Mitochondrial DNA replication is critical for maintaining mtDNA copy number to generate sufficient cellular energy that is required for development and for functional cells. In early development, mtDNA copy number is strictly regulated at different stages, and, as a result, the establishment of the mtDNA set point is required for sequential cell lineage commitment. The failure to establish the mtDNA set point results in incomplete differentiation or embryonic arrest. The regulation of mtDNA copy number during differentiation is closely associated with cellular gene expression, especially with the pluripotency network, and DNA methylation profiles. The findings from cancer research highlight the relationship between mitochondrial function, mtDNA copy number and DNA methylation in regulating differentiation. DNA methylation at exon 2 of DNA polymerase gamma subunit A (POLGA) has been shown to be a key factor, which can be modulated to change the mtDNA copy number and cell fate of differentiating and tumour cells. The present review combines multi-disciplinary data from mitochondria, development, epigenetics and tumorigenesis, which could provide novel insights for further research, especially for developmental disorders and cancers.

Introduction

Mitochondria are double-membraned organelles primarily responsible for the generation of ATP as well as several other vital cellular functions. Each mitochondrion hosts multiple copies of the mitochondrial genome (mtDNA), which is a circular double-stranded genome encoding key subunits of the electron transfer chain (ETC). There are programmed changes to mtDNA copy number during cellular development and tumorigenesis, which are directly regulated by a group of nuclear-encoded regulators. It has also been demonstrated that changes to mtDNA copy number are associated with the expression of key regulators of cell fate and the DNA methylation status at exon 2 of the catalytic subunit of DNA polymerase gamma (POLGA). These processes are strictly regulated to establish the mtDNA set point, which provides differentiating cells with the potential to increase mtDNA copy number in a cell-specific manner.

Mitochondria and OXPHOS

The mitochondrion is a double-membraned organelle present in nearly all mammalian cells. Mitochondria are associated with a variety of cellular and metabolic activities such as cell signalling, ion homoeostasis, cell differentiation and apoptosis [1,2]. They are primarily described as the powerhouses of cellular metabolism because they are the major generators of cellular energy (ATP) through the aerobic process of oxidative phosphorylation (OXPHOS) [3,4] (Figure 1).

mtDNA interactions.

Figure 1.
mtDNA interactions.

mtDNA replication and transcription take place inside the mitochondria. mtDNA replication is dependent on nDNA-encoded replication factors including the mitochondrial single-stranded-binding protein (mtSSB), Twinkle, POLGA, POLG accessory subunit (POLGB) and DNA topoisomerase I mitochondrial (TOP1MT). They translocate into the mitochondria. A group of upstream regulators including STAT3, HIF1α, SIRT1, MYC, PGC1α/β, NRF1/2, ERRα/β/γ, SIRT3 and PPARα/β, participate in regulating gene expression of the direct regulators of mtDNA transcription and replication. The ETC consists of five complexes, which contribute to the generation of ATP through OXPHOS.

Figure 1.
mtDNA interactions.

mtDNA replication and transcription take place inside the mitochondria. mtDNA replication is dependent on nDNA-encoded replication factors including the mitochondrial single-stranded-binding protein (mtSSB), Twinkle, POLGA, POLG accessory subunit (POLGB) and DNA topoisomerase I mitochondrial (TOP1MT). They translocate into the mitochondria. A group of upstream regulators including STAT3, HIF1α, SIRT1, MYC, PGC1α/β, NRF1/2, ERRα/β/γ, SIRT3 and PPARα/β, participate in regulating gene expression of the direct regulators of mtDNA transcription and replication. The ETC consists of five complexes, which contribute to the generation of ATP through OXPHOS.

The ETC is located on the inner membrane of the mitochondrion. It comprises five enzyme complexes (Figure 1): Complex I (NADH dehydrogenase; ND), Complex II (succinate dehydrogenase; SD), Complex III (cytochrome c reductase; CYTB), Complex IV (cytochrome c oxidase; COX) and Complex V (ATP synthase). Additionally, coenzyme Q (CoQ) and cytochrome c (CytC) are two electron carriers that are extensively involved [3]. Briefly, Complex I and Complex II accept electrons from the electron donors and pass them on to CoQ. The electrons are then transferred through Complex III, CytC and Complex IV and finally accepted by O2. During this process, a pH gradient across the inner membrane forms due to the protons being pumped out by the complexes. Potential energy stored in the pH gradient is then transformed into biochemical energy, in the form of ATP, through the process of transferring the protons back into the mitochondrial matrix through Complex V [3]. It is noted that the process described above constitutes aerobic respiration. This is a far more efficient process than anaerobic respiration, as it produces 32–36 molecules of ATP per molecule of glucose compared with two molecules of ATP from glycolysis [4].

The mitochondrial genome

The mitochondrion hosts a genome, mtDNA, whose replication and expression apparatuses are distinct from those of the nuclear genome. mtDNA is a circular double-stranded DNA molecule that is 16.6 kb in size in humans (Figure 2). It consists of a heavy (H) and a light (L) strand. The mitochondrial genome encodes 37 genes which include 22 tRNAs, two rRNAs (12S and 16S rRNA) and 13 polypeptides of the ETC complex enzymes [5] (Figure 2). These include genes encoding seven subunits of Complex I (ND1–ND6 and ND4L), one Complex III subunit (CYTB), three Complex IV subunits (COX I, II and III) and two Complex V subunits (ATPase6 and ATPase8) (Figure 2). As for the other subunits of ETC complexes, they are nuclear DNA (nDNA)-encoded proteins that are imported into the mitochondrial matrix. Therefore, efficient and functional OXPHOS requires a well-established synergy between the nucleus and the mitochondria to meet the demands of a specialized cell for the production of energy.

The human mitochondrial genome.

Figure 2.
The human mitochondrial genome.

The human mitochondrial genome is a double-stranded circular genome, which is ∼16.6 kb in size. It encodes 22 tRNAs, two rRNAs and 13 subunits of the ETC complexes. The 13 subunits are seven subunits of Complex I (NADH dehydrogenase, ND1–ND6 and ND4L), one Complex III subunit (cytochrome c reductase, CYTB), three Complex IV subunits (cytochrome c oxidase, COX I, II and III) and two Complex V subunits (ATPase6 and ATPase8). The main non-coding region contains the D-loop region, the origin replication for the H strand (OH) and the promoters of transcription for the H and L strands (HSP and LSP). The origin of replication for the L strand (OL) is located two-thirds of the genome downstream from OH, between the tRNA-Cys and tRNA-Asn genes.

Figure 2.
The human mitochondrial genome.

The human mitochondrial genome is a double-stranded circular genome, which is ∼16.6 kb in size. It encodes 22 tRNAs, two rRNAs and 13 subunits of the ETC complexes. The 13 subunits are seven subunits of Complex I (NADH dehydrogenase, ND1–ND6 and ND4L), one Complex III subunit (cytochrome c reductase, CYTB), three Complex IV subunits (cytochrome c oxidase, COX I, II and III) and two Complex V subunits (ATPase6 and ATPase8). The main non-coding region contains the D-loop region, the origin replication for the H strand (OH) and the promoters of transcription for the H and L strands (HSP and LSP). The origin of replication for the L strand (OL) is located two-thirds of the genome downstream from OH, between the tRNA-Cys and tRNA-Asn genes.

The mitochondrial genome is highly compact with only two non-coding regions and no introns. The main non-coding region contains the displacement loop region (D-loop). It also consists of the origin of replication for the H strand (OH), and the promoters of transcription for both the H and L strands (HSP1, HSP2 and LSP) (Figure 2). The origin of replication for the L strand is located in the other non-coding region that is located two-thirds of the genome downstream of OH, between the genes tRNA-Cys and tRNA-Asn [5]. Genes encoding tRNAs are interspersed between genes encoding rRNAs and the ETC subunits, especially on the L strand [6]. Also, the termination codons are not encoded in some of the mitochondrial genes, but are created post-transcriptionally by the poly(A) tail mechanism [6], which means that the initial transcript normally ends with either a U or a UA, and polyadenylation can add adenines after these transcripts to form a UAA termination codon.

Another key feature that distinguishes the mitochondrial genome from the nuclear genome is that there are multiple copies of the mitochondrial genome per mitochondrion, and also per cell, as each cell also possesses multiple mitochondria [7]. Indeed, different cell types have different levels of mtDNA copy number depending on their demands for cellular energy [8]. mtDNA copy number could be as high as several thousand in cells such as muscle and neurons, whereas it is much lower in fibroblasts, spleen cells and stem cells [8]. Moreover, the mitochondrial genome can be present in a heteroplasmic manner, which means that both wild-type and mutated forms can coexist in the same cell [9]. It is thought that mtDNA mutations are a constant phenomenon mostly due to the primary targeting of reactive oxygen species (ROS). This is despite the mtDNA-specific replicase DNA polymerase gamma (POLG) having one of the lowest error rates of the eukaryotic polymerases for its exonucleolytic proofreading functions [10]. There is also the presence of diverse repair systems in the mitochondrion including recombinational repair [10]. Nevertheless, the mutations remain mostly silent or have no effect on phenotype, as it has been demonstrated that a mutation level of over 70% is normally required to lead to the onset of one of the many mitochondrial diseases [11].

In mitochondria, mtDNA molecules are organized into DNA–protein complexes similar to nucleoid structures in prokaryotes which supports the theory that the mitochondrion evolved from a proteobacterial partner that invaded eukaryotes [12]. The mitochondrial nucleoid consists of more than 20 binding proteins, which comprise many of the factors responsible for the transcription and replication of mtDNA. Owing to the limited coding capacity of mtDNA, these factors are nuclear-encoded and imported into the mitochondrial matrix.

mtDNA copy number is strictly controlled by its transcription and replication, which take place in mitochondria. mtDNA transcription and replication are driven by a group of nDNA-encoded mitochondrial specific factors (Figure 1), which includes a group of factors that bind directly with mtDNA, such as POLG and mitochondrial transcription factor A (TFAM), and a group of indirect factors, which regulate the gene expression of those direct factors. The best characterized of the indirect factors are the nuclear respiratory factors (NRF1 and NRF2) and the peroxisome-proliferator-activated receptor γ (PPARγ) co-activator 1α (PGC1α) family (Figure 1). It is noted that the regulation of mtDNA transcription and replication requires the coordinated control of all the factors and is at a complex level. As the related data have been well summarized and reviewed by others [1318], we are not covering this topic here. However, it is important to point out that the regulation of mtDNA copy number involves numerous factors, which have cell- and developmental-stage-specific expression profiles and are associated with other cellular functions.

