Transcription Of Mitochondrial Genome Research Articles

Accumulation of DNA G-quadruplex in mitochondrial genome hallmarks mesenchymal senescence.

Searching for biomarkers of senescence remains necessary and challenging. Reliable and detectable biomarkers can indicate the senescence condition of individuals, the need for intervention in a population, and the effectiveness of that intervention in controlling or delaying senescence progression and senescence-associated diseases. Therefore, it is of great importance to fulfill the unmet requisites of senescence biomarkers especially when faced with the growing global senescence nowadays. Here, we established that DNA G-quadruplex (G4) in mitochondrial genome was a reliable hallmark for mesenchymal senescence. Via developing a versatile and efficient mitochondrial G4 (mtG4) probe we revealed that in multiple types of senescence, including chronologically healthy senescence, progeria, and replicative senescence, mtG4 hallmarked aged mesenchymal stem cells. Furthermore, we revealed the underlying mechanisms by which accumulated mtG4, specifically within respiratory chain complex (RCC) I and IV loci, repressed mitochondrial genome transcription, finally impairing mitochondrial respiration and causing mitochondrial dysfunction. Our findings endowed researchers with the visible senescence biomarker based on mitochondrial genome and furthermore revealed the role of mtG4 in inhibiting RCC genes transcription to induce senescence-associated mitochondrial dysfunction. These findings depicted the crucial roles of mtG4 in predicting and controlling mesenchymal senescence.

Lncrna Rmrp Regulate Mitochondrial Function in Relapsed and Refractory Acute Myeloid Leukemia

Background: The prognosis of relapsed and refractory acute myeloid leukemia (R/R AML) is extremely poor, primarily due to the development of chemotherapy resistance during the treatment, which leads to its relapse and refractory disease. Extensive studies have established that various dysfunctions in the mitochondria of AML cells play a significant role in promoting chemotherapy resistance and the recurrence of the disease. In our previous investigation, we successfully demonstrated the substantial enrichment of the long non-coding RNA (lncRNA) RMRP in the mitochondria. However, the specific function of RMRP in the mitochondria of R/R AML remains unexplored. The objective of this project is to uncover the function of RMRP in R/R AML mitochondria, aiming to provide innovative insights and a theoretical foundation for surmounting chemotherapy resistance in R/R AML. Methods: In order to investigate the factors that play a crucial role in R/R AML, we employed mass spectrometry (MS) to conduct a screening of factors associated with AML chemoresistance. We utilized quantitative real-time polymerase chain reaction (RT-qPCR) to assess the expression of lncRNA RMRP in both newly diagnosed AML (ND AML) and R/R AML patients. Furthermore, we knocked down the RMRP expression level by transfection with siRNA-NC and siRNA-RMRP. Additionally, we analyzed the correlation between lncRNA RMRP and mitochondria. To explore the involvement of lncRNA RMRP in regulating mitochondrial function in R/R AML, we performed mitochondrial oxidative phosphorylation metabolic function assays subsequent to the inhibition of lncRNA RMRP. Moreover, Western blot analysis was employed to evaluate the relative protein expression levels in AML cell lines. Results: We enrolled 23 AML patients (ND AML, n=12; R/R AML, n=11), and the result of MS shows that R/R AML group expressed higher level of mitochondria related protein. The qPCR results showed that R/R AML had higher expression level of RMRP ( P<0.0001) and mitochondrial genome genes ( P<0.0001) than ND AML patients. The result of co-cultured of BMSCs and AML suggests that mitochondrial transmission of BMSCs to AML existed in AML patients with high RMRP expression level( P<0.0001). The Seahorse metabolomics results suggest that RMRP knock down attenuated Spare Respiratory Capacity ( P=0.01) and Maximal Respiration ( P=0.01), increased the Cytarabine and Doxorubicin sensitivity, and induced alterations in the mitochondrial CpG1 and CpG9 methylation. Knockdown of RMRP is also accompanied by a decrease in mtDNA copy number ( P=0.10, 0.42, and 0.0074, respectively for mtDNA gene MT-ND2, MT-ND3, and MT-CO1), and mitochondrial genome transcription. Under transmission electron microscope (TEM), mitochondria were engulfed by lysosomes in RMRP knockdown AML cells. And results of Western blot showed that the autophagy markers such as LC3B, BNIP3L/Nix, NDP52 was upregulated, and SQSTM1, Parkin was downregulated in RMRP knockdown AML cells, suggesting that RMRP may induce mitophagy through the BNIP3L/Nix pathway, PINK1/Parkin pathway, and NDP52 pathway. Conclusion: In R/R AML, RMRP increased chemotherapy resistance and RMRP high expression was associated with multiple mitochondrial function abnormalities, including altered mitochondrial metabolism, reduced mitophagy, and altered mtDNA epigenetics.

Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling.

Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. Here, we investigate this by examining Drosophila macrophage-like cells called hemocytes. We used knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. We find that this reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. We also find that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, we show that genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. Our data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, our findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis.

Altered H3K4me3 profile at the TFAM promoter causes mitochondrial alterations in preadipocytes from first-degree relatives of type 2 diabetics

BackgroundFirst-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR.ResultsSequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression.ConclusionsHistone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk.Graphical abstract

Identification of mitochondrial RNA polymerase as a potential therapeutic target of osteosarcoma

POLRMT (RNA polymerase mitochondrial) is essential for transcription of mitochondrial genome encoding components of oxidative phosphorylation process. The current study tested POLRMT expression and its potential function in osteosarcoma (OS). The Cancer Genome Atlas (TCGA) cohorts and Gene Expression Profiling Interactive Analysis (GEPIA) database both show that POLRMT transcripts are elevated in OS tissues. In addition, POLRMT mRNA and protein levels were upregulated in local OS tissues as well as in established and primary human OS cells. In different OS cells, shRNA-induced stable knockdown of POLRMT decreased cell viability, proliferation, migration, and invasion, whiling inducing apoptosis activation. CRISPR/Cas9-induced POLRMT knockout induced potent anti-OS cell activity as well. Conversely, in primary OS cells ectopic POLRMT overexpression accelerated cell proliferation and migration. In vivo, intratumoral injection of adeno-associated virus-packed POLRMT shRNA potently inhibited U2OS xenograft growth in nude mice. Importantly, levels of mitochondrial DNA, mitochondrial transcripts and expression of respiratory chain complex subunits were significantly decreased in U2OS xenografts with POLRMT shRNA virus injection. Together, POLRMT is overexpressed in human OS, promoting cell growth in vitro and in vivo. POLRMT could be a novel therapeutic target for OS.

The requirement of mitochondrial RNA polymerase for non-small cell lung cancer cell growth

POLRMT (RNA polymerase mitochondrial) is responsible for the transcription of mitochondrial genome encoding key components of oxidative phosphorylation. This process is important for cancer cell growth. The current study tested expression and potential functions of POLRMT in non-small cell lung cancer (NSCLC). TCGA cohorts and the results from the local lung cancer tissues showed that POLRMT is overexpressed in human lung cancer tissues. In both primary human NSCLC cells and A549 cells, POLRMT silencing (by targeted lentiviral shRNAs) or knockout (through CRSIPR/Cas9 gene editing method) potently inhibited cell viability, proliferation, migration, and invasion, and induced apoptosis activation. On the contrast, ectopic overexpression of POLRMT using a lentiviral construct accelerated cell proliferation and migration in NSCLC cells. The mtDNA contents, mRNA levels of mitochondrial transcripts, and subunits of respiratory chain complexes, as well as S6 phosphorylation, were decreased in POLRMT-silenced or -knockout NSCLC cells, but increased after ectopic POLRMT overexpression. In vivo, intratumoral injection of POLRMT shRNA adeno-associated virus (AAV) potently inhibited NSCLC xenograft growth in severe combined immune deficiency mice. The mtDNA contents, mRNA levels of mitochondria respiratory chain complex subunits, and S6 phosphorylation were decreased in POLRMT shRNA AAV-injected NSCLC xenograft tissues. These results show that POLRMT is a novel and important oncogene required for NSCLC cell growth in vitro and in vivo.

Elevated TEFM expression promotes growth and metastasis through activation of ROS/ERK signaling in hepatocellular carcinoma

TEFM (transcription elongation factor of mitochondria) has been identified as a novel nuclear-encoded transcription elongation factor in the transcription of mitochondrial genome. Our bioinformatics analysis of TCGA data revealed an aberrant over-expression of TEFM in hepatocellular carcinoma (HCC). We analyzed its biological effects and clinical significance in this malignancy. TEFM expression was analyzed by quantitative real-time PCR, western blot, and immunohistochemistry analysis in HCC tissues and cell lines. The effects of TEFM on HCC cell growth and metastasis were determined by cell proliferation, colony formation, flow cytometric cell cycle and apoptosis, migration, and invasion assays. TEFM expression was significantly increased in HCC tissues mainly caused by down-regulation of miR-194-5p. Its increased expression is correlated with poor prognosis of HCC patients. TEFM promoted HCC growth and metastasis both in vitro and in vivo by promoting G1–S cell transition, epithelial-to-mesenchymal transition (EMT), and suppressing cell apoptosis. Mechanistically, TEFM exerts its tumor growth and metastasis promoting effects at least partly through increasing ROS production and subsequently by activation of ERK signaling. Our study suggests that TEFM functions as a vital oncogene in promoting growth and metastasis in HCC and may contribute to the targeted therapy of HCC.

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.

Quantification of Mitochondrial Genome Transcription in Male Reproductive Cells by Real-Time PCR.

Mitochondria are organelles that play a key role in the regulation of cell energy metabolism, biosynthesis, and cell death. Mitochondria are also involved in important physiological processes, such as the three-carboxylic acid cycle, the oxidation of fatty acids and amino acids, and calcium ion homeostasis. The energy demands of male germ cells during mitosis and meiosis are higher than those of somatic cells, suggesting that mitochondria play a critical role in sperm. Mitochondria are nanotoxicity targets in male germ cells. The level of mitochondrial genome transcription is reflective of mitochondrial function and can therefore be used as a quantitative index for evaluating nanotoxicity. This study describes how to use real-time PCR to evaluate the effects of nanomaterials on mitochondrial genome transcription in male reproductive cells.

Structure, evolution and phylogenetic informativeness of eelpouts (Cottoidei: Zoarcales) mitochondrial control region sequences

Control region (CR) is a major non-coding domain of mitochondrial DNA in vertebrates which contains the promoters for replication and transcription of mitochondrial genome along with the binding sites for metabolic machinery and, hence, is a vital element for the integrity of mitochondrial genome as a biological replicator. The origin and diversity of structural elements within CR have been intensively studied in recent years with the involvement of new diverse taxa. In this paper, we provide new data on the nucleotide and structural patterns of CR evolution and phylogenetic suitability among eelpouts (Cottoidei: Zoarcales). To achieve this, we carried out a comparative phylogenetic and structural analysis of 29 CR sequences belonging to the long shanny Stichaeus grigorjewi together with nine sequences of other eelpouts taxa representing four families in contrast to mitochondrial protein-coding fragments. The CR organization within S. grigorjewi, as well as in all other eelpouts, is consistent with the common three-domain structure known from most vertebrates. We found a hidden CR variation constrains on the landscape level and a lack of nucleotide saturation. Finally, our results demonstrate the advantage of the length variation in CR sequences for phylogenetic reconstructions among eelpouts.

PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs

Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehydemediated crosslinking and immunoprecipitation-sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.

ESTROGEN REGULATION OF MITOCHONDRIAL RESPIRATION IS CELL TYPE AND ER SUBTYPE SPECIFIC

Late onset AD has an approximately 20-year prodromal period, during which brain glucose hypometabolism can be detected in at risk groups and is predictive of disease progression. Loss of estrogen during the perimenopausal transition is associated with deficits in brain glucose metabolism and mitochondrial function, which may explain the two-fold greater lifetime risk of LOAD in females than males. In postmenopausal women, estrogen replacement therapy can improve brain glucose metabolism and recognition, and in ovariectomized rodents, estrogen treatment preserved mitochondrial respiration capacity, supporting the role of E2 as a master regulator of brain bioenergetics via mitochondrial function. However, the underlying mechanism is not fully elucidated. Interestingly, in neuronal cells, estrogen receptor beta (ERβ), but not estrogen receptor alpha (ERα), was demonstrated to co-localize with mitochondria, and has a functional role in ATP production, suggesting potential differential contributions of ERα and ERβ to regulation of mitochondrial bioenergetics. To identify cell type specific responses to estrogen treatment, we used rat embryonic primary neurons and astrocytes. To identify the contribution of ER subtypes, cells were treated with either ERα selective antagonist (MPP), or ERβ selective antagonist (PHTPP) prior to E2 treatment. Cellular respiratory capacity was determined by XF24 metabolic flux analyzer, and Oxygen consumption rate (OCR) was used as an indicator for mitochondrial oxidative phosphorylation. To determine whether ERs regulate mitochondrial respiration via regulation of transcription of mitochondrial genome, gene expression of the 13 mitochondrial protein encoding genes will be determined by real-time rtPCR. Ongoing analysis also explores how mitochondrial genetic variances affect treatment effect of selective estrogen modulators on perimenopausal symptoms. Estrogen increased both ATP production and maximum respiration in neurons, but only ATP production in astrocytes. ERα antagonist prevented E2 mediated enhancement of mitochondrial respiration in astrocytes but not neurons, whereas ERβ antagonist abolished E2 mediated enhancement of mitochondrial respiration in both cell types. E2 regulation of mitochondrial respiration is cell type and ER subtype specific, possibly through differential regulation of transcription of the mitochondrial genome. Acknowledgement: This work was supported by NIA R01 AG032236 to RDB; NIA P01AG026572 to RDB, Project 1 to RDB & EC.

The Parkinson's disease-associated protein DJ-1 plays a positive nonmitochondrial role in endocytosis in Dictyostelium cells.

ABSTRACTThe loss of function of DJ-1 caused by mutations in DJ1 causes a form of familial Parkinson's disease (PD). However, the role of DJ-1 in healthy and in PD cells is poorly understood. Even its subcellular localization in mammalian cells is uncertain, with both cytosolic and mitochondrial locations having been reported. We show here that DJ-1 is normally located in the cytoplasm in healthy Dictyostelium discoideum cells. With its unique life cycle, straightforward genotype-phenotype relationships, experimental accessibility and genetic tractability, D. discoideum offers an attractive model to investigate the roles of PD-associated genes. Furthermore, the study of mitochondrial biology, mitochondrial genome transcription and AMP-activated protein kinase-mediated cytopathologies in mitochondrial dysfunction have been well developed in this organism. Unlike mammalian systems, Dictyostelium mitochondrial dysfunction causes a reproducible and readily assayed array of aberrant phenotypes: defective phototaxis, impaired growth, normal rates of endocytosis and characteristic defects in multicellular morphogenesis. This makes it possible to study whether the underlying cytopathological mechanisms of familial PD involve mitochondrial dysfunction. DJ-1 has a single homologue in the Dictyostelium genome. By regulating the expression level of DJ-1 in D. discoideum, we show here that in unstressed cells, DJ-1 is required for normal rates of endocytic nutrient uptake (phagocytosis and, to a lesser extent, pinocytosis) and thus growth. Reduced expression of DJ-1 had no effect on phototaxis in the multicellular migratory ‘slug’ stage of the life cycle, but resulted in thickened stalks in the final fruiting bodies. This pattern of phenotypes is distinct from that observed in Dictyostelium to result from mitochondrial dyfunction. Direct measurement of mitochondrial respiratory function in intact cells revealed that DJ-1 knockdown stimulates whereas DJ-1 overexpression inhibits mitochondrial activity. Together, our results suggest positive roles for DJ-1 in endocytic pathways and loss-of-function cytopathologies that are not associated with impaired mitochondrial function.

Interactions between mitochondria and the transcription factor myocyte enhancer factor 2 (MEF2) regulate neuronal structural and functional plasticity and metaplasticity.

The classical view of mitochondria as housekeeping organelles acting in the background to simply maintain cellular energy demands has been challenged by mounting evidence of their direct and active participation in synaptic plasticity in neurons. Time-lapse imaging has revealed that mitochondria are motile in dendrites, with their localization and fusion and fission events regulated by synaptic activity. The positioning of mitochondria directly influences function of nearby synapses through multiple pathways including control over local concentrations of ATP, Ca(2+) and reactive oxygen species. Recent studies have also shown that mitochondrial protein cascades, classically associated with apoptosis, are involved in neural plasticity in healthy cells. These findings link mitochondria to the plasticity- and metaplasticity-associated activity-dependent transcription factor myocyte enhancer factor 2 (MEF2), further repositioning mitochondria as potential command centres for regulation of synaptic plasticity. Intriguingly, MEF2 and mitochondrial functions appear to be intricately intertwined, as MEF2 is a target of mitochondrial apoptotic caspases and, in turn, MEF2 regulates mitochondrial genome transcription essential for production of superoxidase and hydrogen peroxidase. Here, we review evidence supporting mitochondria as central organelles controlling the spatiotemporal expression of neuronal plasticity, and attempt to disentangle the MEF2-mitochondria relationship mediating these functions.

The prokaryotic ClpQ protease plays a key role in growth and development of mitochondria inPlasmodium falciparum

The ATP-dependent ClpQY system is a prokaryotic proteasome-like multi-subunit machinery localized in the mitochondrion of malaria parasite. The ClpQY machinery consists of ClpQ threonine protease and ClpY ATPase. In the present study, we have assessed cellular effects of transient interference of PfClpQ protease activity in Plasmodium falciparum using a trans-dominant negative approach combined with FKBP degradation domain system. A proteolytically inactive mutant PfClpQ protein [PfClpQ(mut)] fused with FKBP degradation domain was expressed in parasites, which gets stabilized by Shield1 drug treatment. We show that the inactive PfClpQ(mut) interacts with wild-type PfClpQ and associates within multi-subunit complex in the parasite. Stabilization of the PfClpQ(mut) and its association in the protease machinery caused dominant negative effect in the transgenic parasites, which disrupted the growth cycle of asexual blood stage parasites. The mitochondria in these parasites showed abnormal morphology, these mitochondria were not able to grow and divide in the parasite. We further show that the dominant negative effect of PfClpQ(mut) disrupted transcription of mitochondrial genome encoded genes, which in turn blocked normal development and functioning of the mitochondria.

Mitochondrial DNA: A Blind Spot in Neuroepigenetics.

Neuroepigenetics, which includes nuclear DNA modifications such as 5-methylcytosine and 5-hydoxymethylcytosine and modifications of nuclear proteins such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and the functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here we summarize for the first time evidence supporting the existence of these mechanisms and we propose the term "mitochondrial epigenetics" to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics - a gap that we expect to close in the near future.

Nuclear Respiratory Factor 1 Controls Myocyte Enhancer Factor 2A Transcription to Provide a Mechanism for Coordinate Expression of Respiratory Chain Subunits

Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6A(H) and COX7A(H) lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5'-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6A(H) promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 --> MEF2A --> COX(H) transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1alpha in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4.

Endocrine regulation of mitochondrial activity: involvement of truncated RXRalpha and c-Erb Aalpha1 proteins.

The importance of mitochondrial activity has recently been extended to the regulation of developmental processes. Numerous pathologies associated with organelle's dysfunctions emphasize their physiological importance. However, regulation of mitochondrial genome transcription, a key element for organelle's function, remains poorly understood. After characterization in the organelle of a truncated form of the triiodothyronine nuclear receptor (p43), a T3-dependent transcription factor of the mitochondrial genome, our purpose was to search for other mitochondrial receptors involved in the regulation of organelle transcription. We show that a 44 kDa protein related to RXRalpha (mt-RXR), another nuclear receptor, is located in the mitochondrial matrix. We found that mt-RXR is produced after cytosolic or intramitochondrial enzymatic cleavage of the RXRalpha nuclear receptor. After mitochondrial import and binding to specific sequences of the organelle genome, mt-RXR induces a ligand-dependent increase in mitochondrial RNA levels. mt-RXR physically interacts with p43 and acts alone or through a heterodimerical complex activated by 9-cis-retinoic acid and T3 to increase RNA levels. These data indicate that hormonal regulation of mitochondrial transcription occurs through pathways similar to those that take place in the nucleus and open a new way to better understand hormone and vitamin action at the cellular level.

Thyroid hormone action in mitochondria.

Triiodothyronine (T3) is considered a major regulator of mitochondrial activity. In this review, we show evidence of the existence of a direct T3 mitochondrial pathway, and try to clarify the respective importance of the nuclear and mitochondrial pathways for organelle activity. Numerous studies have reported short-term and delayed T3 stimulation of mitochondrial oxygen consumption. Convincing data indicate that an early influence occurs through an extra-nuclear mechanism insensitive to inhibitors of protein synthesis. Although it has been shown that diiodothyronines could actually be T3 mediators of this short-term influence, the detection of specific T3-binding sites, probably corresponding to a 28 kDa c-Erb Aalpha1 protein of the inner membrane, also supports a direct T3 influence. The more delayed influence of thyroid hormone upon mitochondrial respiration probably results from mechanisms elicited at the nuclear level, including changes in phospholipid turnover and stimulation of uncoupling protein expression, leading to an increased inner membrane proton leak. However, the involvement of a direct mitochondrial T3 pathway leading to a rapid stimulation of mitochondrial protein synthesis has to be considered. Both pathways are obviously involved in the T3 stimulation of mitochondrial genome transcription. First, a 43 kDa c-Erb Aalpha1 protein located in the mitochondrial matrix (p43), acting as a potent T3-dependent transcription factor of the mitochondrial genome, induces early stimulation of organelle transcription. In addition, T3 increases mitochondrial TFA expression, a mitochondrial transcription factor encoded by a nuclear gene. Similarly, the stimulation of mitochondriogenesis by thyroid hormone probably involves both pathways. In particular, the c-erb Aalpha gene simultaneously encodes a nuclear and a mitochondrial T3 receptor (p43), thus ensuring coordination of the expression of the mitochondrial genome and of nuclear genes encoding mitochondrial proteins. Recent studies concerning the physiological importance of the direct mitochondrial T3 pathway involving p43 led to the conclusion that it is not only involved in the regulation of fuel metabolism, but also in the regulation of cell differentiation. As the processes leading to or resulting from differentiation are energy-consuming, p43 coordination of metabolism and differentiation could be of significant importance in the regulation of development.

Mitochondrial DNA content and mitochondrial gene transcriptional activities in the early development of loach and goldfish.

The mitochondrial DNA (mtDNA) content of the mature eggs and embryos of loach and goldfish at early developmental stages were detected by means of dot hybridization. The transcription of mitochondrial cytochrome oxidase subunit I and II (COI and COII) genes during their early development was also detected by Northern hybridization. The experimental results showed that the mtDNA content of the mature egg as well as that of the embryos during the period from fertilized egg up to hatching stage in both fishes is maintained at a constant level, giving an average value of 7.40 x 107 molecules or 1.33 ng for every embryo in loach and an average value of 1.87 x 10(8) molecules or 3.31 ng for every embryo in goldfish. In both fish embryos, the COI and COII transcripts declined gradually after fertilization until late-blastula stage and then increased in early gastrula stage. This indicated that the transcription of mitochondrial genomes of these two freshwater fishes, which belong to different families, might be activated at the beginning of gastrulation. The steady-state amounts of mitochondrial messenger transcripts existing in the embryos during the early development in both fishes seemed to be regulated by both their half-lives and the transcriptional level of the mitochondrial genomes. The results showed that the transcription of the mitochondrial genome in the early developmental process in loach and goldfish was not regulated by a gene dosage mechanism.