Notch1 sets the molecular clock for T cell development

Abstract Notch1 signaling is instructive for T cell development, but how this single factor orchestrates commitment remains unclear. Here, by integrating multi-omics platforms, we identified 46 transcription factors (TF) that are directly regulated by NOTCH1 via super-enhancers during T cell development. Among these, two ETS factors, FLI1 and ERG, formed an “early transcriptional network”, with NOTCH1 that is crucial for T cell lineage specification. Remarkably, we showed that the NOTCH-ETS complex repressed KLF factors to regulate proliferation and developmental progression, allowing NOTCH1 dependent induction of the “late T cell transcriptional network”. This late network established the expression of T cell lineage specific genes and favored chromatin contraction at the TCR locus. Collectively, our study provides a mechanistic explanation for how Notch1 signaling progressively shapes its own transcriptional network through a finely timed activation as well as repression of lineage specific genes thereby attenuating proliferation, facilitating chromatin looping, and ultimately enabling T cell development.