Live-cell super-resolution imaging of enhancer-mediated gene bursting

About This Grant

Abstract The overall theme of my research program is to understand how enhancers and transcription machineries organize in the 3D genome to regulate the target gene expression. We plan to address this problem from two aspects: 1) at the single-cell level, we will characterize the function and dynamics of transcriptional condensates in mouse embryonic stem cells and investigate the specificity of the condensate, the formation and dissolution mechanisms, and their activity regulated by genome architecture; 2) at the developmental level, we will study the mechanism of how transcriptional condensates achieve differential regulation of target genes during development using the beta-globin regulation in mouse erythroleukemia cells as a model. In the past years, we have made significant contributions in the first direction, and we excited to extend our research to a new direction. New methods were developed and insights generated from these studies form the foundation of this proposal. Theme1: In eukaryotes, RNA Pol II, Mediator and transcription activators form transcription-dependent condensates in live mouse embryonic stem cells and enhance super-enhancer controlled gene bursting in a proximity-dependent manner. However, molecular mechanisms of how condensates function on gene bursting within the 3D genome is not fully resolved. The long-term goal of this theme is to characterize the condensate specificity, to investigate their mechanisms of formation and regulation by enhancers/enhancer RNAs, and to develop a full mechanistic understanding of how they interact with their target regulatory elements. In the next five years, we plan to 1) test whether transcriptional condensate can co-activate a reporter gene with different promoter specificity; 2) develop a labeling strategy to image and study the roles of enhancer RNAs in condensate function; 3) selectively manipulate chromosomal interactions and test how condensate-gene interactions and gene bursting change. Theme 2: The dynamics and functions of transcriptional condensate have not been explored extensively due to their close-to-diffraction-limit size. The long-term goal of this theme is to characterize the composition, dynamics and functions of transcriptional condensates and their effect on beta-globin gene switching during erythroid differentiation, aiming to test the function of condensates across different systems. In the next five years, we plan to: 1) test whether and how transcriptional condensate dynamically controls beta-globin gene bursting; 2) develop a method to probe the condensate composition and its changes during erythroid differentiation. We envision that our study will provide significant insights and open new research directions on gene regulation.