Project Summary Transcription is a critical yet a complex process that requires precise regulation to ensure normal development. A large number of molecules and regulatory elements in the genome should interact in combinatorial manner to determine when and where a gene needs to be turned on and off. Such complex nature of the process makes it challenging to understand the comprehensive mechanisms of transcriptional regulation. Recent studies with state-of-the-art live imaging techniques provide clear evidence that transcription activity is dynamic, consisting of a series of sequential and stochastic bursts. While it is suggested that transcriptional bursting is a general property of gene expression across many species, the underlying cause of transcriptional bursting remains largely unknown. The experiments described in the proposal explore the potential role of unstable enhancer-promoter interactions as a cause of the transcriptional bursting. This research project proposes to test two aspects of the relationship between enhancer-promoter interactions and transcription. First, it will be determined whether transcriptional bursts arise from unstable enhancer- promoter interactions. While some studies with cultured cells suggested that bursts might arise from unstable assembly of pre-initiation complex or competitive binding of nucleosomes, there has been no effort to investigate the role of enhancers in this process. Drosophila embryos will be used as a model system, since extensive studies have identified hundreds of enhancers, and live imaging permits the detailed visualization of de novo transcription in development. The system is ideally suited for testing the hypothesis that stochastic enhancer-promoter interactions influence the frequency, duration, or amplitudes of transcriptional bursts. Second, the traditional looping models of enhancer-promoter interactions will be explored. It is generally assumed that enhancers that are bound to their cognate activators somehow loop to the target promoter. This looping process recruits the pre-initiation complex and allows the release of Pol II to start transcription. The preliminary results, however, suggest that this physical looping might not be required for transcription. A plasmid construct where two target promoters are regulated by a single shared enhancer was used to show that both promoters exhibit coordinated transcriptional bursts, turning on and off with similar dynamics. This provides a counter-example to the traditional looping models, whereby two reporter genes would display sequential bursts due to separate enhancer-promoter looping interactions. With combination of transgenesis, live imaging, and optogenetics, this research project proposes to force interactions of single enhancer- promoter loops and analyze the effect on transcription dynamics with quantitative image processing. The accomplishment of the proposed studies will provide a dynamic view of the nature of enhancer-promoter communication and how it can contribute to transcriptional regulation in eukaryotes.