Abstract
In brief
This review discusses advances in the knowledge of epigenetic mechanisms regulating mitochondrial DNA and the relationship with reproductive biology.
Abstract
Initially perceived simply as an ATP producer, mitochondria also participate in a wide range of other cellular functions. Mitochondrial communication with the nucleus, as well as signaling to other cellular compartments, is critical to cell homeostasis. Therefore, during early mammalian development, mitochondrial function is reported as a key element for survival. Any mitochondrial dysfunction may reflect in poor oocyte quality and may impair embryo development with possible long-lasting consequences to cell functions and the overall embryo phenotype. Growing evidence suggests that the availability of metabolic modulators can alter the landscape of epigenetic modifications in the nuclear genome providing an important layer for the regulation of nuclear-encoded gene expression. However, whether mitochondria could also be subjected to such similar epigenetic alterations and the mechanisms involved remain largely obscure and controversial. Mitochondrial epigenetics, also known as ‘mitoepigenetics’ is an intriguing regulatory mechanism in mitochondrial DNA (mtDNA)-encoded gene expression. In this review, we summarized the recent advances in mitoepigenetics, with a special focus on mtDNA methylation in reproductive biology and preimplantation development. A better comprehension of the regulatory role of mitoepigenetics will help the understanding of mitochondrial dysfunction and provide novel strategies for in vitro production systems and assisted reproduction technologies, as well as prevent metabolic related stress and diseases.
Introduction
Cellular metabolism consists of a complex set of biochemical reactions that occur in living cells in order to produce the energy and substrates that sustain life. Metabolism is notably susceptible to environmental changes, and that is why monitoring bioenergetics represents a challenge but becomes an important way to understand physiological and pathophysiological conditions.
In reproductive sciences, the importance of metabolic processes has been studied for many years in the context of preparation of gametes for in vitro fertilization and embryo production. Most of these studies, however, have been aimed at practical applications, such as culture media creation and manipulation, which do not always address the comprehension of basic cell biology functioning.
As a result, basic knowledge is limited, but we know that early mammalian gametes and embryos present a very peculiar metabolism in comparison with somatic/differentiated cells. In different species, metabolic requirements vary according to the developmental stage of the embryo, particularly during first cleavages and up to the blastocyst stage (Krisher et al. 2015, Milazzotto et al. 2022b). During this peri-implantation stage, there is a remarkable shift in the way cells utilize metabolites to produce the energy and the substrates that will be used for cell division, growth, and differentiation. The main causes of metabolic fluctuations comes from the availability of enzymes and substrates, meaning that if one of those has limited availability, the cell might divert from its preferable metabolic pathway toward the one that is most available at the moment (Warzych & Lipinska 2020).
Balance is vital for the survival of any cell, but this seems to be particularly important during initial development. In 2016, Leese et al. have described the existence of an optimal range of metabolic activity, a ‘Goldilocks zone’, in reference to the range in which oocytes and embryos will develop at their maximum capacity (not too little, not too much, and ‘just the right amount’) (Leese et al. 2016). This homeostatic environment is not observed either in vivo or in vitro during embryo growth and differentiation, and that is the reason why so many adaptation mechanisms are necessary to keep these embryos growing. Regulation of intracellular reactive oxygen species (ROS), initiation of stress responses, apoptosis, calcium regulation, and optimized energy production are some examples of the mechanisms used by embryos to sustain the trajectory of initial development. It is noteworthy that most of these adaptations happen, at least in parts, inside mitochondria, making them key players for the maintenance of metabolic homeostasis and basic cell function (Harvey 2019, Chakrabarty & Chandel 2022).
It is important to realize that because of their plasticity and high adaptability, embryos are capable of surviving even in non-optimal conditions. This survival at any cost, however, does have consequences, and these changes can be passed to daughter cells and create phenotypes that are perpetuated in the next generations through epigenetic inheritance (Perez & Lehner 2019). Therefore, in this article, we review how mitochondrial metabolism is critical to the successful initiation of the developmental program. We also discuss mitochondria’s central role in the modulation of the epigenetic profile during early embryonic development.
