bims-camemi Biomed News
on Mitochondrial metabolism in cancer
Issue of 2023‒07‒09
forty-four papers selected by
Christian Frezza, Universität zu Köln



  1. Cell Death Dis. 2023 Jul 06. 14(7): 403
      Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.
    DOI:  https://doi.org/10.1038/s41419-023-05927-5
  2. Nat Cell Biol. 2023 Jul 03.
      Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.
    DOI:  https://doi.org/10.1038/s41556-023-01180-2
  3. Cell Metab. 2023 Jun 28. pii: S1550-4131(23)00215-2. [Epub ahead of print]
      Tumor cell phenotypes and anti-tumor immune responses are shaped by local metabolite availability, but intratumoral metabolite heterogeneity (IMH) and its phenotypic consequences remain poorly understood. To study IMH, we profiled tumor/normal regions from clear cell renal cell carcinoma (ccRCC) patients. A common pattern of IMH transcended all patients, characterized by correlated fluctuations in the abundance of metabolites and processes associated with ferroptosis. Analysis of intratumoral metabolite-RNA covariation revealed that the immune composition of the microenvironment, especially the abundance of myeloid cells, drove intratumoral metabolite variation. Motivated by the strength of RNA-metabolite covariation and the clinical significance of RNA biomarkers in ccRCC, we inferred metabolomic profiles from the RNA sequencing data of ccRCC patients enrolled in 7 clinical trials, and we ultimately identifyied metabolite biomarkers associated with response to anti-angiogenic agents. Local metabolic phenotypes, therefore, emerge in tandem with the immune microenvironment, influence ongoing tumor evolution, and are associated with therapeutic sensitivity.
    Keywords:  cancer metabolism; immune microenvironment; immunometabolism; imputation; intratumoral heterogeneity; metabolites; metabolomics; renal cell carcinoma; transcriptomics
    DOI:  https://doi.org/10.1016/j.cmet.2023.06.005
  4. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00762-3. [Epub ahead of print]42(7): 112751
      Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to profound epigenetic changes and the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The extent to which chromatin remodeling shapes this anti-oxidant response is currently unknown. Here, we explored the effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the remodeled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor that regulates anti-oxidant response genes and subsequent metabolic rewiring cooperating without direct interaction with the anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides additional insights into the molecular mechanisms behind cell responses to fumarate accumulation and potentially provides further avenues for therapeutic intervention for HLRCC.
    Keywords:  CP: Cancer; CP: Molecular biology; FOXA2; NRF2; anti-oxidant response; fumarate hydratase
    DOI:  https://doi.org/10.1016/j.celrep.2023.112751
  5. Trends Cell Biol. 2023 Jul 05. pii: S0962-8924(23)00125-3. [Epub ahead of print]
      Ferroptosis is a form of necrotic cell death characterized by iron-dependent lipid peroxidation culminating in membrane rupture. Accumulating evidence links ferroptosis to multiple cardiac diseases and identifies mitochondria as important regulators of ferroptosis. Mitochondria are not only a major source of reactive oxygen species (ROS) but also counteract ferroptosis by preserving cellular redox balance and oxidative defense. Recent evidence has revealed that the mitochondrial integrated stress response limits oxidative stress and ferroptosis in oxidative phosphorylation (OXPHOS)-deficient cardiomyocytes and protects against mitochondrial cardiomyopathy. We summarize the multiple ways in which mitochondria modulate the susceptibility of cells to ferroptosis, and discuss the implications of ferroptosis for cardiomyopathies in mitochondrial disease.
    Keywords:  Gpx4; ferroptosis; integrated stress response; mitochondrial cardiomyopathy
    DOI:  https://doi.org/10.1016/j.tcb.2023.06.002
  6. PLoS Genet. 2023 Jul 03. 19(7): e1010793
      Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.
