Use this URL to cite or link to this record in EThOS:
Title: Quantitative analysis of dynamic DNA modifications in pluripotent stem cells and primordial germ cells
Author: Linnett, Sarah
ISNI:       0000 0004 7657 131X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
Availability of Full Text:
Access from EThOS:
Access from Institution:
Almost 65 years have passed since the discovery of 5 methylcytosine (5mC). DNA methylation has since been shown to be involved in gene expression, genomic imprinting, X chromosome inactivation, the repression of retrotransposons and cell differentiation. In the context of mammalian embryonic development, DNA methylation undergoes genome-wide reprogramming in primordial germ cells (PGCs). Despite a huge research effort, the mechanism underlying DNA demethylation in PGCs is unclear. Limited progress is largely due to technical challenges. Many widely used analytical techniques require large amounts of biological material and are therefore inappropriate for studies using small numbers of cells isolated from developing embryos. Our understanding of the mechanisms behind the developmental reprogramming processes may therefore be greatly enhanced by the use of complementary model systems and alternative technologies. The experiments described herein focus on the development and optimisation of a number of in vitro and in vivo systems to explore the nature of epigenetic DNA modifications, including 5-methylcytosine, and the mechanisms that dictate dynamic changes in the epigenetic landscape. The development and validation of sensitive LC-MS methods facilitates the quantification of DNA modifications, potentially enables the discovery of novel modifications, and by combining LC-MS with isotope tracing, may fundamentally challenge our current understanding of the stable nature of DNA methylation. Ultimately, we envisage that these approaches will form a versatile and informative platform to study genome-wide epigenetic reprogramming processes in an in vivo context. In many respects, loss of 5-methylcytosine in migratory PGCs mirrors the loss of 5-methylcytosine in mESCs upon transition from serum-containing to 2i medium. Accordingly, I used this accessible system to probe the contribution of DNA methyltransferases, putative DNA demethylases, their cofactors and binding partners to the loss of 5-methylcytosine in nascent PGCs. I show that loss of Dnmt3a isoform 2 is necessary for 2i-induced DNA demethylation. Furthermore, this loss is dependent on RNA-induced silencing. More generally, miRNAs appear to be necessary for the epigenetic reprogramming of mESCs to the naive ground state. It is possible that the same miRNAs are involved in DNA demethylation in migratory PGCs. I also show that retinol has an unexpected effect on the rate and extent of DNA demethylation. How this finding translates in the context of primordial germ cell development is unknown. It does, however, highlight both a potential source of variation in the published data and the need to understand the interplay between cell metabolism and epigenetic reprogramming.
Supervisor: Hajkova, Petra Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral