Characterization of Notch transcription complex sequence paired sites

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and are linked to the Notch-responsiveness of a few genes, but their overall contribution to Notch-dependent gene regulation is unknown. To address this issue, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay, and applied insights from these in vitro studies to Notch-“addicted” leukemia cells. We find that SPSs contribute to the regulation of approximately a third of direct Notch target genes. While originally described in promoters, SPSs are present mainly in long-range enhancers. Our work provides a general method for identifying sequence-paired sites in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells. All samples were run in triplicate and all samples were treated either with DMSO or a gamma secretase inhibitor (GSI). The samples were T6E cells transfected with the empty MigR1 vector, a dominant negative MAML (DNMAML), intracellular Notch 1 (ICN1), or a dimerization defective intracellular Notch 1(R1984A).