Control of Imprinting by the Arabidopsis DEMETER Gene

描述(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代来源表达。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，就像印记某些哺乳动物基因的额外胚胎膜一样，介导着从母亲到胚胎的营养转移。这里我们展示了德米特(DME)调节胚乳印迹的过程。DME有一个DNA糖基酶结构域，可以在体外从DNA中去除5-甲基胞嘧啶。DME在雌配子体的中央细胞中表达，雌配子体是胚乳的祖先。在中央细胞和胚乳中，印记的MEA Polycomb和FWA转录因子基因的母体等位基因表达需要DME。异位表达的DME诱导了MEA和FWA的转录，对MEA启动子的分析表明，DME诱导了多个位置的缺口。DME DNA糖基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激活了中央细胞中印记基因的母体表达。我们将进行以下实验，以了解印记的机制。1)确定DME是否通过从FWA启动子的直接重复序列中剔除5-甲基胞嘧啶来调节FWA印迹。2)通过描绘MEA印迹所必需的DNA序列和确定DME在体外切除的修饰碱基的范围，阐明DME用于调控MEA的DNA甲基化非依赖性机制。3)通过鉴定DME直接激活其转录的新的靶基因，识别受DME调控的印迹遗传回路。4)通过克隆抑制DME突变表型的基因，鉴定与DME基因相互作用的蛋白质。5)通过识别与DME结合的蛋白质，确定DME是否在复合体中发挥调节基因转录的功能。6)通过鉴定将DME转录限制在中央细胞的DNA调控序列和转录因子，来描述植物基因印迹系统的上游成分。7)评估与DME相关的DNA糖基酶的功能。这些实验将为新的DNA糖基酶蛋白用于调节基因印迹、转录和繁殖的机制提供深入的见解。

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.