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[
International Worm Meeting,
2019]
Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitro methylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.
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[
International Worm Meeting,
2019]
Transcription initiation is a dynamic, multi-step process, requiring the ordered recruitment of multiple factors to the core promoter. The highly dynamic nature of transcription means that at any given moment, a particular promoter might be in one of multiple states in different cells, resulting in heterogeneous signals from a population of cells. Most techniques that study transcription initiation, such as chromatin-IP or GRO-seq provide population average measurements of the transcriptional process. To really understand transcription regulation, it is necessary to examine changes in promoter state at the single molecule level. To achieve this, we applied to C. elegans a technique known as "dual enzyme Single Molecule Footprinting (dSMF)". The method involves in vitromethylation of chromatin in intact nuclei with bacterial CpG and GpC methyltransferases. Closed chromatin bound by proteins is protected from methylation, whereas accessible DNA is methylated. After bisulfite conversion, specific amplicons, or genome-wide DNA libraries are sequenced to identify footprints of accessible DNA. Correlation of dSMF data with publicly available genome wide datasets shows that dSMF footprints coincide with nucleosome free regions in promoters and other genomic regions such as HOT sites. To identify what the different footprints represent in terms of promoter states in the transcription cycle, we then inhibit individual steps of the transcription cycle either with chemicals or by blocking engineered cyclin-dependent kinases with ATP analogs. Upon mapping the promoter occupancy states in transcription inhibited conditions, we are able to correlate the different promoter footprints with specific protein complexes present during the different steps of transcription initiation. Our data strengthen our understanding of how chromatin and gene structure regulate gene expression at the level of transcription initiation.
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Ham S, Park J, Park HH, Jung Y, Lee Y, Kim SS, Jeong DE, Kim E, Annibal A, Antebi A, Kim Y, Lee SV, Park S, Kwon S
[
Nat Commun,
2023]
Accumulating evidence indicates that mitochondria play crucial roles in immunity. However, the role of the mitochondrial Krebs cycle in immunity remains largely unknown, in particular at the organism level. Here we show that mitochondrial aconitase, ACO-2, a Krebs cycle enzyme that catalyzes the conversion of citrate to isocitrate, inhibits immunity against pathogenic bacteria in C. elegans. We find that the genetic inhibition of
aco-2 decreases the level of oxaloacetate. This increases the mitochondrial unfolded protein response, subsequently upregulating the transcription factor ATFS-1, which contributes to enhanced immunity against pathogenic bacteria. We show that the genetic inhibition of mammalian ACO2 increases immunity against pathogenic bacteria by modulating the mitochondrial unfolded protein response and oxaloacetate levels in cultured cells. Because mitochondrial aconitase is highly conserved across phyla, a therapeutic strategy targeting ACO2 may eventually help properly control immunity in humans.
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[
Worm Breeder's Gazette,
1993]
Fluoroacetic acid which inhibits aconitase, an enzyme in both the Krebs and glyoxylate cycles, was discovered to be a potent and specific inhibitor of reproduction in a toxicity test using the nematode Caenorhabditis elegans. Fluoroacetic acid reduced reproduction in the second generation by 50% at concentrations 3000 times lower than the LC(50) of 76 mM. Four concentrations (1.7, 4.2,8.5, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations the survival rates were unaffected, and first generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. To determine whether fluoroacetic acid inhibited reproduction because it interfered with both the Krebs cycle and the glyoxylate cycle, malonic acid which inhibits the Krebs cycle and itaconic acid which inhibits the glyoxylate cycle were tested individually and in combination against C. elegans. If the potent reproduction effect of fluoroacetic acid relative to lethality is caused by inhibiting both the Krebs and glyoxylate cycles, the combination of malonic acid and itaconic acid would be expected to produce results qualitatively similar to those obtained with fluoroacetic acid. For the mixture, a large ratio of the LC(50) to EC(50) for reproduction would indicate synergy. Concentration-response curves for the survival assay and the reproduction assay were developed for each test compound and combination of compounds. The probit method was used to calculate the LC(50) and EC(50) for reproduction. The ratio of the LC(50) to EC(50) was calculated for each and is presented in the Table. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid, possibly one that is specific to reproduction and possibly one which affects aconitase.
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[
J Nematol,
1993]
Fluoroacetic acid is known to lead to inhibition of aconitase and block both the Krebs and glyoxylate cycles. In this study, we discovered it to be a potent and specific inhibitor of reproduction in a bioassay using the nematode Caenorhabditis elegans. Fluoroacetic acid added to the growth medium reduced reproduction in the second generation by 50% at concentrations 3,000 times lower than the concentrations that reduced 24-hour survival by 50%. Four concentrations (2, 4, 8, and 17 mM) of fluoroacetic acid were tested thoroughly. At the two lower concentrations, the survival rates were unaffected, and first-generation reproduction was greatly reduced but not completely eliminated. Survival was reduced at the higher concentrations. Malonate, which inhibits the Krebs cycle, and itaconate, which inhibits the glyoxylate cycle, were tested individually and in combination. The combination did not specifically inhibit reproduction, suggesting another mode of action for fluoroacetic acid. Fluoroacetic acid shows promise as a tool in studies requiring age synchrony.
