Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Regulation of Tumor Initiation by the Mitochondrial Pyruvate Carrier.

Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.

SDHAF4 promotes mitochondrial succinate dehydrogenase activity and prevents neurodegeneration.

Succinate dehydrogenase (SDH) occupies a central place in cellular energy production, linking the tricarboxylic cycle with the electron transport chain. As a result, a subset of cancers and neuromuscular disorders result from mutations affecting any of the four SDH structural subunits or either of two known SDH assembly factors. Herein we characterize an evolutionarily conserved SDH assembly factor designated Sdh8/SDHAF4, using yeast, Drosophila, and mammalian cells. Sdh8 interacts specifically with the catalytic Sdh1 subunit in the mitochondrial matrix, facilitating its association with Sdh2 and the subsequent assembly of the SDH holocomplex. These roles for Sdh8 are critical for preventing motility defects and neurodegeneration in Drosophila as well as the excess ROS generated by free Sdh1. These studies provide insights into the mechanisms by which SDH is assembled and raise the possibility that some forms of neuromuscular disease may be associated with mutations that affect this SDH assembly factor.

The LYR factors SDHAF1 and SDHAF3 mediate maturation of the iron-sulfur subunit of succinate dehydrogenase.

Disorders arising from impaired assembly of succinate dehydrogenase (SDH) result in a myriad of pathologies, consistent with its unique role in linking the citric acid cycle and electron transport chain. In spite of this critical function, however, only a few factors are known to be required for SDH assembly and function. We show here that two factors, Sdh6 (SDHAF1) and Sdh7 (SDHAF3), mediate maturation of the FeS cluster SDH subunit (Sdh2/SDHB). Yeast and Drosophila lacking SDHAF3 are impaired in SDH activity with reduced levels of Sdh2. Drosophila lacking the Sdh7 ortholog SDHAF3 are hypersensitive to oxidative stress and exhibit muscular and neuronal dysfunction. Yeast studies revealed that Sdh6 and Sdh7 act together to promote Sdh2 maturation by binding to a Sdh1/Sdh2 intermediate, protecting it from the deleterious effects of oxidants. These studies in yeast and Drosophila raise the possibility that SDHAF3 mutations may be associated with idiopathic SDH-associated diseases.

Coordinated metabolic transitions during Drosophila embryogenesis and the onset of aerobic glycolysis.

Rapidly proliferating cells such as cancer cells and embryonic stem cells rely on a specialized metabolic program known as aerobic glycolysis, which supports biomass production from carbohydrates. The fruit fly Drosophila melanogaster also utilizes aerobic glycolysis to support the rapid growth that occurs during larval development. Here we use singular value decomposition analysis of modENCODE RNA-seq data combined with GC-MS-based metabolomic analysis to analyze the changes in gene expression and metabolism that occur during Drosophila embryogenesis, spanning the onset of aerobic glycolysis. Unexpectedly, we find that the most common pattern of co-expressed genes in embryos includes the global switch to glycolytic gene expression that occurs midway through embryogenesis. In contrast to the canonical aerobic glycolytic pathway, however, which is accompanied by reduced mitochondrial oxidative metabolism, the expression of genes involved in the tricarboxylic cycle (TCA cycle) and the electron transport chain are also upregulated at this time. Mitochondrial activity, however, appears to be attenuated, as embryos exhibit a block in the TCA cycle that results in elevated levels of citrate, isocitrate, and α-ketoglutarate. We also find that genes involved in lipid breakdown and ß-oxidation are upregulated prior to the transcriptional initiation of glycolysis, but are downregulated before the onset of larval development, revealing coordinated use of lipids and carbohydrates during development. These observations demonstrate the efficient use of nutrient stores to support embryonic development, define sequential metabolic transitions during this stage, and demonstrate striking similarities between the metabolic state of late-stage fly embryos and tumor cells.

Transcriptional regulation of xenobiotic detoxification in Drosophila.

Living organisms, from bacteria to humans, display a coordinated transcriptional response to xenobiotic exposure, inducing enzymes and transporters that facilitate detoxification. Several transcription factors have been identified in vertebrates that contribute to this regulatory response. In contrast, little is known about this pathway in insects. Here we show that the Drosophila Nrf2 (NF-E2-related factor 2) ortholog CncC (cap 'n' collar isoform-C) is a central regulator of xenobiotic detoxification responses. A binding site for CncC and its heterodimer partner Maf (muscle aponeurosis fibromatosis) is sufficient and necessary for robust transcriptional responses to three xenobiotic compounds: phenobarbital (PB), chlorpromazine, and caffeine. Genetic manipulations that alter the levels of CncC or its negative regulator, Keap1 (Kelch-like ECH-associated protein 1), lead to predictable changes in xenobiotic-inducible gene expression. Transcriptional profiling studies reveal that more than half of the genes regulated by PB are also controlled by CncC. Consistent with these effects on detoxification gene expression, activation of the CncC/Keap1 pathway in Drosophila is sufficient to confer resistance to the lethal effects of the pesticide malathion. These studies establish a molecular mechanism for the regulation of xenobiotic detoxification in Drosophila and have implications for controlling insect populations and the spread of insect-borne human diseases.

