Mechanisms and Epigenetic Effectors of Cellular Reprogramming Factor Activity

描述(申请人提供)：本研究提案的目标是利用合成的染色质模板来促进Oct4、Sox2、Klf4和c-Myc(OSKM)多能调节子的生化和生物物理特性。当OSKM异位表达在体细胞中时，OSKM协同作用将细胞命运重新编程为多能性，导致自我更新诱导的多潜能干细胞(IPSCs)可以分化为三种生殖层的细胞类型。在再生医学和定制细胞治疗领域，IPSCs是一个无价的工具，因为它们在功能上与胚胎干细胞(ESCs)没有区别，并绕过了所有与ESCs相关的伦理问题。此外，从患有复杂遗传综合征的患者中产生的IPSCs可以分化为受苦细胞类型，以提供对特定疾病的发病机制的无与伦比的洞察，并提供一种与治疗性筛查兼容的模型。目前，在典型的重编程实验中，只有不到1%的起始细胞群体会达到多能性，这一过程需要进行实质性的优化，才能充分实现IPSCs的临床应用。 OSKM与基因组结合，在那里它们通过招募各种转录调节因子到染色质来影响表观遗传修饰和控制基因表达。毫不奇怪，OSKM在IPSCs和ESCs中的定位模式非常相似，并且在未能实现多能性的细胞中观察到错误的定位。有趣的是，某些表观遗传标记能够通过扰乱OSKM活动而成为重新编程过程的障碍。尽管全基因组研究继续强调表观遗传信号在OSKM定位和功能中的重要性，但缺乏描述组蛋白修饰和OSKM之间的串扰的机械性信息。我建议通过结合多肽合成、蛋白质工程、定点蛋白质修饰和荧光光谱学等技术，在体外重述重编程因子与核小体的相互作用。这些研究将在单核小体和核小体阵列的背景下阐明组蛋白标记对OSKM结合和功能的影响。此外，我还将设计针对表观遗传障碍的多价Oct4、Sox2和Klf4蛋白质，这些蛋白质将被用来产生ipscs。本研究的具体目的是：1)体外研究OSKM结合对核小体稳定性和染色质结构的影响。确定关键的表观遗传修饰在OSKM结合和功能中的作用，以及3.设计能够克服重编程表观遗传障碍的嵌合因子。这项拟议的研究旨在描述OSKM结合和功能背后的有价值的机制信息，这对于开发能够克服表观遗传障碍并在更一致的基础上产生高质量IPSCs的重新编程策略至关重要。

DESCRIPTION (provided by applicant): The goal of this research proposal is to utilize synthetically generated chromatin templates to facilitate the biochemical and biophysical characterization of the Oct4, Sox2, Klf4, and c-Myc (OSKM) pluripotency regulators. When ectopically expressed in somatic cells, OSKM act synergistically to reprogram cell fate to pluripotency, resulting in self-renewing induced pluripotent stem cells (iPSCs) that can differentiate into cell types of the three germ layers. iPSCs are an invaluable tool in the fields f regenerative medicine and custom cell therapies because they are functionally indistinguishable from embryonic stem cells (ESCs) and circumvent all ESC-related ethical concerns. Furthermore, iPSCs that are generated from patients with complex genetic syndromes can be differentiated into the afflicted cell type to afford unparalleled insight into the pathogenesis ofa given disease and to provide a model that is compatible with therapeutic screening. Currently, less than 1% of the starting cell population will reach pluripotency in a typical reprogramming experiment, and the process needs to be substantially optimized to fully realize the clinical applications of iPSCs. OSKM bind to the genome, where they influence epigenetic modifications and control gene expression by recruiting a variety of transcriptional regulators to chromatin. Not surprisingly, OSKM localization patterns in iPSCs and ESCs are highly similar and mislocalization is observed in cells that fail to achieve pluripotency. Interestingly, certain epigenetic marks are able to act as barriers to the reprogramming process by disrupting OSKM activity. Despite the fact that genome-wide studies continue to emphasize the importance of epigenetic signatures in OSKM localization and function, there is a lack of mechanistic information describing the crosstalk between histone modifications and OSKM. I propose to recapitulate reprogramming factor-nucleosome interactions in vitro by combining techniques in peptide synthesis, protein engineering, site-specific protein modification, and fluorescence spectroscopy. These studies will elucidate the effects that histone marks have on OSKM binding and function in the context of mononucleosomes and nucleosome arrays. Additionally, I will engineer multivalent Oct4, Sox2 and Klf4 proteins that target epigenetic barriers, which will be used to generate iPSCs. The specific aims of this research are: 1) characterize the effect of OSKM binding on nucleosome stability and chromatin structure in vitro, 2.) determine the role of key epigenetic modifications in OSKM binding and function, and 3.) design chimeric factors that can overcome epigenetic barriers to reprogramming. This proposed research is designed to delineate valuable mechanistic information underlying OSKM binding and function, which is crucial for the development of reprogramming strategies that can overcome epigenetic barriers and generate high quality iPSCs on a more consistent basis.