    DOI:  https://doi.org/10.1371/journal.pgen.1010793
  7. Biochem J. 2023 Jul 12. 480(13): 909-919
      Energy and metabolic homeostasis at the level of the whole body are dictated by the balance between nutrient intake/utilization, bioenergetic potential, and energy expenditure, which are tightly coupled with fed/fast cycles and circadian oscillation. Emerging literature has highlighted the importance of each of these mechanisms that are essential to maintain physiological homeostasis. Lifestyle changes predominantly associated with altered fed-fast and circadian cycles are well established to affect systemic metabolism and energetics, and hence contribute to pathophysiological states. Therefore, it is not surprising that mitochondria have emerged as being pivotal in maintaining physiological homeostasis through daily oscillations/fluctuations in nutrient inputs and light-dark/sleep-wake cycles. Moreover, given the inherent association between mitochondrial dynamics/morphology and functions, it is important to understand the phenomenological and mechanistic underpinnings of fed-fast and circadian cycles dependent remodeling of mitochondria. In this regard, we have summarized the current status of the field in addition to providing a perspective vis-a-vis the complexity of cell-autonomous and non-cell-autonomous signals that dictate mitochondrial dynamics. We also highlight the lacunae besides speculating on prospective efforts that will possibly redefine our insights into the diurnal orchestration of fission/fusion events, which are ultimately coupled to the mitochondrial output.
    Keywords:  circadian clock; fed–fast cycles; fission/fusion; metabolic sensing; mitochondrial biogenesis; mitochondrial functions
    DOI:  https://doi.org/10.1042/BCJ20220378
  8. J Vis Exp. 2023 06 16.
      Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.
    DOI:  https://doi.org/10.3791/65444
  9. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00735-0. [Epub ahead of print]42(7): 112724
      The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
    Keywords:  ATF4; CHAC1; CP: Cell biology; GSH; NRF2; SLC7A11; integrated stress response; melanoma
    DOI:  https://doi.org/10.1016/j.celrep.2023.112724
  10. Mol Cell. 2023 Jul 06. pii: S1097-2765(23)00473-2. [Epub ahead of print]83(13): 2149-2151
      
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.026
  11. Free Radic Biol Med. 2023 Jul 05. pii: S0891-5849(23)00511-7. [Epub ahead of print]
      Mitochondria are vital for energy production and Redox homeostasis, yet knowledge of relevant mechanisms remains limited. Here, through a genome-wide CRISPR-Cas9 knockout screening, we have identified DMT1 as a major regulator of mitochondria membrane potential. Our findings demonstrate that DMT1 deficiency increased the activity of mitochondrial complex I and reduced that of complex III. Enhanced complex I activity leads to increased NAD+ production, which activates IDH2 by promoting its deacetylation via SIRT3, This results in higher levels of NADPH and GSH, which improve antioxidant capacity during Erastin-induced ferroptosis. Meanwhile, loss of complex III activity impairs mitochondrial biogenesis and promotes mitophagy, contributing to suppression of ferroptosis. Thus, DMT1 differentially regulates activities of mitochondrial complex I and III to cooperatly suppress Erastin-induced ferroptosis. Furthermore, NMN, an alternative method of increasing mitochondrial NAD+, exhibits similar protective effects against ferroptosis by boosting GSH in a manner similar to DMT1 deficiency, shedding a light on potential therapeutic strategy for ferroptosis-related pathologies.
    Keywords:  DMT1; Ferroptosis; Mitochondria NAD(+); REDOX homeostasis
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.06.023
  12. Nature. 2023 Jul 05.
      Cancer cells evade T cell-mediated killing through tumour-immune interactions whose mechanisms are not well understood1,2. Dendritic cells (DCs), especially type-1 conventional DCs (cDC1s), mediate T cell priming and therapeutic efficacy against tumours3. DC functions are orchestrated by pattern recognition receptors3-5, although other signals involved remain incompletely defined. Nutrients are emerging mediators of adaptive immunity6-8, but whether nutrients affect DC function or communication between innate and adaptive immune cells is largely unresolved. Here we establish glutamine as an intercellular metabolic checkpoint that dictates tumour-cDC1 crosstalk and licenses cDC1 function in activating cytotoxic T cells. Intratumoral glutamine supplementation inhibits tumour growth by augmenting cDC1-mediated CD8+ T cell immunity, and overcomes therapeutic resistance to checkpoint blockade and T cell-mediated immunotherapies. Mechanistically, tumour cells and cDC1s compete for glutamine uptake via the transporter SLC38A2 to tune anti-tumour immunity. Nutrient screening and integrative analyses show that glutamine is the dominant amino acid in promoting cDC1 function. Further, glutamine signalling via FLCN impinges on TFEB function. Loss of FLCN in DCs selectively impairs cDC1 function in vivo in a TFEB-dependent manner and phenocopies SLC38A2 deficiency by eliminating the anti-tumour therapeutic effect of glutamine supplementation. Our findings establish glutamine-mediated intercellular metabolic crosstalk between tumour cells and cDC1s that underpins tumour immune evasion, and reveal glutamine acquisition and signalling in cDC1s as limiting events for DC activation and putative targets for cancer treatment.
