Gene positioning and dynamic chromatin organization of the human genome

人类基因组的基因定位和动态染色质组织

• 批准号: 10714346
• 负责人: Li-Chun Tu
• 金额: $ 38.33万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714346
• 关键词: 3-Dimensional; Architecture; Cell Nucleus; Cell Proliferation; Cell physiology; Cells; Chromatin; Chromosome Territory; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; DNA; Data; Enhancers; Essential Genes; Foundations; Gene Expression; Gene Order; Genes; Genetic Transcription; Genome; Genome Stability; Human Genome; Imaging Techniques; Individual; Interphase Chromosome; Maps; Mediating; Microtubule Polymerization; Molecular Conformation; Morphology; Movement; Mus; Nuclear; Permeability; Physiological; Proteins; RNA; RNA analysis; Research; Resolution; Speed; Technology; Temperature; Time; biophysical model; cell type; embryonic stem cell; genetic information; genomic locus; imaging approach; improved; live cell imaging; non-invasive imaging; promoter; transcriptome sequencing

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.

项目摘要 包括真核基因组的核包装不仅存储遗传信息，而且还存储 介导细胞型特异性基因表达。分层基因组组织受到严格监管 精确控制细胞功能。相间染色体占据不同的核空间，保守的基因组 建筑被称为染色体领土。在过去的二十年中，技术进步揭示了 基因组三维结构的许多新方面。但是，了解 在细胞核中定位和动员染色体基因座和领土的机制需要高分辨率 实时研究在生理条件下。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七个基因组基因座的高分辨率活细胞成像技术 同时地。最近，我们用小细胞可渗透的RNA相互作用代替了荧光蛋白 改善亮度并将标签尺寸降低> 100倍的分子。我们的初步数据显示 染色质的令人惊讶的动态和结构方面：（1）同源和非同源染色体 Loci以不同的速度和不同的方向移动； （2）连续大规模染色体结构域 在不压力条件下在几分钟内重新排列，称为染色体形态动力学； （3） 染色体构象对温度敏感。 （4）转化和未转化的细胞具有 独特的染色体构象。在小鼠胚胎干细胞中，启动子和增强子的迁移率 与特定基因的转录活性相关；但是，染色质迁移率与 转录活性知之甚少，有争议。在我们的初步结果的基础上，我们建议 研究四个关键概念：（i）染色体DNA如何在单个染色体领土中组织起来，（ii） 哪些因素驱动染色体形态动力学，（iii）活性基因如何相对于非 - 转录的DNA区域以制作基因组的景观，以及（iv）染色质运动如何与 细胞核中的转录活性。我们将考虑到转录，温度和微管 聚合以识别控制染色体动力学的因素。非侵入成像的整合 生物物理模型和RNA-seq数据的方法将提供有关机械和机械和 实时染色质动力学和基因定位在单个染色体水平的功能基础 核。