Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells

Mitotic errors can activate cyclic GMP–AMP synthase (cGAS) and induce type I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of antimitotic drugs to perturb mitosis in human fibroblasts and measured abnormal nuclear morphologies, cGAS localization, and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2′3′-cGAMP to fibroblasts, and in mouse cells. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and antitumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Download Full-text

Chromatin Bridges, not Micronuclei, Activate cGAS after Drug-induced Mitotic Errors in Human Cells

10.1101/2021.02.02.429360 ◽

2021 ◽

Cited By ~ 1

Author(s):

Patrick J. Flynn ◽

Peter D. Koch ◽

Timothy J. Mitchison

Keyword(s):

Chromosome Segregation ◽

Type I Interferon ◽

Human Cells ◽

Type I ◽

Drug Induced ◽

Chromatin Bridges

SummaryMitotic errors can activate cGAS and induce type-I interferon (IFN) signaling. Current models propose that chromosome segregation errors generate micronuclei whose rupture activates cGAS. We used a panel of anti-mitotic drugs to perturb mitosis in fibroblasts and measured abnormal nuclear morphologies, cGAS localization and IFN signaling in the subsequent interphase. Micronuclei consistently recruited cGAS without activating it. Instead, IFN signaling correlated with formation of cGAS-coated chromatin bridges that were selectively generated by microtubule stabilizers and MPS1 inhibitors. cGAS activation by chromatin bridges was suppressed by drugs that prevented cytokinesis. We confirmed cGAS activation by chromatin bridges in cancer lines that are unable to secrete IFN by measuring paracrine transfer of 2′3-cGAMP to fibroblasts. We propose that cGAS is selectively activated by self-chromatin when it is stretched in chromatin bridges. Immunosurveillance of cells that fail mitosis, and anti-tumor actions of taxanes and MPS1 inhibitors, may depend on this effect.

Download Full-text

Faculty Opinions recommendation of Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717970193.793472513 ◽

2013 ◽

Author(s):

Shinji Makino ◽

Krishna Narayanan

Keyword(s):

Cyclic Gmp ◽

Type I Interferon ◽

Type I ◽

Dna Sensor ◽

Interferon Pathway

Download Full-text

Faculty Opinions recommendation of Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717970193.793478726 ◽

2013 ◽

Author(s):

Shigekazu Nagata

Keyword(s):

Cyclic Gmp ◽

Type I Interferon ◽

Type I ◽

Dna Sensor ◽

Interferon Pathway

Download Full-text

Transcriptional activation of the mouse Mx gene by type I interferon.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4770 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4770-4774 ◽

Cited By ~ 49

Author(s):

P Staeheli ◽

P Danielson ◽

O Haller ◽

J G Sutcliffe

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Type I Interferon ◽

Type I ◽

Mrna Synthesis ◽

Polyadenylated Rna ◽

Mrna Concentration ◽

Mouse Cells ◽

Ifn Treatment

Mouse cells of the Mx+ genotype accumulate Mx mRNA in response to type I interferon (IFN). Nuclear runoff experiments show that IFN stringently regulates Mx gene expression at the level of transcription. Mx mRNA synthesis peaks about 3 h after IFN treatment, and within 5 h, Mx mRNA concentration rises from undetectable levels to about 0.1% of polyadenylated RNA.

Download Full-text

Plasmacytoid Dendritic Cells Mediate Myocardial Ischemia/Reperfusion Injury by Secreting Type I Interferons

Journal of the American Heart Association ◽

10.1161/jaha.121.020754 ◽

2021 ◽

Author(s):

Lina Lai ◽

Aimee Zhang ◽

Boris Yang ◽

Eric J. Charles ◽

Irving L. Kron ◽

...

Keyword(s):

Myocardial Ischemia ◽

Infarct Size ◽

Reperfusion Injury ◽

Cyclic Gmp ◽

Type I Interferon ◽

Ischemia Reperfusion ◽

Interferon Α ◽

Type I ◽

Myocardial Ischemia Reperfusion Injury ◽

Myocardial Ischemia Reperfusion

Background We previously demonstrated that ischemically injured cardiomyocytes release cell‐free DNA and HMGB1 (high mobility group box 1 protein) into circulation during reperfusion, activating proinflammatory responses and ultimately exacerbating reperfusion injury. We hypothesize that cell‐free DNA and HMGB1 mediate myocardial ischemia‐reperfusion injury by stimulating plasmacytoid dendritic cells (pDCs) to secrete type I interferon (IFN‐I). Methods and Results C57BL/6 and interferon alpha receptor‐1 knockout mice underwent 40 minutes of left coronary artery occlusion followed by 60 minutes of reperfusion (40′/60′ IR) before infarct size was evaluated by 2,3,5‐Triphenyltetrazolium chloride–Blue staining. Cardiac perfusate was acquired in ischemic hearts without reperfusion by antegrade perfusion of the isolated heart. Flow cytometry in pDC‐depleted mice treated with multiple doses of plasmacytoid dendritic cell antigen‐1 antibody via intraperitoneal injection demonstrated plasmacytoid dendritic cell antigen‐1 antibody treatment had no effect on conventional splenic dendritic cells but significantly reduced splenic pDCs by 60%. pDC‐depleted mice had significantly smaller infarct size and decreased plasma interferon‐α and interferon‐β compared with control. Blockade of the type I interferon signaling pathway with cyclic GMP‐AMP synthase inhibitor, stimulator of interferon genes antibody, or interferon regulatory factor 3 antibody upon reperfusion similarly significantly attenuated infarct size by 45%. Plasma levels of interferon‐α and interferon‐β were significantly reduced in cyclic GMP‐AMP synthase inhibitor‐treated mice. Infarct size was significantly reduced by >30% in type I interferon receptor monoclonal antibody–treated mice and interferon alpha receptor‐1 knockout mice. In splenocyte culture, 40′/0′ cardiac perfusate treatment stimulated interferon‐α and interferon‐β production; however, this effect disappeared in the presence of cyclic GMP‐AMP synthase inhibitor. Conclusions Type I interferon production is stimulated following myocardial ischemia by cardiogenic cell‐free DNA/HMGB1 in a pDC‐dependent manner, and subsequently activates type I interferon receptors to exacerbate reperfusion injury. These results identify new potential therapeutic targets to attenuate myocardial ischemia‐reperfusion injury.

Download Full-text

Editing of the Human TRIM5 Gene to Introduce Mutations with the Potential to Inhibit HIV-1

10.1101/185298 ◽

2017 ◽

Author(s):

Caroline Dufour ◽

Alix Claudel ◽

Nicolas Joubarne ◽

Natacha Merindol ◽

Tara Maisonnet ◽

...

Keyword(s):

Cell Line ◽

Type I Interferon ◽

Human Cells ◽

Restriction Factor ◽

Type I ◽

Dna Transfection ◽

Homology Directed Repair ◽

Cell Clones ◽

Overall Efficiency ◽

Hiv 1

ABSTRACTThe type I interferon (IFN-I)-inducible human restriction factor TRIM5α inhibits the infection of human cells by specific nonhuman retroviruses, such as N-MLV and EIAV, but does not generally target HIV-1. However, the introduction of two aminoacid substitutions, R332G and R355G, in the human TRIM5α (huTRIM5α) domain responsible for retroviral capsid recognition leads to efficient HIV-1 restriction. Using a DNA transfection-based CRISPR-Cas9 genome editing protocol, we successfully mutated TRIM5 to its HIV-1-restrictive version by homology-directed repair (HDR) in HEK293T cells. Nine clones bearing at least one HDR-edited TRIM5 allele containing both mutations were isolated (5.6% overall efficiency), whereas another one contained only the R332G mutation. Of concern, several of these HDR-edited clones contained on-target undesired mutations, and none had all the alleles corrected. We observed a lack of HIV-1 restriction in the cell clones generated, even when cells were stimulated with IFN-I prior to infection. This, however, was partly explained by the unexpectedly low potential for TRIM5α-mediated restriction activity in this cell line. Our study demonstrates the feasibility of editing the TRIM5 gene to in human cells and identifies the main challenges to be addressed in order to use this approach to confer protection from HIV-1.

Download Full-text