TET mediated 5’hydroxymethylation in the pathogenesis of non alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) now affects around one in four adults in the human population and parallels the global increase in obesity. Within the spectrum of NAFLD, simple steatosis is associated with insulin resistance and type 2 diabetes while progression to steatohepatitis (NASH) is associated with an increased risk of liver cirrhosis and all-cause mortality. The molecular pathology of NAFLD is incompletely understood, however observational studies in human cohorts suggest the regulation of DNA methylation may play a role. 5-hydroxymethylcytosine (5hmC) is a cytosine modification generated from 5- methylcytosine (5mC) by the Ten eleven translocase isoenzymes (Tets) as part of a demethylation process. The aim of this project was to examine the role of Tet enzyme activity on the pathogenesis and progression of NAFLD. Detailed characterisation of two established murine dietary interventions allowed the selection of a NAFLD mouse model which broadly recapitulated the metabolic, histological and transcriptional features of human disease. Using DNA immunoprecipitation coupled with whole genome next generation sequencing and RNA micro expression arrays I examined the effect of high fat diet feeding (HFD) on hepatic DNA 5hmC levels within annotated gene regions. Whilst the global 5hmC profile was not altered by HFD, there was profound genic enrichment of 5hmC in upregulated mediators of cholesterol synthesis and transport (Lss, Sc4mol, Fdps, Hsd17b7, Cyp17a1, Mvd, Cyp1a2, Dhcr7 and Apoa4) with no enrichment in genes with other pathological functions (drug detoxification, inflammation, cell cycle regulation). Induced peaks of 5hmC enrichment were subsequently abolished following rescue of the NAFLD phenotype by conversion to control diet. Cross species validation was performed in vitro utilising embryonic stem cell derived hepatocytes challenged with a cocktail of high energy substrates. My in vivo findings were broadly replicated with specific 5hmC enrichment in genes synthesising lipotoxic molecules (PLIN2, CIDEC, APOA4, ACADVL, HMGCS2, APOA5, CYP2J2, IGFBP1, PPAP2C, ACSL1, APOC3, ANGPTL4, NRG1) with no enrichment in upregulated genes of alternative function. To determine whether or not the 5hmC enrichment seen is of functional relevance, I studied Tet1-/- C57BL/6J mice. Tet1-/- mice are grossly normal in appearance, however loss of Tet1 conferred a striking resistance to diet induced obesity with reduced body fat mass, improved insulin-sensitivity and near complete absence of NAFLD compared to wild type littermates. Furthermore, the HFD fed Tet1-/- liver transcriptome showed a ‘protective’ profile, with suppression of genes for lipid synthesis, inflammation and fibrosis. Thus, in multiple cross-species models of NAFLD, over nutrition induces genic hydroxymethylation specifically within activated genes driving the synthesis and transport of lipid molecules. Such changes are reversible with resolution of the NAFLD phenotype strengthening functional association. Tet1 deficiency conveys an obesity and NAFLD resistant phenotype. I therefore introduce Tet1 mediated hydroxymethylation as a novel mechanism for NAFLD pathogenesis.