Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II

Steurer, Barbara; Janssens, Roel C.; Geverts, Bart; Geijer, Marit E; Wienholz, Franziska; Theil, Arjan F.; Chang, Jiang; Dealy, Shannon; Pothof, Joris; Cappellen, Wiggert A. van; Houtsmuller, Adriaan B.; Marteijn, Jürgen A.

doi:10.1073/pnas.1717920115

Cited by 181 publications

(265 citation statements)

References 56 publications

(121 reference statements)

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“…To further explore the relationship between global chromatin dynamics and transcriptional activity, we examined the dynamics of RNA polymerase II (RPB1-Dendra2; RNA Pol II) in live U2OS cell nuclei ( Figure 4a) at different transcriptional states. Hi-D analysis resolved three mobility populations of RNA Pol II (Figure 4b), which is consistent with previous studies that revealed three kinetically different groups of RNA Pol II based on their chromatin-binding times [28,29]. The three dynamic populations in actively transcribing cells (grown in normal condition) exhibited significantly higher diffusion constants as in transcriptionally less active cells (serum-starved cells) ( Figure 4b).…”

Section: Transcription Status Modulates Chromatin Diffusion Processessupporting

confidence: 89%

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

Shaban

Barth

Bystricky

2018

Preprint

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ABSTRACTBulk chromatin motion has not been analysed at high resolution. We present Hi-D, a method to quantitatively map dynamics of chromatin and abundant nuclear proteins for every pixel simultaneously over the entire nucleus from fluorescence image series. Hi-D combines reconstruction of chromatin motion, and classification of local diffusion processes by Bayesian inference. We show that DNA dynamics in the nuclear interior are spatially partitioned into 0.3 – 3 μm domains in a mosaic-like pattern, uncoupled from chromatin compaction. This pattern was remodelled in response to transcriptional activity. Hi-D can be applied to any dense and bulk structures opening new perspectives towards understanding motion of nuclear molecules.

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Section: Transcription Status Modulates Chromatin Diffusion Processessupporting

confidence: 89%

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

Shaban

Barth

Bystricky

2018

Preprint

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“…To strength our proposal, we treated cells with a CDK7 inhibitor THZ1, which suppresses initiation and pausing (Kwiatkowski et al , ; Steurer et al , ). THZ1 treatment markedly decreased heat shock‐induced expression of HSP70 mRNA (Appendix Fig S6E).…”

Section: Resultsmentioning

confidence: 99%

The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment

et al. 2019

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The recruitment of RNA polymerase II (Pol II) to core promoters is highly regulated during rapid induction of genes. In response to heat shock, heat shock transcription factor 1 (HSF1) is activated and occupies heat shock gene promoters. Promoter‐bound HSF1 recruits general transcription factors and Mediator, which interact with Pol II, but stress‐specific mechanisms of Pol II recruitment are unclear. Here, we show in comparative analyses of HSF1 paralogs and their mutants that HSF1 interacts with the pericentromeric adaptor protein shugoshin 2 (SGO2) during heat shock in mouse cells, in a manner dependent on inducible phosphorylation of HSF1 at serine 326, and recruits SGO2 to the HSP70 promoter. SGO2‐mediated binding and recruitment of Pol II with a hypophosphorylated C‐terminal domain promote expression of HSP70, implicating SGO2 as one of the coactivators that facilitate Pol II recruitment by HSF1. Furthermore, the HSF1‐SGO2 complex supports cell survival and maintenance of proteostasis in heat shock conditions. These results exemplify a proteotoxic stress‐specific mechanism of Pol II recruitment, which is triggered by phosphorylation of HSF1 during the heat shock response.

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“…This discrepancy may be explained by recent experiments, including live imaging in vivo which showed that most 5' bound Pol II is rapidly turned over (Erickson et al 2018;Kamieniarz-Gdula and Proudfoot 2019;Steurer et al 2018) This could be associated with cycles of abortive initiation rather than stable pausing. Over time, such transiently associated Pol II may be detected by ChIP but would not necessarily produce scaRNAs which require transcriptionally engaged Pol II.…”

Section: Discussionmentioning

confidence: 97%

“…As turnover of Pol II during recruitment/initiation is approximately two orders of magnitude faster than in gene bodies (J. Li et al 2019;Steurer et al 2018) this could result in a higher level of fixation in promoter versus gene bodies resulting in skewed increases in pausing indices which does not truly reflect the process of transcription which is readily seen using scaRNAseq and nascent-RNA-seq.…”

Section: Discussionmentioning

confidence: 99%

“…However, recently it has also been suggested that the high density of Pol II as revealed by ChIP-seq may reflect a dynamic process of premature termination (Erickson et al 2018). This is supported by in vivo imaging studies which reveal the majority of Pol II is associated with chromatin for seconds (Steurer et al 2018) and modeling which revealed that the turnover of Pol II on housekeeping genes was comparable to highly "paused" genes such as HSPA1 (Krebs et al 2017). Whilst it is clear from many studies that Pol II is often found near the TSS the question remains to what extent does this accumulation contribute to changes in gene expression?…”

Section: Differential Gene Expression Is Predominantly Regulated Via mentioning

confidence: 95%

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Enhancers predominantly regulate gene expression in vivo via transcription initiation

Larke

Nojima

Telenius

et al. 2019

Preprint

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Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

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Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II

Cited by 181 publications

References 56 publications

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

Hi-D: Nanoscale mapping of nuclear dynamics in single living cells

The pericentromeric protein shugoshin 2 cooperates with HSF 1 in heat shock response and RNA Pol II recruitment

Enhancers predominantly regulate gene expression in vivo via transcription initiation

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