Control of Imprinting by the Arabidopsis DEMETER Gene

描述（由申请人提供）：哺乳动物和植物中的基因组印记导致基因根据其亲本来源表达。在哺乳动物中，印记基因影响产前发育，行为和人类疾病。在植物中，胚乳是印记的关键部位，并且就像某些哺乳动物基因被印记的额外的胚胎膜一样，介导从母体到胚胎的营养转移。在这里，我们显示DEMETER（DME）介导胚乳印迹。DME具有在体外从DNA切除5-甲基胞嘧啶的DNA糖基化酶结构域。DME在雌配子体的中央细胞（胚乳的祖先）中表达。DME是中央细胞和胚乳中印迹MEA Polycomb和FWA转录因子基因的母体等位基因表达所必需的。异位DME表达诱导MEA和FWA转录，MEA启动子的分析揭示DME诱导的多个位点的切口。DME DNA糖基化酶活性位点的突变证实了碱基切除活性对于DME在体内的功能是必不可少的。因此，DME激活中央细胞中印记基因的母体表达。我们将进行以下实验来了解印记的机制。1)确定DME是否通过从FWA启动子的正向重复序列中切除5-甲基胞嘧啶来调节FWA印迹。2)阐明DNA甲基化的独立机制，DME使用的MEA调节通过描绘DNA序列必需的MEA印记和确定的范围内的DME在体外切除的修改后的碱基。3)通过识别DME直接激活其转录的新靶基因来识别DME调控的印迹遗传电路。4)通过克隆抑制DME突变体表型的基因来鉴定与DME发生遗传相互作用的蛋白质。5)通过鉴定与DME结合的蛋白质，确定DME是否在复合物中起调节基因转录的作用。6)通过鉴定限制DME转录到中央细胞的DNA调控序列和转录因子，描绘植物基因印迹系统的上游组分。7)评估与DME相关的DNA糖基化酶的功能。这些实验将提供有关新的DNA糖基化酶蛋白调节基因印迹，转录和繁殖的机制的见解。

DESCRIPTION (provided by applicant): 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.