# A single cluster of RNA Polymerase II molecules is stably associated with active genes

## TL;DR

RNA Polymerase II clusters remain stably bound to active genes during transcription in fly embryos, with cluster intensity reflecting transcriptional activity.

## Contribution

The study shows that RNA Polymerase II clusters form through transcription initiation and represent engaged polymerases, not phase separation.

## Key findings

- RNA Polymerase II clusters are stably associated with active gene loci during transcription.
- Cluster intensity is strongly correlated with transcriptional output.
- Cluster formation depends on transcription initiation and not higher-order mechanisms like phase separation.

## Abstract

In eukaryotic nuclei, transcription is associated with the clustering of RNA Polymerase II (RNAPII) molecules. The mechanisms underlying cluster formation, their interactions 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 depend on transcriptional activity when not constrained by interphase duration. We show that single clusters are stably associated with active gene loci during transcription and that cluster intensities are strongly correlated with transcriptional output. Collectively, our data and simulations on cluster formation kinetics show that RNAPII clusters reflect local accumulations of transcriptionally engaged polymerases and do not form through higher-order mechanisms such as phase separation.

Live molecular-scale imaging in fly embryos shows that a single RNA Polymerase II cluster stays bound to active genes during transcription. Cluster formation requires initiation, and intensity represents the number of engaged polymerases at a gene.