Molecular and Architectural Mechanisms of Reprogramming to Pluripotency

描述（由申请人提供）：本计划的长期目标是确定在细胞转化为多能性和维持多能性过程中转录因子、染色质结构和高阶基因组组织之间相互作用的基本机制。尽管转录因子和miRNA具有将细胞转化为多能性的显著能力，但目前不确定的随机参数限制了该过程的效率。此外，不同的多能系具有不同的终末分化能力，并且我们对决定体外分化与体内分化相比如何的参数知之甚少。了解多能性诱导和维持以及那些限制分化的分子机制将允许增强该过程，这反过来将有助于比目前技术更有效地使用小的人类活检样品。为此，该计划的四个项目提出了以下问题：1）在重编程为多能性期间，分化细胞基因组如何在细胞核内重组，重组的哪些方面是重要的，以及是什么控制了多能细胞中的基因组组织？2)异位多能性转录因子如何进入沉默的、染色质化的靶位点以激活内源性多能性网络，以及如何增强该过程？3)在多能细胞中需要适当地建立什么调节回路，以允许它们随后分化为完全成熟的后代？4)多能细胞中存在哪些分化能力的标志，它们是如何建立的？通过在一个单一的程序中寻求这些问题的答案，我们可以获得一个时间分辨的，综合的观点，即核基因组的不同方面协调变化的机制，以适当地将细胞转化为多能性，以及细胞返回体细胞状态的过程。我们还预计，我们的研究所揭示的协调机制将为直接细胞重编程提供见解，而不依赖于多能性。行政和生物信息学核心和一个共享网站将为这些项目提供综合服务，以实现最佳质量、效率和数据共享。行政核心利用现有的高通量测序，微阵列和干细胞核心在各自的机构。该项目和核心领导人在干细胞生物学、分化、转录和染色质/表观遗传学的相关领域具有互补的专业知识，并拥有长期的互动合作和出版记录。该计划提供了独特的实验协同作用，以实现资金公告的目标。

DESCRIPTION (provided by applicant): The long-term objective of this Program is to determine the fundamental mechanisms underlying the interplay between transcription factors, chromatin structure, and higher-order genomic organization during the cellular conversion to and maintenance of pluripotency. Despite the remarkable ability of transcription factors and miRNAs to convert cells to pluripotency, undefined stochastic parameters presently limit the efficiency of the process. In addition, different pluripotent lines have different capacities for terminal differentiation and we poorly understand parameters that determine how well in vitro differentiation compares to in vivo differentiation. Understanding the molecular mechanisms in pluripotency induction and maintenance as well as those limiting differentiation will allow enhancements of the process that, in turn, will facilitate the use of small human biopsy samples much more efficiently than present techniques allow. To this end, the four projects of the Program ask: 1) How is the differentiated cell genome reorganized within the nucleus, during reprogramming to pluripotency, what aspects of reorganization are important, and what controls genome organization in pluripotent cells? 2) How do ectopic pluripotency transcription factors gain access to silent, chromatinized target sites to activate the endogenous pluripotency network, and how can the process be enhanced? 3) What regulatory circuits need to be properly established within pluripotent cells to allow their subsequent differentiation to fully mature progeny? 4) What marks of the competence to differentiate exist in pluripotent cells and how do they get established? By seeking answers to these questions in a single Program, we can obtain a time-resolved, integrated view of the mechanisms by which different aspects of the nuclear genome change coordinately to properly convert a cell to pluripotency and the process by which cells return to the somatic state. We also anticipate that the coordinate mechanisms unveiled by our studies will provide insights into direct cell reprogramming, independent of pluripotency. Administrative and Bioinformatics Cores and a shared Web site will support the projects with integrated services for optimal quality, efficiency, and data-sharing. The Administrative Core leverages existing high throughput sequencing, microarray, and stem cell cores at the respective institutions. The Project and Core leaders have complementary expertise in the relevant areas of stem cell biology, differentiation, transcription and chromatin/ epigenetics and have a long-standing record of interactive collaborations and publications. The plan provides unique experimental synergies that address the objectives of the funding announcement.