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.

  • Article
  • Published:

Nonprocessive methylation by Dot1 leads to functional redundancy of histone H3K79 methylation states

Nature Structural & Molecular Biology volume 15, pages 550–557 (2008)Cite this article

Abstract

Whereas mono-, di- and trimethylation states of lysines on histones typically have specific functions, no specific functions have been attributed so far to the different methylation states of histone H3 Lysine 79 (H3K79) generated by Dot1. Here we show that Dot1, in contrast to other known histone methyltransferases, introduces multiple methyl groups via a nonprocessive mechanism. The kinetic mechanism implies that the H3K79 methylation states cannot be generated independently, suggesting functional redundancy. Indeed, gene silencing in yeast, which is dependent on Dot1, relied on global H3K79 methylation levels and not on one specific methylation state. Furthermore, our findings suggest that histone H2B ubiquitination affects H3K79 trimethylation by enhancing synthesis of all H3K79 methylation states. Our results suggest that multiple methylation of H3K79 leads to a binary code, which is expected to limit the possibilities for regulation by putative demethylases or binding proteins.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Dot1 is a nonprocessive methyltransferase in vitro.
Figure 2: Dot1 is a nonprocessive methyltransferase in vivo.
Figure 3: Dot1-G401A is an active site mutant with reduced catalytic activity.
Figure 4: The functions of Dot1 in telomeric silencing depend on the overall levels of H3K79 methylation.
Figure 5: Trimethylation of histone H3K79 is not required for DNA-damage signaling.
Figure 6: H2B ubiquitination regulates all H3K79 methylation states and does not act via the putative ubiquitin binding domain of Dot1.

Similar content being viewed by others

References

  1. Bhaumik, S.R., Smith, E. & Shilatifard, A. Covalent modifications of histones during development and disease pathogenesis. Nat. Struct. Mol. Biol. 14, 1008–1016 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Li, B., Carey, M. & Workman, J.L. The role of chromatin during transcription. Cell 128, 707–719 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Cheng, X., Collins, R.E. & Zhang, X. Structural and sequence motifs of protein (histone) methylation enzymes. Annu. Rev. Biophys. Biomol. Struct. 34, 267–294 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Klose, R.J. & Zhang, Y. Regulation of histone methylation by demethylimination and demethylation. Nat. Rev. Mol. Cell Biol. 8, 307–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Shi, Y. & Whetstine, J.R. Dynamic regulation of histone lysine methylation by demethylases. Mol. Cell 25, 1–14 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D. & Patel, D.J. 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 

  7. 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 

  8. 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 

  9. 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 

  10. Okada, Y. et al. hDOT1L links histone methylation to leukemogenesis. Cell 121, 167–178 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C.M. & Green, M.R. An elaborate pathway required for Ras-mediated epigenetic silencing. Nature 449, 1073–1077 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang, W., Xia, X., Reisenauer, M.R., Hemenway, C.S. & Kone, B.C. Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCα in an aldosterone-sensitive manner. J. Biol. Chem. 281, 18059–18068 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. 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 

  14. Lacoste, N., Utley, R.T., Hunter, J.M., Poirier, G.G. & Cote, J. Disruptor of telomeric silencing-1 is a chromatin-specific histone H3 methyltransferase. J. Biol. Chem. 277, 30421–30424 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. San Segundo, P.A. & Roeder, G.S. Role for the silencing protein Dot1 in meiotic checkpoint control. Mol. Biol. Cell 11, 3601–3615 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ng, H.H., Ciccone, D.N., Morshead, K.B., Oettinger, M.A. & Struhl, K. Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: a potential mechanism for position-effect variegation. Proc. Natl. Acad. Sci. USA 100, 1820–1825 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Giannattasio, M., Lazzaro, F., Plevani, P. & Muzi-Falconi, M. The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1. J. Biol. Chem. 280, 9879–9886 (2005).

    Article  CAS  PubMed  Google Scholar 

  18. Wysocki, R. et al. Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol. Cell. Biol. 25, 8430–8443 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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 

  20. Katan-Khaykovich, Y. & Struhl, K. Heterochromatin formation involves changes in histone modifications over multiple cell generations. EMBO J. 24, 2138–2149 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Liang, G., Klose, R.J., Gardner, K.E. & Zhang, Y. Yeast Jhd2p is a histone H3 Lys4 trimethyl demethylase. Nat. Struct. Mol. Biol. 14, 243–245 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Tu, S. et al. Identification of histone demethylases in Saccharomyces cerevisiae. J. Biol. Chem. 282, 14262–14271 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Botuyan, M.V. et al. Structural basis for the methylation state-specific recognition of histone H4–K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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 

  26. Briggs, S.D. et al. Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418, 498 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. 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 

