Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility [TT-seq]

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cell fate decisions by regulating gene activity. However, despite years of study, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examined the response to human Notch1 across a time course of activation, using genomic assays of nascent RNA and chromatin accessibility. These data revealed that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII), in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility. Indeed, we found that open chromatin is established at Notch-responsive regulatory elements prior to Notch signaling, through SWI/SNF-mediated remodeling. Notch activation, however, elicited no further chromatin opening at these loci. Together, these studies reveal that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation. To determine which genes are induced by Notch signaling in SC2 cells, and how SWI/SNF inhibition affects gene expression in the Notch response, Notch signaling was induced in SC2 cells by GSI washout. Cells were harvested at 1 and 4 hours post Notch activation, and immediately after mock GSI washout, and harvested for TT-seq sample preparation. Mock washout control samples were also harvested at 1 and 4 hours. For each of these time points, cells were either treated with SWI/SNF inhibitor (1 µM BRM014) or 0.01% DMSO at the time of washout or mock-washout. There are 10total conditions, and two replicates of the full experiment.