THE ROLE OF DNA REPAIR IN DNA METHYLATION DYNAMICS

The mammalian epigenome is globally reprogrammed at two stages of development; this involves the erasure and re-establishment of DNA methylation by both passive and active mechanisms, including DNA repair pathways, and occurs concurrently with an increase in developmental potency. In addition to Uhrf1 and the Tet enzymes, the interplay between activation induced cytidine deaminase (AID) and the DNA repair machinery has been implicated in epigenetic reprogramming of various in vivo and in vitro systems including mouse primordial germ cells, zygotes and induced pluripotent stem cells. AID deaminates cytosine to uracil and can also deaminate methylcytosine, whereas the primary role of UNG is to maintain the integrity of the genome through erasure of uracil. In this thesis, I have aimed to investigate the role of DNA repair in demethylation. To do this I have focused on the specific role of AID and UNG in the demethylation of a static system – primed serum ESCs and a dynamic system – serum to 2i (naïve) to epiblast-like ES cells. As the role of both AID and UNG involves genomic uracil, the central theme of my thesis is the impact of accumulation of uracil on DNA methylation levels in the genome. Therefore, my first aim was to develop a quantitative method to detect low levels of genomic uracil in DNA firstly, by mass spectrometry and secondly, by whole genome sequencing. In Chapter Three, I show that the impact of deamination during DNA preparation can be minimised, such that the level of genomic ESC uracil can be accurately determined as around 12,000 uracil per genome and that, as anticipated, Ung null ESCs have almost twice the genomic uracil content of wildtype ESCs. Secondly, I address the main question which is the impact of uracil accumulation on methylation levels. In order to do this, I generate two cell lines: Ung knockout and Aid over expressing, both of which should result in an increase in genomic uracil. I demonstrate that while over expression of Aid stimulates demethylation in static system and in a dynamic demethylating system, the impact of Ung knockout is less clear. In (static) serum ESCs, loss of Ung results in hypomethylation however, in order to transition to 2i (naïve) ESCs, a process which involves demethylation of the genome, it appears the Ung is required as loss of this gene inhibits proper demethylation. As such, I conclude that UNG-mediated DNA repair functions alongside passive demethylation, by reduction of UHRF1 levels, to demethylate 2i ESCs. To probe the mechanism by which accumulation of uracil in the genome alters methylation levels, I investigate the impact of Ung KO and Aid OE on global levels of DNA damage. I show that both cell lines have a greater incidence of double strand breaks compared to a wild type cell line, and accordingly, upregulate their DNA damage response pathway and the expression of certain repair genes. I suggest that increasing genomic levels of uracil causes genomic instability and that DNA demethylation occurs as a consequence of the repair of extensive DNA damage. More broadly, I suggest that ESCs are uniquely poised, due to their heightened DNA damage response, to use uracil as an intermediate of DNA demethylation. Interestingly, I also note that the biological impact on serum ESCs of loss of Ung appears to be an increase in pluripotency.MRC funded PhD via The Babraham Institut