Quantifying out-of-equilibrium transcriptional bursting in living fly embryos

Gene expression is intrinsically dynamic. A series of molecular events, including RNA polymerase II (Pol II) loading, underlie the regulation of gene (or transcriptional) activation. However, knowledge about the dynamics of these molecular events remains elusive. Here, we present an optimized two-photon microscope to measure gene activity of individual loci in living fly embryos. We achieve single RNA detection sensitivity, and combined with statistical analysis we construct time serieses of single Pol II loading events from the transcriptional output. This leads, for the first time, to the observation of non-steady state transcriptional bursting. We quantified the kinetic parameters of Pol II loading events, such as waiting time distributions of Pol II loading, time-dependent transcription rates, and durations of transcriptional bursting. With a novel inference framework, we extract the non-stationary bursting parameters from the transcriptional output. Applying our approach to multiple genes, we found common regulatory strategies that are shared among different genes, regardless of space and time during embryo development, opening the path to universal features of out-of-equilibrium gene control.