Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells

Abstract

Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell's ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

“…Despite these plausible scenarios, we cannot exclude the possibility that a small fraction of RNAP II molecules do form functional clusters. In fact, our observations are in line with the recent report in living cells of transient RNAP II assembly to short-lived clusters (41). The evanescent nature of this assembly might result in a small subset of molecules colocalizing with each other at a given moment, whereas on a global scale the majority of the RNAP II molecules remain unclustered.…”

Interallelic interaction and gene regulation in budding yeast

“…Recent studies indicate that pol II tends to form concentrated foci in the nucleus, and such pol II "clustering" may present a rate-limiting step for transcription activation (39). Because the Δ1/Δ2pr has less Gal4 binding capacity, we speculate that they are not as effective as the WT promoter in the initial recruitment of general transcription factors and pol II, causing the delayed activation.…”

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…The results strongly support a functional link between Mig1 and Msn2 TF clusters and target gene expression; a biological role of multivalent TFs for enhancing intersegmental transfer had been elucidated previously in simulations [9] but unobserved experimentally until our discoveries here, and so our findings impact on the longstanding question of how TFs might find their targets in the genome so efficiently. Clustering of a range of nuclear factors has been observed previously using single-molecule techniques, such as transient RNA Polymerase II cluster dynamics in living cells using time-correlated PALM (tc-PALM) [73,74]. Also functional nuclear protein clusters have been seen [75] and the Bicoid transcription factor in fruit fly embryos has been observed to form clusters mediated in part mediated by intrinsically disordered peptide sequences [76].…”