Malignancy and NF-κB signalling strengthen coordination between expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes

Background Mitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. The mammalian mitochondrial genome encodes for 13 mitochondrial proteins, and the remaining mitochondrial proteins are encoded by the nuclear genome. Little is known about how coordination between the expression of the two sets of genes is achieved. Results Correlation analysis of RNA-seq expression data from large publicly available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalisation techniques based on total read counts, such as FPKM or TPM, produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, with important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalisation (MRN) or trimmed mean of M values (TMM) methods. Using these normalisations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database. Conclusions We show that a subset of healthy tissues with high expression of NF-κB show significant coordination, suggesting a role for NF-κB in ensuring balanced expression between mitochondrial and nuclear genes. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer.

Malignancy and NF-kB signalling strengthen coordination between the expression of mitochondrial and nuclear-encoded oxidative phosphorylation genes

Mitochondria are ancient endosymbiotic organelles crucial to eukaryotic growth and metabolism. Mammalian mitochondria carry a small genome containing thirteen protein-coding genes with the remaining mitochondrial proteins encoded by the nuclear genome. Little is known about how coordination between the two sets of genes is achieved. Correlation analysis of RNA-seq expression data from large publicly-available datasets is a common method to leverage genetic diversity to infer gene co-expression modules. Here we use this method to investigate nuclear-mitochondrial gene expression coordination. We identify a pitfall in correlation analysis that results from the large variation in the proportion of transcripts from the mitochondrial genome in RNA-seq data. Commonly used normalization techniques based on total read count (such as FPKM or TPM) produce artefactual negative correlations between mitochondrial- and nuclear-encoded transcripts. This also results in artefactual correlations between pairs of nuclear-encoded genes, thus having important consequences for inferring co-expression modules beyond mitochondria. We show that these effects can be overcome by normalizing using the median-ratio normalization (MRN) or trimmed mean of M values (TMM) methods. Using these normalizations, we find only weak and inconsistent correlations between mitochondrial and nuclear-encoded mitochondrial genes in the majority of healthy human tissues from the GTEx database. However, a subset of healthy tissues with high expression of NFkB show significant coordination supporting a role for NFkB in retrograde signalling. Contrastingly, most cancer types show robust coordination of nuclear and mitochondrial OXPHOS gene expression, identifying this as a feature of gene regulation in cancer.

Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

Oxidative Phosphorylation Regulated By PGC-1α Promotes Multiple Myeloma Progression

Introduction: With the deeper understanding of metabolic reprograming, more and more studies highlight that many tumors heavily rely on mitochondrial oxidative phosphorylation (OXPHOS) for bioenergetics and biosynthesis, and targeting OXPHOS appears to be a promising approach for cancer therapy. Despite multiple metabolism alterations occur in multiple myeloma (MM), the role of OXPHOS in MM is rarely focused on. Many metabolic pathways are orchestrated by certain master co-regulators through transcriptional programs. The expression of OXPHOS gene set was reported to be regulated by peroxisome proliferator activated receptor gamma coactivator 1α (PGC-1α) in some other tissues, which serves as a transcriptional coactivator. Our previous studies demonstrated that PGC-1α was overexpression in MM cell line RPMI-8226 and knockdown of PGC-1α inhibited proliferation. Herein, we aimed to investigate the significance of OXPHOS in MM and clarify whether PGC-1α is the core regulator of OXPHOS in MM. Methods: Transcriptome data of GSE6477, GSE13591, GSE47552 were downloaded from the Gene Expression Omnibus (GEO) database and then removed batch effects. Gene set variation analysis (GSVA) were used to identify pathways enriched in MM. Differential expression analysis was performed and the PGC-1α expression value was extracted. Gene set enrichment analysis (GSEA) was adopted to explore biological functions involved by PGC-1α. RT-PCR was conducted to reflect the influence of PGC-1α inhibitor on OXPHOS gene set. Mitochondrial morphology was exhibited by transmission electron microscopy analysis. Cell counting kit-8 assay was employed to determine cell viability and flow cytometry analysis was used to assess apoptosis. Results: Integrated bioinformatics analysis revealed that OXPHOS pathway was significantly upregulated in MM than in normal donors (Figure A), and aberrantly overexpression of OXPHOS gene set was confirmed in our MM samples at mRNA level (Figure B-C). Moreover, we also demonstrated that aberrantly overexpression of OXPHOS genes in MM were generally associated with poorer survival (Figure D-F), indicating that OXPHOS appears as a potential oncogenic pathway for MM progression. Meanwhile, bioinformatics results showed that PGC-1α was upregulated in 247 MM patients from GEO database (Figure G). In addition, our results indicated that the overexpression of PGC-1α prevailed among 5 MM cell lines with different inherited backgrounds and our several MM samples (Figure H-I). GSEA analysis showed that gene sets of OXPHOS, TCA cycle, respiratory electron transport, ATP synthesis, were all significantly enriched in MM patients with high PGC-1α expression (Figure J-L). The inhibition of PGC-1α have exerted significant inhibition effect on the transcription of OXPHOS gene set in MM. In accordance with that OXPHOS is a major source of ATP, PGC-1α inhibitor resulted in the reduction of ATP levels of MM cells. Meanwhile, treatment of PGC-1α inhibitor triggered shriveled mitochondria with decreased volume, disorganized cristae, and increased electron density of matrix, indicative of impaired mitochondria OXPHOS. In vitro experiments suggested that PGC-1α inhibitor robustly inhibited proliferation of MM cells in a time and dose-dependent manner, with little effect on normal hematopoietic cells. Besides, PGC-1α inhibitor induced MM cells apoptosis significantly. Conclusions: Our investigations reported for the first time that OXPHOS regulated by PGC-1α may serve as a potential mechanism for MM progression. Besides, this study may provide new strategies for the treatment of MM from the perspective of OXPHOS and its core regulator. Figure Disclosures No relevant conflicts of interest to declare.