The mtDNA set point

In order to meet the very different energy demands of a variety of cell types, mtDNA copy number is strictly regulated during development. This is, perhaps, best exemplified in embryonic stem cells (ESCs), which acquire mtDNA copy number in a cell-specific manner during differentiation through the strict regulation of mtDNA replication. This equips mature cells with the appropriate number of mtDNA copies in order that they are able to perform their specialized functions [1921] (Figure 3).

Very early on in development, the primordial germ cells, which are the first indicative germ cells, are laid down and they possess approximately 200 copies of mtDNA per cell [22,23]. As these cells mature during the extensive process of oogenesis to form fertilizable oocytes, namely metaphase II oocytes, mtDNA copy number increases significantly to more than 180,000 copies per cell. Indeed, there is a ‘critical threshold’ of mtDNA copy number to enable the metaphase II oocyte to proceed through fertilization [2427] (Figure 3). Failure to surpass this threshold results in either unsuccessful fertilization or embryos arresting during pre-implantation development. However, those oocytes that mature with insufficient mtDNA copy number, namely mtDNA-deficient oocytes, can be rescued through supplementation with exogenous populations of mtDNA [28]. This improves fertilization rates and successful development to the blastocyst stage [29], the final stage of pre-implantation development when the embryo first differentiates into two cellular compartments [24,28]. These cellular compartments comprise the inner cell mass (ICM), which gives rise to the embryo proper and then the fetus, and the trophectoderm, which forms the placenta. Indeed, mitochondrial supplementation is being introduced clinically to promote embryo development, especially in older patients when their embryos either fail to fertilize or they arrest very early during development [30].

Synchronous changes to mtDNA copy number and DNA methylation during early development.

Figure 3.
Synchronous changes to mtDNA copy number and DNA methylation during early development.

mtDNA copy number increases during oogenesis, peaks at fertilization, and decreases through to the blastocyst stage. Surpassing the threshold of mtDNA copy number (red broken line) during oogenesis is critical for fertilization. mtDNA replication is initiated in the trophectoderm, whereas the ICM continues to reduce mtDNA copy number and establishes the mtDNA set point before differentiation. Once cells commit to a specific lineage, they replicate mtDNA in a cell-specific manner so that they have sufficient mtDNA to transcribe essential subunits of the ETC to produce ATP and perform their specialized functions. Synchronous changes to DNA methylation are also observed to regulate gene expression required for development. TET enzymes reduce parental DNA methylation until the blastocyst stage. De novo methylation by DNMT3a and DNMT3b is initiated in the blastocyst. DNMT1 then works to maintain cell-specific DNA methylation profiles.

Figure 3.
Synchronous changes to mtDNA copy number and DNA methylation during early development.

mtDNA copy number increases during oogenesis, peaks at fertilization, and decreases through to the blastocyst stage. Surpassing the threshold of mtDNA copy number (red broken line) during oogenesis is critical for fertilization. mtDNA replication is initiated in the trophectoderm, whereas the ICM continues to reduce mtDNA copy number and establishes the mtDNA set point before differentiation. Once cells commit to a specific lineage, they replicate mtDNA in a cell-specific manner so that they have sufficient mtDNA to transcribe essential subunits of the ETC to produce ATP and perform their specialized functions. Synchronous changes to DNA methylation are also observed to regulate gene expression required for development. TET enzymes reduce parental DNA methylation until the blastocyst stage. De novo methylation by DNMT3a and DNMT3b is initiated in the blastocyst. DNMT1 then works to maintain cell-specific DNA methylation profiles.

Once fertilization has taken place, mtDNA copy number peaks between fertilization and activation of the embryonic genome [25,31,32], a transcriptional event that enables the embryo to initiate transcription of its own gene products, which means that it is no longer dependent on products carried over from the oocyte. The timing of embryonic genome activation is species-specific, but usually occurs between the two- and eight-cell stages [31,33]. Nevertheless, this mtDNA replication event is usually completed by the two-cell embryo stage. Subsequent mtDNA replication is restricted until the blastocyst stage [25,28,34]. As cell division progresses in an exponential manner in the early embryo, mtDNA copy number is diluted out significantly up to the blastocyst stage. Indeed, this appears to be a key developmental process as it is now evident from studies in human embryos cultured in vitro that they actively secrete, or shed, mtDNA into the surrounding culture medium. To this extent, it has been hypothesized that this is an indicator of better quality embryo development, as it enhances development to the blastocyst stage and promotes implantation [35]. This outcome is supported by the knowledge that too many copies of mtDNA at the blastocyst stage can lead to aneuploidy and failure of the hatched blastocyst to implant [36].

Pluripotency defines a naïve state that provides the cell with the potential to differentiate into all cell types of the body [37]. Pluripotency is regulated by gene markers that include OCT4, SOX2 and NANOG, which collectively form the pluripotent network, and are expressed in the ICM and undifferentiated embryonic stem cells [38,39]. Indeed, failure to express the markers of the pluripotent network results in loss of pluripotent potential and leads to embryonic death or differentiation [3941]. Consequently, the trophectodermal cells are the first cells to have undergone differentiation, and they lose their pluripotent potential and express lineage specific markers, such as CDX2 and GATA3 [4244]. In line with the onset of differentiation, the trophectodermal cells initiate mtDNA replication [25,28,34] (Figure 3). On the other hand, the ICM cells retain low mtDNA copy number and restrict mtDNA replication so that copy number is further reduced with each cell division [19,44,45] (Figure 3).

As the ICM consists of embryonic cells that are pluripotent, the continual dilution of mtDNA copy number contributes to the establishment of the mtDNA set point, which is required to establish lineage commitment [46]. The mtDNA set point is characterized by low mtDNA copy number. This can range from as few as 20 to 200 copies, which is a similar state as that of the primordial germ cells [22,23]. This provides the template for subsequent differentiation events in order that specialized cells acquire the specific numbers of mtDNA copies required to undertake their specialized functions, as is the case for primordial germ cells that differentiated into mature fertilizable oocytes [20,4749] (Figure 3). Indeed, it is of great importance for a pluripotent cell to establish the mtDNA set point to maintain pluripotency. Partial knockdown of PolgA, the major mtDNA replication factor, in undifferentiated ESCs leads to reduced mtDNA copy number, down-regulation of Oct4 expression and the onset of early differentiation [19].

There is a close association between pluripotency, mtDNA and mitochondrial function during embryo development, and the consequences of failing to establish the mtDNA set point can be understood from the perspective of induced pluripotency [49]. Induced pluripotent stem cells (iPSCs) are derived from differentiated cells, such as fibroblasts, which undergo reprograming, or dedifferentiation, to re-establish pluripotency. However, the degree of reprogramming that takes place is a key issue. iPSCs may express pluripotent markers; however, they can still have incomplete pluripotent potential due to the carry-over of epigenetic memory from the cells of origin, which leads them to differentiate into certain cell lineages instead of all cell types [50,51]. Furthermore, in some cases, they spontaneously start to express lineage-specific markers, but mtDNA replication is restricted when compared with differentiated ESCs, which eventually leads to failed differentiation [49]. Indeed, it has been shown that iPSCs, which failed to complete the process of reprogramming, attempt to increase mtDNA copy number during differentiation but this then stalls, a situation that we refer to as ‘pseudo-differentiation’ [8]. Thus complete pluripotency requires not only a genetically and epigenetically reprogrammed nucleus, but also the establishment of the mtDNA set point to synchronize the amount of mtDNA available to enable cells to perform their specialized functions when fully differentiated.

Cancer cells help to explain the importance of mtDNA to the process of differentiation. Cancer cells are highly proliferative cells, some of which can be induced to differentiate [8]. Indeed, the differentiated status of a cancer cell is widely used to grade cancers [52,53]. A more differentiated cancer cell is more like a normal somatic cell that is non-cancerous in nature, and it will grow at a slower rate than an undifferentiated cancer cell, as is the case for mature differentiated cells [52,53]. A variety of mitochondrial dysfunctions have been discovered in cancer cells, which are tightly related to mtDNA and highly likely to contribute to their failure to undergo differentiation and their ability to adopt a pseudo-differentiated state [8,54]. As an example, glioblastoma multiforme cells can be induced to initiate the process of differentiation. However, the process stalls and is marked by asynchronous increases in mtDNA copy number and perturbed cell-specific gene expression patterns [8]. Undifferentiated cancer cells share some characteristics associated with pluripotency, including cellular energy mechanisms and the regulation of mtDNA replication when they fail to differentiate [8,21]. We now review some of the related mitochondrial dysfunctions identified in tumorigenesis to bring new insights to the understanding and the importance of mtDNA in the overall picture of development.

Mitochondrial dysfunction in tumorigenesis associated with the mtDNA set point

The Warburg effect

The Warburg hypothesis states that cancer cells primarily rely on aerobic glycolysis instead of OXPHOS for energy production even in the presence of oxygen (Figure 4). In 1924, Warburg initially asserted the hypothesis that the primary cause of cancer was the damage to mitochondria and, subsequently, insufficient metabolic respiration [55]. With the finding of oncogenic mutations as the root cause of cancer [56], the Warburg effect currently describes the metabolic features of cancer cells. Indeed, not only do cancer cells utilize aerobic glycolysis for energy production but embryonic cells do so in a similar manner [57]. Low mtDNA copy number in cancer and pluripotent cells limits energy production through OXPHOS, and this is thought to aid cellular proliferation [58]. Even though aerobic glycolysis is not as efficient as OXPHOS in generating large amounts of ATP, one explanation is that it incorporates nutrients and metabolizes them into other organic elements (e.g. nucleotides, amino acids and lipids) to support the requirements to build and generate new cells when in a proliferative mode [58]. Although impaired mitochondrial function is not found in most cancers to be the direct cause mediated by the Warburg effect [58], recent studies have extended the hypothesis to a variety of mitochondrial dysfunctions in cancer cells, which might contribute to or result from the failure to establish the mtDNA set point [59].

