Mechanisms and Epigenetic Effectors of Cellular Reprogramming Factor Activity

细胞重编程因子活性的机制和表观遗传效应器

• 批准号: 8714612
• 负责人: Glen Liszczak
• 金额: $ 4.99万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714612
• 关键词: Binding; Biochemical; Biological Assay; Biological Models; Cell Therapy; Cells; Chimera organism; Chromatin; Chromatin Structure; Clinical; Complex; Custom; DNA Binding; DNA-Binding Proteins; Development; Disease; Dissociation; Due Process; EP300 gene; Engineering; Enzymes; Epigenetic Process; Ethics; Exhibits; Fibrinogen; Fibroblasts; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; GKLF protein; Gene Expression; Generations; Genetic; Genetic Transcription; Genome; Germ Layers; Goals; Histones; Human; In Vitro; Lead; Length; Ligation; Modeling; Modification; Monitor; Nucleosomes; Pathogenesis; Patients; Pattern; Peptide Synthesis; Population; Post-Translational Protein Processing; Process; Property; Protein Chemistry; Protein Engineering; Proteins; Protocols documentation; Reader; Recruitment Activity; Regenerative Medicine; Relative (related person); Research; Research Proposals; Role; Site; Somatic Cell; Syndrome; Techniques; Therapeutic; Variant; base; biophysical properties; c-myc Genes; cell type; chromatin modification; clinical application; design; embryonic stem cell; engineering design; fluorophore; gain of function; genome wide association study; heterochromatin-specific nonhistone chromosomal protein HP-1; histone modification; in vitro Assay; in vivo; induced pluripotent stem cell; insight; particle; pluripotency; public health relevance; research study; screening; self-renewal; stem cell technology; tool

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.

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 Oct4、Sox2、Klf4 和 c-Myc (OSKM) 多能性调节因子的生化和生物物理表征。当在体细胞中异位表达时，OSKM 协同作用，将细胞命运重新编程为多能性，从而产生自我更新的诱导多能干细胞 (iPSC)，该细胞可以分化为三个胚层的细胞类型。 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) 没有区别，并且规避了所有与 ESC 相关的伦理问题。此外，从患有复杂遗传综合征的患者身上产生的 iPSC 可以分化为受影响的细胞类型，从而为特定疾病的发病机制提供无与伦比的洞察力，并提供与治疗筛选兼容的模型。目前，在典型的重编程实验中，只有不到1%的起始细胞群能够达到多能性，并且需要对过程进行大幅优化才能完全实现iPSC的临床应用。 OSKM 与基因组结合，通过向染色质招募各种转录调节因子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 活性来充当重编程过程的障碍。尽管全基因组研究继续强调表观遗传特征在 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。我建议通过结合肽合成、蛋白质工程、位点特异性蛋白质修饰和荧光光谱技术来概括重编程因子与核小体的体外相互作用。这些研究将阐明组蛋白标记对单核小体和核小体阵列中 OSKM 结合和功能的影响。此外，我将设计针对表观遗传障碍的多价 Oct4、Sox2 和 Klf4 蛋白，这些蛋白将用于生成 iPSC。本研究的具体目标是：1) 表征 OSKM 结合对体外核小体稳定性和染色质结构的影响，2.) 确定关键表观遗传修饰在 OSKM 结合和功能中的作用，以及 3.) 设计能够克服重编程的表观遗传障碍。这项拟议的研究旨在描绘 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生成高质量 iPSC 的重编程策略至关重要。