Metabolism of the preimplantation embryo
Mammalian pre-implantation development is a complex and tightly regulated process that depends on a number of internal and external factors. It starts with the release of the oocyte, followed by fertilization and formation of a single-cell zygote. Then, the speed of the following events varies between species, but, in general, the zygote undergoes a series of three to four rounds of division, followed by the compaction step around the eight-cell stage in mice (Lo & Gilula 1979) or the 16- to 32-cell stage in bovine, for example (Soom et al. 1997). During the first cleavages, the metabolism of the blastomeres is slow and basically sustained by the depletion of transcripts and proteins that were produced and stored during oocyte maturation (Zhang & Smith 2015). At this point, the energy requirements are low; thus, the high ATP:ADP ratio maintains the inhibition of phosphofructokinase, limiting glucose oxidation. Pyruvate and lactate (obtained either from culture media or from oviductal fluid) are the main substrates used for energy generation via glycolysis (Guerif et al. 2013, Gardner & Harvey 2015). Previous studies have demonstrated that in the absence of pyruvate, murine embryos arrest at the two-cell stage (Aoki et al. 1997) and at morula stage in bovine (Takahashi & First 1992).
Development also requires the activation of transcription, a step referred to as ‘Embryonic Genome Activation’, which in bovine happens around the 8- to 16-cell stage (Graf et al. 2014). It is expected that such a critical moment requires not only a lot more energy but also structural changes and epigenetic remodeling to make possible the switch from maternal/paternal genome to the embryo genome (Graf et al. 2014). To support these and other cellular events such as the synthesis of biomass, cell proliferation and blastocoel formation, and hatching (Hamatani et al. 2006, Jeanblanc et al. 2008), the embryo gradually increases the consumption of glucose, oxygen, and the overall oxidative metabolism, which is notably more efficient than glycolysis (Gardner & Harvey 2015, Lane & Gardner 2005).
With the expansion of the blastocoel, the first step of cell differentiation also happens, resulting in a blastocyst that contains the inner cell mass (ICM; progenitor cells of the embryo itself) and trophectoderm cells that will form the extra-embryonic tissues (Nagaraj et al. 2017, Carreiro et al. 2021). Interestingly, these two cell types present different preferred metabolic pathways, reflecting their own special needs. For example, the ICM cells are mostly glycolytic, while the trophectoderm cells mostly oxidize glucose via oxidative phosphorylation. Interestingly, only half of the glucose is consumed that way. The other half is converted to lactate, even in the presence of oxygen, in a pathway originally known as aerobic glycolysis (Warburg effect) (Gardner & Harvey 2015). Although this may sound counterproductive, it is a mechanism that ensures the activity of the pentose–phosphate pathway to generate glutathione (a powerful antioxidant), as well as reducing equivalents (NAD+/NADPH) that will be used for biosynthesis of macromolecules (Gardner 2015, Gardner & Harvey 2015).
In addition to their important role as structural units in membrane formation and cell signaling pathways, lipids are also a potential source of energy for the embryo through the beta-oxidation of fatty acids. Fatty acids either remain free in the cytoplasm or are accumulated during oocyte maturation and are stored within of lipid droplets, in the form of triacylglycerols (Sutton-McDowall et al. 2012), although their exact content varies widely among species (Kajdasz et al. 2020). Either way, fatty acids can be transported to mitochondria via the carnitine palmitoyl–transferase shuttle and then oxidized via beta-oxidation, which is a fast and direct way of obtaining energy (Dunning et al. 2010).
Previous studies have demonstrated that lipid composition differs also between in vivo and in vitro derived embryos (Romek et al. 2010), as well as between different stages of development (de Lima et al. 2018). In most studies, the lipid content remains constant up to the morula stage and then decreases substantially until blastocyst formation, probably due to an intensification of beta-oxidation to support the increase in energy demand for blastocoel expansion (Sudano et al. 2016).
Although the preferred metabolic pathways to produce energy are variable between species (Milazzotto et al. 2022b), one thing is clear: embryos are dynamic and present high ability to adjust their metabolism to the environment surrounding them. The concept of embryo plasticity has been vastly explored, and modifications in temperature, osmolality, composition, and pH of the culture media have been extensively investigated (Coticchio et al. 2021). Another study recently described in detail how embryonic metabolism is capable of shifting in response to common stressors, individually or combined (de Lima et al. 2020). The study demonstrated that bovine embryos present heterogeneous metabolic responses depending on early phenotypes (e.g. cleavage kinetics) and culture environment (e.g. glucose and oxygen concentration). A lower oxygen concentration (5%), for example, boosted the metabolism of embryos cultured in lower glucose concentrations (0.6 mM or 2 mM), but the same improvement was not observed when higher glucose concentration was used (5 mM).
Although the embryo can adapt to environmental challenges and nutrient imbalance, the adaptive processes come at a cost. The energetic demands of blastocyst formation require an appropriate regulation, as well as nutrient-sensing pathways. In this scenario, the roles of mitochondria as drivers of these processes are only emerging, as it becomes clear that this is critical for embryo competence.
