Won Jaejoon, Kim Tae Kook
Biomedical Research Center, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea.
Methods Mol Biol. 2006;325:273-83. doi: 10.1385/1-59745-005-7:273.
Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.
染色质作为真核生物遗传信息的模板，会受到多种翻译后修饰的影响，这些修饰主要作用于组蛋白的N端，如乙酰化、甲基化、磷酸化和泛素化。不同的组蛋白修饰会对新的组蛋白产生协同或拮抗的相互作用亲和力，进而决定染色质转录活性或沉默状态之间的动态转变。除了转录，包括DNA复制、DNA修复和重组在内的许多生物学过程都受到与染色质相关的因子的调控。染色质免疫沉淀（ChIP）技术为我们提供了一种精妙的工具，通过蛋白质与蛋白质、蛋白质与DNA的甲醛交联，然后对固定材料进行免疫沉淀并检测相关DNA，来研究结构或调节蛋白与DNA之间的相互作用及其在体内调节多种细胞过程中的作用。在这里，我们以人类端粒酶逆转录酶启动子的ChIP分析为例来说明整个实验过程。
