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Page 4 of 14 Lozano-Rosas et al. Hepatoma Res 2018;4:19 I http://dx.doi.org/10.20517/2394-5079.2018.48

DNMT enzymes use SAM as the methyl donor. However, the importance of mtDNA in methylation not
only lies in the mitochondrial function but it can modify the overall epigenetic state of the cell. In fact,
it has been observed that the decrease of mtDNA leads to altered levels of methylation in the genomic
DNA, which is normalized once the mtDNA content is reestablished . In 2013, Bellizzi et al. studied the
[33]
[35]
methylation patterns in mitochondrial cytosines in humans and mice and the effect of suppressing DNMT1,
DNMT3a, and 3b. In general, a preponderant methylation of CpG dinucleotides and its inhibition was found
in knockout mice without affecting methylation in non-CpG sites .
[35]
The DNA methylation pattern of the human mitochondrial genome remains relatively constant; however,
there are some loci that are differentially methylated in different tissues and over time . For example,
[36]
subunit 6 of NADH dehydrogenase, a crucial subunit for the assembly of complex I, is suppressed due to
hypermethylation by an increase in the expression of DNMT1 . It is also known that the 12s rRNA gene
[33]
is methylated by the rRNA methyltransferase-related transcription factor 1 (mt-TFB1) . This epigenetic
[37]
regulation is important for ribosomal biogenesis and mitochondrial translation and has been related to
aging; this modification alters the efficiency of the ETC by hampering the assembly of complex I . In 2016,
[38]
Liu et al. determined methylation of the human mitochondrial genome in blood and saliva samples; by
[39]
bisulfite pyrosequencing, 9 regions in human mtDNA were detected including a D-loop, 12S rRNA, 16S
rRNA, ND1, COXI, ND3, ND4, ND5, CYTB.

Hydroxymethylation is another important epigenetic modification described for mtDNA, where 5mC is
oxidized into 5-hmC by the TET family of methylcytosine dioxygenases. The presence of TET1 and TET2
has been described in neuronal mitochondrial fractions [32,40] . TET proteins are members of the family of
2-oxoglutarate-dependent dioxygenases (2-OGDO) that can oxidize 5mC to generate 5hmC, 5-formylcytosine
(5fC), and 5-carboxylcytosine (5caC), mediating DNA demethylation by oxidation in cooperation with
the BER repair pathway . The reaction is dependent on the presence of oxygen, 2-oxoglutarate, and Fe .
2+
[40]
2-Oxoglutarate is a key metabolite in the Krebs cycle that occurs in the matrix of mitochondria; on the other
hand, succinate and fumarate, also TCA intermediates, are potent inhibitors of 2-OGDO enzymes, in this
manner TCA cycle controls the DNA and histone methylation and, thus, controls gene expression .
[40]

The kind of hydroxymethylation of cytosines in mitochondria has also been reported . The profile of
[33]
modifications in the D-loop region (a non-coding region that acts as a promoter for the H and L strands of
the mtDNA and contains transcription and replication elements), and the similarity of this profile in cells
of similar tissues and how the profile is different in cells of different tissues have been described recently .
[41]
Given the relevance of mitochondrial DNA and its regulation at different levels, recent studies have also
proposed mitochondrial DNA methylation as a potential biomarker .
[42]
The central role of microRNAs in modulating mitochondria
Other regulators that have recently been studied in mitochondria are microRNAs (miRNAs) . miRNAs
[43]
are small non-coding RNAs, implicated in gene post-transcriptional regulation and the conformation of
genetic expression patterns with physiological relevance. They derive from longer RNAs, primary miRNAs
(pri-miRNAs), and are sequentially cleaved by ribonuclease III (RNAse III) enzymes or processed for pre-
miRNA splicing and RNA degradation pathways .
[44]
It has been found that both pre-miRNAs and mature miRNAs can be found in mitochondria, suggesting
that this organelle can synthesize them and keep them active in their transcriptional machinery or export
them to the cytosol. Likewise, the possibility arises that the miRNAs processed in mitochondria regulate
the expression of genes related to the function of the same organelle . On the other hand, to mention some
[45]
examples, miR-181c-5p regulates mitochondrial energy metabolism through mt-COX1 mRNA; although