Li Pingxin, Hu Jing, Wang Yanming
Center for Eukaryotic Gene Regulation, University Park, PA, USA.
Methods Mol Biol. 2012;809:473-88. doi: 10.1007/978-1-61779-376-9_31.
Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. Here, we describe assays to study histone citrullination by PAD4 in vitro and in vivo. We also describe approaches to measure histone citrullination levels at gene promoters using chromatin immunoprecipitation assay and analyze the effects of PAD4 inhibitor on cell cycle and apoptosis by flow cytometry. These methods would be useful techniques to study this unique histone modification.
组蛋白翻译后修饰在调节染色质结构和基因表达中发挥着重要作用。组蛋白修饰之一，即组蛋白瓜氨酸化，由一种名为肽基精氨酸脱氨酶4（PAD4，也称为PADI4）的酶催化，该酶将组蛋白精氨酸（Arg）和单甲基精氨酸残基都转化为瓜氨酸。最近的研究发现，组蛋白瓜氨酸化抵消了组蛋白精氨酸甲基化的作用，并作为一种抑制标记来关闭基因表达。在这里，我们描述了在体外和体内研究PAD4介导的组蛋白瓜氨酸化的实验方法。我们还描述了使用染色质免疫沉淀法测量基因启动子处组蛋白瓜氨酸化水平的方法，并通过流式细胞术分析PAD4抑制剂对细胞周期和细胞凋亡的影响。这些方法将是研究这种独特组蛋白修饰的有用技术。
