Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells

doi:10.1073/pnas.0701635104

Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells

*Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and
^†Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved June 12, 2007 (received for review February 22, 2007)

Abstract

siRNAs targeted to gene promoters can direct epigenetic modifications that result in transcriptional gene silencing in human cells. It is not clear whether the antisense strand of the siRNAs bind directly to DNA or to a sense-stranded RNA transcript corresponding to the known promoter region. We present evidence that an RNA polymerase II expressed mRNA containing an extended 5′ untranslated region that overlaps the gene promoter is required for RNA-directed epigenetic modifications and transcriptional silencing of the RNA-targeted promoter. These promoter-associated RNAs were detected by their hybridization to the antisense strand of the complementary promoter-directed siRNA. Antisense phosphorothioate oligodeoxynucleotides were used to degrade the promoter-associated RNA transcripts, the loss of which abrogated the effect of siRNA-mediated transcriptional gene silencing, as well as the complexing of the siRNA with the silent state histone methyl mark and the promoter-associated RNA. These data demonstrate that low-copy promoter-associated RNAs transcribed through RNAPII promoters are recognized by the antisense strand of the siRNA and function as a recognition motif to direct epigenetic silencing complexes to the corresponding targeted promoters to mediate transcriptional silencing in human cells.

Footnotes

^‡To whom correspondence should be addressed. E-mail: kmorris{at}scripps.edu
Author contributions: K.V.M. designed research; J.H., D.K., and K.V.M. performed research; K.V.M. contributed new reagents/analytic tools; J.H. and K.V.M. analyzed data; and K.V.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ503424, DQ642693, and EF362804).
This article contains supporting information online at www.pnas.org/cgi/content/full/0701635104/DC1.
Abbreviations:

ODN,

oligodeoxynucleotide;

RT,

reverse transcription;

R/T,

room temperature;

TGS,

transcriptional gene silencing.
Freely available online through the PNAS open access option.