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Transcriptome profiling of cybrids cells with and without m.14487T>C mutation

ABSTRACT: Transcriptome profiling is a sensitive strategy to uncover the change of mitochondrial to nucleus signaling due to the minor alternation of mitochondrial OXPHOS function. Therefore, we hypothesized that even a minor alternation of OXPHOS function can also remold transcriptome profiles in m.14487T>C mutant cybrid when compared with control cybrids. Our data suggest that a comparable mitochondrial OXPHOS function between m.14487T and m.14487C cybrids is not because of compensatory boost of mitochondrial biogenesis in m.14487C cybrids.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131739 | GEO | 2020/05/19

REPOSITORIES: GEO

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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).

Project description:Background: Transcription control of mitochondrial metabolism is essential for cellular function. A better understanding of this process will aid the elucidation of mitochondrial disorders, in particular of the many genetically unsolved cases of oxidative phosphorylation (OXPHOS) deficiency. Yet, to date only few studies have investigated nuclear gene regulation in the context of OXPHOS deficiency. In this study, we combined RNA sequencing of human complex I-deficient patient cells across 32 conditions of perturbed mitochondrial metabolism, with a comprehensive analysis of gene expression patterns, co-expression calculations and transcription factor binding sites. Results: Our analysis shows that OXPHOS genes have a significantly higher co-expression with each other than with other genes, including mitochondrial genes. We found no evidence for complex-specific mRNA expression regulation in the tested cell types and conditions: subunits of different OXPHOS complexes are similarly (co-)expressed and regulated by a common set of transcription factors. However, we did observe significant differences between the expression of OXPHOS complex subunits compared to assembly factors, suggesting divergent transcription programs. Furthermore, complex I co-expression calculations identified 684 genes with a likely role in OXPHOS biogenesis and function. Analysis of evolutionarily conserved transcription factor binding sites in the promoters of these genes revealed almost all known OXPHOS regulators (including GABP, NRF1/2, SP1, YY1, E-box factors) and a set of six yet uncharacterized candidate transcription factors (ELK1, KLF7, SP4, EHF, ZNF143, and EL2). Conclusions: OXPHOS genes share an expression program distinct from other mitochondrial genes, indicative of targeted regulation of this mitochondrial sub-process. Within the subset of OXPHOS genes we established a difference in expression between subunits and assembly factors. Most transcription regulators of genes that co-express with complex I are well-established factors for OXPHOS biogenesis. For the remaining six factors we here suggest for the first time a link with transcription regulation in OXPHOS deficiency. RNA-SEQ of whole cell RNA in 2 control and 2 complex I deficient patient fibroblast cell lines treated with 4 compounds in duplicate, resulting in a total of 2x2x4x2=32 samples

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Transcriptome profiling of cybrids cells with and without m.14487T>C mutation

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