The regulatory role of chromatin interaction in pluripotency and differentiation

描述(申请人提供)：多能干细胞是一种可再生的细胞来源，具有分化为成人体内任何特化细胞的能力。这些特性使多能细胞成为研究正常和功能失调的生物网络的易操作的工具。 发展的背景。因此，通过药物测试、疾病模型、组织工程和再生医学，多能细胞有可能极大地影响人类健康。利用这种潜力需要对支配多能性和指导分化的生物学有详细的了解。指导这些过程的机制已经在蛋白质和RNA水平上进行了广泛的研究；然而，表观遗传调控的作用直到最近才开始受到关注。表观遗传调控的一个很大程度上未被探索的方面是相对于遥远的基因组区域和调控元件的基因的空间组织。我们的中心假设是，对多能性至关重要的基因组区域在细胞核内相互关联，并在诱导分化和重新编程时发生变化。绘制DNA相互作用及其动力学图将解决多能细胞中的这一调控系统，并可能为研究和治疗目的提供一种操纵这些细胞的手段。具体目标：这项提案的第一个目标是绘制多能细胞中关键多能性基因的远程DNA相互作用图。第二个目标是跟踪不同发育阶段的染色质相互作用，并探索重新编程期间DNA相互作用的动态。第三个目的是确定介导染色质相互作用的新的蛋白质成分，并应用生物化学来表征这些蛋白质。研究设计：我们建议进行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。具体地说，我们将使用改进的环形染色体构象捕获(M4C)来检测多能性干细胞中关键的多能性基因Oct4和Sox2的DNA相互作用。我们还将通过在重新编程的中间阶段采样细胞来追踪诱导多能性后的染色质组织。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。通过有针对性地取消这些监管机构，我们将确立它们在多能性方面的职能作用。这些数据，再加上现有的基因表达、染色质修饰和芯片结果，将无与伦比地详细揭示在多能性、分化和重新编程过程中发挥作用的表观遗传调控机制。赞助实验室有许多资源可用于实现这些目标，包括m4C和深度测序的仪器和技术培训。还可通过主办机构获得资源，用于计算支助和大规模数据分析。该培训计划包括许多通过尖端研究、拨款撰写和学生指导实现职业发展的机会。

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.