Regulation and Function of DNA Demethylation in Arabidopsis

描述（由申请人提供）：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超甲基化与某些疾病相关，包括癌症的发作和进展。我们发现拟南芥DEMETER（DME）蛋白通过DNA去甲基化调节印迹。DME与在碱基切除DNA修复途径中切除受损/错配碱基的DNA糖基化酶有关。DME切除被胞嘧啶取代的5-甲基胞嘧啶。DME主要在中央细胞中表达，中央细胞是支持胚胎发育的胎盘样胚乳的祖细胞。DME去甲基化并激活胚乳中印记基因的母体等位基因表达。拟南芥有三个DME-LIKE（DML）基因：ROS1、DML2和DML3，它们在植物中广泛表达。像DME一样，它们切除5-甲基胞嘧啶，并具有DNA糖基化酶结构域，侧翼是活性所必需的额外保守结构域。DME和DML蛋白特异性地使至少180个基因的5 '和3'末端去甲基化。ROS1使抗病基因去甲基化，并且是抵抗细菌病原体假单胞菌所必需的。我们将进行以下实验来了解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的表达是如何被限制在中央细胞，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关联直接使DNA去甲基化，并阐明伴随DNA去甲基化的染色质结构变化。3)为了了解DNA去甲基化如何调节抗病性，我们将阐明被ROS 1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为DNA甲基化在基因印迹和疾病调控中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