    DOI:  https://doi.org/10.1038/s41586-023-06299-8
  13. PLoS One. 2023 ;18(7): e0286525
      Reprogramming metabolism is of great therapeutic interest for reducing morbidity and mortality during sepsis-induced critical illness. Disappointing results from randomized controlled trials targeting glutamine and antioxidant metabolism in patients with sepsis have begged a deeper understanding of the tissue-specific metabolic response to sepsis. The current study sought to fill this gap. We analyzed skeletal muscle transcriptomics of critically ill patients, versus elective surgical controls, which revealed reduced expression of genes involved in mitochondrial metabolism and electron transport, with increases in glutathione cycling, glutamine, branched chain, and aromatic amino acid transport. We then performed untargeted metabolomics and 13C isotope tracing to analyze systemic and tissue specific metabolic phenotyping in a murine polymicrobial sepsis model. We found an increased number of correlations between the metabolomes of liver, kidney, and spleen, with loss of correlations between the heart and quadriceps and all other organs, pointing to a shared metabolic signature within vital abdominal organs, and unique metabolic signatures for muscles during sepsis. A lowered GSH:GSSG and elevated AMP:ATP ratio in the liver underlie the significant upregulation of isotopically labeled glutamine's contribution to TCA cycle anaplerosis and glutamine-derived glutathione biosynthesis; meanwhile, the skeletal muscle and spleen were the only organs where glutamine's contribution to the TCA cycle was significantly suppressed. These results highlight tissue-specific mitochondrial reprogramming to support liver energetic demands and antioxidant synthesis, rather than global mitochondrial dysfunction, as a metabolic consequence of sepsis.
    DOI:  https://doi.org/10.1371/journal.pone.0286525
  14. Curr Opin Chem Biol. 2023 Jul 04. pii: S1367-5931(23)00100-X. [Epub ahead of print]76 102362
      Mass spectrometry imaging (MSI) is an emerging technology in cancer metabolomics. Desorption electrospray ionization (DESI) and matrix-assisted laser desorption ionization (MALDI) MSI are complementary techniques to identify hundreds of metabolites in space with close to single-cell resolution. This technology leap enables research focusing on tumor heterogeneity, cancer cell plasticity, and the communication signals between cancer and stromal cells in the tumor microenvironment (TME). Currently, unprecedented knowledge is generated using spatial metabolomics in fundamental cancer research. Yet, also translational applications are emerging, including the assessment of spatial drug distribution in organs and tumors. Moreover, clinical research investigates the use of spatial metabolomics as a rapid pathology tool during cancer surgeries. Here, we summarize MSI applications, the knowledge gained by this technology in space, future directions, and developments needed.
    Keywords:  Cancer cell plasticity; Cancer research; Labelled isotope tracing; Mass spectrometry imaging; Metabolite identification; Metabolomics; Tumor heterogeneity; Tumor microenvironment (TME)
    DOI:  https://doi.org/10.1016/j.cbpa.2023.102362
  15. Cell Chem Biol. 2023 Jun 22. pii: S2451-9456(23)00186-1. [Epub ahead of print]
      Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.
