Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and ...nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.
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•Nucleosomes suppress DNA-induced cGAMP synthesis by cGAS•During mitotic arrest, cGAS promotes a slow buildup of IRF3 phosphorylation•Phospho-IRF3 promotes mitotic apoptosis independently of transcription induction•Xenograft experiments and patient data indicate a role for cGAS in Taxol chemotherapy
Activation of the cytoplasmic DNA sensor cGAS is inhibited by nucleosomes during normal mitosis, but during prolonged mitotic arrest, cGAS functions to promote cell death via slowly accumulating phosphorylation of IRF3.
Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. ...Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.
The antitumor efficacy of cancer immunotherapy can correlate with the presence of certain bacterial species within the gut microbiome. However, many of the molecular mechanisms that influence host ...response to immunotherapy remain elusive. In this study, we show that members of the bacterial genus
improve checkpoint inhibitor immunotherapy in mouse tumor models. Active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in nonprotective
was sufficient to promote immunotherapy response, and its activity required the peptidoglycan sensor NOD2. Notably, SagA-engineered probiotics or synthetic muropeptides also augmented anti-PD-L1 antitumor efficacy. Taken together, our data suggest that microbiota species with specialized peptidoglycan remodeling activity and muropeptide-based therapeutics may enhance cancer immunotherapy and could be leveraged as next-generation adjuvants.
5-Methylcytosine (5mC) and DNA methyltransferases (DNMTs) are broadly conserved in eukaryotes but are also frequently lost during evolution. The mammalian SNF2 family ATPase HELLS and its plant ...ortholog DDM1 are critical for maintaining 5mC. Mutations in HELLS, its activator CDCA7, and the de novo DNA methyltransferase DNMT3B, cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, a genetic disorder associated with the loss of DNA methylation. We here examine the coevolution of CDCA7, HELLS and DNMTs. While DNMT3, the maintenance DNA methyltransferase DNMT1, HELLS, and CDCA7 are all highly conserved in vertebrates and green plants, they are frequently co-lost in other evolutionary clades. The presence-absence patterns of these genes are not random; almost all CDCA7 harboring eukaryote species also have HELLS and DNMT1 (or another maintenance methyltransferase, DNMT5). Coevolution of presence-absence patterns (CoPAP) analysis in Ecdysozoa further indicates coevolutionary linkages among CDCA7, HELLS, DNMT1 and its activator UHRF1. We hypothesize that CDCA7 becomes dispensable in species that lost HELLS or DNA methylation, and/or the loss of CDCA7 triggers the replacement of DNA methylation by other chromatin regulation mechanisms. Our study suggests that a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance is broadly inherited from the last eukaryotic common ancestor.
N
-methyladenosine is the most prominent RNA modification in mammals. Here, we study mouse skin embryogenesis to tackle m6A's functions and physiological importance. We first landscape the m6A ...modifications on skin epithelial progenitor mRNAs. Contrasting with in vivo ribosomal profiling, we unearth a correlation between m6A modification in coding sequences and enhanced translation, particularly of key morphogenetic signaling pathways. Tapping physiological relevance, we show that m6A loss profoundly alters these cues and perturbs cellular fate choices and tissue architecture in all skin lineages. By single-cell transcriptomics and bioinformatics, both signaling and canonical translation pathways show significant downregulation after m6A loss. Interestingly, however, many highly m6A-modified mRNAs are markedly upregulated upon m6A loss, and they encode RNA-methylation, RNA-processing and RNA-metabolism factors. Together, our findings suggest that m6A functions to enhance translation of key morphogenetic regulators, while also destabilizing sentinel mRNAs that are primed to activate rescue pathways when m6A levels drop.
Learning and memory require activity-induced changes in dendritic translation, but which mRNAs are involved and how they are regulated are unclear. In this study, to monitor how depolarization ...impacts local dendritic biology, we employed a dendritically targeted proximity labeling approach followed by crosslinking immunoprecipitation, ribosome profiling and mass spectrometry. Depolarization of primary cortical neurons with KCl or the glutamate agonist DHPG caused rapid reprogramming of dendritic protein expression, where changes in dendritic mRNAs and proteins are weakly correlated. For a subset of pre-localized messages, depolarization increased the translation of upstream open reading frames (uORFs) and their downstream coding sequences, enabling localized production of proteins involved in long-term potentiation, cell signaling and energy metabolism. This activity-dependent translation was accompanied by the phosphorylation and recruitment of the non-canonical translation initiation factor eIF4G2, and the translated uORFs were sufficient to confer depolarization-induced, eIF4G2-dependent translational control. These studies uncovered an unanticipated mechanism by which activity-dependent uORF translational control by eIF4G2 couples activity to local dendritic remodeling.
Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. ...Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.
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•Upon injury, IL-24 is induced specifically in epithelial stem cells at wound edges•Il24 is regulated by hypoxia and STAT3, independent of microbes, B cells, or T cells•IL-24 acts in autocrine and paracrine signaling to regulate proliferation and metabolism•Epithelial stem cells sense tissue damage and orchestrate organ-level repair
Epithelial stem cells sense injury signals to activate an IL-24-mediated tissue repair pathway that is molecularly distinct but functionally parallel to pathogen-induced IFN signaling in innate immunity.
Obesity is a major risk factor for adverse outcomes in breast cancer; however, the underlying molecular mechanisms have not been elucidated. To investigate the role of crosstalk between mammary ...adipocytes and neoplastic cells in the tumor microenvironment (TME), we performed transcriptomic analysis of cancer cells and adjacent adipose tissue in a murine model of obesity-accelerated breast cancer and identified glycine amidinotransferase (Gatm) in adipocytes and Acsbg1 in cancer cells as required for obesity-driven tumor progression. Gatm is the rate-limiting enzyme in creatine biosynthesis, and deletion in adipocytes attenuated obesity-driven tumor growth. Similarly, genetic inhibition of creatine import into cancer cells reduced tumor growth in obesity. In parallel, breast cancer cells in obese animals upregulated the fatty acyl-CoA synthetase Acsbg1 to promote creatine-dependent tumor progression. These findings reveal key nodes in the crosstalk between adipocytes and cancer cells in the TME necessary for obesity-driven breast cancer progression.
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•Gatm in peritumoral adipocytes promotes breast cancer progression in obesity•Acsbg1 in breast cancer cells promotes breast cancer progression in obesity•Creatine is a metabolite linking these pathways in obesity-dependent tumor growth•Acsbg1 and Gatm in breast cancer patients are linked to disease grade and outcome
Obesity is a major risk factor for adverse outcomes in breast cancer. Maguire, Ackerman et al. reveal Gatm and Acsbg1 as molecular regulators of obesity-driven breast cancer progression. They further show that in obesity creatine is a key metabolite in the crosstalk between adipocytes and breast tumors.
A solid-state sensor embedded microfluidic chip is demonstrated for the detection of glucose, urea and creatinine in human serum. In the presented device, magnetic powder-containing enzyme-carrying ...alginate microbeads are immobilized on the surface of an electrolyte-insulator-semiconductor (EIS) sensor by means of a step-like obstacle in the microchannel and an external magnetic force. The sample is injected into the microchannel and reacts with the enzyme contained within the alginate beads; prompting the release of hydrogen ions. The sample concentration is then evaluated by measuring the resulting change in the voltage signal of the EIS sensor. The reaction time and alginate bead size are optimized experimentally using a standard glucose solution. The experimental results show that the device has a detection range of 2–8mM, 1–16mM and 10−2–10mM for glucose, urea and creatinine, respectively. Furthermore, it is shown that the device is capable of sequentially measuring all three indicators in a human serum sample. Finally, it is shown that the measured values of the glucose, urea and creatinine concentrations obtained using the device deviate from those obtained using a commercial kit by just 5.17%, 6.22% and 13.53%, respectively. This method can be extended to sequentially measure multiple blood indicators in the sample chip by replacing different types of enzyme in alginate bead and can address the enzyme preservation issue in the microfluidic device. Overall, the results presented in this study indicate that the microfluidic chip has significant potential for blood monitoring in point-of-care applications.
► A solid-state sensor integrated with a microfluidic chip is demonstrated. ► An enzyme delivery and immobilization approach is proposed using alginate microbeads. ► Multi-target detection is accomplished by alginate bead containing different enzymes.
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