scholarly journals High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo

2021 ◽  
Vol 4 (11) ◽  
pp. e202101034
Author(s):  
Nina A Bonekamp ◽  
Min Jiang ◽  
Elisa Motori ◽  
Rodolfo Garcia Villegas ◽  
Camilla Koolmeister ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Mitochondrial Rna ◽  
Mtdna Copy Number ◽  
Specific Expression ◽  
Mitochondrial Transcription Factor ◽  
Mouse Tissues ◽  
Animal Metabolism ◽  
Very High

Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.

scholarly journals Targeting reduced mitochondrial DNA quantity as a therapeutic approach in pediatric high-grade gliomas

2019 ◽  
Vol 22 (1) ◽  
pp. 139-151 ◽  
Author(s):  
Han Shen ◽  
Man Yu ◽  
Maria Tsoli ◽  
Cecilia Chang ◽  
Swapna Joshi ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Glucose Metabolism ◽  
Mitochondrial Function ◽  
Copy Number ◽  
Pyruvate Dehydrogenase Kinase ◽  
High Grade ◽  
Mtdna Copy Number ◽  
Mitochondrial Oxidation ◽  
High Grade Gliomas

Abstract Background Despite increased understanding of the genetic events underlying pediatric high-grade gliomas (pHGGs), therapeutic progress is static, with poor understanding of nongenomic drivers. We therefore investigated the role of alterations in mitochondrial function and developed an effective combination therapy against pHGGs. Methods Mitochondrial DNA (mtDNA) copy number was measured in a cohort of 60 pHGGs. The implication of mtDNA alteration in pHGG tumorigenesis was studied and followed by an efficacy investigation using patient-derived cultures and orthotopic xenografts. Results Average mtDNA content was significantly lower in tumors versus normal brains. Decreasing mtDNA copy number in normal human astrocytes led to a markedly increased tumorigenicity in vivo. Depletion of mtDNA in pHGG cells promoted cell migration and invasion and therapeutic resistance. Shifting glucose metabolism from glycolysis to mitochondrial oxidation with the adenosine monophosphate–activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) or the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA) significantly inhibited pHGG viability. Using DCA to shift glucose metabolism to mitochondrial oxidation and then metformin to simultaneously target mitochondrial function disrupted energy homeostasis of tumor cells, increasing DNA damage and apoptosis. The triple combination with radiation therapy, DCA and metformin led to a more potent therapeutic effect in vitro and in vivo. Conclusions Our results suggest metabolic alterations as an onco-requisite factor of pHGG tumorigenesis. Targeting reduced mtDNA quantity represents a promising therapeutic strategy for pHGG.

199 EVIDENCE FOR A NOVEL PERTURBATION IN CLONED FETUSES: MITOCHONDRIAL DNA DEPLETION

2007 ◽  
Vol 19 (1) ◽  
pp. 216
Author(s):  
S. Hiendleder ◽  
D. Bebbere ◽  
S. E. Ulbrich ◽  
V. Zakhartchenko ◽  
M. Weppert ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Nuclear Gene ◽  
Complement C3 ◽  
Mtdna Copy Number ◽  
Vitro Fertilization ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Dna Depletion

