Genomic imprinting in mammals and plants causes genes to be expressed according to their parental origin. In mammals, imprinted genes influence prenatal development, behavior, and human disease. In plants, the endosperm is a critical site for imprinting and, like the extra embryonic membranes where certain mammalian genes are imprinted, mediates nutrient transfer from mother to embryo. Here we show DEMETER (DME) mediates endosperm imprinting. DME has a DNA glycosylase domain that excises 5-methylcytosine from DNA in vitro. DME is expressed in the central cell of the female gametophyte, the progenitor of the endosperm. DME is required for maternal allele expression of the imprinted MEA Polycomb and FWA transcription factor genes in the central cell and endosperm. Ectopic DME expression induces MEA and FWA transcription and analysis of the MEA promoter reveals DME-induced nicking at multiple sites. Mutagenesis of the DME DNA glycosylase active site verifies that base excision activity is essential for DME function in vivo. Thus, DME activates maternal expression of imprinted genes in the central cell. We will perform the following experiments to understand the mechanisms of imprinting. 1) Determine if DME regulates FWA imprinting by excising 5-methylcytosine from direct repeats in the FWA promoter. 2) Elucidate the DNA methylation-independent mechanism used by DME to regulate MEA by delineating DNA sequences essential for MEA imprinting and determining the range of modified bases excised by DME in vitro. 3) Identify imprinted genetic circuits regulated by DME by identifying novel target genes whose transcription is directly activated by DME. 4) Identify proteins that genetically interact with DME by cloning genes that suppress dme mutant phenotypes. 5) Determine if DME functions in a complex to regulate gene transcription by identifying proteins that bind to DME. 6) Delineate upstream components of the plant gene imprinting system by identifying DNA regulatory sequences and transcription factors that restrict DME transcription to the central cell. 7) Assess the function of DNA glycosylases related to DME. These experiments will provide insights about the mechanisms used by novel DNA glycosylase proteins to regulate gene imprinting, transcription, and reproduction.
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