DME-INTERACTING PROTEIN 1 REGULATES DEMETHYLATOIN AND REPRODUCTION IN ARABIDOPSIS

描述（由申请人提供）：胞嘧啶甲基化是哺乳动物和植物中沉默转座子和逆转录病毒、控制基因组印记和调节生殖的主要机制之一。DNA甲基化的改变与人类的白血病、癌症和先天性综合征等疾病有关。拟南芥是研究表观遗传机制的一个很好的系统，这些表观遗传机制在哺乳动物和植物之间是进化上保守的。在拟南芥中，DEMETER（DME）属于DNA糖基化酶家族，其在碱基切除DNA修复中切除受损或错配的碱基。DME去甲基化并激活印记基因的母系等位基因。然而，一个关键的问题没有得到回答：DME或DML DNA糖基化酶如何找到它们在基因组中去甲基化的靶点？最近，我们在酵母双杂交筛选中分离了DME相互作用蛋白1（DIP 1）。DIP 1是一种具有RING结构域的泛素结合酶7相互作用蛋白4。有趣的是，我们发现DIP 1/dip 1杂合植物的自花授粉后代中有50%的种子败育。当DIP 1/dip 1雌性植物与野生型雄性植物杂交时，遗传母本dip 1突变等位基因的种子败育。为了更好地理解DIP 1如何调节DNA去甲基化和印迹，将执行以下具体目标：1）确定DIP 1是否结合印迹基因的启动子并靶向DME以使DNA去甲基化。使用凝胶迁移试验，我们将检查DIP 1是否与印迹MEDEA（MEA）（Polycomb基因）的启动子结合。我们将研究DIP 1是否调节MEA表达、印迹和甲基化。2)分析DIP 1的分子功能。我们将研究RING结构域蛋白DIP 1是否具有E3泛素连接酶活性，如果是，DME和DMLs是否是DIP 1介导的泛素蛋白水解途径的底物。3)阐明DIP 1调控DNA去甲基化和基因表达的机制。我们将探讨DIP 1是否通过另一种DME相互作用蛋白组蛋白H1的泛素化来调节MEA印迹，这一事件可能导致MEA启动子中特异性的DNA甲基化和/或组蛋白H3甲基化。使用遗传上易于处理的拟南芥系统进行的拟议实验将阐明通过RING结构域蛋白将泛素化与DNA去甲基化、基因印迹和基因表达联系起来的机制-这些过程在进化上是保守的，并且是人类疾病（如癌症）病理学的组成部分。特异性靶向DNA去甲基化的机制将为未来靶向人类疾病中特定DNA超甲基化位点的基因治疗的发展提供有价值的见解。这笔赠款将用于培训本科生和研究生在表观遗传学和分子生物学在圣刘易斯大学的科学发现。PI将积极参与外展工作，以促进圣路易斯K-12年级的科学培训和教育计划。公共卫生相关性使用表观遗传学上易处理的拟南芥系统执行的拟议的具体目标将阐明通过RING结构域蛋白将泛素化与DNA去甲基化、基因组印记和基因表达联系起来的机制-这些过程在进化上是保守的，并且是人类疾病（如癌症、白血病和先天性综合征）病理学的组成部分。特异性靶向DNA去甲基化的机制将为未来靶向人类疾病中特定DNA超甲基化位点的基因治疗的发展提供重要的见解。

DESCRIPTION (provided by applicant): Cytosine methylation is one of the major mechanisms to silence transposons and retroviruses, control genomic imprinting, and regulate reproduction in both mammals and plants. Altered DNA methylation is associated with diseases including leukemia, cancer and congenital syndromes in humans. Arabidopsis thaliana is an excellent system to investigate epigenetic mechanisms, which are evolutionarily conserved between mammals and plants. In Arabidopsis, DEMETER (DME) belongs to a family of DNA glycosylases that excise damaged or mispaired bases in the base excision DNA repair. DME demethylates and activates the maternal allele of imprinted genes. However, one key question is not answered: how do DME or DML DNA glycosylases find their targets to demethylate in the genome? Recently, we isolated DME-INTERACTING PROTEIN 1 (DIP1) in the yeast two-hybrid screen. DIP1 is a putative ubiquitin-conjugating enzyme 7-interacting protein 4 that has a RING domain. Interestingly, we found 50% seed abortion in the self-pollinated progeny of the DIP1/dip1 heterozygous plant. The seed inheriting a maternal dip1 mutant allele is aborted when the DIP1/dip1 female plant was crossed with the wild-type male. To better understand how DIP1 regulates DNA demethylation and imprinting, the following specific aims will be executed: 1) Determine if DIP1 binds to the promoter of imprinted genes and targets DME to demethylate DNA. Using the gel shift assay, we will examine if DIP1 binds to the promoter of imprinted MEDEA (MEA), a Polycomb gene. We will investigate if DIP1 regulates MEA expression, imprinting, and methylation. 2) Analyze molecular functions of DIP1. We will examine if the RING domain protein DIP1 has an E3 ubiquitin ligase activity, and if so, whether DME and DMLs are substrates for the DIP1-mediated ubiquitin proteolytic pathway. 3) Elucidate the mechanism used by DIP1 in regulating DNA demethylation and gene expression. We will explore if DIP1 regulates MEA imprinting through ubiquitination of another DME-interacting protein, histone H1, an event that might lead to DNA methylation and/or histone H3 methylation specifically in the MEA promoter. The proposed experiments carried out using the genetically tractable Arabidopsis system will elucidate mechanisms that link ubiquitination via a RING domain protein to DNA demethylation, gene imprinting, and gene expression - processes that are evolutionarily conserved and integral to the pathology of human diseases such as cancer. Mechanisms amenable to specific targeting of DNA demethylation will provide valuable insights for the development of future gene therapies for targeting specific DNA hypermethylation loci in human diseases. This grant will be used to train undergraduate and graduate students for scientific discovery in epigenetics and molecular biology at Saint Louis University. PI will participate actively in outreach efforts to foster scientific training and education programs in grades K-12 in St. Louis. PUBLIC HEALTH RELEVANCE The proposed specific aims executed using the epigenetically tractable Arabidopsis thaliana system will elucidate mechanisms that link ubiquitination via a RING domain protein to DNA demethylation, genomic imprinting, and gene expression - processes that are evolutionarily conserved and integral to the pathology of human diseases such as cancer, leukemia, and congenital syndromes. Mechanisms amenable to specific targeting of DNA demethylation will provide significant insights for the development of future gene therapies for targeting specific DNA hypermethylation loci in human diseases.