Mitochondrial function and dysfunction in tumour cells and during development.

Figure 4.
Mitochondrial function and dysfunction in tumour cells and during development.

Tumour cells mainly rely on aerobic glycolysis even in the presence of oxygen, a process known as the Warburg effect. Pluripotent cells also utilize glycolysis to produce ATP probably due to limited numbers of mtDNA copy. Mitochondrial biogenesis involves formation of cristae and a membrane potential. Mature mitochondria in cells exhibit various activities including fusion and fission, mtDNA mutations, cellular apoptosis and mitophagy, which could contribute to developmental disorders and cancers.

Figure 4.
Mitochondrial function and dysfunction in tumour cells and during development.

Tumour cells mainly rely on aerobic glycolysis even in the presence of oxygen, a process known as the Warburg effect. Pluripotent cells also utilize glycolysis to produce ATP probably due to limited numbers of mtDNA copy. Mitochondrial biogenesis involves formation of cristae and a membrane potential. Mature mitochondria in cells exhibit various activities including fusion and fission, mtDNA mutations, cellular apoptosis and mitophagy, which could contribute to developmental disorders and cancers.

Pathological DNA mutations

Sporadic nDNA mutations or abnormal expression levels of the nDNA-encoded factors of mtDNA transcription and replication lead to dysfunctional mtDNA transcription and replication, which could result in reduced oxidative capacity and energy production. Once the reduced level affects cellular metabolism, clinical symptoms are often observed. It is known that mutations to POLG could lead to depletion of mtDNA and promote breast cancer [60]. In addition, frequent truncating mutations of Tfam have been demonstrated to induce mtDNA depletion and apoptotic resistance in a mouse model of colorectal cancer [61]. Thus these mutations could perturb the establishment of the mtDNA set point, which then limits the differentiation potential of tumour cells.

The impact of ROS production and mitochondrial metabolites on gene expression

ROS are a group of by-products from cellular metabolism that can readily oxidize other molecules. ROS are frequently generated in the mitochondria [62]. Increased ROS results from cellular stress and can translate the stress into cellular signalling pathways for certain responses [63]. It has been demonstrated that increased ROS could trigger mitophagy and apoptosis to either reduce the number of mitochondria, therefore reducing mtDNA copy number, or trigger cell death dependent on the extent of the increase [64,65]. Low levels of ROS are beneficial for healthy metabolism through post-translational modification of phosphatases and kinases [66], whereas excessively high levels of ROS generate lethal levels of oxidative stress and lead to cell death by causing damage to DNA [67], triggering senescence [68] and apoptosis [69].

Elevated ROS has been identified in cancer cells that are induced by oncogenes to promote tumorigenesis. However, to maintain the elevated level at a beneficial level rather than at the excessively high level, it is tightly controlled by aberrant gene expression and metabolism in cancer, as cancer cells can produce higher levels of antioxidants to balance the redox level to a level beneficial for tumorigenesis [64]. It has been reported that PGC1α can up-regulate the expression of antioxidant enzymes to maintain redox balance [70]. The tumour-suppressor gene p53 is also known to interact with NRF2 to up-regulate the expression of antioxidants [71]. The oncogene MYC regulates antioxidant levels by promoting glutamine uptake and glutaminolysis [72]. Furthermore, down-regulation of TFAM has recently been found to inhibit the tumorigenesis of non-small-cell lung cancer by activating ROS-mediated c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signalling and reduce cellular energy [73]. These findings related to the mtDNA replication and transcription factors provide functional explanations as to how the failure to establish the mtDNA set point blocks the differentiation of cancer cells and promotes tumorigenesis, but this has yet to be tested experimentally.

Mutations to mtDNA

The mitochondrial genome encodes several subunits of the ETC complexes and other functional RNAs. Therefore, reduced function in the ETC complexes resulting from homoplasmic mtDNA mutations can lead to OXPHOS inefficiency. Mutations in the mtDNA-encoded ND genes and COX1 have been identified in primary tumours and it is suggested that they promote tumour progression [7478]. This will contribute to the inefficiency within the ETC, as more electrons are passed through the complexes resulting in the production of more ROS that causes further mtDNA damage or mutation [74] (Figure 4). Nevertheless, mechanistic evidence is needed to determine whether mtDNA mutations are a part of tumorigenesis or just a part of mitochondrial dysfunction in cancer. Even though the levels of mtDNA mutations are not directly related to mtDNA copy number, they affect the cell's respiratory capacity. If the levels of mtDNA mutations affect the function of the mtDNA-encoded ETC subunits, it therefore suggests that the establishment of the mtDNA set point is disturbed functionally. Consequently, mtDNA copy number should be assessed in the context of mitochondrial function and whether a cell's set point is modulated to compensate for this.

The control of the number of mitochondria

Mitochondrial biogenesis is activated through environmental stimuli or when cells are under redox stress [7982]. Mitochondrial biogenesis is a complicated procedure requiring mtDNA replication and synthesis of ETC subunits [81,82]. In early differentiation, mitochondrial maturation also involves the formation of cristae and a higher membrane potential for normal mitochondrial functions [83,84] (Figure 4). The Sirtuin family, especially Sirtuin 1 and 3 (encoded by SIRT1/SIRT3) have been shown to be involved in mitochondrial biogenesis, in mitochondrial transcription as the target of PGC1α, and have a role in suppressing ROS production [85,86]. Moreover, the ETC subunits, as well as other mitochondrial structural proteins, are encoded by both the chromosomal and mitochondrial genomes. Thus the process of mitochondrial biogenesis is highly dependent on the ordered and effective co-operation between the hierarchical network of the factors from both the nucleus and the mitochondria. In cancer, some oncogenes and tumour suppressors have been found to affect this network to alter the rate of biogenesis. The tumour suppressor p53 has been demonstrated to repress the transcription of the main initiator of mitochondrial biogenesis, PGC1α, when telomere function is compromised [87], and therefore causes deregulation of the downstream pathway through cofactors of PGC1α, namely PPARγ, NRF1/2 and oestrogen-related receptor (ERR) α/β and, extensively, to key mitochondrial regulators, such as TFAM and POLGA [81]. In addition, the oncogene MYC could promote mitochondrial biogenesis by up-regulating expression of PGC1β [8890] (Figure 1). The cell proliferation factor signal transducer and activator of transcription 3 (STAT3) also directly binds to the D-loop region of mtDNA to regulate its transcription. More importantly, STAT3 promotes mitochondrial transcription to optimize energy production which is used to promote the proliferation of ESCs [91]. STAT3 also targets hypoxia-induced factor 1α (HIF1α), the master transcription regulator of the genes that respond to hypoxia, which also interacts with PGC1α to promote mitochondrial biogenesis [92,93]. Increased mitochondrial biogenesis in cancer cells is probably a response to offset lower energy production resulting from the Warburg effect and to balance out the elevated levels of ROS.

On the other hand, the number of mitochondria is also controlled by mitochondria-specific autophagy, known as mitophagy. Mitophagy describes the process of degradation of mitochondria by the autophagosome (Figure 4). Mitophagy is triggered when the mitochondrial membrane is depolarized, which, in most cases, results from mitochondrial fission (discussed below) [64,94]. Mitophagy has tumour-suppressor function as the loss of the key regulators of mitophagy, parkin and BNIP3/NIX, promotes tumorigenesis dependent on the type and the stage of the cancers [9597]. BNIP3/NIX can be induced by hypoxia as the tumour cell microenvironment involves the regulation of p53 [96]. In ESCs, cellular function and pluripotency are also sensitive to the microenvironment, especially in tissue culture. It has been shown that the lack of a growth factor, Gfer, can trigger mitophagy and loss of pluripotency, which could result in reduced mtDNA copy number and failure to establish the mtDNA set point [98].

The dynamics of mitochondrial fission and fusion happen at a balanced rate in normal cells, which closely responds to hypoxia, energy metabolism, apoptosis and other cellular stress [99] (Figure 4). Mitochondrial fission is driven by the GTPase dynamin-related protein 1 (DRP1) with co-factors including MFF1 and FIS1. Mitochondrial fusion requires the GTPases mitofusin 1/2 located on the outer membrane and OPA1 on the inner membrane of mitochondria [99]. Mitochondrial fission is important for mitosis to distribute the organelles evenly into daughter cells, whereas fusion has been found to promote OXPHOS and cell growth probably by increasing the density of the ETC complexes and improving the formation of the ETC supercomplexes [100]. Evidence of increased fission rate has been reported in cancer cells and has been related to the increases in ROS and mitophagy, which again could lead to the reduction in mtDNA copy number [64]. Moreover, it has been noted that mitochondrial dynamics might play vital roles in apoptosis, as observed in interactions between the key regulators of fusion/fission and apoptosis including oncogenic BCL-2 family members [101,102]. As apoptotic evasion is one of the hallmarks of tumorigenesis, these findings have shown the potential contribution of mitochondrial dynamics to tumorigenesis. However, direct experimental results need to be analysed to determine the cellular mechanisms in which the abnormal mitochondrial dynamics identified in tumorigenesis contribute to or resulted from the abnormal regulation of mtDNA copy number and stalled differentiation.

In summary, due to the fundamental role of mitochondria in cellular metabolism and bioenergetics, it is not surprising to see such a variety of mitochondrial dysfunctions associated with tumorigenesis (Figure 4). However, most of the explanations are not clear enough to clarify how mitochondrial dysfunction mechanistically relates to tumorigenesis and failure to complete differentiation. As it has been shown that, by resetting mtDNA copy number and thus the mtDNA set point, cellular differentiation could be promoted [8], these dysfunctions probably result from or contribute to the failure to establish the mtDNA set point. As reviewed above, the interactions between oncogenes/tumour suppressors and mitochondrial replication and transcription factors are present at multiple levels, which result in a variety of metabolic changes during tumorigenesis. Cancers derived from different cell types exhibit different mitochondrial dysfunctions with different metabolic pathways being perturbed. Moreover, the mtDNA set point is essential to determining cell fate. These findings highlight that the regulation of mtDNA copy number co-operates tightly with other metabolic pathways to regulate various cellular functions, which, if perturbed, could lead to tumorigenesis.

