Mitochondrial DNA Methylation Research Articles

RACGAP1 modulates ECT2-Dependent mitochondrial quality control to drive breast cancer metastasis

Most cancer deaths are due to the colonization of tumor cells in distant organs. More evidence indicates that overexpression of RACGAP1 plays a critical role in cancer metastasis. However, the underlying mechanism still remains poorly understood. Here we found that RACGAP1 promoted breast cancer metastasis through regulating mitochondrial quality control. Overexpression of RACGAP1 in breast cancer cells led to the fragmentation of mitochondria, increased mitophagy intensity, mitochondrial turnover, and aerobic glycolysis ATP production. We showed that RACGAP1 promoted mitochondrial fission through recruiting ECT2 during anaphase and subsequently had activated ERK-DRP1 pathway. We further demonstrated the phosphorylation of RACGAP1 is essential for its ability of binding with ECT2 and its downstream effects. RACGAP1 overexpression also increased the expression of PGC-1a, a key mitochondrial biogenesis regulator, presumably by the increased mitophagy intensity induced by RACGAP1. PGC-1a increased the enrichment of DNMT1 in mitochondria, mitochondrial DNMT1 augmented mitochondrial DNA methylation and upregulated mitochondrial genome transcription. Our data indicated that RACGAP1 simultaneously facilitated mitophagy and mitochondrial biogenesis through regulating DRP1 phosphorylation and PGC-1a expression, eventually improved mitochondrial quality control in breast cancer cells. Our study provided a new angle in understanding the RACGAP1-overexpression related malignancy in breast cancer patients.

Mitochondrial DNA Methylation Is Higher in Acute Coronary Syndrome Than in Stable Coronary Artery Disease.

Decreased mitochondrial DNA copy number (mtDNA-CN) has been associated with coronary artery disease (CAD). We aimed to clarify the difference between stable CAD (SCAD) and acute coronary syndrome (ACS) regarding mtDNA-CN and the DNA methylation ratio in regions influencing the regulation of mitochondrial biogenesis. Using quantitative real-time polymerase chain reaction, mtDNA-CN was measured in peripheral blood leukocytes sampled from 50 patients with SCAD and 50 with ACS. We then conducted bisulfite modification of DNA followed by methylation-specific polymerase chain reaction to quantify mtDNA methylation in the mitochondrial D-loop region (mtDLR) and nuclear DNA methylation in the promoter region of nuclear peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) gene. Compared to patients with SCAD, those with ACS had significantly lower relative mtDNA-CN (0.89±0.24 vs. 1.00±0.28, p=0.013) and higher DNA methylation ratio of the mtDLR (1.11±0.24 vs. 1.00±0.25, p=0.027) Conclusion: Our findings suggest that increased DNA methylation in the mtDLR, which translates into reduced mtDNA content, may affect the clinical phenotype of CAD.

Mitochondrial gene COX2 methylation and downregulation is a biomarker of aging in heart mesenchymal stem cells.

The mitochondria have been proven to be involved in processes of aging; however, the mechansims through which mitoepigenetics affect the cytological behaviors of cardiomyocytes during the aging process are not yet fully understood. In the present study, two senescence models were constructed, replicative senescence (RS) and stress-induced premature senescence (SIPS), using human heart mesenchymal stem cells (HMSCs). First, the differences in age-related gene expression levels and telomere length were compared between the HMSCs in the RS and SIPS models by PCR. Subsequently, protein expression and the mitochondrial DNA (mtDNA) methylation status of cytochrome c oxidase subunit II (COX2) was measured by western blot analysis and bisulfite genomic sequencing (BSP). Finally, the value of the DNA methyltransferase (Dnmt) inhibitor, 5-aza-2′-deoxycytidine (AdC), in delaying the senescence of HMSCs was evaluated. It was found that the p16, p27 and p53 mRNA expression levels increased in the senescent cells, whereas p21 mRNA expression did not. It was also found that telomere shortening only occurred in the RS model, but not in the SIPS model. Along with the senescence of HMSCs, COX2 gene methylation increased and its protein expression level significantly decreased. It was demonstrated that AdC inhibited COX2 methylation and downregulated COX2 expression. The addition of exogenous COX2 or the administration of AdC promoted cell proliferation and delayed cell aging. On the whole, the present study demonstrates that COX2 methylation and downregulation are biomarkers of HMSC senescence. Thus, COX2 may have potential for use as a therapeutic target of cardiovascular diseases and this warrants further investigation.

