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Title: The effect of replication impediments on differentiation
Author: Eldridge, Cara Bernadette
ISNI:       0000 0004 7968 5961
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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In this thesis I set out to answer the question 'is differentiation robust to replication impediments?'. Prior work in the group has focussed on the epigenetic impact of replication impediments in the terminally differentiated DT40 chicken cell line. I wanted to find out whether these impediments could impact the fundamental biological process of embryonic development. In this work the BOBSC human induced pluripotent stem cell line was differentiated to definitive endoderm while replication was perturbed using DNA damaging agents, low dose hydroxyurea and G-quadruplex secondary structure stabilisation. DNA damage was induced both in the undifferentiated state and during differentiation. When damage was induced during differentiation, greater levels of cell death were seen and there was a larger increase in cells in G2/M phase of the cell cycle compared with treatment in the undifferentiated state. The efficiency of differentiation was observed to negatively correlate with the dose of DNA damaging agent. During unperturbed differentiation, the levels of DNA damage response proteins, including p53, were found to decrease. However, when DNA damage was induced during differentiation the level of p53 increased. In order to understand whether the upregulation of p53 was preventing differentiation, TP53-/- cells were differentiated in the presence of DNA damaging agents. These cells were found to differentiate as efficiently as wildtype untreated cells. This indicates that p53 prevents differentiation in the presence of DNA damage, termed here a 'differentiation checkpoint'. The level of γH2A.X was found to be markedly increased 50 hours into unperturbed differentiation. This correlates with the point at which the cells transition from being epithelial to mesenchymal. The role of this rise is not known but may correspond to massive transcriptional changes that occur during this transition. G-quadruplex-binding ligands were also shown to alter the course of differentiation, either by a p53-dependent mechanism or by a separate, possibly G-quadruplex specific, mechanism. This also occurred in REV1-/- cells, a specialised polymerase known to play a role in G-quadruplex-processing. This work has led to the conclusion that replication impediments are able to alter the course of definitive endoderm differentiation in human induced pluripotent stem cells.
Supervisor: Sale, Julian Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: differentiation ; G-quadruplex ; DNA damage response ; induced pluripotent stem cell ; stem cell ; endoderm