Daxinger Lucia, Kanno Tatsuo, Bucher Etienne, van der Winden Johannes, Naumann Ulf, Matzke Antonius J M, Matzke Marjori
Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.
EMBO J. 2009 Jan 7;28(1):48-57. doi: 10.1038/emboj.2008.260. Epub 2008 Dec 11.
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.
我们利用转基因系统来研究拟南芥转录基因沉默过程中RNA指导的DNA甲基化（RdDM）的扩散。使用该系统的正向和反向遗传学方法描绘了一条次级小干扰RNA（siRNA）生物合成的逐步途径，以及甲基化从上游增强子元件向下游序列的单向扩散。反式作用的、发夹结构衍生的初级siRNA在靶增强子区域诱导初级RdDM，这一过程独立于与增强子相关的“新生”RNA。初级RdDM是该途径中的关键步骤，因为它吸引了次级siRNA产生机制，包括RNA聚合酶IV、RNA依赖的RNA聚合酶2和Dicer样蛋白3（DCL3）。这些因子在一个涉及新生RNA的周转途径中发挥作用，新生RNA通常在未沉默的植物中稳定积累，从而产生顺式作用的次级siRNA，诱导下游区域的甲基化。在沉默缺陷突变体的正向遗传筛选中鉴定出DCL3，这表明指导甲基化严格需要24个核苷酸的siRNA。DNA甲基化扩散的类似逐步过程可能发生在哺乳动物基因组中，这些基因组在上游调控区域广泛转录。