  28. 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 

  29. Wang, H. et al. mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression. Mol. Cell 12, 475–487 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Schneider, J. et al. Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression. Mol. Cell 19, 849–856 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Dehe, P.M. et al. Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation. J. Biol. Chem. 281, 35404–35412 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Dou, Y. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 13, 713–719 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Fingerman, I.M., Li, H.C. & Briggs, S.D. A charge-based interaction between histone H4 and Dot1 is required for H3K79 methylation and telomere silencing: identification of a new trans-histone pathway. Genes Dev. 21, 2018–2029 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Schwabish, M.A. & Struhl, K. Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 24, 10111–10117 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Altaf, M. et al. Interplay of chromatin modifiers on a short basic patch of histone H4 tail defines the boundary of telomeric heterochromatin. Mol. Cell 28, 1002–1014 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. van Leeuwen, F. & Gottschling, D.E. Assays for gene silencing in yeast. Methods Enzymol. 350, 165–186 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Rusche, L.N., Kirchmaier, A.L. & Rine, J. The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. Annu. Rev. Biochem. 72, 481–516 (2003).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Zhang, X. et al. Structural basis for the product specificity of histone lysine methyltransferases. Mol. Cell 12, 177–185 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Tamaru, H. et al. Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat. Genet. 34, 75–79 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Patnaik, D. et al. Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase. J. Biol. Chem. 279, 53248–53258 (2004).

    Article  CAS  PubMed  Google Scholar 

  42. Collins, R.E. et al. In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J. Biol. Chem. 280, 5563–5570 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Dirk, L.M. et al. Kinetic manifestation of processivity during multiple methylations catalyzed by SET domain protein methyltransferases. Biochemistry 46, 3905–3915 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Eskeland, R. et al. The N-terminus of Drosophila SU(VAR)3–9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43, 3740–3749 (2004).

    Article  CAS  PubMed  Google Scholar 

  45. Chin, H.G., Patnaik, D., Esteve, P.O., Jacobsen, S.E. & Pradhan, S. Catalytic properties and kinetic mechanism of human recombinant Lys-9 histone H3 methyltransferase SUV39H1: participation of the chromodomain in enzymatic catalysis. Biochemistry 45, 3272–3284 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. McBride, A.E. & Silver, P.A. State of the Arg: protein methylation at arginine comes of age. Cell 106, 5–8 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Janzen, C.J., Hake, S.B., Lowell, J.E. & Cross, G.A. Selective di- or trimethylation of histone H3 lysine 76 by two DOT1 homologs is important for cell cycle regulation in Trypanosoma brucei. Mol. Cell 23, 497–507 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  50. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Krijgsveld, J., Gauci, S., Dormeyer, W. & Heck, A.J. In-gel isoelectric focusing of peptides as a tool for improved protein identification. J. Proteome Res. 5, 1721–1730 (2006).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Gottschling (Fred Hutchinson Cancer Research Center) and R. Gardner (University of Washington) for plasmids and reagents, H. Hilkmann for peptide synthesis, K. Verzijlbergen (The Netherlands Cancer Institute (NKI)) for the antibody against the C terminus of histone H3 and E. Battaglia for help with initial experiments. We thank X. Cheng, D. Gottschling, P. Borst, T. Sixma, B. van Steensel and members of the van Leeuwen laboratory for critical reading of the manuscript and helpful discussions. F.v.L. was a Special Fellow of the Leukemia and Lymphoma Society and was supported by NOE The Epigenome of the EU 6th framework program. This work was supported by the Netherlands Proteomics Centre.

Author information

Authors and Affiliations

  1. Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066, CX, The Netherlands

    Floor Frederiks, Tibor van Welsem & Fred van Leeuwen

  2. Department of Biomolecular Mass Spectrometry, Faculty of Science, Utrecht University, Sorbonnelaan 16, Utrecht, 3584, CA, The Netherlands

    Manuel Tzouros, Gideon Oudgenoeg & Jeroen Krijgsveld

  3. Division of Tumor Biology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066, CX, The Netherlands

    Maarten Fornerod

Authors
  1. Floor Frederiks
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manuel Tzouros
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gideon Oudgenoeg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tibor van Welsem
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maarten Fornerod
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeroen Krijgsveld
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fred van Leeuwen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

F.F. was responsible for strain constructions, sample preparations, western blots, nonradioactive in vitro assays, silencing assays, DNA-damage checkpoint assays and antibody generation and characterization; M.T. and G.O. developed and performed MS analyses under supervision of J.K.; T.v.W. performed the initial characterization of the Dot1-G401A mutant; M.F. developed the binomial distributive methylation model; F.v.L. devised and supervised the project; F.F. and F.v.L. wrote the paper.

Corresponding author

Correspondence to Fred van Leeuwen.

Supplementary information

Supplementary Text and Figures

Supplementary Figues 1–5 and Supplementary Table 1 (PDF 1094 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Frederiks, F., Tzouros, M., Oudgenoeg, G. et al. Nonprocessive methylation by Dot1 leads to functional redundancy of histone H3K79 methylation states. Nat Struct Mol Biol 15, 550–557 (2008). https://doi.org/10.1038/nsmb.1432

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1432

This article is cited by

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