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, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the 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. • mCG is rapidly restored during replication, whereas mCHG remains asymmetric • Asymmetric mCHH methylation is depleted in actively dividing cells • Epialleles are stably maintained in cell cultures via siRNA and H3K9me2 • Loss of siRNA during cell division may lead to demethylation when the cell cycle arrests Borges et al. use immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine DNA methylation dynamics during the plant cell cycle. Comparative methylome analysis suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and somaclonal variation. [ABSTRACT FROM AUTHOR]