Abstract
In eukaryotic nuclei, transcription is associated with discrete foci of RNA Polymerase II (RNAPII) molecules. How these clusters interact with genes and their impact on transcriptional activity remain heavily debated. Here we take advantage of the naturally occurring increase in transcriptional activity during Zygotic Genome Activation (ZGA) in Drosophila melanogaster embryos to characterize the functional roles of RNAPII clusters in a developmental context. Using single-molecule tracking and lattice light-sheet microscopy, we find that RNAPII cluster formation depends on transcription initiation and that cluster lifetimes are reduced upon transcription elongation. We show that single clusters are stably associated with active gene loci during transcription and that cluster intensities are strongly correlated with transcriptional output. Our data suggest that prior to ZGA, RNAPII clusters prime genes for activation, whereas after ZGA, clusters are composed mostly of elongating molecules at individual genes.