Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo (original) (raw)

Nature volume 442, pages 1054–1057 (2006)Cite this article

Abstract

Transactivator–promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells1 owing to rapid and reversible disruption of activator–promoter complexes by molecular chaperones2,3,4,5,6, or a slower, ubiquitin–proteasome-pathway-mediated turnover of DNA-bound activator7,8,9. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein–DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression8,10,11,12. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4–promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4–promoter complexes ‘lock in’ and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator–promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.

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

Access options

Subscribe to this journal

Receive 51 print issues and online access

$199.00 per year

only $3.90 per issue

Buy this article

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

Additional access options:

Similar content being viewed by others

References

  1. McNally, J. G., Müller, W. G., Walker, D., Wolford, R. & Hager, G. L. The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287, 1262–1265 (2000)
    Article ADS CAS Google Scholar
  2. Freeman, B. C. & Yamamoto, K. R. Continuous recycling: a mechanism for modulatory signal transduction. Trends Biochem. Sci. 26, 285–290 (2001)
    Article CAS Google Scholar
  3. Elbi, C. et al. Molecular chaperones function as steroid receptor nuclear mobility factors. Proc. Natl Acad. Sci. USA 101, 2876–2881 (2004)
    Article ADS CAS Google Scholar
  4. Fletcher, T. M. et al. ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling. Mol. Cell. Biol. 22, 3255–3263 (2002)
    Article CAS Google Scholar
  5. Freeman, B. C., Felts, S. J., Toft, D. O. & Yamamoto, K. R. The p23 molecular chaperones act at a late step in intracellular receptor action to differentially affect ligand efficacies. Genes Dev. 14, 422–434 (2000)
    CAS PubMed PubMed Central Google Scholar
  6. Freeman, B. C. & Yamamoto, K. R. Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 296, 2232–2235 (2002)
    Article ADS CAS Google Scholar
  7. Métivier, R. et al. Estrogen receptor-α directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751–763 (2003)
    Article Google Scholar
  8. Reid, G. et al. Cyclic, proteasome-mediated turnover of unliganded and liganded ERα on responsive promoters is an integral feature of estrogen signaling. Mol. Cell 11, 695–707 (2003)
    Article CAS Google Scholar
  9. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A. & Brown, M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843–852 (2000)
    Article CAS Google Scholar
  10. Nawaz, Z. & O'Malley, B. W. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription? Mol. Endocrinol. 18, 493–499 (2004)
    Article CAS Google Scholar
  11. Lipford, J. R., Smith, G. T., Chi, Y. & Deshaies, R. J. A putative stimulatory role for activator turnover in gene expression. Nature 438, 113–116 (2005)
    Article ADS CAS Google Scholar
  12. Muratani, M., Kung, C., Shokat, K. M. & Tansey, W. P. The F box protein Dsg1/Mdm30 is a transcriptional coactivator that stimulates Gal4 turnover and cotranscriptional mRNA processing. Cell 120, 887–899 (2005)
    Article CAS Google Scholar
  13. Fankhauser, C. P., Briand, P. A. & Picard, D. The hormone binding domain of the mineralocorticoid receptor can regulate heterologous activities in cis. Biochem. Biophys. Res. Commun. 200, 195–201 (1994)
    Article CAS Google Scholar
  14. Picard, D. Posttranslational regulation of proteins by fusions to steroid-binding domains. Methods Enzymol. 327, 385–401 (2000)
    Article CAS Google Scholar
  15. Louvion, J. F., Havaux-Copf, B. & Picard, D. Fusion of GAL4–VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast. Gene 131, 129–134 (1993)
    Article CAS Google Scholar
  16. Wehrman, T. S., Casipit, C. L., Gewertz, N. M. & Blau, H. M. Enzymatic detection of protein translocation. Nature Methods 2, 521–527 (2005)
    Article CAS Google Scholar
  17. Siddiqui, A. H. & Brandriss, M. C. The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences. Mol. Cell. Biol. 9, 4706–4712 (1989)
    Article CAS Google Scholar
  18. Lohr, D., Venkov, P. & Zlatanova, J. Transcriptional regulation in the yeast GAL gene family: a complex genetic network. FASEB J. 9, 777–787 (1995)
    Article CAS Google Scholar
  19. Lee, D. H. & Goldberg, A. L. Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J. Biol. Chem. 271, 27280–27284 (1996)
    Article CAS Google Scholar
  20. Arndt, K. & Winston, F. An unexpected role for ubiquitylation of a transcriptional activator. Cell 120, 733–734 (2005)
    Article CAS Google Scholar
  21. Sprague, B. L. & McNally, J. G. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15, 84–91 (2005)
    Article CAS Google Scholar
  22. Yao, J., Munson, K. M., Webb, W. W. & Lis, J. T. Dynamics of heat shock factor association with native gene loci in living cells. Nature doi:10.1038/nature05025 (this issue)
  23. Valley, C. C. et al. Differential regulation of estrogen-inducible proteolysis and transcription by the estrogen receptor-α N terminus. Mol. Cell. Biol. 25, 5417–5428 (2005)
    Article CAS Google Scholar
  24. Gonzalez, F., Delahodde, A., Kodadek, T. & Johnston, S. A. Recruitment of a 19S proteasome subcomplex to an activated promoter. Science 296, 548–550 (2002)
    Article ADS CAS Google Scholar

Download references

Acknowledgements

This research was supported by the National Institutes of Health and the NHLBI Proteomics Initiative of the National Heart, Lung and Blood Institute, NIH. K.N. was supported by an NIH Cardiology Training Grant Fellowship. ER(LBD)-encoding plasmids were a gift from D. Picard.

Author information

Author notes

  1. Stephen Albert Johnston
    Present address: Center for Innovations in Medicine, Biodesign Institute, Arizona State University, 1001 S. McAllister Ave, Tempe, Arizona, 85287-5001, USA

Authors and Affiliations

  1. Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Texas, 75390-9185, Dallas, USA
    Kip Nalley, Stephen Albert Johnston & Thomas Kodadek

Authors

  1. Kip Nalley
    You can also search for this author inPubMed Google Scholar
  2. Stephen Albert Johnston
    You can also search for this author inPubMed Google Scholar
  3. Thomas Kodadek
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toThomas Kodadek.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Methods, Supplementary Figure Legends and additional references. (DOC 39 kb)

Supplementary Figures

This file contains Supplementary Figures 1–6. (PPT 1039 kb)

Rights and permissions

About this article

Cite this article

Nalley, K., Johnston, S. & Kodadek, T. Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.Nature 442, 1054–1057 (2006). https://doi.org/10.1038/nature05067

Download citation

This article is cited by

Editorial Summary

Seen the Movie Yet?

Salivary glands of Drosophila larvae contain giant polytene chromosomes whose characteristic banded structure is readily visible by light microscopy. These chromosomes are usually visualized by breaking the nuclei, then 'spreading' the chromosomes in two dimensions. But using two-photon laser-scanning microscopy, they can now be examined in living salivary gland tissue in three dimensions and in real time. The above image is a 3D-reconstructed view of polytene nuclei where DNA is stained red and transcription factor HSF is shown green, before (top) and after heat shock. This technique reveals the dynamics of the interaction between DNA and a model transcription factor, as illustrated in a movie in Supplementary Information. Nalley et al., using different methods, draw similar conclusions about the dynamics of transcription factor Gal4.

Associated content