Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Anal Chem. 2013 Jul 2;85(13):6485-90. doi: 10.1021/ac401036k. Epub 2013 Jun 14.
Epigenetic modification such as DNA methylation and histone modification have important roles in gene regulation. Epigenetic modification can be altered by environmental influences and are related to diseases. Therefore, epigenetic modifications may serve as biomarkers. In this study, we developed a convenient histone modification detection system by combining a chromatin immunoprecipitation (ChIP) and bioluminescence resonance energy transfer (BRET)-based homogeneous PCR product detection system using zinc finger fused to luciferase (ZF-luciferase) with DNA intercalating dye (ChIP-ZF-BRET assay). The ChIP-ZF-BRET assay comprises the following 3 steps: (1) ChIP, (2) PCR amplification of the target genomic region, which includes a zinc-finger recognition site, and (3) homogeneous detection of the PCR product by BRET using ZF-luciferase and fluorescent DNA intercalating dye. Using this system, we conveniently and accurately detected target histone modification at the androgen receptor gene promoter region in LNCaP and Du145 cells. The system can be applicable to DNA methylation detection using a methyl-CpG-binding domain protein or methylcytidine antibody instead of histone modification antibodies. Therefore, it may be useful and convenient for simultaneous detection of histone modification and DNA methylation in clinical diagnoses.
表观遗传修饰，如 DNA 甲基化和组蛋白修饰，在基因调控中起着重要作用。表观遗传修饰可以通过环境影响而改变，并且与疾病有关。因此，表观遗传修饰可以作为生物标志物。在本研究中，我们开发了一种方便的组蛋白修饰检测系统，该系统结合了染色质免疫沉淀（ChIP）和基于生物发光共振能量转移（BRET）的均相 PCR 产物检测系统，使用融合到荧光素酶的锌指（ZF-荧光素酶）与 DNA 嵌入染料（ChIP-ZF-BRET 测定法）。ChIP-ZF-BRET 测定法包括以下 3 个步骤：（1）ChIP；（2）PCR 扩增靶基因组区域，该区域包括锌指识别位点；（3）使用 ZF-荧光素酶和荧光 DNA 嵌入染料通过 BRET 对 PCR 产物进行均相检测。使用该系统，我们方便、准确地检测了 LNCaP 和 Du145 细胞中雄激素受体基因启动子区域的靶组蛋白修饰。该系统可用于使用甲基-CpG 结合结构域蛋白或甲基胞嘧啶抗体代替组蛋白修饰抗体进行 DNA 甲基化检测。因此，它可能在临床诊断中同时检测组蛋白修饰和 DNA 甲基化时非常有用和方便。
