Mitochondrial DNA Methylation Research Articles

Circulating Cell Free Nuclear DNA, Mitochondrial DNA and Global DNA Methylation: Potential Noninvasive Biomarkers for Breast Cancer Diagnosis

Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.

Profiles of mitochondrial DNA methylation in the ridgetail white prawn, Exopalaemon carinicauda (Holthuis, 1950) (Decapoda, Palaemonidae) under heavy metal stress from chromium and cadmium

Abstract For the purpose of studying the epigenetic characteristics of the mitochondrial genome of the ridgetail white prawn, Exopalaemon carinicauda (Holthuis, 1950) under Cd2+ and Cr6+ heavy metal stress, the mitochondrial DNA methylation of E. carinicauda was analysed by bisulfite sequencing PCR (BSP). Many methylation sites were found at the 3′ end sequence of COX3 and at the starting region sequence of ND3, while only a few methylation sites were found at the 3′ end sequence of ND5. The mitochondrial genome was inferred to regulate the energy metabolism through the methylation process. In addition, under Cd2+ stress, mitochondrial DNA methylation was more common, and found during all stress periods (3, 6, 12, 24, 48, 72 and 96 h), while under Cr6+ stress, mitochondrial DNA methylation was less common, mainly occurring after 48 hours of stress. The sensitivity of the mitochondrial genome response to Cd2+ stress was inferred to be greater than that to Cr6+. This study revealed for the first time that methylation occurs in the mitochondrial genome of E. carinicauda in response to heavy metal stress.

Human mitochondrial DNA is extensively methylated in a non-CpG context.

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.

Abstract P2042: Alteration of Mitochondrial Deoxyribonucleic Acid Methylation and Nuclear Transcription Factors Reduces Mitochondrial Biogenesis in Aged Hypertensive Kidney

Accumulating evidence suggests that epigenetic mechanisms contribute to aging and target organ injury during hypertension. Aberrant DNA methylation of mitochondrial genes can affect mitochondrial copy numbers, replication and metabolism. Recently, the presence of mitochondrial DNA (mtDNA) methylation was reported; however, its functional significance in aging and hypertension-induced chronic kidney disease is unknown. We hypothesized that alteration in mtDNA methylation aggravates hypertension injury in aged kidney by disrupting mitochondrial homeostasis leading to reduced mitochondrial biogenesis and dysfunction. Hydrogen sulfide (H 2 S) was shown to modulate chromatin recently, we therefore investigated whether H 2 S can act as an epigenetic modifying agent to protect renal mitochondria and improve renal function. Aged (72-75 weeks) and young (10-12 weeks) wild type (WT, C57BL/6J) mice were treated without or with angiotensin-II (Ang II, 1000 ng/Kg/d) and H 2 S donor, GYY4137 (0.5 mg/Kg/d, i.p.), for 28 d. Ang-II increased mean blood pressure in aged mice compared to young (136.54±3.55 vs. 108.68±2.86 mmHg) and reduced glomerular filtration rate (688.3±24.83 vs. 1029.3±30.8 μL/min/100 g b.w.). H 2 S treatment reduced mean BP (122.34±3.47) and increased GFR (962.7±21.5) in aged mice receiving Ang II. In aged mice, querying renal mitochondrial genes for methylation status revealed increased methylation of mt-ND1 and reduction of mt-CO1 and mt-CO2 and H 2 S treatment reversed the changes. Methylation of mt-Cytb was undetected and mt-Dloop was low in Ang II treated aged mice and increased with H 2 S treatment. Ang II treated aged mice exhibited decreased PGC-1α and nuclear transcription factors, NRFI, NRF2, ERRα and TFAM, indicating reduced mitochondrial biogenesis compared to young mice and H 2 S restored their levels. ATP synthase activity, ATP generation was reduced in aged mice receiving Ang II compared to young and increased with H 2 S treatment. Together, our results suggest that alteration in mtDNA methylation and impaired nuclear DNA genes is associated with reduced mitochondrial replication and disrupted metabolism and H 2 S therapy protects the aged kidney from hypertension-induced damage by restoring mitochondrial homeostasis.

