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Characterisation of the Set7 lysine methyltransferase in proliferation, survival and transcriptional regulation
thesisposted on 14.02.2017, 01:59 by Keating, Samuel T
Epigenetic mechanisms underlying the transcriptional regulation of eukaryotic cells have been a recent focus of intensive research. Post-translational histone modifications define the dynamic structural adaptation of regions of chromatin to confer transcriptionally permissive or repressive configurations. Several studies have demonstrated an association between histone H3 mono-methylated at lysine 4 (H3K4me1) and transcriptional activation. The Set7 lysine methyltransferase can catalyse this reaction. However, since the initial characterisation of this enzyme, the physiological substrate of Set7 has been questioned predominantly due to weakened methyltransferase activity towards nucleosomal histones. Despite this discordance, Set7 and the associated H3K4 methyltransferase activity have been thoroughly demonstrated to be required for the transcriptional activation of several discrete genes. In addition to chromatin modification, increasingly studies have focused on the role of Set7 in post-translational methylation of lysine residues within non-histone peptides, namely transcription factors. Methylation of numerous Set7 substrates impacts transcriptional of downstream targets through various mechanisms. Thus it appears that Set7 can regulate transcriptional events through both H3K4me1-dependent and independent mechanisms. Difficulties arise in discriminating transcriptional changes causatively associated with altered H3K4 methylation from those derived from transcription factor methylation and altered stability and/or transactivity. Confounding this issue are not only the number of confirmed Set7 substrates that may individually regulate transcriptional events, but also the likelihood that there exists a plethora of uncharacterised transcription factors that are regulated by Set7-mediated methylation. To this end, defining the genes that are subject to regulation by Set7 by each mechanism is paramount to moving towards a complete understanding of Set7-mediated transcriptional control. This study investigated the global epigenetic effects of Set7 depletion with respect to growth, survival and extended biochemical analysis of H3K4me1 enrichment of human endothelial cells. The emergence of massive parallel sequencing technology has enabled investigators to examine mechanisms of epigenetic gene regulation with unprecedented detail at a genome-wide level. The first transcriptome profiling of Set7-depleted microvascular endothelial cells and gene ontology analysis was conducted to identify biological pathways deregulated by Set7 loss. Deregulated genes associated with cell cycle regulation and apoptosis were identified that could contribute to the observed phenotype of Set7 knockdown cells. Additionally, this study demonstrates that Set7 distinguishes core and nucleosomal histones and is capable of writing modifications associated with transcriptional activation. Bioinformatic analysis of the transcriptome profile identified five previously described Set7 substrates that were significantly connected to the dataset. A further 10 transcription factors devoid of prior association to Set7 function were identified as highly connected to deregulated genes. To link Set7 function to the activity of transcription factors identified by this investigation, two consensus formulas for the prediction of Set7 methylation sites were applied to amino acid sequences of transcription factors not previously associated with Set7 function. The screen uncovered 28 potential Set7 methylation sites across nine transcription factor peptide sequences. This novel method of transcription factor analysis in models of Set7 knockdown complements the growing list of studies that have addressed this aspect of Set7-mediated transcriptional regulation. Defining the transcription factors that interact with Set7 in endothelial and other cell types is critical for a complete understanding of the role of the methyltransferase in transcriptional regulation in human health and disease.