Medical Biology - Theses
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ItemNo Preview AvailableDevelopmental Control of Hox Genes by the Epigenetic Regulator SMCHD1( 2023-05)Epigenetic processes govern transcription to ensure precise timing and location of gene expression during development. Structural Maintenance of Chromosomes Hinge Domain containing 1 (SMCHD1) plays a key role in the epigenetic silencing of the inactive X chromosome (Xi) and several autosomal clustered gene families, including the Hox clus- ters. SMCHD1 also facilitates ultra-long-range chro- matin interactions on the autosomes and the Xi, and CTCF binding is increased in its absence, suggesting that SMCHD1 functionally opposes CTCF. To elucidate SMCHD1’s mechanism of Hox gene silencing, I used a range of genomic and transcriptomic techniques to profile gene expression and chromatin architecture in vivo and in vitro from several Smchd1 mutant mouse lines. Recent work has shown that maternal SMCHD1 contributes to the silencing of H3K27me3- controlled non-canonical imprinted genes. As Hox genes are also marked by H3K27me3, I asked whether maternal SMCHD1 plays a role in their regulation. I found that knocking out Smchd1 in the oocyte caused an overexpression of Hox genes and homeotic transformations in the axial skeleton of the post-implantation embryo. This is the first case of a maternal effect on Hox genes in mammals, as genetically identical offspring with SMCHD1 in the oocyte exhibited wildtype axial patterning and Hox expression. I also demonstrated that Hox gene upregulation in the absence of maternal SMCHD1 did not coincide with detectable changes in Polycomb marks H3K27me3 and H2AK119ub, DNA methylation or the chromatin architecture of the Hox clusters. I therefore propose that SMCHD1 is required to set up an as yet undefined chromatin state preimplantation that persists through development to ensure correct timing of Hox expression post-implantation. Evidence from the Blewitt Lab and others has shown that SMCHD1 is reliant upon the H2AK119 ubiquitination capacity of PRC1 to bind to the Xi. To test whether this is also the case for SMCHD1’s autosomal targets, including the Hox genes, I carried out H2AK119ub ChIP-seq in Smchd1 wildtype and null cells and found no difference in H2AK119ub between the genotypes, suggesting that SMCHD1 does not act upstream of H2AK119ub. I also optimised SMCHD1 ChIP-seq and identified 14,923 SMCHD1-bound loci, most of which are DNA-binding transcription factors with important roles in development. Three-quarters of these SMCHD1 targets were also enriched for H2AK119ub, suggesting that that SMCHD1 may act downstream of PRC1 to regulate their shared targets. Taken together, I propose that both maternal and zygotic SMCHD1 regulate Hox gene expression by acting downstream of Polycomb to form ultra-long-range chromatin in- teractions that functionally oppose CTCF to prevent precocious Hox gene activation. Maternal SMCHD1 has an additional unique role of setting up a mitotically heritable chromatin state preimplantation that ensures correct timing of Hox gene expression post-implantation.
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ItemComputational tools for long-read DNA methylation analysis and benchmarking complex single-cell genomics pipelines( 2023-03)Developing new high-throughput assaying techniques necessitates the development of novel bioinformatics software that can not only extract insight from newly generated data types, but also evaluate the efficacy of newly developed tools. To this end, I created the NanoMethViz package to enhance the exploratory data analysis of DNA methylation data obtained from ONT long-read sequencing through the provision of data management and visualisation tools. The application of this software to female mouse placenta and neural stem cell samples enabled the study of methylation patterns in the context of X-inactivation. Additionally, the proliferation of single-cell analysis techniques and the need for comprehensive pipeline-level benchmarking led me to create the CellBench package, which can automatically execute complete combinations of methods to fully characterise the performance of single-cell analysis pipelines. This package establishes a benchmarking framework for combinations of methods that promotes modular code without duplication, resulting in readable, reproducible, and extensible pipeline benchmarking code. Both packages are open source and available through the R/Bioconductor repository, providing useful support for researchers who are working with these emerging and quickly advancing genomic technologies.
