Fine-structure analysis of ribosomal protein gene transcription.
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Journal: 2006/August - Molecular and Cellular Biology
ISSN: 0270-7306
Abstract:
The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.
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Mol Cell Biol 26(13): 4853-4862
PMID: 16782874

Fine-Structure Analysis of Ribosomal Protein Gene Transcription

Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461, Department of Molecular Biology and NCCR Program “Frontiers in Genetics,” University of Geneva, Sciences III, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland2
Corresponding author. Mailing address: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3022. Fax: (718) 430-8574. E-mail: ude.uy.mocea@renraw.
Deceased.
Received 2005 Dec 12; Revised 2006 Jan 30; Accepted 2006 Apr 17.
Copyright © 2006, American Society for Microbiology

Abstract

The ribosomal protein genes of Saccharomyces cerevisiae, responsible for nearly 40% of the polymerase II transcription initiation events, are characterized by the constitutive tight binding of the transcription factor Rap1. Rap1 binds at many places in the yeast genome, including glycolytic enzyme genes, the silent MAT loci, and telomeres, its specificity arising from specific cofactors recruited at the appropriate genes. At the ribosomal protein genes two such cofactors have recently been identified as Fhl1 and Ifh1. We have now characterized the interaction of these factors at a bidirectional ribosomal protein promoter by replacing the Rap1 sites with LexA operator sites. LexA-Gal4(AD) drives active transcription at this modified promoter, although not always at the correct initiation site. Tethering Rap1 to the promoter neither drives transcription nor recruits Fhl1 or Ifh1, showing that Rap1 function requires direct DNA binding. Tethering Fhl1 also fails to activate transcription, even though it does recruit Ifh1, suggesting that Fhl1 does more than simply provide a platform for Ifh1. Tethering Ifh1 to the promoter leads to low-level transcription, at the correct initiation sites. Remarkably, activation by tethered LexA-Gal4(AD) is strongly reduced when TOR kinase is inhibited by rapamycin. Thus, TOR can act independently of Fhl1/Ifh1 at ribosomal protein promoters. We also show that, in our strain background, the response of ribosomal protein promoters to TOR inhibition is independent of the Ifh1-related protein Crf1, indicating that the role of this corepressor is strain specific. Fine-structure chromatin mapping of several ribosomal protein promoters revealed that histones are essentially absent from the Rap1 sites, while Fhl1 and Ifh1 are coincident with each other but distinct from Rap1.

Abstract

The 138 genes encoding the 79 ribosomal proteins (RP) of Saccharomyces cerevisiae are arguably the most coordinately regulated cluster of genes, spread throughout the yeast genome (7, 11). It was originally thought that the basis for much of this regulation lay in the presence of binding sites for the protein Rap1 upstream of a large majority of the RP genes (17, 21).

However, Rap1 is a protein of many functions (reviewed by references 28 and 29). It is the primary transcription factor for the glycolytic genes and several translation factor genes. It acts as the major duplex DNA binding protein of telomeres. It nucleates the silencing of the HML and HMR mating-type loci. Genome-wide chromatin immunoprecipitation (ChIP) analysis revealed that Rap1 binds to about 5% of yeast genes and participates in the activation of 37% of RNA polymerase II transcripts in exponentially growing yeast cells (21). There is good evidence that the initial step of Rap1 is to clear nucleosomes from a patch of DNA (28, 40) and that the second step is to recruit specific factors to carry out the appropriate function. It is now clear that for the RP genes these factors are Fhl1 and Ifh1, which are found almost exclusively at RP genes. Gcr1 and Gcr2 are present at many glycolytic enzyme genes. Sir3, Sir4, and others are recruited for silencing at the silent MAT loci and telomeres (19, 21, 34).

ChIP analysis of the RP genes showed that both Rap1 and Fhl1 are constitutively found at the promoters. Only occupancy by Ifh1 is correlated with active transcription, suggesting that Ifh1 plays a central role in the regulation of RP gene transcription (26, 33, 34, 39). Rap1 is one of the DNA binding proteins for which many consensus sequences have been suggested (29). Interestingly, the Rap1-binding sequences at RP gene promoters, termed RPG boxes, are quite different from those at the telomeres, while those at glycolytic gene promoters appear to be in between. Yet the basis for specificity remains obscure, although it has been suggested that Rap1 undergoes distinct conformational changes as a result of binding to somewhat different sequences (29).

