Live-imaging and optogenetic dissection of transcriptional repression in living embryos

Transcriptional repression is essential for establishing and maintaining gene expression patterns. However, how repressors control gene expression at the single-cell level over time remains elusive. Here, we utilized optogenetics and live-imaging tools to analyze transcriptional repression in the early fruit fly embryo. Our analysis reveals that repression is achieved through all-or-nothing silencing activities controlled by a sharp response function. As a result, the repressor modulates the fraction of silenced cells rather than the mRNA production rate. We then applied optogenetic perturbations to demonstrate the reversible nature of silencing, with both silencing and reactivation occurring with an average response time of 2.5-3 minutes. Further analysis reveals that we can use a two-state model, including promoter ON and promoter OFF states, to recapitulate observed silencing/reactivation dynamics. The repressor controls the bursting frequency, which indicates that the repressor can only act on transcription when the promoter is switched off. We have provided a framework for further understanding and rational control of the eukaryotic transcriptional repression.