Abstract
Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein–DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i.e., ribosomal DNA (rDNA).
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References
Gilmour DS, Lis JT (1984) Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci 81(14):4275–4279
Solomon MJ, Varshavsky A (1985) Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc Natl Acad Sci 82(19):6470–6474
Hecht A, Strahl-Bolsinger S, Grunstein M (1996) Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature 383(6595):92–96
Henegariu O, Heerema NA, Dlouhy SR, Vance GH, Vogt PH (1997) Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques 23(3):504–511
Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M, Brown PO (2001) Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409(6819):533–538
Horak CE, Snyder M (2002) ChIP-chip: a genomic approach for identifying transcription factor binding sites. Methods Enzymol 350:469–483
Euskirchen G, Snyder M (2004) A plethora of sites. Nat Genet 36(4):325–326
Johnson DS, Mortazavi A, Myers RM, Wold B (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316(5830):1497–1502
Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, Séraphin B (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10):1030–1032
Huang J, Moazed D (2003) Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing. Genes Dev 17(17):2162–2176
Huang J, Brito IL, Villén J, Gygi SP, Amon A, Moazed D (2006) Inhibition of homologous recombination by a cohesin-associated clamp complex recruited to the rDNA recombination enhancer. Genes Dev 20(20):2887–2901
Straight AF, Shou W, Dowd GJ, Turck CW, Deshaies RJ, Johnson AD, Moazed D (1999) Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity. Cell 97(2):245–256
Shou W, Seol JH, Shevchenko A, Baskerville C, Moazed D, Chen ZW, Jang J, Shevchenko A, Charbonneau H, Deshaies RJ (1999) Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97(2):233–244
Visintin R, Hwang ES, Amon A (1999) Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature 398(6730):818–823
Stegmeier F, Huang J, Rahal R, Zmolik J, Moazed D, Amon A (2004) The replication fork block protein Fob1 functions as a negative regulator of the FEAR network. Curr Biol 14(6):467–480
Hoppe GJ, Tanny JC, Rudner AD, Gerber SA, Danaie S, Gygi SP, Moazed D (2002) Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation. Mol Cell Biol 22(12):4167–4180
Acknowledgments
We thank the late Beth Jones for providing the SF10 yeast strain. This work was supported by an NIH training grant (T32 GM007226; J. Huang); an EMBO long-term postdoctoral fellowship and a Swiss National Science Foundation advanced postdoctoral fellowship (N. Iglesias); the Ellison Medical Foundation (D. Moazed); and NIH grants GM061641 and GM079535 (D. Moazed). D. Moazed is an investigator of the Howard Hughes Medical Institute.
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Huang, J., Iglesias, N., Moazed, D. (2017). Evaluation of the Nucleolar Localization of the RENT Complex to Ribosomal DNA by Chromatin Immunoprecipitation Assays. In: Monje-Casas, F., Queralt, E. (eds) The Mitotic Exit Network. Methods in Molecular Biology, vol 1505. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6502-1_15
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DOI: https://doi.org/10.1007/978-1-4939-6502-1_15
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