Dynamics of chromosome organization and chromatin states in single cells

摘要 细胞分化涉及基因表达、染色质状态和细胞因子的紧密耦合变化。 染色体的亚核排列。了解和控制差异化 需要了解每个过程如何在同一单元内动态发生，并且 他们如何相互影响。现有技术可以提供基因组规模分析 一些染色体位置的相互作用或空间组织。然而，我们却缺少一个 同时重建原子核整体动力学的通用框架 跨越所有三个级别。我们实验室最近的工作开辟了实现这种目标的可能性 耦合分析。我们先追踪，后识别的方法允许许多 DNA 同时被识别 在活细胞中进行追踪。 RNA 和 DNA seqFISH 允许大量转录本和 DNA 位点 在单个固定细胞中成像，MEMOIR 允许从中恢复谱系信息 端点测量。在这个项目中，我们建议将实时成像、多重 RNA、 DNA、免疫荧光测量以及 MEMOIR 谱系追踪以捕获整个 染色体位点和染色质状态的基因组动力学。使用小鼠胚胎干细胞 （mESC）作为模型系统，我们将研究从多能状态到早期 2- 细胞（2C）样状态，显示出剧烈的染色体重排和新生的变化 基因表达模式。此外，我们还将研究X失活的染色体动力学 根据初步观察，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 单细胞中染色体动力学和染色质状态同时发生。 “轨道优先”和 ID later”方法允许在活细胞中追踪大量基因座。合并后的 多重免疫荧光的 MEMOIR 方法使我们能够推断染色质的动力学 状态转换。利用这些工具来研究 X 失活和 2C 状态转换将 展示这种方法解决广泛的细胞命运决定的能力 问题。我们还将开发整合基因组学的分析和可视化工具（SPRITE） 和成像数据。该项目开发的技术可以很容易地应用于人类 细胞系并被 4DN 联盟的其他实验室采用。

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.