基因调控动力学机制的单细胞定量研究

Gene expression is fundamental to life. Regulation between different genes via their expression products forms the so-called gene regulatory network (GRN). Driving distinct cellular behaviors at any given moment, GRN is a focus of modern biological research. Due to the lack of quantitative measurement techniques, a precise mechanistic understanding of the dynamics of complex GRN, especially a precise picture of the regulation of individual gene copies in a single cell, is still missing. In this proposed work, by combining immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), we will apply our recently developed fluorescence imaging, image analysis, and stochastic modeling tools to study, with single-cell/single-molecule resolution, the dynamics of the segmentation gene network of the developing Drosophila melanogaster embryo. By quantifying the mRNAs and regulatory factors of multiple genes in the network, we will characterize the regulatory effect of, and mutual interactions between, different gene regulatory modules, transcription factors, as well as other regulatory factors. We will also develop a new quantitative imaging approach to measuring the regulation process of individual gene copies in live cells. With these, we will build up a precise dynamical theory for the complex network. Our research will help understand the role of gene regulation in embryo development and will provide a general experimental and theoretical framework to decipher gene regulation in other biological systems.

基因表达是细胞中的基本生命活动。不同基因通过其表达产物相互调控而形成复杂的基因调控网络是生物体实现各种行为的基础，也是当代生命科学的重要研究对象。由于定量测量手段的缺乏，人们对于复杂基因调控网络的精确运行机制，特别是单个细胞内单个基因的调控过程仍然知之甚少。本项目将运用和发展申请人近期开发的一套基于免疫荧光和单分子荧光原位杂交（smFISH）的光学测量、图像分析和理论方法，在单细胞、单分子尺度研究掌管果蝇胚胎发育的分段基因网络的运行机制。通过定量测量网络中多种基因的mRNA和调控因子，申请人将探索基因调控过程中不同调控序列模块、转录因子及其他调控因子的相互作用和调控效应；发展新的定量测量方法对活细胞中单个基因的调控过程进行实时观测；在此基础上建立适用于复杂基因网络的精确动力学理论。本项目的研究将有助于理解基因调控在胚胎发育过程中的作用，并为其他生命现象中的基因调控研究提供实验和理论方法。

本项目关注生物体复杂基因调控网络的动力学机制，以果蝇胚胎等生物体系为研究对象，利用单细胞、单分子定量荧光显微测量技术和随机动力学理论模型开展了一系列研究，取得如下成果：1）定量解析了果蝇胚胎分段基因网络中典型基因的调控序列上不同调控模块运行和相互作用的微观机制；2）揭示了典型分段基因的调控过程中不同转录因子及其他调控因子之间相互作用的动力学规律；3）开发了针对单个基因位点表达及染色质动力学的活细胞定量观测技术；4）建立了适用于复杂基因调控过程的通用随机动力学框架和计算平台；5）发展改进了基于杂交链式反应的高灵敏度RNA定量探测方法；6）解析了单细胞内多基因拷贝相互作用的转录动力学。目前本项目已正式发表SCI论文4 篇，另有1篇论文正在Nature Communications期刊评审和修改。本项目的成果推动了对复杂基因调控网络动力学机制的理解，为进一步研究奠定了基础。