Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction

Journal name:
Nature Chemical Biology
Volume:
7,
Pages:
113–119
Year published:
DOI:
doi:10.1038/nchembio.501
Received
Accepted
Published online

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.

At a glance

Figures

  1. H2B ubiquitylation impairs fiber folding.
    Figure 1: H2B ubiquitylation impairs fiber folding.

    (a) Ubiquitylated H2B appears incompatible with nucleosome stacking. Upper panel: model of a 30-nm chromatin fiber (1ZBB, ref. 9). Lower panel: the structure of a tetranucleosome unit extracted from the fiber and rotated by 38.1o (ref. 9). The sites of ubiquitin attachment are shown in red, whereas the fluorescein labeling site is shown in green. (b) Sedimentation coefficient distributions for unmodified and uH2BSS containing chromatin arrays (black and red symbols) are determined by sedimentation velocity experiments and van Holde–Weischet analysis at 0 and 1 mM Mg2+ (solid and open symbols). Inset: ubiquitin is attached via disulfide-based coupling chemistry26. (c) S20,w values of unmodified (black) and uH2BSS containing arrays (red) are shown as a function of Mg2+ concentration. Error bars, s.d. (n = 3).

  2. H2B ubiquitylation increases chromatin accessibility in vitro and in vivo.
    Figure 2: H2B ubiquitylation increases chromatin accessibility in vitro and in vivo.

    (ac), Unmodified arrays (ar) (lane 1) and mononucleosomes (mn) (lane 2) or H2B ubiquitylated arrays (lane 3) and mononucleosomes (lane 4) were used as substrates for hDot1L methyltransferase assays with 3H-SAM at 1 mM Mg2+. Histones were separated by SDS-PAGE (a), stained with Coomassie Brilliant Blue and (b) probed for 3H-methyl incorporation by fluorography. (c) Quantification of methylation was performed by p81 filter binding and liquid scintillation counting. Student's two-tailed t-test: *, P = 0.0001; Error bars, s.e.m. (n = 9); c.p.m., counts per minute. The fluorography represents two different exposures of the film: 24 h, left panel, and 3 h, right panel. For verification of the modification site and full gels, see Supplementary Figure 3. (d–g) Chromatin from micrococcal nuclease digested nuclei of NIH/3T3 fibroblasts was successively extracted with increasing concentrations of NaCl. (d) DNA purified from MNase digested nuclei (lane 1), from the supernatant after centrifugation of the nuclei (lane 2), from 80, 150 and 600 mM salt extracted chromatin (lanes 3–5) and from the insoluble pellet (lane 6) was separated on an agarose gel and stained with ethidium bromide. Histones were acid extracted, and equal amounts were separated by SDS-PAGE and either (e) stained with Coomassie Brilliant Blue or (f) analyzed by western blotting with antibodies against uH2B and H4 K16ac. For full gels see Supplementary Figure 3. (g) uH2B and H4 K16ac levels from two independent experiments were quantified by densitometry. Error bars, s.e.m. (n = 2).

  3. A fluorescence method to monitor chromatin fiber folding reveals conformational heterogeneity at intermediate chromatin fiber compaction.
    Figure 3: A fluorescence method to monitor chromatin fiber folding reveals conformational heterogeneity at intermediate chromatin fiber compaction.

    (a) Compaction upon addition of divalent cations results in a loss of fluorescence steady-state anisotropy (SSA) because of internucleosomal homo-FRET between fluorescein moieties. (b) Experimental SSA data obtained from fluorescently labeled 12-mer nucleosomal arrays as a function of Mg2+ concentration. Error bars, s.d. (n = 3). (c) Representative structures of arrays in different compaction states. S20,w and SSA values are calculated from the structures as described in the supplementary information. The two middle structures share the same sedimentation constant, whereas the conformationally heterogenous chain configuration (red) leads to more FRET than the conformationally homogenous chain (blue). S, Svedberg unit. (d) Calculated SSA values from 500 randomly generated array conformations from either heterogenous (red) or homogenous (blue) chains.

  4. uH2B and acH4 have distinct effects on fiber compaction and higher-order structure formation.
    Figure 4: uH2B and acH4 have distinct effects on fiber compaction and higher-order structure formation.