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[
Front Genet,
2015]
Indy (I'm Not Dead Yet) encodes the fly homolog of a mammalian SLC13A5 plasma membrane transporter. INDY is expressed in metabolically active tissues functioning as a transporter of Krebs cycle intermediates with the highest affinity for citrate. Decreased expression of the Indy gene extends longevity in Drosophila and C. elegans. Reduction of INDY or its respective homologs in C. elegans and mice induces metabolic and physiological changes similar to those observed in calorie restriction. It is thought that these physiological changes are due to altered levels of cytoplasmic citrate, which directly impacts Krebs cycle energy production as a result of shifts in substrate availability. Citrate cleavage is a key event during lipid and glucose metabolism; thus, reduction of citrate due to Indy reduction alters these processes. With regards to mammals, mice with reduced Indy (mIndy(-/-)) also exhibit changes in glucose metabolism, mitochondrial biogenesis and are protected from the negative effects of a high calorie diet. Together, these data support a role for Indy as a metabolic regulator, which suggests INDY as a therapeutic target for treatment of diet and age-related disorders such as Type II Diabetes and obesity.
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[
Mech Ageing Dev,
2003]
The dauer larva, a non-feeding and developmentally arrested stage of the free-living nematode Caenorhabditis elegans, is morphologically and physiologically specialized for survival and dispersal during adverse growth conditions. The ability of dauer larvae to live several times longer than the continuous developmental life span has been attributed in part to a repressed metabolism. We used serial analysis of gene expression (SAGE) profiles from dauer larvae and mixed growing stages to compare expression patterns for genes with known or predicted roles in glycolysis, gluconeogenesis, glycogen metabolism, the Krebs and glyoxylate cycles, and selected fermentation pathways. Ratios of mixed:dauer transcripts indicated non-dauer enrichment that was consistent with previously determined adult:dauer enzyme activity ratios for hexokinase (glycolysis), phosphoenolpyruvate carboxykinase and fructose 1,6-bisphosphatase (gluconeogenesis), isocitrate dehydrogenase (NADP-dependent), and isocitrate lyase-malate synthase (glyoxylate cycle). Transcripts for the majority of Krebs cycle components were not differentially represented in the two profiles. Transcript abundance for pyruvate kinase, alcohol dehydrogenase, a putative cytosolic fumarate reductase, two pyruvate dehydrogenase components, and a succinyl CoA synthetase alpha subunit implied that anaerobic pathways were upregulated in dauer larvae. Generation of nutritive fermentation byproducts and the moderation of oxidative damage are potential benefits of a hypoxic dauer interior.
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[
J Pharmacol Exp Ther,
2015]
NaCT (SLC13A5) is a Na(+)-coupled transporter for Krebs cycle intermediates and is expressed predominantly in the liver. Human NaCT is relatively specific for citrate compared with other Krebs cycle intermediates. The transport activity of human NaCT is stimulated by Li(+), whereas that of rat NaCT is inhibited by Li(+). We studied the influence of Li(+) on NaCTs cloned from eight different species. Li(+) stimulated the activity of only NaCTs from primates (human, chimpanzee, and monkey); by contrast, NaCTs from nonprimate species (mouse, rat, dog, and zebrafish) were inhibited by Li(+). Caenorhabditis elegans NaCT was not affected by Li(+). With human NaCT, the Li(+)-induced increase in transport activity was associated with the conversion of the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type. H(+) was able to substitute for Li(+) in eliciting the stimulatory effect. The amino acid Phe500 in human NaCT was critical for Li(+)/H(+)-induced stimulation. Mutation of this amino acid to tryptophan (F500W) markedly increased the basal transport activity of human NaCT in the absence of Li(+), but the ability of Li(+) to stimulate the transporter was almost completely lost with this mutant. Substitution of Phe500 with tryptophan in human NaCT converted the transporter from a low-affinity/high-capacity type to a high-affinity/low-capacity type, an effect similar to that of Li(+) on the wild-type NaCT. These studies show that Li(+)-induced activation of NaCT is specific for the transporter in primates and that the region surrounding Phe500 in primate NaCTs is important for the Li(+) effect.
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[
Biomol Ther (Seoul),
2016]
Alpha-ketoglutarate (AKG) is a key molecule in the Krebs cycle determining the overall rate of the citric acid cycle of the organism. It is a nitrogen scavenger and a source of glutamate and glutamine that stimulates protein synthesis and inhibits protein degradation in muscles. AKG as a precursor of glutamate and glutamine is a central metabolic fuel for cells of the gastrointestinal tract as well. AKG can decrease protein catabolism and increase protein synthesis to enhance bone tissue formation in the skeletal muscles and can be used in clinical applications. In addition to these health benefits, a recent study has shown that AKG can extend the lifespan of adult Caenorhabditis elegans by inhibiting ATP synthase and TOR. AKG not only extends lifespan, but also delays age-related disease. In this review, we will summarize the advances in AKG research field, in the content of its physiological functions and applications.
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[
Reprod Toxicol,
2019]
Mitochondrial toxicity has been proposed as a potential cause of developmental defects in humans. We evaluated 51 organophosphate and carbamate pesticides using the U.S. EPA ToxCast and Tox21 databases. Only a small number of them bind directly to cholinesterases in the parent form. The hydrophobicity of organophosphate pesticides is correlated significantly to TSPO binding affinity, mitochondrial membrane potential reduction in HepG2 cells, and developmental toxicity in Caenorhabditis elegans and Danio rerio (p < 0.05). Structural analysis suggests that in some cases the Krebs cycle is a potential target of organophosphate and carbamate exposure at early life stages. The results support the hypothesis that mitochondrial effects of some organophosphate pesticides-particularly those that require enzymatic activation to the oxon form-may augment the documented effects of disruption of acetylcholine signaling. This study provides a proof of concept for applying new approach methodologies to interrogate mechanisms of action for cumulative risk assessment.