Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors.
Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors.
复制标题
DOI：
10.1016/j.ymeth.2011.02.010
复制
发表时间：
2011-07
期刊：
影响因子：
4.8
通讯作者：
Sun, Zu-Wen
中科院分区：
文献类型：
--
作者：
Chandrasekharan, Mahesh B.;Huang, Fu;Sun, Zu-Wen
关键词：
通过组蛋白H2 B赖氨酸123单泛素化调节组蛋白H3赖氨酸4和79甲基化是进化上保守的跨组蛋白串扰机制，其证明了组蛋白泛素化在细胞内的功能作用。参与这些修饰的建立和动态调节的调节酶、因子和过程及其全基因组分布模式已经在许多模型系统中确定。在理解这种跨组蛋白串扰的快速进展已经取得了使用染色质生物学的标准实验工具，在芽殖酵母（酿酒酵母），一个高度听话的模式生物。在这里，我们提供了一套修改和完善的实验程序，可用于获得进一步的见解，在芽殖酵母这种串扰管理的基本机制。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。本文提出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、非组蛋白蛋白及其修饰的稳态水平。提供了改进的染色质免疫沉淀和双重免疫沉淀方案，以测量蛋白质及其修饰形式在特定染色质区域或基因座处的占据和分布。还描述了一种快速简便的方法来测量整体蛋白质丰度和天然染色质上蛋白质-蛋白质和蛋白质-DNA相互作用的变化。
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.
登录
查看更多内容
影响因子：
--
作者：
Aparicio, Oscar;Geisberg, Joseph V;Struhl, Kevin
通讯作者：
Struhl, Kevin
影响因子：
3.3
作者：
Geng, Fuqiang;Tansey, William P.
通讯作者：
Tansey, William P.
影响因子：
5.3
作者：
Chandrasekharan, Mahesh B.;Huang, Fu;Sun, Zu-Wen
通讯作者：
Sun, Zu-Wen
影响因子：
16
作者：
Ng, HH;Robert, F;Struhl, K
通讯作者：
Struhl, K
影响因子：
4.8
作者：
Huang, Fu;Chandrasekharan, Mahesh B.;Sun, Zu-Wen
通讯作者：
Sun, Zu-Wen