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

Pol II Micro Clusters In higher eukaryotes, messenger RNA (mRNA) synthesis is thought to involve foci of clustered RNA polymerase II (Pol II) called transcription factories. However, clustered Pol II have not been resolved in living cells, raising the debate about their existence in vivo and what role, if any, they play in nuclear organization and regulation of gene expression. Cisse et al. (p. 664, published online 4 July; see the Perspective by Rickman and Bickmore) developed single-molecule in vivo analyses revealing the distribution and dynamics of Pol II clustering in living cells. Pol II clusters were smaller than the diffraction limit (<250 nm). Transient dynamics of the Pol II clusters, and correlation with changes in transcription, pointed to a role in transcription initiation rather than in elongation. A single-cell quantitative method reveals changes in the distribution of proteins with single-molecule sensitivity. [Also see Perspective by Rickman and Bickmore] 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.