Structural basis of histone H2A–H2B recognition by the essential chaperone FACT

Abstract

Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair1,2,3,4,5,6. Key to the broad functions of FACT is its recognition of histones H2A–H2B (ref. 2). However, the structural basis for how histones H2A–H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3–H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A–H2B heterodimer. A novel ‘U-turn’ motif scaffolded onto a Rtt106-like module7,8,9,10 embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3–H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance ‘nucleosome breathing’ by re-organizing the first 30 base pairs of nucleosomal histone–DNA contacts. Our snapshot of the engagement of the chaperone with H2A–H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The histone chaperone complex FACT recognizes the histone H2A-H2B heterodimer through the Spt16M domain of Spt16.
Figure 2: A conserved, hydrophobic groove in the U-turn motif of Spt16M interacts with a hydrophobic patch of H2B.
Figure 3: Multiple interactions support histone binding by FACT, but Spt16M-mediated contacts are key to chaperoning function.
Figure 4: The heterodimerization domain of FACT mediates interaction with the DNA replication machinery.

Accession codes

Accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession codes 4KHA (Spt16M–H2A–H2B), 4KHO (Spt16M) and 4KHB (Spt16D–Pob3N).

References

  1. 1

    Orphanides, G., Wu, W. H., Lane, W. S., Hampsey, M. & Reinberg, D. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400, 284–288 (1999)

  2. 2

    Belotserkovskaya, R. et al. FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093 (2003)

  3. 3

    Mason, P. B. & Struhl, K. The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo. Mol. Cell. Biol. 23, 8323–8333 (2003)

  4. 4

    Wittmeyer, J., Joss, L. & Formosa, T. Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha. Biochemistry 38, 8961–8971 (1999)

  5. 5

    Kaplan, C. D., Laprade, L. & Winston, F. Transcription elongation factors repress transcription initiation from cryptic sites. Science 301, 1096–1099 (2003)

  6. 6

    Lejeune, E. et al. The chromatin-remodeling factor FACT contributes to centromeric heterochromatin independently of RNAi. Curr. Biol. 17, 1219–1224 (2007)

  7. 7

    Su, D. et al. Structural basis for recognition of H3K56-acetylated histone H3–H4 by the chaperone Rtt106. Nature 483, 104–107 (2012)

  8. 8

    Zunder, R. M., Antczak, A. J., Berger, J. M. & Rine, J. Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing. Proc. Natl Acad. Sci. USA 109, E144–E153 (2012)

  9. 9

    Kemble, D. J. et al. Structure of the Spt16 middle domain reveals functional features of the histone chaperone FACT. J. Biol. Chem. 288, 10188–10194 (2013)

  10. 10

    Liu, Y. et al. Structural analysis of Rtt106p reveals a DNA binding role required for heterochromatin silencing. J. Biol. Chem. 285, 4251–4262 (2010)

  11. 11

    Orphanides, G., LeRoy, G., Chang, C. H., Luse, D. S. & Reinberg, D. FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92, 105–116 (1998)

  12. 12

    VanDemark, A. P. et al. The structure of the yFACT Pob3-M domain, its interaction with the DNA replication factor RPA, and a potential role in nucleosome deposition. Mol. Cell 22, 363–374 (2006)

  13. 13

    Stuwe, T. et al. The FACT Spt16 ‘peptidase’ domain is a histone H3–H4 binding module. Proc. Natl Acad. Sci. USA 105, 8884–8889 (2008)

  14. 14

    VanDemark, A. P. et al. Structural and functional analysis of the Spt16p N-terminal domain reveals overlapping roles of yFACT subunits. J. Biol. Chem. 283, 5058–5068 (2008)

  15. 15

    Winkler, D. D., Muthurajan, U. M., Hieb, A. R. & Luger, K. Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events. J. Biol. Chem. 286, 41883–41892 (2011)

  16. 16

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

  17. 17

    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, 57–66 (2008)

  18. 18

    Koopmans, W. J. A., Buning, R., Schmidt, T. & van Noort, J. spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes. Biophys. J. 97, 195–204 (2009)

  19. 19

    Andrews, A. J., Chen, X., Zevin, A., Stargell, L. A. & Luger, K. The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. Mol. Cell 37, 834–842 (2010)

  20. 20

    Cho, U.-S. & Harrison, S. C. Recognition of the centromere-specific histone Cse4 by the chaperone Scm3. Proc. Natl Acad. Sci. USA 108, 9367–9371 (2011)

