The regulatory role of chromatin interaction in pluripotency and differentiation

染色质相互作用在多能性和分化中的调节作用

• 批准号: 8714180
• 负责人: Justin Brumbaugh
• 金额: $ 5.51万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714180
• 关键词: Adult; Architecture; Area; Basic Science; Binding; Biochemistry; Biological; Biology; Cell Nucleus; Cells; Cellular biology; ChIP-seq; Chromatin; Chromatin Structure; Chromosomes; Complex; Coupled; DNA; Data; Data Analyses; Development; Disease model; Distant; Epigenetic Process; Gene Expression; Gene Expression Regulation; Gene Structure; Genes; Genetic Transcription; Genomics; Goals; Health; Human; Immunoprecipitation; Institution; Knock-out; Knowledge; Laboratories; Learning; Maps; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Mentors; Methods; Molecular Conformation; Monitor; Multipotent Stem Cells; Play; Pluripotent Stem Cells; Process; Proteins; RNA; Regenerative Medicine; Regulation; Regulatory Element; Relative (related person); Research; Research Design; Research Project Grants; Resources; Role; Sampling; Site; Source; Staging; Stem cells; Students; System; Technology; Testing; Therapeutic Human Experimentation; Tissue Engineering; Training; Training Programs; Western Blotting; Work; Writing; adult stem cell; career development; cell type; chromatin modification; cohesin; deep sequencing; drug testing; embryonic stem cell; genome-wide; improved; induced pluripotent stem cell; instrumentation; interest; large-scale database; meetings; multipotent cell; nerve stem cell; novel; overexpression; pluripotency; public health relevance; research study; self-renewal; stem cell biology; tool; trait

DESCRIPTION (provided by applicant): Pluripotent stem cells are a renewable cell source with the capacity to differentiate into any specialized cell in the adult body. These traits make pluripotent cells a tractable tool for studying normal and dysfunctional biological networks in the context of development. Therefore, pluripotent cells have the potential to greatly impact human health through drug testing, disease modeling, tissue engineering, and regenerative medicine. Harnessing this potential requires a detailed understanding of the biology that governs pluripotency and directs differentiation. The mechanisms that guide these processes have been studied extensively at the protein and RNA level; however, the role of epigenetic regulation has only recently come into focus. A largely unexplored aspect of epigenetic regulation is the spatial organization of genes relative to distant genomic regions and regulatory elements. It is our central hypothesis that genomic regions important for pluripotency associate in the nucleus and change upon induction of differentiation and reprogramming. Charting DNA interaction and its dynamics will resolve this regulatory system in pluripotent cells and may provide a means to manipulate these cells for research and therapeutic purposes. Specific Aims: The first aim in this proposal will map long-range DNA interaction for key pluripotency genes in pluripotent cells. The second aim tracks chromatin interactions at various stages of development and also explores the dynamics of DNA interaction during reprogramming. The third aim identifies novel protein components that mediate chromatin interaction and applies biochemistry to characterize these proteins. Study Design: We propose research that combines expertise in stem cell biology with emerging genomic technology. Specifically, we will use modified circular chromosome conformation capture (m4C) to examine DNA interaction for key pluripotency genes Oct4 and Sox2 in pluripotent and multipotent stem cells. We will also track chromatin organization following the induction of pluripotency by sampling cells at intermediate stages of reprogramming. By characterizing proteins that mediate chromatin structure, we will determine key epigenetic regulators. Through targeted knockdown of these regulators, we will establish their functional role in pluripotency. These data, coupled to existing gene expression, chromatin modification, and ChIP results will reveal in unparalleled detail the epigenetic regulatory mechanisms at play during pluripotency, differentiation, and reprogramming. The sponsoring laboratory has numerous resources available to accomplish these goals, including instrumentation and technical training for m4C and deep sequencing. Resources are also available through the host institution for computational support and large-scale data analysis. The training program includes numerous opportunities for career development through cutting edge research, grant writing, and student mentoring.

描述（由申请人提供）：多能干细胞是可再生细胞来源，具有分化成人体内任何专门细胞的能力。这些特征使多能细胞成为研究正常和功能障碍生物网络的可拖动工具 发展的背景。因此，多能细胞有可能通过药物测试，疾病建模，组织工程和再生医学来极大地影响人类健康。利用这一潜力需要详细了解控制多能性并指导分化的生物学。指导这些过程的机制已在蛋白质和RNA水平上进行了广泛的研究。但是，表观遗传调节的作用直到最近才开始焦点。表观遗传调节的一个很大程度上未开发的方面是基因相对于遥远的基因组区域和调节元素的空间组织。我们的核心假设是，基因组区域对核中多能性助理很重要，并在诱导分化和重编程后变化。图表DNA相互作用及其动力学将解决多能细胞中的调节系统，并可能提供一种操纵这些细胞的方法，以进行研究和治疗目的。具体目的：该提案中的第一个目标将绘制多能细胞中关键多能基因的远程DNA相互作用。第二个目标跟踪开发各个阶段的染色质相互作用，还探讨了重编程过程中DNA相互作用的动力学。第三个目标确定了介导染色质相互作用并应用生物化学来表征这些蛋白质的新型蛋白质成分。研究设计：我们提出了将干细胞生物学专业知识与新兴基因组技术结合起来的研究。具体而言，我们将使用经过修改的圆形染色体构象捕获（M4C）检查多能和多能干细胞中关键多能基因OCT4和SOX2的DNA相互作用。我们还将通过在重编程的中间阶段在中间阶段对细胞进行采样后，跟踪染色质组织。通过表征介导染色质结构的蛋白质，我们将确定关键的表观遗传调节剂。通过针对这些调节剂的目标敲低，我们将确定其在多能性中的功能作用。这些数据与现有基因表达，染色质修饰和芯片结果结合在一起，将在多能性，分化和重新编程过程中无与伦比的细节揭示表观遗传调节机制。赞助实验室拥有许多可用于实现这些目标的资源，包括用于M4C和深入测序的工具和技术培训。还可以通过主机机构获得计算支持和大规模数据分析的资源。该培训计划包括通过尖端研究，赠款写作和学生指导来进行职业发展的许多机会。