| Summary: | A dissertation submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, 2020 === Vitamin D is a fat-soluble hormone that is principally obtained from the sun, but also from the diet. The biologically active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), synthesized within the body upon UVB exposure, plays a central role in cell growth and differentiation, and has been reported to be essential in maintaining a healthy epigenome, with reported effects on histone modification and DNA methylation. DNA methylation refers to the addition of a methyl group on the 5thcarbon of a cytosine ring, generally in the sequence context of a CpG dinucleotide. Healthy cells are known to have hypomethylated CpG islands in regulatory regions of tumour suppressor genes, while cancer cells are characterised by global hypomethylation and hypermethylated regulatory regions of tumour suppressor genes. This global hypomethylated state results in dysregulated gene expression and chromosomal instability, while promoter hypermethylation in tumour suppressor genes inhibits expression. Methylation patterns are established and maintained by DNA methyltransferase (DNMT) 3A and 3B, and DNMT1, respectively. The demethylation process involves the ten-eleven translocation enzymes (TETs), including TET1, TET2 and TET3. The interplay between DNMTs and TETs are essential in establishing and maintaining methylation patterns. Notably, vitamin D deficiency (< 20ng/mL) has been associated with breast cancer susceptibility and fatality. Vitamin D supplementation has also been associated with increased global methylation in leukocytes and has been reported to interact with DNA methylation to influence breast cancer risk. However, the detailed molecular mechanism linking vitamin D to DNA methylation is still unclear and in vitro evidence supporting the observed cohort correlations is lacking. Given the genomic effects of vitamin D, it was hypothesized that vitamin D increase DNA methylation by increasing DNMT expression or inhibiting TET expression. The aim of this study was to assess the molecular mechanism governing vitamin D-induced changes in global DNA methylation in MCF-7 breast cancer cells. In vitro cell culture models for MCF-7 cells and embryonic kidney cells (HEK293 – serving as a control) were supplemented with or without 10 nM or 100 nM 1,25(OH)2D3. After 18 h, DNA was extracted from the cells for methylation and hydroxymethylation analysis, and RNA for the gene expression study. 10 nM 1,25(OH)2D3 significantly induced DNMT3B expression level (P < 0.010), while decreasing TET3 expression (P < 0.010), to significantly increase global methylation level in MCF-7 cells (P < 0.050). Additionally, 10 nM 1,25(OH)2D3 significantly increased the tumour suppressor-targeting TET1 and TET2 expression in MCF-7 cells, suggesting that the reported anticancer effects of vitamin D may be related to demethylation of tumour suppressor genes as a results of vitamin D induced TET expression Therefore, the results suggest that vitamin D increases global DNA methylation in MCF-7 breast cancer cells by decreasing TET3 and increasing DNMT3B expression to increase de novo methylation, thereby increasing genome stability, while decreasing promoter methylation of tumour suppressor genes by enhancing TET1 and TET2 expression. Importantly, vitamin D had minimal effects on the global methylation levels in the embryonic kidney cells, with no change in TET2 and TET3 expression, or DNMT3 level, suggesting that vitamin D acts specifically to correct aberrant hypomethylation. Thus maintaining a sufficient vitamin D status could be key in promoting good health and well-being === CK2021
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