Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.
Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.
The mitochondrial genome is a circular, double-stranded structure of approximately 16.5-kilo base (kb) long, constituting of a heavy and a light strand. The mitochondrial genome is present in multiple copies within each cell, maternally-inherited, and encodes essential components of the respiratory chain complexes1. Similar to bacterial genomes and unlike the nuclear genome, the mitochondrial genome is organized in numerous secondary and tertiary structures, such as in coiled and supercoiled structures2, which can render access difficult during sequencing experiments3.
In the nucleus, methylation of the DNA is an extensively studied epigenetic mark that plays a role in numerous processes, notably in the regulation of gene expression. In mammalian genomes, DNA methylation occurs primarily on the 5-position of the pyrimidine ring of deoxycytidines, mostly on CG dinucleotides (or CpG). Cytosine methylation is found at 70% of all CpG in the genome of somatic cells and accounts for ~1% of total DNA bases4. DNA methylations has also been described in non-CpG contexts, such as CpA, CpT, and CpC and exist in various amounts in nuclear DNA, with values up to 25% of all methylated cytosines in embryonic stem cells5,6,7.
While cytosine methylation of the nuclear genome is widely accepted, the existence of mitochondrial DNA (mtDNA) methylation is still controversial. The first study investigating mtDNA methylation was performed in cultured cells where mtDNA methylation was readily detected, although at lower levels compared to nuclear DNA8. In both human and murine cells, mtDNA methylation was also detected at low levels (2-5%). Using assays relying on 5 methylcytosine capture such as methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR, mtDNA methylation was also detected in various mouse and human and cells lines9,10,11,12. Using antibodies against 5-methylcytosine in an ELISA assay or mass spectrometry, substantial levels of DNA methylation were detected from purified mitochondrial fractions13,14,15,16. However, most of the assays in the aforementioned studies used techniques that were not designed to provide absolute quantification of DNA methylation at the single base-resolution.
Quantitative and resolutive DNA methylation analysis can be achieved by a technique named "bisulfite sequencing", which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil in single-stranded DNA context17. Using bisulfite sequencing, a constellation of studies has detected the presence of cytosine methylation at various levels. Methylation mtDNA in the D-loop region, the 12S or the 16S region was readily detected in human18,19,20,21,22,23 and mouse24 tissues and cells, however, with an intriguing variability, of 1-20% of total cytosines across studies.
In comparison to these numerous studies, only a few studies, including from our group, have disputed the presence of mtDNA methylation3,25,26,27 or questioned the biological relevance of very low levels of mtDNA levels (below 2%)28. Recently, we reported the observation of a potential bisulfite-sequencing artifact in whole mitochondrial bisulfite sequencing3. We provided evidence that the secondary structure of mitochondrial DNA could lead to false positives in bisulfite sequencing, thereby overestimating methylation levels. We provide here a protocol to prevent an artifact of bisulfite-conversion of mtDNA. This protocol uses a simple enzymatic digestion of DNA to disrupt mtDNA secondary structures and allow full access to bisulfite following a bisulfite sequencing protocol. In addition, we provide an accompanying bioinformatic pipeline for the analysis of bisulfite sequencing.
1. Restriction Enzyme Treatment
2. Bisulfite Conversion
3. Design of Bisulfite Sequencing Primers
4. Bisulfite Converted PCR and Gel Extraction
5. Bisulfite Sequencing Library Preparation
6. Next Generation Sequencing
7. Computational Analysis – Estimate Methylation Levels
8. Computational Analysis – Test of differences
Two steps in this protocol are crucial when investigating mtDNA methylation. 1) The opening of the secondary structure and 2) the design of mitochondrial DNA-specific primers.
By digesting the human genomic DNA with the restriction enzyme BamHI (Figure 1), the mitochondrial DNA structure will be cut at nucleotide position 14,258 and the secondary structure will be opened.
The possible impairment of bisulfite conversion with an intact mtDNA secondary structure is very likely to overestimate the mtDNA unconversion rate. By bisulfite sequencing, the mtDNA unconversion rate was investigated, in undigested and digested total DNA from human skeletal muscle cells, at 5 different regions of the mitochondrial genome: the displacement Loop (D-Loop), tRNA phenylalanine and 12S ribosome (tRNA-F+12S), 16S ribosome (16S), NADH dehydrogenase 5 (ND5) and cytochrome b (CYTB) mRNA-encoding gene (Figure 2). Unconversion rate ranged from 0 – 15.1% across all investigated regions for undigested mtDNA. However, when the DNA was digested prior to bisulfite conversion, the unconversion rate dropped to a maximum of 1% across all regions (Figure 3). Thus, illustrating the importance of BamHI digestion prior to bisulfite conversion to avoid overestimation of mtDNA methylation levels.
