Molecular mechanisms regulating chromatin looping in time and space

项目概要/摘要 CTCF和cohesin将哺乳动物基因组组织成拓扑关联结构域 （TADs）通过将染色质片段折叠成环。由于两个DNA基因座优先在一个染色体内相互作用， TADs通过调节增强子-启动子接触来关键性地调节基因表达。符合其 在基因组折叠和基因调控中起着至关重要的作用，CTCF和粘附素亚单位是其中最重要的 在人类癌症中经常突变的蛋白质，也在神经系统疾病中发挥重要作用。 了解CTCF和粘附素的失调如何导致染色质成环失调， 疾病中的基因表达首先需要深入了解CTCF和粘附素 在生理条件下调节循环。汉森博士先前已经建立了小鼠干细胞系 其中CTCF和粘附素被内源性标记。他发现使用2D超分辨率成像， 和粘附素在细胞核中形成小的共定位簇。这一观察结果提出了一种可能性， CTCF和cohesin将染色质环连接在一起。在K99阶段，汉森博士将对此进行调查 目的1中的假设，通过使用3D超- 分辨率成像在前所未有的分辨率和聚类机制，使用正交 生物化学方法此外，染色质循环的动力学目前尚不清楚。为了解决这个 在我们的理解差距，汉森博士将建立一个系统，以可视化染色质循环在活细胞中， Aim 2的K99期，并阐明干细胞中染色质成环的动力学。 有了这些信息和这些进展，汉森博士将进行机械和 R 00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 和急性消耗扰动实验，以了解染色质循环的动力学是如何 在Aim 2的R 00阶段进行功能调节。其次，他将在目标3中的K99工作的基础上， 了解CTCF和cohesin簇的功能。 汉森博士的长期目标是成为一家研究机构的独立首席研究员 并了解染色质循环的分子机制，以及这种机制在细胞中是如何失调的。 疾病为了帮助他实现这一目标，汉森博士将由他的导师和科学顾问指导 以马克思K99阶段的培训将扩展汉森博士的技能，包括3D超级 分辨率成像，干细胞分化，显微镜的建设和深化他的粘蛋白生物学的知识。 此外，汉森博士将提高他的写作，教学，指导和管理技能在K99 相位研究和培训的完成将极大地促进汉森博士向独立的过渡 作为一名独立调查员的成功

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.