Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of mtDNA-depleted (Rho) mouse embryonic fibroblasts

ABSTRACT: Genome-wide transcriptional profiles of parental and mtDNA-depleted mouse embryonic fibroblasts. Analysis revealed a downregulation of ChrM genes. Mcl1 knockout mouse embryonic fibroblasts (transformed with SV40 large T antigen) were cultured for 7 days with Ethidium Bromide (100 ng/mL) in DMEM supplemented with Glucose (4.5 g/L), Uridine (50 ug/mL) and Sodium Pyruvate (100 ug/mL). Depletion of mtDNA was assessed by quantitative real-time PCR. Total RNA was isolated and up to 250 ng of total RNA per sample was labelled and hybridized to Illumina Mouse WG6V2 expression BeadChip platform. Three replicate samples were available for each population.

ORGANISM(S): Mus musculus

SUBMITTER: Matthew Ritchie

PROVIDER: E-GEOD-59972 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Gene expression analysis of mtDNA-depleted (Rho) mouse embryonic fibroblasts

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