Dynamic of DNA methylation during Arabidopsis reproduction

Cytosine methylation is a key epigenetic mark in many organisms, important for both transcriptional control and genome integrity. While relatively stable during somatic growth, DNA methylation is reprogrammed genome-wide during mammalian reproduction. Reprogramming is essential for zygotic totipotency, and to prevent transgenerational inheritance of epimutations. The extent of DNA methylation reprogramming in plants however remains unclear. Here, we developed sensors reporting with single-cell resolution CG and non-CG methylation in Arabidopsis. Live imaging during reproduction revealed distinct and sex-specific dynamics for both contexts. We found that CHH methylation in the egg cell depends on DRM2 and Pol V, two main actors of RNA-directed DNA methylation, but does not depend on Pol IV. Our sensors provide insight into global DNA methylation dynamics at the single cell level with high temporal resolution, and offer a powerful tool to track CG and non-CG methylation both during development and in response to environmental cues in all organisms with methylated DNA. Overall design: ChIP-seq experiments conducted using transgenic lines expressing either CG or non-CG binding proteins. mCG in WT (Col(O)), mCHH and drm1-drm2-cmt2-cmt3, plus inputs.胞嘧啶甲基化（Cytosine methylation）是众多生物中一类关键的表观遗传标记（epigenetic mark），对转录调控与基因组完整性均具有重要意义。尽管在体细胞生长过程中相对稳定，但DNA甲基化（DNA methylation）在哺乳动物生殖过程中会发生全基因组范围的重编程。该重编程过程对于合子全能性（zygotic totipotency）的维持以及阻断表观突变（epimutations）的跨代遗传均不可或缺。然而，植物中DNA甲基化重编程的程度仍未明确。本研究开发了可在拟南芥（Arabidopsis）中以单细胞分辨率（single-cell resolution）检测CG与非CG甲基化的传感工具。生殖过程中的活细胞成像（live imaging）结果显示，两类甲基化模式均呈现出独特且具有性别特异性的动态变化。本研究发现，卵细胞中的CHH甲基化依赖于RNA指导的DNA甲基化（RNA-directed DNA methylation）的两个核心因子DRM2与Pol V，但不依赖于Pol IV。本研究开发的传感工具可在单细胞水平上以高时间分辨率（high temporal resolution）解析全局DNA甲基化动态变化，同时可为所有携带甲基化DNA的生物，在发育过程及响应环境信号时追踪CG与非CG甲基化提供一款高效实用的研究工具。实验设计概要：采用分别表达CG结合蛋白与非CG结合蛋白的转基因株系开展染色质免疫共沉淀测序（ChIP-seq）实验；样本涵盖野生型（WT）哥伦比亚生态型（Col(O)）中的mCG、mCHH以及drm1-drm2-cmt2-cmt3突变体材料，并设置输入对照样本。