Modulation of Epigenetic Mark 5-hydroxymethylcytosine in Intestinal Organoid Cultures

Chromatin regulation plays a role in establishing and maintaining cell identity, and is generally highly dynamic between stem and differentiated cells. The intestinal epithelium is a monolayer of cells that is replaced every 3-5 days, and is thus a rapidly self-renewing and differentiating tissue. It is not currently understood how the integrity of the intestinal stem cell (ISC) genome is maintained or how the identity of a differentiated cell is dictated. Vital to chromatin regulation and gene expression are epigenetic modifications, such as methylation. The addition of methyl groups to the nucleotide cytosine silences genes, while demethylation allows gene expression. Demethylation pathways produce the epigenetic mark 5-hydroxymethylcytosine (5-hmC) as an intermediate. The conversion of 5-methylcytosine (5mC) to 5-hmC is accomplished by ten-eleven translocation (TET) enzymes, which require co-factors, such as the micronutrient alpha-ketoglutarate (a key intermediate in the Krebs cycle). Assessing the global 5-hmC abundance among distinct cellular populations will elucidate this epigenetic mark’s role in determination of cell fate. To investigate this aim, murine intestinal organoid cultures were exposed to varying dosages of dimethyl 2-oxoglutarate, a cell permeable form of alpha-ketoglutarate, in order to modulate TET activity and thus 5-hmC abundance. Through dot blot analysis of 5-hmC abundance, the ability to modulate the epigenetic mark was assessed. Results indicate that exposure of intestinal organoids to dimethyl 2-oxoglutarate leads to phenotypic differences compared to controls, but these differences cannot be attributed to 5-hmC abundance. Understanding the differences in 5-hmC across cellular populations will provide insight into the role of epigenetic modifications in regulating gene expression. Furthermore, the impact of micronutrients on the genome emphasizes the significance of cellular environment for cellular identity.Bachelor of Scienc