Publication Date

Fall 2018

Degree Type


Degree Name

Master of Science (MS)


Biological Sciences


Brandon White


Hes1, Notch, Notch response element, RBPJ-k, sequence-paired site, transcription factor

Subject Areas

Molecular biology


The Notch signaling pathway is a one of few fundamentally conserved signal transduction pathways critical for metazoan cellular development. Upon ligand activation, the Notch intracellular domain (NICD) translocates to the nucleus and forms a transcription complex with C-binding promoter factor-1 (RBPJ-k/CBF-1/Suppressor of Hairless) and Mastermind-like protein (MAM). The DNA-binding factor, RBPJ-k, binds to a response element containing a consensus sequence of RTGRGAR (where R is G or A). When RBPJ-k interacts with the NICD and MAM, Notch target genes are activated. The most well-characterized gene for Notch is Hes1. Hes1 contains four Notch response elements (NREs), labeled NRE 1-4. Of the four, NRE 2 and NRE 4 form what has been termed a sequence-paired site (SPS), identified as critical for transcription of Notch-dependent genes. Not all NREs are formed into an SPS, and it is hypothesized that a different transcriptional cofactor is recruited to the NREs versus the SPS to stabilize protein-DNA complexes. Base pair mutations in the Hes1 promoter were tested for binding to nuclear extract and purified RBPJ-k using an electrophoretic mobility shift assay (EMSA). Results were insufficient to determine Notch complex binding; however, the internal guanines were determined critical for RBPJ-k binding to NRE 2 and NRE 4. Additionally, despite its context in Hes1, NRE 3 also showed binding to RBPJ-k. Taken together, these results confirm that the NREs in the SPS are required for RBPJ-k structure formation and raise questions about the roles of the other single NREs.