Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Visualization of gene activity in living cells

Nature Cell Biology volume 2, pages 871–878 (2000)Cite this article

Abstract

Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Experimental design.
Figure 2: Characterization of isolated clones.
Figure 3: Visualization of the genetic locus.
Figure 4: Comparison of different methods of fixation and staining.
Figure 5: Changes in chromatin organization during gene activation.
Figure 6: The open chromatin structure is static.
Figure 7: Relationship between the integrated locus and PML bodies.

Similar content being viewed by others

References

  1. Spector, D. L. Macromolecular domains within the cell nucleus. Annu. Rev. Cell Biol. 9, 265–315 ( 1993).

    Article  CAS  Google Scholar 

  2. Lamond, A. I. & Earnshaw, W. C. Structure and function in the nucleus. Science 280, 547– 553 (1998).

    Article  CAS  Google Scholar 

  3. Misteli, T. & Spector, D. L. Applications of the green fluorescent protein in cell biology and biotechnology. Nature Biotechnol. 15, 961–964 (1997).

    Article  CAS  Google Scholar 

  4. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  5. Ellenberg, J., Lippincott-Schwartz, J. & Presley, J. F. Dual-colour imaging with GFP variants . Trends Cell. Biol. 9, 52– 56 (1999).

    Article  CAS  Google Scholar 

  6. Belmont, A. S. & Straight, A.F. In vivo visualization of chromosomes using lac operator–repressor binding. Trends Cell. Biol. 8, 121–124 ( 1998).

    Article  CAS  Google Scholar 

  7. Robinett, C. C. et al. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition . J. Cell Biol. 135, 1685– 1700 (1996).

    Article  CAS  Google Scholar 

  8. Li, G., Sudlow, G. & Belmont, A. S. Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: precise choreography of condensation/decondensation and nuclear positioning. J. Cell Biol. 140, 975–989 (1998).

    Article  CAS  Google Scholar 

  9. Tumbar, T., Sudlow, G. & Belmont, A. S. Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J. Cell Biol. 145 , 1341–1354 (1999).

    Article  CAS  Google Scholar 

  10. Straight, A. F., Belmont, A. S., Robinett, C. C. & Murray, A. W. GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr. Biol. 6, 1599–1608 (1996).

    Article  CAS  Google Scholar 

  11. Minshull, J. et al. Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast. Curr. Biol. 6, 1609–1620 (1996).

    Article  CAS  Google Scholar 

  12. Miyazawa, S. et al. Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol. Cell. Biol. 9 , 83–91 (1989).

    Article  CAS  Google Scholar 

  13. Miller, J. H. & Reznikoff, W. A. The Operon, 17–220 (Cold Spring Harbor Laboratory Press, New York, 1980).

    Google Scholar 

  14. Maul, G. G., Negorev, D., Bell, P. & Ishov, A. M. Properties and assembly mechanisms of ND10, PML bodies, or PODs. J. Struct. Biol. 129, 278–287 ( 2000).

    Article  CAS  Google Scholar 

  15. Zhong, S., Salomoni, P. & Pandolfi, P. P. The transcriptional role of PML and the nuclear body . Nature Cell Biol. 2, E85– E90 (2000).

    Article  CAS  Google Scholar 

  16. Weintraub, H. & Groudine, M. Chromosomal subunits in active genes have an altered conformation. Science 193, 848–856 (1976).

    Article  CAS  Google Scholar 

  17. Paranjape, S. M., Kamakaka, R. T. & Kadonaga, J. T. Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annu. Rev. Biochem. 63, 265–297 (1994).

    Article  CAS  Google Scholar 

  18. Armstrong, J. A. & Emerson, B. M. Transcription of chromatin: these are complex times. Curr. Opin. Genet. Dev. 8, 165–172 ( 1998).

    Article  CAS  Google Scholar 

  19. Kuo, M. H. & Allis, C. D. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20, 615–626 (1998).

    Article  CAS  Google Scholar 

  20. Pederson, T. Chromatin structure and gene transcription: nucleosomes permit a new synthesis . Int. Rev. Cytol. 55, 1– 21 (1978).

    Article  CAS  Google Scholar 

  21. Manuelidis, L. Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences. Proc. Natl Acad. Sci. USA 81, 3123–3127 ( 1984).

    Article  CAS  Google Scholar 

  22. Manuelidis, L. & Borden, J. Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction. Chromosoma 96 , 397–410 (1988).

    Article  CAS  Google Scholar 

  23. Ferguson, M. & Ward, D. C. Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei. Chromosoma 101, 557–565 (1992).

    Article  CAS  Google Scholar 

  24. Dietzel, S. et al. Three-dimensional distribution of centromeric or paracentromeric heterochromatin of chromosomes 1, 7, 15, and 17 in human lymphocyte nuclei studied with light microscopic axial tomography. Bioimaging 3, 121–133 (1995).

    Article  CAS  Google Scholar 

  25. Abney, J. R., Cutler, B., Fillbach, M. L., Axelrod, D. & Scalettar, B. A. Chromatin dynamics in interphase nuclei and its implications for nuclear structure. J. Cell Biol. 137, 1459–1468 (1997).

    Article  CAS  Google Scholar 

  26. Marshall, W. F. et al. Interphase chromosomes undergo constrained diffusional motion in living cells. Curr. Biol. 7, 930– 939 (1997).

    Article  CAS  Google Scholar 

  27. Manuelidis, L. Individual interphase chromosome domains revealed by in-situ hybridization . Hum. Genet. 71, 288–293 (1985).