Mitochondria: characteristics and function
Mitochondria are double-membraned organelles also known as the ‘powerhouses of the cell’. Extensive research over the past decades established two main functions of mitochondria: (i) production of ATP to fuel biochemical reactions and (ii) generation of biosynthetic intermediates (carbohydrates, proteins, lipids, and nucleic acids) (Chakrabarty & Chandel 2022). But beyond canonical roles, mitochondria can also act in signaling pathways, a fact that brought a dramatic shift in our view of mitochondria’s relationship with cell function. Some examples of non-canonical roles include cytochrome C release and caspase activation in programmed cell death, ROS-mediated signaling, and activation of cascades, leading to immune response (Harvey 2019).
Mitochondria’s outer membrane faces the cytosol, and the folds of the inner membrane protrude into the matrix (cristae). The bulk of the electron transport chain (ETC) complex occurs in the cristae, while the tricarboxylic acid (TCA) cycle enzymes and mitochondrial genome are found in the matrix (Chakrabarty & Chandel 2022). Mitochondria also contain their own genome. The mitochondrial DNA (mtDNA) is a highly condensed, maternally inherited genome, consisting of a 16 kb circular sequence that encodes for 13 proteins components of the ETC complex, 22 transfer RNAs, and 2 ribosomal RNAs (D’Souza & Minczuk 2018).
Although it does not contain histones, mtDNA is packaged into nucleoprotein structures named nucleoids (Farge & Falkenberg 2019). The packaging of mtDNA can be monomeric (a single mtDNA molecule) or multimeric (multiple copies), and both types have been described in different cell types (Gilkerson et al. 2013, Menger et al. 2021). The formation of these nucleoids relies mostly on the presence of the transcription initiation factor known as TFAM (Gilkerson et al. 2013). The literature describes that TFAM is abundant and capable of coating the entire mtDNA molecule creating U-shape turns, particularly at promoter regions. This means that in order to transcribe, repair, or replicate, interacting proteins (e.g. topoisomerases) must manipulate the structure of the mtDNA molecule to gain access to the sequence (Menger et al. 2021).
Differently from nuclear DNA, which contains at least one promoter per gene, mtDNA contains three promoter regions: light strand promoter (LSP) for genes located in the light strand and heavy strand promoter (HSP)1 and HSP2 for the genes located in the heavy strand, meaning that the whole mitochondrial genome is transcribed as long polycistronic transcripts that need multiple processing steps in order to be functional (D’Souza & Minczuk 2018, Stoccoro & Coppedè 2021).
Communication between mitochondria and nucleus
The mitochondrial genome codes only for 13 proteins; however, more than 2000 proteins can be found in the mitochondrial compartment. Most of them are products of nuclear DNA that were synthetized in the cytoplasm and then translocated into the mitochondria. The nucleus-to-mitochondria communication is relatively well studied, but information on the retrograde communication in the mitochondria-to-nucleus direction is still lacking. (Soledad et al. 2019)
Mitochondria generate a wide range of retrograde signals to the nucleus, reflecting metabolic status and mitochondrial activity. When submitted to cellular stress, mitochondria can change these signals, altering the expression of nuclear genes and ultimately leading to metabolic reprogramming for cellular adaptation (defense mechanism) or death. In mammalian cells, good examples of the retrograde response are the mTOR, AMPK signaling, and ROS-dependent pathways (Morita et al. 2015, Soledad et al. 2019).
The mTOR signaling pathway is activated under conditions of energetic stress and is a master nutrient-sensing mechanism. The activation of mTOR pathway can modulate protein synthesis from the nuclear genome and induce mitochondrial biogenesis (Kahn et al. 2005, Soledad et al. 2019). The AMPK pathway is activated mainly by the increase of AMP/ATP ratio and promotes the activation of PGC1A, which in turn stimulates cellular metabolism and mitochondrial biogenesis (Kahn et al. 2005).
ROS are normally produced during aerobic metabolism, and their production is tightly balanced by scavenging mechanisms (Agarwal et al. 2022). Increases in ROS levels can activate transcription factors such as nuclear factor erythroid 2-related factor 2, which triggers the expression of antioxidant genes (Nguyen et al. 2009), for example. Other cellular stresses, such as glucose deprivation, hypoxia, inhibition of the TCA/oxidative phosphorylation, and hyperosmotic stress, can also trigger a retrograde response in mitochondria through the abovementioned mechanisms (D’Souza & Minczuk 2018).
More recently, mitochondria have been put in a central position as one of the most important pieces of the epigenetic puzzle in early development, because several mitochondrial metabolites have a role as signaling molecules and can be involved in the regulation of cytosolic and nuclear enzymes, including the ones responsible for epigenetic regulation.
