Direct observation of Notch signaling–induced transcription hubs mediating gene-expression responses

Developmental decisions rely on cells making accurate transcriptional responses to signals they receive, as with Notch pathway activity. Local condensates or transcription factor hubs are a proposed mechanism for facilitating gene activation by nuclear complexes. To investigate their importance in endogenous Notch signaling, we deployed multicolor live imaging to measure Notch transcription-complex enrichment at a target gene locus in combination with the transcription dynamics. The coactivator Mastermind (Mam) was present in signaling-dependent nuclear foci during Notch active developmental stages. Tracking these highly dynamic Mam hubs together with transcription in the same nucleus revealed that their appearance precedes and correlates with the profile of transcription and becomes stabilized if transcription is inhibited. Manipulations to signaling levels had concordant effects on hub intensities and transcription profiles, altering their probability and amplitude. Together, the results argue that signaling induces the formation of transcription hubs whose properties are instrumental in the quantitative gene-expression response to Notch activation.