The effect of DNA methylation on mtDNA copy number during differentiation

What is DNA methylation?

DNA methylation is the only known covalent modification of DNA [103]. Typically, DNA methylation occurs at 5΄ of the pyrimidine ring of cytosine (5 mC) followed by guanine (CpG dinucleotide) in mammalian DNA (Figure 5). It is known that over 28 million CpGs exist in the human genome. In plants, it can also occur at non-CpG cytosines [104]. There is also emerging evidence showing that non-CpG DNA methylation can occur at CpA and CpT dinucleotides in mammalian cells, such as embryonic and neuronal cells [105,106].

DNA methylation and demethylation mechanisms.

Figure 5.
DNA methylation and demethylation mechanisms.

Cytosine in CpG dinucleotides can be methylated to 5 mC by DNMT enzymes: DNMT1 maintains DNA methylation patterns, DNMT3a/b create novel DNA methylation sites. 5 mC can be demethylated by TET enzymes to 5 hmC. DNA methylation inhibitors, such as 5-Azacytidine (5-Aza), inhibit DNMT1, whereas vitamin C (VitC) enhances TET activity.

Figure 5.
DNA methylation and demethylation mechanisms.

Cytosine in CpG dinucleotides can be methylated to 5 mC by DNMT enzymes: DNMT1 maintains DNA methylation patterns, DNMT3a/b create novel DNA methylation sites. 5 mC can be demethylated by TET enzymes to 5 hmC. DNA methylation inhibitors, such as 5-Azacytidine (5-Aza), inhibit DNMT1, whereas vitamin C (VitC) enhances TET activity.

Methyl groups are added to cytosines by DNA methyltransferase (DNMT) enzymes [107] (Figure 5). There are two types of DNMT enzymes either working to maintain DNA methylation during DNA replication or initiating DNA methylation at a novel DNA site. Maintenance of DNA methylation is carried out by DNMT1 along with DNA replication, whereas DNMT3A and DNMT3B are believed to function as the de novo methylation DNMT enzymes adding methyl groups at new cytosine sites [108] (Figure 5). 5 mC can be demethylated to 5-hydroxymethylcytosine (5 hmC) by ten-eleven translocation methylcytosine dioxygenase (TET; Figure 5) [109]. The TET family, consisting of TET1, TET2 and TET3, is known to oxidize 5 mC to 5 hmC [109,110].

CpG dinucleotides usually appear in repeat clusters termed CpG islands (CGIs). The widely accepted definition of CGI was given by Gardiner-Garden and Frommer in 1987 as a 200-bp sequence of DNA with a CG content of more than 50% and the observed/expected CpG ratio (O/E ratio) is more than 0.6 [111]. Where the O/E ratio is calculated by the formula [111]:

 
formula

CGIs contain high CpG components, which could be 5-fold higher than the other regions. The estimated number of CGIs is 30 000 in the human genome. On average, CGIs are ∼1 kb in length with 7% being CpG dinucleotides [111,112].

Chemical agents can also inhibit DNA methylation or induce DNA demethylation. They have been widely used in DNA methylation research. 5-Azacytidine is a chemical analogue of cytosine that inhibits DNA methylation at low concentrations [113]. It blocks DNA methyltransferease, DNMT1, which causes global DNA demethylation [114] (Figure 5). Recent research has revealed that vitamin C can also induce DNA demethylation by enhancing the activity of the TET1 enzymes and thus improving the conversion of 5 mC into the DNA demethylated state of 5 hmC [115] (Figure 5).

In mammalian cells, the methylome is mainly composed of genomic regions of low CpG densities, whereas regions of higher CpG densities, such as CGIs, are mainly unmethylated. DNA methylation profiles vary significantly between different tissue types reflecting different gene expression profiles and biological functions [116]. DNA regions with a different methylation status can result in different biological consequences.

Biological function of DNA methylation in different gene regions

DNA methylation plays vital roles in embryonic development, maintenance of pluripotency, genomic imprinting and X chromosome inactivation by regulating transcription and stabilizing chromosomes and chromatin structure [117119]. It is a dispensable epigenetic system in the regulation of gene expression. Most often, DNA methylation is best characterized by silencing transcription which occurs at CpG islands in gene promoter regions. Through access to high-throughput methylome datasets, it has been suggested that DNA methylation seems to occur at intragenic (within gene-coding) regions, and at regulatory intergenic (between gene-coding) regions, which appear to regulate gene expression patterns differently.

More than 50% of human genes possess CGIs in their promoter regions [120]. As mentioned above, most of them remain unmethylated in somatic cells. Some suppressed genes have been shown with methylated CGIs in their promoter regions [116]. Hypermethylated promoters have been well characterized to block binding of transcription factors and to silence gene expression. Aberrant methylation of promoter-located CGIs, especially hypermethylated promoters, plays important roles in repressing tumour-suppressor genes [121,122]. Interestingly, nearly all of the known mitochondrial replication and transcription factors have a CGI in their promoter regions, which indicates that their gene expression is probably affected by the DNA methylation status across their CGI regions. DNA hypermethylation across the promoters of PPARGC1A (encoding PGC1α) has been shown to be mediated by DNMT3B and leads to the reduction of mitochondrial content in muscle cells in diabetic patients [123]. Methylation in the promoter region of TFAM also has an impact on mitochondrial function in insulin resistance in peripheral leucocytes [124].

Methylome datasets of single-base resolution have shown that fewer CGIs exist but more DNA methylation occurs in intragenic regions [125,126]. Intragenic methylation also presents in a strong tissue-specific manner. However, the function of intragenic methylation shows diversity and has also been referred to as the DNA methylation paradox [127]. It is believed that high DNA methylation is important for blocking transcription of the repetitive DNA elements and suppressing initiation from spurious promoters within gene regions [122,128]. However, a non-monotonic relationship is observed between intragenic methylation and gene expression [129]. Both positive and negative correlations exist between DNA methylation at exons and gene expression in different cell types [125]. Evidence has also shown that DNA methylation across exons is higher than in introns [130]. The transition of DNA methylation at exon/intron boundaries leads to a proposed function for maintaining transcriptional efficiency by regulating splicing and elongation [130]. Moreover, the dynamics of DNA methylation across gene regions might also be related to the transcriptional unit including the accessibility of chromatin and the relative density of the nucleosomes, DNMT1 and human RNA polymerase II (POL II) [129]. In an inactive gene region, chromatin is closed and dense nucleosomes block DNMT1 from mediating DNA methylation, whereas when chromatin partially opens, DNMT1 methylates gene regions along with POL II to execute transcription; a completely opened chromatin is fully occupied with POL II for transcription, which interferes with the access of DNMT1.

Overall, there is still a question mark over the relationship between methylome patterns and transcriptional profiles. When talking about DNA methylation patterns, inter- (between gene-coding regions) and intra- (within gene-coding regions) genetically, it is necessary to take gene expression and other regulatory mechanisms into consideration and investigate them all thoroughly based on the particular cell types and their corresponding cell functions.

The effect of DNA methylation on mtDNA copy number during differentiation

During cell differentiation and embryonic development, the DNA methylation profile is re-established. After fertilization, TET enzymes become activate and erase the parental DNA methylation levels significantly [131] (Figure 3). By the blastocyst stage, the extensively demethylated genome of the ICM starts to be re-methylated by DNMT3a and DNMT3b up to their specific levels and maintained by DNMT1 afterwards during DNA replication [132] (Figure 3). The changes that occur during differentiation are believed to be predominantly responsible for transcriptional changes specified for different cell fates. It has been reported that in vivo liver differentiation in human ESCs is characterized by DNA demethylation over time [133]. In a DNA methylation study targeting genome-wide promoter regions of mouse ESCs, it was reported that the DNA–protein interactions between the promoter regions and OCT4, NANOG, Polycomb proteins and histone modifications (H3K27 trimethylation) are correlated with the DNA methylation status of the cell [134]. Increasing studies on DNA methylation in embryonic cells have provided insights into how developmental processes are modulated by DNA methylation and more and more cell lineages are characterized by their DNA methylation signatures.

As mentioned before, mtDNA copy number is also reset to establish the mtDNA set point starting at the blastocyst stage leading towards specified mtDNA copy number for different cell lineages (Figure 3). It has been questioned whether there is a relationship between the coinciding changes in DNA methylation and mtDNA copy number. A recent study revealed that the levels of DNA methylation increased at exon 2 of PolgA in mouse ESCs as they underwent differentiation into neurons [20]. Furthermore, the level of DNA methylation varies at exon 2 of PolgA in a tissue-specific manner. Statistical analysis suggested that PolgA expression was negatively correlated with its intragenic DNA methylation at exon 2 and the corresponding mtDNA copy number, which indicates that changes to DNA methylation could modulate mtDNA copy number by regulating the gene expression of the primary mtDNA replication factor POLGA. Similar findings are also observed in the differentiation process from human ESCs to neural stem cells and finally into astrocytes [21]. High DNA methylation was observed in pluripotent and multipotent cells that correlated with low POLGA expression and low mtDNA copy number. In terminally differentiated astrocytes, however, DNA methylation at exon 2 of POLGA decreased with higher mtDNA copy number. Interestingly, cancer cell lines, such as the glioblastoma multiforme HSR-GBM1 cells, which failed to differentiate, are unable to demethylate exon 2 of POLGA [21]. However, in the presence of DNA demethylation agents such as 5-Aza, the high DNA methylation status at exon 2 of POLGA was decreased promoting mtDNA replication and cell differentiation. These findings highlight the important role of DNA methylation in modulating mtDNA copy number, both of which act as vital determinants in cell differentiation and development (Figure 6). Likewise, partial depletion of mtDNA in cancer cells changes the methylation status at exon 2 of POLGA and affects the tumorigenic capacity of these cells. Nevertheless, when tumours form, mtDNA copy number is reinstated to original levels, suggesting that cells do, indeed, have an ‘in built’ set point [21,135].