Residential air pollution measures are associated with perturbations in a placental gene coexpression network

Background: A growing body of evidence has linked air pollution exposure to human health effects. Early life may pose a particularly vulnerable period, where impacts on the developing organ systems can reverberate across the lifespan. A number of studies suggest that the placenta plays a critical role in transmitting air pollution exposure to the developing fetus. These include observed associations between various air pollutants and candidate placental features, such as mitochondrial DNA content, DNA methylation and telomere length. However, gaps remain in delineating the role of the placenta as a conveyor of air pollution-related health effects, including a comprehensive profiling of placental processes impacted by intrauterine air pollution exposure. In this study, we examined alterations in a placental transcriptome-wide network in relation to ambient fine particulate matter (PM2.5) and black carbon (BC) exposure. Methods: We evaluated placental RNAseq data (n=200) generated from study participants enrolled in the Rhode Island Child Health Study. Residential PM2.5 and BC exposure levels were estimated using a hybrid model incorporating land-use regression and satellite remote sensing. Differential gene expression analysis was performed using the DESeq2 R package. A transcriptome-wide gene coexpression network was generated using the WGCNA R package. Results: Testing each gene independently, we observed no significant differences in expression levels between the highest and lowest tertile of exposure for PM2.5 or BC. Mapping PM2.5 levels to the generated gene coexpression network, we observed significant correlations (p<0.05) to gene-sets enriched for DNA conformation change, cell adhesion and histone modification. BC levels were significantly correlated to gene-sets enriched for cellular respiration, gonadotropin secretion, protein targeting and organogenesis. Conclusion: The current study represents the most comprehensive assessment of genome-wide changes in placental gene expression and residential air pollution measures to date, revealing alterations in placental processes associated with PM2.5 and BC levels.

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism.

Equineembryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. Experiments comparing in vitro and in vivo produced embryos. Oocytes (n=641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10mmol/L glucose respectively; n=87); 5-17 (5 and 17.5mmol/L; n=66); and 10-17 (10 and 17.5mmol/L; n=117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.

Mitochondrial-induced Epigenetic Modifications: From Biology to Clinical Translation.

Mitochondria are maternally inherited semi-autonomous organelles that play a central role in redox balance, energy metabolism, control of integrated stress responses, and cellular homeostasis. The molecular communication between mitochondria and the nucleus is intricate and bidirectional in nature. Though mitochondrial genome encodes for several key proteins involved in oxidative phosphorylation, several regulatory factors encoded by nuclear DNA are prominent contributors to mitochondrial biogenesis and function. The loss of synergy between this reciprocal control of anterograde (nuclear to mitochondrial) and retrograde (mitochondrial to nuclear) signaling, triggers epigenomic imbalance and affects mitochondrial function and global gene expressions. Recent expansions of our knowledge on mitochondrial epigenomics have offered novel perspectives for the study of several non-communicable diseases including cancer. As mitochondria are considered beacons for pharmacological interventions, new frontiers in targeted delivery approaches could provide opportunities for effective disease management and cure through reversible epigenetic reprogramming. This review focuses on recent progress in the area of mitochondrial-nuclear cross-talk and epigenetic regulation of mitochondrial DNA methylation, mitochondrial micro RNAs, and post-translational modification of mitochondrial nucleoid-associated proteins that hold major opportunities for targeted drug delivery and clinical translation.

Mitoepigenetics: Methylation of mitochondrial DNA is strand-biased in bovine oocytes and embryos.

