Regulation and Function of DNA Demethylation in Arabidopsis

描述(申请人提供)：哺乳动物和植物中的DNA甲基化(5-甲基胞嘧啶)使转座子、逆转录病毒沉默，并调节基因印迹。在哺乳动物中，DNA高甲基化与某些疾病有关，包括癌症的发生和发展。我们发现，拟南芥DME蛋白通过DNA去甲基化来调节印迹。DME与DNA糖基酶有关，该酶在碱基切除DNA修复途径中切除受损/错配的碱基。二甲基醚去除5-甲基胞嘧啶，用胞嘧啶取代。DME主要在中央细胞表达，中央细胞是支持胚胎发育的胎盘状胚乳的祖细胞。DME去甲基化并激活胚乳中印记的基因的母体等位基因表达。拟南芥有三个类DME(DML)基因：ROS1、DML2和DML3，它们在植物中广泛表达。和DME一样，它们去除了5-甲基胞嘧啶，并有一个DNA糖基酶结构域，两侧是对活性至关重要的额外保守结构域。DME和DML蛋白特异性地使至少180个基因的5‘和3’端去甲基化。ROS1使抗病基因去甲基化，是抵抗细菌病原体丁香假单胞菌所必需的。我们将进行以下实验，以了解DNA去甲基化在基因印迹调控中的机制和功能。1)我们将阐明DME的表达如何局限于中央细胞，识别DME印记的新基因，并分离调控DME表达、靶向和/或DNA去甲基化的基因突变。2)为了了解DNA去甲基化的分子机制，我们将鉴定和分析DME结合蛋白的功能，确定DME是否通过与其靶基因的物理结合直接使DNA去甲基化，并阐明伴随着DNA去甲基化的染色质结构的变化。3)为了了解DNA去甲基化如何调控抗病，我们将阐明由ROS1去甲基化的植物防御基因的功能。植物和动物都有控制DNA甲基化的相关机制。有关DNA去甲基化的基本机制的信息将对DNA甲基化在基因印迹和疾病调节中所起的关键作用提供有价值的见解

DESCRIPTION (provided by applicant): DNA methylation (5-methylcytosine) in mammals and plants silences transposons, retroviruses, and regulates gene imprinting. In mammals, DNA hypermethylation is associated with certain diseases including the onset and progression of cancer. We discovered that the Arabidopsis DEMETER (DME) protein regulates imprinting by DNA demethylation. DME is related to DNA glycosylases that excise damaged/mispaired bases in the base excision DNA repair pathway. DME excises 5-methylcytosine that is replaced with cytosine. DME is expressed primarily in the central cell, the progenitor of the placenta-like endosperm that supports embryo development. DME demethylates and activates maternal allele expression of genes that are imprinted in the endosperm. Arabidopsis has three DME-LIKE (DML) genes: ROS1, DML2, and DML3, which are broadly expressed in the plant. Like DME, they excise 5-methylcytosine and have a DNA glycosylase domain flanked by additional conserved domains that are essential for activity. DME and DML proteins specifically demethylate the 5' and 3' ends of at least 180 genes. ROS1 demethylates disease resistance genes and is required for resistance to a bacterial pathogen, Pseudomonas syringae. We will carry out the following experiments to understand the mechanisms and functions of DNA demethylation in the regulation of gene imprinting. 1) We will elucidate how expression of DME is restricted to the central cell, identify new genes imprinted by DME, and isolate mutations in genes that regulate DME expression, targeting, and/or DNA demethylation. 2) To understand the molecular mechanisms of DNA demethylation, we will identify and analyze the function of DME-binding proteins, determine if DME directly demethylates DNA by physical association with its target genes, and elucidate what changes in chromatin structure accompany DNA demethylation. 3) To understand how DNA demethylation regulates disease resistance, we will elucidate the function of plant defense genes that are demethylated by ROS1. Plants and animals have related mechanisms for controlling DNA methylation. Information about the basic mechanisms of DNA demethylation will provide valuable insights into the critical role DNA methylation plays in the regulation of gene imprinting and disease