**4.2. The DNA methylation of chromosomal DNAs**

modulate the PAR synthesis/degradation ratio at the transcription level. The accumulation of

ratio would not only reduce the activities but also the expression of enzymes that function

in a mitochondria-independent manner. This metabolic change would be observed as the

**3.2. The regulation of TFs and nucleotide binding proteins by poly(ADP-ribosyl)ation**

PARP inhibitors, such as talazoparib, niraparib, rucaparib, olaparib, and veliparib, are clinically used for the treatment of cancer, especially when *BRCA1* and *BRCA2* gene mutations

regulates interaction with DBC1, which is deleted in breast cancer 1, which is a known SIRT1

and PARP1, leading to the suppression of its activity. This might partly explain why DNA repair declines with aging [91]. The poly(ADP-ribosyl)ation of proteins not only initiates the response to DNA damage, but also regulates the transcription of specific genes [92]. The poly(ADP-ribosyl)ation of C/EBPβ by PARP-1 modulates its transcriptional activity to enhance the expression of the genes encoding factors that regulate adipogenesis [93]. A recent study showed that the poly(ADP-ribosyl)ation of an RNA-binding protein HuR by PARP1 stabilizes *Cxcl2* gene transcripts [94]. Moreover, the poly(ADP-ribosyl)ation of FoxO3 suppresses its transcriptional activity and leads to cardiac hypertrophy [95]. Taken together,

late transcription to respond to DNA damage-induced signals. Thus, it should be noted that PARP inhibitors not only limit the DNA damage response to lead to the death of cancerous

Epigenetic alterations are frequently found in cancer, implying that "cancer is an epigenetic disease" [96]. It has been hypothesized that epigenetic and/or transcriptional changes play a role in determining the chromatin state in tumor cells [97]. Epigenetic regulation is mainly driven by modifications of chromosomal DNAs and histone proteins [98]. The biological relevance between cellular metabolites and the gene expression has been proposed as the RNA/enzyme/metabolite (REM) networking system [99]. The metabolites, NAD+

S-adenosylmethionine (AdoMet), and acetyl-CoA, are the substrates for poly (ADP-ribosyl) ation, methylation, and acetylation, respectively [76], suggesting that these metabolic state-

**4. Epigenetic alterations in chromosomal DNAs and proteins**

dependent molecules play important roles in the epigenetic regulation.

thesize PAR polymer in the nuclei or mitochondria. Thus, the decrease in the NAD+

when DNA damage eventually activates PARP, NAD+

are present [89]. They all interact with the NAD+

inhibitor protein [90]. A decrease in the NAD+

poly(ADP-ribosyl)ation, which consumes NAD+

cells, but also reduce the consumption of NAD+

PARP1 and PARP2. A recent study indicated that the NAD+

"Warburg effect" in cancer cells [5, 6].

 molecules in cells might be transiently caused by mitochondrial dysfunction, which is usually accompanied by insufficient OXPHOS or aberrant respiration [87, 88]. However,


molecules will be consumed to syn-


will upregulate the interaction between DBC1

as a substrate for PAR synthesis, may regu-

molecules to modulate the transcription of


/NADH

,

NAD+

in the NAD+

112 Mitochondrial Diseases

specific genes.

The methylation of promoter regions of specific genes in human chromosomes can be used as biomarkers in various cancers [116]. The DNA methylation reaction is catalyzed by methyltransferases (DNMTs), which utilize AdoMet as a methyl group donor [117]. A recent study showed that intragenic DNA methylation, which is carried out by Dnmt3b in mouse embryonic stem cells, protects the gene body from the entry of spurious RNA pol II and the initiation of cryptic transcription [118]. The extended data showed that the ETS factor binding regions are sensitive to the knock out of the *Dnmt3b* gene, suggesting that the occupation of the GGAA (TTCC)-motifs by GGAA-motif binding proteins could be epigenetically controlled by methylation. Furthermore, the regulation of demethylation by ten-eleven translocation (TET)-family enzymes [119], the activity of which is reduced by hypoxia, should not be ignored. Hypoxia-induced hyper methylation has been demonstrated to occur on the promoter regions of the DNA repair factor-encoding genes, including *BRCA1*, *FANCD2*, *FANCF*, *POLL*, and *UNG* [120]. Of note, the duplicated GGAA-motifs are contained in these gene promoters [53]. A methylation sensitive SELEX analysis showed that ETS-binding was inhibited by mCpG, though NFAT, which also recognizes the GGAA-core motif sequence and preferentially binds to methylated DNA [121]. The observation suggests that GGAA-motif recognizing TFs could be classified into two groups according to their preference to DNA methylation.

