Our “STREAMING-tag” paper is now available on bioRxiv!
In this study, we revealed spatiotemporal relationships between regulatory factors and transcriptional activity!
Transcription dynamically switches between ON and OFF states, but the regulatory mechanism is not fully understood. Mediator, BRD4, and non-phosphorylated RNA polymerase II (RNAPII) has been shown to form clusters around gene regions in the ON state.
In the ON state, gene loci can be visualized by the MS2 system, but this is not the case in the OFF state. To understand the regulatory mechanism of transcriptional bursting, it is necessary to understand what is happening not only in the ON state but also in the OFF state.
Therefore, we established the “STREAMING-tag” system, which can quantify gene transcriptional activity and subcellular localization of genes near the TSS with minimal effect on gene function. See the paper for details!
We applied this system to Nanog in mouse ES cells, and found that clusters of RPB1, the largest subunit of RNAPII, and BRD4, a coactivator, are dynamically associated with the TSS of Nanog during transcriptional bursting.
We also visualized the organization of specifically phosphorylated forms of RNAPII (i.e., Ser5ph and Ser2ph in the initiation and elongation complexes, respectively) using newly developed genetically encoded probes, mintbodies, developed by Kimura Hiroshi lab at Tokyo Tech!!
The TSS region of Nanog was more closely associated with the cluster of Ser5-phosphorylated RNAPII than with the Ser2-phosphorylated form. This implies that the STREAMING-tag system can visualize the TSS-proximal region of Nanog.
In contrast to RNAPII and BRD4, clusters of MED19 and MED22, the Mediator subunits, were near the TSS, regardless of the transcriptional state, may serve as a scaffold for the formation of new initiation clusters containing BRD4 and hypophosphorylated RNAPII.
Consequently, the STREAMING-tag system revealed 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.
Finally, I would like to thank everyone who was involved in this work. In particular, I would like to thank wonderful postdoc Hiroaki for his hard work, which was the main driving force behind this work.
Thanks also to the grad students Seiru, Manabu, and Hitoshi who contributed to the work. I would also like to thank Kimura-san for not only developing mintbody, but also for making it a deep research with thoughtful discussions!!