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Title: Exploration of the potential roles of DNA methylation in controlling cellular function in human and mouse differentiated cells
Author: O'Neill, Karla M.
Awarding Body: Ulster University
Current Institution: Ulster University
Date of Award: 2013
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DNA methylation has been implicated in a range of cellular processes such as X chromosome inactivation, imprinting, the silencing of developmentally important genes and in maintaining cellular viability. However, not all of these roles are equally well supported and much of the functional work has been done in mouse alone. I took advantage of a normal but immortalised human hTERT -1604 cell line to examine the effects of depleting the maintenance methyltransferase DNMTl and carried out comparative work in other cell lines and in mouse. Several studies reported increased microsatellite instability (MSI) following loss of DNMTl, even at microsatellite repeats lacking DNA methylation. I present data which demonstrates that MSI following loss of DMMTI is indirect and due to parallel reductions in multiple components of the mismatch repair system (MMR), the latter of which is responsible for detecting and repairing post-replicative DNA mismatches. Reductions occur in large part via PARP- dependent activation of the DNA damage response (DDR), since inhibition of the latter protein led to reduced DNA damage signalling and partial restoration of MLH I. Secondly, I examined DNA methylation levels in stable c1onally-derived cells following recovery from DNMTI depletion. All clones demonstrated loss of imprinting but displayed recovered levels of methylation at repetitive DNA elements and at a number of germline genes. Such data, in combination with experiments involving pharmacological inhibition or transient genetic depletion of DNMTI has helped me to indicate a novel role for this epigenetic modification in the silencing of a group of germ line- and testis-specific genes in human. Finally, I show that methylation at ribosomal DNA genes in differentiated cells is unlikely to be important developmentally, since levels appear to vary within the same tissues from various strains of mouse. Furthermore, rDNA transcription does not appear to be dependent on DNA methylation. In conclusion, the use of novel human cell models and transient depletion, together with benchmarking to well-known targets, has allowed me to help discriminate between gene targets which are directly controlled by DNA methylation and those which are secondarily affected by the loss of the maintenance enzyme, with important implications for our understanding of epigenetic control.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available