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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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.
The authors declare no competing financial interests.
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)
Dose response profiles for inhibition of hPR by agRNA PR9 compared to other knock-down strategies. (PDF 1077 kb)
Effect of estradiol treatment on the ability of agRNAs to inhibit hPR gene expression. (PDF 499 kb)
CpG sites near the hPR-B transcription start site are not methylated. (PDF 1597 kb)
CpG sites near the transcription start site of AR are not methylated after treatment with agRNAs. (PDF 1947 kb)
Effect of inhibiting hPR expression on cell morphology. (PDF 1322 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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