Journal
EMBO JOURNAL
Volume 28, Issue 1, Pages 48-57Publisher
WILEY
DOI: 10.1038/emboj.2008.260
Keywords
Dicer-like3; methylation spreading; Pol IV; RNA-directed DNA methylation; secondary siRNAs
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Funding
- European Science Foundation (ESF)
- European Commission [ERAS-CT-2003-980409]
- DG Research
- Austrian Fonds zur Forderung der wissenschaftlichen Forschung (FWF) [I26-B03, P20707-B03]
- European Union [HPRN-CT2002-00257]
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We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.
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