MtDNA-acquisition
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Project description:Integrity of mitochondrial DNA (mtDNA), encoding several subunits of the respiratory chain, is essential to maintain mitochondrial fitness. Mitochondria, as a central hub for metabolism, are affected in a wide variety of human diseases but also during normal ageing, where mtDNA integrity is compromised. Mitochondrial quality control mechanisms work at different levels, and mitophagy and its variants are critical to remove dysfunctional mitochondria and mtDNA to maintain cellular homeostasis. Understanding the mechanisms governing a selective turnover of mutation-bearing mtDNA without affecting the entire mitochondrial pool is fundamental to design therapeutic strategies against mtDNA diseases and ageing. Here, we show that mtDNA damage after expressing a dominant negative version of the mitochondrial helicase Twinkle, or by chemical means, leads to an exacerbated mtDNA turnover. mtDNA removal depends on lysosomal function and requires the autophagy protein Atg5 but is independent of canonical mitophagy or autophagy. Using proximity labelling, we demonstrated that the area of influence of mitochondrial nucleoids differs upon mtDNA damage, which induces mitochondrial membrane remodelling and endosomal recruitment in close proximity to mitochondrial nucleoid sub compartments. Targeting of nucleoids is controlled by the mitochondrial transmembrane proteins ATAD3 and SAMM50, which together with the endosomal trafficking protein VPS35, orchestrate endosomal nucleoid engulfment. SAMM50 acts as a gatekeeper to avoid mtDNA release to the cytoplasm and facilitating mtDNA transfer to VPS35. Lastly, we show that stimulation of lysosomal activity by rapamycin selectively removes mtDNA deletions in vivo, without affecting mtDNA copy number. With these results, we unveil the molecular players of a new complex mechanism specifically targeting and removing mutant mtDNA which occurs outside the mitochondrial network, a process with multiple potential benefits to understand human mtDNA related diseases, either inherited, acquired or due to normal ageing.
2022-11-07 | PXD023939 | Pride
Project description:Mitochondrial DNA (mtDNA) breaks are deleterious lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain ill-defined. Using mitochondrial targeted restriction enzymes, we show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Furthermore, mtDNA damage activates the integrated stress response (ISR) through phosphorylation of eIF2α by the OMA1-DELE1-HRI pathway. Electron microscopy reveals concomitant defects in mitochondrial membranes and cristae ultrastructure. Notably, inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, thereby implicating this stress response in mitigating mitochondrial dysfunction following mtDNA damage. Last, we provide evidence suggesting that ATAD3A, a membrane-anchored protein that interacts with nucleoids, relays the signal from mtDNA breaks to the inner mitochondrial membrane. Altogether, our study fully delineates the sequence of events linking damaged mitochondrial genomes with the cytoplasm and uncovers an unanticipated role for the ISR in response to mitochondrial genome instability.
2022-10-04 | GSE214512 | GEO
Project description:Geminin is a small nucleoprotein that neuralizes ectoderm in the Xenopus embryo. Geminin promotes neural fate acquisition of mouse embryonic stem cells: Geminin knockdown during neural fate acquisition decreased expression of neural precursor cell markers (Pax6, Sox1), while increasing expression of Pitx2, Lefty1 and Cited2, genes involved in formation of the mouse node. Here we differentiated mouse embryonic stem cells into embryoid bodies to study Geminin's ability to repress primitive streak mesendoderm fate acquisition. We used microarrays to define the sets of genes that are regulated by Geminin during cell fate acquisition in embryoid bodies, using Dox-inducible Geminin knockdown or overexpression mouse embryonic stem cell lines. ES cell lines for Geminin over-expression (GemOE) were treated without or with Dox from day 3 to day 5 of EB differentiation and were collected on days 4 or 5 for microarray analysis. Gem knockdown (KD) ES cell lines were treated without or with Dox from day 0 to day 4 of EB differentiation and were collected on day 4 for microarray analysis.
2014-07-01 | E-GEOD-39676 | biostudies-arrayexpress
Project description:Mitochondria contain a 16kb-dsDNA genome encoding 13 proteins essential for respiration, whereas its regulatory mechanism and potential role in cancer development remain elusive. Although Methyl-CpG-binding protein (MBD) proteins are essential for nuclear transcription, their role in mitochondrial DNA (mtDNA) transcription is unknown. Here, we report that the MBD2c splicing variant translocates into mitochondria to mediate mtDNA transcription and increase mitochondrial respiration in triple negative breast cancer (TNBC) cells. Specifically, MBD2c binds D-loop regions in mtDNA to recruit SIRT3, which in turn deacetylates TFAM, a primary mitochondrial transcription factor, and activates its function. TFAM activation subsequently enhances transcription of the whole mitochondrial genome. Furthermore, MBD2c overexpression recovered the decreased mtDNA-encoded RNA and protein levels induced by the DNA synthesis inhibitor, cisplatin (CDDP), in vitro and in vivo, preserving mitochondrial gene expression and respiration, consequently enhancing TNBC cells drug resistance and proliferation. These data collectively demonstrate that MBD2c positively regulates mtDNA transcription, thus connecting epigenetic regulation by deacetylation with cancer cell metabolism, suggesting druggable targets to overcome resistance.
