SRP181126 PRJNA516357 GSE125431 Nuclear GFP ChIP-seq from human Monocyte Derived Dendritic Cells transduced with GFP-NLS-cGAS or GFP-NLS Cytosolic DNA activates cyclic GMP-AMP (cGAMP) synthase (cGAS), an innate immune sensor pivotal in anti-microbial defense, senescence, auto-immunity and cancer. cGAS is considered 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, but cGAMP levels are 200-fold lower than following transfection with exogenous DNA. Using cGAS ChIP-seq and a GFP-cGAS knock-in mouse, we find nuclear cGAS enrichment on centromeric satellite DNA, confirmed by imaging, and to a lesser extent with 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. Overall design: Three independent donors were analyzed. Donor 1 was transduced only with GFP-NLS-cGAS. Donor 2 and 3 were transduced with GFP-NLS-cGAS and GFP-NLS. GSE125431 pubmed 30811988 parent_bioproject PRJNA516454