    Keywords:  TurboID; chemoproteomics; cysteine; cysteine oxidation; lipopolysaccharide; macrophages; mitochondria; proximity labeling
    DOI:  https://doi.org/10.1016/j.chembiol.2023.06.008
  16. bioRxiv. 2023 Jun 13. pii: 2023.06.13.544768. [Epub ahead of print]
      Mitochondria play a crucial role in the regulation of cellular metabolism and signalling. Mitochondrial activity is modulated by the processes of mitochondrial fission and fusion, which are required to properly balance respiratory and metabolic functions, transfer material between mitochondria, and remove damaged or defective mitochondria. Mitochondrial fission occurs at sites of contact between the endoplasmic reticulum (ER) and mitochondria, and is dependent on the formation of mitochondria- and ER-associated actin filaments that drive the recruitment and activation of the fission GTPase DRP1. On the other hand, the role of mitochondria- and ER-associated actin filaments in mitochondrial fusion remains unknown. Here we show that preventing the formation of actin filaments on either mitochondria or the ER using organelle-targeted Disassembly-promoting, encodable Actin tools (DeActs) blocks both mitochondrial fission and fusion. We show that fusion but not fission is dependent on Arp2/3, and both fission and fusion are dependent on INF2 formin-dependent actin polymerization. Together, our work introduces a novel method for perturbing organelle-associated actin filaments, and demonstrates a previously unknown role for mitochondria- and ER-associated actin in mitochondrial fusion.
    DOI:  https://doi.org/10.1101/2023.06.13.544768
  17. Epigenetics Chromatin. 2023 Jul 06. 16(1): 29
      Super-enhancers are large, densely concentrated swaths of enhancers that regulate genes critical for cell identity. Tumorigenesis is accompanied by changes in the super-enhancer landscape. These aberrant super-enhancers commonly form to activate proto-oncogenes, or other genes upon which cancer cells depend, that initiate tumorigenesis, promote tumor proliferation, and increase the fitness of cancer cells to survive in the tumor microenvironment. These include well-recognized master regulators of proliferation in the setting of cancer, such as the transcription factor MYC which is under the control of numerous super-enhancers gained in cancer compared to normal tissues. This Review will cover the expanding cell-intrinsic and cell-extrinsic etiology of these super-enhancer changes in cancer, including somatic mutations, copy number variation, fusion events, extrachromosomal DNA, and 3D chromatin architecture, as well as those activated by inflammation, extra-cellular signaling, and the tumor microenvironment.
    Keywords:  Cancer; Enhancers; Extrachromosomal DNA; Inflammation; Insulators; Non-coding mutations; Phase separation; Super-enhancers; Therapeutic resistance; Topologically associated domain; Tumor microenvironment
    DOI:  https://doi.org/10.1186/s13072-023-00502-w
  18. Cells. 2023 May 17. pii: 1409. [Epub ahead of print]12(10):
      The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca2+-activated, high-conductance channels in the inner membrane of mitochondria from a variety of eukaryotes led to renewed interest in the permeability transition (PT), a permeability increase mediated by the PT pore (PTP). The PT is a Ca2+-dependent permeability increase in the inner mitochondrial membrane whose function and underlying molecular mechanisms have challenged scientists for the last 70 years. Although most of our knowledge about the PTP comes from studies in mammals, recent data obtained in other species highlighted substantial differences that could be perhaps attributed to specific features of F-ATP synthase and/or ANT. Strikingly, the anoxia and salt-tolerant brine shrimp Artemia franciscana does not undergo a PT in spite of its ability to take up and store Ca2+ in mitochondria, and the anoxia-resistant Drosophila melanogaster displays a low-conductance, selective Ca2+-induced Ca2+ release channel rather than a PTP. In mammals, the PT provides a mechanism for the release of cytochrome c and other proapoptotic proteins and mediates various forms of cell death. In this review, we cover the features of the PT (or lack thereof) in mammals, yeast, Drosophila melanogaster, Artemia franciscana and Caenorhabditis elegans, and we discuss the presence of the intrinsic pathway of apoptosis and of other forms of cell death. We hope that this exercise may help elucidate the function(s) of the PT and its possible role in evolution and inspire further tests to define its molecular nature.
