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

ABSTRACT Eukaryotic transcription begins with the binding of transcription factors (TFs), which promotes the subsequent recruitment of coactivators and pre-initiation complexes. It is commonly assumed that these factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. Here we performed live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), in early Drosophila embryos. We show that these factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. We present evidence that Zelda acts with the acetyltransferase dCBP to nucleate dBrd4 hubs, which then trigger pre-transcriptional clustering of RNAPII; continuous transcriptional elongation then disperses clusters of dBrd4 and RNAPII. Our results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biochemical complexes that culminate in a self-limiting burst of transcription.