Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Methods Mol Biol. 2024;2842:225-252. doi: 10.1007/978-1-0716-4051-7_12.
Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.
表观遗传学研究面临着染色质修饰网络高度复杂和紧密调控的挑战。尽管已经描述了许多染色质介导的基因调控的分离机制，但缺乏全面分析特定过程作为更大表观基因组网络一部分的可靠方法。为了通过一个有助于捕捉和描述转录调控复杂性的系统扩展方法工具箱，我们在这里描述了一种用于生成转录活性的稳定报告系统的稳健方案，并总结了它们的应用。该系统允许诱导染色质调节剂募集到荧光报告基因，然后使用流式细胞术检测转录变化。报告基因整合到内源性染色质环境中，从而能够检测所研究的染色质调节剂对内源性共因子的调节依赖性。该系统允许在单细胞水平上进行简单和动态的读出，并能够补偿转录的细胞间变化。该系统的模块化设计使得可以轻松调整该方法，以在广泛的细胞系面板中研究不同的染色质调节剂。我们还总结了该技术在表征不同染色质效应物的沉默速度、去除激活组蛋白修饰、分析表观遗传修饰的稳定性和可逆性、研究小分子对染色质效应物的影响以及功能效应物-共调节剂关系方面的应用。所提出的方法允许在活细胞中研究表观遗传效应蛋白对转录调控的复杂性。
