A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation

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Abstract
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.