Genome-Wide Identification and Characterization of Notch Transcriptional Complex-Binding Sequence Paired Sites in Leukemia Cells

Abstract

Notch transcription complexes (NTCs) drive target gene expression by binding two distinct types of genomic response elements, NTC monomer sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and linked to rapid responses to Notch in 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 at least 16% of functional NTC binding sites are SPSs. While originally described in promoters, SPSs are present mainly in enhancers and contribute to regulation of both coding and non-coding RNAs. 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 mammalian gene regulation.