    (a) Chromatin fiber compaction as a function of Mg2+ concentration was assessed by measuring SSA for nucleosomal arrays containing unmodified histones (black), uH2BSS (red), acH4 (green) or both uH2BSS and acH4 (blue). (b) Reconstituted arrays containing uH2BSS were chemically deubiquitylated by reduction of the disulfide bond with DTT. Compaction was then determined at various Mg2+ concentrations by measuring SSA (black). For comparison, the SSA traces of unmodified and ubiquitylated arrays are shown in gray and red, respectively. Error bars, s.d. (n = 2–4). (c) Chromatin fiber compaction upon Mg2+ addition was determined by measuring SSA for arrays containing increasing amounts of uH2BSS. The relative SSA change at 1 mM Mg2+ is shown as a function of uH2BSS content. The solid line is a linear fit to the data. Error bars, s.d. (n = 4–6). (d) Nucleosomal arrays containing unmodified histones (black), uH2BSS (red), acH4 (green) or both acH4 and uH2BSS (blue) were incubated in the presence of indicated concentrations of Mg2+, and oligomers were removed by centrifugation. The amount of arrays remaining in solution was determined by UV absorption. Errors bars, s.e.m. (n = 2–4).

  5. Fiber decompaction is a specific property of the ubiquitin protein.
    Figure 5: Fiber decompaction is a specific property of the ubiquitin protein.

    (a) Surface rendering of the X-ray structure of ubiquitin (PDB: 1UBQ, left)49 and the NMR structure of Hub1 (PDB: 1M94, right, including the C-terminal RGG residues)50. The color code shows hydrophobic (gray), hydrophilic (green), positively charged (blue) and negatively charged (red) residues. (b) Compaction behavior of nucleosomal arrays containing unmodified histones (gray), uH2BSS (red) or hub1-H2BSS in the absence (yellow) or presence (black) of DTT was determined by SSA. Error bars, s.d. (n = 2–4). (c) Oligomerization of nucleosomal arrays containing unmodified histones (gray), uH2BSS (red) or hub1-H2BSS (yellow) was assessed by determining Mg2+-dependent solubility. Error bars, s.e.m. (n = 3–4). (d) A model of the distinct effects of uH2B and acH4 on fiber folding (for details see text).

