PROJECT SUMMARY Genomic instability, referring to a heightened frequency of mutations and DNA damage, is a hallmark of cancer. A major consequence of cancer-intrinsic genomic instability is the activation of innate immune response, which can confer an antitumor state to cells. However, many cancers co-opt regulatory pathways to escape immune surveillance. Our long-term goal is to elucidate the molecular mechanisms of how innate immune consequences of genomic instability are regulated. Particularly, our group studies the immunogenic potential of cytosolic DNA, a molecular signal that arises from mitotic and nuclear aberrations. Cytosolic DNA activates a DNA sensing pathway termed cGAS-STING to trigger the type I interferon response. Three prime repair exonuclease 1 (TREX1) suppresses this pathway by degrading cytosolic DNA. Indeed, many cancers upregulate TREX1 to accommodate cytosolic DNA without immune activation. Yet, TREX1-null mutations are linked to inflammatory disorders like Aicardi-Goutières syndrome (AGS). Further, frameshift mutations that cause ectopic TREX1 nuclear localization are linked to retinal vasculopathy with cerebral leukodystrophy (RVCL). Therefore, TREX1 plays a complex role in human diseases through degradation of cytosolic DNA, but molecular mechanisms of TREX1 function are poorly understood. The goals of my project are to characterize the TREX1- specific elements, the polyproline helix (PPII) and the C-terminal intrinsically disordered region (C-IDR), in the context of immune regulation (Aim 1); and to dissect the impacts of ectopic TREX1 localization in the nucleus (Aim 2). My working hypothesis is that these TREX1-specific non-catalytic domains are essential for recruiting protein partners that prime DNA substrates for digestion by TREX1, thus enhancing cGAS-STING regulation. Further, I expect that PPII induces de novo interactions when TREX1 is ectopically expressed in the nucleus. I propose to test my working hypothesis using molecular and cellular approaches in human cells to quantify the activation of cGAS-STING, to identify protein-interacting domains of TREX1, and to examine the behavior of nuclear TREX1. This project will characterize novel TREX1 structural features that confer its unique immunosuppressive role, as well as explain how ectopic TREX1 localization can induces disease. It will also identify novel TREX1 interactors, contributing to the discovery of molecular targets in cancer therapy.