Investigating the impact of pharmacological exposures on the developing epigenome

Abstract

Epigenetic processes are important for establishing and maintaining cell-type-specific and dynamic gene expression patterns. Genomic imprinting is an epigenetic process found in eutherian and metatherian mammals, which results in parent-of-origin specific expression. There are currently over 100 described imprinted genes in the mammalian genome and these genes have been reported to play pivotal and diverse roles in physiological processes. Failure to correctly control imprinted gene expression has been demonstrated to be causal for developmental syndromes, such as Beckwith-Weidemann syndrome, and strongly associated with disease and life-long health issues in humans, including cancer, mental disabilities and diabetes. Imprinted loci strongly rely on epigenetic marks for the correct control of gene expression. Imprinting can thus be utilised as a barometer for epigenetic status following environmental or pharmacological exposures. In this thesis, I characterise the impact of pharmacological exposures on imprinted gene regulation in vitro. 5-azacytidine (AZA) and decitabine (DAC) are both cytidine nucleoside analogues used in the treatment of acute myeloid leukaemia. Following incorporation into DNA, both drugs have been demonstrated to induce depletion of DNA methyltransferases (DNMTs), removal of DNA methylation and induction of DNA damage. I show that treatment of mouse embryonic stem cells (mESCs) with 5-azacytidine and decitabine, whilst both causing DNA hypomethylation, leads to differing effects on imprinted gene expression and long-term cell survival. I then investigate the potential underlying causes of these differences, beginning with the impact of AZA on RNA methylation in mESCs. I show that genetic perturbation of the RNA methylation machinery leads to disruption of imprinted gene expression, indicating a potential link between RNA modifications and the control of imprinted gene transcripts. Lastly, I present a novel reporter system to allow both transient and sustained disruption to imprinted loci to be distinguished following pharmacological or environmental exposures. Overall, my work emphasises the need for a greater understanding of the impact of exposures on the developing epigenome and presents imprinted genes as sensitive barometers for detecting alterations to the epigenome during development.