Control of Imprinting by the Arabidopsis DEMETER Gene

拟南芥 DEMETER 基因对印记的控制

• 批准号: 6702802
• 负责人: ROBERT L FISCHER
• 金额: $ 28.88万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702802
• 关键词: Arabidopsis; DNA methylation; N glycosidase; active sites; binding sites; gel mobility shift assay; gene expression; genetic regulation; genetic transcription; genomic imprinting; laboratory rabbit; mutant; phenotype; reporter genes; yeast two hybrid system

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.

描述（由申请人提供）：哺乳动物和植物的基因组印记导致基因根据其亲本起源表达。在哺乳动物中，印迹基因影响产前发育、行为和人类疾病。在植物中，胚乳是印记的关键部位，就像某些哺乳动物基因被印记的胚胎外膜一样，介导营养物质从母体向胚胎的转移。在这里我们发现DEMETER （DME）介导胚乳印迹。DME具有DNA糖基化酶结构域，可在体外从DNA中切除5-甲基胞嘧啶。DME在雌性配子体的中心细胞表达，雌性配子体是胚乳的祖先。二甲醚是中心细胞和胚乳中印迹MEA Polycomb和FWA转录因子基因的母系等位基因表达所必需的。异位DME表达诱导MEA和FWA转录，MEA启动子分析显示DME在多个位点诱导切口。二甲醚DNA糖基酶活性位点的突变验证了碱基切除活性对二甲醚在体内的功能至关重要。因此，二甲醚激活母体在中枢细胞中印迹基因的表达。我们将进行以下实验来了解印迹的机制。1)确定DME是否通过从FWA启动子的直接重复序列中切除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范围，阐明DME调控MEA的DNA甲基化非依赖性机制。3)通过鉴定二甲醚直接激活转录的新靶基因，鉴定二甲醚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。5)通过鉴定与二甲醚结合的蛋白，确定二甲醚是否在一个复合体中起调节基因转录的作用。6)通过鉴定限制DME转录到中心细胞的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)评估与二甲醚相关的DNA糖基酶的功能。这些实验将提供新的DNA糖基酶蛋白用于调节基因印迹、转录和繁殖的机制的见解。