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

Stochastic properties of phage promoter Full understanding of regulated gene expression requires characterization of stochastic variation in the activity of individual promoters. To avoid cell-to-cell variability and variation between the activity of specific gene copies, Sepúlveda et al. investigated the behavior of the lysogeny maintenance promoter of phage lambda in individual Escherichia coli cells. They measured the concentration of transcription factor and the actual number of mRNAs produced, and used mathematical modeling to discern the stochastic activity of the regulated promoter. The promoter underwent switching between configurations that occurred more rapidly than the lifetime of mRNA molecules produced, and individual copies of the same gene functioned independently in the same cell. Such studies can reveal new aspects of systems that have been well studied by more conventional techniques. Science, this issue p. 1218 Precise quantitation reveals the properties of a phage gene promoter. In vivo mapping of transcription-factor binding to the transcriptional output of the regulated gene is hindered by probabilistic promoter occupancy, the presence of multiple gene copies, and cell-to-cell variability. We demonstrate how to overcome these obstacles in the lysogeny maintenance promoter of bacteriophage lambda, PRM. We simultaneously measured the concentration of the lambda repressor CI and the number of messenger RNAs (mRNAs) from PRM in individual Escherichia coli cells, and used a theoretical model to identify the stochastic activity corresponding to different CI binding configurations. We found that switching between promoter configurations is faster than mRNA lifetime and that individual gene copies within the same cell act independently. The simultaneous quantification of transcription factor and promoter activity, followed by stochastic theoretical analysis, provides a tool that can be applied to other genetic circuits.

[1]  A. D. Kaiser,et al.  Control of λ Repressor Synthesis , 1971 .

[2]  M. Ptashne.  A Genetic Switch , 1986 .

[3]  I. Dodd,et al.  Octamerization of lambda CI repressor is needed for effective repression of P(RM) and efficient switching from lysogeny. , 2001, Genes & development.

[4]  J. Elf,et al.  Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation , 2014, Nature Genetics.

[5]  M. Ptashne,et al.  Genes and Signals , 2001 .

[6]  B. Pulverer.  Getting specific: Sequence-specific DNA-binding transcription factors , 2005 .

[7]  Huiyi Chen,et al.  Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli , 2015, Molecular systems biology.

[8]  Jie Xiao,et al.  Transcription-Factor-Mediated DNA Looping Probed by High-Resolution, Single-Molecule Imaging in Live E. coli Cells , 2013, PLoS biology.

[9]  P. Swain,et al.  Gene Regulation at the Single-Cell Level , 2005, Science.

[10]  K. Nordström,et al.  Copy‐number control of the Escherichia coli chromosome: a plasmidologist's view , 2006, EMBO reports.

[11]  Lun Cui,et al.  Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping , 2013, Proceedings of the National Academy of Sciences.

[12]  Ido Golding,et al.  Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization , 2013, Nature Protocols.

[13]  Laura Finzi,et al.  Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro , 2011, Proceedings of the National Academy of Sciences.

[14]  C. Zurla,et al.  The effect of nonspecific binding of lambda repressor on DNA looping dynamics. , 2012, Biophysical journal.

[15]  E. Segal,et al.  Predicting expression patterns from regulatory sequence in Drosophila segmentation , 2008, Nature.

[16]  M. Khammash,et al.  The finite state projection algorithm for the solution of the chemical master equation. , 2006, The Journal of chemical physics.

[17]  A. Coulon,et al.  Eukaryotic transcriptional dynamics: from single molecules to cell populations , 2013, Nature Reviews Genetics.

[18]  Eric Mjolsness,et al.  Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy , 2011, Nature Protocols.

[19]  Haidong Feng,et al.  Stochastic expression dynamics of a transcription factor revealed by single-molecule noise analysis , 2012, Nature Structural &Molecular Biology.

[20]  Terence Hwa,et al.  Combinatorial transcriptional control of the lactose operon of Escherichia coli , 2007, Proceedings of the National Academy of Sciences.

[21]  E. Cox,et al.  Gene location and DNA density determine transcription factor distributions in Escherichia coli , 2012, Molecular systems biology.

[22]  Eric H Davidson,et al.  Properties of developmental gene regulatory networks , 2008, Proceedings of the National Academy of Sciences.

[23]  A. Lévine,et al.  Cellular levels of the prophage λ and 434 repressors , 1979 .

[24]  E. Davidson.  The Regulatory Genome: Gene Regulatory Networks In Development And Evolution , 2006 .

[25]  J. Zerubia,et al.  Gaussian approximations of fluorescence microscope point-spread function models. , 2007, Applied optics.

[26]  Eric H. Davidson,et al.  Gene Regulatory Networks for Development: What They Are, How They Work, and What They Mean , 2006 .

[27]  Heng Xu,et al.  COMBINING PROTEIN AND mRNA QUANTIFICATION TO DECIPHER TRANSCRIPTIONAL REGULATION , 2015, Nature Methods.

[28]  M. Elowitz,et al.  Functional roles for noise in genetic circuits , 2010, Nature.

[29]  D. Tranchina,et al.  Stochastic mRNA Synthesis in Mammalian Cells , 2006, PLoS biology.

[30]  Haw Yang,et al.  DNA looping can enhance lysogenic CI transcription in phage lambda , 2008, Proceedings of the National Academy of Sciences.

[31]  M. Khammash,et al.  Systematic Identification of Signal-Activated Stochastic Gene Regulation , 2013, Science.

[32]  Rob Phillips,et al.  Promoter architecture dictates cell-to-cell variability in gene expression , 2014, Science.

[33]  F. Neidhardt,et al.  Escherichia Coli and Salmonella: Typhimurium Cellular and Molecular Biology , 1987 .

[34]  Rapid confirmation of single copy lambda prophage integration by PCR. , 1994, Nucleic acids research.

[35]  Ian B. Dodd,et al.  Cooperativity in long-range gene regulation by the λ CI repressor , 2004 .

[36]  D. Gillespie.  A General Method for Numerically Simulating the Stochastic Time Evolution of Coupled Chemical Reactions , 1976 .

[37]  Nir Friedman,et al.  Linking stochastic dynamics to population distribution: an analytical framework of gene expression. , 2006, Physical review letters.

[38]  G. K. Ackers,et al.  Energetics of subunit dimerization in bacteriophage lambda cI repressor: linkage to protons, temperature, and KCl. , 1991, Biochemistry.

[39]  S. Itzkovitz,et al.  Bursty gene expression in the intact mammalian liver. , 2015, Molecular cell.

[40]  Carla Coltharp,et al.  Super-resolution imaging of the bacterial division machinery. , 2013, Journal of visualized experiments : JoVE.

[41]  L. A. Sepúlveda,et al.  Lysogen stability is determined by the frequency of activity bursts from the fate-determining gene , 2010, Molecular systems biology.

[42]  R. Segev,et al.  GENERAL PROPERTIES OF THE TRANSCRIPTIONAL TIME-SERIES IN ESCHERICHIA COLI , 2011, Nature Genetics.

[43]  Marshall J. Levesque,et al.  Visualizing SNVs to quantify allele-specific expression in single cells , 2013, Nature Methods.

[44]  N. Kleckner,et al.  Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps , 2011, Proceedings of the National Academy of Sciences.

[45]  Arkady B. Khodursky,et al.  Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Ido Golding,et al.  Genetic Determinants and Cellular Constraints in Noisy Gene Expression , 2013, Science.

[47]  M. A. Shea,et al.  The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation. , 1985, Journal of molecular biology.