Global mitochondrial DNA (mtDNA) methylation has been recently described in bovine and showed particular signatures in both gametes and embryos. Here, we investigated the distribution of mtDNA methylation through strand-specific mapping of methylation sites to gain perspective on how epigenetic mechanisms can be involved in mitochondrial function. We demonstrate that in both oocytes and embryos, the frequency of methylation is biased towards the light strand (L-strand), particularly in the gene bodies and in the region containing the L-strand promoter (LSP). Methylation is not restricted to CpG nucleotides and is not symmetrical on both strands. This configuration reinforces the hypothesis of a specific epigenetic regulation of mtDNA, which is an important observation for the understanding of how mitochondrial function is regulated.

A New Approach to Identify the Methylation Sites in the Control Region of Mitochondrial DNA.

Mitochondrial DNA (mtDNA) methylation has the potential to be used as a biomarker of human development or disease. However, mtDNA methylation procedures are costly and time-consuming. Therefore, we developed a new approach based on an RT-PCR assay for the base site identification of methylated cytosine in the control region of mtDNA through a simple, fast, specific, and low-cost strategy. Total DNA was purified, and methylation was determined by RT-PCR bisulfite sequencing. This procedure included the DNA purification, bisulfite treatment and RT-PCR amplification of the control region divided into three subregions with specific primers. Sequences obtained with and without the bisulfite treatment were compared to identify the methylated cytosine dinucleotides. Furthermore, the efficiency of C to U conversion of cytosines was assessed by including a negative control. Interestingly, mtDNA methylation was observed mainly within non-Cphosphate- G (non-CpG) dinucleotides and mostly in the regions containing regulatory elements, such as OH or CSBI, CSBII, and CSBIII. This new approach will promote the generation of new information regarding mtDNA methylation patterns in samples from patients with different pathologies or that are exposed to a toxic environment in diverse human populations.

Mitochondrial DNA methylation misleads global DNA methylation detected by antibody-based methods

Cytosine methylation is the leading epigenetic modification on DNA playing a role in gene regulation. Methylation can occur in cytosines of any nucleic acids in cytosol (as mitochondrial DNA, mtDNA) and in nuclear DNA (ncDNA). mtDNA exists as multiple copies within numerous mitochondria. This suggests that the number of mitochondria and mtDNA copy number can indicate the presence of a significant amount of DNA methylation within total DNA methylation detected. However, immunofluorescence method does not have a step to discriminate the staining between ncDNA and mtDNA. Antibodies used in immunological methods are methylation-specific but not selective for DNA type and they can bind to methylated cytosines in any DNA within the specimen. Current study aimed to understand whether mtDNA methylation interferes with the detection of nuclear DNA methylation by immunofluorescence and affinity enrichment (ELISA) in different mammalian cells. Experiments were performed to distinguish methylation between mtDNA and ncDNA. Immunofluorescence showed that there was no significant difference in the detected amount of methylation between mitochondrial and nuclear DNA. But ELISA revealed that up to 25% of cellular methylation was derived from mitochondria. This suggests that significant contamination of mtDNA methylation with ncDNA methylation can result in overestimation of the quantitative level of nuclear methylation.

Polymorphisms of genes required for methionine synthesis and DNA methylation influence mitochondrial DNA methylation.

Aim: Impaired methylation of the mitochondrial DNA and particularly in the regulatory displacement loop (D-loop) region, is increasingly observed in patients with neurodegenerative disorders. The present study aims to investigate if common polymorphisms of genes required for one-carbon metabolism (MTHFR, MTRR, MTR and RFC-1) and DNA methylation reactions (DNMT1, DNMT3A and DNMT3B) influence D-loop methylation levels. Materials & methods: D-loop methylation data were available from 133 late-onset Alzheimer's disease patients and 130 matched controls. Genotyping was performed with PCR-RFLP or high resolution melting techniques. Results: Both MTRR 66A > G and DNMT3A -448A > G polymorphisms were significantly associated with D-loop methylation levels. Conclusion: This exploratory study suggests that MTRR and DNMT3A polymorphisms influence mitochondrial DNA methylation; further research is required to better address this issue.

