Methodology for Accurate Detection of Mitochondrial DNA Methylation

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 cells^5,6,7.

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 lines^9,10,11,12. Using antibodies against 5-methylcytosine in an ELISA assay or mass spectrometry, substantial levels of DNA methylation were detected from purified mitochondrial fractions^13,14,15,16. 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 human^{18,19,20,21,22,23} 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 methylation^3,25,26,27 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.

Protokół

1. Restriction Enzyme Treatment

1. Linearize mtDNA by treating total human DNA with the restriction enzyme BamHI, that cuts at position 14258 in the human mitochondrial DNA.
   NOTE: For mouse DNA, use the restriction enzyme BglII under identical conditions as below.
2. For each sample where mtDNA methylation is to be assessed, prepare one 0.2 mL reaction tube and add the following mix:3 μg genomic DNA (quantified by fluorometry), 15 μL Buffer 3, 3 μL BamHI and water up to of 150 μL
3. Place tubes in a thermal cycler for 4 h at 37 °C.

2. Bisulfite Conversion

1. Convert BamHI-treated DNA using sodium bisulfite as described elsewhere²⁹.
2. Use 200-500 ng BamHI-treated DNA for each conversion reaction in a total volume of 20 μL. The DNA recovery is 50-70%.
3. Elute the Bisulfite-converted DNA in 10 μL elution buffer.
4. Quantify the single-stranded DNA by fluorometry.
5. Optional: Store bisulfite-converted DNA at -20 °C before proceeding the remaining of the protocol. For long-term storage, store bisulfite-converted DNA at -80 °C

3. Design of Bisulfite Sequencing Primers

1. Design primers for bisulfite sequencing using the online tools Methprimer³⁰ and BiSearch^31,32.
   NOTE: Methprimer and BiSearch allow to search for primer binding sequences on genomic sequences where cytosines are converted to thymines, similarly to post-bisulfite treatment.
2. For optimal and unbiased amplification, avoid CpG dinucleotides in the primers and design the amplicon size between 100-300 bp.
3. Ensure that designed primers are specific to the mitochondrial DNA using BiSearch's function Primer Search. Insert the already designed bisulfite converted primers, tick the bisulfite box and include a reference genome and PCR parameters.
   NOTE: A list of possible PCR products will be displayed.
4. Copy the top 1-5 primer sequences and paste them into an ordering form for oligonucleotide synthesis.
   NOTE: A list of human and mouse mtDNA primer sequences that has been validated in the lab is displayed in Table 1.
5. Test primers using bisulfite converted PCR following agarose gel electrophoresis as described in step 4. Test and optimize all primer pairs separately and ensure the correct amplicon size appears on the agarose gel.
   NOTE: If multiplexing primers are needed, add multiple primers to one single PCR reaction and visualize the PCR products on agarose gel electrophoresis or, a more resolutive method such as polyacrylamide gel electrophoresis, to distinguish products of similar size.

4. Bisulfite Converted PCR and Gel Extraction

1. To amplify the regions of interest, perform PCR using a hot start Taq polymerase. Briefly, mix 100 ng converted DNA, 500 µM sense and anti-sense primers, 1 µL dNTP mix (10 µM of each dNTP), and polymerase at 7.5 U/reaction in a total volume of 50 µL. Run PCR with the following conditions: 5 min at 95 °C (60 s 94 °C; 60 s 55 °C*; 60 s 72 °C) 35 cycles; 10 min 72 °C. Use primers either separately or multiplexed.
2. Optional: When multiplexing primers, use 200 ng converted DNA and the following cycling conditions: 5 min 95 °C (60 s 94 °C; 90 s 55 °C*; 90 s 72 °C) 35 cycles; 10 min 72 °C.
   NOTE: Temperature may vary depending on the melting temperature of primers.
3. Subject PCR products to gel electrophoresis on 2% agarose at 100 volts.
4. Under gentle UV light, extract PCR products with a scalpel and add to microtubes. Do not expose PCR products to excessive UV light to avoid DNA damage. Avoid exposure time over 30 s.
5. Purify the PCR products using gel purification as described previously³.
6. Quantify the purified DNA from step 4.5 using fluorometry. Proceed to step 5 or store samples at -20°C.