At the RP genes, it has been proposed that Rap1 recruits not only TAFs, which in turn recruit TATA binding protein to the RP genes that have characteristically poor TATA boxes (27), but also Esa1, which could acetylate either histone H4 or another participant in transcriptional activation (31). Yet, Esa1 probably provides little specificity since by ChIP analysis it is found upstream of many actively transcribed genes (30, 32).

While genome-wide ChIP analysis revealed that Rap1, Fhl1, and Ifh1 are recruited to a majority of the RP gene promoters (19, 34, 39), neither the basis for the recruitment nor the role played by the factors in transcription of RP genes was clear. Furthermore, the binding sites for Fhl1 and for Ifh1 are elusive. When assayed in vitro, neither Fhl1 nor Ifh1 binds RP promoters, either by itself or in the presence of Rap1 (33). Ifh1 appears to be recruited to RP promoters through its interaction with the “forkhead-associated” (FHA) domain of Fhl1 (9, 26, 33, 34). However, the story must be more complex. Although the FHA domain of Fhl1 can recruit Ifh1 to serve as a transcriptional activator of a GAL-based artificial reporter, a nearly full length Fhl1 recruits nearly as much Ifh1, but very little transcription ensues (34). Indeed, Fhl1 has been proposed as a repressor of RP gene transcription (5, 14). Furthermore, there is no direct evidence that Ifh1 functions as a transcriptional activator in the context of an RP gene promoter.

Utilizing a minimally engineered promoter that drives the transcription of two RP genes oriented head-to-head, we have found that for Rap1 to recruit Fhl1 and Ifh1 and to activate transcription, it must bind DNA directly. Furthermore, Rap1 binding to sites from glycolytic genes, within the context of the RP genes, recruits neither Fhl1 nor Ifh1. Recruitment of Ifh1 by Fhl1 tethered to the promoter is also insufficient to drive transcription, although tethered Ifh1 alone does. By high-resolution ChIP analysis, we find that Fhl1 and Ifh1 are recruited to the RP promoters at a location distinct from the Rap1-binding sites.

Acknowledgments

We dedicate this paper to the memory of Stephan Schawalder, a most promising young scientist.

We are grateful to Rodolfo Negri for discussions and access to unpublished data and to Dietmar Martin and Mike Hall for providing strains and advice.

This research was supported in part by grants from the Human Frontiers Program (to both D.S. and J.R.W.), by the NIH (GM-25532 to J.R.W. and CAI-3330 to the Albert Einstein Cancer Center), by the Swiss National Fund (through the ESF euroDYNA program to D.S.), and by funds provided by the Canton of Geneva (to D.S.).

