Whole genome bisulfite sequencing of nrpd1 mutant in C24 background [methyl-seq]

Arabidopsis ROS1 is the first genetically characterized DNA demethylase in eukaryotes. Dysfunction of ROS1 leads to increase in DNA methylation level at thousands of genomic loci. However, the features of ROS1 targets are not well understood. In this study, we identified and characterized ROS1 target loci in Arabidopsis Col-0 and C24 ecotypes. Most ROS1 targets are transposable elements (TEs) and intergenic regions. Compared to other TEs, ROS1-targeted TEs are closer to protein coding genes, suggesting a role for ROS1 in preventing the spreading of DNA methylation from highly methylated TEs to nearby genes. Interestingly, we found that unlike general TEs, ROS1 targets are associated with an enrichment of H3K18ac and H3K27me3, and depletion of H3K27me and H3K9me2. We investigated the antagonism between ROS1 and RNA-directed DNA methylation (RdDM) by identifying and characterizing thousands of genomic regions regulated by both ROS1 and RdDM. Unexpectedly, we uncovered thousands of previously unidentified RdDM targets by analyzing the DNA methylome of ros1/nrpd1 double mutant plants. In addition, we show that ROS1 also antagonizes RdDM-independent DNA methylation at more than a thousand genomic loci. Our results provide significant insights into the genome-wide effects of both ROS1-mediated active DNA demethylation and RNA-directed DNA methylation as well as their interaction in plants. Overall design: Using whole genome bisulfite sequencing to provide single-base resolution of DNA methylation status in nrpd1 mutant (C24 background)

拟南芥ROS1是真核生物中首个经遗传鉴定的DNA去甲基化酶（DNA demethylase）。ROS1功能缺失会导致数千个基因组位点的DNA甲基化水平升高，但目前人们对ROS1靶位点的特征仍缺乏深入了解。本研究在拟南芥Col-0和C24生态型中鉴定并表征了ROS1的靶位点。大多数ROS1靶位点为转座元件（transposable elements, TEs）和基因间区域。与其他转座元件相比，ROS1靶向的转座元件更靠近蛋白质编码基因，这提示ROS1可能参与阻止高度甲基化转座元件的DNA甲基化扩散至邻近基因。有趣的是，我们发现与普通转座元件不同，ROS1的靶位点富集H3K18ac和H3K27me3修饰，同时缺失H3K27me和H3K9me2修饰。我们通过鉴定并表征数千个受ROS1和RNA指导的DNA甲基化（RNA-directed DNA methylation, RdDM）共同调控的基因组区域，探究了ROS1与RdDM之间的拮抗作用。出乎意料的是，通过分析ros1/nrpd1双突变体植株的DNA甲基化组，我们发现了数千个此前未被鉴定的RdDM靶位点。此外，我们证实ROS1还能在超过一千个基因组位点上拮抗不依赖RdDM的DNA甲基化。本研究结果为植物中ROS1介导的主动DNA去甲基化与RNA指导的DNA甲基化的全基因组效应及其相互作用提供了重要见解。整体实验设计：采用全基因组亚硫酸氢盐测序技术，获取nrpd1突变体（C24遗传背景）中单碱基分辨率的DNA甲基化状态信息。