Abstract
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.
Highlights
Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, trichostatin A (TSA)) is a pharmacological agent endowed with potent histone deacetylase inhibitory activity (HDI or HDACI), promoting histone acetylation with broad effects on epigenetic signature [1].TSA-mediated HDAC inhibition contributes to chromatin relaxation, allowing transcription factors to access the DNA molecule within the chromatin structure
In order to validate our experimental system, the morphology of ES14 cells treated with either TSA or DMSO for 24 h was analyzed by phase-contrast microscopy
We examined lysine residues’ acetylation and methylation global profiles of H3 and H4 histones from E14 mouse embryonic stem cells treated with either trichostatin A (TSA) or dimethylsulfoxide (DMSO) by using a newly developed limited-proteolysis mass-spectrometry protocol
Summary
Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) is a pharmacological agent endowed with potent histone deacetylase inhibitory activity (HDI or HDACI), promoting histone acetylation with broad effects on epigenetic signature [1].TSA-mediated HDAC inhibition contributes to chromatin relaxation, allowing transcription factors to access the DNA molecule within the chromatin structure. Oxo-2,4-heptadienamide, TSA) is a pharmacological agent endowed with potent histone deacetylase inhibitory activity (HDI or HDACI), promoting histone acetylation with broad effects on epigenetic signature [1]. TSA was reported to promote morphology and gene expression changes in embryonic stem cells (ESCs) [2]. Inhibition of histone deacetylases drives cells toward differentiation by regulating the expression of differentiation- and pluripotency-associated genes. Many studies demonstrated that TSA enhances cell differentiation [4,5,6] by negatively regulating the expression of stemness markers [7], such as Oct4 [8], and simultaneously activating differentiation markers, such as Pdx1 [9]. The molecular mechanism of TSA-mediated regulation of gene activation and repression is very likely related to post-translational modifications of histones. Because of its biochemical activities, TSA has some activities as an anticancer drug [10,11] or as a cell differentiation promoter [12]
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