Using Chromatin Immunoprecipitation in Toxicology: A Step-by-Step Guide to Increasing Efficiency, Reducing Variability, and Expanding Applications

Shaun D. McCullough, Doan M. On, Emma C. Bowers
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引用次数: 10

Abstract

Histone modifications work in concert with DNA methylation to regulate cellular structure, function, and response to environmental stimuli. More than 130 unique histone modifications have been described to date, and chromatin immunoprecipitation (ChIP) allows for the exploration of their associations with the regulatory regions of target genes and other DNA/chromatin-associated proteins across the genome. Many variations of ChIP have been developed in the 30 years since its earliest version came into use, which makes it challenging for users to integrate the procedure into their research programs. Furthermore, the differences in ChIP protocols can confound efforts to increase reproducibility across studies. The streamlined ChIP procedure presented here can be readily applied to samples from a wide range of in vitro studies (cell lines and primary cells) and clinical samples (peripheral leukocytes) in toxicology. We also provide detailed guidance on the optimization of critical protocol parameters, such as chromatin fixation, fragmentation, and immunoprecipitation, to increase efficiency and improve reproducibility. Expanding toxicoepigenetic studies to more readily include histone modifications will facilitate a more comprehensive understanding of the role of the epigenome in environmental exposure effects and the integration of epigenetic data in mechanistic toxicology, adverse outcome pathways, and risk assessment. © 2017 by John Wiley & Sons, Inc.

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在毒理学中使用染色质免疫沉淀:逐步提高效率,减少可变性和扩大应用的指南
组蛋白修饰与DNA甲基化协同作用,调节细胞结构、功能和对环境刺激的反应。迄今为止,已有超过130种独特的组蛋白修饰被描述,染色质免疫沉淀(ChIP)允许探索它们与靶基因和基因组中其他DNA/染色质相关蛋白的调控区域的关联。自最早的版本投入使用以来,30年来已经开发了许多不同的ChIP,这使得用户将该程序集成到他们的研究计划中具有挑战性。此外,ChIP方案的差异可能会混淆提高研究可重复性的努力。这里介绍的简化ChIP程序可以很容易地应用于毒理学中各种体外研究(细胞系和原代细胞)和临床样本(外周白细胞)的样本。我们还提供了关键方案参数优化的详细指导,如染色质固定、碎片化和免疫沉淀,以提高效率和提高可重复性。扩大毒性表观遗传学研究,使其更容易包括组蛋白修饰,将有助于更全面地了解表观基因组在环境暴露效应中的作用,并将表观遗传学数据整合到机械毒理学、不良后果途径和风险评估中。©2017 by John Wiley &儿子,Inc。
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