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      Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

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          Abstract

          The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ∼2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.

          Author Summary

          There is an astonishing amount of variation in the number of mRNA and protein molecules generated from particular genes between genetically identical single cells grown in the same environment. Particularly for mRNA, the large variation seen from these “noisy” genes is consistent with the idea of transcriptional bursting where transcription occurs in random, intermittent periods of high activity. There is considerable experimental support for transcriptional bursting, and it is a primary feature of stochastic models of gene expression that account for variation. Still, it has long been recognized that variation, especially in protein levels, can occur because of global differences between genetically identical cells. We show that in budding yeast, mRNA variation is driven to a large extent by differences in the transcriptional activity of a noisy gene between different phases of the cell cycle. These differences are not because of specific cell-cycle regulation, and in some cases transcription appears restricted to certain phases, leading to pulses of mRNA production. These results raise new questions about the origins of transcriptional bursting and how the statistics of gene expression are regulated in a global way by the cell cycle.

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          Most cited references51

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          Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.

          A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.
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            Gene regulation at the single-cell level.

            The quantitative relation between transcription factor concentrations and the rate of protein production from downstream genes is central to the function of genetic networks. Here we show that this relation, which we call the gene regulation function (GRF), fluctuates dynamically in individual living cells, thereby limiting the accuracy with which transcriptional genetic circuits can transfer signals. Using fluorescent reporter genes and fusion proteins, we characterized the bacteriophage lambda promoter P(R) in Escherichia coli. A novel technique based on binomial errors in protein partitioning enabled calibration of in vivo biochemical parameters in molecular units. We found that protein production rates fluctuate over a time scale of about one cell cycle, while intrinsic noise decays rapidly. Thus, biochemical parameters, noise, and slowly varying cellular states together determine the effective single-cell GRF. These results can form a basis for quantitative modeling of natural gene circuits and for design of synthetic ones.
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              Noise in protein expression scales with natural protein abundance.

              Noise in gene expression is generated at multiple levels, such as transcription and translation, chromatin remodeling and pathway-specific regulation. Studies of individual promoters have suggested different dominating noise sources, raising the question of whether a general trend exists across a large number of genes and conditions. We examined the variation in the expression levels of 43 Saccharomyces cerevisiae proteins, in cells grown under 11 experimental conditions. For all classes of genes and under all conditions, the expression variance was approximately proportional to the mean; the same scaling was observed at steady state and during the transient responses to the perturbations. Theoretical analysis suggests that this scaling behavior reflects variability in mRNA copy number, resulting from random 'birth and death' of mRNA molecules or from promoter fluctuations. Deviation of coexpressed genes from this general trend, including high noise in stress-related genes and low noise in proteasomal genes, may indicate fluctuations in pathway-specific regulators or a differential activation pattern of the underlying gene promoters.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Comput Biol
                PLoS Comput. Biol
                plos
                ploscomp
                PLoS Computational Biology
                Public Library of Science (San Francisco, USA )
                1553-734X
                1553-7358
                July 2013
                July 2013
                25 July 2013
                : 9
                : 7
                : e1003161
                Affiliations
                [1]Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                Brandeis University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CJZ KQ NM. Performed the experiments: CJZ KQ JZ. Analyzed the data: CJZ KQ JZ NM. Contributed reagents/materials/analysis tools: CJZ KQ NM. Wrote the paper: CJZ KQ NM. Designed the software used in analysis: CJZ KQ NM.

                Article
                PCOMPBIOL-D-13-00074
                10.1371/journal.pcbi.1003161
                3723585
                23935476
                f3ee531d-adea-4136-b90d-072ca47018c3
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 January 2013
                : 14 June 2013
                Page count
                Pages: 12
                Funding
                This work was funded by a Monash Graduate Fellowship (to KQ) and GM095733, BBBE 103316, and MIT startup funds (to NM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Computational Biology
                Systems Biology
                Genetics
                Gene Expression
                Molecular Cell Biology
                Cell Growth

                Quantitative & Systems biology
                Quantitative & Systems biology

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