Machleidt Thomas, Robers Matthew B, Hermanson Spencer B, Dudek Jeanne M, Bi Kun
Life Technologies Corporation, Discovery and ADMET Systems, Madison, WI 53719, USA.
J Biomol Screen. 2011 Dec;16(10):1236-46. doi: 10.1177/1087057111422943. Epub 2011 Oct 4.
Posttranslational modifications such as phosphorylation, acetylation, and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone-modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. The authors have developed high-throughput LanthaScreen cellular assays for Histone H3 site-specific modifications. These assays use cells expressing green fluorescence protein-tagged Histone H3 transiently delivered via BacMam and terbium-labeled anti-Histone H3 modification-specific antibodies. Robust time-resolved Förster resonance energy transfer signals were detected for H3 lysine-9 acetylation and dimethylation (H3K9me2), serine-10 phosphorylation, K4 di- and trimethylation, and K27 trimethylation. Consistent with previous reports, hypoxic stress increased K4 methylation levels, and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly, with little effects on other modifications. To demonstrate the utility of this assay platform in screening, the K9 acetylation assay was used to profile the Enzo Epigenetics Library. Twelve known HDAC inhibitors were identified as hits and followed up in a dose-response format. In conclusion, this assay platform enables high-throughput cell-based analysis of diverse types of posttranslational modifications of Histone H3.
磷酸化、乙酰化和甲基化等翻译后修饰在调节组蛋白的结构和功能中发挥着重要作用，而组蛋白的结构和功能又反过来调节基因表达、DNA修复和复制。诸如去乙酰化酶、甲基转移酶和去甲基化酶等组蛋白修饰酶已被作为各种疾病的治疗靶点。然而，以高通量细胞为基础的形式检测这些酶的活性仍然具有挑战性。作者开发了用于组蛋白H3位点特异性修饰的高通量镧系元素筛选细胞分析方法。这些分析方法使用通过杆状病毒颗粒瞬时递送的表达绿色荧光蛋白标记的组蛋白H3的细胞，以及铽标记的抗组蛋白H3修饰特异性抗体。对于H3赖氨酸-9乙酰化和二甲基化（H3K9me2）、丝氨酸-10磷酸化、K4二甲基化和三甲基化以及K27三甲基化，检测到了强大的时间分辨荧光共振能量转移信号。与先前的报道一致，缺氧应激增加了K4甲基化水平，甲基转移酶G9a抑制剂UNC-0638显著降低了K9me2水平，而对其他修饰影响很小。为了证明该分析平台在筛选中的实用性，使用K9乙酰化分析对Enzo表观遗传学文库进行了分析。鉴定出12种已知的HDAC抑制剂为命中物，并以剂量反应形式进行后续研究。总之，该分析平台能够以高通量细胞为基础对组蛋白H3的多种类型翻译后修饰进行分析。
