Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA.
Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA; Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California at Los Angeles, Los Angeles, CA 90095, USA.
Cell. 2019 Feb 21;176(5):1068-1082.e19. doi: 10.1016/j.cell.2019.01.029. Epub 2019 Feb 7.
The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.
植物中的 RNA 指导的 DNA 甲基化 (RdDM) 途径通过胞嘧啶 DNA 甲基化来控制基因表达。操纵 RdDM 的能力将揭示 DNA 甲基化控制基因表达的机制和应用。在这里，我们鉴定了多种 RdDM 蛋白，当它们与人工锌指 (ZF-RdDM) 连接时，能够在拟南芥中靶向甲基化和沉默。我们通过测试它们在不同突变体中靶向甲基化的能力来研究它们在 RdDM 途径中的作用顺序。我们还评估了数千个 ZF-RdDM 靶标中的异位 siRNA 生物发生、RNA 聚合酶 V (Pol V) 募集、靶向 DNA 甲基化和基因表达变化。我们发现，同时靶向 RdDM 途径的两个臂，siRNA 生物发生和 Pol V 募集，显著增强了靶向甲基化。这项工作定义了 RdDM 成分如何建立 DNA 甲基化，并为通过靶向 DNA 甲基化进行表观遗传基因调控提供了新的策略。
