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MASTERMIND-LIKE 1-DEPENDENT NOTCH TARGET GENE ACTIVATION REQUIRES A SEQUENCE-PAIRED SITE AND A TATA BOX by Cassandra Agbayani Ramos Notch signaling plays an important role in mammalian cellular proliferation, apoptosis, and differentiation. To activate target genes, the Notch intracellular domain (NICD) forms a complex with CBF1 and Mastermind-like protein (MAM). The Notch activation complex binds to a response element containing the consensus sequence RTGRGAR (R= purine) on target gene promoters. The promoter of one well characterized target gene, Hes1, contains four Notch response elements (NREs). Sitedirected mutagenesis and reporter gene assays were used to examine the activation of Hes1 by mutating individual and combinations of NREs. Results indicated that NREs 2 and 4 are important for Hes1 activity, confirming previous results that this sequencepaired site (SPS) is an important feature of Notch target genes. Orientation and spacing between elements on the SPS were mutated in these studies. Other mutations included the Hes1 TATA box and the spacing between the SPS and TATA box. Additionally, a charged residue on NICD was mutated; this residue is believed to be responsible for Notch complex dimers. These results showed that SPS spacing and orientation as well as dimerization of Notch complexes are important for the promoter activity of Hes1. The TATA box was found to be necessary for promoter activation, and this activity is independent of spacing between the SPS and TATA box up to 173 base pairs. Taken together, these findings suggest that NICDand MAM-mediated activation of target genes requires orientationand spacing-dependent SPS and TATA box elements.

MASTERMIND-LIKE 1-Dependent Notch Target Gene Activation Requires a Sequence-Paired Site and a TATA Box