Distribution and dynamics of mitochondrial DNA methylation in oocytes, embryos and granulosa cells

Comparison of mitochondrial DNA (mtDNA) methylation patterns in oocytes, blastocysts and ovarian granulosa cells indicates hitherto unsuspected dynamics. Oocytes and blastocysts recovered from cows subjected to ovarian stimulation and from non-stimulated abattoir ovaries were analyzed using bisulphite transformation of DNA followed by whole genome sequencing. The cow is a recognized as a good model for human oocyte and pre-implantation development. The number of mtDNA copies is high in oocytes (200,000–400,000) and early embryos, resulting in very high coverage (>3000x) and very low p values for each of 716 cytosine-based nucleosides. Methylation ratio was lowest in oocytes, following by blastocysts then granulosa cells and was not restricted to CG sites but was found also at CHG and CHH sites. The initial methylation pattern is conserved during the first week of life but not in somatic cells. RNA analysis of mitochondria encoded genes showed a significant inverse correlation between methylation and expression for almost all sequences. Methylation was more extensive in somatic tissues from mature animals than in immature pre-pubertal animals. Our findings suggest that mtDNA methylation might play a programming role during gametogenesis and would be subject to epigenetic regulation according to environment and/or maternal maturity.

Platelet mitochondrial DNA methylation: Markers of cardiovascular disease predisposition in overweight and obese individuals

AbstractObesity‐related non‐communicable diseases are global health issues. More than 50% of men and women are overweight or obese in the UK, which is associated with increased risk of cardiovascular disease (CVD) among other non‐communicable diseases. Early detection of the risk of CVD development within high‐risk groups is urgently needed to reduce the burden of this disease. Several mechanisms underlie the increased risk of CVD in overweight and obese individuals, yet non‐invasive molecular markers for risk prediction are not readily available. With the support of funding from the British Nutrition Foundation Drummond Pump Priming Award, we examined platelet mitochondrial DNA methylation in overweight and obese participants at baseline in relation to future CVD incidence. In this article, the findings from the project supported by the British Nutrition Foundation and future directions are described.

11 C]Choline as a Novel PET/CT Biomarker of Liver Cirrhosis: A Prospective Pilot Study

Aim: Choline plays a role in hepatic mitochondrial impairment, oxidative stress and DNA methylation levels of genes involved in lipid metabolism. The aim of this study was to compare findings between patients with liver cirrhosis and subjects with a normal liver on a [11C]choline PET/CT. Methods: This prospective pilot study was conducted between the years 2012-2016. The cohort included 14 patients with prostate cancer (reference group) and 11 patients with cirrhosis attending a tertiary medical center. Demographic, clinical and laboratory data were obtained from the medical files. All participants underwent a dynamic [11C]choline PET/CT (Discovery ST, GE Medical Systems, Milwaukee WI). We compared the maximal standard uptake values (SUVmax) and the area under the curve (AUC) at 1110 seconds in both groups. Results: The mean age of the cirrhosis group (63.4% men) was 68.4 ± 10.7 and the control group, 69.7 ± 7.3 years. The mean SUVmax was significantly higher in the cirrhosis group than in the controls (right lobe, 10.06 ± 12 vs. 6.3 ± 1.6, P=0.011; left lobe, 8.6 ± 11.6 vs. 5.4 ± 0.9, P=0.024; spleen 17.99 ± 27.8 vs. 13.4 ± 2.6, P=0.027; kidney, 35.9 ± 59.5 vs. 19.3 ± 4.8, P=0.025). The corresponding AUC values at 1110 seconds was significantly distinguished between the groups (right lobe, 13538 ± 20020 vs. 8427.3 ± 1557.9, P=0.026; left lobe 12304 ± 18871 vs. 6878.9 ± 1294.3, P=0.024; spleen, 12875 ± 17930 vs. 8263.9 ± 1279.2, P=0.023; kidney, 24623 ± 36025 vs. 13667 ± 3873.9, P=0.032). No correlations were found between the clinical characteristics and the imaging-derived parameters in the patients with cirrhosis. Conclusions: Our findings suggest a role for [11C]choline PET/CT as a noninvasive biomarker of cirrhosis. Further larger-scale studies are needed to confirm these observations.

