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

染色质相关因子 Dppa2 对多能状态的表观遗传控制

• 批准号: 8631207
• 负责人: Natalia B Ivanova
• 金额: $ 32.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8631207
• 关键词: Affect; Alzheimer' s Disease; Binding; Blast Cell; Blood; Cell Culture System; Cell Differentiation process; Cell Lineage; Cell Maintenance; Cells; ChIP-seq; Chromatin; Chromatin Modeling; Complex; DNA Methylation; Data; Derivation procedure; Development; Developmental Gene; Disease; Ensure; Epigenetic Process; Fibroblasts; Gene Activation; Gene Expression; Gene Expression Profile; Genes; Genomics; Germ Layers; Goals; Hematopoietic; Histones; Homologous Gene; In Vitro; Individual; Knowledge; Link; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Memory; Methodology; Mission; Modeling; Modification; Molecular; Mus; Neurodegenerative Disorders; Parkinson Disease; Patients; Pattern; Population; Process; Proteins; Proteomics; Psyche structure; Public Health; Regenerative Medicine; Regulation; Regulatory Element; Reporting; Research; Role; Shotguns; Somatic Cell; Source; Spinal cord injury; Staging; Stem cells; Testing; Therapeutic Human Experimentation; Translating; Work; base; burden of illness; chromatin modification; disability; embryonic stem cell; functional genomics; histone modification; human disease; improved; in vivo; induced pluripotent stem cell; innovation; novel; novel strategies; overexpression; pluripotency; programs; promoter; protein complex; public health relevance; regenerative therapy; self-renewal; small hairpin RNA; transcriptome sequencing

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.

项目说明 尽管在破译胚胎干细胞(ESC)自我更新方面取得了显著进展，但分子 赋予ESCs形成广泛差异化衍生品能力的电路仍然很差 明白了。这种知识鸿沟的持续存在限制了我们有效和安全地操纵- 晚期多能细胞用于治疗和研究目的。我们的长期目标是更好地理解警戒- 负责维持多能性状态的磁性机制。通过利用基于shRNA的功能 基因组学方法在我们以前的研究中，我们发现了新的染色质相关因子Dppa2 对维持胚胎干细胞的发育潜能至关重要。此外，我们还演示了强制 在细胞重编程过程中，Dppa2的表达促进了多能性状态的获得。目标是 在这个应用中是为了确定Dppa2是如何建立和维持多能性的表观遗传格局的 州政府。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发育的启动子上保持独特的表观遗传学特征。 确保这些基因在分化诱导和再分化过程中被适当激活的基因。 编程。拟议研究的基本原理是，对Dppa2功能的深入了解具有 转化为新的策略以加强干细胞的维持和多核细胞来源的潜力 帐篷牢房。这一假设将通过追求三个具体目标来检验：1)在以下位置识别表观遗传足迹 ESC和EpiSCs中Dppa2结合的稳定启动子。确定这些足迹在Dppa2中如何更改- 耗尽的细胞；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，Dppa2结合，组蛋白 将比较野生型和Dppa2基因的修饰、DNA甲基化模式和基因表达。 耗尽的ESCs和EpiSCs之后，由Dppa2复合体调节染色质的机制模型将 被开发出来。在第二个目标下，Dppa2相关蛋白复合体将得到纯化，蛋白质同源性降低。 通过质谱学终止，以及单个基因被shRNAs失活，以确定 在Dppa2结合的基因组座位上，这些相互作用的伙伴参与染色质的调节。目标机制(S) 承认也将在这一目标下进行调查。在第三个目标下，IPSCs将由纤维- 有或没有Dppa2的原始细胞以及它们在体外和体内形成生殖层衍生物的能力将被分析。 将进行分子分析，以确定Dppa2复合体在复制过程中的功能。 语法过程。这项拟议的研究意义重大，因为预计它将在垂直方向上推进，并在未来 对发育潜能及其在染色质水平的调节的理解。这样的知识已经 增强干细胞维护和分化的潜力--这是新的和创新的AP的关键步骤 治疗各种疾病的方法。