It is generally assumed that DNA methylation changes at genomic regions targeted by the de novo RNA-directed DNA methylation (RdDM) pathway are unstable. Here, we show that RdDM targets in Arabidopsis can be classified into two groups on the basis of whether there is remethylation following the restoration of NRPD1 function in nrpd1 mutant plants—remethylable loci and non-remethylable loci. In contrast to the remethylable loci, the non-remethylable loci contain higher levels of the euchromatic marks of trimethylation at Lys 4 of histone H3 (H3K4me3), which interferes with the recruitment of the RdDM molecular machinery, and acetylation at Lys 18 of histone H3 (H3K18ac), which helps to recruit the DNA demethylase ROS1 to antagonize RdDM. Here, using targeted methylation erasure by CRISPR–dCas9–TET1, we demonstrate that methylated CG (mCG) and mCHG (where H represents A, C or T) are memory marks that are required for targeting the RdDM machinery to remethylable loci. Our results show that histone and DNA methylation marks are critical in determining the ability of RdDM target loci to form stable epialleles, and contribute to understanding the formation and transmission of epialleles.

通常认为，从头RNA指导的DNA甲基化（RdDM）途径靶向的基因组区域的DNA甲基化变化是不稳定的。在这里，我们表明，在RdDM目标拟南芥能是否有再次甲基化NRPD1功能的恢复后的基础上分为两组nrpd1突变植物-可甲基化的基因座和不可甲基化的基因座。与可甲基化的位点相反，不可甲基化的位点在组蛋白H3（H3K4me3）的Lys 4处含有较高水平的三甲基化的常色标记，这会干扰RdDM分子机制的募集以及在组蛋白H3的Lys 18处的乙酰化。 （H3K18ac），有助于募集DNA脱甲基酶ROS1拮抗RdDM。在这里，使用CRISPR–dCas9–TET1的靶向甲基化擦除，我们证明了甲基化CG（mCG）和mCHG（其中H代表A，C或T）是将RdDM机械靶向可甲基化位点所需的记忆标记。我们的结果表明，组蛋白和DNA甲基化标记对于确定RdDM目标基因座形成稳定等位基因的能力至关重要，