Association between mitochondrial metabolism and epigenetic modifications
It is well established that epigenetic changes may influence gene expression by causing temporary or heritable alterations on the DNA and/or associated histones (Portela & Esteller 2010). Modifications to the epigenome are reversible and do not interfere directly with the DNA sequence (Gibney & Nolan 2010). The epigenome is also responsible for establishing and maintaining cellular differentiation and phenotype. In early embryos, the epigenome is almost completely remodeled to ensure the acquisition of a totipotent state and ability to differentiate into all cell types (Ross & Canovas 2016). The most common epigenetic modifications include DNA and RNA methylation, post-translational modification of histones, and non-coding RNAs. Epigenetic remodeling and the different types of epigenetic modifications have been extensively studied and reviewed elsewhere (Ross & Canovas 2016, Stoccoro & Coppedè 2021).
Studies done mostly in embryonic stem cells and cancer have recently introduced the concept of metaboloepigenetics (Donohoe & Bultman 2012), given that some epigenetic marks related to the control of gene expression (e.g. DNA methylation, histone methylation, and acetylation) are also subjected to mitochondrial related signaling. In other words, the availability of specific nutrients can modulate metabolic pathways and directly affect the activity of enzymes that act as epigenetic modifiers (Wallace & Fan 2010, Zhang et al. 2019, Ispada et al. 2020) (Fig. 1). For example, alpha-ketoglutarate (α-KG) from the TCA cycle, as well as succinate and glutamine, can modulate histone and DNA demethylation catalyzed by ten–eleven translocation (TET) and Jumanji (JMJ) demethylases (Tsukada et al. 2006, TeSlaa et al. 2016). Acetyl-CoA generated from pyruvate or from beta-oxidation of fatty acids can act as a cofactor for histone acetyltransferases (HATs) (Martínez-Reyes & Chandel 2020). One-carbon metabolism provides methionine, which is required for the generation of S-adenosylmethionine (SAM) that, in turn, acts as a methyl donor for histone methyl transferase and DNA methyltransferases (DNMTs) (Xu & Sinclair 2015). Therefore, perturbations in mitochondrial activity have the potential to significantly affect cell functioning in the short (histones) and long (DNA) term via epigenetic modifications of the chromatin.
Regulation of nuclear epigenome by mitochondrial metabolism. Mitochondrial metabolism controls the availability of cofactors (S-adenosyl methionine, acetyl-CoA, and NADH/NAD+), as well as other TCA intermediates (citrate, succinate, and α-ketoglutarate), which can act as key substrates for DNA methyltransferases/demethylases (DNMTs/TETs), as well as for chromatin modifier enzymes such as histone acetyl transferases (HATs), histone methyl transferases (HMTs), and histone demethylases (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
Regulation of nuclear epigenome by mitochondrial metabolism. Mitochondrial metabolism controls the availability of cofactors (S-adenosyl methionine, acetyl-CoA, and NADH/NAD+), as well as other TCA intermediates (citrate, succinate, and α-ketoglutarate), which can act as key substrates for DNA methyltransferases/demethylases (DNMTs/TETs), as well as for chromatin modifier enzymes such as histone acetyl transferases (HATs), histone methyl transferases (HMTs), and histone demethylases (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
Regulation of nuclear epigenome by mitochondrial metabolism. Mitochondrial metabolism controls the availability of cofactors (S-adenosyl methionine, acetyl-CoA, and NADH/NAD+), as well as other TCA intermediates (citrate, succinate, and α-ketoglutarate), which can act as key substrates for DNA methyltransferases/demethylases (DNMTs/TETs), as well as for chromatin modifier enzymes such as histone acetyl transferases (HATs), histone methyl transferases (HMTs), and histone demethylases (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
One-carbon metabolism
The pathway known as one-carbon metabolism is a network of biochemical reactions that integrates the folate and methionine cycles and is present in the cytoplasm, mitochondria and nucleus. The main function of 1C metabolism is to produce and transfer carbon units that will be used in the de novo synthesis of purines as well as the remethylation of methionine (Xu & Sinclair, 2015). Methionine plays key roles in nuclear functions (polyamines) and maintenance of cellular redox status (glutathione) (Lauinger & Kaiser, 2021), but ultimately, it contributes to the production of SAM, which is the main methyl donor for DNA, RNA, and HMTs (Xu & Sinclair, 2015).
