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Characterization of Complex Regulatory Networks and Identification of Promoter Regulatory Elements in Yeast: “In Silico” and “Wet-Lab” Approaches

  • Nuno P. Mira
  • Miguel C. Teixeira
  • Isabel Sá-CorreiaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 809)

Abstract

Transcription is the first step in the flow of biological information from genome to proteome and its tight regulation is a crucial checkpoint in most biological processes occurring in all living organisms. In eukaryotes, one of the most important mechanisms of transcriptional regulation relies on the activity of transcription factors which, upon binding to specific nucleotide motifs (consensus) present in the promoter region of target genes, modulate the activity of RNA polymerase II activating and/or repressing gene transcription. The identification of binding sites for these transcription factors is crucial to the understanding of transcriptional regulation at the molecular level and to the prediction of putative target genes for each transcription factor. However, transcription regulation cannot simply be reduced to transcription factor-gene associations. Frequently, the transcript level of a given gene is determined by a multitude of activators and/or repressors resulting in intertwined and complex regulatory networks. Two case studies dedicated to the study of transcriptional regulation in the experimental model Saccharomyces cerevisiae are presented in this chapter. The computational tools available in YEASTRACT information system are explored in both studies, to identify the regulatory elements that serve as functional DNA-binding sites for a transcription factor (Rim101p), and to characterize the regulatory network underlying the transcriptional regulation of a given yeast gene (FLR1). A set of easily accessible experimental approaches that can be used to confirm the predictions of the bioinformatic analysis is also detailed.

Key words

Transcriptional regulation Transcription factors cis-Regulatory elements Transcriptional regulatory network Bioinformatics Saccharomyces cerevisiae YEASTRACT 

Notes

Acknowledgments

Work on gene and genome-wide transcription regulation in yeast in our laboratory is financially supported by FEDER and Fundação para a Ciência e Tecnologia (FCT) (contracts PTDC/AGR-ALI/102608/2008, PTDC/EBB-EBI/108517/2008 and PTDC/BIO/72063/2006). N.P.M. acknowledges a postdoctoral grant (SFRH/BPD/46982/2008) from FCT.

