École Polytechnique Fédérale de Lausanne, SB ISIC LCBM, Station 6, CH-1015 Lausanne, Switzerland.
École Polytechnique Fédérale de Lausanne, SB ISIC LCBM, Station 6, CH-1015 Lausanne, Switzerland.
Methods. 2020 Dec 1;184:112-124. doi: 10.1016/j.ymeth.2020.01.018. Epub 2020 Jan 28.
In eukaryotic cells, the genome is packaged into chromatin and exists in different states, ranging from open euchromatic regions to highly condensed heterochromatic regions. Chromatin states are highly dynamic and are organized by an interplay of histone post-translational modifications and effector proteins, both of which are central in the regulation of gene expression. For this, chromatin effector proteins must first search the nucleus for their targets, before binding and performing their role. A key question is how chromatin effector proteins search, interact with and alter the different chromatin environments. Here we present a modular fluorescence based in vitro workflow to directly observe dynamic interactions of effector proteins with defined chromatin fibres, replicating different chromatin states. We discuss the design and creation of chromatin assemblies, the synthesis of modified histones, the fabrication of microchannels and the approach to data acquisition and analysis. All of this with the aim to better understand the complex in vivo relationship between chromatin structure and gene expression.
在真核细胞中，基因组被包装成染色质，并存在于不同的状态，从开放的常染色质区域到高度浓缩的异染色质区域。染色质状态是高度动态的，由组蛋白翻译后修饰和效应蛋白的相互作用来组织，这两者在基因表达的调控中都起着核心作用。为此，染色质效应蛋白必须首先在核内搜索其靶标，然后才能结合并发挥作用。一个关键问题是，染色质效应蛋白如何搜索、相互作用并改变不同的染色质环境。在这里，我们提出了一种基于荧光的模块化体外工作流程，用于直接观察效应蛋白与定义的染色质纤维的动态相互作用，复制不同的染色质状态。我们讨论了染色质组装体的设计和创建、修饰组蛋白的合成、微通道的制造以及数据采集和分析的方法。所有这些都是为了更好地理解染色质结构和基因表达之间复杂的体内关系。