The mtDNA set point is critical for differentiation of pluripotent cells and tumour cells.

Figure 6.
The mtDNA set point is critical for differentiation of pluripotent cells and tumour cells.

During differentiation, mtDNA copy number is strictly regulated in a cell-specific manner accompanied by changes in the DNA methylation status at exon 2 of POLGA. Cancer cells fail to complete differentiation and adopt a pseudo-differentiated state. However, they can complete differentiation by resetting mtDNA copy number by DNA demethylation.

Figure 6.
The mtDNA set point is critical for differentiation of pluripotent cells and tumour cells.

During differentiation, mtDNA copy number is strictly regulated in a cell-specific manner accompanied by changes in the DNA methylation status at exon 2 of POLGA. Cancer cells fail to complete differentiation and adopt a pseudo-differentiated state. However, they can complete differentiation by resetting mtDNA copy number by DNA demethylation.

Interestingly, the mtDNA set point can be modulated by mtDNA genotype. In cells that have had their mtDNA replaced with mtDNA from different genotypes, either in tumour cell [135] or ESC [136] models, not only are chromosomal gene expression patterns changed, but mtDNA copy number is modulated in response along with DNA methylation patterns at exon 2 of POLGA [21]. This indicates that the establishment of the mtDNA set point is a two way process where interactions between the nuclear and mitochondrial genomes reflect adaptation to the environment and cellular phenotype.

Conclusion

mtDNA is a critical part of the cellular energy-production system. Nevertheless, its impact is no longer limited to powering cellular function. mtDNA copy number changes during cellular development which affects fertilization rates and embryo viability. The establishment of the mtDNA set point is essential for the potential of a cell to complete differentiation. During differentiation, mtDNA copy number increases in a lineage-specific manner as dictated by a cell's specialized cellular functions. The changes to mtDNA copy number are determined not only by the expression of mtDNA transcription and replication factors but also by stage-specific markers, as is the case during pluripotency, which it is very tightly regulated. The synchronous changes to DNA methylation during development, especially at exon 2 of POLGA, provide insights into our understanding of the cross-talk between the nucleus and the mitochondria; in other words, the changes to gene expression profiles and mtDNA copy number. These findings can be combined further to understand tumorigenesis and developmental disorders. Aberrant DNA methylation profiles found in early development and tumorigenesis interfere with the expression of POLGA, which destabilizes the mtDNA set point and the expansion of mtDNA copy number during differentiation. As a great variety of genes and regulators are involved in regulating mtDNA as well as general mitochondrial function, it is highly likely that similar mechanisms exist that have an impact on development and tumorigenesis.

Abbreviations

5-Aza, 5-azacitidine; CGI, CpG island; CoQ, coenzyme Q; COX, cytochrome c oxidase; CytB, cytochrome c reductase; CytC, cytochrome c; D-loop, displacement loop; DNMT, DNA methyltransferase; ERR, oestrogen-related receptor; ESC, embryonic stem cell; ETC, electron transfer chain; HIF1α, hypoxia-induced factor 1α; 5 hmC, 5-hydroxymethylcytosine; 5 mC, 5-methylcytosine; ICM, inner cell mass; iPSC, induced pluripotent stem cell; ND, NADH dehydrogenase; nDNA, nuclear DNA; NRF, nuclear respiratory factor; O/E ratio, observed/expected CpG ratio; OXPHOS, oxidative phosphorylation; PGC1α, peroxisome-proliferator-activated receptor γ co-activator 1α; POLG, DNA polymerase gamma; POLGA, POLG subunit A; POL II, RNA polymerase II; PPAR, peroxisome-proliferator-activated receptor; ROS, reactive oxygen species; SIRT, sirtuin; STAT3, signal transducer and activator of transcription 3; TET, ten-eleven translocation methylcytosine dioxygenase; TFAM, mitochondrial transcription factor A.

