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Analysis of SUC2 Promoter Structure by Nucleosome Scanning

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Transcriptional Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 809))

Abstract

Chromatin remodeling is a key mode of transcriptional regulation, and studying the nucleosome positioning at promoters is an important means to understand how genes are regulated. Nucleosome scanning is a convenient method to study nucleosome positioning. Yeast cells are converted to spheroplasts and nuclei are isolated. The nuclei are then digested by micrococcal nuclease to yield mononucleosome-sized DNA. Using a set of overlapping primers that cover the entire promoter, quantitative real-time PCR is performed using the mononucleosome DNA as the template. The nucleosome enrichment for each primer is calculated to yield a map of nucleosome occupancy across the promoter.

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References

  1. Erkina, T. Y., and Erkine, A. M. (2006) Displacement of histones at promoters of Saccharomyces cerevisiae heat shock genes is differentially associated with histone H3 acetylation. Mol Cell Biol 26, 7587–600.

    Google Scholar 

  2. Rando, O. J., and Chang, H. Y. (2009) Genome-Wide Views of Chromatin Structure. Annual Review of Biochemistry 78, 245–271.

    Google Scholar 

  3. Hogan, C., and Varga-Weisz, P. (2007) The regulation of ATP-dependent nucleosome remodelling factors. Mutat Res 618, 41–51.

    Google Scholar 

  4. Varga-Weisz, P. D., and Becker, P. B. (2006) Regulation of higher-order chromatin structures by nucleosome-remodelling factors. Current Opinion in Genetics & Development 16, 151–156.

    Google Scholar 

  5. Carlson, M., Taussig, R., Kustu, S., and Botstein, D. (1983) The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence. Mol Cell Biol 3, 439–47.

    Google Scholar 

  6. Taussig, R., and Carlson, M. (1983) Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res 11, 1943–54.

    Google Scholar 

  7. Matallana, E., Franco, L., and Perez-Ortin, J. E. (1992) Chromatin structure of the yeast SUC2 promoter in regulatory mutants. Mol Gen Genet 231, 395–400.

    Google Scholar 

  8. Perez-Ortin, J. E., Estruch, F., Matallana, E., and Franco, L. (1987) Fine analysis of the chromatin structure of the yeast SUC2 gene and of its changes upon derepression. Comparison between the chromosomal and plasmid-inserted genes. Nucleic Acids Res 15, 6937–56.

    Google Scholar 

  9. Gavin, I. M., and Simpson, R. T. (1997) Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure. EMBO J 16, 6263–71.

    Google Scholar 

  10. Hirschhorn, J. N., Brown, S. A., Clark, C. D., and Winston, F. (1992) Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure. Genes Dev 6, 2288–98.

    Google Scholar 

  11. Sudarsanam, P., Cao, Y., Wu, L., Laurent, B. C., and Winston, F. (1999) The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, Gcn5. EMBO J 18, 3101–6.

    Google Scholar 

  12. Wu, L., and Winston, F. (1997) Evidence that Snf-Swi controls chromatin structure over both the TATA and UAS regions of the SUC2 promoter in Saccharomyces cerevisiae. Nucleic Acids Res 25, 4230–4.

    Google Scholar 

  13. Bu, Y., and Schmidt, M. C. (1998) Identification of cis-acting elements in the SUC2 promoter of Saccharomyces cerevisiae required for activation of transcription. Nucleic Acids Res 26, 1002–9.

    Google Scholar 

  14. Treitel, M. A., and Carlson, M. (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc Natl Acad Sci USA 92, 3132–6.

    Google Scholar 

  15. DeLillo, N., Romero, C., Lin, H., and Vancura, A. (2003) Genetic evidence for a role of phospholipase C at the budding yeast kinetochore. Mol Genet Genomics 269, 261–70.

    Google Scholar 

  16. Yu, Y., Eriksson, P., and Stillman, D. J. (2000) Architectural transcription factors and the SAGA complex function in parallel pathways to activate transcription. Mol Cell Biol 20, 2350–7.

    Google Scholar 

  17. Odom, A. R., Stahlberg, A., Wente, S. R., and York, J. D. (2000) A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 287, 2026–9.

    Google Scholar 

  18. York, J. D., Odom, A. R., Murphy, R., Ives, E. B., and Wente, S. R. (1999) A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 285, 96–100.

    Google Scholar 

  19. York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., and York, J. D. (2005) Inositol diphosphate signaling regulates telomere length. J Biol Chem 280, 4264–9.

    Google Scholar 

  20. Flick, J. S., and Thorner, J. (1993) Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Sacchar­omyces cerevisiae. Mol Cell Biol 13, 5861–76.

    Google Scholar 

  21. Romero, C., Desai, P., DeLillo, N., and Vancura, A. (2006) Expression of FLR1 transporter requires phospholipase C and is repressed by Mediator. J Biol Chem 281, 5677–85.