The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth.

Metabolism must be coordinated with development to provide the appropriate energetic needs for each stage in the life cycle. Little is known, however, about how this temporal control is achieved. Here, we show that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. dERR mutants die as larvae with low ATP levels and elevated levels of circulating sugars. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells, supporting the dramatic growth that occurs during larval development. This study shows that dERR plays a central role in carbohydrate metabolism, demonstrates that a proliferative metabolic program is used in normal developmental growth, and provides a molecular context to understand the close association between mammalian ERR family members and cancer.

Med24 and Mdh2 are required for Drosophila larval salivary gland cell death.

The steroid hormone ecdysone triggers the rapid destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here, we show that mutations in Mdh2 and Med24 block caspase cleavage and larval salivary gland cell death. Mdh2 encodes a predicted malate dehydrogenase that localizes to mitochondria. Consistent with this proposed function, Mdh2 mutants have significantly lower levels of ATP and accumulate late-stage citric acid cycle intermediates, suggesting that the cell death defects arise from a deficit in energy production. Med24 encodes a component of the Mediator transcriptional coactivator complex. Unexpectedly, however, expression of the key death regulator genes is normal in Med24 mutant salivary glands. This study identifies novel mechanisms for controlling the destruction of larval tissues during Drosophila metamorphosis and provides new directions for our understanding of steroid-triggered programmed cell death.

Functional interactions between the Moses corepressor and DHR78 nuclear receptor regulate growth in Drosophila.

Expression of the Drosophila orphan nuclear receptor DHR78 is regulated by the steroid hormone ecdysone and is required for growth and viability during larval stages. In contrast to our understanding of its biological functions, however, relatively little is known about how DHR78 acts as a transcription factor. Here we show that DHR78 is an obligate partner for Moses (Middleman of seventy-eight signaling), a SAM (sterile alpha motif) domain-containing cofactor that requires DHR78 for its stability. Unlike other nuclear receptor cofactors, Moses has no obvious interaction domains and displays a unique binding specificity for DHR78. Moses acts as a corepressor, inhibiting DHR78 transcriptional activity independently of histone deacetylation. Consistent with their close association, DHR78 and Moses proteins are coexpressed during development and colocalize to specific genomic targets in chromatin. Moses mutants progress normally through early larval stages, like DHR78 mutants, but display an opposite overgrowth phenotype, with hypertrophy of adult tissues. Genetic interactions between DHR78 and moses result in a similar phenotype, suggesting that the relative dose of Moses and DHR78 regulates growth and prevents cancer. The tight functional association between DHR78 and Moses provides a new paradigm for understanding the molecular mechanisms by which cofactors modulate nuclear receptor signaling pathways.

The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis.

BACKGROUND: The steroid hormone 20-hydroxyecdysone (20E) triggers the major developmental transitions in Drosophila, including molting and metamorphosis, and provides a model system for defining the developmental and molecular mechanisms of steroid signaling. 20E acts via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and Ultraspiracle, to directly regulate target gene transcription. RESULTS: Here we identify the genomic transcriptional response to 20E as well as those genes that are dependent on EcR for their proper regulation. We show that genes regulated by 20E, and dependent on EcR, account for many transcripts that are significantly up- or downregulated at puparium formation. We provide evidence that 20E and EcR participate in the regulation of genes involved in metabolism, stress, and immunity at the onset of metamorphosis. We also present an initial characterization of a 20E primary-response regulatory gene identified in this study, brain tumor (brat), showing that brat mutations lead to defects during metamorphosis and changes in the expression of key 20E-regulated genes. CONCLUSION: This study provides a genome-wide basis for understanding how 20E and its receptor control metamorphosis, as well as a foundation for functional genomic analysis of key regulatory genes in the 20E signaling pathway during insect development.

Powered by gas--a ligand for a fruit fly nuclear receptor.

The difficulty in identifying ligands for nuclear hormone receptors remains an obstacle to understanding their function. For example, in the fruit fly Drosophila melanogaster, only one of its nuclear receptors has a known ligand. In this issue of Cell, report that the fruit fly E75 nuclear receptor contains heme in its ligand binding pocket and that the oxidation state of this molecule controls E75 activity. They also show that E75-heme responds to the small diatomic gases, nitric oxide and carbon monoxide. This study sheds light on how heme, gas signaling, and nuclear receptors interact to control metabolic and developmental pathways.