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）：多能干细胞是一种可再生细胞来源，具有分化成成人体内任何特化细胞的能力。这些特征使多能细胞成为研究正常和功能失调的生物网络的易于处理的工具。 发展的背景。因此，多能细胞有潜力通过药物测试、疾病建模、组织工程和再生医学极大地影响人类健康。利用这种潜力需要详细了解控制多能性和指导分化的生物学。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；然而，表观遗传调控的作用最近才引起人们的关注。表观遗传调控的一个很大程度上未被探索的方面是基因相对于遥远基因组区域和调控元件的空间组织。我们的中心假设是，对于多能性重要的基因组区域在细胞核中关联并在诱导分化和重编程时发生变化。绘制 DNA 相互作用及其动态图将解决多能细胞中的这一调节系统，并可能提供一种操纵这些细胞用于研究和治疗目的的方法。具体目标：该提案的第一个目标是绘制多能细胞中关键多能性基因的长程 DNA 相互作用图谱。第二个目标是追踪发育各个阶段的染色质相互作用，并探索重编程过程中 DNA 相互作用的动态。第三个目标是确定介导染色质相互作用的新型蛋白质成分，并应用生物化学来表征这些蛋白质。研究设计：我们提出将干细胞生物学专业知识与新兴基因组技术相结合的研究。具体来说，我们将使用改良的环状染色体构象捕获 (m4C) 来检查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取样细胞来追踪多能性诱导后的染色质组织。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因子。通过有针对性地敲除这些调节因子，我们将确定它们在多能性中的功能作用。这些数据与现有的基因表达、染色质修饰和 ChIP 结果相结合，将以无与伦比的细节揭示多能性、分化和重编程过程中发挥作用的表观遗传调控机制。赞助实验室拥有大量可用于实现这些目标的资源，包括 m4C 和深度测序的仪器和技术培训。还可以通过主办机构获得计算支持和大规模数据分析的资源。培训计划包括通过前沿研究、资助写作和学生指导提供大量职业发展机会。