Article | Published:

Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Nature Chemical Biology volume 7, pages 113119 (2011) | Download Citation

Abstract

Regulation of chromatin structure involves histone posttranslational modifications that can modulate intrinsic properties of the chromatin fiber to change the chromatin state. We used chemically defined nucleosome arrays to demonstrate that H2B ubiquitylation (uH2B), a modification associated with transcription, interferes with chromatin compaction and leads to an open and biochemically accessible fiber conformation. Notably, these effects were specific for ubiquitin, as compaction of chromatin modified with a similar ubiquitin-sized protein, Hub1, was only weakly affected. Applying a fluorescence-based method, we found that uH2B acts through a mechanism distinct from H4 tail acetylation, a modification known to disrupt chromatin folding. Finally, incorporation of both uH2B and acetylated H4 resulted in synergistic inhibition of higher-order chromatin structure formation, possibly a result of their distinct modes of action.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    & Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure. Proc. Natl. Acad. Sci. USA 97, 127–132 (2000).

  2. 2.

    , , & Spontaneous access to DNA target sites in folded chromatin fibers. J. Mol. Biol. 379, 772–786 (2008).

  3. 3.

    et al. Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber. Nat. Struct. Mol. Biol. 16, 534–540 (2009).

  4. 4.

    , , & Dynamics and function of compact nucleosome arrays. Nat. Struct. Mol. Biol. 16, 938–944 (2009).

  5. 5.

    Conformational dynamics of the chromatin fiber in solution: determinants, mechanisms, and functions. Annu. Rev. Biophys. Biomol. Struct. 31, 361–392 (2002).

  6. 6.

    , , , & Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251–260 (1997).

  7. 7.

    , , & Chromatin fiber folding: requirement for the histone H4 N-terminal tail. J. Mol. Biol. 327, 85–96 (2003).

  8. 8.

    et al. A charged and contoured surface on the nucleosome regulates chromatin compaction. Nat. Struct. Mol. Biol. 14, 1105–1107 (2007).

  9. 9.

    , , & X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436, 138–141 (2005).

  10. 10.

    & The language of covalent histone modifications. Nature 403, 41–45 (2000).

  11. 11.

    et al. Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311, 844–847 (2006).

  12. 12.

    et al. 30 nm chromatin fibre decompaction requires both H4–K16 acetylation and linker histone eviction. J. Mol. Biol. 381, 816–825 (2008).

  13. 13.

    , , & mof, a putative acetyl transferase gene related to the Tip60 and MOZ human genes and to the SAS genes of yeast, is required for dosage compensation in Drosophila. EMBO J. 16, 2054–2060 (1997).

  14. 14.

    & Histone 2B can be modified by the attachment of ubiquitin. Nucleic Acids Res. 8, 4671–4680 (1980).

  15. 15.

    et al. Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol. Cell. Biol. 25, 637–651 (2005).

  16. 16.

    et al. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat. Cell Biol. 10, 483–488 (2008).

  17. 17.

    et al. RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell 137, 459–471 (2009).

  18. 18.

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

  19. 19.

    et al. The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression. Genes Dev. 22, 2664–2676 (2008).

  20. 20.

    , & Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability. Proc. Natl. Acad. Sci. USA 106, 16686–16691 (2009).

  21. 21.

    et al. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 125, 703–717 (2006).

  22. 22.

    , , , & H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol. Cell 31, 57–66 (2008).

  23. 23.

    , & Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, 501–504 (2000).

  24. 24.

    & Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104–108 (2002).

  25. 25.

    et al. Control of DNA methylation and heterochromatic silencing by histone H2B deubiquitination. Nature 447, 735–738 (2007).

  26. 26.

    , , & Disulfide directed histone ubiquitylation reveals plasticity in hDot1L stimulation. Nat. Chem. Biol. 6, 267–269 (2010).

  27. 27.

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

  28. 28.

    , , , & Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008).

  29. 29.

    , , , & Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460–469 (2009).

  30. 30.

    , & Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin. Biochemistry 28, 958–963 (1989).

  31. 31.

    & Chromatin structure of erythroid-specific genes of immature and mature chicken erythrocytes. Biochem. J. 263, 179–186 (1989).

  32. 32.

