Dynamics of chromosome organization and chromatin states in single cells

摘要 细胞分化涉及基因表达、染色质状态和 染色体的亚核排列。理解和控制差异化 需要了解这些过程中的每一个是如何在同一个细胞内动态发生的 它们如何相互影响。现有的技术可以提供基因组规模的分析 少数染色体位置的相互作用或空间组织。然而，我们一直缺乏一个 同时重建原子核整体动力学的概括性框架 横跨所有三个层面。我们实验室最近的工作为实现这一目标开辟了可能性 耦合分析。我们的先追踪后识别方法允许同时检测多个DNA 在活细胞中进行追踪。RNA和DNA seqFISH允许大量转录本和DNA基因座 在单个固定细胞中成像，回忆录允许从 端点测量。在这个项目中，我们建议将实时成像、多路复用的RNA、 DNA，和免疫荧光测量，以及回忆录血统追踪，以捕捉整个- 染色体基因座和染色质状态的基因组动力学。利用小鼠胚胎干细胞 (MESCs)作为一个模型系统，我们将研究从多能态到更早的2- 细胞(2C)样状态，显示出染色体的剧烈重排和新生变化 基因表达模式。此外，我们还将研究X失活的染色体动力学 根据最初的观察，姐妹X染色体在 失活过程的早期阶段。这两个生物学问题都需要跟踪 单细胞中的染色体动力学和染色质状态同时存在。《轨道先行》 稍后ID“方法允许在活细胞中跟踪大量的基因座。合并后的 多重免疫荧光回忆法允许我们推断染色质的动力学 状态转换。将这些工具用于研究X失活和2C状态转换将 展示此方法在解决广泛的小区命运决策方面的能力 问题。我们还将开发分析和可视化工具，以整合基因组学(Sprite) 和成像数据。该项目开发的技术可以很容易地在人类身上实现 细胞系，并被4DN联盟中的其他实验室采用。

SUMMARY Cellular differentiation involves tightly coupled changes in gene expression, chromatin state, and sub-nuclear arrangements of chromosomes. Understanding and controlling differentiation requires understanding how each of these processes occurs dynamically within the same cell and how they influence one another. Existing techniques can provide genome scale analysis of interactions or spatial organization of a few chromosomal positions. However, we have lacked a generalizable framework for simultaneous reconstruction of the overall dynamics of the nucleus across all three levels. Recent work from our labs has opened up the possibility of achieving such coupled analysis. Our track first, identify later approach allows many DNA to be simultaneously tracked in living cells. RNA and DNA seqFISH allows a large number of transcripts and DNA loci to be imaged in single fixed cells and MEMOIR allows lineage information to be recovered from endpoint measurements. In this project, we propose to combine live imaging, multiplexed RNA, DNA, and immunofluorescence measurements, and MEMOIR lineage tracking to capture whole- genome dynamics of chromosomal loci and chromatin states. Using mouse embryonic stem cells (mESCs) as a model system, we will study the transition from the pluripotent state to an earlier 2- cell (2C) like state which shows drastic chromosome re-arrangement and changes in nascent gene expression patterns. In addition, we will study the chromosomal dynamics of X-inactivation based on the initial observations that sister X chromosomes are in contact with each other during early phases of the inactivation process. Both of these biological questions require tracking chromosomal dynamics and chromatin state simultaneously in single cells. The “Track First and ID later” approach allows a large number of loci to be tracked in living cells. The combined MEMOIR approach with multiplex immunofluorescence allows us to infer the kinetics of chromatin states transitions. Bringing these tools to study X inactivation and 2C state transition will demonstrate the capability of this approach for addressing a broad range of cell fate decision questions. We will also develop analysis and visualization tools to integrate genomics (SPRITE) and imaging data. The technology developed in this project can be readily implemented in human cell lines and adopted by other labs in the 4DN consortium.