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Identifying Myc Interactors

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The Myc Gene

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1012))

Abstract

In this chapter, we discuss in detail two essential methods used to evaluate the interaction of Myc with another protein of interest: co-immunoprecipitation (Co-IP) and in vitro pull-down assays. Co-IP is a method that, by immunoaffinity, allows the identification of protein–protein interactions within cells. We provide methods to conduct Co-IPs from whole-cell extracts as well as cytoplasmic and nuclear-enriched fractions. By contrast, the pull-down assay evaluates whether a bait protein that is bound to a solid support can specifically interact with a prey protein that is in solution. We provide methods to conduct in vitro pull-downs and further detail how to use this assay to distinguish whether a protein–protein interaction is direct or indirect. We also discuss methods used to screen for Myc interactors and provide an in silico strategy to help prioritize hits for further validation using the described Co-IP and in vitro pull-down assays.

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References

  1. Blackwood EM, Eisenman RN (1991) Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 251:1211–1217

    Article  PubMed  CAS  Google Scholar 

  2. Cheng SW, Davies KP, Yung E et al (1999) c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function. Nat Genet 22:102–105

    Article  PubMed  CAS  Google Scholar 

  3. Peukert K, Staller P, Schneider A et al (1997) An alternative pathway for gene regulation by Myc. EMBO J 16:5672–5686

    Article  PubMed  CAS  Google Scholar 

  4. Sakamuro D, Elliott KJ, Wechsler-Reya R et al (1996) BIN1 is a novel MYC-interacting protein with features of a tumour suppressor. Nat Genet 14:69–77

    Article  PubMed  CAS  Google Scholar 

  5. Wang Q, Zhang H, Kajino K et al (1998) BRCA1 binds c-Myc and inhibits its transcriptional and transforming activity in cells. Oncogene 17:1939–1948

    Article  PubMed  CAS  Google Scholar 

  6. Hirst M, Ho C, Sabourin L et al (2001) A two-hybrid system for transactivator bait proteins. Proc Natl Acad Sci U S A 98:8726–8731

    Article  PubMed  CAS  Google Scholar 

  7. Huang A, Ho CS, Ponzielli R et al (2005) Identification of a novel c-Myc protein interactor, JPO2, with transforming activity in medulloblastoma cells. Cancer Res 65:5607–5619

    Article  PubMed  CAS  Google Scholar 

  8. Johnson S, Stockmeier CA, Meyer JH et al (2011) The reduction of R1, a novel repressor protein for monoamine oxidase A, in major depressive disorder. Neuropsychopharmacology 36:2139–2148

    Article  PubMed  CAS  Google Scholar 

  9. Kalkat M, Wasylishen AR, Kim SS et al (2011) More than MAX: discovering the Myc interactome. Cell Cycle 10:374–375

    Article  PubMed  CAS  Google Scholar 

  10. Bader GD, Donaldson I, Wolting C et al (2001) BIND–the biomolecular interaction network database. Nucleic Acids Res 29:242–245

    Article  PubMed  CAS  Google Scholar 

  11. Reguly T, Breitkreutz A, Boucher L et al (2006) Comprehensive curation and analysis of global interaction networks in Saccharomyces cerevisiae. J Biol 5:11

    Article  PubMed  Google Scholar 

  12. Xenarios I, Rice DW, Salwinski L et al (2000) DIP: the database of interacting proteins. Nucleic Acids Res 28:289–291

    Article  PubMed  CAS  Google Scholar 

  13. Eskin E, Sharan R, Halperin E (2006) A note on phasing long genomic regions using local haplotype predictions. J Bioinform Comput Biol 4:639–647

    Article  PubMed  CAS  Google Scholar 

  14. Hermjakob H, Montecchi-Palazzi L, Lewington C et al (2004) IntAct: an open source molecular interaction database. Nucleic Acids Res 32:D452–D455

    Article  PubMed  CAS  Google Scholar 

  15. Zanzoni A, Montecchi-Palazzi L, Quondam M et al (2002) MINT: a molecular INTeraction database. FEBS Lett 513:135–140

    Article  PubMed  CAS  Google Scholar 

  16. Cerami EG, Bader GD, Gross BE et al (2006) cPath: open source software for collecting, storing, and querying biological pathways. BMC Bioinformatics 7:497

    Article  PubMed  Google Scholar 

  17. Brown KR, Jurisica I (2007) Unequal evolutionary conservation of human protein interactions in interologous networks. Genome Biol 8:R95

    Article  PubMed  Google Scholar 

  18. Razick S, Magklaras G, Donaldson IM (2008) iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics 9:405

    Article  PubMed  Google Scholar 

  19. Orchard S, Kerrien S, Abbani S et al (2012) Protein interaction data curation: the International Molecular Exchange (IMEx) consortium. Nat Methods 9:345–350

