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

  • bioRxiv. 2025 Sep 16:2025.02.10.637507. doi: 10.1101/2025.02.10.637507.
Apratim Mukherjee  1  2 ,  Manya Kapoor  2  3 ,  Kareena Shankta  2  4 ,  Samantha Fallacaro  2  5 ,  Raymond D Carter  2  6 ,  Gabriela Hayward-Lara  2  5 ,  Puttachai Ratchasanmuang  2  7 ,  Yara I Haloush  2 ,  Mustafa Mir  1  2  7  8
Affiliations
  • 1. Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania; Philadelphia, PA 19104, USA.
  • 2. Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia; Philadelphia, PA 19104, USA.
  • 3. Department of Bioengineering, University of Pennsylvania; Philadelphia, PA 19104, USA.
  • 4. Roy and Diana Vagelos Program in Life Sciences and Management, University of Pennsylvania; Philadelphia, PA 19104, USA.
  • 5. Developmental, Stem Cell, and Regenerative Biology Graduate Group, Perelman School of Medicine; Philadelphia, PA 19104, USA.
  • 6. Biochemistry, Biophysics, and Chemical Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania; Philadelphia, PA 19104, USA.
  • 7. Howard Hughes Medical Institute, Children's Hospital of Philadelphia; Philadelphia, PA 19104, USA.
  • 8. Epigenetics Institute, University of Pennsylvania Perelman School of Medicine; Philadelphia, PA 19104, USA.
PMID: 39990393 DOI: 10.1101/2025.02.10.637507
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.

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