DME-INTERACTING PROTEIN 1 REGULATES DEMETHYLATOIN AND REPRODUCTION IN ARABIDOPSIS

描述（由申请人提供）：在哺乳动物和植物中，胞嘧啶甲基化是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甲基化改变与人类白血病、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，在哺乳动物和植物之间具有进化保守性。在拟南芥中，DEMETER （DME）属于一个DNA糖基酶家族，在碱基切除DNA修复中切除受损或错配的碱基。二甲醚去甲基化并激活印迹基因的母体等位基因。然而，有一个关键问题没有得到回答：DME或DML DNA糖基酶是如何在基因组中找到去甲基化的目标的？最近，我们在酵母双杂交筛选中分离到了dme相互作用蛋白1 （DIP1）。DIP1是一种假定的泛素偶联酶7相互作用蛋白4，具有RING结构域。有趣的是，我们在DIP1/ DIP1杂合植物的自花后代中发现50%的种子败育。当dip1 /dip1雌性植株与野生型雄性植株杂交时，遗传母体dip1突变等位基因的种子流产。为了更好地了解DIP1如何调节DNA去甲基化和印迹，将执行以下具体目标：1)确定DIP1是否与印迹基因的启动子结合并靶向DME使DNA去甲基化。使用凝胶移位法，我们将检查DIP1是否结合到印迹MEDEA （MEA）的启动子上，这是一种多梳基因。我们将研究DIP1是否调控MEA的表达、印迹和甲基化。2)分析DIP1的分子功能。我们将研究环结构域蛋白DIP1是否具有E3泛素连接酶活性，如果有，DME和dml是否是DIP1介导的泛素蛋白水解途径的底物。3)阐明DIP1调控DNA去甲基化和基因表达的机制。我们将探索DIP1是否通过另一种与dme相互作用的蛋白组蛋白H1的泛素化来调节MEA印迹，这一事件可能导致MEA启动子中的DNA甲基化和/或组蛋白H3甲基化。利用遗传易感拟南芥系统进行的拟议实验将阐明通过环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，并且与癌症等人类疾病的病理是不可缺少的。特异性靶向DNA去甲基化的机制将为未来开发针对人类疾病中特异性DNA超甲基化位点的基因疗法提供有价值的见解。该基金将用于培养圣路易斯大学本科生和研究生在表观遗传学和分子生物学方面的科学发现。PI将积极参与推广工作，促进圣路易斯K-12年级的科学培训和教育项目。利用表观遗传易感拟南芥系统执行的拟议特定目标将阐明通过环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，是癌症、白血病和先天性综合征等人类疾病病理的组成部分。特异性靶向DNA去甲基化的机制将为未来开发针对人类疾病中特异性DNA超甲基化位点的基因疗法提供重要见解。

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.