Interleukin-22 (IL-22) Binding Protein Constrains IL-22 Activity, Host Defense, and Oxidative Phosphorylation Genes during Pneumococcal Pneumonia

ABSTRACT Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide, and interleukin-22 (IL-22) helps contain pneumococcal burden in lungs and extrapulmonary tissues. Administration of IL-22 increases hepatic complement 3 and complement deposition on bacteria and improves phagocytosis by neutrophils. The effects of IL-22 can be tempered by a secreted natural antagonist, known as IL-22 binding protein (IL-22BP), encoded by Il22ra2. To date, the degree to which IL-22BP controls IL-22 in pulmonary infection is not well defined. Here, we show that Il22ra2 inhibits IL-22 during S. pneumoniae lung infection and that Il22ra2 deficiency favors downregulation of oxidative phosphorylation (OXPHOS) genes in an IL-22-dependent manner. Il22ra2−/− mice are more resistant to S. pneumoniae infection, have increased IL-22 in lung tissues, and sustain longer survival upon infection than control mice. Transcriptome sequencing (RNA-seq) analysis of infected Il22ra2−/− mouse lungs revealed downregulation of genes involved in OXPHOS. Downregulation of this metabolic process is necessary for increased glycolysis, a crucial step for transitioning to a proinflammatory phenotype, in particular macrophages and dendritic cells (DCs). Accordingly, we saw that macrophages from Il22ra2−/− mice displayed reduced OXPHOS gene expression upon infection with S. pneumoniae, changes that were IL-22 dependent. Furthermore, we showed that macrophages express IL-22 receptor subunit alpha-1 (IL-22Ra1) during pneumococcal infection and that Il22ra2−/− macrophages rely more on the glycolytic pathway than wild-type (WT) controls. Together, these data indicate that IL-22BP deficiency enhances IL-22 signaling in the lung, thus contributing to resistance to pneumococcal pneumonia by downregulating OXPHOS genes and increasing glycolysis in macrophages.

Oxidative-phosphorylation genes from the mitochondrial and nuclear genomes co-express in all human tissues except for the ancient brain

In humans, oxidative phosphorylation (OXPHOS), the cellular energy producer, harbors ∼90 nuclear DNA (nDNA)- and mitochondrial DNA (mtDNA)-encoded subunits. Although nDNA- and mtDNA-encoded OXPHOS proteins physically interact, their transcriptional regulation profoundly diverges, thus questioning their co-regulation. To address mtDNA-nDNA gene co-expression, we analyzed ∼8,500 RNA-seq Gene-Tissue-Expression (GTEx) experiments encompassing 48 human tissues. We found overall positive cross-tissue mtDNA-nDNA OXPHOS gene co-expression. Nevertheless, alternatively-spliced variants, as well as certain OXPHOS genes, did not converge into the main OXPHOS gene cluster, suggesting tissue-specific flavor of OXPHOS gene expression. Finally, unlike non-brain body sites, and neocortex and cerebellum (‘mammalian’ brain), negative mito-nuclear expression correlation was found in the hypothalamus, basal ganglia and amygdala (‘ancient brain’). Analyses of co-expression, DNase-seq and ChIP-seq experiments identified candidate RNA-binding genes and CEBPb as best explaining this phenomenon. We suggest that evolutionary convergence of the ‘mammalian’ brain into positive mtDNA-nDNA OXPHOS co-expression reflects adjustment to novel bioenergetics needs.