RNA polymerase II clusters form in line with surface condensation on regulatory chromatin

Abstract

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid–liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super‐resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid‐phase condensation at concentrations that are too low for canonical liquid–liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface‐associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding.

Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications

“…Finally, the complexity of chromatin-based condensates involving a variety of distinct biomolecular species, as opposed to purely protein-based condensates, also raises concerns as to whether LLPS is still a sufficiently accurate mechanistic model for describing these systems, or if certain modifications are needed to account for the size and structural features of chromatin, as well as the distinct types of interactions involved. For example, the finding that Pol II clusters adopt a variety of shapes in zebrafish is consistent with a model in which regulatory chromatin provides surfaces for liquid condensation at concentrations too low for LLPS to occur and points to an alternative surface condensation mechanism distinct from canonical LLPS [ 163 ]. In other cases where chromatin bridging is necessary to initiate condensate formation, PPPS is perhaps more suitable as an alternative mechanism [ 46 ], although definitive experimental evidence for PPPS in actual biological systems remains very limited to date.…”

Spatially coherent diffusion of human RNA Pol II depends on transcriptional state rather than chromatin motion

“…Transcription by RNA Polymerase II (Pol II) takes place for all protein-coding genes in eukaryotic genomes and is vital for many physiological processes [3]. Transcription often takes place in so-called transcription factories, domains of clustered transcription factors, whose formation has been explained by liquid-liquid phase separation [4][5][6]. These transcription factories are highly dynamic macromolecular that permit transcription initiation and elongation [7,8].…”

RNA polymerase II and PARP1 shape enhancer-promoter contacts

“…Given its stable attachment to DNA through the transcription bubble, it is possible that paused Pol II may serve as one of the tethers connecting promoter or enhancer DNA into an enhancerpromoter interaction. Under a hub model, paused Pol II initiated from multiple TSSs within a transcription initiation domain 36 may serve to keep both enhancer and promoter DNA tethered to the hub 79,80 . Indeed, paused Pol II tethering enhancers into a hub may serve as one way in which enhancer-templated RNAs (eRNAs) have a sequence-independent biological function 81 .…”