    Article  CAS  Google Scholar 

  28. Janevski, J., Park, P. C. & De Boni, U. Organization of centromeric domains in hepatocyte nuclei: rearrangement associated with de novo activation of the vitellogenin gene family in Xenopus laevis. Exp. Cell Res. 217 , 227–239 (1995).

    Article  CAS  Google Scholar 

  29. Bartholdi, M. F. Nuclear distribution of centromeres during the cell cycle of human diploid fibroblasts. J. Cell Sci. 99, 255– 263 (1991).

    PubMed  Google Scholar 

  30. Funabiki, H., Hagan, I., Uzawa, S. & Yanagida, M. Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J. Cell Biol. 121, 961– 976 (1993).

    Article  CAS  Google Scholar 

  31. LaSalle, J. M. & Lalande, M. Homologous association of oppositely imprinted chromosomal domains. Science 272, 725–728 (1996).

    Article  CAS  Google Scholar 

  32. Borden, J. & Manuelidis, L. Movement of the X chromosome in epilepsy. Science 242, 1687 –1691 (1988).

    Article  CAS  Google Scholar 

  33. Guldner, H. H., Szostecki, C., Grotzinger, T. & Will, H. IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis . J. Immunol. 149, 4067– 4073 (1992).

    CAS  PubMed  Google Scholar 

  34. Koken, M. H. et al. The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion. EMBO J. 13, 1073 –1083 (1994).

    Article  CAS  Google Scholar 

  35. Korioth, F., Gieffers, C., Maul, G. G. & Frey, J. Molecular characterization of NDP52, a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment. J. Cell Biol. 130, 1–13 (1995).

    Article  CAS  Google Scholar 

  36. Maul, G. G., Yu, E., Ishov, A. M. & Epstein, A. L. Nuclear domain 10 (ND10) associated proteins are also present in nuclear bodies and redistribute to hundreds of nuclear sites after stress. J. Cell. Biochem. 59, 498–513 (1995).

    Article  CAS  Google Scholar 

  37. Ishov, A. M. & Maul, G. G. The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition. J. Cell Biol. 134, 815–826 ( 1996).

    Article  CAS  Google Scholar 

  38. Maul, G. G., Jensen, D. E., Ishov, A. M., Herlyn, M. & Rauscher, F. J. III Nuclear redistribution of BRCA1 during viral infection. Cell Growth Differ. 9 , 743–755 (1998).

    CAS  PubMed  Google Scholar 

  39. Gongora, C. et al. Molecular cloning of a new interferon-induced PML nuclear body-associated protein. J. Biol. Chem. 272, 19457–19463 (1997).

    Article  CAS  Google Scholar 

  40. Chen, C. & Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7, 2745–2752 (1987).

    Article  CAS  Google Scholar 

  41. Mintz, P. J., Patterson, S. D., Neuwald, A. F., Spahr, C. S. & Spector, D. L. Purification and biochemical characterization of interchromatin granule clusters. EMBO J. 18, 4308–4320 ( 1999).

    Article  CAS  Google Scholar 

  42. Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning (Cold Spring Harbor Laboratory Press, New York, 1989).

    Google Scholar 

  43. Spector, D. L., Goldman, R. D. & Leinwand, L. A. Cells: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1998).

    Google Scholar 

Download references

Acknowledgements

We thank T. Misteli for discussions and P. Sacco-Bubulya and N. Saitoh for reviewing the manuscript. pW7-C3 (=pCFP-C3), which encoded CFP, was provided by R. Tsien, EV-124 was provided by M. Wilkinson and pUHD10-4B was provided by J. Skowronski, and monoclonal antibody 5E10 was obtained from R. van Driel. S.M.J. is supported by an NIH/NCI training grant 5T32CA09311. D.L.S. is funded by a grant from NIGMS (NIH 498100).

Author information

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, 11724 , New York, USA

    Toshiro Tsukamoto, Noriyo Hashiguchi, Susan M. Janicki & David L. Spector

  2. Department of Life Science, Himeji Institute of Technology, 3-2-1 Koto, Kamigori, 678-1297, Hyogo, Japan

    Toshiro Tsukamoto & Noriyo Hashiguchi

  3. Department of Cell and Structural Biology, University of Illinois, Urbana, 61801, Illinois, USA

    Tudorita Tumbar & Andrew S. Belmont

Authors
  1. Toshiro Tsukamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Noriyo Hashiguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Susan M. Janicki
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tudorita Tumbar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew S. Belmont
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David L. Spector
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David L. Spector.

Supplementary information

Movie 1

Visualization of gene expression Clone 2 cells were transiently transfected with EYFP/lac repressor and pTet-On and plated onto a coverslip fitted for a FCS2 live-cell chamber (Bioptechs, Butler, Pennsylvania). An image was acquired using OpenLab software (Improvision, Boston, Massachusetts); doxycline then was perfused into the live-cell chamber. Images were acquired every 10 minutes in the YFP channel for the first 2 h, after which images were obtained in bothe the YFP and CFP channels at 20-min intervals. The entire movie was taken over a 7-h period and is played back at 2,880 x. (MOV 3600 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tsukamoto, T., Hashiguchi, N., Janicki, S. et al. Visualization of gene activity in living cells. Nat Cell Biol 2, 871–878 (2000). https://doi.org/10.1038/35046510

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35046510

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing