Endogenous Real Time Imaging Reveals Dynamic Chromosomal Mobility During ER-Mediated Transcriptional Burst Events [ChIP-seq]

Enhancers serve as the major activators of mammalian signal-dependent transcriptional programs, exemplified by responses to liganded nuclear receptors, where their acute initial activation, is characterized by alternating periods of target gene “bursting” and “non-busting”, followed by a protracted chronic period of diminished transcriptional activation. Linking these responses to alterations in induced enhancer condensates and locus motility in real time would provide insights into these dynamic regulatory events. Here, endogenous live cell imaging reveals the altered chromosomal dynamics during estrogen receptor  (ER)-dependent target gene bursting/post-bursting and chronic activation events. We first observed that acute stimulation of ER results in increased frequency of bursting compared to the unstimulated state, but bursting actually diminishes with prolonged, chronic E2 stimulation. Second, while acute ligand-dependent activation results in higher levels of mobility of the chromosomal locus than in the unstimulated condition, this mobility further increases during chronic E2 stimulation. Simultaneous DNA/RNA endogenous live imaging of enhancer regulated target gene transcription revealed that the increased mobility of acutely ER-stimulated loci observed during the bursting phase was, unexpectedly, further increased in the subsequent non-burst phase, revealing that the transcription burst event is not the mechanism underlying increased motility. Single molecule tracking (SMT) of ER showed that the relatively high-burst, lower-mobility acute state was indeed enriched for high-viscosity, 1,6-hexanediol-sensative ER molecules. Interestingly, the increased proximity of E2-stimulated loci to interchromatin granules remained unchanged between the bursting and non-bursting phases of acute activation. Together these findings support a dynamic, multi-state condensate model for enhancer-regulated transcriptional programs.