scholarly journals HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation

Life ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 22
Author(s):  
Vanessa Cristina de Oliveira ◽  
Kelly Cristine Santos Roballo ◽  
Clésio Gomes Mariano Junior ◽  
Sarah Ingrid Pinto Santos ◽  
Fabiana Fernandes Bressan ◽  
...  
Keyword(s):  
Copy Number ◽  
Control Cell ◽  
Mtdna Copy Number ◽  
Mitochondrial Transcription Factor ◽  
Cell Clones ◽  
Key Factor ◽  
Mitochondrial Transcription Factor A ◽  
Vitro Model ◽  
Mitotracker Red

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67–96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

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.

scholarly journals Mitochondrial transcription factor A regulation of mitochondrial degeneration in experimental diabetic neuropathy

2015 ◽  
Vol 309 (2) ◽  
pp. E132-E141 ◽  
Author(s):  
Krish Chandrasekaran ◽  
Muragundla Anjaneyulu ◽  
Tatsuya Inoue ◽  
Joungil Choi ◽  
Avinash Rao Sagi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Diabetic Neuropathy ◽  
Nerve Conduction ◽  
Copy Number ◽  
Sensory Nerve ◽  
Mtdna Copy Number ◽  
Mitochondrial Transcription ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A ◽  
Intraepidermal Nerve Fiber

Oxidative stress-induced mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in peripheral neurons is considered to be important in the development of diabetic neuropathy. Mitochondrial transcription factor A (TFAM) wraps mtDNA and promotes mtDNA replication and transcription. We studied whether overexpression of TFAM reverses experimental peripheral diabetic neuropathy using TFAM transgenic mice (TFAM Tg) that express human TFAM (hTFAM). Levels of mouse mtDNA and the total TFAM (mouse TFAM + hTFAM) in the dorsal root ganglion (DRG) increased by approximately twofold in the TFAM Tg mice compared with control (WT) mice. WT and TFAM Tg mice were made diabetic by the administration of streptozotocin. Neuropathy end points were motor and sensory nerve conduction velocities, mechanical allodynia, thermal nociception, and intraepidermal nerve fiber density (IENFD). In the DRG neurons, mtDNA copy number and damage to mtDNA were quantified by qPCR, and TFAM levels were measured by Western blot. Mice with 16-wk duration of diabetes developed motor and sensory nerve conduction deficits, behavioral deficits, and intraepidermal nerve fiber loss. All of these changes were mostly prevented in diabetic TFAM Tg mice and were independent of changes in blood parameters. Mice with 16 wk of diabetes had a 40% decrease in mtDNA copy number compared with nondiabetic mice ( P < 0.01). Importantly, the mtDNA copy number in diabetic TFAM Tg mice reached the same level as that of WT nondiabetic mice. In comparison, there was upregulation of mtDNA and TFAM in 6-wk diabetic mice, suggesting that TFAM activation could be a therapeutic strategy to treat peripheral neuropathy.

scholarly journals Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

2015 ◽  
Vol 112 (36) ◽  
pp. 11288-11293 ◽  
Author(s):  
Christian Kukat ◽  
Karen M. Davies ◽  
Christian A. Wurm ◽  
Henrik Spåhr ◽  
Nina A. Bonekamp ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Copy ◽  
Mtdna Copy Number ◽  
Mitochondrial Nucleoid ◽  
Superresolution Microscopy ◽  
Mitochondrial Transcription Factor ◽  
Multistep Process ◽  
Mitochondrial Nucleoids ◽  
Mitochondrial Transcription Factor A

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

A human mitochondrial transcriptional activator can functionally replace a yeast mitochondrial HMG-box protein both in vivo and in vitro

1993 ◽  
Vol 13 (3) ◽  
pp. 1951-1961
Author(s):  
M A Parisi ◽  
B Xu ◽  
D A Clayton
Keyword(s):  
Transcription Factor ◽  
Mitochondrial Dna ◽  
Nuclear Gene ◽  
Transcriptional Activator ◽  
Yeast Mitochondria ◽  
Yeast Protein ◽  
Mitochondrial Transcription ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A

Human mitochondrial transcription factor A is a 25-kDa protein that binds immediately upstream of the two major mitochondrial promoters, thereby leading to correct and efficient initiation of transcription. Although the nature of yeast mitochondrial promoters is significantly different from that of human promoters, a potential functional homolog of the human transcriptional activator protein has been previously identified in yeast mitochondria. The importance of the yeast protein in yeast mitochondrial DNA function has been shown by inactivation of its nuclear gene (ABF2) in Saccharomyces cerevisiae cells resulting in loss of mitochondrial DNA. We report here that the nuclear gene for human mitochondrial transcription factor A can be stably expressed in yeast cells devoid of the yeast homolog protein. The human protein is imported efficiently into yeast mitochondria, is processed correctly, and rescues the loss-of-mitochondrial DNA phenotype in a yeast abf2 strain, thus functionally substituting for the yeast protein. Both human and yeast proteins affect yeast mitochondrial transcription initiation in vitro, suggesting that the two proteins may have a common role in this fundamental process.

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&lt;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.

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 &lt; 0.05) and muscle (P &lt; 0.01), and on fetus (P &lt; 0.001), heart (P &lt; 0.001), and liver (P &lt; 0.001) weights. The mtDNA amount in all tissues from IVF-fetuses was normal, but mtDNA levels in liver (-23%; P &lt; 0.05) and muscle (-24%; P &lt; 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 &lt; 0.001), but only the NT-fetuses were affected by disproportionate hepatomegaly and cardiomegaly with 31% and 49% increases (ANCOVA; P &lt; 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 &gt; 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 Mitochondria directly influence fertilisation outcome in the pig

Reproduction ◽  
2006 ◽  
Vol 131 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Shahinaz H El Shourbagy ◽  
Emma C Spikings ◽  
Mariana Freitas ◽  
Justin C St John
Keyword(s):  
Copy Number ◽  
Mtdna Copy Number ◽  
Oocyte Maturity ◽  
Oocyte Competence ◽  
Cresyl Blue ◽  
Significant Difference ◽  
Mtdna Replication ◽  
Mitochondrial Number ◽  
Glucose 6 Phosphate Dehydrogenase

The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell’s major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB+) and BCB negative (BCB−) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB− oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB− oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages.

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