Live-cell characterization of transcriptional condensates

Transcription involves many factors and chromatin elements that need to locally act in a coordinated fashion. However, the underlying spatiotemporal organization and regulation of these are incompletely understood. Recent work supports a model in which factors involved with transcription can form phase-separated biomolecular condensates. Although these transcriptional condensates (TCs) are known to be large and dynamic multicomponent clusters and thought to compartmentalize gene expression, little is known about their properties and functions in live cells. Here, we set out to establish a platform for studying transcriptional condensates directly in live cells under near-physiological conditions. We use multigene endogenous fluorescent tagging with high-throughput live-cell and super-resolution imaging techniques to determine TC properties. In combination with systematic perturbations, we characterize TCs, elucidate factor and chromatin dependencies, and investigate how TCs can react to their environment. Our investigations show that in response to environmental changes as well as specific cellular perturbations the abundance, size, and dynamics of TCs are altered. These experiments also unravel novel and unanticipated dependencies, which we now want to understand in more detail. In conclusion, our live cell and endogenous approach enables a thorough characterization of transcriptional condensates. The results obtained have provided insights into the dynamic properties of TCs and are thereby relevant to the regulation of gene expression in space and time.