Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Sepúlveda, Leonardo A.; Xu, Heng; Zhang, Jing; Wang, Mengyu; Golding, Ido

doi:10.1126/science.aad0635

Cited by 119 publications

(151 citation statements)

References 43 publications

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“…Furthermore, the largely changeable k A , relative to the other parameters ( k R and k I ), is consistent with the suggested kinetic scheme under the gene-nonspecific constraint, in which only k off (10 −5 ∼10 2 s −1 ) is varied to change the gene expression level6. In addition, our model is compatible with the model that promoter structure and state control the kinetics of transcriptional bursting1023 by the modulation of transcription initiation rate ( k I ). The k I is a critical parameter to control not only the mean of mRNA distribution but also the Fano factor as our experimental data suggest.…”

Section: Discussionsupporting

confidence: 85%

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

2016

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Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

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Section: Discussionsupporting

confidence: 85%

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

2016

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“…At the same time, recent work has also attempted to link transcription factor localization and binding to transcription. Using cutting edge microscopy (Elf et al, 2007; Hammar et al, 2012) and probing technology (Shah and Tyagi, 2013), efforts to relate the binding of transcription factors to transcription itself are just now shedding light on these relationships (Larson et al, 2011b; Sepúlveda et al, 2016; Shah and Tyagi, 2013; Xu et al, 2015), and single cell reporters of methylation are on the horizon as well (Stelzer et al, 2015). The advent of genome-wide techniques for measuring transcription factor binding provide a glimpse of what the future holds in this regard (Buenrostro et al, 2015; Cusanovich et al, 2015).…”

Section: How Cells Are Different From Each Other: Let Me Count the Waysmentioning

confidence: 99%

What’s Luck Got to Do with It: Single Cells, Multiple Fates, and Biological Nondeterminism

2016

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The field of single cell biology has morphed from a philosophical digression at its inception to a playground for quantitative biologists, to a major area of biomedical research. The last several years have witnessed an explosion of new technologies, allowing us to apply ever more of the modern molecular biology toolkit to single cells. Conceptual progress, however, has been comparatively slow. Here, we provide a framework for classifying both the origins of the differences between individual cells and the consequences of those differences. We discuss how the concept of “random” differences is context dependent, and propose that rigorous definitions of inputs and outputs may bring clarity to the discussion. We also categorize ways in which probabilistic behavior may influence cellular function, highlighting studies that point to exciting future directions in the field.

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“…Uncovering such dynamics is not only fundamental to comprehending how transcription is orchestrated, but also essential to interpret the behaviors of gene regulatory networks – due to the resulting complex temporal evolution of transcript numbers (2,4,17–22). Those kinetic steps are hard to detect experimentally, especially when unstable protein complexes and unknown transient interactions are involved (23–26). Recently, the steps of the holoenzyme σ 54 -RNA polymerase (σ 54 RNAP) associating with promoter DNA have been dissected (27,28).…”

Section: Introductionmentioning

confidence: 99%

Kinetics of transcription initiation directed by multiplecis-regulatory elements on theglnAp2promoter

2016

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Transcription initiation is orchestrated by dynamic molecular interactions, with kinetic steps difficult to detect. Utilizing a hybrid method, we aim to unravel essential kinetic steps of transcriptional regulation on the glnAp2 promoter, whose regulatory region includes two enhancers (sites I and II) and three low-affinity sequences (sites III-V), to which the transcriptional activator NtrC binds. By structure reconstruction, we analyze all possible organization architectures of the transcription apparatus (TA). The main regulatory mode involves two NtrC hexamers: one at enhancer II transiently associates with site V such that the other at enhancer I can rapidly approach and catalyze the σ54-RNA polymerase holoenzyme. We build a kinetic model characterizing essential steps of the TA operation; with the known kinetics of the holoenzyme interacting with DNA, this model enables the kinetics beyond technical detection to be determined by fitting the input-output function of the wild-type promoter. The model further quantitatively reproduces transcriptional activities of various mutated promoters. These results reveal different roles played by two enhancers and interpret why the low-affinity elements conditionally enhance or repress transcription. This work presents an integrated dynamic picture of regulated transcription initiation and suggests an evolutionarily conserved characteristic guaranteeing reliable transcriptional response to regulatory signals.

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Measurement of gene regulation in individual cells reveals rapid switching between promoter states

Cited by 119 publications

References 43 publications

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

What’s Luck Got to Do with It: Single Cells, Multiple Fates, and Biological Nondeterminism

Kinetics of transcription initiation directed by multiplecis-regulatory elements on theglnAp2promoter

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