Advertisement

Silencing in Yeast

Identification of Clr4 Targets
  • Sergey V. Ivanov
  • Alla V. Ivanova
Part of the Methods in Molecular Biology book series (MIMB, volume 317)

Abstract

Efficient handling of multiple reactions is a crucial prerequisite for productive RNA differential display (DD) analysis. To identify transcriptional targets of the histone H3 Lys9-specific methyltransferase Clr4, we applied a multiformat modification of DD to compare between clr4 + and clr4 transcriptomes of Schizosaccaromyces pombe. As a result, 14 differentially expressed bands were identified among 720 polymerase chain reaction (PCR) studied. The content of these bands was then analyzed by cloning, sequencing, and Northern analysis. In the final stage of verification, four Clr4 targets were isolated based on their expression in six Clr4 chromo and SET domain mutant strains. The step-by-step description of the multiformat DD provided below includes RNA purification, cDNA synthesis, 96-well PCR, electrophoretic separation of PCR products, isolation of DNA fragments from differentially expressed bands, and verification of candidate genes by Northern analysis.

Key Words

RNA differential display clr4 silencing yeast histone-specific methyltransferase transcriptional regulator 

Notes

Acknowledgments

We thank Dr. W. Modi for advising on gel loading strategy. This work was supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract no. NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US government.

References

  1. 1.
    Allshire, R. C. (1996) Transcriptional silencing in the fission yeast: a manifestation of higher order chromatin structure and function, in Epigenetic Mechanisms of Gene Expression (Russo V.E.A., Martienssen R.A., and Riggs A.D, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY, pp. 443–466.Google Scholar
  2. 2.
    Klar, A.J.S., Ivanova, A. V., Dalgaard, J. C., Bonaduce, M. J., and Grewal, S.I.S. (1998) Multiple epigenetic events regulate mating-type switching of fission yeast, in Epigenetics (Chadwick D.J. and Casdew G., eds.), Wiley and Sons, Chichester, UK, pp. 7–103.Google Scholar
  3. 3.
    Grewal, S. I. and Klar, A.J.S. (1997) A recombinationally repressed region between mat2 and mat3 loci shares homology to centromeric repeats and regulates directionality of mating-type switching in fission yeast. Genetics 146, 1221–1238.PubMedGoogle Scholar
  4. 4.
    Rea, S., Eisenhaber, F., O’Carroll, D., et al. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599.PubMedCrossRefGoogle Scholar
  5. 5.
    Nakayama, J., Rice, J. C., Strahl, B. D., et al. (2001) Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113.PubMedCrossRefGoogle Scholar
  6. 6.
    Liang, P. and Pardee, A. B. (1997) Differential display: a general protocol in Differential Display Methods and Protocols (Liang P. and Pardee A.B., eds.), Humana Press, Totowa, N J, pp. 3–11.CrossRefGoogle Scholar
  7. 7.
    Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 18, 3091–3092.PubMedCrossRefGoogle Scholar
  8. 8.
    Sambrook, J. and Russell, D. W. (2001) Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  9. 9.
    Ivanova, A. V., Bonaduce, M. J., Ivanov, S. V., and Klar, A. J. (1998) The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nat. Genet. 19, 192–195.PubMedCrossRefGoogle Scholar
  10. 10.
    Ivanova, A. V. and Ivanov, S. V. (2002) Differential display analysis of gene expression in yeast. Cell. Mol. Life Sci. 59, 1241–1245.PubMedCrossRefGoogle Scholar
  11. 11.
    Allshire, R. C., Nimmo, E. R., Ekwall, K., Javerzat, J. P., and Cranston, G. (1995). Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 9, 218–233.PubMedCrossRefGoogle Scholar
  12. 12.
    Okazaki, K., Okayama, H., and Niwa, O. (2000) The polyubiquitin gene is essential for meiosis in fission yeast. Exp. Cell Res. 254, 143–152.PubMedCrossRefGoogle Scholar
  13. 13.
    Naresh, A., Saini, S., and Singh, J. (2003) Identification of Uhp1, a ubiquitinated histone-like protein, as a target/mediator of Rhp6 in mating-type silencing in fission yeast. J. Biol Chem. 278, 9185–9194.PubMedCrossRefGoogle Scholar
  14. 14.
    Ivanov, S. V., Kuzmin, I., Wei, M. H., et al. (1998) Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc. Natl. Acad. Sci. USA 95, 12,596–12,601.PubMedCrossRefGoogle Scholar
  15. 15.
    Plisov, S. Y., Ivanov, S. V., Yoshino, K., et al. (2000) Mesenchymal-epithelial transition in the developing metanephric kidney: gene expression study by differential display. Genesis 27, 22–31.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Sergey V. Ivanov
    • 1
  • Alla V. Ivanova
    • 1
  1. 1.SAIC-FrederickNCI Center for Cancer ResearchFrederick

Personalised recommendations