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Combinatorial profiling of chromatin binding modules reveals multisite discrimination

Nature Chemical Biology volume 6, pages 283290 (2010) | Download Citation

Abstract

Specific interactions between post-translational modifications (PTMs) and chromatin-binding proteins are central to the idea of a 'histone code'. Here, we used a 5,000-member, PTM-randomized, combinatorial peptide library based on the N terminus of histone H3 to interrogate the multisite specificity of six chromatin binding modules, which read the methylation status of Lys4. We found that Thr3 phosphorylation, Arg2 methylation and Thr6 phosphorylation are critical additional PTMs that modulate the ability to recognize and bind histone H3. Notably, phosphorylation of Thr6 yielded the most varied effect on protein binding, suggesting an important regulatory mechanism for readers of the H3 tail. Mass spectrometry and antibody-based evidence indicate that this previously uncharacterized modification exists on native H3, and NMR analysis of ING2 revealed the structural basis for discrimination. These investigations reveal a continuum of binding affinities in which multisite PTM recognition involves both switch- and rheostat-like properties, yielding graded effects that depend on the inherent 'reader' specificity.

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Acknowledgements

We thank G. Barrett-Wilt and G. Sabat (University of Wisconsin Biotechnology Center) for their help with MALDI-TOF mass spectrometry. We are grateful to G. Case (University of Wisconsin Peptide Synthesis Facility) for helpful conversations and advice on peptide synthesis. We thank G. Musco (Dulbecco Telethon Institute), Y. Shi (Harvard Medical School), W. Yang (National Institute of Health) and R.-M. Xu (New York University) for expression plasmids used in this study. We thank S. Bednarek (University of Wisconsin-Madison) for the GST-specific antibody. This work was supported by the US National Institutes of Health (grant GM059785 to J.M.D. and predoctoral fellowship T32GM008505 to S.S.O.) and an American Society for Mass Spectrometry research award (B.A.G.).

Author information

Author notes

    • Adam L Garske
    •  & Samuel S Oliver

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.

    • Adam L Garske
  2. Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin, USA.

    • Samuel S Oliver
    • , Elise K Wagner
    •  & John M Denu
  3. Department of Pharmacology, University of Colorado Denver School of Medicine, Aurora, Colorado, USA.

    • Catherine A Musselman
    •  & Tatiana G Kutateladze
  4. Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.

    • Gary LeRoy
    •  & Benjamin A Garcia

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Contributions

A.L.G., S.S.O. and J.M.D. designed the library, screened the proteins and analyzed the results. S.S.O. executed the ITC validation experiments, and E.K.W. performed and analyzed the western blots. C.A.M. and T.G.K. carried out the NMR analysis. B.A.G. and G.L. performed the mass spectrometry experiments for H3T6ph identification. Initial manuscript drafts were written by A.L.G., S.S.O. and J.M.D. All authors participated in writing the final document.

Competing interests

J.M.D. and A.L.G. have a US patent pending (number 11/585,625) that describes the construction and uses of PTM peptide libraries.

Corresponding author

Correspondence to John M Denu.

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    Supplementary Methods, Supplementary Tables 1–7 and Supplementary Figures 1–6

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DOI

https://doi.org/10.1038/nchembio.319

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