Molecular mechanisms regulating chromatin looping in time and space

调节染色质时间和空间循环的分子机制

• 批准号: 10076878
• 负责人: Anders Sejr Hansen
• 金额: $ 24.9万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10076878
• 关键词: 3-Dimensional; Acute; Address; Advisory Committees; Affect; Binding; Biochemical; Biology; Cell Cycle; Cell Differentiation process; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Complex; DNA; Development; Disease; Educational process of instructing; Enhancers; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genome; Goals; Hand; Human; Image; Institution; Interdisciplinary Study; Knock-in; Knowledge; Malignant Neoplasms; Mathematics; Mentors; Microscope; Mitotic; Molecular; Mus; Mutate; Mutation; Neuronal Differentiation; Neurons; Nuclear; Organizational Change; Outcome; Phase; Physiological; Play; Principal Investigator; Proteins; Research; Research Personnel; Research Training; Resolution; Role; Sister Chromatid; Structure; System; Testing; Therapeutic Intervention; Time; Time Study; Training; Work; Writing; analysis pipeline; cohesin; cohesion; embryonic stem cell; experience; experimental study; falls; genetic regulatory protein; genome editing; genome-wide; improved; mammalian genome; nerve stem cell; nervous system disorder; promoter; skills; stem cell differentiation; stem cells; success

Project summary/Abstract CTCF and cohesin causally organize mammalian genomes into topologically associating domains (TADs) by folding chromatin segments into loops. Since two DNA loci preferentially interact inside a TAD, TADs critically regulate gene expression by regulating enhancer-promoter contacts. Consistent with their crucial role in genome folding and gene regulation, CTCF and cohesin sub-units are among the most frequently mutated proteins in human cancers and also play prominent roles in neurological disorders. Understanding how dysregulation of CTCF and cohesin causes dysregulation of chromatin looping and gene expression in disease first requires a deep mechanistic understanding of how CTCF and cohesin regulate looping under physiological conditions. Dr. Hansen has previously established mouse stem cell lines where CTCF and cohesin are endogenously tagged. He found using 2D super-resolution imaging that CTCF and cohesin form small co-localizing clusters in the nucleus. This observation raises the possibility that clusters of CTCF and cohesin hold together chromatin loops. During the K99 phase, Dr. Hansen will investigate this hypothesis in Aim 1 by elucidating the detailed 3D nuclear organization of CTCF and cohesin using 3D super- resolution imaging at unprecedented resolution and the mechanism of clustering using an orthogonal biochemical approach. Moreover, the dynamics of chromatin looping are currently unknown. To address this gap in our understanding, Dr. Hansen will set up a system to visualize chromatin looping in live cells during the K99 phase of Aim 2 and elucidate the dynamics of chromatin looping in stem cells. With this information and these developments in hand, Dr. Hansen will then perform mechanistic and functional studies in the R00 phase. First, Dr. Hansen will use stem cell differentiation, induced gene activation and acute depletion perturbation experiments to understand how the dynamics of chromatin looping are functionally regulated during the R00 phase of Aim 2. Second, he will build on his K99 work in Aim 3 to understand the function of CTCF and cohesin clusters. Dr. Hansen's long-term goal is to become an independent principal investigator at a research institution and to understand the molecular mechanisms underlying chromatin looping and how this is dysregulated in disease. To help him achieve this goal, Dr. Hansen will be guided by his mentors and Scientific Advisory Committee. Training in the mentored K99 phase will expand Dr. Hansen's skill-set to include 3D super- resolution imaging, stem cell differentiation, microscope building and deepen his knowledge of cohesin biology. Moreover, Dr. Hansen will improve his writing, teaching, mentoring and management skills during the K99 phase. Completion of the research and training will greatly facilitate Dr. Hansen's transition to independence and success as an independent investigator.

项目概要/摘要 CTCF 和粘连蛋白将哺乳动物基因组因果组织成拓扑关联域 （TAD）通过将染色质片段折叠成环来实现。由于两个 DNA 位点优先在 TAD 内相互作用， TAD 通过调节增强子-启动子接触来严格调节基因表达。符合他们的 CTCF 和粘连蛋白亚基在基因组折叠和基因调控中发挥着至关重要的作用，是最重要的亚基之一 人类癌症中经常发生突变的蛋白质，并且在神经系统疾病中也发挥着重要作用。 了解 CTCF 和粘连蛋白的失调如何导致染色质环和染色质的失调 疾病中的基因表达首先需要深入了解 CTCF 和粘连蛋白如何发挥作用 在生理条件下调节循环。 Hansen博士此前已建立了小鼠干细胞系 其中 CTCF 和粘连蛋白是内源标记的。他发现使用 2D 超分辨率成像 CTCF 和粘连蛋白在细胞核中形成小的共定位簇。这一观察结果提出了聚类的可能性 CTCF 和粘连蛋白将染色质环结合在一起。在 K99 阶段，汉森博士将对此进行调查 通过使用 3D super- 阐明 CTCF 和粘连蛋白的详细 3D 核组织，实现目标 1 中的假设 以前所未有的分辨率成像以及使用正交的聚类机制 生化方法。此外，染色质环的动力学目前尚不清楚。为了解决这个问题 为了弥补我们理解上的差距，汉森博士将建立一个系统来可视化活细胞中染色质循环的过程 目标 2 的 K99 相并阐明干细胞中染色质环的动态。 有了这些信息和这些进展，汉森博士将进行机械和 R00 阶段的功能研究。首先，汉森博士将利用干细胞分化，诱导基因激活 和急性耗竭扰动实验，以了解染色质循环的动态变化 在目标 2 的 R00 阶段进行功能调节。其次，他将在目标 3 中的 K99 工作基础上 了解 CTCF 和粘连蛋白簇的功能。 汉森博士的长期目标是成为研究机构的独立首席研究员 并了解染色质环化的分子机制以及染色质环化是如何失调的 疾病。为了帮助他实现这一目标，汉森博士将得到他的导师和科学咨询的指导 委员会。在 K99 指导阶段的培训将扩展 Hansen 博士的技能组合，包括 3D 超级 分辨率成像、干细胞分化、显微镜构建并加深了他对粘连蛋白生物学的了解。 此外，汉森博士将在K99期间提高他的写作、教学、指导和管理技能 阶段。研究和培训的完成将极大地促进汉森博士向独立的过渡 以及作为独立调查员的成功。