    Keywords:  ATP synthase; adenine nucleotide translocase; calcium signaling; cell death; channels; mitochondria; permeability transition
    DOI:  https://doi.org/10.3390/cells12101409
  19. Mol Cell. 2023 Jun 26. pii: S1097-2765(23)00429-X. [Epub ahead of print]
      K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.
    Keywords:  Ago2; CLIP-seq; K-Ras; casein kinase; colon; colorectal cancer; miRNA
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.008
  20. Nat Biotechnol. 2023 Jul 06.
      Characterization of somatic mutations at single-cell resolution is essential to study cancer evolution, clonal mosaicism and cell plasticity. Here, we describe SComatic, an algorithm designed for the detection of somatic mutations in single-cell transcriptomic and ATAC-seq (assay for transposase-accessible chromatin sequence) data sets directly without requiring matched bulk or single-cell DNA sequencing data. SComatic distinguishes somatic mutations from polymorphisms, RNA-editing events and artefacts using filters and statistical tests parameterized on non-neoplastic samples. Using >2.6 million single cells from 688 single-cell RNA-seq (scRNA-seq) and single-cell ATAC-seq (scATAC-seq) data sets spanning cancer and non-neoplastic samples, we show that SComatic detects mutations in single cells accurately, even in differentiated cells from polyclonal tissues that are not amenable to mutation detection using existing methods. Validated against matched genome sequencing and scRNA-seq data, SComatic achieves F1 scores between 0.6 and 0.7 across diverse data sets, in comparison to 0.2-0.4 for the second-best performing method. In summary, SComatic permits de novo mutational signature analysis, and the study of clonal heterogeneity and mutational burdens at single-cell resolution.
    DOI:  https://doi.org/10.1038/s41587-023-01863-z
  21. Cell Metab. 2023 Jun 25. pii: S1550-4131(23)00218-8. [Epub ahead of print]
      An epidemic of obesity has affected large portions of the world, increasing the risk of developing many different age-associated diseases, including cancer, cardiovascular disease, and diabetes. In contrast with the prevailing notion that "a calorie is just a calorie," there are clear differences, within and between individuals, in the metabolic response to different macronutrient sources. Recent findings challenge this oversimplification; calories from different macronutrient sources or consumed at different times of day have metabolic effects beyond their value as fuel. Here, we summarize discussions conducted at a recent NIH workshop that brought together experts in calorie restriction, macronutrient composition, and time-restricted feeding to discuss how dietary composition and feeding schedule impact whole-body metabolism, longevity, and healthspan. These discussions may provide insights into the long-sought molecular mechanisms engaged by calorie restriction to extend lifespan, lead to novel therapies, and potentially inform the development of a personalized food-as-medicine approach to healthy aging.
    Keywords:  calorie restriction; fasting; isoleucine; ketogenesis; methionine; protein restriction; time-restricted feeding
    DOI:  https://doi.org/10.1016/j.cmet.2023.06.008
  22. Trends Cancer. 2023 Jul 01. pii: S2405-8033(23)00106-1. [Epub ahead of print]
      The microphthalmia/transcription factor E (MiT/TFE) transcription factors (TFs; TFEB, TFE3, MITF, and TFEC) play a central role in cellular catabolism and quality control and are subject to extensive layers of regulation that influence their localization, stability, and activity. Recent studies have highlighted a broader role for these TFs in driving diverse stress-adaptation pathways, which manifest in a context- and tissue-dependent manner. Several human cancers upregulate the MiT/TFE factors to survive extreme fluctuations in nutrients, energy, and pharmacological challenges. Emerging data suggest that reduced activity of the MiT/TFE factors can also promote tumorigenesis. Here, we outline recent findings relating to novel mechanisms of regulation and activity of MiT/TFE proteins across some of the most aggressive human cancers.
    Keywords:  TFE3; TFEB; autophagy; cancer; lysosome; mTORC1; metabolism
    DOI:  https://doi.org/10.1016/j.trecan.2023.06.005
  23. Nat Cell Biol. 2023 Jul 06.
      Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.
    DOI:  https://doi.org/10.1038/s41556-023-01171-3
  24. Trends Cell Biol. 2023 Jun 21. pii: S0962-8924(23)00086-7. [Epub ahead of print]
      Ferroptosis is an iron-dependent lethal mechanism that can be activated in disease and is a proposed target for cancer therapy. Ferroptosis is defined by the overwhelming accumulation of membrane lipid peroxides. Ferroptotic lipid peroxidation is initiated on internal membranes and then appears at the plasma membrane, triggering lethal ion imbalances and membrane permeabilization. Sensitivity to ferroptosis is governed by the levels of peroxidizable polyunsaturated lipids and associated lipid metabolic enzymes. A different network of enzymes and endogenous metabolites restrains lipid peroxidation by interfering with the initiation or propagation of this process. This emerging understanding is informing new approaches to treat disease by modulating lipid metabolism to enhance or inhibit ferroptosis.
    Keywords:  MUFA; PUFA; cell death; ferroptosis; lipid; metabolism
    DOI:  https://doi.org/10.1016/j.tcb.2023.05.003
  25. Plant Physiol Biochem. 2023 Jun 22. pii: S0981-9428(23)00373-X. [Epub ahead of print]201 107862
      Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.
    Keywords:  (13)C-labelling analysis; Gluconeogenesis; Glutamate; Metabolic network; Metabolic regulation; Phosphoenolpyruvate carboxylase; TCA cycle
    DOI:  https://doi.org/10.1016/j.plaphy.2023.107862
  26. Elife. 2023 Jul 07. pii: e84077. [Epub ahead of print]12
      Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.
    Keywords:  FAD; cell biology; gluconeogenesis; inborn errors of metabolism; metabolism; mouse; nuclear receptor
    DOI:  https://doi.org/10.7554/eLife.84077
  27. Nat Commun. 2023 07 05. 14(1): 3966
      KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention.
    DOI:  https://doi.org/10.1038/s41467-023-39591-2
  28. Nature. 2023 Jul 05.
      Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.
    DOI:  https://doi.org/10.1038/s41586-023-06303-1
  29. Cell Rep. 2023 Jun 30. pii: S2211-1247(23)00719-2. [Epub ahead of print]42(7): 112708
      Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine the impact of autophagic modulation in the context of inflammatory conditions are lacking. Here, using mice with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production during a model of macrophage activation syndrome and in adherent-invasive Escherichia coli (AIEC) infection. Moreover, loss of functional autophagy through conditional deletion of Beclin1 in myeloid cells significantly enhances innate immunity in these contexts. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.
    Keywords:  CP: Immunology; RNF128; TBK1 signaling; autophagy; glutamine metabolism; glutathione metabolism; infection; inflammation; macrophages; proteogenomics
    DOI:  https://doi.org/10.1016/j.celrep.2023.112708
  30. Cell Death Discov. 2023 Jul 05. 9(1): 226
      STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.
    DOI:  https://doi.org/10.1038/s41420-023-01507-w
  31. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00747-7. [Epub ahead of print]42(7): 112736
      Patients with von Hippel-Lindau disease (vHL) are at risk of developing spatially and temporally multiple clear cell renal cell carcinomas (ccRCCs), which offers a valuable opportunity to analyze inter- and intra-tumor heterogeneity of genetic and immune profiles within the same patient. Here, we perform whole-exome and RNA sequencing, digital gene expression, and immunohistochemical analyses for 81 samples from 51 ccRCCs of 10 patients with vHL. Inherited ccRCCs are clonally independent and have less genomic alterations than sporadic ccRCCs. Hierarchical clustering of transcriptome profiles shows two clusters with distinct immune signatures: immune hot and cold clusters. Interestingly, not only samples from the same tumors but also different tumors from the same patients tend to show a similar immune signature, whereas samples from different patients frequently exhibit different signatures. Our findings reveal the genetic and immune landscape of inherited ccRCCs, demonstrating the relevance of host factors in shaping anti-tumor immunity.
    Keywords:  CP: Cancer; clear cell renal cell carcinoma; copy-number alteration; hereditary cancer syndrome; heterogeneity; immune profile; mutation; tumor microenvironment; von Hippel-Lindau disease
    DOI:  https://doi.org/10.1016/j.celrep.2023.112736
  32. Nat Cell Biol. 2023 Jul 03.
      The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.
    DOI:  https://doi.org/10.1038/s41556-023-01170-4
  33. Cell Death Dis. 2023 Jul 06. 14(7): 401
      Sepsis involves endothelial cell (EC) dysfunction, which contributes to multiple organ failure. To improve therapeutic prospects, elucidating molecular mechanisms of vascular dysfunction is of the essence. ATP-citrate lyase (ACLY) directs glucose metabolic fluxes to de novo lipogenesis by generating acetyl-Co-enzyme A (acetyl-CoA), which facilitates transcriptional priming via protein acetylation. It is well illustrated that ACLY participates in promoting cancer metastasis and fatty liver diseases. Its biological functions in ECs during sepsis remain unclear. We found that plasma levels of ACLY were increased in septic patients and were positively correlated with interleukin (IL)-6, soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule 1 (sVCAM-1), and lactate levels. ACLY inhibition significantly ameliorated lipopolysaccharide challenge-induced EC proinflammatory response in vitro and organ injury in vivo. The metabolomic analysis revealed that ACLY blockade fostered ECs a quiescent status by reducing the levels of glycolytic and lipogenic metabolites. Mechanistically, ACLY promoted forkhead box O1 (FoxO1) and histone H3 acetylation, thereby increasing the transcription of c-Myc (MYC) to facilitate the expression of proinflammatory and gluco-lipogenic genes. Our findings revealed that ACLY promoted EC gluco-lipogenic metabolism and proinflammatory response through acetylation-mediated MYC transcription, suggesting ACLY as the potential therapeutic target for treating sepsis-associated EC dysfunction and organ injury.
    DOI:  https://doi.org/10.1038/s41419-023-05932-8
  34. Nat Cell Biol. 2023 Jul 06.
      Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.
    DOI:  https://doi.org/10.1038/s41556-023-01167-z
  35. Nat Commun. 2023 07 03. 14(1): 3921
      The increasing integration between biological and digital interfaces has led to heightened interest in utilizing biological materials to store digital data, with the most promising one involving the storage of data within defined sequences of DNA that are created by de novo DNA synthesis. However, there is a lack of methods that can obviate the need for de novo DNA synthesis, which tends to be costly and inefficient. Here, in this work, we detail a method of capturing 2-dimensional light patterns into DNA, by utilizing optogenetic circuits to record light exposure into DNA, encoding spatial locations with barcoding, and retrieving stored images via high-throughput next-generation sequencing. We demonstrate the encoding of multiple images into DNA, totaling 1152 bits, selective image retrieval, as well as robustness to drying, heat and UV. We also demonstrate successful multiplexing using multiple wavelengths of light, capturing 2 different images simultaneously using red and blue light. This work thus establishes a 'living digital camera', paving the way towards integrating biological systems with digital devices.
    DOI:  https://doi.org/10.1038/s41467-023-38876-w
  36. EMBO Rep. 2023 Jul 04. e57499
      Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.
    Keywords:  GSK3β; cholesterol; endoplasmic reticulum; mitochondria; tau protein
    DOI:  https://doi.org/10.15252/embr.202357499
  37. Curr Biol. 2023 Jun 23. pii: S0960-9822(23)00777-7. [Epub ahead of print]
      Competition drives rapid evolution, which, in turn, alters the trajectory of ecological communities. These eco-evolutionary dynamics are increasingly well-appreciated, but we lack a mechanistic framework for identifying the types of traits that will evolve and their trajectories. Metabolic theory offers explicit predictions for how competition should shape the (co)evolution of metabolism and size, but these are untested, particularly in eukaryotes. We use experimental evolution of a eukaryotic microalga to examine how metabolism, size, and demography coevolve under inter- and intraspecific competition. We find that the focal species evolves in accordance with the predictions of metabolic theory, reducing metabolic costs and maximizing population carrying capacity via changes in cell size. The smaller-evolved cells initially had lower population growth rates, as expected from their hyper-allometric metabolic scaling, but longer-term evolution yielded important departures from theory: we observed improvements in both population growth rate and carrying capacity. The evasion of this trade-off arose due to the rapid evolution of metabolic plasticity. Lineages exposed to competition evolved more labile metabolisms that tracked resource availability more effectively than lineages that were competition-free. That metabolic evolution can occur is unsurprising, but our finding that metabolic plasticity also co-evolves rapidly is new. Metabolic theory provides a powerful theoretical basis for predicting the eco-evolutionary responses to changing resource regimes driven by global change. Metabolic theory needs also to be updated to incorporate the effects of metabolic plasticity on the link between metabolism and demography, as this likely plays an underappreciated role in mediating eco-evolutionary dynamics of competition.
