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Title: Investigating the epigenetic mechanism behind transgenerational inheritance in mice with abnormal folate metabolism
Author: Blake, Georgina Emma Tallulah
ISNI:       0000 0004 7961 9366
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Exposure to environmental stressors can impact our health and that of future generations even when they are not similarly exposed. How disease risk is inherited is unclear. My thesis focuses on a mouse model of transgenerational inheritance in which folate metabolism is disrupted by a mutation in Methionine synthase reductase (Mtrr^gt). Remarkably, Mtrr⁺/gt heterozygosity leads to an increased likelihood of a wide spectrum of congenital malformations in their wildtype offspring for at least four generations. Folate metabolism is required for DNA synthesis and cellular methylation. Folate and its metabolism have also been linked to spermatogenesis and male fertility. My thesis aims to explore three possible mechanisms for the transgenerational inheritance of congenital malformations in the Mtrr^gt model: germ cell morphology and function abnormalities, genetic instability and altered germ cell epigenetic patterns. We first considered if the Mtrr^gt mutation affected testes morphology, spermatogenesis or sperm parameters. These were largely normal in Mtrr^gt males. Next, we performed whole genome DNA sequencing of Mtrr embryos to determine whether abnormal folate metabolism affects genetic stability. Importantly, the frequency of structural variants and single nucleotide polymorphisms (SNPs) were similar in C57Bl/6 control and Mtrr^gt/gt embryos indicating that the Mtrr^gt mutant genome is relatively stability. We did however identify an increase in SNP and SV frequency at the Mtrr locus, linked to the generation of the Mtrr^gt mice in a 129/P2 genetic background prior to backcrossing into the C57Bl/6 background. Subsequently, we identified a large number of differentially methylated regions (DMRs) in sperm DNA of Mtrr⁺/+, Mtrr⁺/gt, and Mtrr^gt/gt males compared to C57Bl/6 control sperm. Few DMRs were associated with underlying SNPs or SVs. However, no sperm DMRs identified in Mtrr⁺/gt males persisted in embryonic or adult tissues of the wildtype F1 or F2 generations. Despite this, we identified two genes, Hira and Rn45s, that were adjacent to DMRs and were misexpressed in the F2 and F3 generation embryos. This suggested that abnormal DNA methylation in sperm may influence gene expression two generations later despite reprogramming events. Additionally, we identified a number of differentially expressed small non-coding RNAs in Mtrr⁺/gt and Mtrr^gt/gt sperm compared to C57Bl/6 control sperm. Overall, a complete understanding of the mechanisms behind transgenerational inheritance of phenotypes in the Mtrr^gt model remains elusive. Unravelling the mechanisms of transgenerational epigenetic inheritance could have important implications for the future prediction and prevention of human diseases.
Supervisor: Watson, Erica ; Ferguson-Smith, Anne Sponsor: Wellcome Trust ; St John's College ; Cambridge Philosophical Society
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Folate metabolism ; DNA methylation ; Epigenetics ; sncRNA ; Genetic stability ; Mtrr ; Transgenerational inheritance ; Epigenetic Inheritance