DNA methylation maintenance consolidates RNA ‐directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana

Summary In plants, 24 nucleotide short interfering RNA s serve as a signal to direct cytosine methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter function, this RNA ‐directed DNA methylation may result in transcriptional gene silencing. In a genetic screen for factors involved in RNA ‐directed transcriptional silencing of a Pro NOS – NPTII reporter transgene in Arabidopsis thaliana , we captured alleles of DOMAINS REARRANGED METHYLTRANSFERASE  2 , the gene encoding the DNA methyltransferase that is mainly responsible for de novo DNA methylation in the context of RNA ‐directed DNA methylation. Interestingly, methylation of the reporter gene Pro NOS was not completely erased in these mutants, but persisted in the symmetric CG context, indicating that RNA ‐directed DNA methylation had been consolidated by DNA methylation maintenance. Taking advantage of the segregation of the transgenes giving rise to Pro NOS short interfering RNA s and carrying the Pro NOS – NPTII reporter in our experimental system, we found that Pro NOS DNA methylation maintenance was first evident after two generations of ongoing RNA ‐directed DNA methylation, and then increased in extent with further generations. As Pro NOS DNA methylation had already reached its final level in the first generation of RNA ‐directed DNA methylation, our findings suggest that establishment of DNA methylation at a particular region may be divided into distinct stages. An initial phase of efficient, but still fully reversible, de novo DNA methylation and transcriptional gene silencing is followed by transition to efficient maintenance of cytosine methylation in a symmetric sequence context accompanied by persistence of gene silencing.