Regulation and Function of Active DNA Demethylation in Arabidopsis

描述(申请人提供)：DNA甲基化是哺乳动物发育的重要调节因子。DNA甲基转移酶和甲基胞嘧啶结合蛋白基因的突变会导致遗传病，DNA甲基化中断是癌症的标志。DNA去甲基化缺陷与中枢神经系统的紊乱、肿瘤的发展以及未能建立胚胎多能性有关。此外，有缺陷的DNA去甲基化阻碍了诱导多能干细胞的体外衍生，这是一项有潜力的技术 用于临床细胞、组织、器官的再生。尽管大多数常见的模式生物缺乏DNA甲基化，但开花植物，包括拟南芥，与哺乳动物共享高度保守的甲基化和去甲基化途径。植物和哺乳动物都使用DNA甲基化来调节基因组印记--表观遗传标记和仅来自从父本或母本继承的等位基因的基因表达。我们发现，由拟南芥DME DNA糖基酶催化的DNA去甲基化只发生在伴生细胞、中央细胞和营养细胞中，这些细胞分别毗邻配子、卵子和精子。一个精子使中央细胞受精，产生胚乳，这是一种营养丰富的胚外组织。第二个精子使卵子受精，形成胚胎。我们发现，基因印迹发生在胚乳中，这是由中央细胞中的DNA去甲基化建立的。此外，我们最近的结果表明，中央细胞和营养细胞基因组的DNA去甲基化产生了小RNA，这些小RNA通过RNA指导的DNA甲基化途径移动到相邻的配子并沉默转座元件。在目标1中，我们将测试我们的模型，即DME介导的伴生细胞中的DNA去甲基化可以加强配子中的TE沉默。在目标2中，我们将阐明染色质结构和靶向DNA去甲基化之间的关键关系。在目标3中，我们将识别DNA去甲基化所需的新基因。我们的研究将导致对DNA去甲基化在植物有性繁殖过程中催化的表观遗传重编程的详细理解。

DESCRIPTION (provided by applicant): DNA methylation is a crucial regulator of mammalian development. Mutations in DNA methyltransferases and methylcytosine binding protein genes cause genetic disease, and disrupted DNA methylation is a hallmark of cancer. Defects in DNA demethylation are associated with disorders of the central nervous system, tumor development, and failure to establish embryonic pluripotency. In addition, defective DNA demethylation prevents the in vitro derivation of induced pluripotent stem cells, a technology with the potential to be used in the clinic for the regeneration of cells, tissues and organs. Whereas most common model organisms lack DNA methylation, flowering plants, including Arabidopsis thaliana, share highly conserved methylation and demethylation pathways with mammals. Both plants and mammals use DNA methylation to regulate genomic imprinting - epigenetic marking and resultant gene expression only from an allele inherited from either the male or the female parent. We discovered that DNA demethylation catalyzed by the Arabidopsis DME DNA glycosylase occurs exclusively in companion cells, the central cell and the vegetative cell, which are adjacent to the gametes, the egg and sperm, respectively. One sperm fertilizes the central cell to generate the endosperm, a nutritive extra-embryonic tissue. A second sperm fertilizes the egg to form the embryo. We showed that gene imprinting occurs in the endosperm, which is established by DNA demethylation in the central cell. Moreover, our recent results suggest that DNA demethylation of central cell and vegetative cell genomes generates small RNAs that move to the adjacent gametes and silence transposable elements by the RNA-directed DNA methylation pathway. In Aim 1, we will test our model that DME-mediated DNA demethylation in companion cells functions to reinforce TE silencing in gametes. In Aim 2, we will elucidate the critical relationship between chromatin structure and targeted DNA demethylation. In Aim 3, we will identify new genes required for DNA demethylation. Our studies will lead to a detailed understanding of the epigenetic reprogramming catalyzed by DNA demethylation during plant sexual reproduction.