Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

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详细了解植物细胞中从头RNA指导的DNA甲基化初始阶段的动力学。

胞嘧啶的甲基化是进化上保守的表观遗传标记，对于许多类群中染色质活性的控制至关重要。它主要起抑制作用，引起转录基因沉默。在植物中，从头DNA甲基化主要通过RNA指导的DNA甲基化途径建立。即使所涉及的蛋白质机制描述得比较好，但初始阶段的过程仍然是秘密的。我们显示了从头DNA甲基化动力学的第一个详细描述。由于流行的工厂模型系统无法提供以短时间间隔按时间序列收集同质响应物料的可能性，我们开发了一种基于烟草BY-2细胞系的便捷系统，可诱导产生siRNA（来自RNA发夹），将甲基化机制引导至控制GFP报告基因的CaMV 35S启动子。这些品系非常同步地响应，并且高水平的启动子特异性siRNA触发了快速的启动子甲基化，诱导后12小时观察到的首次增加。启动子中先前存在的CG甲基化不会影响甲基化动力学。各个胞嘧啶的环境反应不同。CHH甲基化在两天内达到约80％的峰值，然后下降，而CG和CHG甲基化则需要更多的时间，而CHG在10天后达到100％的水平。根据不存在传递性siRNA的情况，甲基化的扩散仅在靶区域外最小。24 nt siRNA的比例低且稳定，这表明Pol IV不参与初始阶段。我们的结果表明，从头DNA甲基化是一个快速过程，几乎随启动子特异性siRNA的出现而立即启动，并且与目标位置上甲基胞嘧啶的先前存在无关。甲基化是精确靶向的，其动力学取决于胞嘧啶序列的背景。逐渐增加的甲基化导致平滑，逐渐抑制启动子活性，在2天内被完全抑制。我们的结果表明，从头DNA甲基化是一个快速过程，几乎随启动子特异性siRNA的出现而立即启动，并且与目标位置上甲基胞嘧啶的先前存在无关。甲基化是精确靶向的，其动力学取决于胞嘧啶序列的背景。逐渐增加的甲基化导致平滑，逐渐抑制启动子活性，在2天内被完全抑制。我们的结果表明，从头DNA甲基化是一个快速过程，几乎随启动子特异性siRNA的出现而立即启动，并且与目标位置上甲基胞嘧啶的先前存在无关。甲基化是精确靶向的，其动力学取决于胞嘧啶序列的背景。逐渐增加的甲基化导致平滑，逐渐抑制启动子活性，在2天内被完全抑制。