Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA

[Updated 26.06.2018]

I have always tried to avoid touching the field of nuclear DNA methylation or any epigenetic stuff. So many claims in the field seem so implausible that there is probably plentiful of BS out there but not being a specialist in the field makes it difficult to critically assess the literature. I do, however, have an interest in mitochondrial DNA (mtDNA) methylation. It is an interesting niche because there are almost 40 years of data showing that it is ineligible/non-existent but still some researchers consider it a subject worthy of research.

The short story, as shown recently by Matsuda et al (Matsuda et al. 2018), is that once you control you experiments properly, there is no mtDNA methylation.

Now to the longer version of the story. DNA methylation can be detected in various ways, including restriction enzymes, antibodies, mass spectrometry, and bisulfite sequencing (van der Wijst & Rots, 2015). These methods have different caveats as explained in the previous reference. For instance, bisulfite sequencing is known to be sensitive to mtDNA topology changes and the other methods can actually detect methylation of nuclear mitochondrial sequences (NUMTs). Pawar and Eide had recently a nice publication trying to assess the signal-to-noise ratio of various methylation detection methods (Pawar & Eide, 2017). They showed how the choice of buffer for restriction enzyme digestions can have a large effect on the results, how restriction enzyme based methods have 3-5% background noise level, how linearization of mtDNA is essential in bisulfite based methods and how antibody-based methods should use proper negative controls.

I tried to assemble a (probably non-exhaustive) timeline of both negative and positive mtDNA methylation studies. Unfortunately, most publications reporting positive findings do not take into consideration the methodological recommendations coming from several methods papers (Matsuda et al. 2018, Owa et al. 2018, Mechta et al. 2017, Pawar et al. 2017, Liu et al. 2016).

Figure 1: The history of mtDNA methylation research. Multiple publications have reported either the absence (upper part) or presence (lower part) of mtDNA methylation. Most studies claiming the presence of mtDNA methylation have questionable methods.

Paper	Method	Shortcoming
Shmookler Reis et al. 1983	Restriction enzyme	Below 5% detection threshold
Shock et al. 2011	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Infantino et al. 2011	Mass spectrometry	mdC levels below 0.25% could come from nuclear DNA
Chestnut et al. 2011	Immunofluorescence microscopy	Only some mdC punctae in the cytoplasm co-localize with mitochondrial SOD2
Chen et al. 2012	ELISA kit	No controls for mtDNA purity
Dzitoyeva et al. 2012	ELISA kit	No controls for mtDNA purity
Kelly et al. 2012	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Sun et al. 2013	Aba-seq	E. coli instead of PCR-amplified mtDNA as negative control
Bellizzi et al. 2013	Bisulphite	mtDNA was not linearized
Pirola et al. 2013	Bisulphite	mtDNA was not linearized
Wong et al. 2013	Bisulphite	mtDNA was not linearized
Ghosh et al. 2014	Bisulphite	Unclear whether mtDNA was linearized
Jia et al. 2016	Bisulphite	mtDNA was not linearized
Mawlood et al. 2016	Bisulphite	mtDNA was not linearized
Janssen et al. 2017	Bisulphite	mtDNA was not linearized
Saini et al. 2017	Restriction enzyme	Below 5% detection threshold
Ren et al. 2017	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Dewall et al. 2017	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Yu et al. 2017	Bisulphite	mtDNA was not linearized
Kim et al. 2018	Bisulphite	mtDNA was not linearized
Sanyal et al. 2018	Bisulphite	mtDNA was not linearized

So in the end only one of the papers detecting mtDNA methylation seemed valid. It is interesting to note however, that the only valid paper (Infantino et al. 2011) quantified mtDNA methylation using mass spectrometry and their results suggested the levels of mdC were <25%. Another paper also using mass spectrometry saw much lower mdC levels in mtDNA (0.3-0.5%) and concluded mtDNA methylation to be absent (Matsuda et al. 2018). The problem is that in order to measure mtDNA methylation levels by mass spectrometry, one has to first obtain highly pure mtDNA. This is extremely difficult. Even when one uses one of the best protocols out there to obtain pure mtDNA (Kennedy et al. 2014) there will always be nuclear DNA contaminations detectable by Illumina sequencing and that contamination will increase the detected amount of mdC.

So the take-home message method-wise is.

1) Restriction enzyme methods

The background noise level of this method is 3-5% so if you measure mtDNA methylation below this level, you are working with noise instead of signal.

            Optimize the buffer conditions for digestion.

2) Antibody methods

There is a reason why western blot results are not reproducible and the reason is crappy antibodies. Similarly, antibodies supposed to detect only methylated dC bind all kinds of sequences and as a negative control you should always have the same DNA without methylation. In case on mitochondria, one should have PCR amplified mtDNA as the negative control.

3) Bisulfite methods

MtDNA has a topology which will affect bisulfite conversion efficiency. Therefore, one has to linearize mtDNA before this chemical modification or you will be just detecting artefacts.

4) Mass spectrometry approaches

You will always have nuclear DNA contamination in your purified mtDNA. I would like to see a study where one would actually quantify the amount of this background using Illumina sequencing instead of trying to amplify a single nuclear gene by PCR.

In the end I think we can stop researching mtDNA methylation. It is just not there.

PS: Please everybody stop referencing the Rebelo et al (Rebelo et al. 2009) paper as proof of mtDNA methylation. Obviously you either did not read the paper or did not understand it.

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