Kubicek Stefan, O'Sullivan Roderick J, August E Michael, Hickey Eugene R, Zhang Qiang, Teodoro Miguel L, Rea Stephen, Mechtler Karl, Kowalski Jennifer A, Homon Carol Ann, Kelly Terence A, Jenuwein Thomas
Research Institute of Molecular Pathology, Vienna Biocenter, Dr Bohrgasse 7, A-1030 Vienna, Austria.
Mol Cell. 2007 Feb 9;25(3):473-81. doi: 10.1016/j.molcel.2007.01.017.
Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity, we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds, seven hits were identified. Of those, one inhibitor, BIX-01294 (diazepin-quinazolin-amine derivative), does not compete with the cofactor S-adenosyl-methionine, and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays, transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly, chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin.
组蛋白赖氨酸甲基化在染色质结构域的组织和基因表达调控中发挥着重要作用。为了分析其功能并调节其活性，我们以重组G9a作为靶酶，筛选针对组蛋白赖氨酸甲基转移酶（HMTases）的特异性抑制剂。从一个包含125,000种预选化合物的化学文库中，鉴定出了7个活性化合物。其中，一种抑制剂BIX-01294（二氮杂萘-喹唑啉-胺衍生物）不与辅因子S-腺苷甲硫氨酸竞争，并在体外选择性地损害G9a HMTase和H3K9me2的生成。在细胞试验中，用BIX-01294短暂孵育几种细胞系会降低总体H3K9me2水平，去除抑制剂后该水平得以恢复。重要的是，在几个G9a靶基因处进行的染色质免疫沉淀表明，在抑制剂处理的小鼠胚胎干细胞和成纤维细胞中，启动子近端H3K9me2出现可逆性降低。我们的数据鉴定出了一种具有生物活性的HMTase抑制剂，它能够在哺乳动物染色质中对H3K9me2标记进行短暂调节。