The reported mtDNA turnover and plasticity of mtDNA copy number in mammalian zygotes and early embryos (McConnel and Petrie 2004 Reprod. Biomed. Online 9, 418–424) have revealed a potential for adverse effects of in vitro embryo techniques on mtDNA and mitochondrial function. We explored the effects of in vitro fertilization (IVF) and somatic cell nuclear transfer cloning (NT) on relative mtDNA amount and phenotype in viable bovine fetuses recovered 80 days after the initiation of embryonic development (Hiendleder et al. 2004 Biol. Reprod. 71, 217–223). We sampled brain, liver, and skeletal muscle to represent all 3 embryonic germ layers, and compared IVF-fetuses (n = 24), NT-fetuses (n = 23), and fetuses generated by in vivo insemination (controls, n = 24). This experimental approach allowed us to distinguish abnormalities specific to cloning from more general consequences of in vitro embryo manipulation. We analyzed relative mtDNA amounts by real-time quantitative PCR (qPCR) and amplified a segment of the mtDNA control region that was normalized against the nuclear gene complement C3. ANOVA (SPSS 13.0) of qPCR data and phenotypic parameters revealed significant effects of fetus group on mtDNA amount in liver (P < 0.05) and muscle (P < 0.01), and on fetus (P < 0.001), heart (P < 0.001), and liver (P < 0.001) weights. The mtDNA amount in all tissues from IVF-fetuses was normal, but mtDNA levels in liver (-23%; P < 0.05) and muscle (-24%; P < 0.01) of NT-fetuses were significantly lower than in controls. Fetuses derived from IVF- or NT-embryos were similar in weight and displayed fetal overgrowth (+19% and +22%; P < 0.001), but only the NT-fetuses were affected by disproportionate hepatomegaly and cardiomegaly with 31% and 49% increases (ANCOVA; P < 0.001) in their respective organ weights. This further partitioned NT-fetuses from IVF-fetuses and identified symptoms that are also encountered in mitochondrial DNA depletion syndromes (MDDS): a phenotypically heterogeneous group of human disorders characterized by loss of mtDNA from various tissues during development and associated respiratory chain dysfunction. The MDDS phenotypes have mainly been classified into a hepatocerebral (MIM 251880) or myopathic (MIM 609560) form, and neonates and infants display a spectrum of abnormalities, including hepatomegaly and cardiomegaly, that are similar or identical to phenotypic abnormalities commonly encountered in cloned mammals. Reduced mtDNA amounts in NT-fetuses could stem from perturbation of mtDNA during the reported turnover period, or be a secondary effect of epigenetic change in nuclear-encoded genes involved in mtDNA replication and stability. Mitochondrial transcription factor A (TFAM) is regulated by CpG methylation in vitro, but our real-time RT-PCR quantification of TFAM transcript in liver and muscle of a subset of NT- and control fetuses failed to detect significant differences (P > 0.10). In conclusion, our observed reduction of mtDNA amount in cloned fetuses provides the molecular basis for a mitochondrial perspective on pathological phenotypes of cloned mammals, and may explain similarities to mitochondrial disease in human.

scholarly journals mtDNA in the Pathogenesis of Cardiovascular Diseases

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Lili Wang ◽  
Qianhui Zhang ◽  
Kexin Yuan ◽  
Jing Yuan
Keyword(s):  
Heart Failure ◽  
Cardiovascular Disease ◽  
Mitochondrial Dna ◽  
Copy Number ◽  
Heart Diseases ◽  
Mtdna Copy Number ◽  
Cardiac Inflammation ◽  
Ischemic Heart Diseases ◽  
Metabolic Functions ◽  
Underlying Mechanisms

The incidence rate of cardiovascular disease (CVD) has been increasing year by year and has become the main cause for the increase of mortality. Mitochondrial DNA (mtDNA) plays a crucial role in the pathogenesis of CVD, especially in heart failure and ischemic heart diseases. With the deepening of research, more and more evidence showed that mtDNA is related to the occurrence and development of CVD. Current studies mainly focus on how mtDNA copy number, an indirect biomarker of mitochondrial function, contributes to CVD and its underlying mechanisms including mtDNA autophagy, the effect of mtDNA on cardiac inflammation, and related metabolic functions. However, no relevant studies have been conducted yet. In this paper, we combed the current research status of the mechanism related to the influence of mtDNA on the occurrence, development, and prognosis of CVD, so as to find whether these mechanisms have something in common, or is there a correlation between each mechanism for the development of CVD?

scholarly journals A Case-control Study on the Association of Mitochondrial Transcription Factor a Gene +35G/C Polymorphism and Mitochondrial DNA Copy Number with the Risk of Endometriosis in Indian Women

2021 ◽  
pp. 60-67
Author(s):  
Himabindu Beeram ◽  
Tumu Venkat Reddy ◽  
Suresh Govatati ◽  
Swapna Siddamalla ◽  
Mamata Deenadayal ◽  
...  
Keyword(s):  
Copy Number ◽  
Case Control Study ◽  
Case Control ◽  
Mtdna Copy Number ◽  
Chain Reaction ◽  
Indian Women ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A ◽  
Polymerase Chain ◽  
Control Study

Aim: The Mitochondrial transcription factor A (TFAM) and mitochondrial (mt) DNA copy number variations are known to contribute in disease development. Genetic factors play an important role in the development of endometriosis. Therefore, this case–control study aimed to analyze the association of TFAM+35G/C polymorphism and mitochondrial copy number with the risk of endometriosis in Indian women. Study Design: This study was carried out on 418 subjects including 200 endometriosis cases and 218 controls. Methodology: Genotyping of TFAM +35G/C polymorphism (rs1937) was carried out by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Quantification of mtDNA copy number was carried out using a real time quantitative polymerase chain reaction (qRT-PCR). Place and Duration of Study: Department of Biochemistry, Osmania University, 2014 to 2020. Results: TFAM genotype as well as allele distributions were all in Hardy-Weinberg equilibrium. The results indicated a significant reduction of GG genotype frequency (P=0.009), high ‘C’ allele frequency (P=0.017) and significantly decreased mtDNA copy number in endometriosis cases compared to controls (P= 0.0001). Conclusion: Present study revealed a statistically significant association of decreased GG genotype of TFAM +35G/C polymorphism and mtDNA copy number with the risk of developing endometriosis in Indian women.

Mitochondrial DNA replication is initiated at blastocyst formation in equine embryos

2019 ◽  
Vol 31 (3) ◽  
pp. 570 ◽  
Author(s):  
W. Karin Hendriks ◽  
Silvia Colleoni ◽  
Cesare Galli ◽  
Damien B. B. P. Paris ◽  
Ben Colenbrander ◽  
...  
Keyword(s):  
Dna Replication ◽  
Copy Number ◽  
Oocyte Quality ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Mtdna Copy Number ◽  
Blastocyst Development ◽  
Mt Dna ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Number

Intracytoplasmic sperm injection is the technique of choice for equine IVF and, in a research setting, 18–36% of injected oocytes develop to blastocysts. However, blastocyst development in clinical programs is lower, presumably due to a combination of variable oocyte quality (e.g. from old mares), suboptimal culture conditions and marginal fertility of some stallions. Furthermore, mitochondrial constitution appears to be critical to developmental competence, and both maternal aging and invitro embryo production (IVEP) negatively affect mitochondrial number and function in murine and bovine embryos. The present study examined the onset of mitochondrial (mt) DNA replication in equine embryos and investigated whether IVEP affects the timing of this important event, or the expression of genes required for mtDNA replication (i.e. mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and single-stranded DNA binding protein (SSB)). We also investigated whether developmental arrest was associated with low mtDNA copy number. mtDNA copy number increased (P<0.01) between the early and expanded blastocyst stages both invivo and invitro, whereas the mtDNA:total DNA ratio was higher in invitro-produced embryos (P=0.041). Mitochondrial replication was preceded by an increase in TFAM but, unexpectedly, not mtPOLB or SSB expression. There was no association between embryonic arrest and lower mtDNA copy numbers.

264 MITOCHONDRIAL DNA COPY NUMBER IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS

2011 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
P. Pawlak ◽  
E. Pers-Kamczyc ◽  
D. Lechniak-Cieslak
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Published Data ◽  
Mtdna Copy Number ◽  
Final Size ◽  
Domestic Species ◽  
Mitochondrial Dna Copy Number ◽  
Wide Range

In many domestic species (pig, cow, sheep), oocytes from prepubertal females show impaired quality when compared with those from adult animals. Incomplete cytoplasmic maturation is thought to be the main factor responsible for reduced developmental competence of embryos derived from prepubertal oocytes. The status of ooplasm maturation is also reflected by the copy number of mitochondrial DNA (mtDNA). Because replication of mtDNA ceases when oocytes reach their final size and occurs again at the blastocyst stage, the mtDNA copy number is a proved marker of oocyte quality in the pig (El Shourbagy et al. 2006 Reproduction 131, 233–245). The number of mtDNA copies in the grown oocyte is crucial to sustain the first embryonic divisions. To increase the rate of good-quality blastocysts, oocytes of domestic animals have been evaluated by the brilliant cresyl blue test (BCB). According to El Shourbagy et al. (2006), more competent BCB+ oocytes possess higher copy number of mtDNA (on average 222 446) than do their BCB– counterparts (115 352). However, there are no published data on the variation in mtDNA copy number in oocytes derived from ovaries of prepubertal (NCL) and cyclic (CL) gilts. Ovaries of NCL and CL gilts were collected in a local slaughterhouse. Cumulus–oocyte complexes (COC) were aspirated from nonatretic follicles 2 to 6 mm in diameter and evaluated morphologically. Only COC with a proper morphology were subjected to the BCB test. A group of non-BCB-treated COC served as control. Four groups of COC were collected: BCB+ (CL, NCL) and control (CL, NCL). Follicular cells attached to oocytes were removed by pipetting, and completely denuded gametes were individually frozen in liquid nitrogen. Analysis of the mtDNA copy number included isolation of the total DNA followed by amplification of the Cytochrome b (CYTB) gene by real-time PCR (one copy per one mitochondrial genome). Differences in mtDNA copy number among experimental groups were evaluated by Student’s t-test. To date, 30 BCB+ oocytes have been analysed individually (15 CL and 15 NCL). The analysed parameter varied in a wide range from 79 852 to 522 712 copies in CL oocytes and from 52 270 to 287 852 copies in NCL oocytes. Oocytes from cyclic gilts contained significantly more mtDNA copies (on average 267 524) than did gametes of prepubertal females (179 339; P < 0.05). The data on the mtDNA copy number in the control oocytes are currently under investigation. The preliminary results indicate that impaired oocytes quality of prepubertal gilts may be also attributed to the reduced copy number of mtDNA. This project was sponsored by MSHE Poland (grant no. 451/N-COST/2009/0).

scholarly journals Increased gene copy number of DEFA1/DEFA3 worsens sepsis by inducing endothelial pyroptosis

2019 ◽  
Vol 116 (8) ◽  
pp. 3161-3170 ◽  
Author(s):  
QiXing Chen ◽  
Yang Yang ◽  
JinChao Hou ◽  
Qiang Shu ◽  
YiXuan Yin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Copy Number ◽  
Treatment Options ◽  
Gene Copy Number ◽  
High Copy Number ◽  
Gene Copy ◽  
Dependent Manner ◽  
Barrier Dysfunction ◽  
Specific Expression

Sepsis claims an estimated 30 million episodes and 6 million deaths per year, and treatment options are rather limited. Human neutrophil peptides 1–3 (HNP1–3) are the most abundant neutrophil granule proteins but their neutrophil content varies because of unusually extensive gene copy number polymorphism. A genetic association study found that increased copy number of the HNP-encoding gene DEFA1/DEFA3 is a risk factor for organ dysfunction during sepsis development. However, direct experimental evidence demonstrating that these risk alleles are pathogenic for sepsis is lacking because the genes are present only in some primates and humans. Here, we generate DEFA1/DEFA3 transgenic mice with neutrophil-specific expression of the peptides. We show that mice with high copy number of DEFA1/DEFA3 genes have more severe sepsis-related vital organ damage and mortality than mice with low copy number of DEFA1/DEFA3 or wild-type mice, resulting from more severe endothelial barrier dysfunction and endothelial cell pyroptosis after sepsis challenge. Mechanistically, HNP-1 induces endothelial cell pyroptosis via P2X7 receptor-mediating canonical caspase-1 activation in a NLRP3 inflammasome-dependent manner. Based on these findings, we engineered a monoclonal antibody against HNP-1 to block the interaction with P2X7 and found that the blocking antibody protected mice carrying high copy number of DEFA1/DEFA3 from lethal sepsis. We thus demonstrate that DEFA1/DEFA3 copy number variation strongly modulates sepsis development in vivo and explore a paradigm for the precision treatment of sepsis tailored by individual genetic information.

Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle’ Hypothesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3116-3116
Author(s):  
Myung-Geun Shin ◽  
Hye Ran Kim ◽  
Hyeoung-Joon Kim ◽  
Hoon Kook ◽  
Tai Ju Hwang ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Control Region ◽  
Copy Number ◽  
Regulatory Region ◽  
Mtdna Control Region ◽  
Vicious Cycle ◽  
Mtdna Copy Number ◽  
Length Heteroplasmy ◽  
Region Length ◽  
D Loop

Abstract Abstract 3116 Poster Board III-53 Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle’ (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. Disclosures No relevant conflicts of interest to declare.

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