ABSTRACT One of the most astonishing feats in all of biology is the process by which chromatin, the complex of DNA, histone proteins and associated factors, becomes compacted from a length of 2 m into nuclei that measure just microns across. Unsurprisingly, our understanding of the principles that govern this process is in its infancy. My lab became interested in this process after we identified the Ferguson-Bonni ANAPC7 neurodevelopmental syndrome caused by mutation of the degradative ubiquitin ligase Anaphase-Promoting Complex (APC). We discovered that Ki-67 was the major APC7-dependent target in post-mitotic neurons, where it accumulated in constitutive heterochromatin. Subsequent experiments in complementary mouse mutants demonstrated a much broader role for the APC in regulating the composition of neuronal heterochromatin, where we also observed dramatic accumulation of the chromosome passenger complex (CPC) and its product phosphorylated histone 3 (H3S10ph). Like Ki-67, the contributions of the CPC and H3S10ph to neurologic disease had been previously unknown. Although these observations established an APC-dependent ubiquitination-phosphoprotein axis in neurodevelopmental chromatin regulation, how Ki-67 and H3S10ph control heterochromatin remained poorly defined. We hypothesize that Ki-67 and H3S10ph control liquid-liquid phase separation (LLPS), a physicochemical process driven by multivalent interactions of disordered proteins like Ki-67 and phosphoproteins like H3S10ph. Studies from other labs showed that LLPS of constitutive heterochromatin requires the small protein HP1α, which interacts with Ki-67. Data from my lab showed that neuronal Ki-67 undergoes rapid relocalization away from heterochromatin upon disruption of LLPS, indicating Ki-67’s association with constitutive heterochromatin requires LLPS. This compelling series of observations leads us to the central hypothesis of this proposal: LLPS of heterochromatin-associated APC targets is required for neurodevelopment and when perturbed contributes to the pathogenesis of APC-related neurodevelopmental disorders. In Aim 1, we will explore the role of Ki-67 in the formation of heterochromatin in neurons using imaging and molecular analyses of a novel mouse model of Ki-67 mutation. In Aim 2, we will employ elegant in vitro assays to determine how Ki- 67 enhances LLPS of HP1α and nucleosome arrays. In Aim 3, we will explore the liquid-like behavior of APC substrates Ki-67 and the CPC by imaging heterochromatin dynamics in live neurons. Armed with novel in vivo genetic systems, powerful in vitro assays, and world-class expertise in the wet lab and computational methods, we are poised to generate fundamental insight into chromatin regulation by ubiquitin and LLPS in neurons.