Heritable epigenetic variation of DNA methylation targets in plants

DNA methylation marks regulate gene expression and genome structure. Stability and dynamics of DNA methylation patterns are influenced by four major factors including de novo methylation, maintenance methylation, passive loss of methylation and active demethylation. Maintenance methylation functions are still well conserved among plants and animals, which separated more than 1.5 billion years ago. In contrast, demethylation mechanisms differ considerably among plants and mammals. Interfering with DNA methylation and demethylation systems could be a source of heritable epigenetic variation if DNA methylation changes are introduced that transcend into stable heritable gene expression changes. The high tolerance of plants to DNA methylation changes makes them an ideal experimental system to exploit DNA methylation and demethylation systems. In this study, four strategies have been developed and tested for their capacity to induce heritable epigenetic variation by interfering with DNA methylation and demethylation systems. These strategies included a chemical treatment with a DNA methylation inhibitor, genetic demethylation using a mutant deficient in the maintenance methyltransferase MET1 and transgenic approaches to over-express MET1 and to express the human TET3 demethylase. While chemical demethylation only generated non-heritable changes, inactivating MET1 induced stable DNA methylation and expression changes at specific loci. Expression of the human TET3 protein also induced locus-specific loss of methylation but the efficiency of demethylation varied in individual transformants independent of TET3 level, which suggests that demethylation is locus-specific but stochastic.