5. Bisulfite Sequencing Library Preparation

1. Perform library preparation of bisulfite-converted PCR products.
   Optional: multiplex PCR products at this stage.
2. Perform end-repair using up to 100 ng of PCR product. Add 3 µL End prep enzyme and 6.5 µL End repair reaction buffer (10x) to 55.5 µL PCR product in a 0.2 mL tube. Place and incubate tube in a thermal cycler for 30 min at 20 °C followed by 30 min at 65 °C.
3. Ligate sequencing adaptors by mixing the end-repaired DNA with 15 µL Blunt/TA ligase mix, 2.5 µL Adaptor and 1 µL Ligation enhancer. If using <100 ng PCR product as input, dilute adaptor 10 times.
4. Place the mix (step 5.3) in a thermal cycler and incubate 15 min at 20 °C. Pause the thermal cycler and add 3 µl USER Enzyme to the tube. Mix and place the tube back in the Thermal cycler and incubate 15 min at 37 °C.
5. Size select the adaptor-ligated DNA by using Solid Phase Reversible Immobilization (SPRI) beads with varying ratios of beads to DNA depending on PCR product size.
6. Add 13.5 µL H₂O to adaptor-ligated DNA from step 5.4 and 55 µL resuspended SPRI beads. Incubate at room temperature for 5 min and place tube on a magnetic stand. When the solution is clear, transfer the supernatant to a new tube and discard tube containing the beads.
   NOTE: The supernatant contains the adaptor-ligated DNA and large unwanted fragments are bound to the discarded beads.
7. Add 25 µL resuspended SPRI beads to the supernatant from step 5.6, mix and incubate for 5 min at room temperature. Place the tube on the magnetic stand and discard the supernatant when the solution is clear.
   NOTE: The discarded supernatant contains unwanted DNA, whereas the adaptor-ligated DNA is bound to beads.
8. While the tube is on the magnetic stand, add 200 µL 80% ethanol (freshly prepared) to wash the beads. Incubate 30 s and remove and discard the supernatant. Repeat this step for a total of 2 washes.
9. Air dry beads for 5 min while the tube is on magnetic stand with an open lid.
10. Remove the tube from the magnet, elute the DNA in 23 µL elution buffer and mix. Incubate at room temperature for 2 min.
11. Place the tube on a magnetic stand and when the solution is clear, transfer 2 µL to a 96-well PCR plate for the pre-PCR amplification (step 5.14).
12. Transfer the rest approximately 21 µL to a new 0.2 mL tube for PCR amplification (step 5.17).
    NOTE: Be sure NOT to transfer any beads as beads can inhibit enzymatic reactions of next steps.
13. Proceed to step 5.14 or store samples at -20°C.
14. Perform pre-PCR amplification by RT-qPCR to estimate the number of cycles needed to amplify the adaptor-ligated DNA. Per reaction, add the following in the 96-well PCR plate containing 2 µL DNA (step 5.11): 0.4 µL Index primer, 0.4 µL universal PCR primer, 7.2 µL H₂O, 10 µL RT-qPCR master mix.
15. Place the plate in a real-time PCR machine and subject the plate to the following conditions: 30 s at 98 °C (10 s 98 °C; 75 s 65 °C) 20 cycles; 5 min 65 °C.
16. Estimate the number of amplification cycles needed for each sample by subtracting one Ct-value to the Ct-value of the pre-amplification RT-qPCR corresponding to the end of the linear phase of the PCR reaction.
17. Amplify the adaptor-ligated DNA from step 5.12 by mixing: 21 µL DNA, 25 µL PCR master mix, 1 µL Index primer, 1 µL Universal PCR primer and 2 µL H₂O. In a thermal cycler, subject each sample to the following conditions: 30 s at 98 °C (10 s 98 °C; 75 s 65 °C) [calculated Ct from step 5.16] cycles; 5 min 65 °C.
    NOTE: Remember to only use one Index primer per sample to allow for multiplexing. The index is inserted during the PCR.
18. Purify the amplified DNA SPRI beads and elute in 22 µL elution buffer.
19. Control library quality by gel electrophoresis or alternative method. Look for correct library peak sizes (PCR product(s) size plus adaptor).
20. Estimate the average base-pair size of the library product(s) by smear analysis: In advanced global settings, double-click on "Table" under smear analysis. Define the region from 100-1000 bp and click OK. Under Region Table, the defined region appears and library average size (bp) is calculated.
21. Quantify libraries by fluorometry. Proceed to step 6 or store libraries at -20 °C.

