Repression of CHROMOMETHYLASE 3 Prevents Epigenetic Collateral Damage in Arabidopsis

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Moreover, CMT3-induced ectopic methylation of genes undergoing transcriptional activation can reduce their corresponding transcript levels. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants. Methyl cytosine sequencing libraries were generated from 2-4 biological replicates of bent cotyledon stage embryos or seedlings from four genotypes. mRNA-seq libraries were generated from 9 biological replicates of bent cotyledon embryos from three genotypes.

DNA甲基化（DNA methylation）在演化过程中形成了沉默诱变型转座因子（transposable elements, TEs）的功能，同时通常会规避对内源基因的靶向作用。在包括植物胚胎发生在内的细胞周期动态活跃阶段，防止DNA甲基转移酶（DNA methyltransferases）对基因进行异位甲基化的机制，理应具备核心重要性。然而，目前学界对胚胎发生过程中如何精准调控DNA甲基转移酶活性，以避免诱发潜在有害且有丝分裂稳定的基因表观突变，几乎一无所知。本研究发现，微RNA（microRNA）介导的铬甲基化酶3（CHROMOMETHYLASE 3, CMT3）抑制作用，以及CMT3偏好的染色质特征，可防止胚胎发生期间数千个基因发生异位甲基化，且该甲基化状态可维持数周之久。此外，CMT3诱导的正在经历转录激活的基因发生异位甲基化，可降低其对应的转录本水平。因此，对CMT3的抑制可避免内源基因遭受表观遗传附带损伤。本研究还提出了一个模型，或可帮助调和有关几乎所有开花植物中普遍存在的基因体甲基化功能的争议观点。甲基胞嘧啶测序文库的构建，使用了4种基因型的弯子叶期胚胎或幼苗的2-4个生物学重复样本。信使RNA测序（mRNA-seq）文库的构建，则使用了3种基因型的弯子叶期胚胎的9个生物学重复样本。