DNA methylation in cytosine residues is a conserved epigenetic mark that is relevant for genome defense and stability, development and stress responses in plants. The transgenerational stability of DNA methylation changes is important in setting up genomic DNA methylation patterns and in the formation and transmission of epialleles. 

De novo DNA methylation in plants is mediated by the RNA-directed DNA methylation (RdDM) pathway. However, it remains to be determined whether and how DNA methylation changes at RdDM target loci are transgenerationally inherited, and if these loci can become stable epialleles.

A research team led by Prof. ZHU Jiankang from the Shanghai Center for Plant Stress Biology of the Chinese Academy of Sciences reported that not all loci exhibiting hypomethylation in RdDM mutants can be reverted to wild type-like state upon restoration of RdDM function, and thus can form stable epialleles. This study was published online in Nature Plants on June 8.

RNA polymerase IV (Pol IV) dependent 24-siRNA production is a key step for RNA-directed DNA methylation. The researchers classified RdDM targets in Arabidopsis into two groups, remethylableloci (CD) and non-remethylable loci (ND), based on whether there is re-methylation following restoration of NRPD1 (largest subunit of Pol IV) function in thenrpd1 mutant plants.

Using genetic approaches, they found that the higher levels of the euchromatic marks H3K4me3 at ND loci could interfere with the recruitment of the RdDM machinery, and higher H3K18ac could help to recruit the DNA demethylase ROS1 to antagonize RdDM at ND loci.

Furthermore, using targeted methylation erasure by the CRISPR/dCas9-TET1cd system, the researchers demonstrated that the remaining CG and CHG methylation in thenrpd1 mutant are memory marks required for targeting the RdDM machinery to CD loci.

These discoveries demonstrate that histone and DNA methylation marks are critical in determining the capacity of RdDM target loci to form stable epialleles, and thus contribute to understanding the formation and transmission of epialleles.