Absence of mitochondrial DNA methylation in mouse oocyte maturation, aging and early embryo development

Mitochondrial DNA (mtDNA) is important for oxidative phosphorylation; dysfunctions can play a role in many mitochondrial diseases and can also affect the aging of cells and individuals. DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression. While recent studies have revealed the existence of mtDNA methylation there are still controversies about mtDNA methylation due to the special structure of mtDNA. Mitochondria and DNA methylation are both essential for regulating oocyte maturation and early embryo development, but whether mtDNA methylation changes during this process is unknown. By employing bisulfite sequencing, we found that in the process of mouse oocyte maturation, postovulatory oocyte aging, and early embryo development, all analyzed mitochondrial genes, including 16S-CpGI, DCR, ND6, 12S, and ATP8, lacked 5’mC. Thus, mtDNA methylation does not occur in the oocyte and early embryo.

Effects of air pollution on mitochondrial function, mitochondrial DNA methylation, and mitochondrial peptide expression

Mitochondrial DNA is sensitive to damage by exogenous reactive oxygen sources, including traffic-related air pollution (TRAP). Given the important role for mitochondria in human disease, we hypothesized that prenatal air pollution exposure may be associated with mitochondrial dysfunction and that mitochondrial-derived peptides (MDPs) might protect against these effects. In in vitro studies, 24-hour exposure to nanoparticulate matter (nPM) increased oxidation of mtDNA, decreased mitochondrial consumption rate (OCR), and decreased mtDNAcn in SH-SY5Y cells. Addition of MDPs rescued these effects to varying degrees. Liver tissue taken from C57Bl/6 males exposed for 10 weeks to nPM had lower OCR, lower mtDNAcn and higher MDP levels, similar to in vitro studies. In newborn cord blood, MDP levels were positively associated with prenatal TRAP exposures. Moreover, DNA methylation of two distinct regions of the D-Loop in the mitochondria genome was associated with levels of several MDPs. Our in vitro and in vivo data indicate that TRAP can directly affect mitochondrial respiratory function and mtDNAcn. Treatment of cells with MDPs can counteract TRAP induced-effects. Lastly, we present evidence that suggests MDPs may be regulated in part by mitochondrial DNA methylation in humans.

Cytosine methylation of mitochondrial DNA at CpG sequences impacts transcription factor A DNA binding and transcription

In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (5mCpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which 5mCpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of 5mCpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether 5mCpG alters any TFAM-dependent activities. 5mCpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, 5mCpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, 5mCpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that 5mCpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro.

Aanat Knockdown and Melatonin Supplementation in Embryo Development: Involvement of Mitochondrial Function and DNA Methylation.

