Co-targeting RNA Polymerases IV and V promotes efficient de novo DNA methylation in Arabidopsis [WGBS]

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. 45 WGBS

植物体内的RNA指导的DNA甲基化（RNA-directed DNA methylation，RdDM）通路通过胞嘧啶DNA甲基化调控基因表达。操纵RdDM通路的能力，将有助于揭示DNA甲基化调控基因表达的机制与应用价值。本研究中，我们鉴定出多种RdDM蛋白，当将其拴系至人工锌指（artificial zinc finger，ZF）以构建ZF-RdDM融合系统时，该系统可在拟南芥中靶向介导甲基化与基因沉默。我们通过检测这些蛋白在不同突变体中靶向甲基化的能力，解析了它们在RdDM通路中的作用时序。此外，我们还在数千个ZF-RdDM靶向位点处，评估了异位小干扰RNA（small interfering RNA，siRNA）生成、RNA聚合酶V（RNA Polymerase V，Pol V）招募、靶向DNA甲基化以及基因表达的变化情况。研究发现，同时靶向RdDM通路的两个分支——siRNA生成与Pol V招募，可显著提升靶向甲基化的效率。本研究阐明了RdDM组分建立DNA甲基化的具体机制，并为通过靶向DNA甲基化实现表观遗传基因调控提供了全新策略。本数据集包含45份全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing，WGBS）数据。