2024-04-18 | GSE263933 | GEO
Project description:Mitochondrial DNA (mtDNA) breaks are toxic lesions that lead to degradation of mitochondrial genomes and subsequent reduction in mtDNA copy number. The signaling pathways activated in response to mtDNA damage remain poorly understood. We reasoned that factors that sense mtDNA damage are likely to be recruited to nucleoids shortly after break formation, prompting us to develop a proteomic-based approach to survey the interactome of mitochondrial nucleoids. Specifically, we combined proximity-dependent biotin labeling with Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and tandem mass spectrometry. We used mitochondrial targeted restriction enzymes for the proximity probe and show that cells with mtDNA breaks exhibit reduced respiratory complexes, loss of membrane potential, and mitochondrial protein import defect. Notably, mtDNA breaks activate the integrated stress response (ISR) through phosphorylation of eIF2a by the OMA1-DELE1-HRI pathway. Inhibition of the ISR exacerbates mitochondrial defects and delays the recovery of mtDNA copy number, hinting at a role for the ISR in mitigating mitochondrial dysfunction following mtDNA damage. Electron microscopy reveals defective mitochondrial membranes and cristae ultrastructure. Last, we highlight a potential role for ATAD3A - a protein that is anchored to the inner mitochondrial membrane while interacting with nucleoids - in relaying the signal from damaged mtDNA to the mitochondrial inner membrane. Altogether, our study delineates the sequence of events linking damaged mitochondrial genomes to the cytoplasmic stress response. The mass spectrometry raw files for the combined SILAC and proximity-dependent biotin labeling experiment are deposited here.
2022-10-05 | MSV000090459 | MassIVE
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:The goal of the study was to understand whether mitochondrial-driven epigenetic changes regulate gene expression. Mitochondrial metabolism has been implicated in epigenetics but the extent to which this impacts gene expression is unclear. Here we show that loss of mitochondrial DNA (mtDNA) results in locus-specific alterations in histone acetylation, DNA methylation and expression of a subset of genes. Most of these changes are rescued by restoring mitochondrial electron transport in a way that maintains the oxidative tricarboxylic acid cycle, but not reactive oxygen species or ATP production, or by modulating the mitochondrial pool of acetyl-CoA. Changes in acetyl-CoA and histone acetylation precede overt mitochondrial dysfunction and significant changes in gene expression and DNA methylation. This suggests that acetyl-CoA levels signal mitochondrial status to the nucleus. Differentially expressed genes with altered histone marks or DNA methylation regulate amino acid degradation, which likely compensates for the changes in acetyl-CoA and one carbon metabolism. These have the potential to further affect methylation reactions, redox control and nucleotide levels. These results illustrate the extent to which mitochondria impact cell physiology through epigenetic remodeling.
2018-03-21 | GSE99834 | GEO
Project description:The goal of the study was to understand whether mitochondrial-driven epigenetic changes regulate gene expression. Mitochondrial metabolism has been implicated in epigenetics but the extent to which this impacts gene expression is unclear. Here we show that loss of mitochondrial DNA (mtDNA) results in locus-specific alterations in histone acetylation, DNA methylation and expression of a subset of genes. Most of these changes are rescued by restoring mitochondrial electron transport in a way that maintains the oxidative tricarboxylic acid cycle, but not reactive oxygen species or ATP production, or by modulating the mitochondrial pool of acetyl-CoA. Changes in acetyl-CoA and histone acetylation precede overt mitochondrial dysfunction and significant changes in gene expression and DNA methylation. This suggests that acetyl-CoA levels signal mitochondrial status to the nucleus. Differentially expressed genes with altered histone marks or DNA methylation regulate amino acid degradation, which likely compensates for the changes in acetyl-CoA and one carbon metabolism. These have the potential to further affect methylation reactions, redox control and nucleotide levels. These results illustrate the extent to which mitochondria impact cell physiology through epigenetic remodeling.
2018-03-21 | GSE100249 | GEO
Project description:The goal of the study was to understand whether mitochondrial-driven epigenetic changes regulate gene expression. Mitochondrial metabolism has been implicated in epigenetics but the extent to which this impacts gene expression is unclear. Here we show that loss of mitochondrial DNA (mtDNA) results in locus-specific alterations in histone acetylation, DNA methylation and expression of a subset of genes. Most of these changes are rescued by restoring mitochondrial electron transport in a way that maintains the oxidative tricarboxylic acid cycle, but not reactive oxygen species or ATP production, or by modulating the mitochondrial pool of acetyl-CoA. Changes in acetyl-CoA and histone acetylation precede overt mitochondrial dysfunction and significant changes in gene expression and DNA methylation. This suggests that acetyl-CoA levels signal mitochondrial status to the nucleus. Differentially expressed genes with altered histone marks or DNA methylation regulate amino acid degradation, which likely compensates for the changes in acetyl-CoA and one carbon metabolism. These have the potential to further affect methylation reactions, redox control and nucleotide levels. These results illustrate the extent to which mitochondria impact cell physiology through epigenetic remodeling.
2018-03-21 | GSE100133 | GEO
Project description:The goal of the study was to understand whether mitochondrial-driven epigenetic changes regulate gene expression. Mitochondrial metabolism has been implicated in epigenetics but the extent to which this impacts gene expression is unclear. Here we show that loss of mitochondrial DNA (mtDNA) results in locus-specific alterations in histone acetylation, DNA methylation and expression of a subset of genes. Most of these changes are rescued by restoring mitochondrial electron transport in a way that maintains the oxidative tricarboxylic acid cycle, but not reactive oxygen species or ATP production, or by modulating the mitochondrial pool of acetyl-CoA. Changes in acetyl-CoA and histone acetylation precede overt mitochondrial dysfunction and significant changes in gene expression and DNA methylation. This suggests that acetyl-CoA levels signal mitochondrial status to the nucleus. Differentially expressed genes with altered histone marks or DNA methylation regulate amino acid degradation, which likely compensates for the changes in acetyl-CoA and one carbon metabolism. These have the potential to further affect methylation reactions, redox control and nucleotide levels. These results illustrate the extent to which mitochondria impact cell physiology through epigenetic remodeling.
2018-03-21 | GSE100132 | GEO