Signaling activated by nucleolar localised Notch4 Intracellular Domain underlies protection from genomic damage

The assembly of signaling hierarchies and their spatiotemporal organization together, contribute to diverse signaling outcomes. This is evident in the Notch pathway, which regulates an array of cellular processes, despite a small number of core components. Here, we describe a Notch4 activated signaling cascade, dependent on the nucleolar localization of the Notch4 Intracellular Domain (NIC4), that protects cells from genotoxic damage. Localization was assessed by immune-staining for endogenous Notch4 and visualization by confocal microscopy, in breast cancer cell lines. Live-cell, imaging-based, biophysical analysis of NIC4-GFP expressing cells, indicated unhindered mobility between the nucleolus and nucleoplasm and a stable nucleolar pool of NIC4-GFP. RNAi-mediated ablations, coupled with analysis of recombinant forms of NIC4 with modifications of its nucleolar localization sequence, confirmed nucleolar localization and identified the nucleolar proteins, Nucleolin and Fibrillarin, as key intermediates in the NIC4-activated signaling cascade. The transcriptional control of ribosome biogenesis (47s and 45s pre-rRNA transcription), emerged as another unexpected consequence of the subcellular distribution of NIC4. Taken together, this study describes intrinsic features of NIC4 that confer spatial flexibility and expand the repertoire of Notch4 signaling.