Stable transgenerational epigenetic inheritance requires a DNA methylation-sensing circuit

Eukaryotic genomes must maintain stable inheritance of epigenetic states. In plants, DNA methylation patterns are faithfully inherited over many generations but it is unknown how the dynamic activities of cytosine DNA methyltransferases and 5-methylcytosine DNA glycosylases, which remove 5-methylcytosine by base excision repair, interact to maintain epigenetic homeostasis. Here we show that a methylation-sensing gene regulatory circuit centered on a 5-methylcytosine DNA glycosylase gene is required for long-term epigenetic fidelity in Arabidopsis. Disrupting this circuit causes widespread methylation losses and abnormal phenotypes that progressively worsen over generations. In heterochromatin, these losses are counteracted such that methylation returns to a normal level over four generations. However, thousands of loci in euchromatin progressively lose DNA methylation between generations and remain unmethylated. We conclude that actively maintained equilibrium between methylation and demethylation activities is required to ensure long-term stable inheritance of epigenetic information. Overall design: Whole genome bisulfite sequencing was used to examine DNA methylation in wild-type (Col-0), ros1, rdr2, and rdr2; ros1 mutant plants. In addition, whole genome bisulfite sequencing was performed on multiple generations of “broken rheostat (BR)” lines, in which expression of the DNA glycosylase ROS1 is decoupled from the methylation state of the genome. “BRmut” denotes control lines in which ROS1 is mutated. T(n) denotes the generation of each BR or BRmut line sample