Methylation profiling by bisulfite sequencing analysis of the mtDNA Non-Coding Region in replicative and senescent Endothelial Cells

• CG and CH sites of mtDNA are methylated to different extent.
• 5mC in mtDNA NCR are not randomly distributed, but tend to cluster together.
• Methylation might start from preferential cytosines and spread to the neighbouring.
• Methylation profile varies in senescent vs. proliferative cells.
• 5mC distribution and pattern variation may suggest a role in 7S DNA synthesis.

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5mC clusters and a biased distribution of 5mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5mC in D-loop formation, rather than in mitochondrial gene expression.