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: 8851409
• 负责人: Glen Liszczak
• 金额: $ 5.24万
• 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/8851409
• 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 analysis; 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）：本研究计划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编程为多能性，从而产生自我更新的诱导多能干细胞（iPSCs），可以分化为三种胚层的细胞类型。iPSCs是再生医学和定制细胞治疗领域的宝贵工具，因为它们在功能上与胚胎干细胞（ESCs）无法区分，并且规避了所有与胚胎干细胞相关的伦理问题。此外，由复杂遗传综合征患者产生的iPSCs可以分化为受影响的细胞类型，从而对特定疾病的发病机制提供无与伦比的见解，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%的起始细胞群能达到多能性，为了充分实现iPSCs的临床应用，这一过程需要进行大量优化。