(42e) Investigating the Role of DNA-Protein Binding in Regulating Developmental Gene Expression in Drosophila embryos

It is known that an enhancer regulates transcriptional activity of its gene through activators and repressors by establishing the spatiotemporal expression of a target gene. Since enhancers may contain multiple binding sites for the same transcription factors (TFs), it is not yet understood how each binding site affects the overall transcriptional competency of the enhancer. In this study, we have implemented the MS2-MCP live imaging technique to quantitatively analyze dynamic transcriptional activity of a region of the snail distal enhancer. Through systematically modulating 3 Dorsal (Dl) and 1 Twist (Twi) binding domains, we found that the mutations in these binding sites caused a drastic reduction in the maximum intensity, resulting in a reduction in total mRNA production. We characterized the synergistic capabilities of each binding site by quantifying the contribution of each binding site to the total transcriptional dynamics. While the timing of activation was not substantially affected in the mutated constructs, the bursting dynamics and nuclei activation were altered to varying degrees. Using mathematical modeling, we determined the time spent in the on and off states as well as other kinetic parameters. Through this we identified distinct mechanisms by which TFs regulate proper gene expression during development.