Publication Date

Summer 2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Advisor

J B. White

Keywords

Hes1, Mastermind-like, Notch, sequence-paired site, TATA box, transcription initiation

Subject Areas

Molecular biology

Abstract

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). Site-directed 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 sequence-paired 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 NICD- and MAM-mediated activation of target genes requires orientation- and spacing-dependent SPS and TATA box elements.

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