Funding

This work was funded by the Australian National Health and Medical Research Council [grant number GNT1041471] and the Victorian Government's Operational Infrastructure Support Program.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Duchen
,
M.R.
(
1999
)
Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death
.
J. Physiol.
516
,
1
17
doi:
2
Rossi
,
C.S.
,
Bielawski
,
J.
and
Lehninger
,
A.L.
(
1966
)
Separation of H+ and OH− in the extramitochondrial and mitochondrial phases during Ca++-activated electron transport
.
J. Biol. Chem.
241
,
1919
1921
PMID:
[PubMed]
3
Mitchell
,
P.
(
1961
)
Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism
.
Nature
191
,
144
148
doi:
4
Brown
,
G.C.
(
1992
)
Control of respiration and ATP synthesis in mammalian mitochondria and cells
.
Biochem. J.
284
,
1
13
doi:
5
Anderson
,
S.
,
Bankier
,
A.T.
,
Barrell
,
B.G.
,
de Bruijn
,
M.H.L.
,
Coulson
,
A.R.
,
Drouin
,
J.
et al. 
(
1981
)
Sequence and organization of the human mitochondrial genome
.
Nature
290
,
457
465
doi:
6
Ojala
,
D.
,
Montoya
,
J.
and
Attardi
,
G.
(
1981
)
tRNA punctuation model of RNA processing in human mitochondria
.
Nature
290
,
470
474
doi:
7
Satoh
,
M.
and
Kuroiwa
,
T.
(
1991
)
Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell
.
Exp. Cell Res.
196
,
137
140
doi:
8
Dickinson
,
A.
,
Yeung
,
K.Y.
,
Donoghue
,
J.
,
Baker
,
M.J.
,
Kelly
,
R.D.W.
,
McKenzie
,
M.
et al. 
(
2013
)
The regulation of mitochondrial DNA copy number in glioblastoma cells
.
Cell Death Differ.
20
,
1644
1653
doi:
9
Shoubridge
,
E.A.
and
Wai
,
T.
(
2007
)
Mitochondrial DNA and the mammalian oocyte
.
Curr. Top. Dev. Biol.
77
,
87
111
doi:
10
Alexeyev
,
M.
,
Shokolenko
,
I.
,
Wilson
,
G.
and
LeDoux
,
S.
(
2013
)
The maintenance of mitochondrial DNA integrity: critical analysis and update
.
Cold Spring Harb. Perspect. Biol.
5
,
a012641
doi:
11
Schon
,
E.A.
,
DiMauro
,
S.
and
Hirano
,
M.
(
2012
)
Human mitochondrial DNA: roles of inherited and somatic mutations
.
Nat. Rev. Genet.
13
,
878
890
doi:
12
Gray
,
M.W.
,
Burger
,
G.
and
Lang
,
B.F.
(
1999
)
Mitochondrial evolution
.
Science
283
,
1476
1481
doi:
13
Falkenberg
,
M.
and
Larsson
,
N.G.
(
2007
)
DNA replication and transcription in mammalian mitochondria
.
Annu. Rev. Biochem.
76
,
679
699
doi
:10.1146/annurev.biochem.76.060305.152028
14
Leigh-Brown
,
S.
,
Enriquez
,
J.A.
and
Odom
,
D.T.
(
2010
)
Nuclear transcription factors in mammalian mitochondria
.
Genome Biol.
11
,
215
doi:
15
Holt
,
I.J.
and
Reyes
,
A.
(
2012
)
Human mitochondrial DNA replication
.
Cold Spring Harb. Perspect. Biol.
4
,
a012971
doi
:10.1101/cshperspect.a012971
16
Gustafsson
,
C.M.
,
Falkenberg
,
M.
and
Larsson
,
N.G.
(
2016
)
Maintenance and expression of mammalian mitochondrial DNA
.
Annu. Rev. Biochem.
85
,
133
160
doi:
17
Young
,
M.J.
and
Copeland
,
W.C.
(
2016
)
Human mitochondrial DNA replication machinery and disease
.
Curr. Opin. Genet. Dev.
38
,
52
62
doi:
18
Kaguni
,
L.S.
and
Oliveira
,
M.T.
(
2016
)
Structure, function and evolution of the animal mitochondrial replicative DNA helicase
.
Crit. Rev. Biochem. Mol. Biol.
51
,
53
64
doi:
19
Facucho-Oliveira
,
J.M.
,
Alderson
,
J.
,
Spikings
,
E.C.
,
Egginton
,
S.
and
St. John
,
J.C.
(
2007
)
Mitochondrial DNA replication during differentiation of murine embryonic stem cells
.
J. Cell Sci.
120
,
4025
4034
doi:
20
Kelly
,
R.D.W.
,
Mahmud
,
A.
,
McKenzie
,
M.
,
Trounce
,
I.A.
and
St John
,
J.C.
(
2012
)
Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A
.
Nucl. Acids Res.
40
,
10124
10138
doi:
21
Lee
,
W.
,
Johnson
,
J.
,
Gough
,
D.J.
,
Donoghue
,
J.
,
Cagnone
,
G.L.M.
,
Vaghjiani
,
V.
et al. 
(
2015
)
Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny
.
Cell Death Dis.
6
,
e1664
doi:
22
Cree
,
L.M.
,
Samuels
,
D.C.
,
de Sousa Lopes
,
S.C.
,
Rajasimha
,
H.K.
,
Wonnapinij
,
P.
,
Mann
,
J.R.
et al. 
(
2008
)
A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes
.
Nat. Genet.
40
,
249
254
doi:
23
Wai
,
T.
,
Teoli
,
D.
and
Shoubridge
,
E.A.
(
2008
)
The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes
.
Nat. Genet.
40
,
1484
1488
doi:
24
El Shourbagy
,
S.H.
,
Spikings
,
E.C.
,
Freitas
,
M.
and
St John
,
J.C.
(
2006
)
Mitochondria directly influence fertilisation outcome in the pig
.
Reproduction
131
,
233
245
doi:
25
Spikings
,
E.C.
,
Alderson
,
J.
and
St John
,
J.C.
(
2007
)
Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development
.
Biol. Reprod.
76
,
327
335
doi:
26
Reynier
,
P.
,
May-Panloup
,
P.
,
Chretien
,
M.-F.
,
Morgan
,
C.J.
,
Jean
,
M.
,
Savagner
,
F.
et al. 
(
2001
)
Mitochondrial DNA content affects the fertilizability of human oocytes
.
Mol. Hum. Reprod.
7
,
425
429
doi:
27
Santos
,
T.A.
,
El Shourbagy
,
S.
and
St. John
,
J.C.
(
2006
)
Mitochondrial content reflects oocyte variability and fertilization outcome
.
Fertil. Steril.
85
,
584
591
doi:
28
May-Panloup
,
P.
,
Vignon
,
X.
,
Chrétien
,
M.-F.
,
Heyman
,
Y.
,
Tamassia
,
M.
,
Malthièry
,
Y.
and
Reynier
,
P.
(
2005
)
Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors
.
Reprod. Biol. Endocrinol.
3
,
65
doi:
29
Cagnone
,
G.L.M.
,
Tsai
,
T.S.
,
Makanji
,
Y.
,
Matthews
,
P.
,
Gould
,
J.
,
Bonkowski
,
M.S.
et al. 
(
2016
)
Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency
.
Sci. Rep.
6
,
23229
doi:
30
Chappel
,
S.
(
2013
)
The role of mitochondria from mature oocyte to viable blastocyst
.
Obstet. Gynecol. Int.
2013
,
1
10
183024
doi:
31
Aoki
,
F.
,
Worrad
,
D.M.
and
Schultz
,
R.M.
(
1997
)
Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo
.
Dev. Biol.
181
,
296
307
doi:
32
St John
,
J.C.
,
Amaral
,
A.
,
Bowles
,
E.
,
Oliveira
,
J.F.
,
Lloyd
,
R.
,
Freitas
,
M.
et al. 
(
2006
)
The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells
.
Methods Mol. Biol.
331
,
347
–374
doi
:10.1345/1-59785-046-4:347
33
Telford
,
N.A.
,
Watson
,
A.J.
and
Schultz
,
G.A.
(
1990
)
Transition from maternal to embryonic control in early mammalian development: a comparison of several species
.
Mol. Reprod. Dev.
26
,
90
100
doi:
34
McConnell
,
J.M.L.
and
Petrie
,
L.
(
2004
)
Mitochondrial DNA turnover occurs during preimplantation development and can be modulated by environmental factors
.
Reprod. Biomed. Online
9
,
418
424
doi:
35
Stigliani
,
S.
,
Persico
,
L.
,
Lagazio
,
C.
,
Anserini
,
P.
,
Venturini
,
P.L.
and
Scaruffi
,
P.
(
2014
)
Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome
.
Mol. Hum. Reprod.
20
,
1238
1246
doi:
36
Fragouli
,
E.
and
Wells
,
D.
(
2015
)
Mitochondrial DNA assessment to determine oocyte and embryo viability
.
Semin. Reprod. Med.
33
,
401
409
doi:
37
Bradley
,
A.
,
Evans
,
M.
,
Kaufman
,
M.H.
and
Robertson
,
E.
(
1984
)
Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines
.
Nature
309
,
255
256
doi:
38
Loh
,
Y.-H.
,
Wu
,
Q.
,
Chew
,
J.-L.
,
Vega
,
V.B.
,
Zhang
,
W.
,
Chen
,
X.
et al. 
(
2006
)
The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells
.
Nat. Genet.
38
,
431
440
doi:
39
Avilion
,
A.A.
,
Nicolis
,
S.K.
,
Pevny
,
L.H.
,
Perez
,
L.
,
Vivian
,
N.
and
Lovell-Badge
,
R.
(
2003
)
Multipotent cell lineages in early mouse development depend on SOX2 function
.
Genes Dev.
17
,
126
140
doi:
40
Mitsui
,
K.
,
Tokuzawa
,
Y.
,
Itoh
,
H.
,
Segawa
,
K.
,
Murakami
,
M.
,
Takahashi
,
K.
et al. 
(
2003
)
The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells
.
Cell
113
,
631
642
doi:
41
Nichols
,
J.
,
Zevnik
,
B.
,
Anastassiadis
,
K.
,
Niwa
,
H.
,
Klewe-Nebenius
,
D.
,
Chambers
,
I.
et al. 
(
1998
)
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4
.
Cell
95
,
379
391
doi:
42
Niwa
,
H.
,
Toyooka
,
Y.
,
Shimosato
,
D.
,
Strumpf
,
D.
,
Takahashi
,
K.
,
Yagi
,
R.
and
Rossant
,
J.
(
2005
)
Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation
.
Cell
123
,
917
929
doi:
43
Home
,
P.
,
Ray
,
S.
,
Dutta
,
D.
,
Bronshteyn
,
I.
,
Larson
,
M.
and
Paul
,
S.
(
2009
)
GATA3 is selectively expressed in the trophectoderm of peri-implantation embryo and directly regulates Cdx2 gene expression
.
J. Biol. Chem.
284
,
28729
28737
doi:
44
Wray
,
J.
,
Kalkan
,
T.
and
Smith
,
A.G.
(
2010
)
The ground state of pluripotency
.
Biochem. Soc. Trans.
38
,
1027
1032
doi:
45
Evans
,
M.J.
and
Kaufman
,
M.H.
(
1981
)
Establishment in culture of pluripotential cells from mouse embryos
.
Nature
292
,
154
156
doi:
46
St. John
,
J.C.
,
Facucho-Oliveira
,
J.
,
Jiang
,
Y.
,
Kelly
,
R.
and
Salah
,
R.
(
2010
)
Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells
.
Hum. Reprod. Update
16
,
488
509
doi:
47
St. John
,
J.
(
2014
)
The control of mtDNA replication during differentiation and development
.
Biochim. Biophys. Acta
1840
,
1345
1354
doi:
48
St John
,
J.C.
and
Campbell
,
K.H.S.
(
2010
)
The battle to prevent the transmission of mitochondrial DNA disease: is karyoplast transfer the answer?
Gene Ther.
17
,
147
149
doi:
49
Kelly
,
R.D.W.
,
Sumer
,
H.
,
McKenzie
,
M.
,
Facucho-Oliveira
,
J.
,
Trounce
,
I.A.
,
Verma
,
P.J.
and
St. John
,
J.C.
(
2013
)
The effects of nuclear reprogramming on mitochondrial DNA replication
.
Stem Cell Rev. Rep.
9
,
1
15
doi:
50
Polo
,
J.M.
,
Liu
,
S.
,
Figueroa
,
M.E.
,
Kulalert
,
W.
,
Eminli
,
S.
,
Tan
,
K.Y.
et al. 
(
2010
)
Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells
.
Nat. Biotechnol.
28
,
848
855
doi:
51
Polo
,
J.M.
,
Anderssen
,
E.
,
Walsh
,
R.M.
,
Schwarz
,
B.A.
,
Nefzger
,
C.M.
,
Lim
,
S.M.
et al. 
(
2012
)
A molecular roadmap of reprogramming somatic cells into iPS cells
.
Cell
151
,
1617
1632
doi:
52
Umbas
,
R.
,
Schalken
,
J.A.
,
Aalders
,
T.W.
,
Carter
,
B.S.
,
Karthaus
,
H.F.
,
Schaafsma
,
H.E.
et al. 
(
1992
)
Expression of the cellular adhesion molecule E-cadherin is reduced or absent in high-grade prostate cancer
.
Cancer Res.
52
,
5104
5109
PMID:
[PubMed]
53
Elston
,
C.W.
and
Ellis
,
I.O.
(
1991
)
Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up
.
Histopathology
19
,
403
410
doi:
54
St. John
,
J.C.
(
2016
)
Mitochondrial DNA copy number and replication in reprogramming and differentiation
.
Semin. Cell Dev. Biol.
52
,
93
101
doi:
55
Warburg
,
O.
(
1956
)
On the origin of cancer cells
.
Science
123
,
309
314
doi:
56
Shih
,
C.
and
Weinberg
,
R.A.
(
1982
)
Isolation of a transforming sequence from a human bladder carcinoma cell line
.
Cell
29
,
161
169
doi:
57
Krisher
,
R.L.
and
Prather
,
R.S.
(
2012
)
A role for the Warburg effect in preimplantation embryo development: metabolic modification to support rapid cell proliferation
.
Mol. Reprod. Dev.
79
,
311
320
doi:
58
Vander Heiden
,
M.G.
,
Cantley
,
L.C.
and
Thompson
,
C.B.
(
2009
)
Understanding the Warburg effect: the metabolic requirements of cell proliferation
.
Science
324
,
1029
1033
doi:
59
Ward
,
P.S.
and
Thompson
,
C.B.
(
2012
)
Metabolic reprogramming: a cancer hallmark even Warburg did not anticipate
.
Cancer Cell
21
,
297
308
doi:
60
Singh
,
K.K.
,
Ayyasamy
,
V.
,
Owens
,
K.M.
,
Koul
,
M.S.
and
Vujcic
,
M.
(
2009
)
Mutations in mitochondrial DNA polymerase-γ promote breast tumorigenesis
.
J. Hum. Genet.
54
,
516
524
doi:
61
Guo
,
J.
,
Zheng
,
L.
,
Liu
,
W.
,
Wang
,
X.
,
Wang
,
Z.
,
Wang
,
Z.
et al. 
(
2011
)
Frequent truncating mutation of TFAM induces mitochondrial DNA depletion and apoptotic resistance in microsatellite-unstable colorectal cancer
.
Cancer Res.
71
,
2978
2987
doi:
62
Murphy
,
M.P.
(
2009
)
How mitochondria produce reactive oxygen species
.
Biochem. J.
417
,
1
13
doi:
63
D'Autreaux
,
B.
and
Toledano
,
M.B.
(
2007
)
ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis
.
Nat. Rev. Mol. Cell Biol.
8
,
813
824
doi:
64
Frank
,
M.
,
Duvezin-Caubet
,
S.
,
Koob
,
S.
,
Occhipinti
,
A.
,
Jagasia
,
R.
,
Petcherski
,
A.
et al. 
(
2012
)
Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner
.
Biochim. Biophys. Acta
1823
,
2297
2310
doi:
65
Simon
,
H.-U.
,
Haj-Yehia
,
A.
and
Levi-Schaffer
,
F.
(
2000
)
Role of reactive oxygen species (ROS) in apoptosis induction
.
Apoptosis
5
,
415
418
doi:
66
Giannoni
,
E.
,
Buricchi
,
F.
,
Raugei
,
G.
,
Ramponi
,
G.
and
Chiarugi
,
P.
(
2005
)
Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth
.
Mol. Cell. Biol.
25
,
6391
6403
doi:
67
Cooke
,
M.S.
,
Evans
,
M.D.
,
Dizdaroglu
,
M.
and
Lunec
,
J.
(
2003
)
Oxidative DNA damage: mechanisms, mutation, and disease
.
FASEB J.
17
,
1195
1214
doi:
68
Takahashi
,
A.
,
Ohtani
,
N.
,
Yamakoshi
,
K.
,
Iida
,
S.-i.
,
Tahara
,
H.
,
Nakayama
,
K.
et al. 
(
2006
)
Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence
.
Nat. Cell Biol.
8
,
1291
1297
doi:
69
Garrido
,
C.
,
Galluzzi
,
L.
,
Brunet
,
M.
,
Puig
,
P.E.
,
Didelot
,
C.
and
Kroemer
,
G.
(
2006
)
Mechanisms of cytochrome c release from mitochondria
.
Cell Death Differ.
13
,
1423
1433
doi:
70
St-Pierre
,
J.
,
Drori
,
S.
,
Uldry
,
M.
,
Silvaggi
,
J.M.
,
Rhee
,
J.
,
Jäger
,
S.
et al. 
(
2006
)
Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators
.
Cell
127
,
397
408
doi:
71
Faraonio
,
R.
,
Vergara
,
P.
,
Di Marzo
,
D.
,
Pierantoni
,
M.G.
,
Napolitano
,
M.
,
Russo
,
T.
et al. 
(
2006
)
p53 suppresses the Nrf2-dependent transcription of antioxidant response genes
.
J. Biol. Chem.
281
,
39776
39784
doi:
72
Wise
,
D.R.
,
DeBerardinis
,
R.J.
,
Mancuso
,
A.
,
Sayed
,
N.
,
Zhang
,
X.Y.
,
Pfeiffer
,
H.K.
et al. 
(
2008
)
Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction
.
Proc. Natl. Acad. Sci. U.S.A.
105
,
18782
18787
doi:
73
Xie
,
D.
,
Wu
,
X.
,
Lan
,
L.
,
Shangguan
,
F.
,
Lin
,
X.
,
Chen
,
F.
et al. 
(
2016
)
Downregulation of TFAM inhibits the tumorigenesis of non-small cell lung cancer by activating ROS-mediated JNK/p38MAPK signaling and reducing cellular bioenergetics
.
Oncotarget
7
,
11609
–11624
doi
:10.18632/oncotarget.7018
74
Polyak
,
K.
,
Li
,
Y.
,
Zhu
,
H.
,
Lengauer
,
C.
,
Willson
,
J.K.
,
Markowitz
,
S.D.
et al. 
(
1998
)
Somatic mutations of the mitochondrial genome in human colorectal tumours
.
Nat. Genet.
20
,
291
293
doi:
75
Petros
,
J.A.
,
Baumann
,
A.K.
,
Ruiz-Pesini
,
E.
,
Amin
,
M.B.
,
Sun
,
C.Q.
,
Hall
,
J.
et al. 
(
2005
)
mtDNA mutations increase tumorigenicity in prostate cancer
.
Proc. Natl. Acad. Sci. U.S.A.
102
,
719
724
doi:
76
Tseng
,
L.-M.
,
Yin
,
P.-H.
,
Yang
,
C.-W.
,
Tsai
,
Y.-F.
,
Hsu
,
C.-Y.
,
Chi
,
C.-W.
and
Lee
,
H.-C.
(
2011
)
Somatic mutations of the mitochondrial genome in human breast cancers
.
Genes Chromosomes Cancer
50
,
800
811
doi:
77
Wang
,
C.-Y.
,
Wang
,
H.-W.
,
Yao
,
Y.-G.
,
Kong
,
Q.-P.
and
Zhang
,
Y.-P.
(
2007
)
Somatic mutations of mitochondrial genome in early stage breast cancer
.
Int. J. Cancer
121
,
1253
1256
doi:
78
Yeung
,
K.Y.
,
Dickinson
,
A.
,
Donoghue
,
J.F.
,
Polekhina
,
G.
,
White
,
S.J.
,
Grammatopoulos
,
D.K.
et al. 
(
2014
)
The identification of mitochondrial DNA variants in glioblastoma multiforme
.
Acta Neuropathol. Commun.
2
,
1
doi:
79
Attardi
,
G.
and
Schatz
,
G.
(
1988
)
Biogenesis of mitochondria
.
Annu. Rev. Cell Biol.
4
,
289
333
doi:
80
Fernandez-Marcos
,
P.J.
and
Auwerx
,
J.
(
2011
)
Regulation of PGC-1α, a nodal regulator of mitochondrial biogenesis
.
Am. J. Clin. Nutr.
93
,
884S
8890S
doi:
81
Scarpulla
,
R.C.
,
Vega
,
R.B.
and
Kelly
,
D.P.
(
2012
)
Transcriptional integration of mitochondrial biogenesis
.
Trends Endocrinol. Metab.
23
,
459
466
doi:
82
Wenz
,
T.
(
2013
)
Regulation of mitochondrial biogenesis and PGC-1α under cellular stress
.
Mitochondrion
13
,
134
142
doi:
83
Pikó
,
L.
and
Matsumoto
,
L.
(
1976
)
Number of mitochondria and some properties of mitochondrial DNA in the mouse egg
.
Dev. Biol.
49
,
1
10
doi:
84
Wilding
,
M.
,
Dale
,
B.
,
Marino
,
M.
,
di Matteo
,
L.
,
Alviggi
,
C.
,
Pisaturo
,
M.L.
et al. 
(
2001
)
Mitochondrial aggregation patterns and activity in human oocytes and preimplantation embryos
.
Hum. Reprod.
16
,
909
917
doi:
85
Aquilano
,
K.
,
Vigilanza
,
P.
,
Baldelli
,
S.
,
Pagliei
,
B.
,
Rotilio
,
G.
and
Ciriolo
,
M.R.
(
2010
)
Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and sirtuin 1 (SIRT1) reside in mitochondria: possible direct function in mitochondrial biogenesis
.
J. Biol. Chem.
285
,
21590
21599
doi:
86
Kong
,
X.
,
Wang
,
R.
,
Xue
,
Y.
,
Liu
,
X.
,
Zhang
,
H.
,
Chen
,
Y.
et al. 
(
2010
)
Sirtuin 3, a new target of PGC-1α, plays an important role in the suppression of ROS and mitochondrial biogenesis
.
PLoS One
5
,
e11707
doi:
87
Sahin
,
E.
,
Colla
,
S.
,
Liesa
,
M.
,
Moslehi
,
J.
,
Müller
,
F.L.
,
Guo
,
M.
et al. 
(
2011
)
Telomere dysfunction induces metabolic and mitochondrial compromise
.
Nature
470
,
359
365
doi:
88
Kim
,
J.
,
Lee
,
J.H.
and
Iyer
,
V.R.
(
2008
)
Global identification of Myc target genes reveals its direct role in mitochondrial biogenesis and its E-box usage in vivo
.
PLoS One
3
,
e1798
doi:
89
Li
,
F.
,
Wang
,
Y.
,
Zeller
,
K.I.
,
Potter
,
J.J.
,
Wonsey
,
D.R.
,
O'Donnell
,
K.A.
et al. 
(
2005
)
Myc stimulates nuclearly encoded mitochondrial genes and mitochondrial biogenesis
.
Mol. Cell. Biol.