    Google Scholar 

  22. Yoko-o, T., Matsui, Y., Yagisawa, H., Nojima, H., Uno, I., and Toh-e, A. (1993) The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth. Proc Natl Acad Sci USA 90, 1804–8.

    Google Scholar 

  23. Payne, W. E., and Fitzgerald-Hayes, M. (1993) A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation. Mol Cell Biol 13, 4351–64.

    Google Scholar 

  24. Wera, S., Bergsma, J. C., and Thevelein, J. M. (2001) Phosphoinositides in yeast: genetically tractable signalling. FEMS Yeast Res 1, 9–13.

    Google Scholar 

  25. Irvine, R. (2007) Cell signaling. The art of the soluble. Science 316, 845–6.

    Google Scholar 

  26. Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) A conserved family of enzymes that phosphorylate inositol hexakisphosphate. Science 316, 106–9.

    Google Scholar 

  27. Steger, D. J., Haswell, E. S., Miller, A. L., Wente, S. R., and O’Shea, E. K. (2003) Regulation of chromatin remodeling by inositol polyphosphates. Science 299, 114–6.

    Google Scholar 

  28. Shen, X., Xiao, H., Ranallo, R., Wu, W. H., and Wu, C. (2003) Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 299, 112–4.

    Google Scholar 

  29. Lee, Y. S., Mulugu, S., York, J. D., and O’Shea, E. K. (2007) Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science 316, 109–12.

    Google Scholar 

  30. Lin, H., Choi, J. H., Hasek, J., DeLillo, N., Lou, W., and Vancura, A. (2000) Phospholipase C is involved in kinetochore function in Saccharomyces cerevisiae. Mol Cell Biol 20, 3597–607.

    Google Scholar 

  31. Desai, P., Guha, N., Galdieri, L., Hadi, S., and Vancura, A. (2009) Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex. Mol Genet Genomics 281, 511–23.

    Google Scholar 

  32. Sekinger, E. A., Moqtaderi, Z., and Struhl, K. (2005) Intrinsic histone-DNA interactions and low nucleosome density are important for preferential accessibility of promoter regions in yeast. Molecular Cell 18, 735–748.

    Google Scholar 

  33. Bai, L., Charvin, G., Siggia, E. D., and Cross, F. R. (2010) Nucleosome-Depleted Regions in Cell-Cycle-Regulated Promoters Ensure Reliable Gene Expression in Every Cell Cycle. Developmental cell 18, 544–555.

    Google Scholar 

  34. Kondilis-Mangum, H. D., Cobb, R. M., Osipovich, O., Srivatsan, S., Oltz, E. M., and Krangel, M. S. (2010) Transcription-Dependent Mobilization of Nucleosomes at Accessible TCR Gene Segments In Vivo. Journal of Immunology 184, 6970–6977.

    Google Scholar 

  35. Preti, M., Ribeyre, C., Pascali, C., Bosio, M. C., Cortelazzi, B., Rougemont, J., Guarnera, E., Naef, F., Shore, D., and Dieci, G. (2010) The Telomere-Binding Protein Tbf1 Demarcates snoRNA Gene Promoters in Saccharomyces cerevisiae. Molecular Cell 38, 614–620.

    Google Scholar 

  36. Burgos-Rivera, B., Ruzicka, D. R., Deal, R. B., McKinney, E. C., King-Reid, L., and Meagher, R. B. (2008) ACTIN DEPOLYMERIZING FACTOR9 controls development and gene expression in Arabidopsis. Plant Molecular Biology 68, 619–632.

    Google Scholar 

  37. Boukaba, A., Georgieva, E. I., Myers, F. A., Thorne, A. W., Lopez-Rodas, G., Crane-Robinson, C., and Franco, L. (2004) A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene. J Biol Chem 279, 7678–84.

    Google Scholar 

  38. Fleming, A. B., and Pennings, S. (2007) Tup1-Ssn6 and Swi-Snf remodelling activities influence long-range chromatin organization upstream of the yeast SUC2 gene. Nucleic Acids Res 35, 5520–31.

    Google Scholar 

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Authors and Affiliations

  1. Department of Cell Biology, New York University School of Medicine, New York, NY, USA

    Jennifer Chang

  2. Department of Biological Sciences, St. John’s University, Queens, NY, USA

    Ales Vancura

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  1. Jennifer Chang
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  2. Ales Vancura
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Correspondence to Ales Vancura .

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  1. , Department of Biological Sciences, St. John's University, Utopia Parkway 8000, Queens, 11439, New York, USA

    Ales Vancura

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Chang, J., Vancura, A. (2012). Analysis of SUC2 Promoter Structure by Nucleosome Scanning. In: Vancura, A. (eds) Transcriptional Regulation. Methods in Molecular Biology, vol 809. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-376-9_22

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  • DOI: https://doi.org/10.1007/978-1-61779-376-9_22

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-61779-375-2

  • Online ISBN: 978-1-61779-376-9

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