Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation

5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, while mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, while mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small-interfering RNAs and H3K9me2, without gaining CHH methylation. This suggests that spontaneous epialelles that arise in plant cell cultures are stably maintained by small RNA independently of the canonical RNA-directed DNA methylation pathway. In contrast, loci that fail to produce siRNA are targeted for demethylation when cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones. Overall design: Small RNA sequencing of Arabidopsis cell cultures

5-甲基胞嘧啶（5-methyl cytosine）广泛分布于植物基因组的CG与非CG序列环境中。在DNA复制过程中，亲本DNA链的半甲基化可指导新生链形成对称CG甲基化，而非CG甲基化则依赖修饰组蛋白与小RNA引导。本研究利用永生化拟南芥细胞悬浮系，分选处于复制状态的细胞核，解析植物细胞周期进程中的全基因组胞嘧啶甲基化动态变化。研究发现，活跃分裂的培养细胞中选择性保留对称型mCG与mCHG甲基化，而mCHH甲基化则被清除。mCG甲基化在S期会出现短暂的不对称性，但在G2期可快速恢复；而mCHG甲基化在整个细胞周期中始终保持不对称。数百个基因座会获得异位CHG甲基化，同时伴随24-nt小干扰RNA（small-interfering RNAs）与组蛋白H3赖氨酸9二甲基化（H3K9me2）修饰，但未发生CHH甲基化。这表明植物细胞培养中产生的自发表观等位基因（epialleles）可通过小RNA通路稳定维持，不依赖经典RNA指导的DNA甲基化途径。与之相反，无法产生小干扰RNA的基因座在细胞周期停滞时会被靶向去甲基化。对不同组织与细胞类型的甲基化组进行比较分析显示，DNA复制过程中小RNA指导的非CG甲基化缺失，会促进无性繁殖植物的生殖系重编程与表观遗传变异。实验设计：拟南芥细胞培养物的小RNA测序。