用靶向DNA原位纳米分辨成像法在单细胞水平上探索多能性相关基因的染色质三维构像及其对基因表达调控的影响

Three-dimensional (3D) chromatin organization has been revealed to play a role in gene transcription regulation and cell identity determination. Chromosome conformation capture (3C)-based technologies, main approaches in genome organization study, have revealed the existence of topologically-associated domain (TAD) and compartment A/B in genome of mammalian cells. It also showed that some long-range genomic interactions specifically occur in embryonic stem cell (ESC) rather their differentiated cells. In the past three years, we developed a molecular beacon-based nanoresolution imaging method for imaging a non-repetitive genomic sequence as short as 2.5 kb in chromatin of single mammalian cells. Based on this new method as well as other techniques like CRISPR/Cas9-mediated knockout, this proposed study is to directly visualize the interactions between promoter and other elements within Oct4 or Nanog locus in single hESC or mESC, thus obtaining the ESC-specific 3D folding of Oct4 or Nanog locus. Via analyzing the published data from chromatin conformation capture-based technologies and chromatin immuno-precipitation, we will list the potential elements that possibly interact with promoter within Oct or Nanog locus. Then, we will take the advantage of the specific DNA nanoresolution imaging method that has been well-established with sequence resolution of 2.5 kb in our lab, visualize the promoter-genomic element interactions within Oct4 or Nanog locus in single hESCs or mESC as well as their differentiated cells, and obtain the ESC-specific 3D conformation of Oct4 or Nanog locus. Next, we will investigate the relationship between promoter-genomic element interactions and gene expression at single ESCs with the use of the specific DNA nanoresolution imaging, CRISPR/Cas9 knockout, and homogeneous insertion. Finally, we will assess potential effects of 3D conformation of Oct4 or Nanog locus on ESC pluripotency . Taken together, the proposed study would reveal the ESC-specific 3D conformation of Oct4 or Nanog locus as well as its role in gene expression and ESC pluripotency.

人体不同类型细胞的基因组一级序列基本相同，三维折叠方式各不相同；染色质三维折叠参与影响细胞特异的转录及功能。染色体构象捕获系列技术揭示了染色质中拓扑结构域和区室的存在，同时显示了胚胎干细胞（ESC）染色质中存在特异的相互作用，为三维基因组研究奠定基础。我们团队建立了靶向DNA原位纳米分辨成像法，实现对小鼠或人类染色质中短至2.5 kb的非重复的特异DNA片段进行原位观察。在此基础上，本申请研究首先将基于文献确定人类和小鼠ESC的Oct4或Nanog基因座上与启动子可能存在相互作用的一系列位点；在ESC及其分化细胞中，通过靶向DNA原位纳米分辨成像确定Oct4或Nanog基因座上确实与启动子相互作用的位点，并观察同一细胞中两个等位基因座上的相互作用情况，获得Oct4或Nanog基因座在ESC中的三维折叠方式；最后在单细胞水平建立Oct4或Nanog基因座三维折叠与基因表达和细胞多能性的关系。

细胞生物学的发展与光学新技术的发展密不可分，推动生物学研究的一次次飞跃。然而，由于衍射现象的存在，传统光学显微镜的分辨率被限制在横向200 nm和轴向500 nm左右。随机光学重建显微方法 (Stochastic Optical Reconstruction Microscopy，STORM)的出现和快速发展，理论上可以实现定位精度高达一至几纳米，能够满足对细胞内超微结构研究的需要。.胚胎干细胞多能性的维持及状态转变过程中多能性基因及谱系基因的三维结构和表达的研究是了解生命个体发育早期奥秘的重要方向，促进提高人口质量，它的研究亟需新技术的应用。针对这些问题，本项目实现将当前最高分辨率的STORM与胚胎干细胞的多能性维持及转换研究的结合，建立了靶向DNA原位纳米分辨成像法，实现对小鼠或人类染色质中短至2.5 kb的非重复的特异DNA片段进行原位观察。在此基础上，我们将该标记方法与双色超分辨结合，研究胚胎干细胞NANOG基因座不同位点的相互作用以及NANOG启动子与SOX2增强子间的相互作用，并进一步研究诱导分化细胞NANOG基因座不同位点的相互作用。与此同时，我们采用包括RNAseq在内的其它研究手段，开展一系列研究胚胎干细胞多能性转变和谱系基因的初始表达如何受细胞表面信号调控，最后，我进行的ChIP-seqencing 揭示分化mESC基因组序列上转录抑制复合体PRC2结合所受的调节。除此之外，STORM应用于胚胎干细胞研究为该超分辨成像技术提出新的问题和挑战，我们也针对这些问题和挑战开展对STORM在细胞超微结构应用的研究，促进双色三维超分辨成像平台的优化和成熟，为进一步为生物学研究服务提供基础。.本项目部分结果已经发表于2021年Nanophotonics (2022; 11(1): 53–65,本项目负责人是独立通讯作者)；有另外两部分研究工作已完成论文书写、正在投稿之中（这两篇文章本申请人是通讯作者，本基金是第一致谢基金），与此同时本基金产生的一项发明专利已经获得授权，第二项发明专利已经提交，第三、第四发明专利正在修改中，将在2023年初提交。