3 - The EMBO Journal (2009) 28, 4857 www.embojournal.org |&...

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A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation Lucia Daxinger 1 , Tatsuo Kanno 1 , Etienne Bucher 2 , Johannes van der Winden, Ulf Naumann, Antonius JM Matzke and Marjori Matzke* Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria 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 accu- mulates stably in non-silenced plants, to produce cis -act- ing secondary siRNAs that induce methylation in the downstream region. The identi±cation of DCL3 in a for- ward genetic screen for silencing-defective mutants de- monstrated 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. The EMBO Journal (2009) 28, 48–57. doi:10.1038/ emboj.2008.260; Published online 11 December 2008 Subject Categories : chromatin & transcription; RNA Keywords : Dicer-like3; methylation spreading; Pol IV; RNA-directed DNA methylation; secondary siRNAs Introduction Spreading of silent chromatin along a chromosome is a feature of many epigenetic processes in eukaryotes but the mechanisms, including the potential roles of non-coding RNAs, are still under investigation (Talbert and Henikoff, 2006; Clark, 2007; Pauler et al , 2007; Kwon and Workman, 2008). In Fssion yeast, an RNAi-mediated pathway fosters spreading of histone H3 lysine 9 methylation in heterochro- matic DNA repeats (Locke and Martienssen, 2006; Iida et al , 2008; Zhang et al , 2008). The process of RNA-directed DNA methylation (RdDM) in Arabidopsis offers an opportunity to study spreading of cytosine methylation facilitated by the RNAi machinery and siRNAs in euchromatic portions of the genome. RNA-directed DNA methylation is a small RNA-mediated epigenetic modiFcation that is highly developed in ±owering plants (Bei et al , 2007). The hallmarks of RdDM include methylation of cytosines in all sequence contexts (CG, CNG, CNN, where N is A, T or C) and restriction of methyla- tion to the region of RNA–DNA sequence homology. The establishment and maintenance of RdDM require for the most part conventional DNA cytosine methyltransferases, histone- modifying enzymes and nuclear-localized RNAi proteins (Matzke et al , 2007; Chan, 2008). In addition, several plant-
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3 - The EMBO Journal (2009) 28, 4857 www.embojournal.org |&...

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