Regulation and activity of the 5-methylcytosine DNA glycosylase ROS1 contributes to DNA methylation patterning across development

Abstract

DNA methylation patterning is a consequence of opposing activities of DNA methyltransferases and DNA demethylases. A 5-methylcytosine DNA glycosylase, ROS1, removes DNA methylation from the Arabidopsis genome. In flowering plants, two distinct female gametes, the egg cell and the central cell, are fertilized, producing what will become the embryo and the endosperm of the seed. In Arabidopsis, a 5-methylcytosine DNA glycosylase, DME, demethylates regions in the central cell genome, leading to methylation differences between maternally- and paternally-inherited endosperm genomes after fertilization. DME is required for endosperm gene imprinting. Homologues of DME include ROS1, DML2 and DML3. It is unknown whether any of these DNA glycosylases are required for endosperm methylation patterning. We show that ROS1 prevents hypermethylation of paternally-inherited alleles in the endosperm at regions that lack maternal or paternal-allele methylation in wild-type. Thus, ROS1 promotes epigenetic symmetry between genomes in the endosperm by preventing paternal genome hypermethylation. We investigated dynamics of DNA methylation at the edges of transposable elements, where ROS1 is known to prevent spreading of DNA methylation into neighboring regions of the genome. We found that DNA methylation spreading in ros1 mutant is unidirectional, which has implications for the field’s understanding of the mechanism of ROS1 activity at TEs as well as the mechanism of methylation establishment at TEs. We have investigated the regulation of ROS1 expression by interaction of ROS1 and the RdDM pathway at the ROS1 promoter. Using a previously characterized deletion in the ROS1 promoter, we investigated the consequences of ROS1 regulation across the genome in the presence of a wild-type RdDM pathway. Finally, I discuss the implications of the work I have done in understanding the role of ROS1 across plant development and the mechanisms by which DNA methylation is patterned in plants, and propose future directions related to these findings.