Maintenance of epigenetic integrity during nuclear reprogramming

描述(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体组织中获得。这个过程被称为重新编程。由于IPSCs可以分化为任何成体细胞类型，并与其来源的个体完全匹配，重新编程技术从根本上改变了对疾病建模的能力，并导致了个性化细胞治疗的新概念。然而，在重新编程的过程中，ipscs可能会获得有害的表观遗传学异常。这种情况的发生方式仍然鲜为人知。在一个重要的原则证明中，我们先前已经证明，印记的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新编程有效地防止，抗坏血酸是几种经常用于提高重新编程效率的化合物之一。这项研究项目的目标是从机制上理解反复发生的基因特异性和全基因组表观遗传学IPSC异常，以及重新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异常甲基化的基因，以及参与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剂如何促进IPSCs形成过程中的染色质重塑，以及这与这一过程中维持表观遗传完整性的关系。3)我们将系统地确定IPSCs的分子和功能特性在重新编程过程中因暴露于特定的常用化合物而受到持久影响。总之，这些实验将提供更好的理解，了解为什么表观遗传异常被引入IPSCs，以及它们的发生与表观遗传重塑之间的关系，这对成功的重新编程至关重要。此外，我们的工作将揭示化学重新编程的可能好处和风险，从而帮助获得高质量的人类IPSCs，可能只使用化合物。

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.