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Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo

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.

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Figure 1: The competition ChIP assay as applied to the yeast transcription factor Gal4.
Figure 2: Kinetic stability of Gal4–promoter complexes under inducing and non-inducing conditions.
Figure 3: Effect of MG132, an inhibitor of 26S proteasome-mediated proteolysis, on Gal4-mediated gene expression.

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)

    ADS  CAS  Article  Google Scholar 

  2. Freeman, B. C. & Yamamoto, K. R. Continuous recycling: a mechanism for modulatory signal transduction. Trends Biochem. Sci. 26, 285–290 (2001)

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  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)

    CAS  Article  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)

    CAS  Article  Google Scholar 

  14. Picard, D. Posttranslational regulation of proteins by fusions to steroid-binding domains. Methods Enzymol. 327, 385–401 (2000)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  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)

    CAS  Article  Google Scholar 

  20. Arndt, K. & Winston, F. An unexpected role for ubiquitylation of a transcriptional activator. Cell 120, 733–734 (2005)

    CAS  Article  Google Scholar 

  21. Sprague, B. L. & McNally, J. G. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15, 84–91 (2005)

    CAS  Article  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)

    CAS  Article  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)

    ADS  CAS  Article  Google Scholar 

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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.

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Correspondence to Thomas Kodadek.

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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

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