Aims: In addition to pineal gland, many cells, tissues, and organs also synthesize melatonin (N-acetyl-5-methoxytryptamine). Embryos are a group of special cells and whether they can synthesize melatonin is still an open question. However, melatonin application promoted embryo development in many species in in vitro condition. The purpose of this study was to investigate whether embryos can synthesize melatonin; if it is so, what are the impacts of the endogenously produced melatonin on embryo development and the associated molecular mechanisms. These have never been reported previously. Results: Melatonin synthesis was observed at different stages of embryonic development. Aanat (aralkylamine N-acetyltransferase), a rate-limiting enzyme for melatonin production, was found to mostly localize in the mitochondria. Aanat knockdown significantly impeded embryonic development, and melatonin supplementation rescued it. The potential mechanisms might be that melatonin preserved mitochondrial intact and its function, thus providing sufficient adenosine 5'-triphosphate for the embryo development. In addition, melatonin scavenged intracellular reactive oxygen species (ROS) and reduced the DNA mutation induced by oxidative stress. In the molecular level, Aanat knockdown reduced tet methylcytosine dioxygenase 2 (Tet2) expression and DNA demethylation in blastocyst and melatonin supplementation rescued these processes. Innovation: This is the first report to show that embryos synthesize melatonin, and its synthetic enzyme Aanat was located in the mitochondria of embryos. An effect of melatonin is to maintain Tet2 expression and normal methylation status, and thereby promote embryonic development. Conclusion: Embryos can produce melatonin that reduces ROS production, preserves mitochondrial function, and maintains Tet2 expression and the normal DNA methylation.

The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma

BackgroundDifferent cell types possess different copies of mtDNA to support their specific requirements for cellular metabolism. Cell-specific mtDNA copy numbers are established through cell-specific mtDNA replication during cell differentiation. However, cancer cells are trapped in a “pseudo-differentiated” state as they fail to expand mtDNA copy number. Global DNA methylation can regulate this process, as induced DNA demethylation promotes differentiation of cancer cells and expansion of mtDNA copy number.ResultsTo determine the role that mtDNA methylation plays in regulating mtDNA replication during tumorigenesis, we have characterized the patterns of mtDNA methylation using glioblastoma and osteosarcoma tumor models that have different combinations of mtDNA genotypes and copy number against common nuclear genome backgrounds at different stages of tumor progression. To ensure the reliability of the findings, we have applied a robust experimental pipeline including three approaches, namely whole-mtDNA bisulfite-sequencing with mtDNA-genotype-specific analysis, pyrosequencing, and methylated immunoprecipitation against 5mC and 5hmC. We have determined genotype-specific methylation profiles, which were modulated through tumor progression. Moreover, a strong influence from the nuclear genome was also observed on mtDNA methylation patterns using the same mtDNA genotype under different nuclear genomes. Furthermore, the numbers of mtDNA copy in tumor-initiating cells affected mtDNA methylation levels in late-stage tumors.ConclusionsOur findings highlight the influences that the nuclear and mitochondrial genomes have in setting mtDNA methylation patterns to regulate mtDNA copy number in tumorigenesis. They have important implications for assessing global DNA methylation patterns in tumorigenesis and the availability of mtDNA template for mtDNA replication.

Molecular Processes Connecting DNA Methylation Patterns with DNA Methyltransferases and Histone Modifications in Mammalian Genomes.

DNA methylation is an essential part of the epigenome chromatin modification network, which also comprises several covalent histone protein post-translational modifications. All these modifications are highly interconnected, because the writers and erasers of one mark, DNA methyltransferases (DNMTs) and ten eleven translocation enzymes (TETs) in the case of DNA methylation, are directly or indirectly targeted and regulated by other marks. Here, we have collected information about the genomic distribution and variability of DNA methylation in human and mouse DNA in different genomic elements. After summarizing the impact of DNA methylation on genome evolution including CpG depletion, we describe the connection of DNA methylation with several important histone post-translational modifications, including methylation of H3K4, H3K9, H3K27, and H3K36, but also with nucleosome remodeling. Moreover, we present the mechanistic features of mammalian DNA methyltransferases and their associated factors that mediate the crosstalk between DNA methylation and chromatin modifications. Finally, we describe recent advances regarding the methylation of non-CpG sites, methylation of adenine residues in human cells and methylation of mitochondrial DNA. At several places, we highlight controversial findings or open questions demanding future experimental work.

Mitochondrial peptides modulate mitochondrial function during cellular senescence.