    Keywords:  community; density dependence; energy fluxes; energy use; fitness; phytoplankton; species interactions
    DOI:  https://doi.org/10.1016/j.cub.2023.06.026
  38. Mol Cell. 2023 Jun 24. pii: S1097-2765(23)00430-6. [Epub ahead of print]
      Nuclear receptor-binding SET-domain protein 1 (NSD1), a methyltransferase that catalyzes H3K36me2, is essential for mammalian development and is frequently dysregulated in diseases, including Sotos syndrome. Despite the impacts of H3K36me2 on H3K27me3 and DNA methylation, the direct role of NSD1 in transcriptional regulation remains largely unknown. Here, we show that NSD1 and H3K36me2 are enriched at cis-regulatory elements, particularly enhancers. NSD1 enhancer association is conferred by a tandem quadruple PHD (qPHD)-PWWP module, which recognizes p300-catalyzed H3K18ac. By combining acute NSD1 depletion with time-resolved epigenomic and nascent transcriptomic analyses, we demonstrate that NSD1 promotes enhancer-dependent gene transcription by facilitating RNA polymerase II (RNA Pol II) pause release. Notably, NSD1 can act as a transcriptional coactivator independent of its catalytic activity. Moreover, NSD1 enables the activation of developmental transcriptional programs associated with Sotos syndrome pathophysiology and controls embryonic stem cell (ESC) multilineage differentiation. Collectively, we have identified NSD1 as an enhancer-acting transcriptional coactivator that contributes to cell fate transition and Sotos syndrome development.
    Keywords:  H3K36 methylation; NSD1; Pol II pause release; Sotos syndrome; chromatin; enhancers; gene expression; histone methylation; reader domain; stem cells
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.007
  39. Sci Adv. 2023 Jul 07. 9(27): eadf3026
      Oxygen (O2) sensing by the carotid body is critical for maintaining cardiorespiratory homeostasis during hypoxia. Hydrogen sulfide (H2S) signaling is implicated in carotid body activation by low O2. Here, we show that persulfidation of olfactory receptor 78 (Olfr78) by H2S is an integral component of carotid body activation by hypoxia. Hypoxia and H2S increased persulfidation in carotid body glomus cells and persulfidated cysteine240 in Olfr78 protein in heterologous system. Olfr78 mutants manifest impaired carotid body sensory nerve, glomus cell, and breathing responses to H2S and hypoxia. Glomus cells are positive for GOlf, adenylate cyclase 3 (Adcy3) and cyclic nucleotide-gated channel alpha 2 (Cnga2), key molecules of odorant receptor signaling. Adcy3 or Cnga2 mutants exhibited impaired carotid body and glomus cell responses to H2S and breathing responses to hypoxia. These results suggest that H2S through redox modification of Olfr78 participates in carotid body activation by hypoxia to regulate breathing.
    DOI:  https://doi.org/10.1126/sciadv.adf3026
  40. Mol Cell. 2023 Jun 28. pii: S1097-2765(23)00461-6. [Epub ahead of print]
      Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.
    Keywords:  DNA double-strand breaks; insertions; nuclear actin; recombination; transcription, break-induced replication
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.016
  41. J Biol Chem. 2023 Jun 30. pii: S0021-9258(23)02029-X. [Epub ahead of print] 105001
      NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane.
    Keywords:  bioenergetics; chemical biology; complex I; respiratory enzymes; ubiquinone
    DOI:  https://doi.org/10.1016/j.jbc.2023.105001