MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.

Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5 and 3 ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation. DNA glycosylase ͉ epigenetics ͉ genome maintenanceA rabidopsis is a eukaryotic model for DNA methylation studies. In Arabidopsis cytosine methylation is found in all sequence contexts (CG, CNG, and CNN) and is important for genomic imprinting and genome defense against transposable elements (1). Most DNA methylation is located at transposonrich heterochromatic regions (2-4). However, genome-wide mapping of methylation has showed that a significant fraction (20-33%) of genes are methylated (3-5). In general, methylation within Arabidopsis genes is concentrated in the middle and distributed away from 5Ј and 3Ј ends, suggesting that 5Ј and 3Ј methylation is detrimental to gene function (3, 4). The mechanisms that maintain gene ends relatively free of methylation are not known.DNA methylation is often considered a stable epigenetic mark, but enzymatic DNA demethylation is known to occur. In mammals, the paternal pronucleus is actively demethylated immediately after fertilization (6, 7) but the enzymes responsible are unknown (8). In Arabidopsis, DNA demethylation is mediated by the DEMETER (DME) family of bifunctional helixhairpin-helix DNA glycosylases that have both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities (9-13). The DNA glycosylase initiates the base excision repair process by specifically excising 5-methylcytosine through cleavage of the N-glycosylic bond. AP lyase subsequently nicks the DNA, and an AP endonuclease generates a 3Ј-hydroxyl to which a DNA repair polymerase adds an unmethylated cytosine. DNA ligase completes the repair process by sealing the nick.Demethylation by DME is one step in a developmental pathway that establishes genomic imprinting in the Arabidopsis endosperm (9,10,14). Three targets of DME are MEDEA (MEA),...

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known about their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis. This effect is correlated with the down-regulation of key transcriptional gene silencing factors and is partly dependent on an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoter regions, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to TEs/repeats. epigenetics | RNA silencing | MAMP

SUMMARY Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the biofilm matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance, as previous studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid P. aeruginosa biofilms. To our surprise, we showed that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions, but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.