Mechanisms and Epigenetic Effectors of Cellular Reprogramming Factor Activity

描述（由申请人提供）：本研究提案的目标是利用合成生成的染色质模板来促进Oct 4、Sox 2、Klf 4和c-Myc（OSKM）多能性调节因子的生物化学和生物物理表征。当在体细胞中异位表达时，OSKM协同作用以将细胞命运重编程为多能性，从而产生可分化为三个胚层的细胞类型的自我更新诱导多能干细胞（iPSC）。iPSC是再生医学和定制细胞疗法领域的宝贵工具，因为它们在功能上与胚胎干细胞（ESC）无法区分，并且规避了所有与ESC相关的伦理问题。此外，从具有复杂遗传综合征的患者产生的iPSC可以分化成患病的细胞类型，以提供对给定疾病的发病机理的无与伦比的洞察力，并提供与治疗筛选相容的模型。目前，在典型的重编程实验中，只有不到1%的起始细胞群将达到多能性，该过程需要进行实质性优化，以充分实现iPSC的临床应用。 OSKM与基因组结合，在基因组中它们通过将多种转录调节因子募集到染色质来影响表观遗传修饰并控制基因表达。毫不奇怪，iPSC和ESC中的OSKM定位模式高度相似，并且在未能实现多能性的细胞中观察到错误定位。有趣的是，某些表观遗传标记能够通过破坏OSKM活性来作为重编程过程的障碍。尽管全基因组研究继续强调表观遗传标记在OSKM定位和功能中的重要性，但缺乏描述组蛋白修饰和OSKM之间串扰的机制信息。我建议概括重编程因子核小体相互作用在体外结合肽合成技术，蛋白质工程，位点特异性蛋白质修饰，和荧光光谱。这些研究将阐明组蛋白标记对OSKM结合和功能的影响，在单核细胞和核小体阵列的背景下。此外，我将设计多价Oct 4，Sox 2和Klf 4蛋白质，这些蛋白质靶向表观遗传障碍，将用于产生iPSC。本研究的具体目的是：1）表征OSKM结合对体外核小体稳定性和染色质结构的影响。确定关键表观遗传修饰在OSKM结合和功能中的作用，以及3.）设计嵌合因子，克服重编程的表观遗传障碍。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，这对于开发可以克服表观遗传障碍并在更一致的基础上产生高质量iPSC的重编程策略至关重要。

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.