Medical Biology - Theses
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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.