Nuclear GFP ChIP-seq from bone marrow-derived dendritic cells of GFP-cGAS knock-In mice

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. Sample 1 is one mouse and sample 2 is a pool of two mice胞质DNA可激活环鸟苷酸-腺苷酸合酶（cyclic GMP-AMP synthase, cGAS）——这是一类在抗菌防御、细胞衰老、自身免疫及癌症进程中发挥关键作用的天然免疫感受器。传统认知中，cGAS被认为是不依赖序列的DNA感受器，但受细胞区室化限制，其难以接触核内DNA。然而，核被膜属于动态屏障结构，且cGAS本身可定位于细胞核内。本研究鉴定了调控核cGAS定位与激活的关键决定因素。实验结果显示，定位于细胞核的cGAS能够合成cGAMP并诱导树突状细胞产生天然免疫激活反应，但其cGAMP合成水平仅为外源性DNA转染后水平的1/200。本研究通过cGAS染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）以及绿色荧光蛋白（Green Fluorescent Protein, GFP）标记cGAS的敲入小鼠，发现核cGAS在着丝粒卫星DNA上存在显著富集，该结果通过成像实验得到验证；同时，核cGAS在长散在核元件（long interspersed nuclear elements, LINE）上也存在一定程度的富集。cGAS的非酶促N端结构域决定了其核质定位、着丝粒富集特性以及核内cGAS的激活能力。上述研究结果揭示了核cGAS与着丝粒之间存在优先的功能性关联。样本1取自单只小鼠，样本2为两只小鼠的混合样本