  21. 21

    Hu, H. et al. Structure of a CENP-A-histone H4 heterodimer in complex with chaperone HJURP. Genes Dev. 25, 901–906 (2011)

  22. 22

    Hondele, M. & Ladurner, A. G. The chaperone-histone partnership: for the greater good of histone traffic and chromatin plasticity. Curr. Opin. Struct. Biol. 21, 698–708 (2011)

  23. 23

    Zhou, Z. et al. NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B. Nature Struct. Mol. Biol. 15, 868–869 (2008)

  24. 24

    Luger, K., Mäder, 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)

  25. 25

    Killian, J. L., Li, M., Sheinin, M. Y. & Wang, M. D. Recent advances in single molecule studies of nucleosomes. Curr. Opin. Struct. Biol. 22, 80–87 (2012)

  26. 26

    Hall, M. A. et al. High-resolution dynamic mapping of histone-DNA interactions in a nucleosome. Nature Struct. Mol. Biol. 16, 124–129 (2009)

  27. 27

    Xin, H. et al. yFACT induces global accessibility of nucleosomal DNA without H2A–H2B displacement. Mol. Cell 35, 365–376 (2009)

  28. 28

    Hsieh, F.-K. et al. Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc. Natl Acad. Sci.. USA http://dx.doi.org/10.1073/pnas.1222198110 (22 April 2013)

  29. 29

    Bondarenko, V. A. et al. Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol. Cell 24, 469–479 (2006)

  30. 30

    Kulaeva, O. I. et al. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nature Struct. Mol. Biol. 16, 1272–1278 (2009)

  31. 31

    Dyer, P. N. et al. Reconstitution of nucleosome core particles from recombinant histones and DNA. Methods Enzymol. 375, 23–44 (2003)

  32. 32

    Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

  33. 33

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  34. 34

    Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  35. 35

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  36. 36

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

  37. 37

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  38. 38

    Langer, G., Cohen, S. X., Lamzin, V. S. & Perrakis, A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171–1179 (2008)

  39. 39

    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)

  40. 40

    Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

  41. 41

    Sumathi, K., Ananthalakshmi, P., Roshan, M. N. A. M. & Sekar, K. 3dSS: 3D structural superposition. Nucleic Acids Res. 34, W128–W132 (2006)

  42. 42

    Capozzo, C. et al. Gene disruption and basic phenotypic analysis of nine novel yeast genes from chromosome XIV. Yeast 16, 1089–1097 (2000)

Download references

Acknowledgements

We thank J. Basquin, E. Conti, the MPI for Biochemistry and staff at beamlines Swiss Light Source PXII and European Synchrotron Radiation Facility ID23 for crystallographic support, P. Becker, S. Hake, J. Müller and G. Schotta for H3 peptides, and F. Bonneau, P. Cramer, T. Gibson, D. Gilmour, J. Griesenbeck, C. Häring, M. Hothorn, G. Jankevicius, D. Mokranjac, R. Russell, I. Schäfer, K. Scheffzek, C. Schultz, F. Wieland, M. Winter and E. Wolf for discussion. EMBL, LMU Munich, EC FP6 Marie Curie RTN Chromatin Plasticity (to A.G.L.) and Boehringer Ingelheim Fonds (to M.Ho. and F.H.) funded this research.

Author information

Affiliations

Authors

Contributions

Crystallography on Spt16M–H2A–H2B was conducted by M.Ho., M.Ha. and F.H.; T.S. determined the structure of free Spt16M and Spt16D–Pob3N, with assistance from M.Ho. and E.T.Z.; M.Ho. and T.S. conducted biochemical assays; A.B. conducted the chaperoning assay; M.Ho., T.S. and B.N. purified proteins; M.Ho., C.K. and T.S. conducted yeast work; M.Ho. and V.R. carried out ITC; S.A. and E.H. provided C. thermophilum cDNA sequences; M.Ho., T.S., M.Ha., A.B. and A.G.L. designed the study; M.Ha. and A.G.L. supervised the work; M.H.o, M.Ha., A.B. and A.G.L. wrote the manuscript.

Corresponding author

Correspondence to Andreas G. Ladurner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-14 and Supplementary Tables 1-3. (PDF 7699 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hondele, M., Stuwe, T., Hassler, M. et al. Structural basis of histone H2A–H2B recognition by the essential chaperone FACT. Nature 499, 111–114 (2013). https://doi.org/10.1038/nature12242

Download citation

Further reading

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.