Design principles and dynamic gene control in embryonic development

PROJECT SUMMARY Elucidating the mechanism of transcriptional regulation is a complex multivariate problem that requires holistic characterization of all transcriptional machinery including multiple enhancers, promoters, and other architectural proteins. In particular, the development of groundbreaking “seq” techniques has significantly advanced the field by providing a genome-wide frequency of enhancer-promoter interactions and related levels of gene activity. Nevertheless, a critical gap remains because most available genome-wide data lack the temporal and single-cell information needed to grasp the intricate interactions of enhancers and promoters in individual cells. The “average” behavior from a population of cells does not capture the highly variable genome topology that each cell exhibits, nor does a stochastic gene expression stemming from a specific enhancer- promoter interaction. To discern how stochastic transcriptional activity contributes to deterministic developmental processes, there is a critical need to decipher the regulatory logic governing enhancer-promoter dynamics at the single-cell level. Our long-term goal is to identify design principles for precise gene control in embryonic development, using a combination of quantitative live imaging, genetic perturbations, and mathematical modeling. The overall objective of this proposal is to determine regulatory principles underlying enhancer-promoter dynamics that govern transcriptional processes to ensure normal development. Beyond matching an enhancer sequence with the target promoter, we will determine the extent to which a linear enhancer-promoter distance and enhancer’s relative orientation to the promoter impacts the target gene expression and subsequent development for short- and intermediate-range enhancer-promoter interactions. For long-range enhancer-promoter interactions, the functional role of insulator-mediated chromatin looping dynamics on transcriptional stochasticity and developmental robustness will be investigated. Lastly, we will determine the differences in regulatory logic governing multi-enhancer-promoter interactions as compared to one-on-one enhancer-promoter interactions. Since the mechanism of transcriptional regulation is conserved across species, the successful completion of our projects will reveal another layer of gene control underlying the development and diseases of multicellular eukaryotes.