Functional Characterization of the Notch Response Element: Mastermind selectivity and interaction with the TATA box determines activation strength

Abstract

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. Recent studies have shown that a paired CBF1 binding site is important for target gene activation. There are four potential CBF1 binding sites in Hes1 and up to six sites in Hes5. We have undertaken a series of experiments to determine what is required to activate target genes. Site‐directed mutagenesis was used to mutate the different CBF1 binding sites in the Hes1 and Hes5 promoters, change the spacing and orientation between sites, as well as mutate the TATA box and the spacing between the TATA box and the CBF1 sites. Our results confirm previous results indicating that the paired CBF1 sites are the most important in activation of target genes. We also show that the unpaired sites do not contribute significantly to target gene activation. Finally, our results indicate that the ternary Notch complex requires a TATA box to activate target genes. Mutation of the TATA box decreased reporter gene activity to basal levels. Our results suggest that an SPS and TATA are necessary and sufficient to activate Notch responsive genes. Support of research was through NSF‐RUI Grant#1052039 to JBW.