Julia M. Rogers1, Claudia A. Mimoso1, Benjamin J.E. Martin1, Alexandre P. Martin1, Jon C. Aster2,3, Karen Adelman1,3,4 and Stephen C. Blacklow1,4,5 1Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts 02115, USA; 2Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02215, USA; 3Ludwig Center at Harvard, Boston, Massachusetts 02115, USA; 4The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts 02142, USA; 5Department of Cancer Biology, Dana Farber Cancer Institute, Boston, Massachusetts 02215, USA Corresponding authors: stephen_blacklowhms.harvard.edu, karen_adelmanhms.harvard.edu Abstract

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examine the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

Received July 12, 2024. Accepted September 24, 2024.