STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Abstract

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

Transcriptional clusters follow a conserved condensation-dispersal sequence during stem cell differentiation

“…Further, the formation of Pol II clusters in zebrafish embryos could be reproduced by simulations of surface condensation, where regulatory chromatin acts as a condensation surface [30]. Previous work found that, once formed, transcriptional clusters can also exhibit an internal spatial organization, including compartments at the scale of ∼100 nm that correspond to consecutive steps of transcription regulation [9,10,21,74]. Separation of Pol II recruitment and transcript elongation, for example, could be explained in terms of the exclusion of transcribed genes and their RNA transcripts from a liquid phase enriched in recruited Pol II [11,75].…”