Notch induces transcription by stimulating release of paused RNA Pol II without increasing chromatin accessibility [PRO-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 how Notch signaling affects the behavior of RNA Polymerase II, Notch signaling was induced in SC2 cells by GSI washout. Cells were harvested at 15m, 30m, 45m, 1h, 90m, 2h, 3h, and 4h post Notch activation, and harvested for PRO-seq sample preparation. A reference condition was harvested immediately after mock GSI washout, and harvested for PRO-seq sample preparation. There are 9 total conditions, with two replicates of the full experiment, and 3 replicates of the reference condition.