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(2020)ThesisDNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue.
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(2021)ThesisRNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.
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(2021)ThesisUpon fertilisation of vertebrate embryos, the epigenomes of the responsible gametes need to be reconfigured into a state that is compatible with totipotency and zygotic transcriptional programs. Furthermore, the epigenomes of differentiating cells then need to be remodelled again in order to form the complex structures of the body, such as the vastly intricate nervous system. This includes, but is not limited to, the remodelling of DNA methylation, the most abundant DNA modification in vertebrates with critical roles in embryogenesis and neurodevelopment. In mammals, methylation of cytosines in cytosine-guanine dinucleotides (mCG) is almost completely erased after fertilization before it is re-established during gastrulation. Similarly, methylation of cytosines outside the CG context (mCH; H = A,T,C) is diluted in the early mammalian embryo before it is re-established mainly in the nervous system. However, in non-mammalian vertebrates, it appears that no global erasure of mCG takes place, raising questions about their propensity for transgenerational epigenetic inheritance. Additionally, the conservation of mCH in non-mammalian vertebrates is largely unexplored. In this thesis, I look to expand our knowledge on the developmental dynamics, evolutionary conservation and the molecular components of DNA methylome remodelling in vertebrates by studying methylome dynamics in two distantly related teleost species (ray-finned, protruding jawed fish). I functionally explore how DNA methylation is regulated during the development of zebrafish (Danio rerio), medaka (Oryzias latipes), and zebrafish-medaka hybrids, in both the CG and CH context. I employ CRISPR/cas9 technology, whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and RNA sequencing (RNA-seq), to interrogate a wide range of developmental time points and adult tissues. Overall, I have: i) developed a system to functionally test for regulators of developmental DNA methylation; ii) revealed a novel form of developmentally remodelled mCH in zebrafish and medaka which is deposited by the teleost specific DNMT3BA enzyme, iii) demonstrated evolutionary conservation of mammalian-like mCH features in the developing zebrafish nervous system, and iv) shown that DNA methylome dynamics in medaka and zebrafish embryos are highly comparable and compatible during the first 24 hours of zebrafish-medaka hybrid development. Altogether, this work greatly expands our understanding of the form and function of a critical DNA modification during development.
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(2022)ThesisA breakdown in B cell self-tolerance can lead to antibody-mediated autoimmune disease. This thesis aims to explore how B cell tolerance can be broken in two distinct, but complementary projects within the context of the Goodnow somatic mutation hypothesis. In both scenarios, B cells that escape self-tolerance and generate autoantibodies are referred to as “rogue” B cells. First, this thesis aimed to elucidate the precise steps undertaken by expanded rogue B cell clones in patients with chronic Hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis, an autoimmune disease characterised by the production of a rheumatoid factor cryoglobulin autoantibody. The rogue B cell clones in the HCV cryoglobulinemic vasculitis patients were confirmed to be the source of the autoantibody. The rogue B cell clone precursor antibodies failed to bind the HCV envelope glycoprotein E2, yet bound multimerised self-antigen IgG relative to membrane IgM density. These findings disfavour a molecular mimicry hypothesis, and instead indicate IgG immune complexes may be sufficient to drive recruitment of the rogue B cell clone precursors. Finally, the rogue B cells clones were found to carry somatic lymphoma-associated, non-immunoglobulin gene mutations and chromosomal aberrations, predicted to cause hyperactivation of the NF-kB signalling pathway and escape of B cell tolerance. This finding provides additional evidence in support of the Goodnow somatic mutation hypothesis. Second, this thesis examined rogue germinal centre (GC) B cells that arise in the absence of the receptor FAS. Rogue GC B cells loose specificity for the foreign antigen and incidentally generate autoantibodies. However, the accumulation of rogue GC B cells cannot be explained by our current understanding of affinity-based selection in the GC. This work revealed rogue GC B cells, unlike “conventional” GC B cells undergoing affinity maturation to the foreign antigen, can be identified by low expression of CD21 and high expression of B220 (CD21loB220hi). Moreover, rogue GC B cells were found to be rapidly entering cell cycle, enriched for a dark zone phenotype and T-cell dependent, reminiscent of positively selected GC B cells. Thus, rogue GC B cells typically removed by FAS, likely persist in the competitive GC microenvironment despite their loss of BCR specificity to foreign antigen, because they retain the capacity to undergo T-cell dependent positive GC selection.