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

描述(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著进展，但赋予ESC形成广泛分化衍生品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纵用于治疗和研究目的的多能细胞的能力。我们的长期目标是更好地理解负责维持多能性状态的表观遗传机制。通过利用我们先前研究中开发的基于shRNA的功能基因组学方法，我们确定了新的染色质相关因子Dppa2对维持ESCs的发育潜力至关重要。此外，我们还证明了强制表达Dppa2有助于在细胞重编程过程中获得多能性状态。本申请的目的是确定Dppa2如何建立和维持多能状态的表观遗传格局。根据我们自己的初步数据和其他人的工作提出的中心假设是，Dppa2相关蛋白复合体在稳定的发育基因的启动子上保持独特的表观遗传学特征，确保这些基因在诱导分化和重新编程期间正确激活。这项研究的基本原理是，对Dppa2功能的深入了解有可能转化为新的策略，以加强干细胞的维持和多能性细胞的衍生。这一假说将通过追求三个具体目标来检验：1)在ESCs和EpiSCs中识别Dppa2结合的稳定启动子的表观遗传足迹。确定这些足迹是如何在Dppa2缺失的细胞中改变的；2)确定ESCs中Dppa2复合体的组成和功能；以及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，将比较野生型和Dppa2缺失的ESCs和EpiSCs中Dppa2的结合、组蛋白修饰、DNA甲基化模式和基因表达，然后将建立Dppa2复合体调节染色质的机制模型。在第二个目标下，将提纯Dppa2相关蛋白复合体，通过质谱学确定蛋白质的同源性，并通过shRNAs失活单个基因，以确定这些相互作用的伙伴在Dppa2结合的基因组基因座染色质调节中的作用。目标识别机制(S)也将在这一目标下进行研究。在第三个目标下，将从含有或不含有Dppa2的成纤维细胞中产生IPSCs，并将分析它们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复合体在重新编程过程中的功能。这项拟议的研究意义重大，因为它有望纵向推进和扩大对发育潜力及其在染色质水平上的调节的理解。这种知识有可能加强干细胞的维持和分化--这是治疗各种疾病的新的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.