    & Theory and application of fluorescence homotransfer to melittin oligomerization. Biophys. J. 69, 1569–1583 (1995).

  33. 33.

    , , , & The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins. Proc. Natl. Acad. Sci. USA 96, 12477–12481 (1999).

  34. 34.

    et al. Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys. J. 80, 3000–3008 (2001).

  35. 35.

    , , , & Structural rearrangement of CaMKIIalpha catalytic domains encodes activation. Proc. Natl. Acad. Sci. USA 106, 6369–6374 (2009).

  36. 36.

    Excitation energy transfer and its manifestation in isotropic media. Photochem. Photobiol. 38, 487–508 (1983).

  37. 37.

    , , & A chromatin folding model that incorporates linker variability generates fibers resembling the native structures. Proc. Natl. Acad. Sci. USA 90, 9021–9025 (1993).

  38. 38.

    Theory of polarization quenching by excitation transfer. Physica 39, 361–386 (1968).

  39. 39.

    & Chromatin organization re-viewed. Trends Cell Biol. 5, 272–277 (1995).

  40. 40.

    , & Mapping global histone acetylation patterns to gene expression. Cell 117, 721–733 (2004).

  41. 41.

    et al. The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res. 17, 691–707 (2007).

  42. 42.

    , , & Role of a ubiquitin-like modification in polarized morphogenesis. Science 295, 2442–2446 (2002).

  43. 43.

    , , , & How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

  44. 44.

    et al. The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure. Nat. Struct. Mol. Biol. 15, 1122–1124 (2008).

  45. 45.

    , , & Magnesium-dependent association and folding of oligonucleosomes reconstituted with ubiquitinated H2A. J. Biol. Chem. 276, 14597–14601 (2001).

  46. 46.

    UltraScan version 9.9 rev 863. A Comprehensive Data Analysis Software Package for Analytical Ultracentrifugation Experiments (, The University of Texas Health Science Center at San Antonio, Department of Biochemistry, 2009).

  47. 47.

    & Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279–288 (2004).

  48. 48.

    et al. Structure activity analysis of semisynthetic nucleosomes: Mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958–968 (2009).

  49. 49.

    , & Structure of ubiquitin refined at 1.8 A resolution. J. Mol. Biol. 194, 531–544 (1987).

  50. 50.

    et al. Solution structure of the yeast ubiquitin-like modifier protein Hub1. J. Struct. Funct. Genomics 4, 25–30 (2003).

Download references

Acknowledgements

We thank H. Deng and H. Yu (The Rockefeller University) for mass spectrometric analysis of histones, J. Kim and R. Subramanian for assistance with preparing hDot1L, K. Chiang for help with DNA preparation, H. Yang and D. Montiel for help with fluorescence lifetime measurements and A. Ruthenburg, R. Sadeh, P. Moyle and M. Vila-Perelló for assistance with cell experiments and discussions. This work was funded by the US National Institutes of Health (grant number RC2CA148354) and the Starr Cancer Consortium. B.F. was funded by the Swiss National Science Foundation (Nr. PBBSA-118839 and PA00P3_129130/1) and by the Novartis Foundation.

Author information

Author notes

    • Champak Chatterjee

    Present address: Department of Chemistry, University of Washington, Seattle, Washington, USA

Affiliations

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

    • Beat Fierz
    • , Champak Chatterjee
    • , Robert K McGinty
    • , Maya Bar-Dagan
    •  & Tom W Muir
  2. Department of Chemistry, State University of New York Stony Brook, Stony Brook, New York, USA.

    • Daniel P Raleigh

Authors

  1. Search for Beat Fierz in:

  2. Search for Champak Chatterjee in:

  3. Search for Robert K McGinty in:

  4. Search for Maya Bar-Dagan in:

  5. Search for Daniel P Raleigh in:

  6. Search for Tom W Muir in:

Contributions

B.F. and T.W.M. designed the experiments. B.F. performed the biophysical chromatin experiments. B.F. and C.C. performed the methyltransferase assays and cell experiments. B.F., C.C., R.K.M. and M.B.-D. prepared new reagents. B.F., D.P.R. and T.W.M. analyzed the experimental data, and B.F. and T.W.M. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Tom W Muir.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Methods and Supplementary Figures 1–11

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.501