Acknowledgments

REFERENCES

REFERENCES

References

  • 1. Bartel, P. L., C. Chien, R. Sternglanz, and S. Fields. 1993. Using the two-hybrid system to detect protein-protein interactions, p. 153-179. In D. A. Hartley (ed.), Cellular interactions in development: a practical approach. IRL, Oxford, United Kingdom.
  • 2. Beer, M. A., and S. Tavazoie. 2004. Predicting gene expression from sequence. Cell117:185-198. [[PubMed]
  • 3. Bernstein, B. E., C. L. Liu, E. L. Humphrey, E. O. Perlstein, and S. L. Schreiber. 2004. Global nucleosome occupancy in yeast. Genome Biol.5:R62.
  • 4. Boeger, H., J. Griesenbeck, J. S. Strattan, and R. D. Kornberg. 2003. Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell11:1587-1598. [[PubMed]
  • 5. Cherel, I., and PThuriaux. 1995. The IFH1 gene product interacts with a fork head protein in Saccharomyces cerevisiae. Yeast11:261-270. [[PubMed][Google Scholar]
  • 6. Dabeva, M. D., and J. R. Warner. 1987. The yeast ribosomal protein L32 and its gene. J. Biol. Chem.262:16055-16059. [[PubMed]
  • 7. DeRisi, J. L., V. R. Iyer, and P. O. Brown. 1997. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science278:680-686. [[PubMed]
  • 8. Drazinic, C. M., J. B. Smerage, M. C. Lopez, and H. V. Baker. 1996. Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p). Mol. Cell. Biol.16:3187-3196.
  • 9. Durocher, D., and S. P. Jackson. 2002. The FHA domain. FEBS Lett.513:58-66. [[PubMed]
  • 10. Estojak, J., R. Brent, and E. A. Golemis. 1995. Correlation of two-hybrid affinity data with in vitro measurements. Mol. Cell. Biol.15:5820-5829.
  • 11. Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B. Eisen, G. Storz, D. Botstein, and P. O. Brown. 2000. Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell11:4241-4257.
  • 12. Goncalves, P. M., G. Griffioen, R. Minnee, M. Bosma, L. S. Kraakman, W. H. Mager, and R. J. Planta. 1995. Transcription activation of yeast ribosomal protein genes requires additional elements apart from binding sites for Abf1p or Rap1p. Nucleic Acids Res.23:1475-1480.
  • 13. Goncalves, P. M., K. Maurer, G. van Nieuw Amerongen, K. Bergkamp-Steffens, W. H. Mager, and R. J. Planta. 1996. C-terminal domains of general regulatory factors Abf1p and Rap1p in Saccharomyces cerevisiae display functional similarity. Mol. Microbiol.19:535-543. [[PubMed]
  • 14. Hermann-Le Denmat, S., M. Werner, A. Sentenac, and P. Thuriaux. 1994. Suppression of yeast RNA polymerase III mutations by FHL1, a gene coding for a fork head protein involved in rRNA processing. Mol. Cell. Biol.14:2905-2913.
  • 15. Holstege, F. C., E. G. Jennings, J. J. Wyrick, T. I. Lee, C. J. Hengartner, M. R. Green, T. R. Golub, E. S. Lander, and R. A. Young. 1998. Dissecting the regulatory circuitry of a eukaryotic genome. Cell95:717-728. [[PubMed]
  • 16. Kuras, L., and KStruhl. 1999. Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme. Nature399:609-613. [[PubMed][Google Scholar]
  • 17. Lascaris, R. F., W. H. Mager, and R. J. Planta. 1999. DNA-binding requirements of the yeast protein Rap1p as selected in silico from ribosomal protein gene promoter sequences. Bioinformatics15:267-277. [[PubMed]
  • 18. Lee, C. K., Y. Shibata, B. Rao, B. D. Strahl, and J. D. Lieb. 2004. Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet.36:900-905. [[PubMed]
  • 19. Lee, T. I., N. J. Rinaldi, F. Robert, D. T. Odom, Z. Bar-Joseph, G. K. Gerber, N. M. Hannett, C. T. Harbison, C. M. Thompson, I. Simon, J. Zeitlinger, E. G. Jennings, H. L. Murray, D. B. Gordon, B. Ren, J. J. Wyrick, J. B. Tagne, T. L. Volkert, E. Fraenkel, D. K. Gifford, and R. A. Young. 2002. Transcriptional regulatory networks in Saccharomyces cerevisiae. Science298:799-804. [[PubMed]
  • 20. Li, B., C. R. Nierras, and J. R. Warner. 1999. Transcriptional elements involved in the repression of ribosomal protein synthesis. Mol. Cell. Biol.19:5393-5404.
