Wang Nan, Liu Chang
Center for Plant Molecular Biology (ZMBP), University of Tübingen, Tübingen, Germany.
Methods Mol Biol. 2020;2093:115-127. doi: 10.1007/978-1-0716-0179-2_9.
The three-dimensional folding of chromatin contributes to the control of genome functions in eukaryotes, including transcription, replication, chromosome segregation, and DNA repair. In recent decades, many cytological and molecular methods have provided profound structural insights into the hierarchical organization of plant chromatin. With the Hi-C (high-throughput chromosome conformation capture) technique, analyses of global chromatin organization in plants indicate considerable differences across species. However, our knowledge of how chromatin organization at a local level is connected to tissue-specific gene expression is rather limited. This problem can be tackled by performing fluorescence-activated sorting of fixed nuclei followed by Hi-C, which is tailored for a limited number of input nuclei. Here, we describe an approach of isolating Arabidopsis thaliana nuclei with defined endopolyploidy level and subsequent in situ Hi-C library preparation for low-input plant materials. In principle, this method can be applied to any types of fluorescence-labeled nuclei, offering researchers a useful tool to unveil temporal and spatial chromatin dynamics in 3D in a tissue-specific context.
染色质的三维折叠有助于真核生物基因组功能的控制，包括转录、复制、染色体分离和DNA修复。近几十年来，许多细胞学和分子方法为植物染色质的层次组织提供了深刻的结构见解。利用Hi-C（高通量染色体构象捕获）技术，对植物中全局染色质组织的分析表明不同物种之间存在显著差异。然而，我们对局部水平的染色质组织如何与组织特异性基因表达相关联的了解相当有限。这个问题可以通过对固定核进行荧光激活分选，然后进行针对有限数量输入核量身定制的Hi-C来解决。在这里，我们描述了一种分离具有确定的内多倍体水平的拟南芥核，并随后为低输入植物材料制备原位Hi-C文库的方法。原则上，该方法可应用于任何类型的荧光标记核，为研究人员提供了一个有用的工具，以揭示在特定组织背景下三维空间中染色质的时空动态。