The SET protein is an epigenetic regulatory factor that promotes loss of methylation through direct interaction with hypo-acetylated histones [122]. A genome-wide analysis showed that DNA hypermethylation is apparently induced in old male adults, relative to young male adults, suggesting a relationship between DNA methylation and aging [123–125]. Moreover, the methylation and demethylation of the lysine residues of histones might affect the regulation of transcription [126]. In summary, AdoMet, a methyl group donor, plays an important role in epigenetic control.

are known to serve as master regulators of danger signaling to determine cell death or survival [141]. Several mechanisms, including the regulation of the regulators of apoptosis [142, 143] and miRNAs [144], are involved in the induction of apoptosis. The surveillance of the human genomic DNA database indicated the presence of the duplicated GGAA-motifs in the 5′-regulatory regions of the human *PDCD1*, *DFFA*, *BCL2*, *FAS*, *FASL*, *ATG12/AP3S1*, *APOPT1/BAG5*, and *HTRA2/AUP1* genes [13, 53, 54]. These observations suggest that the expression of the apoptosis regulating factor-encoding genes is modulated by the GGAAduplicated sequences. In this context, apoptosis or programmed cell death, which is con-

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trolled by the mitochondria, partly depends on the GGAA-motif binding TFs.

**regulation of their gene expression**

duplicated GGAA-motifs [53, 78].

**5.2. The localization of p53 and other DNA repair factors in the mitochondria and the** 

Recent studies have shown that the p53 protein not only acts as a "guardian of the genome", but also serves as a regulator of metabolism [145, 146]. Moreover, p53 has been reported to accumulate in the mitochondria in response to stress [147]. Besides p53, a number of widely known DNA repair factors, including ATM, BRCA1, PARP, PARG, and RB, localize in the mitochondria or regulate their functions [148–152]. The surveillance of the 5′-upstream regions of these DNA repair factor-encoding genes revealed that they commonly possess

GGAA-motif duplications are found in the bidirectional *APEX1*/*OSGEP* promoter region. The *APEX1* encodes apurinic/apyrimidinic endonuclease 1 (APE1) that regulates both the base excision repair (BER) and the mitochondrial DNA repair systems [75, 153]. The GGAA-duplication is contained in the regulatory region of the head–head configured *ACO2/PHF5A* genes [54]. The *ACO2* gene encodes aconitase, which plays an important role in the TCA cycle to produce citrate and isocitrate, and which also serves as a mitochondrial redox-sensor [154]. Importantly, aconitase and mitochondrial BER enzyme OGG1 (8-oxoguanine DNA glycosylase) cooperatively preserve mitochondrial DNA integrity [155]. We also confirmed that the duplicated GGAA-motifs were present in the 5′-upstream regions of the genes associated with Fanconi's anemia (FA) [53], which encode the DNA repair factors that are shown to regulate nucleotide excision repair and genome stability [156]. Interestingly, it was shown that mitochondrial dysfunction forces FA cells to produce energy by glycolysis [157], suggesting that FA proteins might be involved in the metabolic switch system in cancer cells. Additionally, Cockayne syndrome proteins CSA and CSB, which play roles in nucleotide excision repair (NER), accumulate in the mitochondria under oxidative stress [158]. In KRAS/LKB1-mutant lung cancer cells, carbamoyl phosphate

synthetase-1 (CPS1), which is localized in mitochondria and which eliminates NH4

TFs, supporting the hypothesis that mitochondrial dysfunction causes oncogenesis [8].

urea cycle, also plays a role in controlling the pyrimidine/purine balance to regulate the integrity of nuclear DNAs [159]. In this circumstance, the silencing of the *CPS1* gene expression leads to an incomplete S-phase or apoptotic cell death due to increased DNA damage. As expected, the duplicated GGAA is present in the *CPS1*/*LANCL1* bidirectional promoter region. However, no such element is found near the TSSs of either the *CAD* or *ASS1* genes, which encode cytoplasmic enzymes carbamoyl phosphate syntetase-2 and argininosuccinate synthase, respectively. These observations suggest that expression of the mitochondria-localizing, DNA repair-associated protein-encoding genes could be cooperatively regulated by duplicated GGAA-motif binding

to initiate the

#### **4.3. The acetylation of histones could regulate the generation or progression of cancers**

Acetyl-CoA is required for acetylation on the lysine residue of histones [127]. This process is catalyzed by acetyltransferases (HATs), including KAT2A (GCN5), KAT2B (CAF), KAT5 (ESA1), KAT7 (HBO1), and KAT8 (MOF) [128], which can be classified into two major groups: the GCNT and MYST families [129]. At least 11 enzymes are known to be histone deacetylases (HDACs) [130]. Because the increased or aberrant expression of HDACs has been reported in various cancers, inhibitors or modulators of HDACs are expected to be effective as anticancer drugs [131]. On the other hand, the lysine acetylation is negatively regulated by sirtuin proteins, including SIRT1 [116], which de-acetylate proteins, utilizing NAD+ as an acceptor of the acetyl group [127]. It is hypothesized that a reduction in nutrient levels could induce the accumulation of NAD+ to activate sirtuins. Histone de-acetylation is consistent with the finding that CR mimetics prolong the life span [131–133]. In cancer cells, if mitochondrial dysfunction occurs with a reduction in the NAD+ level or the hindrance of the progression of the TCA cycle, acetyl-CoA might only be converted to citrate to be used as an acetyl group donor for histones in the nuclei. If so, an increase in histone acetylation would occur naturally in the course of oncogenesis. The activation of HDACs in cancer cells might be the response to the aberrant hyper-acetylation of histone proteins that could lead to the abnormal transcription of various genes, including the mitochondrial function-associated genes.

To summarize, key metabolites, NAD+ and acetyl-CoA could regulate DNA methylation and histone acetylation directly or indirectly, and play essential roles in epigenetic control.
