Notch4 but not Notch1 yin-yang dimerization correlates with condensate formation but not transcriptional activity

Notch signaling facilitates direct communication between neighboring cells to largely coordinate cell differentiation mechanisms. Internal regulation of this pathway is far more complex than previously thought, including multiple dimerization states as well as formation of biomolecular condensates. Previous studies have focused on ankyrin mediated head-to-head dimerization in the regulation of Notch signaling. Herein, we focus on the less known DNA independent head-to-tail dimerization state which we refer to as yin-yang dimerization and its connection to the formation of Notch containing biomolecular condensates and transcriptional activity. We show that all four Notch paralogs participate in yin-yang homo and heterodimerization, and that N4ICD displays enhanced yin-yang dimerization compared to N1ICD. Through domain swap experiments, we Found that the N4ICD C-terminus mediates strong yin-yang dimerization, while the N1ICD C-terminus mediates weak yin-yang dimerization. We reveal that all four Notch paralogs form distinct condensates that are capable of colocalization in all combinations and assess the effects of the various domains within the NICD on condensate formation and transcriptional activity. Finally, through serial truncation of both the N4ICD and N1ICD C-termini we provide insight into the role that different amino acid sections within these domains plays in the ability to yin-yang dimerize, form condensates, and induce transcriptional effects. Though much remains to be learned about the specific contributions that the C-terminals of the NICDs has on yin-yang dimerization, condensate formation, and transcriptional output, the data we have laid out provides a framework to direct future research to further detail these properties.