Control of Imprinting by the Arabidopsis DEMETER Gene

拟南芥 DEMETER 基因对印记的控制

基本信息

批准号：
7172249
负责人：
ROBERT L FISCHER
金额：
$ 27.38万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-02-01 至 2008-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7172249
关键词：
Active Sites Alleles Antibodies Arabidopsis Behavior Binding Biological Assay Cells Complex DNA DNA Methylation DNA Repair DNA Sequence DNA glycosylase DNA-(apurinic or apyrimidinic site) lyase DNA-Directed DNA Polymerase Development Direct Repeats Embryo Epigenetic Process Excision Family Female Gametogenesis Gene Targeting Genes Genetic Genetic Transcription Genomic Imprinting In Vitro Left Leucine Zippers Localized Mammals Maps Mediating Membrane Methylation Molecular Genetics Mothers Mutagenesis Mutation Nuclear Nutrient Pathway interactions Pattern Phenotype Plant Components Plant Genes Plants Polycomb Procedures Process Protein Binding Proteins Quantitative Trait Loci Range Regulation Reproduction Seeds Site System Testing Transcription factor genes base gene cloning homeodomain human disease imprint in vivo insight mutant novel polypeptide prenatal influence progenitor promoter repaired research study transcription factor yeast two hybrid system

项目摘要

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烙印会导致基因根据其父母的起源表达。在哺乳动物中，印迹基因会影响产前发育，行为和人类疾病。在植物中，胚乳是印迹的关键部位，就像额外的胚胎膜上印有某些哺乳动物基因的印记一样，介导了从母亲到胚胎的营养转移。在这里，我们显示Demeter（DME）介导了胚乳印记。 DME具有DNA糖基酶结构域，该糖基酶结构域可在体外从DNA中切除5-甲基胞嘧啶。 DME在雌性配子体的中央细胞（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迹的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转录以及MEA启动子的分析揭示了多个位点DME诱导的刻痕。 DME DNA糖基酶活性位点的诱变验证了碱性切除活性对于体内DME功能至关重要。因此，DME激活了中央细胞中印迹基因的母体表达。我们将执行以下实验，以了解印迹的机制。 1）确定DME是否通过从FWA启动子中的直接重复序列中切除5-甲基环肽来调节FWA印迹。 2）阐明了DME使用的DNA甲基化无关机制来调节MEA，通过描述对MEA印记和确定DME在体外切除的修饰碱基的范围所必需的DNA序列。 3）通过识别DME直接通过DME激活的新型靶基因来确定由DME调节的印迹遗传回路。 4）通过克隆抑制DME突变表型的克隆基因来鉴定与DME相互作用的蛋白质。 5）确定DME是否在复合物中起作用以通过鉴定与DME结合的蛋白质来调节基因转录。 6）通过识别限制DME转录到中心细胞的DNA调节序列和转录因子，描述植物基因印记系统的上游成分。 7）评估与DME相关的DNA糖基酶的功能。这些实验将提供有关新型DNA糖基化酶蛋白使用的机制，以调节基因印记，转录和繁殖。