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?
Many cells in the adult use notch signaling to activate specific targets in different cell and disease states. We fall short of understanding how Notch molecules choose the targets they do. Previously, we explored this from the perspective of amino acid composition in the Notch intracellular domains (the ?business end? of the protein). We ruled out composition as a factor differentiating two related proteins with different clinical footprints, Notch1 and Notch2. Our data indicates that much of Notch activity reflects strength (defined here as the ultimate number of intracellular domain of Notch (NICD) molecules reaching the nucleus, integrating ligand-mediated release and nuclear translocation), and duration (half life of NICD/RBPjk/MAML/DNA complexes, integrating cooperativity and stability). We discovered recently that another protein, Runx1, is a frequent binder near Notch bound sites, and Runx1 is needed to regulate half of the cellular transcripts Notch activates. Based on these, and additional findings, we propose to address the following unanswered questions: (1) What is the biochemical basis for Runx1/NICD collaboration? (2) What genomic features distinguish sites Notch- RBP bind to from other RBPj sites? (3) What is the mechanism NICD utilizes to increase target gene expression frequency?
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