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ItemUnravelling the epigenetic modifier Smchd1( 2020)Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic modifier that plays an important role in X chromosome inactivation, autosomal gene silencing, and is also implicated in several diseases in humans (Blewitt et al., 2008, Mould et al., 2013, Gendrel et al., 2013, Lemmers et al., 2012, Gordon et al., 2017, Shaw et al., 2017, Jansz et al., 2018). Thus far the majority of work on Smchd1 has been carried out on its zygotic form. Therefore, I partnered allele-specific genomics with conditional deletion of Smchd1 in the oocyte to test the role of maternally derived Smchd1 in imprinted gene expression. I found that Smchd1 is a novel maternal effect gene involved in genomic imprinting. When Smchd1 is maternally deleted loss of imprinting is observed at ten imprinted genes, without affecting DNA methylation imprints. Additionally, I also discovered seven imprinted genes where zygotic Smchd1 plays a dose dependent role. Interestingly, almost all the imprinted genes affected by the loss of maternal Smchd1, including Xist possess H3K27me3 imprints. This, together with Smchd1’s known role in long range chromatin interactions and function as insulator protein(Chen et al., 2015, Jansz et al., 2018), lead me to hypothesise that maternal Smchd1 may carry out its function secondary to H3K27me3 imprints and establish a chromatin state required for imprinted expression. To narrow down Smchd1’s behaviour in its native environment and how this behaviour relates to its structure and function, I established a series of robust microscopy experiments. I used fluorescence recovery after photo bleaching (FRAP) and Lattice LightSheet microscopy to demonstrate for the first time, Smchd1 dynamics in the inactive X during interphase and mitosis. Through study of our unique gain of function mutant MommeD43 I found that the act of unbinding and reloading likely has a role in Smchd1’s insulation effects. By using 3D-direct stochastic optical reconstruction microscopy (3D-dSTORM) I discovered that Smchd1 is approximately a 40 nm long protein in the nucleus. These novel techniques open up new avenues to explore higher order structures that Smchd1 may form in the nucleus related to its role in chromatin architecture. Together these data not only revealed a new function for maternal Smchd1 but also established novel methods to unravel Smchd1's molecular mechanism.
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ItemFunctional and structural characterisation of the epigenetic regulator, SMCHD1( 2020)Structural Maintenance of Chromosomes Hinge Domain-containing protein 1 (SMCHD1) has been established as an epigenetic regulator, with critical roles in X-chromosome inactivation, autosomal gene silencing and genomic imprinting. Recently, variations in SMCHD1 have been associated with two human conditions: facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). There has therefore been a growing interest in unveiling SMCHD1’s atomic structure and the molecular mechanisms underlying its function in both a healthy and diseased state. To provide a better understanding of Smchd1’s molecular structure and function, I successfully expressed and purified the full-length 2007-amino acid mouse Smchd1 protein. Electron microscopy analyses of the Smchd1 dimer revealed an elongated rod-like structure that displays a high conformational flexibility, similar to that of other structural maintenance of chromosomes (SMC) proteins. This flexibility is largely conferred by the intermediate region of the protein that connects Smchd1’s two functional domains: the N-terminal GHKL ATPase and the C-terminal SMC hinge domain. In follow-up studies of the two individual domains, we revealed the first atomic-resolution structure of Smchd1’s hinge domain, providing a novel insight into its DNA-binding and dimerisation modes. Contrary to previously suggested models describing the DNA interaction mode of canonical SMC proteins, I showed that nucleic acids are not threaded through the central pore region of the Smchd1 hinge domain. Subsequent immunofluorescence studies additionally revealed that the hinge domain targets full-length Smchd1 to chromatin, and that a functional hotspot within the hinge is required for chromatin localisation in cells. SMCHD1’s ATPase domain has been of particular interest due to the identification of disease-related variants that are frequently located within this region of the protein. However, the mechanisms by which some of these pathogenic variants affect SMCHD1 function are poorly understood. Using analytical ultracentrifugation, I demonstrated that the wild-type SMCHD1 ATPase undergoes dimerisation, which was reliant on the inclusion of both the UBL domain and the presence of substrate, ATP. Follow-up cellular studies revealed that Smchd1’s catalytic activity, as well as the presence of the newly- identified UBL domain, are both necessary for the localisation of full-length Smchd1 to chromatin. Together, these studies provide an insight into the molecular basis of Smchd1 function and highlight how chromatin binding may be compromised in human disease. Future studies will further investigate the cellular localisation and dimerisation properties of disease-associated SMCHD1 variants, contributing towards our ongoing drug development program aimed at developing therapeutic treatments for FSHD patients.
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ItemManipulating the humoral immune response using epigenetic modifiers( 2020)The generation of protective antibody is one of the most important parts of the humoral immune response and is the basis for the vast majority of successful vaccination strategies. Antibody is produced by rare populations of differentiated B cells, known as plasmablasts and plasma cells. The differentiation of B cells into antibody secreting cells (ASCs) is complex and highly orchestrated by vast array of mechanisms, including epigenetic regulation. Broadly speaking, epigenetics describes all non-genetic regulation of gene expression. Hence, modifications to the chromatin, but not the underlying DNA sequence, result in altered gene expression. In recent years, epigenetic modifying compounds (EMCs) have emerged as potential therapeutic agents for the treatment of haematological malignancies and immune disorders. However, it is now clear that EMCs also modulate the immune response via both direct and indirect mechanisms. Despite the extensive studies on EMCs, the precise functional role of many of these compounds remains unknown. This thesis explores the effects of two EMCs that have previously been shown to affect the antibody response. Specifically, the Brd4 inhibitor JQ1 and GSK126, an Ezh2 inhibitor. Using quantitative analysis, I examined the effects of each EMC on different parameters that combine to control the magnitude of the antibody response. By combining functional analysis with transcriptomic and epigenomic studies, I investigated the precise molecular mechanism and gene targets of these EMCs. Thus, these studies provide the opportunity to identify novel regulators of antibody secreting response. I showed that JQ1 treatment dampens the antibody secreting response by targeting multiple parameters of B cell function, including cell proliferation and survival. The effects on B cell function were the result of global Brd4 displacement as opposed to previously suggested gene specific mechanisms. In addition, I identified the pro-apoptotic molecule Bim as the molecular target of JQ1 directly responsible for inducing apoptosis in stimulated B cells. Conversely, inhibiting Ezh2 increases B cell differentiation and antibody production of B cells. I showed that Ezh2 inhibition causes global downregulation of H3K27me3 without altering the genome accessibility. Genome-wide studies identified a number of novel regulators of Ezh2 inhibition induced ASC differentiation, including the Blimp-1 target Atoh8. Results from this thesis illustrate the strength of in vitro reductionist systems that combine functional analysis of cell biology with genomics to isolate epigenetic mechanisms that regulate immunity. JQ1 has a significant effect on B cells and has the potential to be used as a therapeutic agent to dampen the antibody secreting responses in autoimmunity, particularly those involving increased antibody production. In contrast, pharmacological inhibition of Ezh2 increases ASC differentiation and antibody production. Thus, it could potentially be used to boost antibody responses that could be applied to treat immunodeficiency or as a differentiation therapy in cancer models.
