Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.
Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.
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DOI：
10.3791/57080
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发表时间：
2017-12-29
期刊：
影响因子：
--
通讯作者：
Green EM
中科院分区：
文献类型：
--
作者：
Jezek M;Jacques A;Jaiswal D;Green EM
在这里，我们描述了一个协议染色质免疫沉淀的修改组蛋白从芽殖酵母酿酒酵母。免疫沉淀的DNA随后用于定量PCR，以询问整个基因组中组蛋白翻译后修饰的丰度和定位。组蛋白翻译后修饰（PTM），如乙酰化，甲基化和磷酸化，是动态调节的一系列酶，添加或删除这些标志物响应于细胞接收的信号。这些PTMS是调控基因表达控制和DNA修复等过程的关键因素。染色质免疫沉淀（chIP）是一种用于解剖整个基因组中许多组蛋白PTM响应于对细胞的不同扰动的丰度和定位的工具方法。在这里，一个通用的方法进行芯片后修饰的组蛋白从芽殖酵母酿酒酵母（S。cerevisiae）中描述。该方法依赖于使用甲醛处理酵母培养物的蛋白质和DNA的交联、通过珠击产生酵母裂解物、通过微球菌核酸酶溶解染色质片段以及组蛋白-DNA复合物的免疫沉淀。纯化与感兴趣的组蛋白标记相关的DNA，并进行定量PCR分析，以评估其在整个基因组的多个基因座处的富集。探讨组蛋白标记H3 K4 me 2和H4 K16 ac在野生型和突变酵母的本地化的代表性实验进行了讨论，以证明数据分析和解释。这种方法适用于各种组蛋白PTM，可以用不同的突变株或在不同的环境应力的存在下进行，使其成为研究不同条件下染色质动力学变化的优秀工具。
Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome. Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.
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影响因子：
5.6
作者：
Jaiswal D;Turniansky R;Green EM
通讯作者：
Green EM
影响因子：
8.8
作者：
Byrum SD;Raman A;Taverna SD;Tackett AJ
通讯作者：
Tackett AJ
DOI：
10.1006/meth.1999.0879
发表时间：
1999-11-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Kuo, MH;Allis, CD
通讯作者：
Allis, CD
影响因子：
10.5
作者：
Briggs, SD;Bryk, M;Allis, CD
通讯作者：
Allis, CD
影响因子：
16
作者：
Rothbart SB;Dickson BM;Raab JR;Grzybowski AT;Krajewski K;Guo AH;Shanle EK;Josefowicz SZ;Fuchs SM;Allis CD;Magnuson TR;Ruthenburg AJ;Strahl BD
通讯作者：
Strahl BD