Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase͞lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.Arabidopsis ͉ demethylation ͉ gene silencing ͉ methylation M ethylation of cytosine at carbon 5 of the pyrimidine ring [5-methylcytosine (5-meC)] is an epigenetic modification that guides formation of transcriptionally silent chromatin and allows transmission of specific patterns of gene activity across cellular divisions (1, 2). In eukaryotes, DNA methylation is detected in protists, fungi, plants, and animals (3) and plays important roles in the establishment of developmental programs (4, 5) and in genome defense against parasitic mobile elements (6). Most of mammalian and plant DNA methylation is restricted to symmetrical CpG sequences, but plants also have significant levels of cytosine methylation in the symmetric context CpNpG (where N is any nucleotide) and even in asymmetric contexts (2, 7). Similarly to other biochemical modifications such as protein phosphorylation and acetylation, DNA methylation is also reversible. Demethylation may take place as a passive process because of lack of maintenance methylation during several cycles of DNA replication or as an active mechanism in the absence of replication (8). In mammalian preimplantation embryos the maternal genome is demethylated by a passive process along cleavage stages, whereas the paternal genome is demethylated by an active mechanism immediately after fertilization (9). In addition to global demethylation, site-specific...

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.