Maintenance of epigenetic integrity during nuclear reprogramming

描述（由申请人提供）：诱导多能干细胞（iPSCs）可以通过强制表达特定的转录因子从成人体细胞组织中获得。这个过程被称为重编程。由于iPSCs可以分化成任何成年细胞类型，并且与它们的来源个体完全匹配，重编程技术从根本上改变了疾病建模的能力，并带来了个性化细胞治疗的新概念。然而，在重编程过程中，iPSCs可以获得有害的表观遗传异常。这发生在人们仍然不太了解的礼仪中。在一项重要的原理证明中，我们之前已经表明，印迹的Dlk1-Dio3簇的DNA超甲基化是一种常见的iPSC正常现象，可以通过在含有抗坏血酸的介质中进行重编程来有效地预防。抗坏血酸是几种经常用于提高重编程效率的化合物之一。本研究项目的目标是建立对复发性基因特异性和全基因组表观遗传iPSC异常的机制理解，以及重编程增强化学物质如何预防或触发它们的发生。利用独特的转基因小鼠模型，我们将追求以下三个目标。1)我们将确定导致Dlk1-Dio3异常高甲基化的基因，以及参与介导抗坏血酸对该基因簇的保护作用的基因。2)我们将进行全基因组研究，以确定几种常用的重编程增强化学物质在iPSCs形成过程中如何促进染色质重塑，以及这与该过程中表观遗传完整性的维持如何相关。3)我们将系统地识别在重编程过程中暴露于特定常用化合物而持久影响的iPSCs的分子和功能特性。总之，这些实验将提供更好的理解为什么表观遗传异常被引入多能干细胞，以及它们的发生与成功重编程至关重要的表观遗传重塑之间的关系。此外，我们的工作将揭示化学重编程可能的益处和风险，从而帮助衍生高质量的人类iPSCs，可能仅通过使用化合物。

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) can be derived from adult somatic tissues by the enforced expression of defined transcription factors. This process is referred to as reprogramming. As iPSCs can differentiate into any adult cell type and are fully matched to the individual they were derived from, reprogramming technology has radically altered the ability to model disease and led to new concepts for personalized cellular therapies. However, iPSCs can acquire detrimental epigenetic abnormalities during the reprogramming process. This occurs in manners that remain poorly understood. In an important proof-of-principle we have previously shown that DNA hypermethylation of the imprinted Dlk1-Dio3 cluster is a frequent iPSC normality that can be efficiently prevented by reprogramming in media containing ascorbic acid, one of several chemical compounds frequently used to increase reprogramming efficiencies. The goal of this research project is to develop a mechanistic understanding of recurrent gene-specific and genome-wide epigenetic iPSC abnormalities and for how reprogramming enhancing chemicals can prevent or trigger their occurrence. Using unique transgenic mouse models, we will pursue the following three aims. 1) We will identify the genes responsible for aberrant hypermethylation of Dlk1-Dio3 as well as those involved in mediating the protective effect of ascorbic acid on this gene cluster. 2) We will conduct genome-wide studies to determine how several frequently used reprogramming enhancing chemicals facilitate chromatin remodeling during iPSCs formation and how this relates to the maintenance of epigenetic integrity during this process. 3) We will systematically identify molecular and functional properties of iPSCs that are affected in a lasting manner by exposure to specific frequently used chemical compounds during the reprogramming process. Together, these experiments will provide a better understanding of why epigenetic abnormalities are introduced into iPSCs and how their occurrence relates to epigenetic remodeling crucial for successful reprogramming. In addition, our work will reveal possible benefits and risks of chemical reprogramming and thereby aid the derivation of high-quality human iPSCs, possibly by the use of chemical compounds alone.