6. Next Generation Sequencing

1. Calculate the nM concentration of libraries using the formula:
   
2. Dilute libraries to the same nM concentration e.g. 2 nM (choose 4 nM, 2 nM, 1 nM, or 0.5 nM depending on library concentrations). Pool all libraries to achieve a pool of 2 nM libraries.
3. Denature and dilute libraries following the sequencing instrument guide. In short: Denature the 2 nM pool by adding 0.2 N NaOH and incubate at room temperature for 5 min. Dilute the denatured pool to a final dilution of 10 pM. Denature and dilute a commercially available Control library to a final dilution of 12.5 pM. To a new tube, mix the 10 pM pool with 20% 12.5 pM Control library.
   NOTE: Bisulfite conversion introduces very low complexity. 20% Control library spike-in will result in better sequencing quality.
4. Run a 150 bp paired-end sequencing using a deep sequencing instrument.

7. Computational Analysis - Estimate Methylation Levels

1. Generate .fastq files (here called sample1_R1.fastq.gz and sample1_R2.fastq.gz), generate a bismark index of the entire genome of interest (here in a folder called bismarkIndex) and make sure that the genomic sequence of chrM is available in fasta format (here in a folder called chrM).
2. Pre-process reads to remove adapters and bias introduced by the primers: Use the tool Trim_galore³³. Remove two nucleotides from the 5' end of both the forward and reverse reads.
   trim_galore --clip_R1 2 --clip_R2 2 -o ./ --trim1 --paired sample1_R1.fastq.gz sample1_R2.fastq.gz
3. Align pre-processed reads to the entire genome using Bismark³⁴
   bismark --dovetail --bam -o ./ ./bismarkIndex / -1 ./sample1_R1_val_1.fq.gz -2 ./sample1_R2_val_2.fq.gz
   NOTE: If another aligner is used, make sure that reads that cannot be uniquely aligned are discarded.
4. Extract reads mapping to the chromosome M, here using Samtools³⁵
   samtools sort -l 0 -O BAM -o sample1_sorted.bam
   sample1_R1_val_1_bismark_bt2_pe.bam
   samtools index sample1_sorted.bam
   samtools view -u sample1_sorted.bam chrM | samtools sort -n -O BAM -o sample1_chrM.bam
5. Extract the methylation information
   bismark_methylation_extractor -p --CX --cytosine_report --comprehensive --genome_folder ./chrM ./sample1_chrM.bam
6. Use the .bedGraph, the .cov or the CX_report.txt files for further analysis. Use the sample1_chrM.CX_report.txt file for visualization and testing.
7. Perform further clipping in the preprocessing step, if multiple regions are interrogated and a primer bias may be visible in the M-bias plots generated during the mapping. Refer to the Bismark documentation for more information.

8. Computational Analysis - Test of differences

1. Ensure that the CX_report.txt file contains 7 columns, chromosome (here chrM), position, strand, number of methylated reads, number of unmethylated reads, C context and surrounding sequence, for every C on chromosome M.
2. As the CX_report covers chrM in its entirety, check the subset to the region(s) which is amplified.
3. Use non-parametric tests for differences between samples, such as a Fishers's exact test for individual C's or a sign-test for an entire region.
   NOTE: Quantification will often be close to 0% methylation, which is the reason why assuming normality is not appropriate.
4. Similarly, for visualization, either visualize quantiles or calculate binomial proportion confidence intervals.

Wyniki

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.

Dyskusje

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 ...

Materiały

Name	Company	Catalog Number	Comments
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