The availability of SAM is directly influenced by the diet since the methyl groups are derived from choline, methionine, or methyl-tetrahydrofolate (Crouse et al. 2022). Modifications to DNA and histone methylation are carefully orchestrated to modulate gene expression and programming; therefore, alterations in the intracellular availability of SAM can result in genome-wide gene expression changes both in the short term (histones) and long term (DNA) (Stover, 2011, Xu & Sinclair, 2015).
The removal of one methyl group from SAM results in SAH (S-adenosylhomocysteine), which then can be hydrolysed into homocysteine and, again, remethylated to methionine to be available for later utilization. SAH is considered an inhibitor of methyltransferases; thus, it is rather the ratio between SAM and SAH that is considered the main indicator for methylation capacity (Milazzotto et al. 2022a).
The TCA cycle
The TCA cycle, also known as citric acid cycle or Krebs cycle, is a basic mitochondrial pathway that is essential for the production of energy and other biosynthetic intermediates (Chakrabarty & Chandel 2022). The TCA is considered a central hub for cell metabolism because multiple substrates are produced and can feed into it. Besides, the tight regulation of the TCA and its constant feedback with oxidative phosphorylation is critical to keep the cellular homeostasis (Martínez-Reyes & Chandel 2020).
Citrate is one of the metabolites produced by the TCA cycle that can be exported into the cytosol and then converted into acetyl-CoA to be used for histone acetylation by HATs enzymes (Harvey, 2019). High glycolytic activity is responsible for the synthesis of acetyl-CoA inside mitochondria, but acetyl-CoA can also be produced in the cytosol (from fatty acids, amino acids, and oxidation of ketone bodies) or inside the nucleus (Wellen & Thompson, 2012).
Another metabolite from the TCA cycle, α-KG, is not only a substrate for dioxygenase enzymes such TETs that regulate demethylation processes at different cellular levels (Chakrabarty & Chandel 2022, Milazzotto et al. 2022a) but also can regulate the activity of JMJ demethylases which remove methyl groups from lysine residues of histones (Tsukada et al. 2006).
α-KG can also bind and block the mitochondrial ATP synthases and inhibit mTOR signaling pathway (Chin et al. 2014). Histone demethylases can also use α-KG as a cofactor to remove methylation from histones residues. In different cell types, α-KG has been shown to promote differentiation, while the accumulation of succinate or the lack of α-KG delays differentiation (TeSlaa et al. 2016, Milazzotto et al. 2020).
Importance of mitochondria in reproductive biology
In reproduction, mitochondria are a prominent source of energy for oocytes, as well as for sperm biogenesis and function (Podolak et al. 2022). However, mitochondria undergo dynamic, stage-specific restructuring and redistribution during early development (Harvey 2019). Initially, during oocyte maturation and up until the morula stage, mitochondria exhibit a more round, hooded morphology with few cristae (Calarco & Brown 1969, Hayashi et al. 2021) and, therefore, a less efficient capacity of oxidation and energy production.
Furthermore, mature oocytes contain approximately 200,000 copies of mtDNA, which represents several order of magnitude greater than the number observed in most somatic cells (Wai et al. 2010, Podolak et al. 2022). This high number observed in oocytes is associated with a limited replication capacity until the embryo reaches the blastocyst stage and where the number of copies per cells comes back to the average somatic cell.
Besides, as mitochondria are exclusively maternally inherited, the quantity of structures present in the mature oocyte at fertilization represents the final progenitor pool that the individual will carry throughout life. Therefore, genetic, structural, or numerical defects that create functional deficiencies will likely have adverse effects on the capacity of the embryo to progress through the preimplantation stages (Van Blerkom 2011).
Several studies have shown that mitochondrial abnormalities can reduce the quality of oocytes and embryos, and contribute to post-implantation failure, long-term cell dysfunction, and disease (Harvey 2019, Chakrabarty & Chandel 2022). The mtDNA copy number is sometimes reported higher in aneuploid embryos, compared with those in chromosomally normal embryos (Fragouli et al. 2015). Furthermore, mitochondrial dysfunction plays an essential role in the mother age-related reduced oocyte competence. In humans and rodents, mitochondria from older individuals shows morphological abnormalities such as swelling, vacuolization, and cristae alterations in comparison with young individuals (Kushnir et al. 2012, Simsek-Duran et al. 2013).
Metaboloepigenetic events in early embryonic development
Growing evidence in the field of reproductive sciences clearly shows an undeniable impact of environmental factors and cellular metabolism on the epigenetic profile (Chaput & Sirard 2020, Peral-Sanchez et al. 2021). During early development, substrate choices are determinants for the fate of the embryo. Cells have to adapt by selecting metabolic pathways for specific moments, and each decision – proliferation, differentiation, and death – is made according to the challenges sensed in the environment (Milazzotto et al. 2022a). For this reason, nutrient and metabolic changes that happen in response to the environment are important for the proper reprogramming of the epigenome and long-term survival (Milazzotto 2022). It is imperative then to identify and understand the differences between specific micronutrient requirements vs global energy requirements that can influence these processes.
