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Type 2 Diabetes Mellitus is a major chronic metabolic disorder in public health. Due to mitochondria’s indispensable role in the body, its dysfunction has been implicated in the development and progression of multiple diseases, including Type 2 Diabetes mellitus. Thus, factors that can regulate mitochondrial function, like mtDNA methylation, are of significant interest in managing T2DM. In this paper, the overview of epigenetics and the mechanism of nuclear and mitochondrial DNA methylation were briefly discussed, followed by other mitochondrial epigenetics. Subsequently, the association between mtDNA methylation with T2DM and the challenges of mtDNA methylation studies were also reviewed. This review will aid in understanding the impact of mtDNA methylation on T2DM and future advancements in T2DM treatment.
Diabetes mellitus (DM) is a chronic metabolic disorder typified by the presence of hyperglycaemia (
T2DM is mainly characterised by insulin resistance, with multiple pieces of evidence showing the key role of mitochondria involvement (
The objectives of this review are to provide an overview on mitochondrial epigenetics, to address the association between mitochondrial epigenetics with T2DM and to conclude by discussing the challenges of mitochondrial epigenetics studies.
The expression of genes is known to depend on the genetic sequence and epigenetic regulation of the gene. Epigenetics refers to the study of inheritable changes which would alter gene expression either transiently or permanently without permanent modifications to the original DNA sequence (
The mechanisms of epigenetics can be mainly categorised into three classes, namely, DNA methylation, post-translational modifications (PTMs) of histones and gene expression regulation by non-coding RNAs (ncRNAs) (
The three major mechanisms of epigenetics. (a) DNA methylation is the formation of a methylated cytosine through the transfer of a methyl group to the fifth carbon of the cytosine residue. When methylation occurs at the promoter region, downregulation of gene expression occurs. (b) The post-translational covalent addition of an acetyl or methyl group at the histone would alter the chromatin structure, resulting in regulation of gene expression. (c) The binding of a non-coding RNA such miRNA to mRNA would either inhibit the translational activity of the mRNA or degrade the mRNA. Adapted and modified from (
Methylation of nuclear DNA (nDNA) typically involves the addition of a methyl group (CH3) from the S-adenosyl methionine to the fifth carbon (C5) of the cytosine residues, which are paired with guanine bases (CpG), leading to the formation of 5-methylcytosine (5mC) and the by-product S-adenosylhomocysteine (
Structure and the phylogeny of known DNA methyltransferases (DNMTs).
DNMT1 was proposed to be the primary enzyme responsible for maintaining normal methylation patterns by restoring hemimethylated sites in CpG sequences to fully methylated sites during replication (
Structure and function of DNA methyltransferases (DNMTs). Adapted from (
Component | DNMT1 | DNMT2 | DNMT3A | DNMT3B | DNMT3L | |
---|---|---|---|---|---|---|
Domain structure | N-terminal | Present | Absent | Present | Present | Present |
C-terminal | Present with slight differences for each DNMT | |||||
Enzymatic activity | Active | Inactive | ||||
Methyltransferase profile | Methylation sites | DNA | tRNA | DNA | DNA | DNA |
Roles | • Maintains methylation patterns during DNA replication and cell proliferation | • Initiates methylation of the tRNA of aspartic acid at the 38 cytosine of the anticodon loop | • Initiates methylation in a distributive manner | • Initiates methylation in a processive manner | • Acts as a regulatory factor and mediates methylation activity of DNMT3 | |
• Localises at replication foci and able to process long stretches of DNA | • DNMT3A1 is predominant in heterochromatic regions of differentiated cells | • Acts as accessory protein in methylation activity | • Express in germ cells and embryonic cells only | |||
• |
• DNMT3A2 is predominant in euchromatic regions of undifferentiated cells |
BAH domain, Bromo-adjacent homology domain.