References

  1. 1.
    Bailey, T. L., Williams, N., Misleh, C., and Li, W. W. (2006) MEME: discovering and analyzing DNA and protein sequence motifs, Nucleic Acids Res 34, W369–373.PubMedCrossRefGoogle Scholar
  2. 2.
    Hughes, J. D., Estep, P. W., Tavazoie, S., and Church, G. M. (2000) Computational identification of cis-regulatory elements associated with groups of functionally related genes in Saccharomyces cerevisiae, J Mol Biol 296, 1205–1214.PubMedCrossRefGoogle Scholar
  3. 3.
    Harbison, C. T., Gordon, D. B., Lee, T. I., Rinaldi, N. J., Macisaac, K. D., Danford, T. W., and al., e. (2004) Transcriptional regulatory code of a eukaryotic genome, Nature 431, 99–104.Google Scholar
  4. 4.
    Nguyen, D. T., Alarco, A. M., and Raymond, M. (2001) Multiple Yap1p-binding sites mediate induction of the yeast major facilitator FLR1 gene in response to drugs, oxidants, and alkylating agents, J Biol Chem 276, 1138–1145.PubMedCrossRefGoogle Scholar
  5. 5.
    Teixeira, M. C., Monteiro, P., Jain, P., Tenreiro, S., Fernandes, A. R., Mira, N. P., Alenquer, M., Oliveira, A., Freitas, A. T., and Sá-Correia, I. (2006) The YEASTRACT database: a tool for the analysis of transcriptional regulatory associations in Saccharomyces cerevisiae, Nucleic Acids Res. 34, D446–D451.PubMedCrossRefGoogle Scholar
  6. 6.
    Monteiro, P., Mendes, N., Teixeira, M. C., d’Orey, S., Tenreiro, S., Mira, N. P., Pais, H., Francisco, A., Carvalho, A., Lourenço, A. B., Sá-Correia, I., Oliveira, A., and Freitas, A. T. (2008) YEASTRACT-DISCOVERER: new tools to improve the analysis of transcriptional regulatory associations in Saccharomyces cerevisiae, Nucleic Acids Res 36(Database issue), D132–136.Google Scholar
  7. 7.
    Penalva, M. A., Tilburn, J., Bignell, E., and Arst, H. N., Jr. (2008) Ambient pH gene regulation in fungi: making connections, Trends Microbiol 16, 291–300.PubMedCrossRefGoogle Scholar
  8. 8.
    Lamb, T. M., and Mitchell, A. P. (2003) The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae, Mol Cell Biol 23, 677–686.PubMedCrossRefGoogle Scholar
  9. 9.
    Lamb, T., and Mitchell, A. P. (2003) The transcription factor Rim101p governs ion tolerance and cell differentiation by direct repression of the regulatory genes NRG1 and SMP1 in Saccharomyces cerevisiae, Mol Cell Biol 23, 677–686.PubMedCrossRefGoogle Scholar
  10. 10.
    Mira, N. P., Lourenço, A. B., Fernandes, A. R., and Sá-Correia, I. (2009) The RIM101 pathway has a role in yeast adaptation and resistance to propionic acid and other weak acids, FEMS Yeast Res.. 9, 202–216.PubMedCrossRefGoogle Scholar
  11. 11.
    Lamb, T. M., Xu, W., Diamond, A., and Mitchell, A. P. (2001) Alkaline response genes of Saccharomyces cerevisiae and their relationship to the RIM101 pathway, J Biol Chem 276, 1850–1856.PubMedCrossRefGoogle Scholar
  12. 12.
    Mira, N. P., Teixeira, M. C., and Sá-Correia, I. (2010) Adaptation and tolerance to weak acid stress in Saccharomyces cerevisiae: a genome-wide view, OMICS: A Journal of Integrative Biology 14, 525–540.Google Scholar
  13. 13.
    Teixeira, M. C., Dias, P. J., Simoes, T., and Sa-Correia, I. (2008) Yeast adaptation to mancozeb involves the up-regulation of FLR1 under the coordinate control of Yap1, Rpn4, Pdr3, and Yrr1, Biochem Biophys Res Commun 367, 249–255.PubMedCrossRefGoogle Scholar
  14. 14.
    Cabrito, T. R., Teixeira, M. C., and Sá-Correia, I. (2009) Global adaptive response and resistance to agricultural fungicides: lessons from yeast and phytopathogenic fungi, In Fungicides: Chemistry, Environmental Impact and Health Effects (Columbus, F., Ed.), Nova Science Publishers, New York, USA.Google Scholar
  15. 15.
    Dias, P. J., Teixeira, M. C., Telo, J. P., and Sá-Correia, I. (2010) Insights into the mechanisms of toxicity and tolerance to the agricultural fungicide mancozeb in yeast, as suggested by a chemogenomic approach, OMICS: A Journal of Integrative Biology 14, 211–227.CrossRefGoogle Scholar
  16. 16.
    Santos, P. M., Simões, T., and Sá-Correia, I. (2009) Insights into yeast adaptive response to the agricultural fungicide mancozeb: A toxicoproteomics approach, Proteomics 3, 657–670.CrossRefGoogle Scholar
  17. 17.
    Bachhawat, N., Ouyang, Q., and Henry, S. A. (1995) Functional Characterization of an Inositol-sensitive Upstream Activation Sequence in Yeast, J. Biol. Chem. 270, 25087–25095.PubMedCrossRefGoogle Scholar
  18. 18.
    Andre, B., Hein, C., Grenson, M., and Jauniaux, J. C. (1993) Cloning and expression of the UGA4 gene coding for the inducible GABA-specific transport protein of Saccharomyces cerevisiae, Mol Gen Genet 237, 17–25.PubMedCrossRefGoogle Scholar
  19. 19.
    Mira, N. P., Henriques, S. F., Keller, G., Matos, R., Arraiano, C., Teixeira, M. C., Winge, D. R., and Sá-Correia, I. (2011) Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress, Nucleic Acids Research, 16, 6896–6907.Google Scholar
  20. 20.
    Hellman, L. M., and Fried, M. G. (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions, Nat Protoc 2, 1849–1861.PubMedCrossRefGoogle Scholar
  21. 21.
    Grably, M., and Engelber, D. (2010) A Detailed Protocol for Chromatin Immunoprecipitation in the Yeast Saccharomyces cerevisiae, In Methods in Molecular Biology, pp 211–224.Google Scholar
  22. 22.
    Teixeira, M. C., Dias, P. J., Monteiro, P., Sala, A., Oliveira, A., Freitas, A. T., and Sá-Correia, I. (2010) Refining current knowledge on the yeast FLR1 regulatory network by combined experimental and computational approaches, Molecular Biosystems 6, 247–281.Google Scholar
  23. 23.
    Miller, J. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, New York.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Nuno P. Mira
    • 1
  • Miguel C. Teixeira
    • 1
  • Isabel Sá-Correia
    • 1
    Email author
  1. 1.Institute for Biotechnology and BioengineeringTechnical University of LisbonLisbonPortugal

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