Previous studies have already correlated aberrant epigenetic patterns with alterations in the microenvironment surrounding the developing embryo. In bovine embryos, for example, the presence of methionine in culture media is necessary for blastocyst development, expansion, and hatching (Bonilla et al. 2010). Another study showed that maternal methionine supplementation reduced transcription and increased methylation of specific genes (Peñagaricano et al. 2013). In sheep, dietary inputs to the methionine/folate cycles during the periconceptional period also leads to alterations to DNA methylation in the offspring (Sinclair et al. 2007). In mice, a diet supplemented with methyl donors induced alteration in specific imprinted genes (Cooney et al. 2002).
In mice (Zhang et al. 2019) and bovine (Ispada et al. 2020), the supplementation of culture media with α-KG during in vitro development interferes with DNA methylation/demethylation dynamics. Still in bovine, the analysis of the effect of the paternal and maternal age (very young bulls and cows) (Morin-Doré et al. 2020, Wu et al. 2020), as well as the exposure to ketone bodies (β-hydroxybutyrate) in post-partum cows, presented evidence that these three conditions share a common metabolic stress (an energy deficit) and have a similar impact on the early embryos: epigenetic alterations, reduction of their own metabolism, and induction of mitochondrial dysfunction (Wu & Sirard 2020). Surprisingly, in vitro culture seems to create a similar picture of embryos being forced into an ‘economy mode’ as an adaptation to suboptimal metabolic environment.
In mice, preimplantation exposure to ketone bodies also affected glucose metabolism and increased histone acetylation in blastocysts that are associated with persistent, female-specific perturbations in fetal development (Whatley et al. 2023). The suboptimal environment of the culture system itself is also sufficient to cause hyperacetylation of histone 3 lysine 9 (Rollo et al. 2017) and H3K4 trimethylation (Wu et al. 2012).
These numerous examples indicate that mitochondria are the critical link between the environment and the epigenome. Initially, this promising field of research was entitled ‘mitoepigenetics’. With the increasing attention mitochondria has been given and the accumulation of new evidences, the term has also been used to refer to epigenetic mechanisms that regulate the mitochondrial genome itself.
Mitoepigenetics: evidences of mtDNA methylation
First reports of mtDNA methylation are from the 1970s and were obtained using radiolabeling (Nass 1973). Subsequent experiments carried out in humans and mouse models estimated between 2 and 5% of 5-methylcytosines in mtDNA (Shmookler Reis & Goldstein 1983, Pollack et al. 1984). Other studies, however, reported the absence of these marks in mitochondria (Mechta et al. 2017). Therefore, until recently, the sole existence of epigenetic mechanisms controlling mtDNA was a controversial topic in research (Hong et al. 2013, Patil et al. 2019), and the main reason behind this controversy was the methodology used for mtDNA methylation detection.
For a while, the use of bisulfite treatment prior to DNA sequencing was the most common method employed to detect mtDNA cytosine methylation. In this case, the characterization of the methylation profile depends on the conversion of 5-methylcytosine to uracil. However, this conversion may be blocked by the secondary and even tertiary structure of circular mtDNA, ultimately leading to an overestimation of mtDNA methylation (Mechta et al. 2017, Owa et al. 2018). To counteract the bias of bisulfite-resistant cytosines, linearization of mtDNA via restriction digestion has been used prior to bisulfite treatment (Owa et al. 2018), as well as the addition of methylated and unmethylated spikes to estimate bisulfite conversion efficiency. Besides, because of the small size of the mitochondrial genome, available tools normally used for methylation measurements in somatic cells need specific adaptations in order to accurately detect and quantify mtDNA methylation levels.
More recently, with the continuous improvements in detection methods and emerging technology, numerous studies demonstrated that similarly to nuclear DNA, mtDNA indeed can be subjected to epigenetic modifications (Dou et al. 2019, Patil et al. 2019, Yue et al. 2022). A strong indicator supporting the existence of this mechanism is that enzymes such as DNMTs and TETs, which are instrumental in DNA methylation/demethylation in the nucleus, have also been visualized in mitochondria (F C Lopes 2020). An isoform of the DNMT1 has a mitochondrial targeting sequence and translocates into mitochondria (Shock et al. 2011) to interact with the mtDNA in the matrix of some tissues such as mouse embryonic fibroblasts, human brain cells, and cancerous cells (Dou et al. 2019). More recently, DNMT3A and DNMT3B were also co-localized to the mitochondrial compartments in implantation and post-implantation embryos in mice (Yue et al. 2022).
Despite the evidences showing that mtDNA could be subjected to methylation by the same enzymes working on nuclear DNA, it should be highlighted that from an epigenetic perspective, mitochondrial genome is very different from nuclear genome. For example, mtDNA lacks CpG islands, as well as a nucleosome–chromatin structure, which are known to be involved in epigenetic regulation (Kowal et al. 2020). Therefore, epigenetic mechanisms as we know might function differently in mitochondria.
Indeed, other evidences of such differences show that contrary to nuclear DNA, mtDNA is most methylated in a non-CpG context, and there is an asymmetrical profile between mtDNA strands, with methylation marks being more present at the light strand, as demonstrated by Dou et al. (2019), Patil et al. (2019), and de Lima & Sirard (2020). It is noteworthy that the L-strand has an important role in mitochondrial function, because 12 out of the 13 mitochondrial genes use this strand as template for transcription (D’Souza & Minczuk, 2018). Furthermore, the presence of hydroxymethyl cytosines (5-hmC) in mtDNA has been observed, and studies suggest that 5-hmC modifications exhibit dynamic characteristics and are concentrated in the upstream regions of gene start sites as well as within the gene body, analogous to nuclear genes (Ghosh et al. 2016).
Another aspect of mtDNA epigenetics is the presence of N6-methyldeoxyadenosine, which is usually found in prokaryotes rather than mammals’ genome. This type of methylation was recently identified in human mtDNA, and its presence is mediated by METLL4, a mutative mammalian methyltransferase (Hao et al. 2020). Studies suggest that adenosine methylation decreases mitochondrial transcription and mtDNA copy number, particularly under hypoxic stress (Hao et al. 2020).
These studies provide clear evidence of a distinct control over mtDNA methylation. Understanding the workings of these mechanisms is crucial to gain a deeper insight into the regulation of mitochondrial function.
Mitoepigenetics: a new regulatory layer
Different cytosine methylation levels have been described in different animal and human tissues, suggesting that there is a specific regulation of mtDNA function via epigenetic mechanisms (Cao et al. 2021, Lüth et al. 2021, Yue et al. 2022). Studies carried out in aging, cancer, obesity, diabetes, and cardiovascular and neurodegenerative diseases (reviewed by Stoccoro & Coppedè 2021) identified cytosine methylation in both gene bodies and in regulatory regions such as the non-coding region known as displacement loop (D-loop), which contains promoters for the heavy and the light strands and transcriptional start sites (e.g. TFAM-binding site), which are responsible for controlling polycistronic mitochondrial gene expression (D’Souza & Minczuk 2018). Therefore, it is increasingly evident that although found in low levels, methylation of mtDNA might be associated with regulation of mitochondrial gene transcription and mtDNA replication.
In that sense, many studies have described a clear correlation between, mtDNA methylation and transcription of mitochondrial genes. It is the case of the condition known as non-alcoholic steatohepatitis (NASH) where methylation of the ND6 gene is inversely correlated with ND6 transcription and protein expression in the liver affected by NASH (Pirola et al. 2013). In humans with diabetic retinopathy, an increase in D-loop methylation was also associated with a decrease in mtDNA transcription in retinal endothelial cells (Mishra & Kowluru 2015). Hypermethylation of gene regions MT-CO1 and MT-CYB was found to be concomitant with higher gene expression levels in oral squamous cells carcinoma (Aminuddin et al. 2020). Numerous examples like these have been extensively reviewed elsewhere (Stoccoro & Coppedè 2021) and reinforce the idea that mitochondrial genome has the capacity to adjust genes and protein levels in response to the environment and cellular state. In order to do that, epigenetic mechanisms seem to be a fundamental part of the equation and help coordinate the crosstalk between the nucleus and mitochondria, allowing the modulation of the metabolic response to different energy demands.
Mitoepigenetics in reproduction
In different reproductive models, non-random patterns of cytosine methylation in mtDNA, predominantly observed in a non-CpG context, have been described (Dou et al. 2019, Patil et al. 2019, Yue et al. 2022). In humans, for example, a study evaluated fetal cord blood from patients with intrauterine growth restriction and preeclampsia, both being pregnancy disorders that lead to placental insufficiency, oxygen/nutrient restriction, and oxidative stress. The authors observed an increased mtDNA content associated with hypomethylation of D-loop and COX1 gene in the pathological groups compared to controls (Novielli et al. 2017). Other studies in humans evaluated the effect of maternal smoking on the placenta and showed hypermethylation in regions of mtDNA, such as D-loop and MT-RNR1 (12S ribosomal RNA) (Armstrong et al. 2016).
