Promoter and Gene-Body RNA-Polymerase II co-exist in partial demixed condensates

Abstract

In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesised to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these phases would mix when present at the same time or remain distinct chemical environments; either as multi-phase condensates or by forming entirely separate condensates. Here we combine particle-based multi-scale simulations and experiments in the model organism C. elegans to characterise the biophysical properties of RNA polymerase II condensates. Both simulations and the in vivo work describe a lower critical solution temperature (LCST) behaviour of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensate stability. Importantly this gradual change in temperature correlates with an incremental transcriptional response to temperature, but is largely uncoupled from the classical stress response. The LCST behaviour of CTD also highlights that these condensates are physio-chemically distinct from heterochromatin condensates. Expanding the simulations we model how the degree of phosphorylation of the disordered C-terminal domain of RNA polymerase II (CTD), which is characteristic for each step of transcription, controls demixing of CTD and pCTD in line with phase separation experiments. We show that the two phases putatively underpinning the initiation of transcription and transcription elongation constitute distinct chemical environments and are in agreement with RNA polymerase II condensates observed in C. elegans embryos by super resolution microscopy. Our analysis reveals how depending on its post-translational modifications and its interaction partners a single protein can adopt multiple morphologies and how partially engulfed condensates promote the selective recruitment of additional factors to the different phases.