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ItemThe Role of Mutant DNMT3a in Ageing and in the Regulation of Normal and Malignant Haematopoiesis( 2020)DNA methyltransferase 3a (DNMT3a) is a de novo DNA methyltransferase that can establish DNA methylation signatures in cells. Recently, germline mutations in DNMT3a were found to cause an intellectual disability and overgrowth disorder named Tatton-Brown-Rahman syndrome and somatic mutations in DNMT3a constitute one of the most common mutations in haematological malignancies. The findings presented in this thesis inform on the role of the most common DNMT3a mutation, R882H, using a novel murine model with an emphasis on ageing, haematopoiesis and hematopoietic malignancies. The mutant Dnmt3a mouse model was created using CRISPR/Cas9 genome editing technology to introduce the most common R882H mutation into the murine Dnmt3a locus at residue R878H (murine homologue of R882). Breeding of Dnmt3aR878H/+ mice revealed an inability of female Dnmt3aR878H/+ mice to deliver healthy offspring. This was a result of a maternal defect as surrogate mice could produce viable Dnmt3aR878H/+ pups through IVF. Dnmt3aR878H/+ mutant mice also had a shorter lifespan compared to their wt littermates when aged. The Dnmt3aR878H/+ aged mice were more susceptible to liver disease that was characterised by extensive hepatocyte steatosis and hepatocyte carcinoma and were also more likely to develop leukaemia with B cell morphology compared to their wt littermates. To determine whether the Dnmt3aR878H/+ mutant mice had defects in haematopoiesis before overt haematological malignancy, the haematopoietic system was analysed under steady state conditions and in haematopoietic competition assays. There was evidence of a defect in early T cell development in the thymus characterised by significantly fewer immature T cell progenitors in Dnmt3aR878H/+ mutant mice compared to their wt littermates. To resolve whether Dnmt3aR878H/+ mutant haematopoietic stem and progenitor cells (HSPCs) had a competitive advantage over wt HSPCs, HSPCs from the Dnmt3aR878H/+ mutant mice were competitively transplanted alongside wt HSPCs into lethally irradiated wt recipient mice. It was shown that Dnmt3aR878H/+ HSPCs and their descendants outcompeted their wt counterparts after 6 months, with some evidence that Dnmt3aR878H/+ HSPCs had already begun to accumulate after 3 months. These findings were extended to show that Dnmt3aR878H/+ HSPC-derived cells can also outcompete wt HSPC-derived cells in other haematopoietic tissues, such as the thymus and spleen. Furthermore, it was also shown that Dnmt3aR878H/+ HSPCs also have an increased serial transplantation capacity compared to their wt counterparts. To better understand how the Dnmt3aR878H mutation promotes the development of haematological malignancies, a model of g-irradiation induced thymic lymphoma was employed where the cancer cell of origin arises from a HSPC. It was shown that Dnmt3aR878H/+ mutant mice developed thymic lymphoma at a significantly faster rate than their wt littermates. Gene expression changes in Dnmt3aR878H/+ HSPCs that might account for their increased predisposition to leukaemogenesis revealed that Dnmt3aR878H/+ LSK cells have an underlying disturbance in Notch signalling and that upon g-irradiation, they have a blunted induction of the p53 signalling network compared to wt HSPCs. Many other cellular pathways were also deregulated in Dnmt3aR878H/+ HSPCs, and they will be the subject of future experiments. Overall, it was shown that heterozygous Dnmt3aR878H mutations cause a vast array of abnormalities including problems in pregnancy, metabolic defects leading to obesity and liver pathologies as well as haematological disturbances leading to an accumulation of HSPCs in the bone marrow and a susceptibility to the development of haematological malignancies.