Stepwise modifications of transcriptional hubs link pioneer factor activity to a burst of transcription.

Binding of transcription factors (TFs) promotes the subsequent recruitment of coactivators and preinitiation complexes to initiate eukaryotic transcription, but this time course is usually not visualized. It is commonly assumed that recruited factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. We use live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), to define a cascade of events upstream of transcriptional initiation in early Drosophila embryos. These factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. Zelda and the acetyltransferase dCBP nucleate dBrd4 clusters, which then trigger pre-transcriptional clustering of RNAPII. Subsequent transcriptional elongation disperses clusters of dBrd4 and RNAPII. Our results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biomolecular condensates that culminates in a self-limiting burst of transcription.