Giaimo Benedetto Daniele, Ferrante Francesca, Borggrefe Tilman
Institute of Biochemistry, University of Giessen;
J Vis Exp. 2017 Jun 17(124):55907. doi: 10.3791/55907.
Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.
信号通路通过在不同水平上调节染色质结构来调控基因表达程序，例如通过组蛋白尾部的翻译后修饰（PTM）、标准组蛋白与组蛋白变体的交换以及核小体去除。这种调控需要信号敏感转录因子（TF）的结合，这些转录因子在定义为增强子的调控元件处募集染色质修饰酶。了解信号级联如何调节增强子活性需要对TF、染色质修饰酶的结合以及特定组蛋白标记和组蛋白变体的占据情况进行全面分析。染色质免疫沉淀（ChIP）分析利用高度特异性抗体免疫沉淀特定的蛋白质/DNA复合物。对纯化DNA的后续分析能够鉴定出被抗体识别的蛋白质所占据的区域。这项工作描述了一种在成熟小鼠T细胞系中高效进行组蛋白ChIP的方案。所展示的方案能够在合理的时间范围内进行ChIP分析，并且具有高重现性。
