染色质三维结构相关蛋白质的研究方法与技术

Research Methods and Techniques of Chromatin 3D Structure-related Proteins

真核生物染色质空间构象的形成和维持受到多种因素的共同调控,其中多种蛋白质在染色质三维结构的形成与维持中具有重要作用,而大量蛋白质在三维基因组中的具体作用机制仍待研究。本文从研究技术、思路及展望三方面分别进行了论述。首先,介绍了染色质三维结构相关蛋白质的研究技术,其中,基于显微荧光成像的技术包括结构光照明显微技术、单分子定位显微技术和受激发射损耗显微技术,它们可以提高染色质三维结构成像分辨率并丰富染色质三维结构成像细节。测序技术包括针对转录因子的染色质免疫共沉淀测序技术,以及针对染色质的染色质构象捕获及其衍生技术。然后,归纳了基于共定位发现染色质三维结构相关目标蛋白质,以及研究其具体作用机制的思路。将染色质三维结构相关的转录因子和辅因子按其作用机制分类,整理了目前已发现的染色质三维结构相关蛋白质,包括直接作用于DNA、与辅因子共同发挥作用、介导相分离、参与环挤出、与非编码RNA共同发挥作用、引起染色质修饰以及G-四链体相关等。最后,讨论了现有研究技术存在的挑战和未来发展趋势,在实验流程、成本以及有偏性等方面指出了现有研究技术的提升空间和发展方向,推动三维基因组学研究向分子层面发展。

The formation and maintenance of the spatial conformation of eukaryotic chromatin are jointly regulated by a variety of factors,among which many proteins play an important role in the formation and maintenance of the three-dimensional(3D)structure of chromatin,and the specific mechanism of action of a large number of proteins in the 3D genome remains to be researched.Here,we discussed research techniques,ideas,and prospects,respectively.Firstly,we summarized the current research techniques for 3D chromatin structure-related proteins.Techniques based on fluorescence microscopy include structured illumination microscopy,single-molecule localization microscopy,and stimulated emission depletion microscopy,which can improve the imaging resolution of chromatin 3D structures and enables more detailed imaging of chromatin 3D structures.Sequencing technologies include chromatin co-immunoprecipitation sequencing technology for transcription factors,chromatin conformation capture technology for chromatin and related technologies.Then,we introduced how to discover 3D chromatin structures based on co-localization and ideas to study related mechanisms based on which we classified,as well as how to study the mechanism of action of proteins related to the 3D structure of chromatin.Mechanisms identified so far include directly acting on deoxyribonucleic acid,cooperating with cofactors,mediating phase separation,participating in ring extrusion,co-operating with non-coding ribonucleic acid,causing chromatin modification and G-quadruplex correlation,etc.Finally,we discuss the challenges and future development trends of existing research technologies,and point out the improvement direction of existing research technologies to promote the development of 3D genomics research at the molecular level,in terms of the experimental process,cost,and bias.