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Investigating the Epigenetic Mechanism Behind Transgenerational Inheritance in Mice with Abnormal Folate Metabolism

  • Blake, Georgina Emma Tallulah
Publication Date
Dec 20, 2018
Apollo - University of Cambridge Repository
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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 ($\it{Mtrr^gt}$). Remarkably, $\it{Mtrr^+/gt}$ 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 $\it{Mtrr^gt}$ model: germ cell morphology and function abnormalities, genetic instability and altered germ cell epigenetic patterns. We first considered if the $\it{Mtrr^gt}$ mutation affected testes morphology, spermatogenesis or sperm parameters. These were largely normal in $\it{Mtrr^gt}$ 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 $\it{Mtrr^gt/gt}$ embryos indicating that the $\it{Mtrr^gt}$ mutant genome is relatively stability. We did however identify an increase in SNP and SV frequency at the $\it{Mtrr}$ locus, linked to the generation of the $\it{Mtrr^gt}$ 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 $\it{Mtrr^+/+}$, $\it{Mtrr^+/gt}$, and $\it{Mtrr^gt/gt}$ males compared to C57Bl/6 control sperm. Few DMRs were associated with underlying SNPs or SVs. However, no sperm DMRs identified in $\it{Mtrr^+/gt}$ males persisted in embryonic or adult tissues of the wildtype F1 or F2 generations. Despite this, we identified two genes, $\it{Hira}$ and $\it{Rn45s}$, 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 $\it{Mtrr^+/gt}$ and $\it{Mtrr^gt/gt}$ sperm compared to C57Bl/6 control sperm. Overall, a complete understanding of the mechanisms behind transgenerational inheritance of phenotypes in the $\it{Mtrr^gt}$ model remains elusive. Unravelling the mechanisms of transgenerational epigenetic inheritance could have important implications for the future prediction and prevention of human diseases. / Wellcome Trust 4-year PhD studentship in Developmental Mechanisms. Additional funding from St John's college and the Cambridge Philosophical Society.

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