Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

A differential proteome screening system for post-translational modification–dependent transcription factor interactions

Abstract

Post-translational modifications (PTMs) of transcription factors alter interactions with co-regulators and epigenetic modifiers. For example, members of the C/EBP transcription factor family are extensively methylated on arginine and lysine residues in short, conserved, modular domains, implying modification-dependent cofactor docking. Here we describe array peptide screening (APS), a systematic and differential approach to detect PTM-dependent interactions in the human proteome using chemically synthesized, biotinylated peptides coupled to fluorophore-labeled streptavidin. Peptides with and without a modified residue are applied in parallel to bacterial expression libraries in an arrayed format. Interactions are detected and quantified by laser scanning to reveal proteins that differentially bind to nonmodified or modified peptides. We have previously used this method to investigate the effect of arginine methylation of C/EBPβ peptides. The method enables determination of PTM-dependent transcription factor interactions, quantification of relative binding affinities and rapid protein classification, all independently of the transactivation potential of peptides or cellular abundance of interactors. The protocol provides a cost-effective alternative to mass spectrometric approaches and takes 3–4 d to complete.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Differential proteomic array peptide screening (APS).
Figure 2: Result of a differential proteomic array screening using nonmodified and asymmetrical R3me2-modified C/EBPβ peptide baits.

Similar content being viewed by others

References

  1. Taverna, S.D. et al. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Huang, J. et al. Repression of p53 activity by Smyd2-mediated methylation. Nature 444, 629–632 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Jansson, M. et al. Arginine methylation regulates the p53 response. Nat. Cell Biol. 10, 1431–1439 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Yamagata, K. et al. Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol. Cell 32, 221–231 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Kowenz-Leutz, E. et al. Crosstalk between C/EBPbeta phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code. EMBO J. 29, 1105–1115 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Edmondson, D.G. & Roth, S.Y. Identification of protein interactions by far western analysis. Curr. Protoc. Protein Sci. 19, 19.7.1–19.7.10 (2001).

    Article  Google Scholar 

  7. Seet, B.T. et al. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7, 473–483 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Takayama, S. & Reed, J.C. Protein interaction cloning by far-Western screening of lambda-phage cDNA expression libraries. Methods Mol. Biol. 69, 171–184 (1997).

    CAS  PubMed  Google Scholar 

  9. Pless, O. et al. G9a-mediated lysine methylation alters the function of CCAAT/enhancer-binding protein-beta. J. Biol. Chem. 283, 26357–26363 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Querfurth, E. et al. Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors. Genes Dev. 14, 2515–2525 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mink, S., Haenig, B. & Klempnauer, K.H. Interaction and functional collaboration of p300 and C/EBPbeta. Mol. Cell Biol. 17, 6609–6617 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chang, C.J., Chen, Y.L. & Lee, S.C. Coactivator TIF1beta interacts with transcription factor C/EBPbeta and glucocorticoid receptor to induce alpha1-acid glycoprotein gene expression. Mol. Cell Biol. 18, 5880–5887 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Leutz, A. et al. Crosstalk between phosphorylation and multi-site arginine/lysine methylation in C/EBPs. Transcription 2, 3–8 (2011).

    Article  PubMed  Google Scholar 

  14. Schulze, W.X. & Mann, M. A novel proteomic screen for peptide-protein interactions. J. Biol. Chem. 279, 10756–10764 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Ruthenburg, A.J., Allis, C.D. & Wysocka, J. Methylation of lysine 4 on histone H3: intricacy of writing and reading a single epigenetic mark. Mol. Cell 25, 15–30 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Ruthenburg, A.J. et al. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Finn, R.D. et al. The Pfam protein families database. Nucleic Acids Res. 38, D211–D222 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Letunic, I., Doerks, T. & Bork, P. SMART 6: recent updates and new developments. Nucleic Acids Res. 37, D229–D232 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Waterhouse, A.M. et al. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gould, C.M. et al. ELM: the status of the 2010 eukaryotic linear motif resource. Nucleic Acids Res. 38, D167–D180 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Bartke, T. et al. Nucleosome-interacting proteins regulated by DNA and histone methylation. Cell 143, 470–484 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kowenz-Leutz, E. & Leutz, A. A C/EBP beta isoform recruits the SWI/SNF complex to activate myeloid genes. Mol. Cell 4, 735–743 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank K.W. Friedrich and U. Englisch from LI-COR Biosciences for technical support and discussions. We thank M. Knoblich for protein purification for subsequent mass spectrometric analysis. O.P. is supported by a grant of the Deutsche Forschungsgemeinschaft (DFG) to A.L. (LE 770/4-1). E.K.-L., G.D. and A.L. are supported by institutional funds from the Helmholtz Association.

Author information

Authors and Affiliations

Authors

Contributions

O.P. and A.L. designed experiments, and O.P. and E.K.-L. performed experiments and analyzed data. G.D. performed mass spectrometric analysis and data evaluation. O.P. and A.L. prepared the manuscript. A.L. supervised the work.

Corresponding author

Correspondence to Achim Leutz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Differential interaction partners on UniPEx library, part 1 (DOC 60 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pless, O., Kowenz-Leutz, E., Dittmar, G. et al. A differential proteome screening system for post-translational modification–dependent transcription factor interactions. Nat Protoc 6, 359–364 (2011). https://doi.org/10.1038/nprot.2011.303

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.303

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing