Transcriptional burst frequency and burst size are equally modulated across the human genome

Dar, Roy D.; Razooky, Brandon S.; Singh, Abhyudai; Trimeloni, Thomas V.; McCollum, James M.; Cox, Chris D.; Simpson, Michael L.; Weinberger, Leor S.

doi:10.1073/pnas.1213530109

Cited by 464 publications

(623 citation statements)

References 46 publications

(66 reference statements)

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“…However, cell-free systems allow the characterization of the intrinsic fluctuations of an individual gene circuit without the confounding extrinsic effects 18 that cannot be avoided in cellular systems. Analysis of noise can offer important physical insights into how genetic circuits are structured and how they function, and has been used in cellular systems to characterize negative 25 and positive 27 autoregulation, extrinsic and intrinsic contributions to expression noise 18 , and transcriptional bursting 45,46 . Here we describe the study of a cell-free expression system in microfluidic devices that enable the simultaneous control of reactor size and reaction initiation times, in order to better understand the roles that confinement and crowding 47,48 have on intrinsic protein expression noise without the complications associated with living cells.…”

Section: Representative Resultsmentioning

confidence: 99%

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred

Caveney

Retterer

et al. 2015

JoVE

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Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

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Section: Representative Resultsmentioning

confidence: 99%

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred

Caveney

Retterer

et al. 2015

JoVE

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“…Triggering of these events in single cells is influenced by fluctuations in protein levels that arise naturally due to noise in gene expression (15)(16)(17)(18). Increasing evidence shows considerable cell-to-cell variation in timing of intracellular events among isogenic cells (19-21), and it is unclear how noisy expression generates this variation.…”

mentioning

confidence: 99%

First-passage time approach to controlling noise in the timing of intracellular events

Ghusinga

Dennehy

Singh

2017

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

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In the noisy cellular environment, gene products are subject to inherent random fluctuations in copy numbers over time. How cells ensure precision in the timing of key intracellular events despite such stochasticity is an intriguing fundamental problem. We formulate event timing as a first-passage time problem, where an event is triggered when the level of a protein crosses a critical threshold for the first time. Analytical calculations are performed for the first-passage time distribution in stochastic models of gene expression. Derivation of these formulas motivates an interesting question: Is there an optimal feedback strategy to regulate the synthesis of a protein to ensure that an event will occur at a precise time, while minimizing deviations or noise about the mean? Counterintuitively, results show that for a stable long-lived protein, the optimal strategy is to express the protein at a constant rate without any feedback regulation, and any form of feedback (positive, negative, or any combination of them) will always amplify noise in event timing. In contrast, a positive feedback mechanism provides the highest precision in timing for an unstable protein. These theoretical results explain recent experimental observations of single-cell lysis times in bacteriophage λ. Here, lysis of an infected bacterial cell is orchestrated by the expression and accumulation of a stable λ protein up to a threshold, and precision in timing is achieved via feedforward rather than feedback control. Our results have broad implications for diverse cellular processes that rely on precise temporal triggering of events.first-passage time | event timing | stochastic gene expression | feedback control | single cell T iming of events in many cellular processes, such as cell-cycle control (1-3), cell differentiation (4, 5), sporulation (6, 7), apoptosis (8, 9), development (10, 11), temporal order of gene expression (12)(13)(14), and so on, depend on regulatory proteins reaching critical threshold levels. Triggering of these events in single cells is influenced by fluctuations in protein levels that arise naturally due to noise in gene expression (15)(16)(17)(18). Increasing evidence shows considerable cell-to-cell variation in timing of intracellular events among isogenic cells (19-21), and it is unclear how noisy expression generates this variation. Characterization of control strategies that buffer stochasticity in event timing are critically needed to understand reliable functioning of diverse intracellular pathways that rely on precision in timing.Mathematically, noise in the timing of events can be investigated via the first-passage time (FPT) framework, where an event is triggered when a stochastic process (single-cell protein level) crosses a critical threshold for the first time. There is already a rich tradition of using such FPT approaches to study timing of events in biological and physical sciences (22-26). Following this tradition, exact analytical expression for the FPT distribution is computed in experimentally valida...

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“…Burst size, that is, the number of transcripts per burst, and burst frequency, that is, the number of transcriptional bursts per time unit are gene specific and appear to depend on the promoter architecture, such as the presence of a CAAT box, a TATA box, the size of the nucleosome-free region as well as the location and number of transcription factor binding sites [7][8][9][10][11][12] . Burst sizes of between 1 and 450 transcripts per burst have been observed followed by periods of inactivity of up to several hours 7,8,[12][13][14][15][16][17][18][19] .…”

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confidence: 99%

Transcriptional refractoriness is dependent on core promoter architecture

Cesbron

Oehler

et al. 2015

Nat Commun

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Genes are often transcribed in random bursts followed by long periods of inactivity. Here we employ the light-activatable white collar complex (WCC) of Neurospora to study the transcriptional bursting with a population approach. Activation of WCC by a light pulse triggers a synchronized wave of transcription from the frequency promoter followed by an extended period (B1 h) during which the promoter is refractory towards restimulation. When challenged by a second light pulse, the newly activated WCC binds to refractory promoters and has the potential to recruit RNA polymerase II (Pol II). However, accumulation of Pol II and phosphorylation of its C-terminal domain repeats at serine 5 are impaired. Our results suggest that refractory promoters carry a physical memory of their recent transcription history. Genome-wide analysis of light-induced transcription suggests that refractoriness is rather widespread and a property of promoter architecture.

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Transcriptional burst frequency and burst size are equally modulated across the human genome

Cited by 464 publications

References 46 publications

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Sealable Femtoliter Chamber Arrays for Cell-free Biology

First-passage time approach to controlling noise in the timing of intracellular events

Transcriptional refractoriness is dependent on core promoter architecture

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