Control of Imprinting by the Arabidopsis DEMETER Gene

拟南芥 DEMETER 基因对印记的控制

• 批准号: 6845297
• 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/6845297
• 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）：哺乳动物和植物中的基因组印记导致基因根据其亲本起源进行表达。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，胚乳是印记的关键位点，就像印记某些哺乳动物基因的胚外膜一样，它介导营养物质从母体到胚胎的转移。在这里，我们显示 DEMETER (DME) 介导胚乳印记。 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 在雌配子体（胚乳的祖细胞）的中央细胞中表达。 DME 是中央细胞和胚乳中印记 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，对 MEA 启动子的分析揭示了 DME 诱导的多个位点的切口。 DME DNA 糖基化酶活性位点的诱变证实碱基切除活性对于 DME 体内功能至关重要。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印记所必需的DNA序列并确定DME在体外切除的修饰碱基范围，阐明DME用于调节MEA的独立于DNA甲基化的机制。 3）通过鉴定其转录被DME直接激活的新靶基因来鉴定受DME调节的印记遗传电路。 4) 通过克隆抑制 dme 突变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 是否在复合物中发挥作用来调节基因转录。 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，描绘植物基因印记系统的上游组件。 7) 评估与DME相关的DNA糖基化酶的功能。这些实验将深入了解新型 DNA 糖基化酶蛋白用于调节基因印记、转录和复制的机制。