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

描述（由申请人提供）：尽管在破译胚胎干细胞（ESC）自我更新方面取得了显着进展，但赋予ESC形成广泛分化衍生物能力的分子电路仍然知之甚少。这种知识差距的持续存在限制了我们有效和安全地操作多能细胞用于治疗和研究目的的能力。我们的长期目标是更好地理解维持多能状态的表观遗传机制。通过利用我们之前研究中开发的基于shrna的功能基因组学方法，我们发现了新的染色质相关因子Dppa2对维持ESCs的发育效力至关重要。此外，我们证明了Dppa2的强制表达有助于在细胞重编程过程中获得多能状态。本应用的目的是确定Dppa2如何建立和维持多能状态的表观遗传景观。基于我们自己的初步数据和其他人的工作，中心假设是dppa2相关蛋白复合物在平衡发育基因的启动子处保持独特的表观遗传特征，确保这些基因在诱导分化和重编程过程中适当激活。这项研究的基本原理是，深入了解Dppa2的功能有可能转化为增强干细胞维持和多能细胞衍生的新策略。这一假设将通过追求三个具体目标来验证：1)确定ESCs和epscs中dppa2结合的平衡启动子的表观遗传足迹。确定这些足迹在Dppa2-缺失细胞中是如何改变的；2)确定ESCs中Dppa2复合物的组成和功能；3)确定Dppa2复合体在细胞重编程过程中的功能。在第一个目标下，Dppa2结合、组蛋白修饰、DNA甲基化模式和基因表达将在野生型和Dppa2缺失的ESCs和EpiSCs中进行比较，之后将开发Dppa2复合物调控染色质的机制模型。在第二个目标下，将纯化dppa2相关蛋白复合物，通过质谱法确定蛋白质身份，并通过shrna灭活单个基因，以确定这些相互作用的伙伴在dppa2结合的基因组位点的染色质调控中的作用。目标识别的机制也将在此目的下进行研究。在第三个目标下，将从含有或不含Dppa2的成纤维细胞中生成iPSCs，并分析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Dppa2复合体在重编程过程中的功能。这项研究具有重要意义，因为它有望在染色质水平上垂直推进和扩展对发育潜能及其调控的理解。这些知识有可能增强干细胞的维持和分化——这是治疗各种疾病的新方法和创新方法的关键步骤。

DESCRIPTION (provided by applicant): 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 manipulat pluripotent cells for therapeutic and research purposes. Our long-term goal is to better understand epigenetic 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 developmental 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 pluripotent cells. This hypothesis will be tested by pursuing three specific aims: 1) Identiy 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 determined 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 reprogramming process. The proposed research is significant, because it is expected to vertically advance and expand 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 approaches to treatment of a variety of diseases.