Accession codes

Referenced accessions

Protein Data Bank

References

  1. Cui, Y. & Bustamante, C. Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure. Proc. Natl. Acad. Sci. USA 97, 127132 (2000).
  2. Poirier, M.G., Bussiek, M., Langowski, J. & Widom, J. Spontaneous access to DNA target sites in folded chromatin fibers. J. Mol. Biol. 379, 772786 (2008).
  3. Kruithof, M. et al. Single-molecule force spectroscopy reveals a highly compliant helical folding for the 30-nm chromatin fiber. Nat. Struct. Mol. Biol. 16, 534540 (2009).
  4. Poirier, M.G., Oh, E., Tims, H.S. & Widom, J. Dynamics and function of compact nucleosome arrays. Nat. Struct. Mol. Biol. 16, 938944 (2009).
  5. Hansen, J.C. Conformational dynamics of the chromatin fiber in solution: determinants, mechanisms, and functions. Annu. Rev. Biophys. Biomol. Struct. 31, 361392 (2002).
  6. 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, 251260 (1997).
  7. Dorigo, B., Schalch, T., Bystricky, K. & Richmond, T.J. Chromatin fiber folding: requirement for the histone H4 N-terminal tail. J. Mol. Biol. 327, 8596 (2003).
  8. Chodaparambil, J.V. et al. A charged and contoured surface on the nucleosome regulates chromatin compaction. Nat. Struct. Mol. Biol. 14, 11051107 (2007).
  9. Schalch, T., Duda, S., Sargent, D.F. & Richmond, T.J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436, 138141 (2005).
  10. Strahl, B.D. & Allis, C.D. The language of covalent histone modifications. Nature 403, 4145 (2000).
  11. Shogren-Knaak, M. et al. Histone H4–K16 acetylation controls chromatin structure and protein interactions. Science 311, 844847 (2006).
  12. Robinson, P.J. et al. 30 nm chromatin fibre decompaction requires both H4–K16 acetylation and linker histone eviction. J. Mol. Biol. 381, 816825 (2008).
  13. Hilfiker, A., Hilfiker-Kleiner, D., Pannuti, A. & Lucchesi, J.C. 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, 20542060 (1997).
  14. West, M.H. & Bonner, W.M. Histone 2B can be modified by the attachment of ubiquitin. Nucleic Acids Res. 8, 46714680 (1980).
  15. Xiao, T. et al. Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol. Cell. Biol. 25, 637651 (2005).
  16. Minsky, N. et al. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat. Cell Biol. 10, 483488 (2008).
  17. Kim, J. et al. RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell 137, 459471 (2009).
  18. Zhu, B. et al. Monoubiquitination of human histone H2B: the factors involved and their roles in HOX gene regulation. Mol. Cell 20, 601611 (2005).
  19. Shema, E. 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, 26642676 (2008).
  20. Chandrasekharan, M.B., Huang, F. & Sun, Z.W. Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability. Proc. Natl. Acad. Sci. USA 106, 1668616691 (2009).
  21. Pavri, R. et al. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II. Cell 125, 703717 (2006).
  22. Fleming, A.B., Kao, C.F., Hillyer, C., Pikaart, M. & Osley, M.A. H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol. Cell 31, 5766 (2008).
  23. Robzyk, K., Recht, J. & Osley, M.A. Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, 501504 (2000).
  24. Sun, Z.W. & Allis, C.D. Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418, 104108 (2002).
  25. Sridhar, V.V. et al. Control of DNA methylation and heterochromatic silencing by histone H2B deubiquitination. Nature 447, 735738 (2007).
  26. Chatterjee, C., McGinty, R.K., Fierz, B. & Muir, T.W. Disulfide directed histone ubiquitylation reveals plasticity in hDot1L stimulation. Nat. Chem. Biol. 6, 267269 (2010).
  27. 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, 1942 (1998).
  28. McGinty, R.K., Kim, J., Chatterjee, C., Roeder, R.G. & Muir, T.W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812816 (2008).
  29. Henikoff, S., Henikoff, J.G., Sakai, A., Loeb, G.B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460469 (2009).
  30. Nickel, B.E., Allis, C.D. & Davie, J.R. Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin. Biochemistry 28, 958963 (1989).
  31. Delcuve, G.P. & Davie, J.R. Chromatin structure of erythroid-specific genes of immature and mature chicken erythrocytes. Biochem. J. 263, 179186 (1989).
  32. Runnels, L.W. & Scarlata, S.F. Theory and application of fluorescence homotransfer to melittin oligomerization. Biophys. J. 69, 15691583 (1995).
  33. Bergström, F., Hagglof, P., Karolin, J., Ny, T. & Johansson, L.B. 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, 1247712481 (1999).
  34. Gautier, I. et al. Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys. J. 80, 30003008 (2001).
  35. Thaler, C., Koushik, S.V., Puhl, H.L. III, Blank, P.S. & Vogel, S.S. Structural rearrangement of CaMKIIalpha catalytic domains encodes activation. Proc. Natl. Acad. Sci. USA 106, 63696374 (2009).
  36. Kawski, A. Excitation energy transfer and its manifestation in isotropic media. Photochem. Photobiol. 38, 487508 (1983).
  37. Woodcock, C.L., Grigoryev, S.A., Horowitz, R.A. & Whitaker, N. A chromatin folding model that incorporates linker variability generates fibers resembling the native structures. Proc. Natl. Acad. Sci. USA 90, 90219025 (1993).
  38. Knox, R. Theory of polarization quenching by excitation transfer. Physica 39, 361386 (1968).
  39. Woodcock, C.L. & Horowitz, R.A. Chromatin organization re-viewed. Trends Cell Biol. 5, 272277 (1995).
  40. Kurdistani, S.K., Tavazoie, S. & Grunstein, M. Mapping global histone acetylation patterns to gene expression. Cell 117, 721733 (2004).
  41. Koch, C.M. et al. The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res. 17, 691707 (2007).
  42. Dittmar, G.A., Wilkinson, C.R., Jedrzejewski, P.T. & Finley, D. Role of a ubiquitin-like modification in polarized morphogenesis. Science 295, 24422446 (2002).
  43. 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, 10251040 (2007).
  44. Lu, X. et al. The effect of H3K79 dimethylation and H4K20 trimethylation on nucleosome and chromatin structure. Nat. Struct. Mol. Biol. 15, 11221124 (2008).
  45. Jason, L.J.M., Moore, S.C., Ausio, J. & Lindsey, G. Magnesium-dependent association and folding of oligonucleosomes reconstituted with ubiquitinated H2A. J. Biol. Chem. 276, 1459714601 (2001).
  46. Demeler, B. UltraScan version 9.9 rev 863. A Comprehensive Data Analysis Software Package for Analytical Ultracentrifugation Experiments (http://www.ultrascan.uthscsa.edu, The University of Texas Health Science Center at San Antonio, Department of Biochemistry, 2009).
  47. Demeler, B. & van Holde, K.E. Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279288 (2004).
  48. McGinty, R.K. et al. Structure activity analysis of semisynthetic nucleosomes: Mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958968 (2009).
  49. Vijay-Kumar, S., Bugg, C.E. & Cook, W.J. Structure of ubiquitin refined at 1.8 A resolution. J. Mol. Biol. 194, 531544 (1987).
  50. Ramelot, T.A. et al. Solution structure of the yeast ubiquitin-like modifier protein Hub1. J. Struct. Funct. Genomics 4, 2530 (2003).

Download references

Author information

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

    • Champak Chatterjee

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 financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (1.5M)

    Supplementary Methods and Supplementary Figures 1–11

Additional data