Analysis of chromatin-immunopurified MeCP2-associated fragments
Analysis of chromatin-immunopurified MeCP2-associated fragments
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DOI：
10.1006/bbrc.2001.6023
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发表时间：
2001-12-07
影响因子：
3.1
通讯作者：
Wolffe, AP
中科院分区：
文献类型：
--
作者：
El-Osta, A;Wolffe, AP
作为分子生物学家，我们正在继续揭示DNA结合蛋白介导基因表达的相互作用。染色质免疫沉淀（ChIP）技术为我们在体内研究染色质结构及其在调节转录、复制和重组中的作用之间的相互作用提供了一种精美的工具。我们描述了一个强大的测定方法，用于识别与染色质相关的分子决定因素。在本文中，我们阐述了ChIP技术，并利用转录沉默-高甲基化多药耐药（MDR1）基因作为甲基- cpg结合蛋白2 （MeCP2）在染色质上定位的平台。根据抑制强烈依赖于内源性启动子甲基化谱的假设，我们证明MDR1是MeCP2的靶标。甲基化的MDR1染色质高度富集McCP2，这与MDR1在转录活跃的细胞中观察到的定位形成鲜明对比。在一个独特的模型系统中，我们讨论了用于免疫纯化染色质上MeCP2抑制复合物的实验方法，并通过竞争性PCR方法定量蛋白质- dna关联。(C) 2001 Elsevier Science。
As molecular biologists, we are continuing to unravel the interactions by which DNA binding proteins mediate the expression of genes. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between chromatin structure and its role in regulating transcription, replication, and recombination in vivo. We describe a robust assay used to identify the molecular determinants associated with chromatin. In this article we illustrate the ChIP technique and use the transcriptionally silent-hypermethylated multidrug resistance (MDR1) gene as the platform for methyl-CpG binding protein 2 (MeCP2) localization on chromatin. Driven by the hypothesis that repression is strongly dependent on the methylation profile of the endogenous promoter, we demonstrate that MDR1 is targeted by MeCP2. Methylated MDR1 chromatin is highly enriched with McCP2 and is in striking contrast to localization observed in cells in which MDR1 is transcriptionally active. In a distinct model system we discuss experimental methods used to immunopurify the MeCP2 repressor complex on chromatin and quantify protein-DNA association by competitive PCR approach. (C) 2001 Elsevier Science.