DNA methylation is a reversible process whereby demethylation occurs, ensuring a gene does not remain repressed permanently. While not as well elucidated as methylation, it is known the methylated sequences can either be demethylated passively or actively, as illustrated in
A schematic diagram of the entire methylation and demethylation processes of DNA. During replication, DNMT 1 ensures the maintenance of normal methylation patterns (9) while DNMT3a and DNMT3b mediates the
Structure and function of Ten-Eleven Translocation proteins (TETs). Adapted from (
Component | TET 1 | TET 2 | TET 3 | |
---|---|---|---|---|
Structure | N-terminal | Contains CXXC domain | Does not contain CXXC domain | Contains CXXC domain |
C-terminal | Contain DBHS domain, cysteine-rich domain and binding sites for Fe (II) and α-ketoglutarate cofactors forming the core catalytic domain | |||
Demethylation | Active | Catalyse oxidation of 5mC to oxidised methylcytosines which is excised by TDG to form abasic sites. Abasic sites will eventually form unmethylated cytosines through BER | ||
Passive | Formation of 5hmC from TET enzymes activity has been proposed to contribute to passive demethylation | |||
Expression in cells | Highly expressed in embryonic stem cell blastocysts and primordial germ cells | Highly expressed in embryonic stem cells, blastocysts and during differentiation | Highly expressed in blastocysts as well as differentiated cells such as oocytes, zygotes, and neurons |
CXXC, Cysteine-rich zinc ion binding domain; DBSH,
Over the past half a century, there have been conflicting findings regarding mtDNA methylation (
The primary methyl donor for mtDNA methylation is also S-adenosyl methionine which is imported into the mitochondria through a carrier (
DNMT3a and DNMT3b were identified in the protein fraction of mitochondria of specific tissues (
TET enzymes were found in mouse neuronal mitochondria indicating their possible role in regulating mtDNA methylation. TETs were demonstrated to be present in mtDNA with differing degrees of abundance of 5hmC depending on the cell type (
In recent years, more groups have identified a high frequency of non-CpG methylation in mtDNA (
Since the first time mtDNA methylation was described, multiple pieces of evidence have indicated the impact of mtDNA methylation on several mitochondrial functions (
Harbouring the origin of replication of heavy-strand and the promoters of both the heavy-strand and light-strand, methylation of the D-loop region will influence mitochondrial gene expression and mtDNA replication (
Replication of mtDNA which determine mtDNA copy number is another process hypothesised to be mediated by mtDNA methylation. This hypothesis was supported by other studies that reported alterations in the levels of mtDNA copy number in response to the upregulation and downregulation of D-loop methylation (
As for the coding regions, the impact of mtDNA methylation on them remains unknown as studies on the differential methylation in these regions remain largely unexplored. Recently, Mposhi et al. (
In summary, methylation of the D-loop region impacts mitochondrial gene expression, whereby hypermethylation of the D-loop results in decreased gene expression levels and
Other epigenetic modifications like PTMs and ncRNAs have been proposed to exist in the mitochondria. Similar to nDNA, PTMs in the mitochondria were found to modulate mitochondrial gene expression through alteration in mitochondrial transcription levels. PTMs in the mitochondria involve mitochondrial proteins such as nucleoid proteins which are integral in the organisation and functioning of the mitochondria (
The ncRNAs involved in mitochondrial epigenetics are predominantly long ncRNAs (lncRNAs) and microRNAs (miRNAs). Both nuclear and mitochondrial genomes encode these ncRNAs, but it is uncertain whether mtDNA-encoded ncRNAs resulted from nuclear mitochondrial DNA or were transcribed in the mitochondria (
As for miRNAs, they were mainly encoded by nDNA and translocated into the mitochondria, but while the mechanism is unknown, some miRNAs were encoded by mtDNA directly (
In short, other epigenetic modifications, such as PTMs and ncRNAs, could regulate mitochondrial gene expression either on a transcriptional or translational level. PTMs of nucleoid proteins could modulate the replication and transcription of mtDNA. Meanwhile, mitochondrial gene expression was altered by ncRNAs at both transcription and translation levels, which subsequently resulted in the regulation of other mitochondrial cellular and biological processes.