In the porcine model, a study explored a possible role of mtDNA methylation in mitochondrial malfunction and decreased oocyte quality (Jia et al. 2016). The authors observed that hypermethylation of the D-loop, as well as other mtDNA sequences, was associated with lower mtDNA content and downregulated expression of mtDNA-encoded genes in oocytes obtained from gilts with polycystic ovaries.
It is important to highlight that in the context of reproductive biology, the oocyte is an interesting model to study the dynamics of mtDNA methylation, as their naturally enriched mitochondria number is critical for successful cytoplasm and nucleus maturation in preparation for fertilization. In that sense, our group recently showed that, in bovine, mtDNA methylation has particular signatures in oocytes obtained from distinct follicular environments (large vs small follicles) that are transmitted to the resulting blastocysts produced in vitro (Sirard 2019). In these samples, the higher density of methylation marks in the control region (D-loop) coincides with the location of both the light- and the heavy-strand promoters (de Lima & Sirard 2020). Interestingly, mtDNA methylation negatively correlated with mitochondrial gene expression, but the consequences to mitochondrial function during preimplantation development remain to be elucidated.
Similar results were observed in mice embryos, where mtDNA methylation levels increased all over the mitochondrial genome, including the D-loop and non-coding regions, during the developmental transition from blastocysts to post-implantation embryos. Furthermore, the same study shows a spatiotemporal coincidence of de novo mtDNA methylation and mitochondrial oxidative stress, indicating that mtDNA might have a protective role against oxidative damage (Yue et al. 2022).
The addition of the previous studies makes a functional hypothesis: cytosine methylation in the mitochondrial genome may have a protective role in the oocytes and early ICM cells (due to the higher ICM glycolytic activity compared to trophectoderm (Gopichandran & Leese 2003). By limiting the transcription of important elements of the electron chain, it may also prevent mitochondria from excessive activation and generation of free radicals that may damage the precious mtDNA that will be transmitted to the next generation of primordial germ cells. At the same time, methylation may serve as a physical protection painted on the mtDNA to block ROS-induced mutations (Fig. 2).
The dynamics of mitochondria during initial development is capable of finely programming the epigenetic landscape during preimplantation events. As metabolic activity increases to meet the energetic demands of embryo genome activation and blastocyst formation, we propose that the presence of cytosine methylation limits the re-activation of mitochondrial transcription and helps physically protecting the mtDNA from damage caused by excessive activation of mitochondrial metabolism and ROS generation (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
The dynamics of mitochondria during initial development is capable of finely programming the epigenetic landscape during preimplantation events. As metabolic activity increases to meet the energetic demands of embryo genome activation and blastocyst formation, we propose that the presence of cytosine methylation limits the re-activation of mitochondrial transcription and helps physically protecting the mtDNA from damage caused by excessive activation of mitochondrial metabolism and ROS generation (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
The dynamics of mitochondria during initial development is capable of finely programming the epigenetic landscape during preimplantation events. As metabolic activity increases to meet the energetic demands of embryo genome activation and blastocyst formation, we propose that the presence of cytosine methylation limits the re-activation of mitochondrial transcription and helps physically protecting the mtDNA from damage caused by excessive activation of mitochondrial metabolism and ROS generation (figure created with BioRender.com).
Citation: Reproduction 166, 1; 10.1530/REP-22-0424
Conclusions and perspectives
The existence of epigenetic modifications influencing mtDNA structure is a hallmark finding. However, in the absence of a comprehensive map of the mitochondrial epigenome, the relationship between the presence of methylation marks and their impact in the regulation of nuclear and mitochondrial gene expression and overall mitochondrial function remains undetermined.
It is clear that our knowledge in mitoepigenetic mechanisms and regulation is still poor, but data obtained so far in numerous models and cell types encourage further exploration of this topic. The use of in vitro systems and assisted reproduction technologies are still not fully optimized to provide the conditions found in the reproductive system, especially facing the dynamic energetic requirements during oocyte development and initial embryonic development. Therefore, the crosstalk between epigenetics and mitochondria is an important aspect of embryonic adaptation and most certainly a good target for further improvement of current methodologies.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this work.
Funding
The authors would like to thank the support of NSERC.
Author contribution statement
CBL and ECS wrote and reviewed the paper. MAS idealized and reviewed the paper.
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