期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 22, 期 4, 页码 -出版社
MDPI
DOI: 10.3390/ijms22042063
关键词
histone PTMs; limited proteolysis; mass spectrometry; TSA; activation of differentiation
资金
- Ministero dell'Istruzione, dell'Universita e della Ricerca Italiana [PRIN 2017AHTCK7]
TSA affects chromatin state by inhibiting histone deacetylase activity, driving cell differentiation and specific gene activation. This study utilized a new, untargeted method to analyze the acetylome and methylome profiling of H3 and H4 histones in ES14 cells.
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.
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