  • 21. Lieb, J. D., X. Liu, D. Botstein, and P. O. Brown. 2001. Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association. Nat. Genet.28:327-334. [[PubMed]
  • 22. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔC(T)) method. Methods25:402-408. [[PubMed]
  • 23. Longtine, M. S., A. McKenzie III, D. J. Demarini, N. G. Shah, A. Wach, A. Brachat, P. Philippsen, and J. R. Pringle. 1998. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast14:953-961. [[PubMed]
  • 24. Lopez, M. C., J. B. Smerage, and H. V. Baker. 1998. Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1. Proc. Natl. Acad. Sci. USA95:14112-14117.
  • 25. Marion, R. M., A. Regev, E. Segal, Y. Barash, D. Koller, N. Friedman, and E. K. O'Shea. 2004. Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression. Proc. Natl. Acad. Sci. USA101:14315-14322.
  • 26. Martin, D. E., A. Soulard, and M. N. Hall. 2004. TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell119:969-979. [[PubMed]
  • 27. Mencia, M., Z. Moqtaderi, J. V. Geisberg, L. Kuras, and K. Struhl. 2002. Activator-specific recruitment of TFIID and regulation of ribosomal protein genes in yeast. Mol. Cell9:823-833. [[PubMed]
  • 28. Morse, RH. 2000. RAP, RAP, open up! New wrinkles for RAP1 in yeast. Trends Genet.16:51-53. [[PubMed][Google Scholar]
  • 29. Pina, B., J. Fernandez-Larrea, N. Garcia-Reyero, and F. Z. Idrissi. 2003. The different (sur)faces of Rap1p. Mol. Genet. Genomics268:791-798. [[PubMed]
  • 30. Pokholok, D. K., C. T. Harbison, S. Levine, M. Cole, N. M. Hannett, T. I. Lee, G. W. Bell, K. Walker, P. A. Rolfe, E. Herbolsheimer, J. Zeitlinger, F. Lewitter, D. K. Gifford, and R. A. Young. 2005. Genome-wide map of nucleosome acetylation and methylation in yeast. Cell122:517-527. [[PubMed]
  • 31. Reid, J. L., V. R. Iyer, P. O. Brown, and K. Struhl. 2000. Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase. Mol. Cell6:1297-1307. [[PubMed]
  • 32. Robert, F., D. K. Pokholok, N. M. Hannett, N. J. Rinaldi, M. Chandy, A. Rolfe, J. L. Workman, D. K. Gifford, and R. A. Young. 2004. Global position and recruitment of HATs and HDACs in the yeast genome. Mol. Cell16:199-209.
  • 33. Rudra, D., Y. Zhao, and J. R. Warner. 2005. Central role of Ifh1p-Fhl1p interaction in the synthesis of yeast ribosomal proteins. EMBO J.24:533-542.
  • 34. Schawalder, S. B., M. Kabani, I. Howald, U. Choudhury, M. Werner, and D. Shore. 2004. Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1. Nature432:1058-1061. [[PubMed]
  • 35. Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics122:19-27.
  • 36. Thomas, B. J., and R. Rothstein. 1989. Elevated recombination rates in transcriptionally active DNA. Cell56:619-630. [[PubMed]
  • 37. Uemura, H., M. Koshio, Y. Inoue, M. C. Lopez, and H. V. Baker. 1997. The role of Gcr1p in the transcriptional activation of glycolytic genes in yeast Saccharomyces cerevisiae. Genetics147:521-532.
  • 38. Vilardell, J., and J. R. Warner. 1994. Regulation of splicing at an intermediate step in the formation of the spliceosome. Genes Dev.8:211-220. [[PubMed]
  • 39. Wade, J. T., D. B. Hall, and K. Struhl. 2004. The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes. Nature432:1054-1058. [[PubMed]
  • 40. Yu, L., and R. H. Morse. 1999. Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae. Mol. Cell. Biol.19:5279-5288.
  • 41. Yuan, G. C., Y. J. Liu, M. F. Dion, M. D. Slack, L. F. Wu, S. J. Altschuler, and O. J. Rando. 2005. Genome-scale identification of nucleosome positions in S. cerevisiae. Science309:626-630. [[PubMed]
  • 42. Zhang, H., D. N. Roberts, and B. R. Cairns. 2005. Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss. Cell123:219-231.
  • 43. Zhao, Y., J. H. Sohn, and J. R. Warner. 2003. Autoregulation in the biosynthesis of ribosomes. Mol. Cell. Biol.23:699-707.
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