Epigenetic control of the pluripotent state by chromatin-associated factor Dppa2

项目描述 尽管在破译胚胎干细胞（ESC）自我更新方面取得了显着进展，但分子生物学研究表明， 赋予胚胎干细胞形成广泛分化衍生物能力的电路仍然很差， 明白这种知识差距的持续存在限制了我们有效和安全地操纵... 晚期多能细胞用于医学和研究目的。我们的长期目标是更好地了解epige- 负责维持多能状态的遗传机制。通过利用基于shRNA的功能性 在我们前期研究的基础上，利用基因组学方法，我们鉴定了一种新的染色质相关因子Dppa 2 对于维持胚胎干细胞的发育潜能至关重要。此外，我们还证明， Dppa 2的表达促进细胞重编程期间多能状态的获得。客观 在本申请中的目的是确定Dppa 2如何建立和维持多能干细胞的表观遗传景观。 状态根据我们自己的初步数据和其他人的工作，中心假设是， dppa 2相关蛋白复合物在平衡发育的启动子处保持独特的表观遗传特征， opmental基因，确保这些基因在诱导分化和再分化过程中的适当激活。 编程.这项研究的基本原理是，深入了解Dppa 2的功能， 有潜力转化为新的策略，以加强干细胞的维护和衍生的pluripo- 帐篷细胞。这一假设将通过追求三个具体目标进行测试：1）确定表观遗传足迹， Dppa 2结合的ESC和EpiSC中的平衡启动子。确定这些足迹在Dppa 2中是如何改变的- 2）确定Dppa 2复合物在ESC中的组成和功能;和3）确定Dppa 2复合物在ESC中的组成和功能。 Dppa 2复合物在细胞重编程过程中的功能。在第一个目标下，Dppa 2结合，组蛋白 修饰，DNA甲基化和基因表达的模式将在野生型和Dppa 2- 耗尽的ESC和EpiSC，之后Dppa 2复合物调节染色质的机制模型将 发展。在第二个目标下，Dppa 2相关蛋白复合物将被纯化，蛋白质身份将被确定。 通过质谱法终止，并通过shRNAs灭活单个基因，以确定 这些相互作用的合作伙伴在染色质调控Dppa 2结合基因组位点。目标机制 在这一目标下，还将对承认进行调查。在第三个目标下，iPSC将从哺乳动物中产生， 分析具有或不具有Dppa 2的胚细胞及其在体外和体内形成胚层衍生物的能力。 将进行分子分析，以确定Dppa 2复合物在生殖过程中的功能。 语法过程这项研究具有重要意义，因为它有望纵向推进， pand了解发育潜能及其在染色质水平上的调节。这样的知识 增强干细胞维持和分化的潜力-这是新的和创新的应用的关键步骤- 致力于多种疾病的治疗。

PROJECT DESCRIPTION Despite the remarkable progress made in deciphering embryonic stem cell (ESC) self-renewal, the molecular circuitry that endows ESCs with the ability to form a broad range of differentiated derivatives remains poorly understood. The continued existence of this gap in knowledge limits our ability to efficiently and safely manipu- late pluripotent cells for theurapeutic and research purposes. Our long-term goal is to better understand epige- netic mechanisms responsible for the maintenance of the pluripotent state. By utilizing shRNA-based functional genomics approach developed in our previous studies, we identified novel chromatin-associated factor Dppa2 as critical for the maintenance of developmental potency in ESCs. Furthermore, we demonstrated that forced expression of Dppa2 facilitates acquisition of the pluripotent state during cellular reprogramming. The objective in this application is to determine how Dppa2 sets up and maintains the epigenetic landscape of the pluripotent state. The central hypothesis, formulated on the basis of our own preliminary data and work by others, is that Dppa2-associated protein complex maintains unique epigenetic signatures at the promoters of poised devel- opmental genes that ensure proper activation of these genes upon induction of differentiation and during re- programming. The rationale for the proposed research is that in-depth understanding of Dppa2 function has the potential to translate into novel strategies to enhance stem cell maintenance and derivation of the pluripo- tent cells. This hypothesis will be tested by pursuing three specific aims: 1) Identify epigenetic footprints at Dppa2-bound poised promoters in ESCs and EpiSCs. Determine how these footprints are altered in Dppa2- depleted cells; 2) Determine the composition and function of the Dppa2 complex in ESCs; and 3) Determine the function of the Dppa2 complex during cellular reprogramming. Under the first aim, Dppa2 binding, histone modifications, patterns of DNA methylation and gene expression will be compared in wild-type and Dppa2- depleted ESCs and EpiSCs after which mechanistic models of chromatin regulation by the Dppa2 complex will be developed. Under the second aim, Dppa2-associated protein complex will be purified, protein identities de- termined by mass-spectrometry, and individual genes inactivated by shRNAs in order to determine the roles of these interacting partners in chromatin regulation at the Dppa2-bound genomic loci. Mechanism(s) of target recognition will also be investigated under this aim. Under the third aim, iPSCs will be generated from fibro- blasts with or without Dppa2 and their ability to form germ layer derivatives in vitro and in vivo will be analyzed. Molecular analyses will be performed in order to define the function of the Dppa2 complex during the repro- gramming process. The proposed research is significant, because it is expected to vertically advance and ex- pand understanding of developmental potency and its regulation at the chromatin level. Such knowledge has the potential to enhance stem cell maintenance and differentiation - critical steps for new and innovative ap- proaches to treatment of a variety of diseases.