Low biological fluctuation of mitochondrial CpG and non-CpG methylation at the single-molecule level

Abstract

Mammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

Nanopore Single-Molecule Sequencing for Mitochondrial DNA Methylation Analysis: Investigating Parkin-Associated Parkinsonism as a Proof of Concept

“…On the one hand, there is evidence for a negative association between mtDNA methylation and gene expression (Sirard, 2019 ) and our results may indicate a higher expression of mtDNA genes in neuron-derived DNA. On the other hand, a previous study reported lower mtDNA CpG methylation levels in cell lines compared to tissue-derived DNA (Goldsmith et al, 2021 ). When exploring iPSC-derived midbrain neurons, lower CpG mtDNA methylation was detected compared to blood and perhaps the reprogramming process also modifies the epigenetics of mtDNA.…”

“…To adjust the detected methylation frequency of the blood- and neuron-derived samples, we calculated the false-positive rate (FPR = Number of called methylated cytosines/Number of all called cytosines) as recommended (Goldsmith et al, 2021 ) from the long-range PCR products. The resulting FPR of Nanopolish and Megalodon for blood- and neuron-derived DNA was subtracted from the methylation frequency of each site.…”

“…Nanopore technology enables the direct sequencing of full-length linearized mtDNA in contrast to the shorter fragments obtained from second-generation sequencing (De Coster and Van Broeckhoven, 2019 ). To our knowledge, there are currently only three studies that have used Nanopore sequencing to investigate mtDNA methylation (Aminuddin et al, 2020 ; Bicci et al, 2021 ; Goldsmith et al, 2021 ). Thus, detecting mtDNA methylation with this novel sequencing technology remains to be thoroughly investigated and validated.…”

Mitochondrial DNA Methylation and Human Diseases

“…However, the authors suggested that mtDNA methylation in the gene bodies promoted the expression of the genes, presumably by affecting the post-transcriptional modifications of polycistronic mitochondrial mRNAs, since in one cell culture, they found hypermethylation of MT-CO1 and MT-CYB genes with concomitant high expression levels [ 74 ]. By using the same technique, increased CpG and CpH mtDNA methylation levels have been observed in head and neck tumor samples when compared to their matched adjacent normal tissues [ 75 ].…”

“…Moreover, a study investigating mtDNA methylation in human cells by means of a third-generation sequencing platform, namely, the nanopore sequencer (MinION, Oxford Nanopore), which is a high-throughput nanopore-based single-molecule device that can directly sequence the entire mtDNA molecule, minimizing potential nucleotide errors introduced by PCR amplification, and preserving epigenetic modifications by avoiding the use of bisulfite treatment, revealed clearly detectable levels of mtDNA methylation [ 74 ]. Likewise, by using high-coverage nanopore sequencing at the single-molecule level, mtDNA methylation was recently investigated in different sample types and biological conditions [ 75 ]. Methylation was overall higher in tissues compared to cell lines, and despite that mtDNA methylation levels were generally low, global and single-base differences were found between cancer tissues and the adjacent healthy tissues.…”