Gene positioning and dynamic chromatin organization of the human genome

项目摘要 构成真核生物基因组的核包装不仅储存遗传信息， 介导细胞类型特异性基因表达。等级基因组组织受到严格的监管， 精确控制细胞功能。间期染色体占据独特的核空间，一个保守的基因组 被称为染色体区域的结构。过去二十年的技术进步揭示了 基因组三维结构的许多新方面。然而，了解 在细胞核中定位和移动染色体位点和区域的机制需要高分辨率 在生理条件下进行真实的研究。在过去的五年里，我们开发了基于CRISPR的 高分辨率活细胞成像技术，使用多种颜色定位和跟踪多达7个基因组位点 同步最近，我们已经用小细胞可渗透的RNA相互作用取代了荧光蛋白。 分子，提高亮度和减少标签的大小超过100倍。我们的初步数据显示 令人惊讶的动态和结构方面的染色质：（1）同源和非同源染色体 基因座以不同的速度和方向移动;（2）大规模的染色体结构域连续 在非应激条件下，在几分钟内重排，称为染色体形态学动态;（3） 染色体构象是温度敏感的;（4）转化和非转化细胞具有 不同的染色体构象在小鼠胚胎干细胞中， 与特定基因的转录活性相关;然而，染色质迁移率如何与 转录活性是知之甚少和有争议的。基于我们的初步结果，我们建议 研究四个关键概念：（i）染色体DNA如何在单个染色体区域中组织，（ii） 什么因素驱动染色体形态动力学，（iii）活性基因相对于非活性基因如何定位， 转录的DNA区域来制作基因组的景观，以及（iv）染色质运动如何与 核中的转录活动。我们将扰乱转录，温度和微管 聚合，以确定控制染色体动力学的因素。集成无创成像 生物物理模型和RNA-seq数据的方法将提供关于生物学机制和生物学特性的新信息。 实时染色质动态和基因定位在单染色体水平的功能基础， 原子核

Project Summary The nuclear package that comprises the eukaryotic genome not only stores genetic information but also mediates cell-type-specific gene expression. The hierarchical genome organization is tightly regulated to precisely control cell functions. Interphase chromosomes occupy distinct nuclear spaces, a conserved genome architecture known as chromosome territories. Technological advances over the last two decades have revealed many new aspects of the three-dimensional architecture of the genome. However, understanding the mechanisms that localize and mobilize chromosomal loci and territories in the nucleus requires high-resolution studies in real time under physiological conditions. In the past five years, we have developed CRISPR-based high-resolution live-cell imaging techniques using multiple colors to localize and track up to seven genomic loci simultaneously. Recently, we have replaced fluorescent proteins with small cell-permeable RNA-interacting molecules that improve brightness and reduce the size of tags by >100-fold. Our preliminary data revealed surprising dynamic and structural aspects of the chromatin: (1) homologous and non-homologous chromosomal loci moved at different speeds and in different directions; (2) large-scale chromosomal domains continuously rearranged in minutes in non-stressed conditions, termed chromosome morphological dynamics; (3) chromosome conformations were temperature-sensitive; and (4) transformed and non-transformed cells had distinct chromosome conformations. In mouse embryonic stem cells, the mobility of promoters and enhancers correlates with transcriptional activity for specific genes; however, how chromatin mobility correlates with transcriptional activity is poorly understood and controversial. Building upon our preliminary results, we propose to investigate four key concepts: (i) how chromosomal DNA is organized in individual chromosome territories, (ii) what factors drive chromosome morphological dynamics, (iii) how active genes are positioned relative to non- transcribed DNA regions to craft the landscape of the genome, and (iv) how chromatin movements correlate with transcriptional activities in the nucleus. We will perturb transcription, temperature, and microtubule polymerization to identify factors that govern chromosome dynamics. Integration of non-invasive imaging approaches with biophysical models and RNA-seq data will provide new information on the mechanistic and functional foundations of real-time chromatin dynamics and gene positioning at the single chromosome level in the nucleus.