Dynamics of chromosome organization and chromatin states in single cells

单细胞染色体组织和染色质状态的动力学

• 批准号: 10456124
• 负责人: Long Cai
• 金额: $ 113.62万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-19 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10456124
• 关键词: Address; Adopted; Antibodies; Biological; Biological Models; CRISPR imaging; Cell Nucleus; Cells; Chromatin; Chromosome Structures; Chromosomes; Color; Coupled; DNA; Data; Elements; Engineering; Epigenetic Process; Event; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genomics; Heterochromatin; Heterogeneity; Human Cell Line; Image; Imaging Device; Imaging technology; Immunofluorescence Immunologic; In Situ; Individual; Introns; Kinetics; Label; Lamins; Link; Measurement; Measures; Messenger RNA; Methods; Molecular Conformation; Mus; Mutagenesis; Nature; Nuclear; Optics; Phase; Positioning Attribute; Process; Protein Dynamics; Proteins; RNA; RNA analysis; Sister; Site; Structure; System; Techniques; Technology; Time; Transcript; Trees; Visualization software; Work; X Chromosome; X Inactivation; base; chromatin modification; chromosomal location; embryonic stem cell; fluorophore; genome-wide; genomic locus; genomic tools; human disease; insight; memory encoding; microscopic imaging; preservation; reconstruction; red fluorescent protein; sample fixation; scale up; spatiotemporal; telomere; tool; whole genome

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.

概括 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个过程如何在同一单元格内动态发生 他们如何互相影响。现有技术可以提供基因组规模分析 几个染色体位置的相互作用或空间组织。但是，我们缺乏 可简单重建原子核的简单重建框架 在所有三个级别上。我们实验室的最新工作打开了实现此类的可能性 耦合分析。我们的曲目首先，识别以后的方法允许许多DNA简单 在活细胞中追踪。 RNA和DNA seq鱼允许大量转录本和DNA基因座 在单个固定单元格中成像和回忆录允许从 端点测量。在这个项目中，我们建议将实时成像，多路复用RNA相结合， DNA和免疫荧光测量以及回忆录谱系跟踪以捕获全部 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-的过渡 单元（2C）类似于显示急剧染色体重新排列和新生变化的状态 基因表达模式。此外，我们将研究X灭活的染色体动力学 根据最初的观察，姐姐X染色体在彼此之间接触 失活过程的早期阶段。这两个生物学问题都需要跟踪 仅在单个细胞中，染色体动力学和染色质状态。 “首先 ID稍后”方法允许在活细胞中跟踪大量基因座。 多重免疫荧光的回忆录方法使我们能够推断染色质的动力学 状态过渡。将这些工具带来研究X失活和2C状态过渡将 证明这种方法可以解决广泛的细胞脂肪决策 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。该项目中开发的技术可以很容易地在人类中实施 细胞系并由其他实验室在4DN财团中采用。