Contamination of nuclear DNA is inevitable when isolating the mitochondrial genome and since almost the entire mitochondrial genome has replicated into the nuclear genome, it has given rise to nuclear insertions of the mitochondrial genome termed NUclear MiTochondrial segments (NUMTs)36. To ensure that the analyzed DNA methylation levels correspond to mitochondrial DNA and not NUMTs, it is of outermost importance to design primers that are specific to the mitochondrial genome. Figure 4 shows how to check the primer specificity and Table 1 displays human and mouse mtDNA primer sequences.
Name | Sequence | Pozisyon | Amplicon size | Annealing temperature | Strand | ||
Human | |||||||
D-Loop | F: AAATCTATCACCCTATTAAC | ||||||
R: GTGGAAATTTTTTGTTATGATGT | 6-298 | 292 | 55°C | Antisense | |||
D-Loop | F: CATAACAAAAAATTTCCACCAAAC | ||||||
R: GGGAAAATAATGTGTTAGTT | 279-458 | 179 | 55°C | Antisense | |||
12S+TF | F: TTTATATAACTTACCTCCTC | ||||||
R: GTGTTTGATGTTTGTTTTTTTTG | 577-765 | 188 | 55°C | Antisense | |||
16S | F: AATAAATTTATAGGTTTTTAAATTATTAAAT | ||||||
R: TAACTAATAAAATCTTAACATATACTACTC | 2763-2873 | 110 | 53°C | Sense | |||
CYTB | F: GGTATTATTTTTTTGTTTGTAATTATAGTA | ||||||
R: CCTCAAATTCATTAAACTAAATCTATCC | 15091-15243 | 152 | 55°C | Sense | |||
Mouse | |||||||
12S | F: ACACATACAAACCTCCATAAAC | ||||||
R: GAGGTATAATTTAGTTAAAT | 111-287 | 176 | 53°C | Antisense | |||
16S | F: AAATTCCAATTCTCCAAACATAC | ||||||
R: TTTGGATTTTTTTTTTAGGT | 1749-1896 | 147 | 53°C | Antisense | |||
CYTB | F: CTACAAAAACACCTAATAACAAAC | ||||||
R: AGTGTATGGTTAAGAAAAGA | 14129-14307 | 178 | 55°C | Antisense | |||
16S | F: AGAGAAATAGAGTTATTTTATAAATAAG | ||||||
R: CTAAAAACAAAATTTTAAATCTTAC | 2576-2727 | 151 | 55°C | Sense | |||
ND5 | F: TTAAGTTAATTAGGTTTGATAATAGTGA | ||||||
R: TAAAACCCTATTAAAAATAATATTCCTATA | 12659-12910 | 251 | 55°C | Sense | |||
CYTB | F: TGGGTTTTTTTTAGGAGTTTGTTTA | ||||||
R: CAATAAAAATACTCCAATATTTCAAATTTC | 14242-14504 | 262 | 55°C | Sense |
Table 1: Primer sequences for human and mouse mtDNA. Note that annealing temperatures of PCR primers vary due to differences in the melting temperature of primers.
Figure 1: Gel electrophoresis of BamHI-digested, or undigested DNA. Genomic DNA from human skeletal muscle was untreated or treated with BamHI for 4h at 37°C and gel electrophoresis was run on a 1.5% agarose gel. Please click here to view a larger version of this figure.
Figure 2: Map of the human mitochondrial genome. Regions of the mitochondrial genome investigated by bisulfite sequencing and the BamHI restriction enzyme site are displayed. The mitochondrial DNA contains 13 mRNAs, 2 ribosomal RNAs, and 22 transfer RNAs. Abbreviations: PH1: heavy-strand promoter 1; PH2: Heavy-strand promote 2; PL: Light-strand promoter; OH: Origin of replication from heavy-strand; OL: Origin of replication from light-strand. Please click here to view a larger version of this figure.
Figure 3: BamHI digestion prior to bisulfite conversion reduces the mtDNA unconversion rate in human skeletal muscle cells. By bisulfite sequencing, undigested and digested mitochondrial DNA unconversion percentage were interrogated at five different regions of the mitochondrial genome in human skeletal muscle cells (N=3). The full square represents CpG sites, and the open square represents non-CpG sites. Results are presented with a min-max interval and a sign test was used to test for significant unconversion differences. D-Loop (6-298) P= 1.83E-42; D-Loop (279-458): P= 4.55E-13; tRNA-F+12S: P= 8.38E-16; 16S: P= 5.91E-06; ND5: P=1.70E-05; CYTB: P= 2.69E-10. D-Loop (6-298) includes the origin of replication and tRNA-F+12S includes heavy strand promoter 2. These data have been published elsewhere 3. Please click here to view a larger version of this figure.
