DNA methylation (5-methylcytosine) in mammals and plants silences transposons, retroviruses, and regulates gene imprinting. In mammals, DNA hypermethylation is associated with certain diseases including the onset and progression of cancer. We discovered that the Arabidopsis DEMETER (DME) protein regulates imprinting by DNA demethylation. DME is related to DNA glycosylases that excise damaged/mispaired bases in the base excision DNA repair pathway. DME excises 5-methylcytosine that is replaced with cytosine. DME is expressed primarily in the central cell, the progenitor of the placenta-like endosperm that supports embryo development. DME demethylates and activates maternal allele expression of genes that are imprinted in the endosperm. Arabidopsis has three DME-LIKE (DML) genes: ROS1, DML2, and DML3, which are broadly expressed in the plant. Like DME, they excise 5-methylcytosine and have a DNA glycosylase domain flanked by additional conserved domains that are essential for activity. DME and DML proteins specifically demethylate the 5' and 3' ends of at least 180 genes. ROS1 demethylates disease resistance genes and is required for resistance to a bacterial pathogen, Pseudomonas syringae. We will carry out the following experiments to understand the mechanisms and functions of DNA demethylation in the regulation of gene imprinting. 1) We will elucidate how expression of DME is restricted to the central cell, identify new genes imprinted by DME, and isolate mutations in genes that regulate DME expression, targeting, and/or DNA demethylation. 2) To understand the molecular mechanisms of DNA demethylation, we will identify and analyze the function of DME-binding proteins, determine if DME directly demethylates DNA by physical association with its target genes, and elucidate what changes in chromatin structure accompany DNA demethylation. 3) To understand how DNA demethylation regulates disease resistance, we will elucidate the function of plant defense genes that are demethylated by ROS1. Plants and animals have related mechanisms for controlling DNA methylation. Information about the basic mechanisms of DNA demethylation will provide valuable insights into the critical role DNA methylation plays in the regulation of gene imprinting and disease [unreadable] [unreadable] [unreadable]