Cellular functions of cGAS-STING signaling

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Cytosolic DNA induces type I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. We found that mammalian cytosolic extracts synthesized cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP) in vitro from adenosine triphosphate and guanosine triphosphate in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP functions as an endogenous second messenger in metazoans and triggers interferon production in response to cytosolic DNA.

Summary DNA is strictly compartmentalised within the nucleus to prevent autoimmunity1; despite this cGAS, a cytosolic sensor of dsDNA, is activated in autoinflammatory disorders and by DNA damage2–6. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localises to micronuclei arising from genome instability in a model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. These micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by their own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis7, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS binds to and is activated by chromatin and, consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA-damage are cell-cycle dependent. Furthermore, by combining live-cell laser microdissection with single cell transcriptomics, we establish that induction of interferon stimulated gene expression occurs in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, cGAS recognition of micronuclei may act as a cell-intrinsic immune surveillance mechanism detecting a range of neoplasia-inducing processes.