Department of Chemical Sciences, University Federico II of Naples, Strada Comunale Cinthia, 26, 80126 Naples, Italy.
CEINGE Advanced Biotechnologies, Via G. Salvatore 486, 80145 Naples, Italy.
Int J Mol Sci. 2021 Feb 19;22(4):2063. doi: 10.3390/ijms22042063.
Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.
曲古抑菌素 A([R-(E,E)]-7-[4-(二甲基氨基)苯基]-N-羟基-4,6-二甲基-7-氧代-2,4-庚二酰胺，TSA)通过其强大的组蛋白去乙酰化酶抑制活性影响染色质状态。干扰赖氨酸残基在组蛋白上乙酰基的去除是许多表观遗传调控过程之一，这些过程控制基因表达。组蛋白去乙酰化酶抑制作用促使细胞向分化阶段发展，有利于特定基因的激活。在本文中，我们使用一种新的非靶向方法研究了 TSA 对 ES14 小鼠胚胎干细胞中 H3 和 H4 赖氨酸乙酰基组和甲基组谱的影响，该方法由胰蛋白酶有限水解实验与 MALDI-MS 和 LC-MS/MS 分析相结合组成。该方法首先在标准鸡核心组蛋白上建立，以研究在酶：底物(E:S)比和水解时间方面的优化条件，然后应用于研究 TSA 刺激的胚胎干细胞(ES14)中 H3 和 H4 组蛋白赖氨酸乙酰化和甲基化状态的全局变化，并针对分化进行研究。所提出的策略在实现一些最显著的表观遗传修饰(如乙酰化和赖氨酸甲基化)的鉴定和相对定量方面被发现非常有效，因此，我们相信它可以在更广泛的研究中成功地用于所有表观遗传修饰的特征描述。