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

Methodology for Accurate Detection of Mitochondrial DNA Methylation.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Methodology for Accurate Detection of Mitochondrial DNA Methylation

MtDNMT1 and DNMT3b cooperate to methylate mitochondrial DNA and regulate mitochondrial transcription

Mitochondria are dynamic modulators of cell physiology, rewiring their metabolic and signaling pathways to allow for rapid and flexible adaptions to environmental changes. Mitochondrial DNA (mtDNA) is packaged into DNA‐protein complexes called nucleoids, and the DNA is epigenetically modified (1,2). We, and others, have demonstrated that mtDNA methylation represents a powerful mechanism for regulating transcription of mtDNA‐encoded oxidative phosphorylation (OXPHOS) genes under conditions of cellular stress (1,3). We identified a mitochondrial (mt) isoform of DNA methyltransferase 1 (mtDNMT1) that translocates to the mt matrix, and binds to mtDNA in critical control regions (1). Our new data demonstrates that mtDNMT1 interacts with several proteins essential for proper functioning of mitochondria, including core components of the nucleoid. These findings place mtDNMT1 at the heart of mitochondrial nucleoid organization, and suggest that mtDNMT1 plays a critical role in regulating nucleoid structure and mitochondrial output, either as a consequence of, or independently from, its methylation activity. We now have evidence that mtDNMT1 is not working alone: DNMT3b works in concert with mtDNMT1 to methylate mtDNA and regulate mt transcription. CRISPR/Cas9‐mediated inactivation of mtDNMT1 and/or DNMT3b activity resulted in significant decrease in mitochondrial methylation across the heavy and light strand promoters of mtDNA, with a concomitant reduction in transcription of several mtDNA‐encoded OXPHOS genes. Cells lacking mtDNMT1 and/or DNMT3b exhibited a striking morphological change, undertaking an elongated, fibroblast‐like appearance with increased migration, reminiscent of cells undergoing epithelial‐to‐mesenchymal transition (EMT), a hallmark of cancer metastasis. These observations implicate mtDNA methylation in the phenotypic plasticity that occurs in response to changing cellular growth conditions, driving the initiation and progression of numerous pathologies. We propose that the mitochondrial DNA methylation machinery may constitute a novel target for therapeutic intervention.Support or Funding InformationMCB 1122018 (NSF) to Taylor, Start‐up funds (VCU Dept of Microbiology and Immunology) to Shock, IRG‐14‐192‐40 (ACS) to ShockThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5’-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

Nuclear and Mitochondrial DNA Methylation Patterns Induced by Valproic Acid in Human Hepatocytes.

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs in the world. Despite its pharmacological importance, it may cause liver toxicity and steatosis through mitochondrial dysfunction. The aim of this study is to further investigate VPA-induced mechanisms of steatosis by analyzing changes in patterns of methylation in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, primary human hepatocytes (PHHs) were exposed to an incubation concentration of VPA that was shown to cause steatosis without inducing overt cytotoxicity. VPA was administered daily for 5 days, and this was followed by a 3 day washout (WO). Methylated DNA regions (DMRs) were identified by using the methylated DNA immunoprecipitation–sequencing (MeDIP-seq) method. The nDNA DMRs after VPA treatment could indeed be classified into oxidative stress- and steatosis-related pathways. In particular, networks of the steatosis-related gene EP300 provided novel insight into the mechanisms of toxicity induced by VPA treatment. Furthermore, we suggest that VPA induces a crosstalk between nDNA hypermethylation and mtDNA hypomethylation that plays a role in oxidative stress and steatosis development. Although most VPA-induced methylation patterns appeared reversible upon terminating VPA treatment, 31 nDNA DMRs (including 5 zinc finger protein genes) remained persistent after the WO period. Overall, we have shown that MeDIP-seq analysis is highly informative in disclosing novel mechanisms of VPA-induced toxicity in PHHs. Our results thus provide a prototype for the novel generation of interesting methylation biomarkers for repeated dose liver toxicity in vitro.

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns.