Transcription profiling of human cells to the absence of mitochondrial DNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Norman Pavelka
PROVIDER: E-GEOD-14900 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Mitochondria have been implicated in insulin resistance and beta cell dysfunction, both of which comprise the core pathophysiology of type 2 diabetes mellitus (T2DM). It has also recently been found that mtDNA haplogroups are distinctively associated with susceptibility to T2DM at least in Koreans and Japanese. To investigate the functional consequences of different mtDNA, we compared gene expression profiles between cybrid clones harboring three different mtDNA haplogroups (D5, F, and N9a). To produce hybrid clones, we fused mtDNA-depleted osteosarcoma cell line (143B TK- rho0) with nucleus-lacking platelets from twelve donors harboring the three haplogroups. A total of twelve cybrid clones from the three mtDNA haplogroups were obtained: D5 (n=3), F (n=5), and N9a (n=4). For each clone, four technical replicates were obtained and hybridized to the array. For rho0 cell, six technical replicates were obtained and hybridized to the array.
2012-03-31 | E-GEOD-26244 | biostudies-arrayexpress
Project description:Mitochondria generate signals of adaptation that regulate nuclear genes expression via retrograde signaling. But this phenomenon is complexified when qualitatively different mitochondria and mitochondrial DNA (mtDNA) coexist within cells. Although this cellular state of heteroplasmy leads to divergent phenotypes clinically, its consequences on cellular function and the cellular transcriptome are unknown. To interrogate this phenomenon, we generated somatic cell cybrids harboring increasing levels of a common mtDNA mutation (tRNALeu(UUR) 3243A>G) and mapped the resulting cellular phenotypes and transcriptional profiles across the complete range of heteroplasmy. Small increases in mutant mtDNAs caused relatively modest defect in mitochondrial oxidative capacity, but resulted in sharp transitions in mitochondrial ultrastructure and in the nuclear and mitochondrial transcriptomes, with the critical functional threshold corresponding to the induction of epigenetic regulatory systems. Principal component analysis underscores how each heteroplasmy level occupies a different "transcriptional space", with low levels heteroplasmy (20-30%) producing a dose-response linear progression in one direction, and mutationload of 50, 60 and 90% producing changes in the opposite direction. Hence, subtle changes in mitochondrial energetics can act through the epigenome to generate the phenotypes of the common “complex” diseases. Cells were generated by transferring the wildtype (3243A) and mutant (3243G) mtDNAs from a heteroplasmic 3243A>G patient’s lymphoblastoid cell line into 143B(TK-) mtDNA-deficient (ρo) cells and selected for transmitochondrial cybrids. Subsequent mtDNA depletion, reamplification, and cloning (Wiseman and Attardi, 1978) resulted in a series of stable cybrids harboring approximately 0, 20, 30, 50, 60, 90, and 100% 3243G mutant mtDNAs. Total RNA extracted from each cell line was then extracted, depleted of rRNA, and measured in sequenced in triplicates.
2014-09-02 | E-GEOD-56158 | biostudies-arrayexpress
Project description:Normal cellular function requires communication between mitochondria and the nucleus, termed mitochondria-to-nucleus retrograde signaling. Interruption of this mechanism has been implicated in the dysregulation of many cancer-related pathways, including cell death programs and tumor suppressor networks. Many proteins are known modulators of retrograde signaling, but whether microRNAs (miRNAs) are also involved is unknown. We conducted a miRNA microarray analysis using RNA from a parental cell line, a Rho0 line lacking mitochondrial DNA (mtDNA) and a Rho0 line with restored mtDNA. We found that miR-663 was down-regulated in the mtDNA-depleted Rho0 line. mtDNA restoration reversed this miRNA to parental levels, suggesting that it is an epigenetically-regulated mediator of retrograde signaling. We further demonstrated by methylation specific PCR and bisulfite sequencing that miR-663 is epigenetically regulated by pharmacological disruption of oxidative phosphorylation (OXPHOS). Restoration of rotenone-suppressed miR-663 expression by N-acetylcysteine suggested that mitochondrial dysfunction–induced reactive oxygen species play a role in epigenetic miR-663 regulation. We noted that miR-663 regulates the expression of nuclear-encoded respiratory chain subunits, e.g. NDUFB8, SDHB, UQCRFS1, and COX4L1. miR-663 also regulated the OXPHOS assembly factors NDUFAF1, SDHAF2, UQCC2, and SCO1 and was required for respiratory supercomplex stability. Furthermore, using luciferase assays, we found that miR-663 directly regulates UQCC2. The miR-663 sponge reduced OXPHOS complex activity and increased in vitro cellular proliferation and promoted tumor development in mice. We also found that increased miR-663 expression in breast tumors consistently correlates with increased patient survival. We provide first evidence for miRNA mediating retrograde signaling, demonstrating its epigenetic regulation and its role in breast tumorigenesis.
2017-10-19 | GSE104277 | GEO
Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).
2013-02-12 | E-GEOD-27545 | biostudies-arrayexpress
Project description:Mitochondrial biogenesis is regulated by signaling pathways sensitive to extracellular conditions and to the internal environment of the cell. We found that deletion of protein phosphatase 2A (PP2A) or of protein phosphatase 6 (PP6) diminishes the nuclear transcriptional response associated with mtDNA damage. Six samples were analyzed to determine message RNA levels.
2013-12-23 | E-GEOD-52242 | biostudies-arrayexpress
Project description:Damage to the mitochondrial genome (mtDNA) severely affects the cell and causes disease. Mutations to mtDNA also accumulate throughout the lifespan of many organisms and may be a proximal cause of aging. There is no effective treatment for ailments caused by mtDNA mutation. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. Experiments using the tractable eukaryote Saccharomyces cerevisiae allow us to investigate the connection between nutrient-sensing and mitochondrial function. We have focused our current studies on the protein kinase A (PKA) pathway, which controls S. cerevisiae behavior according to glucose availability. We found that reduced PKA signaling can lead, in a background-dependent manner, to improved fitness after mtDNA loss. Specifically, over-expression of the cyclic AMP phosphodiesterase Pde2p, removal of PKA isoform Tpk3p, or ablation of other proteins promoting PKA activity leads to improved proliferation of cells deleted of the mitochondrial genome. Over-expression of Pde2p also suppresses the inviability of several mutants that normally cannot survive mtDNA loss. Moreover, Pde2p over-expression diminishes the nuclear transcriptional response to mtDNA damage, further supporting the idea that glucose sensation is harmful for cells lacking the mitochondrial genome. These findings are heavily dependent upon yeast genetic background. Interestingly, robust import of mitochondrial polytopic membrane proteins may be required in order for cells with no mtDNA to receive the full benefits of PKA reduction. Our findings support the idea that perturbation of nutrient-sensing pathways, and specifically the sensation of glucose, may benefit cells with dysfunctional mitochondria. Four experimental conditions were used: BY4743 (WT) cells containing empty pRS426 vector and containing mtDNA because EtBr was not used, BY4743 (WT) cells containing empty pRS426 vector and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, BY4743 (WT) cells overexpressing TIP41 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, and BY4743 (WT) cells overexpressing PDE2 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide. Two replicates were performed for each sample type.
2015-08-07 | E-GEOD-71252 | biostudies-arrayexpress
Project description:The mitochondrial respiratory chain is composed of lipoprotein complexes imbedded in the inner mitochondrial membrane. This chain of enzymes transfers electrons from NADH and FADH2, provided from divers metabolic pathways, to oxygen. It couples the transfer of electrons to the translocation of protons across the membrane. Several clinical syndromes have been associated with respiratory dysfunction caused by mitochondrial or nuclear mutations. A number of mutations in the mitochondrial genes encoding for cytochrome b (CYTB) and cytochrome oxidase (COX 1, 2 and 3) have been linked with diseases. We are using yeast mutants to characterize the deleterious effect of mutations reported in patients on the assembly and catalytic properties of the affected enzymes, and to study the impact of mutations in nuclear genes, such as OXA1, encoding for factors required for the assembly of the respiratory complexes. In this work, we monitored the effects of the mutations causing respiratory defect on the whole genome expression. We compared the change in gene expression in rho0 cells (with a complete deletion of the mitochondrial genome, and by consequence without respiratory chain), in cells with either a single defective enzyme or several, and in cells after prolonged treatment with the bc1 inhibitors myxothiazol or antimycin. The impact of the mutations on the respiratory function ranged from mild to severe. The expression of approx. 350 genes was changed in at least one mutant. Cluster analysis was performed using the Cluster program (Eisen, 1998, PNAS 95:14863). Four groups of genes were studied in more details: Group A, the most repressed genes; Group B, the most over-expressed genes; Group C, genes more repressed in rho0 and Doxa1 cells; and Group D, genes more over-expressed in Doxa1.
