The N-terminal domain of cGAS determines preferential association with centromeric DNA and innate immune activation in the nucleus
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE125475 | GEO | 2019/02/27
REPOSITORIES: GEO
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Project description:Cytosolic DNA activates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity, and cancer. cGAS is considered to be a sequence-independent DNA sensor with limited access to nuclear DNA because of compartmentalization. However, the nuclear envelope is a dynamic barrier, and cGAS is present in the nucleus. Here, we identify determinants of nuclear cGAS localization and activation. We show that nuclear-localized cGAS synthesizes cGAMP and induces innate immune activation of dendritic cells, although cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knockin mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent on LINE elements. The non-enzymatic N-terminal domain of cGAS determines nucleo-cytoplasmic localization, enrichment on centromeres, and activation of nuclear-localized cGAS. These results reveal a preferential functional association of nuclear cGAS with centromeres.
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Project description:The N-terminal domain of cGAS determines preferential association with centromeric DNA and innate immune activation in the nucleus
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Project description:cGAS/STING signaling plays an essential role in sensing cytosolic DNA. cGAS activity is regulated by posttranslational modifications and binding partners. cGAS interactome largely includes mammalian or viral proteins. Whether and how bacterial proteins bind cGAS to modulate innate immunity remain elusive. Here, we found streptavidin, a secreted bacterial protein, selectively bound cGAS to promote DNA-induced cGAS activation and interferon-? production. Mechanistically, streptavidin enhanced DNA binding and cGAS phase separation, therefore facilitating cGAS activation. Using an HSV-1-infected mouse model, we found streptavidin nanoparticles facilitated HSV-1 clearance through improving innate immunity. Considering the clinical usage of streptavidin as an immune stimulant and drug delivery vehicle and its biotechnological usage for biotin-labeled protein purification and detection, our studies not only provide an example for a bacterial protein regulating cGAS activity but also suggest caution needs to be taken when using streptavidin in various applications given to its ability to induce innate immunity.
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Project description:DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoII? is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoII?. CTD SUMOylation did not affect TopoII? activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoII?-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoII?-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoII?-CTD SUMOylation contributes to mitotic centromere activity.
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Project description:Double-stranded DNA is recognized as a danger signal by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), triggering innate immune responses. Palmitoylation is an important posttranslational modification (PTM) catalyzed by DHHC-palmitoyl transferases, which participate in the regulation of diverse biological processes. However, whether palmitoylation regulates cGAS function has not yet been explored. Here, we found that palmitoylation of cGAS at C474 restricted its enzymatic activity in the presence of double-stranded DNA. cGAS palmitoylation was catalyzed mainly by the palmitoyltransferase ZDHHC18 and double-stranded DNA promoted this modification. Mechanistically, palmitoylation of cGAS reduced the interaction between cGAS and double-stranded DNA, further inhibiting cGAS dimerization. Consistently, ZDHHC18 negatively regulated cGAS activation in human and mouse cell lines. In a more biologically relevant model system, Zdhhc18-deficient mice were found to be resistant to infection by DNA viruses, in agreement with the observation that ZDHHC18 negatively regulated cGAS mediated innate immune responses in human and mouse primary cells. In summary, the negative role of ZDHHC18-mediated cGAS palmitoylation may be a novel regulatory mechanism in the fine-tuning of innate immunity.
| S-SCDT-EMBOJ-2021-109272 | biostudies-other
Project description:It is currently unclear whether the GluN2 subtype influences NMDA receptor (NMDAR) excitotoxicity. We report that the toxicity of NMDAR-mediated Ca(2+) influx is differentially controlled by the cytoplasmic C-terminal domains of GluN2B (CTD(2B)) and GluN2A (CTD(2A)). Studying the effects of acute expression of GluN2A/2B-based chimeric subunits with reciprocal exchanges of their CTDs revealed that CTD(2B) enhances NMDAR toxicity, compared to CTD(2A). Furthermore, the vulnerability of forebrain neurons in vitro and in vivo to NMDAR-dependent Ca(2+) influx is lowered by replacing the CTD of GluN2B with that of GluN2A by targeted exon exchange in a mouse knockin model. Mechanistically, CTD(2B) exhibits stronger physical/functional coupling to the PSD-95-nNOS pathway, which suppresses protective CREB activation. Dependence of NMDAR excitotoxicity on the GluN2 CTD subtype can be overcome by inducing high levels of NMDAR activity. Thus, the identity (2A versus 2B) of the GluN2 CTD controls the toxicity dose-response to episodes of NMDAR activity.
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Project description:MCPH1 is a causal gene for the neurodevelopmental disorder, human primary microcephaly (MCPH1, OMIM251200). Most pathogenic mutations are located in the N-terminal region of the gene, which encodes a BRCT domain, suggesting an important function of this domain in brain size determination. To investigate the specific function of the N-terminal BRCT domain in vivo, we generated a mouse model lacking the N'-BRCT domain of MCPH1 (referred as Mcph1-?BR1). These mutant mice are viable, but exhibit reduced brain size, with a thinner cortex due to a reduction of neuroprogenitor populations and premature neurogenic differentiation. Mcph1-?BR1 mice (both male and female) are infertile; however, almost all female mutants develop ovary tumours. Mcph1-?BR1 MEF cells exhibit a defect in DNA damage response and DNA repair, and show the premature chromosome condensation (PCC) phenotype, a hallmark of MCPH1 patient cells and also Mcph1 knockout cells. In comparison with Mcph1 complete knockout mice, Mcph1-?BR1 mice faithfully reproduce all phenotypes, indicating an essential role of the N-terminal BRCT domain for the physiological function of MCPH1 in the control of brain size and gonad development as well as in multiple cellular processes.
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Project description:BackgroundIntervertebral disc degeneration (IDD) is the most common diagnosis of patients with lower back pain. IDD is the underlying lesion of many spinal degenerative diseases; however, the role of cGAS/Sting/NLRP3 pathway and epigallocatechin gallate (EGCG) in the development of IDD remained unclear.MethodsThe expressions of cGAS, Sting and NLRP3 mRNA of intervertebral disc (IVD) samples from IDD patients and controls were detected by RT-PCR. The nucleus pulposus cells (NPCs) were induced by hydrogen peroxide (H2O2) and used as an in-vitro model. Both 5 ?M and 25 ?M EGCG treatment were used to detect the effect of EGCG on the in-vitro model. Cell viability was detected by the MTT method, and cell apoptosis and cell cycle would be detected by flow cytometry. Western blot was used in the detection of the expression of cGAS/Sting/NLRP3 as well as apoptosis-related protein level. ELISA was used in the detection of pro-inflammatory factors, including IL-1?, TNF-?, IL-6 and IL-10.ResultsThe expressions of cGAS, Sting and NLRP3 mRNA were significantly increased in the IVD samples from IDD patients and NLRP3 was associated with cGAS and Sting. Advanced in-vitro study showed that H2O2 significantly increased the expression of cGAS, Sting and NLRP3 protein levels. Advanced experiments showed that EGCG treatment demonstrated significant protective effects in cell viability, apoptosis, cell cycle arrest and inflammatory status through down-regulation of cGAS/Sting/NLRP3 pathway.ConclusionIt was shown that the cGAS, Sting and NLRP3 up-regulation was associated with the incidence of IDD. Our findings also suggest that EGCG treatment would provide anti-apoptosis, anti-inflammation and promote cell viability in H2O2 treatment-incubated NPCs through inhibiting cGAS/Sting/NLRP3 pathway.
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Project description:In human cells chromosome specific aneuploidy was measured. Then, using NGS methods, centromeric features were measured for each chromosome (including centromere length and amount of centromeric proteins). The study aims at testing an association between centromeric features and missegregation rate.
2019-11-26 | GSE132193 | GEO
Project description:Cyclic GMP-AMP synthase (cGAS), a key sensor of intracellular DNA, is essential for eliciting innate immunity against infection, whereas aberrant activation of cGAS by endogenous DNA promotes severe autoimmune diseases. However, it is largely unknown how cGAS expression is regulated during pathogen infection and autoimmunity. Here, we report that during herpes simplex virus type 1 (HSV-1) infection, two microRNAs (miR-23a and miR-23b) whose levels significantly decrease due to their interaction with the lncRNA Oasl2-209 directly regulate the expression of cGAS. Overexpression of miR-23a/b markedly dampens cytosolic DNA-induced innate immune responses, whereas inhibition of miR-23a/b enhances these responses. Mice treated with miR-23a/b agomirs exhibit increased susceptibility to HSV-1 infection. Moreover, cGAS is significantly upregulated in the Trex1-/- mouse autoimmune disease model. Administration of miR-23a/b blunts self DNA-induced autoinflammatory responses in Trex1-/- mice. Collectively, our study not only reveals a novel regulatory mechanism of cGAS expression by miRNAs but also identifies a potential therapy for cGAS-related autoimmune diseases.
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