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Single-RNA counting reveals alternative modes of gene expression in yeast

Nature Structural & Molecular Biology volume 15, pages 1263–1271 (2008)Cite this article

Abstract

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

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Figure 1: Single mRNA–sensitivity FISH.
Figure 2: Quantitative single-molecule, single-cell gene expression analysis.
Figure 3: Expression profiles of constitutively active genes.
Figure 4: Transcriptional loading determines the mode of transcription.
Figure 5: Modeling MDN1 expression kinetics.
Figure 6: Expression profiles of a cell cycle–regulated and a SAGA-controlled gene.
Figure 7: Transcription kinetics of endogenous yeast genes.
Figure 8: Extracting kinetic data from fixed-cell analysis.

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References

  1. Orphanides, G. & Reinberg, D. A unified theory of gene expression. Cell 108, 439–451 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Thomas, M.C. & Chiang, C.-M. Thegeneral transcription machinery and general cofactors. Crit. Rev. Biochem. Mol. Biol. 41, 105–178 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Dieci, G. & Sentenac, A. Detours and shortcuts to transcription reinitiation. Trends Biochem. Sci. 28, 202–209 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Li, B., Carey, M. & Workman, J.L. The role of chromatin during transcription. Cell 128, 707–719 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Saunders, A., Core, L.J. & Lis, J.T. Breaking barriers to transcription elongation. Nat. Rev. Mol. Cell Biol. 7, 557–567 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Struhl, K. Chromatin structure and RNA polymerase II connection: implications for transcription. Cell 84, 179–182 (1996).

    Article  CAS  PubMed  Google Scholar 

  7. Darzacq, X. & Singer, R.H. The dynamic range of transcription. Mol. Cell 30, 545–546 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Iyer, V. & Struhl, K. Mechanism of differential utilization of the His3 TR and TC TATA elements. Mol. Cell. Biol. 15, 7059–7066 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yean, D. & Gralla, J. Transcription reinitiation rate: a special role for the TATA box. Mol. Cell. Biol. 17, 3809–3816 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kaern, M., Elston, T.C., Blake, W.J. & Collins, J.J. Stochasticity in gene expression: from theories to phenotypes. Nat. Rev. Genet. 6, 451–464 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Newman, J.R. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Holstege, F.C. et al. Dissecting the regulatory circuitry of a eukaryotic genome. Cell 95, 717–728 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Elowitz, M.B., Levine, A.J., Siggia, E.D. & Swain, P.S. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Kaufmann, B.B. & van Oudenaarden, A. Stochastic gene expression: from single molecules to the proteome. Curr. Opin. Genet. Dev. 17, 107–112 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Blake, W.J. et al. Phenotypic consequences of promoter-mediated transcriptional noise. Mol. Cell 24, 853–865 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Chubb, J.R., Trcek, T., Shenoy, S.M. & Singer, R.H. Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Newlands, S. et al. Transcription occurs in pulses in muscle fibers. Genes Dev. 12, 2748–2758 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ross, I.L., Browne, C.M. & Hume, D.A. Transcription of individual genes in eukaryotic cells occurs randomly and infrequently. Immunol. Cell Biol. 72, 177–185 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Golding, I., Paulsson, J., Zawilski, S.M. & Cox, E.C. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  23. Bassler, J. et al. Identification of a 60S preribosomal particle that is closely linked to nuclear export. Mol. Cell 8, 517–529 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Akhtar, A. & Gasser, S.M. The nuclear envelope and transcriptional control. Nat. Rev. Genet. 8, 507–517 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Casolari, J.M. et al. Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization. Cell 117, 427–439 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bon, M., McGowan, S.J. & Cook, P.R. Many expressed genes in bacteria and yeast are transcribed only once per cell cycle. FASEB J. 20, 1721–1723 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Velculescu, V.E. et al. Characterization of the yeast transcriptome. Cell 88, 243–251 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Peccoud, J. & Ycart, B. Markovian modeling of gene-product synthesis. Theor. Popul. Biol. 48, 222–234 (1995).

    Article  Google Scholar 

  30. Pedraza, J.M. & Paulsson, J. Effects of molecular memory and bursting on fluctuations in gene expression. Science 319, 339–343 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Mason, P.B. & Struhl, K. Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo. Mol. Cell 17, 831–840 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Spellman, P.T. et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273–3297 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chen, W. & Struhl, K. Saturation mutagenesis of a yeast his3 “TATA element”: genetic evidence for a specific TATA-binding protein. Proc. Natl. Acad. Sci. USA 85, 2691–2695 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Struhl, K. Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms. Mol. Cell. Biol. 6, 3847–3853 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Huisinga, K.L. & Pugh, B.F.A. Genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol. Cell 13, 573–585 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Edwards, A.M., Kane, C.M., Young, R.A. & Kornberg, R.D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro. J. Biol. Chem. 266, 71–75 (1991).

