Fig. 3

Effect of estradiol treatment on the ability of agRNAs to inhibit hPR gene expression. (PDF 499 kb)

Supplementary Fig. 4

CpG sites near the hPR-B transcription start site are not methylated. (PDF 1597 kb)

Supplementary Fig. 5

CpG sites near the transcription start site of AR are not methylated after treatment with agRNAs. (PDF 1947 kb)

Supplementary Fig. 6

Effect of inhibiting hPR expression on cell morphology. (PDF 1322 kb)

Supplementary Methods (PDF 85 kb)

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Janowski, B., Huffman, K., Schwartz, J. et al. Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs. Nat Chem Biol 1, 216–222 (2005). https://doi.org/10.1038/nchembio725

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