基于双酶切的染色体构象捕获新技术开发及其在解析巨噬细胞分化和激活过程中基因组三维构象变化中的应用

The three-dimensional (3D) structure of genomes has important impact on gene functions. Chromosome conformation capture (3C) technology makes it possible to study the 3D structure of the whole genome. However, there are multiple disadvantages in the current 3C-derived methods, such as high noise in the data, tedious in experiment, high cost, no direct assessment of the random ligations. These disadvantages impede the development of 3D genomics. In this project, we are trying to address these disadvantages, and aim to 1) develop a new method for 3D genomics, which is simple to operate, low cost, with low noise, possible to assess the random ligations directly; 2) explore the variations of chromosome conformation of macrophage during the differentiation and the activation after tuberculosis infection; 3) integrate the data from transcriptome, epigenome, DNA methylome, and 3D genome to characterize the variations in chromosome conformation, and define the regulatory function of chromatin interactions for the development, differentiation, and pathology of macrophage cells; 4) dissect the function of the key chromatin interaction anchors for chromosome conformation with CRISPR/Cas9 editing system. The success of this project will promote the development of 3D genomics and will provide theoretical foundations for gene regulation under the conditions of development, differentiation, and pathological stimulation of macrophage cells.

三维基因组学是功能基因组学的研究前沿。基因组三维构象的解析依赖于染色体构象捕获技术。但是传统染色质构象捕获技术存在着噪声高、实验繁琐、实验成本昂贵、难以直接评估随机交互等问题，阻碍了三维基因组学的发展。本项目旨在1）开发一种简单易行、成本低廉 、数据噪音低、可评估随机交互的三维基因组学研究新技术；2）利用该技术解析巨噬细胞不同分化阶段和结核杆菌感染激活后染色体三维构象的动态变化；3）将三维基因组学数据与相应细胞阶段的表达谱、组蛋白修饰、甲基化谱等表观组学数据整合，系统分析基因组三维构象与基因表达、表观修饰之间的内在相关性；4）通过CRISPR/Cas9编辑技术验证关键交互位点对基因组构象的作用。本项目将为三维基因组学提供新的研究工具，为解析细胞在不同分化和病理阶段功能基因组学打下基础，并为结核病的致病机制提供一定依据。

基因组的三维结构，对基因的转录调控、DNA复制、DNA损伤修复等都有着重要的影响。基因组三维构象的解析依赖于染色体构象捕获技术。但是传统染色质构象捕获技术存在着噪声高、实验繁琐、实验成本昂贵、难以直接评估随机交互等问题，阻碍了三维基因组学的发展。针对这些问题，本项目开发了一种简单易行、成本低廉、数据噪音低、可评估随机交互的三维基因组学研究新技术DLO Hi-C，及其分析软件DLO Hi-C Tool；并将DLO Hi-C技术应用于不同的细胞模型，解析基因组三维构象及其表观遗传学特征，系统分析基因组三维构象与基因表达、表观修饰之间的内在相关性。本项目的成果，为三维基因组学研究提供了新的工具，为解析细胞在不同分化和病理阶段功能基因组学打下基础。在本项目的执行过程中，共发表学术论文19篇，培养3名博士研究生和4名硕士研究生。相关杂志包括Nature Genetics、Journal of Genetics and Genomics、Science Advances、Nucleic Acids Research等，并有部分成果的文章在审稿中。