Study of the post-translational modifications of histone H4 by Fourier transform ion cyclotron resonance mass spectrometry.

Abstract

Post-translational modification (PTM) of proteins is known to be a method by which protein function can be regulated. The addition of selected chemical groups at specific amino acid residues can act as a switch by which the function of a modified protein can be attenuated. Histones are a group of proteins which are found in the nucleus of eukaryotic cells and interact with DNA, providing it with a structural foundation upon which the chromosome is built. Histone proteins have numerous sequence variants and are known to be extensively post-translationally modified in a dynamic manner. These modifications have a direct effect on the interacting DNA resulting in increasing or decreasing levels of gene transcription. Advancements in analytical instrumentation, when coupled to high resolution separation techniques permit the analysis of increasingly complex biological mixtures. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) offers unrivalled mass resolving power and mass measurement accuracy, allowing the detailed study of mixtures of intact proteins and their post-translational modifications. These features have been exploited to provide a global view of the PTMs of histone proteins. The work contained within this thesis is a study, by FT-ICR MS, of the modifications of one of the most extensively modified histone proteins; histone H4. Firstly, the modifications of histone H4 were examined after treatment with a potent histone deacetylase inhibitor across several cell lines. The cell lines chosen showed a varying response to treatment with the inhibitor. From the cell lines tested, two which responded differently were further interrogated to elucidate the order in which acetylation occurs in the N-terminal region. Secondly, the modifications of histone H4 were analyzed after exposure to lactic acid over multiple treatment times. Lactic acid is a metabolic by-product, and is of interest when considering the Warburg effect and its role in tumorigenesis. Exposure of cells to levels of lactic acid which can be present under anaerobic conditions (i.e. during intense exercise) showed that lactate is able to inhibit histone de-acetylation. The resulting increase in hyper-acetylated forms of histone H4 could be potentially linked to increased gene expression, a typical observation in tumorigenic cells. Finally, using a mouse model for the neurological condition Rett Syndrome, the posttranslational modifications of histone H4 were investigated. The primary cause of Rett Syndrome is mutation of the DNA binding protein methyl CpG binding protein 2 (MeCP2). MeCP2 has been associated with multiple intracellular functions, one of which is chromatin remodelling. The work carried out showed a link between MeCP2 mutation and tri-methylation of histone H4. In addition, the tri-methylation was not solely identified through the presence of tri-methylated fragments in fragmentation mass spectra. Interestingly, the neutral loss of a methylene group was observed extensively during fragmentation of tri-methylated species. This unreported phenomenon made interpretation of spectra difficult; however, ultimately served as a useful marker for this modification.