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Title: Role of TET1 in adipose tissue
Author: Nicholson, Bonnie May
ISNI:       0000 0004 7969 1376
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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The global obesity epidemic is associated with 2.8 million deaths per year and contributes to the prevalence of numerous cardiometabolic disease risk factors, including non-alcoholic fatty liver disease (NAFLD), hypertension, type 2 diabetes, hyperlipidaemia and stroke. The Ten-Eleven-Translocation enzymes (TET1-3) modify DNA by adding a methyl group to generate 5-hydroxymethylcytosine (5hmC) from 5-methylcytosine (5mC). This DNA methylation mark can alter the nature of the information conveyed by DNA; for example by modulating access of transcription factors. TET1 plays an important role in promoting adipocyte differentiation. I hypothesised that Tet1 gene deficiency impairs adipocyte development and function, thereby counteracting the development of obesity. TET enzymatic activity requires the TCA cycle metabolite α-ketoglutarate (α-KG) as a substrate, and is allosterically inhibited by TCA cycle metabolites succinate and fumarate. Obesity is associated with a number of key metabolic changes including increased levels of glutamate and many constitutive metabolites of the tricarboxylic acid (TCA) cycle (such as α- ketoglutarate, succinate, fumarate, and malate). I therefore additionally hypothesised that TET activity is altered as a consequence of the metabolic perturbations that accompany obesity. To test these hypotheses, male Tet1 gene knockout (Tet1-/-) mice and littermate mice wildtype for the Tet1 allele (Tet1+/+) were fed a high-fat diet (HFD) for 11 weeks. Food intake, adiposity (TD-NMR), energy expenditure (indirect calorimetry) and fat depot mass, including bone marrow adipose tissue (MAT) were measured. RNA-sequencing of adipose tissue gene expression and analysis of 5hmC in adipose tissue DNA was performed. Fat mass of Tet1-/- mice was 50% lower than that of Tet1+/+ littermates after 11 weeks HFD. No changes in MAT were observed. Tet1-/- mice showed a relative reduction in food intake over 11 weeks and a reduction in energy expenditure in the absence of changes in activity levels. RNA-sequencing of mesenteric adipose tissue revealed changes in gene expression related to muscle organ development, synaptic vesicle exocytosis and acyl-coA metabolic processes in Tet1-/- mice. Notably, leptin mRNA and circulating leptin levels were lower in Tet1-/- mice compared to Tet1+/+. Tet1-/- mice challenged with exogenous leptin had a greater reduction in food intake, indicating higher leptin sensitivity. Hydroxymethylated DNA immunoprecipitation-sequencing (hMeDIP-seq) was performed to analyse 5hmC patterns: however, no global differences in 5hmC levels were identified between Tet1-/- and Tet1+/+ mice. Specifically, despite the marked change in leptin mRNA expression, no changes in 5hmC or 5mC were observed within the leptin promoter to indicate causality of DNA methylation changes in altered leptin expression. Brown adipose tissue (BAT) is the main organ of thermogenesis in rodents and has a high energy demand when activated. The recent discovery of active BAT, or an intermediate thermogenic fat called "beige", in human adults has prompted research into the potential of therapeutic BAT activation for the treatment of obesity. A number of epigenetic marks, of which DNA methylation is one, have been associated with activation of thermogenesis in BAT including histone acetylation. However, the potential role of 5mC and 5hmC in BAT function has not been investigated. While histone modifications - most notably histone acetylation in the enhancer regions of Pparg and Ucp1, and putative regulatory regions of Ppara - have been shown to play a role in the transcriptional activation of the thermogenic gene programme, the role of 5mC and 5hmC has not been investigated. I hypothesised that TET1 activity would be altered by changes in metabolite levels in cold-induced thermogenesis, resulting in altered 5hmC. To test this hypothesis, C57BL/6J mice were maintained at 30°C or 4°C for 48 hours, after which adipose tissue was collected. RNA and DNA were extracted from white (WAT), brown (BAT) and beige adipose tissue for RNA-seq and hMeDIP-seq, and the polar phase metabolites were extracted for gas chromatography mass-spectrometry. In mice exposed to 4°C, all metabolites of the tricarboxylic acid (TCA) cycle were significantly increased in WAT and BAT compared to mice housed at 30°C, with the exception of α-KG and succinate in BAT. TET activity was decreased in WAT at 4°C but not in BAT or beige adipose tissue. No global changes in 5hmC were observed in WAT or BAT between the 4°C and 30°C conditions. However, there is a decrease in 5hmC in BAT at 30°C and 4°C, and in WAT at 4°C, at 10% of gene length in a select subset of genes involved in neuromuscular synaptic transmission and perception of chemical stimuli. Many gene pathways were upregulated transcriptionally in all three adipose depots, including mitochondrial function, oxidative phosphorylation and the TCA cycle, but these transcriptional changes are not associated with changes in 5hmC. In summary, this data supports my original hypothesis by showing that Tet1-/- mice have decreased susceptibility to diet-induced obesity due to a reduction in food intake, in association with higher leptin sensitivity. However, in contrast to published data, 5hmC or 5mC modification was not altered at the leptin promoter in adipocytes, and therefore the hypothesis that DNA methylation at the leptin promoter accounts for the observed Tet1-/- phenotype must be rejected. Finally, numerous changes occur in the TCA cycle metabolites in BAT and WAT with cold exposure, which may contribute to the selective decrease in TET activity in WAT. However, no changes are observed in 5hmC in WAT, rejecting the hypothesis that altered TET1 activity in cold exposure results in altered 5hmC. In conclusion, this thesis contends that TET enzymes and 5hmC are not primary drivers of gene expression changes in obesity or cold-induced thermogenesis. However, TET enzymes may act in synergy with other epigenetic changes to alter gene expression in this context, and may remain important contributors to altered adiposity and its metabolic consequences.
Supervisor: Drake, Mandy ; Morton, Nicholas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: TET1 ; 5-hydroxymethylcytosine ; adipose tissue ; obesity ; cold exposure ; epigenetics