Gene positioning and dynamic chromatin organization of the human genome

项目摘要 组成真核基因组的核包不仅存储了遗传信息，而且还 介导特定细胞类型的基因表达。层次化的基因组组织受到严格的监管 精确控制细胞功能。间期染色体占据不同的核空间，是一个保守的基因组 称为染色体领地的建筑。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，理解 在细胞核中定位和移动染色体位置和区域的机制需要高分辨率 在生理条件下实时学习。在过去的五年中，我们开发了基于CRISPR的 使用多种颜色定位和跟踪多达7个基因组座位的高分辨率活细胞成像技术 同时。最近，我们用小分子细胞可穿透的rna相互作用取代了荧光蛋白。 可提高亮度并将标签尺寸缩小100倍的分子。我们的初步数据显示 染色质令人惊讶的动态和结构方面：(1)同源和非同源染色体 基因座以不同的速度和不同的方向移动；(2)大规模的染色体区域连续 在非应激条件下数分钟内重排，称为染色体形态动力学； 染色体构象对温度敏感；以及(4)转化细胞和未转化细胞具有 不同的染色体构象。在小鼠胚胎干细胞中，启动子和增强子的流动性 与特定基因的转录活性相关；然而，染色质迁移率如何与 人们对转录活动知之甚少，也存在争议。根据我们的初步结果，我们建议 为了研究四个关键概念：(I)染色体DNA在单个染色体区域中是如何组织的，(Ii) 驱动染色体形态动态的因素是什么？(Iii)活性基因相对于非活性基因的位置 转录的DNA区域以绘制基因组的图景，以及(Iv)染色质运动如何与 细胞核内的转录活动。我们将干扰转录、温度和微管 聚合，以确定控制染色体动力学的因素。集成非侵入性成像 利用生物物理模型和rna-seq数据的方法将提供有关机制和 实时染色质动力学和单染色体水平基因定位的功能基础 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.