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.

Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation

Nature volume 453, pages 812–816 (2008)Cite this article

Abstract

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans1. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the ‘histone code’ hypothesis, in which histone modifications are integral to choreographing the expression of the genome1,2. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans3, and lysine 123 in yeast4, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5–8), by K79-specific methyltransferase Dot1 (KMT4)9,10,11. However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Semi-synthesis of ubiquitylated H2B.
Figure 2: Semi-synthesis of uH2B and incorporation into chemically defined histone octamers and nucleosomes.
Figure 3: Effects of uH2B on intranucleosomal methylation of H3 K79 by hDot1L.
Figure 4: Methyltransferase assays on dinucleosomes using full-length hDot1L.

References

  1. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007)

    CAS  PubMed  Google Scholar 

  2. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  3. West, M. H. P. & Bonner, W. M. Histone 2B can be modified by the attachment of ubiquitin. Nucleic Acids Res. 8, 4671–4680 (1980)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Robzyk, K., Recht, J. & Osley, M. A. Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, 501–504 (2000)

    Article  ADS  CAS  PubMed  Google Scholar 

  5. Kim, J., Hake, S. B. & Roeder, R. G. The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions. Mol. Cell 20, 759–770 (2005)

    Article  CAS  PubMed  Google Scholar 

  6. Zhu, B. et al. Monoubiquitination of human histone H2B: the factors involved and their roles in HOX gene regulation. Mol. Cell 20, 601–611 (2005)

    Article  CAS  PubMed  Google Scholar 

  7. Ng, H. H., Xu, R. M., Zhang, Y. & Struhl, K. Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J. Biol. Chem. 277, 34655–34657 (2002)

    Article  CAS  PubMed  Google Scholar 

  8. Briggs, S. D. et al. Trans-histone regulatory pathway in chromatin. Nature 418, 498 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  9. Feng, Q. et al. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr. Biol. 12, 1052–1058 (2002)

    Article  CAS  PubMed  Google Scholar 

  10. Ng, H. H. et al. Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev. 16, 1518–1527 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. van Leeuwen, F., Gafken, P. R. & Gottschling, D. E. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 109, 745–756 (2002)

    Article  CAS  PubMed  Google Scholar 

  12. Henry, K. W. & Berger, S. L. Trans-tail histone modifications: wedge or bridge? Nature Struct. Biol. 9, 565–566 (2002)

    Article  CAS  PubMed  Google Scholar 

  13. Sun, Z. W. & Allis, C. D. Ubiquitylation of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104–108 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  14. Krogan, N. J. et al. The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol. Cell 11, 721–729 (2003)

    Article  CAS  PubMed  Google Scholar 

  15. Ezhkova, E. & Tansey, W. P. Proteasomal ATPases link ubiquitylation of histone H2B to methylation of H3. Mol. Cell 13, 435–442 (2004)

    Article  CAS  PubMed  Google Scholar 

  16. Lee, J. S. et al. Histone crosstalk between H2B monoubiquitylation and H3 methylation mediated by COMPASS. Cell 131, 1084–1096 (2007)

    Article  CAS  PubMed  Google Scholar 

  17. Hwang, W. W. et al. A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol. Cell 11, 251–266 (2003)

    Article  Google Scholar 

  18. Wood, A. et al. Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol. Cell 11, 267–274 (2003)

    Article  CAS  PubMed  Google Scholar 

  19. Muralidharan, V. & Muir, T. W. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nature Methods 3, 429–438 (2006)

    Article  CAS  PubMed  Google Scholar 

  20. Chatterjee, C., McGinty, R. K., Pellois, J. P. & Muir, T. W. Auxiliary-mediated site-specific peptide ubiquitylation. Angew. Chem. Int. Edn Engl. 46, 2814–2818 (2007)

    Article  CAS  Google Scholar 

  21. Yan, L. Z. & Dawson, P. E. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 123, 526–533 (2001)

    Article  CAS  PubMed  Google Scholar 

  22. Lowary, P. T. & Widom, J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J. Mol. Biol. 276, 19–42 (1998)

    Article  CAS  PubMed  Google Scholar 

  23. Sawada, K. et al. Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase. J. Biol. Chem. 279, 43296–43306 (2004)

    Article  CAS  PubMed  Google Scholar 

  24. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F. & Richmond, T. J. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260 (1997)