    Article  PubMed  CAS  Google Scholar 

  20. Orchard S, Kerrien S, Jones P et al (2007) Submit your interaction data the IMEx way: a step by step guide to trouble-free deposition. Proteomics 7(Suppl 1):28–34

    Article  PubMed  Google Scholar 

  21. Geraci J, Liu G, Jurisica I (2012) Algorithms for systematic identification of small subgraphs. Methods Mol Biol 804:219–244

    Article  PubMed  CAS  Google Scholar 

  22. Przulj N, Corneil DG, Jurisica I (2004) Modeling interactome: scale-free or geometric? Bioinformatics 20:3508–3515

    Article  PubMed  CAS  Google Scholar 

  23. Przulj N, Corneil DG, Jurisica I (2006) Efficient estimation of graphlet frequency distributions in protein–protein interaction networks. Bioinformatics 22:974–980

    Article  PubMed  CAS  Google Scholar 

  24. Miller AK, Marsh J, Reeve A et al (2010) An overview of the CellML API and its implementation. BMC Bioinformatics 11:178

    Article  PubMed  Google Scholar 

  25. da Huang W, Sherman BT, Tan Q et al (2007) DAVID bioinformatics resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic Acids Res 35:W169–W175

    Article  Google Scholar 

  26. Vastrik I, D'Eustachio P, Schmidt E et al (2007) Reactome: a knowledge base of biologic pathways and processes. Genome Biol 8:R39

    Article  PubMed  Google Scholar 

  27. Kanehisa M, Goto S, Kawashima S et al (2002) The KEGG databases at GenomeNet. Nucleic Acids Res 30:42–46

    Article  PubMed  CAS  Google Scholar 

  28. Pico AR, Kelder T, van Iersel MP et al (2008) WikiPathways: pathway editing for the people. PLoS Biol 6:e184

    Article  PubMed  Google Scholar 

  29. Markham K, Bai Y, Schmitt-Ulms G (2007) Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins. Anal Bioanal Chem 389:461–473

    Article  PubMed  CAS  Google Scholar 

  30. Conacci-Sorrell M, Ngouenet C, Eisenman RN (2010) Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation. Cell 142:480–493

    Article  PubMed  CAS  Google Scholar 

  31. Ponzielli R, Boutros PC, Katz S et al (2008) Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies. Nucleic Acids Res 36:e144

    Article  PubMed  Google Scholar 

  32. Andresen C, Helander S, Lemak A et al (2012) Transient structure and dynamics in the disordered c-Myc transactivation domain affect Bin1 binding. Nucleic Acids Res 40:6353–6366

    Article  PubMed  CAS  Google Scholar 

  33. Beaulieu ME, McDuff FO, Frappier V et al (2012) New structural determinants for c-Myc specific heterodimerization with Max and development of a novel homodimeric c-Myc b-HLH-LZ. J Mol Recognit 25:414–426

    Article  PubMed  CAS  Google Scholar 

  34. Nair SK, Burley SK (2003) X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors. Cell 112:193–205

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to acknowledge the members of the Penn lab, Manpreet Kalkat and Lindsay Lustig, for helpful discussions and critical review of this chapter. This work was supported by Canadian Institutes of Health Research (CIHR #MOP123289, L.Z.P), Ontario Research Fund (GL2-01-030, I. J., L.Z.P.), Canada Foundation for Innovation (CFI #12301 and CFI #203373 I. J.), and Canada Research Chairs Program (CRC #203373 L.Z.P. and CRC #225404, I.J.). Additional support was provided by the Ontario Ministry of Health and Long-Term Care. The views expressed do not necessarily reflect those of the OMOHLTC.

Author information

Authors and Affiliations

  1. Ontario Cancer Institute, The Campbell Family Institute for Cancer Research, Toronto, ON, Canada

    Romina Ponzielli

  2. Department of Medical Biophysics, Ontario Cancer Institute, The Campbell Family Institute for Cancer Research, University of Toronto, Toronto, ON, Canada

    William B. Tu, Igor Jurisica & Linda Z. Penn

  3. Department of Computer Science, Techna Institute, University Health Network, University of Toronto, Toronto, ON, Canada

    Igor Jurisica

Authors
  1. Romina Ponzielli
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  2. William B. Tu
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  3. Igor Jurisica
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  4. Linda Z. Penn
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Editor information

Editors and Affiliations

  1. Fundació Vall d'Hebron Institute of Onco, Barcelona, Spain

    Laura Soucek

  2. Department of Biochemistry Sanger Building, University of Cambridge, Cambridge, United Kingdom

    Nicole M. Sodir

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Ponzielli, R., Tu, W.B., Jurisica, I., Penn, L.Z. (2013). Identifying Myc Interactors. In: Soucek, L., Sodir, N. (eds) The Myc Gene. Methods in Molecular Biology, vol 1012. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-429-6_4

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  • DOI: https://doi.org/10.1007/978-1-62703-429-6_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-428-9

  • Online ISBN: 978-1-62703-429-6

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