Evolutionary perspectives on bee mtDNA from mito-OMICS analyses of a solitary species

The analysis of mitochondrial DNA polymorphism has been applied in multiple organisms to obtain information about species biology, ecology, population dynamics, and evolution. In this manuscript, the complete sequencing and characterization of the mitochondrial genome (mtGenome) of Tetrapedia diversipes are reported and discussed from comparative and evolutionary perspectives among all mtGenomes available for bees so far. The T. diversipes mtGenome is 15,358 bp long and exhibits the typical set of genes and an A+T-rich region of 588 bp. The overall base composition is biased towards A/T (84.3%), with 42.6% A, 41.7% T, 9.8% C, and 5.9% G nucleotides. The obtained data also comprise the mitochondrial DNA methylation and single-nucleotide polymorphic sites of one T. diversipes population. Transcription follows the “tRNA punctuation” model, with at least three primary polycistronic transcripts that are posteriorly processed. Additionally, higher expression rates of the 16S gene suggest the existence of an exclusive transcription site in this region, and the differential expression of the 12S gene between larvae and adults reveals different isoforms for this gene. The sequence order of protein-coding and rRNA genes is conserved across different bee lineages, and differences are restricted to tRNA gene positions. The present results characterize numerous understudied aspects of bee mtGenomes, and a major evolutionary review of this molecule within the group is provided. Therefore, this work is a valuable resource for studying mitochondrial molecular biology and evolution in bees.

Platelet mitochondrial DNA methylation predicts future cardiovascular outcome in adults with overweight and obesity

BackgroundThe association between obesity and cardiovascular disease (CVD) is proven, but why some adults with obesity develop CVD while others remain disease-free is poorly understood. Here, we investigated whether mitochondrial DNA (mtDNA) methylation in platelets is altered prior to CVD development in a population of adults with overweight and obesity.MethodsWe devised a nested case-control study of 200 adults with overweight or obesity who were CVD-free at baseline, of whom 84 developed CVD within 5 years, while 116 remained CVD-free. Platelet mtDNA was isolated from plasma samples at baseline, and mtDNA methylation was quantified in mitochondrially encoded cytochrome-C-oxidase I (MT-CO1; nt6797 and nt6807), II (MT-CO2; nt8113 and nt8117), and III (MT-CO3; nt9444 and nt9449); tRNA leucine 1 (MT-TL1; nt3247 and nt3254); D-loop (nt16383); tRNA phenylalanine (MT-TF; nt624); and light-strand-origin-of-replication (MT-OLR; nt5737, nt5740, and nt5743) by bisulfite-pyrosequencing. Logistic regression was used to estimate the contribution of mtDNA methylation to future CVD risk. ROC curve analysis was used to identify the optimal mtDNA methylation threshold for future CVD risk prediction. A model was generated incorporating methylation at three loci (score 0, 1, or 2 according to 0, 1, or 2–3 hypermethylated loci, respectively), adjusted for potential confounders, such as diastolic and systolic blood pressure, fasting blood glucose, and cholesterol ratio. mtDNA methylation at MT-CO1 nt6807 (OR = 1.08, 95% CI 1.02–1.16; P = 0.014), MT-CO3 nt9444 (OR = 1.22, 95% CI 1.02–1.46, P = 0.042), and MT-TL1 nt3254 (OR = 1.30, 95% CI 1.05–1.61, P = 0.008) was higher at baseline in those who developed CVD by follow-up, compared with those who remained CVD-free. Combined use of the three loci significantly enhanced risk prediction, with hazard ratios of 1.38 (95% CI 0.68–2.78) and 2.68 (95% CI 1.41–5.08) for individuals with score 1 or 2, respectively (P = 0.003). Methylation at these sites was independent of conventional CVD risk factors, including inflammation markers, fasting blood glucose concentration, and blood pressure.ConclusionsMethylations of MT-CO1, MT-CO3, and MT-TL1 are, together, strong predictors of future CVD incidence. Since methylation of these mtDNA domains was independent of conventional CVD risk factors, these markers may represent a novel intrinsic predictor of CVD risk in adults with overweight and obesity.

Cytosine methylation of rice mitochondrial DNA from grain and leaf tissues.

The rice leaf mitochondrial DNA is more methylated compared to the rice grain mitochondrial DNA. The old rice leaf mitochondrial DNA has also a higher methylation level than the young rice leaf mitochondrial DNA. The presence of DNA methylation in rice organelles has not been well characterized. We have previously shown that cytosine methylation of chloroplast DNA is different between leaf and grain, and varies between young and old leaves in rice. However, the variation in cytosine methylation of mitochondrial DNA is still poorly characterized. In this study, we have investigated cytosine methylation of mitochondrial DNA in the rice grain and leaf. Based on CpG, CHG, and CHH methylation analyses, the leaf mitochondrial DNA was found to be more methylated compared to the grain mitochondrial DNA. The methylation of the leaf mitochondrial DNA was also higher in old compared to young leaves. Differences in methylation were observed at different cytosine positions of the mitochondrial DNA between grain and leaf, although there were also positions with a similarlevel of high methylation in all the tissues examined. The differentially methylated cytosine positions in rice mitochondrial DNA were observed mostly in the intergenic region and in some mitochondrial-specific genes involved in ATP production, transcription, and translation. The functional importance of cytosine methylation in the life cycle of rice mitochondria is still to be determined.

Mitochondrial DNA methylation is associated with Mediterranean diet adherence in a population of older adults with overweight and obesity.

AbstractIntroductionAdherence to the Mediterranean dietary pattern (MeDiet) and adiposity, respectively, decreases and increases the risk of multiple common age-related diseases through several mechanisms including inflammation, reactive oxygen species (ROS) production in the mitochondria, and DNA methylation. For example, adverse changes in platelets from obese and overweight adults include hyperaggregability and increased ROS. Since platelets are anuclear, their prothrombotic function is fully orchestrated by the mitochondria and the only DNA present is the mitochondrial DNA (mtDNA). In this study, we tested the hypothesis that MeDiet influences patterns of mtDNA methylation in platelets from older adults with greater adiposity.Material and methodsWe selected 134 participants with overweight or obesity (mean BMI = 35.5 ± 5.1 and age = 62 ± 10 years) from the “Susceptibility to particle health effects, miRNA and exosomes”(SPEHRE) Study. Dietary intake was assessed using a food frequency questionnaire and MeDiet adherence was calculated using the MeDiet Score described by Martínez-González et al.(2012). MtDNA was extracted from platelets, linearized, bisulfite converted and DNA methylation was quantified by pyrosequencing at 13 CpG in seven genes that encode for tRNAs (MT-TF and MT-TL1), regulatory regions (D-Loop and MT-OLR), and subunits of the electron-transport-chain (MT-CO1, MT-CO2, and MT-CO3).ResultsIn these participants, MeDiet score ranged from 3 to 12 (mean = 6.5), with higher scores reflecting greater MeDiet adherence. Regression analysis showed that higher MeDiet score was associated with lower D-loop (β = -0.031, P = 0.019) and higher MT-CO2 CpG1 (β = 0.040, P = 0.023) methylation. No associations were found between MeDiet Score and methylation level at MT-CO1(2 CpGs), MT-CO2(CpG2), MT-CO3(2 CpGs), MT-TL1(2 CpGs), MT-TF(CpG1), MT-OLR(3 CpGs).In addition, there was no association between mtDNA methylation and BMI.DiscussionThe D-loop is critical for mitochondrial function since it initiates mtDNA replication and transcription. Increased D-loop methylation has been associated with reduced mitochondrial functionality, and insulin resistance. Our results suggest that higher adherence to MeDiet lowers D-loop methylation which may protect against obesity-related comorbidities (e.g. insulin resistance).Higher MeDiet scores are associated with MT-CO2 CpG1 hypermethylation. MT-CO2 encodes for a subunit of the Cytochrome-C-oxidase, a highly regulated enzyme involved in the oxidative metabolism. MT-CO2 demethylation, induced by Valproic-Acid administration, has been reported to be associated with increased ROS production. Our results suggest a possible role of MeDiet in mitochondrial ROS regulation via methylation of MT-CO2.For the first time, we observed associations between MeDiet adherence and mtDNA methylation. Validation of these findings in independent cohorts is required.

Possible existence of a system similar to bacterial restriction–modification in plants

The bacterial restriction–modification (R-M) system has evolved as a defense mechanism against infectious phages and other types of integration of foreign DNA fragments into the host chromosome through recombination. In this study, we identified two endonucleases (wheat endonuclease 1-WEN1 and wheat endonuclease 2-WEN2) and the adenine-DNA methyltransferase WAD (involved in mitochondrial DNA methylation) in the vesicular fraction of coleoptiles of wheat Triticum aestivum. WEN1 and WEN2 have multidirectional sensitivity to DNA methylation and to the presence of S-adenosyl-L-methionine and Ca2+ ions, which suggests their participation in the protection of mitochondrial DNA, similar to bacterial DNA. WEN2 has a GAT recognition site, which is part of the methylation site of adenine DNA methyltransferase (WAD-TGATCA) and hydrolyzes the CG (GC) bond upstream of the recognition site only if it is not methylated. Based on the interrelations between the two enzymes, we propose that an R-M system similar to that of the type IV observed in bacteria may exist in plants.

Analysis of microRNA expression profiling during paraquat-induced injury of murine lung alveolar epithelial cells

Paraquat (PQ) as a non-selective heterocyclic herbicide, has been applied worldwide for over a few decades. But PQ is very harmful to humans and rodents. The lung is the main target organ of PQ poisoning. It is an important event that lung epithelial cells are injured during PQ-induced acute lung injury and pulmonary fibrosis. As a regulator of mRNA expression, microRNA (miRNA) may play an important role in the progress. Our study was to investigate the mechanisms of PQ-induced injury of pulmonary epithelial cells through analyzing the profiling of miRNAs and their target genes. As a result, 11 differentially expressed miRNAs were screened, including 1 upregulated miRNA and 10 downregulated miRNAs in PQ-treated murine lung alveolar epithelial cells (MLE-12 cells). The bioinformatic analyses suggested that the target genes of these miRNAs were involved in mitochondrial apoptosis pathway and DNA methylation, and participated in the regulation of PI3K-Akt, mTOR, RAS, TNF, MAPK and other signal pathways which related to oxidative stress and apoptosis. This indicated that miRNAs were an important regulator of oxidative stress and apoptosis during PQ-induced injury of murine lung alveolar epithelial cells. The findings would deepen our understanding of the mechanisms of PQ-induced pulmonary injury and might provide new treatment targets for this disease.

Hypermethylation of mitochondrial DNA in vascular smooth muscle cells impairs cell contractility

Vascular smooth muscle cell (SMC) from arterial stenotic-occlusive diseases is featured with deficiency in mitochondrial respiration and loss of cell contractility. However, the regulatory mechanism of mitochondrial genes and mitochondrial energy metabolism in SMC remains elusive. Here, we described that DNA methyltransferase 1 (DNMT1) translocated to the mitochondria and catalyzed D-loop methylation of mitochondrial DNA in vascular SMCs in response to platelet-derived growth factor-BB (PDGF-BB). Mitochondrial-specific expression of DNMT1 repressed mitochondrial gene expression, caused functional damage, and reduced SMC contractility. Hypermethylation of mitochondrial D-loop regions were detected in the intima-media layer of mouse carotid arteries subjected to either cessation of blood flow or mechanical endothelial injury, and also in vessel specimens from patients with carotid occlusive diseases. Likewise, the ligated mouse arteries exhibited an enhanced mitochondrial binding of DNMT1, repressed mitochondrial gene expression, defects in mitochondrial respiration, and impaired contractility. The impaired contractility of a ligated vessel could be restored by ex vivo transplantation of DNMT1-deleted mitochondria. In summary, we discovered the function of DNMT1-mediated mitochondrial D-loop methylation in the regulation of mitochondrial gene transcription. Methylation of mitochondrial D-loop in vascular SMCs contributes to impaired mitochondrial function and loss of contractile phenotype in vascular occlusive disease.

Epigenetic Modifications in Peripheral Blood as Potential Noninvasive Biomarker of Diabetic Retinopathy.

