Department of Biochemistry and Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, United States.
Methods. 2011 Jul;54(3):304-14. doi: 10.1016/j.ymeth.2011.02.010. Epub 2011 Mar 8.
Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.
组蛋白 H2B 赖氨酸 123 单泛素化调控组蛋白 H3 赖氨酸 4 和 79 甲基化是一种进化上保守的跨组蛋白串扰机制，它证明了组蛋白泛素化在细胞内的功能作用。在许多模型系统中，已经确定了参与这些修饰的建立和动态调节及其全基因组分布模式的调节酶、因子和过程。在芽殖酵母（酿酒酵母）这个高度可操作的模型生物中，利用染色质生物学的标准实验工具，在理解这种跨组蛋白串扰方面取得了快速进展。在这里，我们提供了一套经过修改和完善的实验程序，可以进一步深入了解控制这种芽殖酵母串扰的潜在机制。重要的是，改进的程序及其基本原理也可以应用于其他模式生物。这里介绍的方法提供了一种快速有效的方法来制备富集的蛋白质提取物，以更好地保存和评估组蛋白、非组蛋白和它们的修饰物的稳态水平。提供了改进的染色质免疫沉淀和双重免疫沉淀方案，以测量特定染色质区域或基因座上蛋白质及其修饰形式的占据和分布。还描述了一种快速简便的方法来测量天然染色质上的总蛋白质丰度以及蛋白质-蛋白质和蛋白质-DNA 相互作用的变化。
