| The mechanisms which control the gene expression profile of a particular cell type are critical for maintaining homeostasis and physiological processes. A role for epigenetic processes in the regulation of gene expression of an endothelial cell (EC)-enriched gene, namely endothelial nitric oxide synthase (eNOS), has been previously demonstrated. The extent to which epigenetic mechanisms, particularly proximal promoter DNA methylation, plays a role in regulating other EC-enriched genes is not well understood. To investigate whether proximal promoter DNA methylation was important for regulating the expression of EC-enriched genes, we utilized high-resolution sodium bisulfite genomic sequencing, pyrosequencing analysis and DNA immunoprecipitation to assess the methylation status of several EC-enriched gene promoters. We identified differentially methylated regions (DMRs) at the majority of EC-enriched gene promoters in ECs versus non-ECs. Inhibition of DNA methyltransferase activity resulted in an induction in mRNA expression of EC genes in non-EC types, indicating the importance of proximal promoter DNA methylation in gene silencing. We further investigated the role of the newly rediscovered cytosine modified base, 5-hydroxymethylcytosine (hmC), in the vasculature. We developed a quantitative assay - high performance liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (HPLC-ESI-MS/MS-MRM) - to measure both 5-methylcytosine (mC) and hmC in mouse tissues, during embryonic development and in the vasculature. |
