Notch NICD domains form biomolecular condensates.

Biomolecular condensates are a quickly emerging area of research that strongly impacts how we view the inner workings of the cell itself. Here, we explore the connection between the Notch signaling pathway and nuclear condensate localization. In this study, we hypothesize that activated Notch intracellular domain (NICD) fragments differentially localize into unique nuclear condensates. Using both overexpression and endogenous systems, our results reveal that N1ICD, N2ICD, and N4ICD efficiently localize into subnuclear bodies, whereas N3ICD inefficiently localizes into similar subnuclear puncta. These "notchosomes" appear to be unique because they do not colocalize with markers for other known nuclear puncta. In contrast, we find that N1ICD does colocalize with other known Notch-interacting proteins, including MAML-1, RBPj, AES1, and SKIP1, but not NACK1. Through deletion analysis of the N1ICD C-terminal tail, we identify multiple regions of mouse N1ICD that are necessary for localization into notchosomes, including sequences immediately C-terminal to the ankyrin domain and sequences within the transactivation domain. We also show that N1ICD localization into notchosomes may be important for N1ICD transcriptional activity from some, but not all, Notch-responsive promoters. Collectively, our results show that Notch NICD domains form nuclear localized biomolecular condensates that may be important for transcriptional activity.