Abstract
DNA methylation is an epigenetic modification established during cellular differentiation and undergoes both dramatic changes during development and ageing. During the ‘active’ removal of DNA methylation, the Ten-Eleven Translocation (TET) enzymes catalyse the oxidation of cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) and further derivatives. Recent research has uncovered a striking preference of the TET catalytic domain, meaning certain CG-containing hexamer motifs are targeted for rapid demethylation but other hexamers are left unaffected. While this result is interesting, both the biological consequences of this preference, and how this preference transpires across the lifespan is largely unstudied.
In this thesis, I tested the biological significance of TET-catalytic domain specificity and explored its consequences in the context of ageing. I established that the mouse TET3 catalytic domain (mTET3-CD) construct was a robust and reliable model of active demethylation, showing a significant loss in global methylation following its overexpression that mirrored recently published results. Using the mTET3-CD model, I reported that the overexpression of TET3-CD on an acute timeframe resulted in large scale significant changes in gene expression, the most significant of which seen through an increase of expression of genes with promoters containing E-box binding sites for methylation-sensitive transcription factors like MYC.
Next, I presented the first characterisation of CG-containing hexamer methylation during ageing and showed that there is evidence of at least some TET sequence preference contributing to age associated methylation changes. The iconic E-box hexamer motif CACGTG, which undergoes rapid demethylation in culture, and was here shown to be in the minority of hexamer motifs that lost methylation over the lifespan of sheep. Finally, I investigated the presence of age-related clonal haematopoiesis (ARCH) for the TET2 gene in sheep as a novel model for human ageing and its intersection with TET demethylation biology. Although I was unable to identify characteristic clonal mutations in blood DNA compared to ear punch DNA, likely due to the limitations in sample size and loci examined, I highlight the importance of having animal models of ARCH. A sheep model, in particular, would offer significant benefits for studying ARCH.
Together, these findings contribute to a greater understanding of TET catalytic domain specificity in cells, and its biological consequences for gene expression and ageing. In doing so, my work highlights the view that the TET catalytic domain is not merely a catalytic engine capable of indiscriminate DNA demethylation but possesses genuine target specificity that is inextricably linked to its function.