    Article  ADS  CAS  PubMed  Google Scholar 

  25. Min, J., Feng, Q., Li, Z., Zhang, Y. & Xu, R. M. Structure of the catalytic domain of human Dot1L, a non-SET domain nucleosomal histone methyltransferase. Cell 112, 711–723 (2003)

    Article  CAS  PubMed  Google Scholar 

  26. Shahbazian, M. D., Zhang, K. & Grunstein, M. Histone H2B ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol. Cell 19, 271–277 (2005)

    Article  CAS  PubMed  Google Scholar 

  27. Garcia, B. A. et al. Organismal differences in post-translational modifications in histones H3 and H4. J. Biol. Chem. 282, 7641–7655 (2007)

    Article  CAS  PubMed  Google Scholar 

  28. Zheng, C. & Hayes, J. J. Intra- and inter-nucleosomal protein–DNA interactions of the core histone tail domains in a model system. J. Biol. Chem. 26, 24217–24224 (2003)

    Article  Google Scholar 

  29. Wang, S. S. et al. Facile synthesis of amino acid and peptide esters under mild conditions via cesium salts. J. Org. Chem. 42, 1286–1290 (1977)

    Article  CAS  Google Scholar 

  30. Smith, A. B., Savinov, S. N., Manjappara, U. V. & Chaiken, I. M. Peptide-small molecule hybrids via orthogonal deprotection-chemoselective conjugation to cysteine-anchored scaffolds. Org. Lett. 4, 4041–4044 (2002)

    Article  CAS  PubMed  Google Scholar 

  31. Luger, K., Rechsteiner, T. J., Flaus, A. J., Waye, M. M. & Richmond, T. J. Characterization of nucleosome core particles containing histone proteins made in bacteria. J. Mol. Biol. 272, 301–311 (1997)

    Article  CAS  PubMed  Google Scholar 

  32. Pentelute, B. L. & Kent, S. B. Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation. Org. Lett. 9, 687–690 (2007)

    Article  CAS  PubMed  Google Scholar 

  33. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol. Biol. 119, 1–16 (1999)

    CAS  PubMed  Google Scholar 

  34. Dorigo, B., Schalch, T., Bystricky, K. & Richmond, T. J. Chromatin fiber folding: requirement for the histone H4 N-terminal tail. J. Mol. Biol. 327, 85–96 (2003)

    Article  CAS  PubMed  Google Scholar 

  35. Owen-Hughes, T. et al. Analysis of nucleosome disruption by ATP-driven chromatin remodelling complexes. Methods Mol. Biol. 119, 319–331 (1999)

    CAS  PubMed  Google Scholar 

  36. Ito, T. et al. ACF consists of two subunits, Acf1 and ISWI, that function cooperatively in the ATP-dependent catalysis of chromatin assembly. Genes Dev. 13, 1529–1539 (1999)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. An, W., Kim, J. & Roeder, R. G. Ordered cooperative functions of PRMT1, p300, and CARM1 in transcriptional activation by p53. Cell 117, 735–748 (2004)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge H. Deng and J. Fernandez at The Rockefeller University Proteomics Resource Center for mass spectrometric analysis of methylated peptides. We thank T. J. Richmond for donating the 12_177_601 plasmid. We thank C. D. Allis for contributing the Xenopus histone plasmids for recombinant histone expression. We thank Y. Zhang for donating a plasmid containing hDot1L. We thank B. R. Rosenberg for assistance with phosphorimaging. We thank C. D. Allis, J. Tanny, and K. P. Chiang for discussions. This work was funded by the US National Institutes of Health. R.K.M. was supported by National Institutes of Health MSTP grant GM07739.

Author Contributions R.K.M., J.K. and C.C. did the experimental work; all authors performed project planning, data analysis and manuscript preparation.

Author information

Authors and Affiliations

  1. Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10065, USA ,

    Robert K. McGinty, Champak Chatterjee & Tom W. Muir

  2. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10065, USA ,

    Jaehoon Kim & Robert G. Roeder

Authors
  1. Robert K. McGinty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jaehoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Champak Chatterjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert G. Roeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tom W. Muir
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tom W. Muir.

Supplementary information

Supplementary information

The file contains Supplementary Figures S1-S11 with Legends. (PDF 974 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

McGinty, R., Kim, J., Chatterjee, C. et al. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008). https://doi.org/10.1038/nature06906

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06906

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

Advanced 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