In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.

Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.

Brain tumours account for the most frequent type of solid tumours among children. Despite advances in surgery and chemotherapy, brain tumours are still the main cause of cancer deaths in children. Furthermore, little is known about DNA methylation changes in paediatric astrocytoma. Recent investigations suggest that many tumours are initiated not only by genetic abnormalities, but also caused by epigenetic changes. DNA methylation is a key epigenetic mechanism that controls the regulation of gene expression. Interestingly, unlike DNA mutations, epigenetic abnormalities are reversible. The reversibility of epigenetic abnormalities upon pharmacological unmasking has prompted interest in developing epigenetic therapy with the crucial goal of restoring the expression of aberrantly silenced genes. The focus of this study was to utilise a combination of different microarray strategies to develop an integrative candidate gene approach to identify several novel frequently methylated genes in a cohort of paediatric HGA (High grade glioma) samples. In addition, to investigate the potential of therapeutic efficacy of a DNA methyltransferase inhibitor, 5-Aza-dC in paediatric HGA. There were 147 genes commonly identified to be potentially methylated in IN699 cells using the two different array strategies integration; re-expression array and Illumina Infinium Human Methylation 450k array. Furthermore, using two complementary microarray strategies including methylation 450k array and expression array, this work identified 55 genes that were both methylated and under-expressed in these HGA cultures. Following validation with CoBRA and RT-PCR coupled with the response of hypermethylated promoters to the demethylating agent 5-Aza-dC, six novel genes (CXCL14, PRR5L, ELTD1, ITGA2, KRT8 and NTM) that are frequently silenced in paediatric astrocytoma were identified. This study suggests that re-expression of ii CXCL14 inhibited the colony formation and cell growth and reduces the migration rate significantly in IN699 short term culture and likely have functional significance in the development of paediatric HGA and an excellent candidate gene for further analysis. In parallel, the efficacy of 5-Aza-dC treatment was examined in paediatric HGA aiming to introduce this epigenetic therapy as a potential mechanism in management of this tumours. This study demonstrated that, relatively low dose of 5-Aza-dC sharply reduced the colony formation and inhibited proliferation and not through the apoptotic effect. It is likely that this reduction in proliferation without cell death is due to using relatively low doses that do not acutely kill cells, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Taken together, this work contributes to a more detailed understanding of the effect of epigenetic silencing on paediatric HGA. This investigation also demonstrated the use of epigenetic drug, 5-aza-dC to reverse the gene silencing for the potential treatment of paediatric HGA.

No description available.

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.

DNA methylation is an important epigenetic mark spanning all of life's kingdoms. In humans, DNA methylation has been associated with a wide range of age-related pathologies, including type II diabetes and cancer. More recently, in humans, changes in DNA methylation at specific positions in the genome have been found to be predictive of chronological age. Interestingly, DNA methylation age is also predictive of health status and time-to-death. A better understanding of what these DNA methylation changes represent and whether they might be causative in the ageing process will be important to ascertain. However, at present there is no animal model system with which this process can be studied at a mechanistic level. Furthermore, it is becoming increasingly apparent that many disease states that increase in prevalence with age are not caused by all cells within the individual, but are often the result of changes to a subset of cells. This underscores the importance of studying these processes at the single cell level. The recent advances in single cell sequencing approaches now mean that we can study multiple layers of biology within the same single cell, such as the epigenome and the transcriptome (scM&T-Seq). Unfortunately, we are still only able to probe these important aspects of single cell biology in a static sense. This is a major limitation in the study of ageing because ageing and age-related disease processes are inherently dynamic. As such, it is incumbent upon us to develop approaches to assay single cell biology in a dynamic manner. 
In this thesis, I describe an epigenetic age predictor in the mouse. This predictor is tissue-independent and can accurately predict age (with an error of 3.33 weeks) and can record deviations in biological age upon interventions including ovariectomy and high fat diet both of which are known to reduce lifespan. Next, I describe the analysis of a homogeneous population of muscle satellite cells (MuSCs) that I have interrogated at the single cell level, using single cell combined transcriptome and methylome sequencing (scM&T-seq). I found that with age there was increased global transcriptional variability and increased feature-specific methylome variability. These findings explain the loss of functionality of these cells with age. Lastly, I describe two imaging approaches to study DNA methylation dynamically in single cells. Using these methods, I demonstrate that it is possible to accurately determine methylation status across a wide spectrum of global methylation levels and that by using such approaches novel information about dynamic methylation processes can be obtained. These methods represent the first to study DNA methylation dynamically.

<p>DNA methylation is an epigenetic modification that is nearly ubiquitous. Eukaryotic DNA methylation contributes to the regulation of gene expression and maintaining genome integrity. In mammals, DNA methylation occurs primarily on the C5 carbon of cytosine in a CpG dinucleotide context and is catalyzed by the DNA methyltransferases, DNMT1, DNMT3A and DNMT3B. While <i>dnmt3a</i> and <i>dnmt3b</i> genes are highly homologous, the enzymes have distinct functions. Some previous reports suggested differences in the enzymatic behavior of DNMT3A and 3B, which could affect their biological roles. The goal of my thesis work was to characterize kinetics mechanisms of DNMT3A and 3B, and to identify the similarities and differences in their catalytic properties that contribute to their distinct biological functions. Given the sequence similarity between the enzymes, we asked whether DNMT3B was kinetically similar to DNMT3A. In a series of experiments designed to distinguish between various kinetics mechanisms, we reported that unlike DNMT3A, DNMT3B methylated tandem CpG on DNA in a processive manner. We also reported that the disruption of the R-D interface, critical for the cooperativity of DNMT3A, had no effect on DNMT3B activity, supporting the non-cooperative mechanism of this enzyme. </p> <p>DNMT3A is frequently mutated in numerous cancers. Acute Myeloid Leukemia (AML) is a malignancy of hematopoietic stem cells in which numerous patients exhibit a high frequency of the heterozygous somatic mutation Arg882His in DNMT3A. Through thorough consensus motif building, we discovered a strong similarity in CpG flanking sequence preference between DNMT3A Arg882His variant and DNMT3B enzyme. Moreover, we found that the variant enzyme has the same kinetics mechanism as DNMT3B, indicating a gain-of-function effect caused by the mutation. This change is significant because the variant enzyme can aberrantly methylate DNMT3B targets in AML cells and effect global gene expression. In particular, given that DNMT3B has been shown to have oncogenic properties, this suggests that the Arg882His variant can acquire similar oncogenic properties and drive AML development.</p> <p>Taken together, my thesis work provides novel insights into the relationship between the biochemical properties and the biological functions of DNMT3A and 3B. </p>