    CAS  PubMed  Google Scholar 

  38. O'Brien, T. & Lis, J.T. Rapid changes in Drosophila transcription after an instantaneous heat shock. Mol. Cell. Biol. 13, 3456–3463 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ucker, D.S. & Yamamoto, K.R. Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. Novel assays of transcription rates. J. Biol. Chem. 259, 7416–7420 (1984).

    CAS  PubMed  Google Scholar 

  40. Epshtein, V. & Nudler, E. Cooperation between RNA polymerase molecules in transcription elongation. Science 300, 801–805 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Boireau, S. et al. The transcriptional cycle of HIV-1 in real-time and live cells. J. Cell Biol. 179, 291–304 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kristjuhan, A. & Svejstrup, J.Q. Evidence for distinct mechanisms facilitating transcript elongation through chromatin in vivo. EMBO J. 23, 4243–4252 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Workman, J.L. Nucleosome displacement in transcription. Genes Dev. 20, 2009–2017 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Hereford, L.M. & Rosbash, M. Number and distribution of polyadenylated RNA sequences in yeast. Cell 10, 453–462 (1977).

    Article  CAS  PubMed  Google Scholar 

  45. Wodicka, L., Dong, H., Mittmann, M., Ho, M.H. & Lockhart, D.J. Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotechnol. 15, 1359–1367 (1997).

    Article  CAS  PubMed  Google Scholar 

  46. Iyer, V. & Struhl, K. Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93, 5208–5212 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Arava, Y. et al. Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 100, 3889–3894 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Warner, J.R. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437–440 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Karpova, T.S. et al. Concurrent fast and slow cycling of a transcriptional activator at an endogenous promoter. Science 319, 466–469 (2008).

    Article  CAS  PubMed  Google Scholar 

  50. McNally, J.G. et al. The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287, 1262–1265 (2000).

    Article  CAS  PubMed  Google Scholar 

  51. Zawel, L., Kumar, K.P. & Reinberg, D. Recycling of the general transcription factors during RNA polymerase II transcription. Genes Dev. 9, 1479–1490 (1995).

    Article  CAS  PubMed  Google Scholar 

  52. Yudkovsky, N., Ranish, J.A. & Hahn, S. A transcription reinitiation intermediate that is stabilized by activator. Nature 408, 225–229 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Raser, J.M. & O'Shea, E.K. Control of stochasticity in eukaryotic gene expression. Science 304, 1811–1814 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Belle, A., Tanay, A., Bitincka, L., Shamir, R. & O'Shea, E.K. Quantification of protein half-lives in the budding yeast proteome. Proc. Natl. Acad. Sci. USA 103, 13004–13009 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wang, Y. et al. Precision and functional specificity in mRNA decay. Proc. Natl. Acad. Sci. USA 99, 5860–5865 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Guido, N.J. et al. A bottom-up approach to gene regulation. Nature 439, 856–860 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Yu, J., Xiao, J., Ren, X., Lao, K. & Xie, X.S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).

    Article  CAS  PubMed  Google Scholar 

  58. Harbison, C.T. et al. Transcriptional regulatory code of a eukaryotic genome. Nature 431, 99–104 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Segal, E. et al. A genomic code for nucleosome positioning. Nature 442, 772–778 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Velculescu, V.E. et al. Analysis of human transcriptomes. Nat. Genet. 23, 387–388 (1999).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank S. Burke and S.M. Shenoy for writing scripts for data analysis, and J.R. Warner, E.D. Siggia and M. Keogh for helpful discussions. This work was supported by the US National Institutes of Health (R.H.S.).

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  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Daniel Zenklusen, Daniel R Larson & Robert H Singer

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  1. Daniel Zenklusen
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Contributions

D.Z. initiated the project and performed the experimental work. D.Z. and D.R.L. analyzed the data. D.R.L. wrote the spot-detection program and performed the numerical modeling. R.H.S. supervised the project. D.Z., D.R.L. and R.H.S. wrote the paper.

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Correspondence to Robert H Singer.

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Zenklusen, D., Larson, D. & Singer, R. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15, 1263–1271 (2008). https://doi.org/10.1038/nsmb.1514

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