DNA methylation mark in mitochondrial DNA

DNA methylation, 5-methylcytosine (5-mC), is a key epigenetic modification involved in gene regulation found in the nuclear genome of various organisms. Interestingly, mitochondrial DNA (mtDNA) has also been found to be modified by 5-mC. Research to determine the functional relevance and identify players regulating mtDNA methylation is receiving an ever-increasing interest. This project is focused on understanding the mitochondrial methylation patterns in the normal physiological state using circulating CD4+ and CD8+ T cells as an informative study model. This knowledge might prove useful in understanding what arises during the pathological state. Moreover, mtDNA cellular content differs across tissue types depending on several factors including cellular energy demand which influence mtDNA replication and transcription rate. The objective of this study is to determine variations in mtDNA methylation of CD4+ and CD8+ T cells between healthy individuals as well as the ratio of mitochondrial/nuclear DNA in these cells to estimate whether the changes in methylation could possibly be contributed to changes in their relative mtDNA content. In this study, blood samples from healthy donors were collected and CD4+ and CD8+ T cells were purified for analyses of 5-mC. The methylated DNA fragments enriched by methyl-CpG binding domain were sequenced and subsequent bioinformatic analysis involved preprocessing of the raw data, alignment of reads to the human mitochondrial genome and methylation peak calling. Methylation levels within the mitochondrial D-loop were then quantitatively evaluated by bisulfite-pyrosequencing. The results showed variations in the distribution of 5-mC in the different cell types and individuals, with the exception of 3’ end in the terminal part of D-loop which appeared to be consistently methylated to a certain extent across mtDNA with the average methylation level of ~ 1%. Additionally, quantitative PCR used to measure the ratio of mitochondrial/nuclear DNA showed no association between mtDNA ...