Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

Uchino, Satoshi; Ito, Yoshiyuki; Sato, Yuko; Handa, Tetsuya; Ohkawa, Yasuyuki; Tokunaga, Makio; Kimurâ, Hiroshi

doi:10.1101/2021.04.27.441582

Cited by 3 publications

(2 citation statements)

References 94 publications

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“…6 D shows confocal images of Xenopus GVs isolated into mineral oil under physiological conditions ( 31 ). By injecting the mRNA encoding RNA Pol-II ser2 mintbody ( 32 ), we confirmed the DNA aggregates are the sites of transcriptional assemblies ( SI Appendix , Fig. S5 ).…”

Section: Resultsmentioning

confidence: 58%

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

Javed¹,

Jullien²,

Agarwal³

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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An important characteristic of cell differentiation is its stability. Only rarely do cells or their stem cell progenitors change their differentiation pathway. If they do, it is often accompanied by a malfunction such as cancer. A mechanistic understanding of the stability of differentiated states would allow better prospects of alleviating the malfunctioning. However, such complete information is yet elusive. Earlier experiments performed in Xenopus oocytes to address this question suggest that a cell may maintain its gene expression by prolonged binding of cell type–specific transcription factors. Here, using DNA competition experiments, we show that the stability of gene expression in a nondividing cell could be caused by the local entrapment of part of the general transcription machinery in transcriptionally active regions. Strikingly, we found that transcriptionally active and silent forms of the same DNA template can stably coexist within the same nucleus. Both DNA templates are associated with the gene-specific transcription factor Ascl1, the core factor TBP2, and the polymerase II (Pol-II) ser5 C-terminal domain (CTD) phosphorylated form, while Pol-II ser2 CTD phosphorylation is restricted to the transcriptionally dominant template. We discover that the active and silent DNA forms are physically separated in the oocyte nucleus through partition into liquid–liquid phase-separated condensates. Altogether, our study proposes a mechanism of transcriptional regulation involving a spatial entrapment of general transcription machinery components to stabilize the active form of a gene in a nondividing cell.

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Section: Resultsmentioning

confidence: 58%

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

Javed¹,

Jullien²,

Agarwal³

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…One example would be "transcription factories," defined by immunostaining against RNA Pol II carboxy-terminal domain (CTD) amino acid repeats phosphorylated at specific sites (Ser2, Ser5) in both fixed and living cells (Fig. 1N; Xie and Pombo 2006;Uchino et al 2021). Several hundred to thousands of punctate foci, ∼80-130 nm in diameter, are present per nucleus (Jackson et al 1998;Cook 1999;Eskiw and Fraser 2011), localizing adjacent to many active gene loci (Osborne et al 2004(Osborne et al , 2007Ferrai et al 2010).…”

Section: Less Conventional Suspectsmentioning

confidence: 99%

Nuclear Compartments: An Incomplete Primer to Nuclear Compartments, Bodies, and Genome Organization Relative to Nuclear Architecture

Belmont

2021

Cold Spring Harb Perspect Biol

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This work reviews nuclear compartments, defined broadly to include distinct nuclear structures, bodies, and chromosome domains. It first summarizes original cytological observations before comparing concepts of nuclear compartments emerging from microscopy versus genomic approaches and then introducing new multiplexed imaging approaches that promise in the future to meld both approaches. I discuss how previous models of radial distribution of chromosomes or the binary division of the genome into A and B compartments are now being refined by the recognition of more complex nuclear compartmentalization. The poorly understood question of how these nuclear compartments are established and maintained is then discussed, including through the modern perspective of phase separation, before moving on to address possible functions of nuclear compartments, using the possible role of nuclear speckles in modulating gene expression as an example. Finally, the review concludes with a discussion of future questions for this field.

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A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana

et al. 2021

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Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into Arabidopsis thaliana. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics.

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Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

Cited by 3 publications

References 94 publications

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

DNA-induced spatial entrapment of general transcription machinery can stabilize gene expression in a nondividing cell

Nuclear Compartments: An Incomplete Primer to Nuclear Compartments, Bodies, and Genome Organization Relative to Nuclear Architecture

A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana

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