25
,
6225
6234
doi:
90
Morrish
,
F.
and
Hockenbery
,
D.
(
2014
)
MYC and mitochondrial biogenesis
.
Cold Spring Harb. Perspect. Med.
4
,
a014225
doi:
91
Carbognin
,
E.
,
Betto
,
R.M.
,
Soriano
,
M.E.
,
Smith
,
A.G.
and
Martello
,
G.
(
2016
)
Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency
.
EMBO J.
35
,
618
634
doi:
92
O'Hagan
,
K.A.
,
Cocchiglia
,
S.
,
Zhdanov
,
A.V.
,
Tambuwala
,
M.M.
,
Cummins
,
E.P.
,
Monfared
,
M.
et al. 
(
2009
)
PGC-1α is coupled to HIF-1α-dependent gene expression by increasing mitochondrial oxygen consumption in skeletal muscle cells
.
Proc. Natl. Acad. Sci. U.S.A.
106
,
2188
2193
doi:
93
Pawlus
,
M.R.
,
Wang
,
L.
and
Hu
,
C.J.
(
2014
)
STAT3 and HIF1α cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells
.
Oncogene
33
,
1670
1679
doi:
94
Twig
,
G.
,
Elorza
,
A.
,
Molina
,
A.J.
,
Mohamed
,
H.
,
Wikstrom
,
J.D.
,
Walzer
,
G.
et al. 
(
2008
)
Fission and selective fusion govern mitochondrial segregation and elimination by autophagy
.
EMBO J.
27
,
433
446
doi:
95
Youle
,
R.J.
and
Narendra
,
D.P.
(
2011
)
Mechanisms of mitophagy
.
Nat. Rev. Mol. Cell Biol.
12
,
9
14
doi:
96
Zhang
,
J.
and
Ney
,
P.A.
(
2009
)
Role of BNIP3 and NIX in cell death, autophagy, and mitophagy
.
Cell Death Differ.
16
,
939
946
doi:
97
Cesari
,
R.
,
Martin
,
E.S.
,
Calin
,
G.A.
,
Pentimalli
,
F.
,
Bichi
,
R.
,
McAdams
,
H.
et al. 
(
2003
)
Parkin, a gene implicated in autosomal recessive juvenile parkinsonism, is a candidate tumor suppressor gene on chromosome 6q25-q27
.
Proc. Natl. Acad. Sci. U.S.A.
100
,
5956
5961
doi:
98
Todd
,
L.R.
,
Damin
,
M.N.
,
Gomathinayagam
,
R.
,
Horn
,
S.R.
,
Means
,
A.R.
and
Sankar
,
U.
(
2010
)
Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells
.
Mol. Biol. Cell
21
,
1225
1236
doi:
99
Otera
,
H.
and
Mihara
,
K.
(
2011
)
Molecular mechanisms and physiologic functions of mitochondrial dynamics
.
J. Biochem.
149
,
241
251
doi:
100
Detmer
,
S.A.
and
Chan
,
D.C.
(
2007
)
Functions and dysfunctions of mitochondrial dynamics
.
Nat. Rev. Mol. Cell Biol.
8
,
870
879
doi:
101
Autret
,
A.
and
Martin
,
S.J.
(
2009
)
Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis
.
Mol. Cell
36
,
355
363
doi:
102
Suen
,
D.F.
,
Norris
,
K.L.
and
Youle
,
R.J.
(
2008
)
Mitochondrial dynamics and apoptosis
.
Genes Dev.
22
,
1577
1590
doi:
103
Hotchkiss
,
R.D.
(
1948
)
The quantitative separation of purines, pyrimidines, and nucleosides by paper chromatography
.
J. Biol. Chem.
175
,
315
332
PMID:
[PubMed]
104
Lister
,
R.
,
O'Malley
,
R.C.
,
Tonti-Filippini
,
J.
,
Gregory
,
B.D.
,
Berry
,
C.C.
,
Millar
,
A.H.
and
Ecker
,
J.R.
(
2008
)
Highly integrated single-base resolution maps of the epigenome in Arabidopsis
.
Cell
133
,
523
536
doi:
105
Lister
,
R.
,
Mukamel
,
E.A.
,
Nery
,
J.R.
,
Urich
,
M.
,
Puddifoot
,
C.A.
,
Johnson
,
N.D.
et al. 
(
2013
)
Global epigenomic reconfiguration during mammalian brain development
.
Science
341
,
1237905
doi:
106
Ramsahoye
,
B.H.
,
Biniszkiewicz
,
D.
,
Lyko
,
F.
,
Clark
,
V.
,
Bird
,
A.P.
and
Jaenisch
,
R.
(
2000
)
Non-CpG methylation is prevalent in embryonic stem cells and May be mediated by DNA methyltransferase 3a
.
Proc. Natl. Acad. Sci. U.S.A.
97
,
5237
5242
doi:
107
Cheng
,
X.
(
1995
)
DNA modification by methyltransferases
.
Curr. Opin. Struct. Biol.
5
,
4
10
doi:
108
Okano
,
M.
,
Bell
,
D.W.
,
Haber
,
D.A.
and
Li
,
E.
(
1999
)
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development
.
Cell
99
,
247
257
doi:
109
Shen
,
L.
and
Zhang
,
Y.
(
2012
)
Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation
.
Methods Enzymol.
512
,
93
105
doi:
110
Kohli
,
R.M.
and
Zhang
,
Y.
(
2013
)
TET enzymes, TDG and the dynamics of DNA demethylation
.
Nature
502
,
472
479
doi:
111
Gardiner-Garden
,
M.
and
Frommer
,
M.
(
1987
)
Cpg islands in vertebrate genomes
.
J. Mol. Biol.
196
,
261
282
doi:
112
Deaton
,
A.M.
and
Bird
,
A.
(
2011
)
Cpg islands and the regulation of transcription
.
Genes Dev.
21
,
1074
–1086
doi
:10.1101/gad.2037511
113
Jones
,
P.A.
and
Taylor
,
S.M.
(
1980
)
Cellular differentiation, cytidine analogs and DNA methylation
.
Cell
20
,
85
93
doi:
114
Kelly
,
T.K.
,
De Carvalho
,
D.D.
and
Jones
,
P.A.
(
2010
)
Epigenetic modifications as therapeutic targets
.
Nat. Biotechnol.
28
,
1069
1078
doi:
115
Blaschke
,
K.
,
Ebata
,
K.T.
,
Karimi
,
M.M.
,
Zepeda-Martínez
,
J.A.
,
Goyal
,
P.
,
Mahapatra
,
S.
et al. 
(
2013
)
Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells
.
Nature
500
,
222
226
doi:
116
Eckhardt
,
F.
,
Lewin
,
J.
,
Cortese
,
R.
,
Rakyan
,
V.K.
,
Attwood
,
J.
,
Burger
,
M.
et al. 
(
2006
)
DNA methylation profiling of human chromosomes 6, 20 and 22
.
Nat. Genet.
38
,
1378
1385
doi:
117
Bird
,
A.
(
2002
)
DNA methylation patterns and epigenetic memory
.
Genes Dev.
16
,
6
21
doi:
118
Feil
,
R.
and
Khosla
,
S.
(
1999
)
Genomic imprinting in mammals: an interplay between chromatin and DNA methylation?
Trends Genet.
15
,
431
435
doi:
119
Riggs
,
A.D.
(
1975
)
X inactivation, differentiation, and DNA methylation
.
Cytogenet. Genome Res.
14
,
9
25
doi:
120
Fatemi
,
M.
,
Pao
,
M.M.
,
Jeong
,
S.
,
Gal-Yam
,
E.N.
,
Egger
,
G.
,
Weisenberger
,
D.J.
et al. 
(
2005
)
Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level
.
Nucleic Acids Res.
33
,
e176
doi:
121
Esteller
,
M.
(
2002
)
Cpg island hypermethylation and tumor suppressor genes: a booming present, a brighter future
.
Oncogene
21
,
5427
5440
doi:
122
Maunakea
,
A.K.
,
Chepelev
,
I.
,
Zhao
,
K.
, and
Bruneau
,
B.
(
2010
)
Epigenome mapping in normal and disease states
.
Circ. Res.
107
,
327
339
doi:
123
Barrès
,
R.
,
Osler
,
M.E.
,
Yan
,
J.
,
Rune
,
A.
,
Fritz
,
T.
,
Caidahl
,
K.
et al. 
(
2009
)
Non-CpG methylation of the PGC-1α promoter through DNMT3B controls mitochondrial density
.
Cell Metab.
10
,
189
198
doi:
124
Gemma
,
C.
,
Sookoian
,
S.
,
Dieuzeide
,
G.
,
García
,
S.I.
,
Gianotti
,
T.F.
,
González
,
C.D.
and
Pirola
,
C.J.
(
2010
)
Methylation of TFAM gene promoter in peripheral White blood cells is associated with insulin resistance in adolescents
.
Mol. Genet. Metab.
100
,
83
87
doi:
125
Aran
,
D.
,
Toperoff
,
G.
,
Rosenberg
,
M.
and
Hellman
,
A.
(
2011
)
Replication timing-related and gene body-specific methylation of active human genes
.
Hum. Mol. Genet.
20
,
670
680
doi:
126
Ball
,
M.P.
,
Li
,
J.B.
,
Gao
,
Y.
,
Lee
,
J.-H.
,
LeProust
,
E.M.
,
Park
,
I.-H.
et al. 
(
2009
)
Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells
.
Nat. Biotechnol.
27
,
361
368
doi:
127
Jones
,
P.A.
(
1999
)
The DNA methylation paradox
.
Trends Genet.
15
,
34
37
doi:
128
Walsh
,
C.P.
,
Chaillet
,
J.R.
and
Bestor
,
T.H.
(
1998
)
Transcription of IAP endogenous retroviruses is constrained by cytosine methylation
.
Nat. Genet.
20
,
116
117
doi:
129
Jjingo
,
D.
,
Conley
,
A.B.
,
Yi
,
S.V.
,
Lunyak
,
V.V.
and
Jordan
,
I.K.
(
2012
)
On the presence and role of human gene-body DNA methylation
.
Oncotarget
3
,
462
474
doi:
130
Laurent
,
L.
,
Wong
,
E.
,
Li
,
G.
,
Huynh
,
T.
,
Tsirigos
,
A.
,
Ong
,
C.T.
et al. 
(
2010
)
Dynamic changes in the human methylome during differentiation
.
Genome Res.
20
,
320
331
doi:
131
Guo
,
F.
,
Li
,
X.
,
Liang
,
D.
,
Li
,
T.
,
Zhu
,
P.
,
Guo
,
H.
et al. 
(
2014
)
Active and passive demethylation of male and female pronuclear DNA in the mammalian zygote
.
Cell Stem Cell
15
,
447
458
doi:
132
Santos
,
F.
,
Hendrich
,
B.
,
Reik
,
W.
and
Dean
,
W.
(
2002
)
Dynamic reprogramming of DNA methylation in the early mouse embryo
.
Dev. Biol.
241
,
172
182
doi:
133
Brunner
,
A.L.
,
Johnson
,
D.S.
,
Kim
,
S.W.
,
Valouev
,
A.
,
Reddy
,
T.E.
,
Neff
,
N.F.
et al. 
(
2009
)
Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetal liver
.
Genome Res.
19
,
1044
1056
doi:
134
Fouse
,
S.D.
,
Shen
,
Y.
,
Pellegrini
,
M.
,
Cole
,
S.
,
Meissner
,
A.
,
Van Neste
,
L.
et al. 
(
2008
)
Promoter CpG methylation contributes to ES cell gene regulation in parallel with Oct4/Nanog, PcG complex, and histone H3 K4/K27 trimethylation
.
Cell Stem Cell
2
,
160
169
doi:
135
Lee
,
W.T.Y.
,
Cain
,
J.E.
,
Cuddihy
,
A.
,
Johnson
,
J.
,
Dickinson
,
A.
,
Yeung
,
K.Y.
et al. 
(
2016
)
Mitochondrial DNA plasticity is an essential inducer of tumorigenesis
.
Cell Death Discov.
2
,
16016
doi:
136
Kelly
,
R.D.W.
,
Rodda
,
A.E.
,
Dickinson
,
A.
,
Mahmud
,
A.
,
Nefzger
,
C.M.
,
Lee
,
W.
et al. 
(
2013
)
Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells
.
Stem Cells
31
,
703
716
doi: