Enhancers With Cooperative Notch Binding Sites Are More Resistant To Regulation By The Hairless Co-Repressor

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

Author summaryCell signaling provides a basic means of communication during development. Many signaling pathways, including the Notch pathway, convert extracellular signals into changes in gene expression via transcription factors that bind specific DNA sequences. Importantly, the Notch pathway transcription factor can either form activating complexes upon Notch activation to stimulate gene expression or repression complexes with co-repressors to inhibit gene expression. Prior studies showed that the Notch activation complex binds DNA as either an independent complex on monomer binding sites or as two cooperative complexes (dimer) on paired binding sites. In this study, we used synthetic biology to examine how these two types of DNA sites impact the binding of Notch activation versus repression complexes and the output of Notch target gene expression. Our studies reveal that unlike the Notch activation complex, the repression complex does not cooperatively bind dimer sites. Moreover, our findings support the model that the enhanced stability of the Notch activation complex on dimer sites makes target genes with dimer sites less sensitive to the repression complex than target genes with only monomer sites. Thus, our studies reveal how target genes with different binding sites differ in sensitivity to the ratio of Notch activation to repression complexes.