Project Summary Chromosome inheritance during cell proliferation is fundamental for living systems. Failures in this process cause diverse diseases, such as cancer. Through the development of various innovative methods and the exploitation of the unique Xenopus egg extract cell free system, our research program studies structure, function, and regulation of the nucleosome - the fundamental structural unit for chromosomal DNA - in chromosome inheritance and integrity. 1) Roles of nucleosomes in mitotic chromosome structure, integrity and function. High resolution 3D structural analysis of nucleoprotein complexes on functional chromosomes has been impossible. We propose to combine an innovative cryo-EM method and nucleosome manipulation method that we developed in Xenopus egg extracts to study how nucleosome dynamics and integrity are regulated on mitotic chromosomes. 2) Centromere-associated repeats and DNA methylation. Mutations in DNMT3B, ZBTB24, CDCA7, and HELLS cause Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome. We have demonstrated that CDCA7 is a critical activator for the nucleosome remodeling by HELLS. Expression of HELLS, CDCA7 and its paralog CDCA7L are linked to various cancers. While multiple roles of HELLS, including DNA methylation, loading of macroH2A, and nonhomologous end joining (NHEJ), have been reported, it is not clear how CDCA7 contributes to these diverse processes and how they are related to immunodeficiency and cancers. We will address this question through dissecting the molecular function of CDCA7 and HELLS. 3) Mitotic regulation of cGAS. cGAS is a critical innate immunity pattern receptor targeting pathogenic DNA. cGAS binds to DNA and becomes activated to synthesize cyclic GMP-AMP (cGAMP). cGAMP activates STING, which then triggers signal transduction pathway to promote inflammation. How does cGAS avoid being activated by the host’s chromosomal DNA? We gave an answer to this question by demonstrating that the nucleosome directly binds cGAS to block its DNA-dependent activation. However, when cGAS binds to chromosomes during mitotic arrest, cGAS slowly becomes reactivated and induces apoptosis. Using Xenopus egg extracts where we can precisely control nucleosome assembly, DNA, and cell cycle stages without being confounded by downstream events, we will dissect the mechanism of mitotic suppression and reactivation of cGAS.