Detection of intracellular DNA by the cGAS-STING pathway mediates host defense against DNA viruses, contributes to specific human autoimmune diseases, and can be harnessed to promote anti-tumor immunity. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. We have found that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity, revealing a fundamental mechanism that controls the resting state of cGAS. In this grant application, we have developed innovative new tools to define the cGAS nuclear tethering compartment, quantitate changes in cGAS disposition during DNA virus infection, and explore cGAS- associated nucleic acids before and after its activation in the context of infection and autoimmune disease. Our studies will uncover fundamental new aspects of cGAS biology, will identify new targets for therapeutic modulation of cGAS activity, and will provide new insights into the principles of self/non-self discrimination by intracellular nucleic acid sensors.
We have found that the DNA sensor cGAS, long thought to be a cytosolic protein, is a tightly tethered nuclear protein that is carefully regulated to prevent detection of self-DNA. We will define how nuclear tethering prevents cGAS autoreactivity and enables it to respond to foreign DNA.