PurposeProgression of diabetic retinopathy is related to the duration and severity of hyperglycemia, and after 25 years of diabetes, 90% of patients show some signs of retinopathy. Despite initiation of many retinal molecular/biochemical abnormalities, including mitochondrial damage and epigenetic modifications, the disease remains asympotomatic in the initial stages. Our goal is to examine the utility of DNA methylation as a possible biomarker of diabetic retinopathy.MethodsGenomic DNA (gDNA) was isolated from the buffy coat, isolated from blood of diabetic patients with proliferative (PDR) or no retinopathy (No-DR), and nondiabetic subjects (CONT). Methylation of mitochondrial DNA (mtDNA), especially its D-Loop (the site of mtDNA transcription/replication), was quantified by methylated DNA immunoprecipitation and methyl-specific PCR techniques. Results were confirmed in purified mtDNA. The specific D-Loop region with the highest DNA methylation was identified using five overlapping primers, and DNMT1 binding was quantified by chromatin immunoprecipitation. Promoter DNA methylation of DNA mismatch repair (MLH1) and superoxide scavenging (SOD2) enzymes were also quantified.ResultsCompared to CONT, D-Loop methylation was higher in PDR and No-DR groups, and the D-Loop region responsible for encoding the majority of the mtDNA-encoded genes had significantly higher methylation in the PDR group versus No-DR. Similarly, compared to No-DR, the PDR group also had hypermethylated MHL1 and SOD2 promoters.ConclusionsBlood from PDR patients have higher DNA methylation, than seen in diabetic patients without retinopathy. Thus, DNA methylation can be used as a possible biomarker of diabetic retinopathy.Translational RelevanceDNA methylation status in the blood of diabetic patients could serve as a potential noninvasive biomarker of retinopathy, and also an important readout parameter for testing longitudinal outcome of novel therapeutics for this blinding disease.

Mitochondrial DNA methylation and copy number predict body composition in a young female population

BackgroundSince both genomic and environmental factors are involved in obesity etiology, several studies about the influence of adiposity on both nuclear DNA and mitochondrial DNA methylation patterns have been carried out. Nevertheless, few evidences exploring the usage of buccal swab samples to study mitochondrial DNA epigenetics can be found in literature.MethodsIn this study, mitochondrial DNA from buccal swabs collected from a young Caucasian population (n = 69) have been used to examine potential correlation between mitochondrial DNA copy number and methylation with body composition (BMI, WHtR and bioimpedance measurements).ResultsA negative correlation between mitochondrial DNA copy number and BMI was measured in females (p = 0.028), but not in males. The mean percentage of D-loop methylation is significantly higher in overweight than in lean female subjects (p = 0.003), and a specific CpG located in the D-loop shows per se an association with impaired body composition (p = 0.004). Body composition impairment is predicted by a combined variable including mtDNA copy number and the D-loop methylation (AUC = 0.785; p = 0.009).ConclusionsThis study corroborates the hypothesis that mitochondrial DNA carries relevant information about body composition. However, wider investigations able to validate the usage of mtDNA methylation from buccal swabs as a biomarker are warranted.

Altered mitochondrial DNA methylation and mitochondrial DNA copy number in an APP/PS1 transgenic mouse model of Alzheimer disease

Alzheimer’s disease (AD) is a chronic neurodegenerative disease and mitochondrial impairment is a key feature of AD. The mitochondrial DNA (mtDNA) epigenetic mechanism is a relatively new field compared to nuclear DNA. The relationship between mtDNA epigenetic mechanism and AD hasn’t been established. So we analyzed the mtDNA methylation in D-loop region and 12 S rRNA gene in the hippocampi in amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice by bisulfite pyrosequencing. Mitochondrial DNA copy number and gene expression were studied by quantitative real-time PCR (qRT-PCR). We observed a decrease in the displacement loop (D-loop) methylation and an increase in 12 S rRNA gene methylation, while both the mtDNA copy number and the mitochondrial gene expression were reduced in APP/PS1 transgenic mice. In summary, the present finding suggest that mtDNA methylation may play a role in AD pathology, which warrants larger future investigations.