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Title: Studies of histone modifications in yeast and humans
Author: Hsu, Kuo-Feng
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Epigenetic regulation is a complex biological process that has influence on the structure of chromatin and impacts on the regulation of DNA-associated events, including transcription, DNA replication, and DNA damage repair. Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. The JmjC domain-containing histone demethylases play important roles in the regulation of chromatin structure and consequently gene regulation and biological events. The first aspect of my work was to explore the application of fission yeast to investigating human JmjC histone demethylase (KDM) function. The work focused on the human KDM4A and FBXL11 demethylases and investigation on the role of H3K36 modifications in the survival of wee1 mutant S. pombe. The roles of the JmjC domain function of KDM4A and FBXL11 were also investigated. The results reveal KDM4A mediated demethylation of histone H3K36me3/me2 has synthetic lethality with respect to wee1 mutant S. pombe, whereas FBXL11 mediated demethylation of histone H3K36me2 has more subtle effects. Work on epigenetics is often limited by antibody properties. In the second and third parts of the work described in this thesis, mass spectrometric methods that are capable of histone profiling are described, with the aim of developing methods complementary to immunoblotting. An LC-MS-based method for profiling histones was developed and used for analysis of histone modification under different conditions. Using the high resolution of LC and MS instruments, this method provides robust analysis of modifications to intact histones. Histone profiles for human normal and cancer cell lines were investigated. Subsequently, the method was applied to analyse global changes in histone modification patterns under various conditions, focusing on hypoxia/ hypoxia related stresses. Hypoxia has effects on the activity of JmjC KDMs and the hypoxia-inducible factor (HIF) isoforms levels, so enabling regulation of expression. The effects of 2OG/Fe(II) oxygenase inhibitors of enzymes involved in hypoxic regulation on histone PTM patterns were also investigated. The results reveal that the extent to which hypoxia and hypoxia-mimetics (including inhibition that upregulate HIF-a levels) cause changes to histone profiles vary differently. The LC-MS-based method hence has potential application in drug discovery and in investigations on the role of histone modifications in healthy development and disease.
Supervisor: Schofield, Christopher Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available