Abstract Transcription factors (TFs) activate transcription through a variety of mechanisms, including opening of chromatin by displacing linker histones or repositioning of nucleosomes, promoting looping between regulatory elements, and triggering or enhancing maintenance of RNA polymerase activity. We will address the open question of why few enhancers are selected by a given TF from a multitude of available sites, with a focus on Notch/RBPj. Our previous data indicate that target selection reflects strength (defined here as the ultimate number of intracellular domain of Notch (NICD) molecules reaching the nucleus, which integrates ligand-mediated release and nuclear translocation but is agnostic to differences between Notch and Notch2), and duration (half life of NICD/RBPjk/MAML/DNA complexes, which integrates cooperativity and stability). In this proposal, we leverage novel experimental and computational tools to explore the distinct steps used by the Notch transcriptional complex to select and regulate mammalian target genes. To enable investigation of this question in high resolution, we developed Split DamID (SpDamID, a protein complementation version of DamID(14)) to specifically interrogate target selection by multi-member transcription complexes or factors binding simultaneously near each other in only 100-1000 cells. Adenine methylation at GAmTC occurs only when two halves complement each other on the same chromosome and is blind to complex size, a source of artifacts in ChIP(15). Motif enrichment analyses of NICD bound peaks identified Runx as a frequent collaborator, binding within 100bp of RBPj sites(2). Co-binding of NICD and Runx1 was confirmed by Runx1D/NICDAM SpDamID(2). In combination with other tools, SpDamID enables investigation of the following questions: What is the biochemical basis for Runx1/NICD collaboration? What genomic features distinguish Notch-dependent RBPj peaks from other RBPj sites? What is the mechanism NICD utilizes to increase target gene expression frequency?