The impact of 1α,25(OH)2D3 on genome-wide methylation

Abstract

Vitamin D is a fat-soluble hormone attained through UV exposure and diet. 1α,25(OH)2D3, the biologically active form of vitamin D, bound to its nuclear receptor, the vitamin D receptor, regulates gene expression and has been suggested to influence gene regulatory mechanisms such as the epigenome. As an epigenetic mark, DNA methylation functions to maintain genomic stability and regulate gene expression. Both vitamin D deficiency and aberrant DNA methylation marks is often associated with increased disease risk, suggesting a link between the two features. This study therefore aimed to investigate the impact of 1α,25(OH)2D3 on genome-wide methylation. To this end, genomic DNA was extracted from the THP-1 cells – a highly1α,25(OH)2D3 responsive monocytic cell line, MCF-7 breast cancer cells – a cell line known to show weak responsiveness to1α,25(OH)2D3, and HEK293 cells –an embryonic kidney cell line with highly active methylation machinery. The cells were supplemented with/without 10 nM 1α,25(OH)2D3 and subjected to reduced representation bisulfite sequencing. Read alignment was performed using Bismark aligner and differential methylation analysis was performed using MethylKit (to identify differentially methylated cytosines; DMCs) and eDMR (to identify differentially methylated regions; DMRs). Pathway analysis was conducted using cluster Profiler. 1α,25(OH)2D3 induced differential methylation(q < 0.01, at > 15% difference) in both THP-1 (19 619 DMCs; 52% hypermethylated, 1744 DMRs; 50.2% hypermethylated) and HEK293(78 476 DMCs; 53% hypermethylated, 12352 DMRs; 49.9% hypermethylated) cells. In both THP-1 and HEK293 cells, differential methylation was most concentrated in intergenic and open sea regions and least concentrated in exons. 1α,25(OH)2D3 induced both methylation and demethylation events that were enriched in pathways specific to monocytic and embryonic cell types, as well as specific to the gene expression requirements of the monocytic and embryonic cell state. MCF-7 samples were found to have been contaminated as Msp1 digestion peak profiles did not correspond with human samples and was thus excluded from downstream analyses. Taken together, results from this study suggest that 1α,25(OH)2D3 is plays an active role in altering methylation patters in a cell-type specific manner