2009-02-20 | E-MEXP-585 | biostudies-arrayexpress
Project description:Mitochondrial biogenesis is under the control of two different genetic systems: the nuclear genome (nDNA) and the mitochondrial genome (mtDNA). mtDNA is a circular genome of 16.6 kb encoding 13 of the approximately 90 subunits that form the respiratory chain, the remaining ones being encoded by the nuclear genome (nDNA). Eukaryotic cells are able to monitor and respond to changes in mitochondrial function through alterations in nuclear gene expression, a phenomenon first defined in yeast and known as retrograde regulation. With this experiment we aimed to identify the set of nuclear genes that significantly change their expression level in response to depletion of mtDNA. Keywords: case-control
2009-05-01 | GSE14900 | GEO
Project description:Mitochondrial DNA-depleted human skin fibroblasts (HSF rho0) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-kappaB and STAT3 signaling pathways and by decreased activity of the mitochondrial death pathway, compared to the parent rho+ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor-mediated death pathway remained highly active, and exogenous TRAIL induced higher levels of apoptosis in rho0 cells compared to rho+ HSF. Global gene expression analysis using microarray and quantitative RT-PCR demonstrated that expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, IGFL2), cytokines (IL6, IL17B, IL18, IL19, IL28B) and cytokine receptors (IL1R1, IL21R, IL31RA) were substantially decreased after mitochondrial depletion. Some of these genes were targets of NF-kappaB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation induced expression of several NF-kappaB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in rho+ HSF, but this response was substantially decreased in rho0 HSF. Suppression of the IKK-NF-kappaB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated rho+ HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in rho+ HSF. However, NF-kappaB activation was partially lost in mitochondrial DNA-depleted HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-kappaB targets, further suppressing IL6-JAK2-STAT3, that in concert with NF-kappaB, regulated protection against TRAIL-induced apoptosis. There are 12 total samples, 3 biological replicates each of HSF rho+ and rho0 cells that were not irradiated (control=C) or irradiated (alpha=A). Cells were harvested at 4 hours after treatment.
2011-04-04 | E-GEOD-24945 | biostudies-arrayexpress
Project description:Cancer cells consume large amounts of glucose because of their specific metabolic pathway. However, cancer cells exist in tumor tissue where glucose is insufficient. To survive, cancer cells likely have the mechanism to elude their glucose addiction. Here we show that functional mitochondria are essential if cancer cells are to avoid glucose addiction. Cancer cells with dysfunctional mitochondria, such as mitochondrial DNA-deficient rho0 cells and electron transport chain blocker-treated cells, were highly sensitive to glucose deprivation. Our data demonstrated that this sensitization was caused by failure of the unfolded protein response (UPR), an adaptive response mediated by the endoplasmic reticulum (ER). This study suggests a link between mitochondria and the ER during the UPR under glucose deprivation conditions and that mitochondria govern cell fate, not only through ATP production and apoptosis regulation but also through modulating the UPR for cell survival. Human cancer cell lines (HT-1080, HT-29, and mtDNA-deficient cells derived from these cell lines) were selected for RNA extraction and hybridization on Affymetrix microarrays. We examined the unfolded protein response (UPR), an adaptive response mediated by the endoplasmic reticulum (ER), of cancer cells under stress conditions. Abbreviations List: AA, antimycin A; Bu, buformin; Met, metformin; Phen, phenformin; Rot, rotenone; VST, versipelostatin; TM, tunicamycin; 2DG, 2-deoxyglucose; GS, glucose starvation. Capital S (_S) indicates the supernatant of sample including floating cells.
2010-05-28 | E-GEOD-16157 | biostudies-arrayexpress