Epigenetics, particularly nDNA methylation, is an established field with evidence showing the effect of nDNA methylation on T2DM as well as the impact of T2DM on nDNA methylation. As for the association between mtDNA methylation and T2DM, most of the understanding of their relationship is from indirect inference either through nDNA methylation-T2DM studies or nDNA methylation-obesity studies (
In one experiment, hypermethylation of the
Zheng et al. (
In the case of diabetic retinopathy, retinal cells obtained from cells cultured in high glucose and human donor showed a higher D-loop methylation at their retinal mtDNA compared to the control. Meanwhile, the cultured retinal cells also showed increased methylation at the
In summary, it appears that mtDNA methylation plays a role in the development and progression of T2DM. Hypermethylation of the coding region results in decreased expression due to reduced transcription levels in T2DM (
One of the main limitations of mitochondrial epigenetics study is the isolation of pure mitochondria from cells and clinical samples due to their small size and low abundance. According to Lampl et al. (
Secondly, mtDNA can be isolated either from pure mitochondria or cells. Isolation of mtDNA from pure mitochondria will be ideal for epigenetics study. However, due to the limited amount of mtDNA that can be obtained from pure mitochondria, it is usual to opt for the isolation of mtDNA from cells that contain a mixture of nDNA and mtDNA. This may result in bias involving amplification during the enrichment process, as the higher abundance of nDNA would mask mtDNA during amplification.
Furthermore, identifying mtDNA methylation itself is also challenging due to technical limitations. The gold standard of mtDNA methylation work is bisulphite sequencing, whereby bisulphite conversion coupled with PCR amplification are essential steps for the sequencing (
Currently, as there are contradicting views on the presence of methylated mtDNA, multiple sequencing methods are necessary to confirm the mtDNA methylation status
Additionally, the technical limitations of mtDNA methylation studies are also observed in the non-CpG regions. Most of the commercially available bisulphite conversion kit and primers are biased toward CpG methylation, in which non-CpG nucleotides are usually assumed to be unmethylated (
Mitochondrial epigenetics is a challenging field mainly due to the small size and low abundance of the mitochondria, and its complex network with other cellular organelles. Nonetheless, studies supported the essential role of mitochondrial methylation in modulating mitochondria function. Hypermethylation at the D-loop region is associated with decreased mtDNA copy number of T2DM. Meanwhile, hypermethylation at both the D-loop and coding regions correlates to reduced gene expression. Overall, our finding shows that mtDNA methylation regulates T2DM pathogenesis. Understanding the mitochondrial epigenetics profile may aid in tracing T2DM pathogenesis and evaluating intervention efficacy in T2DM treatment.
YFP contributed to the conception of the review. HL organised the data and drafted the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.
This work was funded by the Malaysia Ministry of Science, Technology and Innovation Grant NMHD0002 and NMHD0003 awarded to YFP.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.
DM, Diabetes mellitus; T2DM, Type 2 Diabetes mellitus; mtDNA, mitochondria DNA; D-loop, displacement loop; nDNA, nuclear DNA; CH3, methyl group; CpG, cytosine-phosphate-guanine (and so on for CpA, CpT and CpC); 5mC, 5-methylcytosine; DNMT, DNA methyltransferase; TET, Ten-eleven translocation methylcytosine dioxygenase; 5hmC, 5-hydroxymethylcytosine; mtDNMT1, mitochondrial-localised DNMT1, post-translational modification, PTM; non-coding RNA, ncRNA; messenger RNA, mRNA; long ncRNA, lncRNA; microRNA, miRNA; C5, fifth carbon, N6-methyldeoxyadenosine; m6A; PCR, polymerase chain reaction;