Figure 4: The use of Bisearch to verify primer specificity towards the mitochondrial genome. A) Bisulfite-converted primer sequences are added to the BiSearch function Primer Search (http://bisearch.enzim.hu/?m=genompsearch). The bisulfite box is ticked, and a reference genome is selected. B) Example of potential PCR products generated on sense and antisense chains. Please click here to view a larger version of this figure.
Here, we provide a bisulfite-sequencing protocol which is specifically designed to interrogate mtDNA methylation. The differences with bisulfite-sequencing protocols used for genomic DNA lies in the utilization of a prior restriction enzyme digestion step and a bioinformatic analysis ruling out false positive arising from NUMT sequences.
We provide a protocol to avoid bisulfite-sequencing artifacts when investigating mtDNA methylation. Bisulfite sequencing artifacts leading to false-positives were previously reported37. Insufficient removal of DNA accessory proteins by proteinase K has been described37. Incomplete denaturation or partial reannealing can lead to stretches of incomplete conversion, possibly by lowering access of bisulfite to single-stranded DNA37. The later artifact could be at play in mtDNA where a secondary structure would impair access to cytosines. To the best of our knowledge, the addition of a restriction enzyme digestion step prior to bisulfite conversion in the context of estimating mtDNA methylation has been first reported in 20163,28. In our hands too, prior digestion with a single-cutter restriction enzyme dramatically lowered detection of unconverted cytosines3,28.
Here, we provide a bioinformatic pipeline to analyze bisulfite sequencing results and to detect bisulfite sequencing artifacts in the context of whole mitochondrial genome bisulfite sequencing. This method originates from our observation that mtDNA non-conversion rate was inversely correlated with sequencing depth3,28. This observation suggests that the secondary and even tertiary structure of mtDNA impairs the fragmentation of mtDNA at specific regions during the construction of sequencing libraries and could also impair full access of sodium bisulfite to mtDNA. Since digestion with a one-cutter enzyme releases secondary and tertiary structures, our results indicate that mtDNA structure is a source of bisulfite artifact and validate the methods described herein for the quantification of cytosine methylation in mtDNA.
The inevitable contamination of nuclear DNA in mitochondrial preparation constitutes a challenge. This leads to contamination in NUMT sequences in sequencing reads, which may bias estimation of cytosine methylation in mtDNA, even when performing targeted and not whole mitochondrial genome bisulfite sequencing. To avoid such bias, the design of unique bisulfite sequencing primers allows to exclude contamination with methylation at NUMTs. NUMTs span certain regions of the human and mouse mitochondrial genome with 100% identity, and it is, therefore, impossible to design unique primers at certain mtDNA regions. When performing whole mitochondrial genome bisulfite sequencing, sequencing long reads can ease discrimination between mtDNA and NUMTs. Aligning reads to the whole genome and only use reads uniquely mapped to the mitochondrial genome can further ensure detection of mtDNA methylation.
Finally, this protocol can be used beyond mitochondrial genomes, for example, in other instances of bisulfite sequencing where the secondary structure of DNA represents a potential source of artifact. The association between methylation levels and sequencing depth can be investigated to assess the need for a digestion with a restriction enzyme prior to bisulfite sequencing of complex DNA sequences.
The authors have nothing to disclose.
The Novo Nordisk Foundation Centre for Basic Metabolic Research is an independent research center at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation.
BamHI | New England BioLabs | # R0136 | |
EZ DNA methylation-lightning kit | Zymo Research | # D5030 | |
Qubit ssDNA assay | Thermo Fisher Scientific | # Q10212 | |
Qubit assay tubes | Thermo Fisher Scientific | # Q32856 | |
HotStarTaq plus DNA polymerase kit | Qiagen | # 203603 | |
QIAquick Gel Extraction Kit | Qiagen | # 28704 | |
NEBNext Ultra DNA Library Kit for Illumina | New England BioLabs | # E7370S | |
NEBNext Multiplex Oligoes for Illumina | New England BioLabs | # E7335S | |
AMPure XP Beads | Beckman Coulter | # A63881 | |
High Sensitivity DNA chip | Agilent | # 5067-4626 | |
Qubit high sensitivity dsDNA assay | Thermo Fisher Scientific | # Q33230 | |
MiSeq reagent kit v2 300 cycles | Illumina | # MS-102-2003 | |
PhiX control v3 | Illumina | # FC-110-3001 | |
Sodium hydroxide | Sigma | # S5881 | |
Thermal cycler C1000 | Biorad | # 1851148 | |
CFX96 Real-Time PCR detection system | Biorad | #1855195 | |
Qubit Fluorometer | Thermo Fisher Scientific | # Q33226 | |
Bioanalyzer 2100 | Agilent | # G2939BA | |
MiSeq instrument | Illumina | # SY-410-1003 |