482A Poster - 06. Regulation of gene expression
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Collin Christensen ¹; Yi Kuang ²; Brian Gebelein ^{1, 2}

Affiliations:
1) University of Cincinnati; 2) Cincinnati Children's Hospital

Keywords:
e. enhancers; d. Notch

Notch signaling regulates distinct cellular fates and processes during the development of nearly every organ in the body. Moreover, Notch-dependent processes are often highly sensitive to Notch signal strength as evidenced by genetic haploinsufficiency across multiple species. For example, decreases in Notch signal strength have been associated with developmental syndromes such as Alagille Syndrome and Adams Oliver Syndrome. However, we still do not fully understand how changes in Notch signal strength affect cell-specific responses. In general, Notch signal strength is determined by ligand induced production of the Notch Intracellular Domain (NICD), which forms a complex with the CSL transcription factor, Suppressor of Hairless (Su(H)) in flies, to induce gene expression via two types of binding sites: monomeric (CSL) and dimeric (Su(H) paired sites, SPS). Unexpectedly, we found that simply inserting synthetic enhancers that couple dimeric SPSs, but not monomeric CSL sites, with binding sites for the Grainyhead pioneer transcription factor resulted in flies that develop wing nicks, a classic Notch-sensitive phenotype. Moreover, this enhancer induced phenotype is highly sensitive to genetic changes in both the Notch pathway and the Cdk8-Mediator complex, which has been previously shown to promote NICD turnover. These findings suggest that Notch dimer sites preferentially promote NICD turnover and that Grainyhead can aid this process by promoting chromatin accessibility. To test this model, we generated a series of enhancers that couple SPS sites with binding sites for other pioneer transcription factors or transcription factors not known to have pioneer activity. Importantly, we found that pioneer factors, such as Zelda and Trithorax-like, could produce wing nicks when placed next to SPS sites, whereas non-pioneer sites such as E-box and UAS sites failed to do so. Our results also revealed that some, but not all, endogenous dimeric SPSs produced wing nicks, suggesting that other factors influence this activity. Genetic dosage experiments in Drosophila further revealed that removing a copy of each of the Cdk8-Mediator components significantly reduced wing nicking. Ongoing studies are focused on testing whether Grainyhead promotes the opening of these synthetic enhancers and on identifying the additional sequences and factors that allow for a subset of SPSs to induce wing nicking phenotypes.