GSE117316
Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation
5-methyl cytosine in plant genomes occurs in all sequence contexts, especially in heterochromatin, transposons and other repeats. During replication, nascent cytosines in symmetric contexts (CG) are guided for methylation by hemi-methyl C on the parental DNA strands of both daughter chromatids. However nascent cytosines in the asymmetric CHH context or in symmetric CHG (where H=A, T or C) use modified histones and 24-nt small interfering RNAs to restore methylation to each daughter chromatid. Here, we use fluorescent activated sorting of EdU-labeled nuclei to examine DNA methylation dynamics in dividing cell cultures of Arabidopsis thaliana. We find that CG methylation becomes transiently asymmetric during late S phase, but is rapidly restored during each cell division, while CHG methylation remains asymmetric and is substantially reduced. Levels of mCHH are extremely low in dividing cells, accompanied by a shift from 24-nt to 21-nt epigenetically activated small RNAs (easiRNAs) and a loss of histone lysine-9 methylation. When cell division arrests, RNA-directed CHH methylation is restored, but only at loci that retained 24-nt siRNAs. Comparisons with methylation patterns in pollen suggest that DNA methylation reprogramming in microspores (G2), sperm cells (S phase) and the vegetative nucleus (which is quiescent) reflect their cell cycle stage.  Our results also account for the loss of non-CG methylation in plants micropropagated as clones, which undergo methylation reprogramming in the absence of fertilization. Overall design: Bisulfite sequencing of Arabidopsis cell culture nuclei