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:

WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity

Abstract

DNA double-stranded breaks present a serious challenge for eukaryotic cells. The inability to repair breaks leads to genomic instability, carcinogenesis and cell death. During the double-strand break response, mammalian chromatin undergoes reorganization demarcated by H2A.X Ser 139 phosphorylation (γ-H2A.X). However, the regulation of γ-H2A.X phosphorylation and its precise role in chromatin remodelling during the repair process remain unclear. Here we report a new regulatory mechanism mediated by WSTF (Williams–Beuren syndrome transcription factor, also known as BAZ1B)—a component of the WICH complex (WSTF–ISWI ATP-dependent chromatin-remodelling complex). We show that WSTF has intrinsic tyrosine kinase activity by means of a domain that shares no sequence homology to any known kinase fold. We show that WSTF phosphorylates Tyr 142 of H2A.X, and that WSTF activity has an important role in regulating several events that are critical for the DNA damage response. Our work demonstrates a new mechanism that regulates the DNA damage response and expands our knowledge of domains that contain intrinsic tyrosine kinase activity.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Tyr 142 of H2A.X is a new phosphorylation mark regulated by DNA damage signals.
Figure 2: The WSTF–SNF2H chromatin remodelling complex is specifically associated with H2A.X nucleosomes in vivo.
Figure 3: WSTF contains a kinase domain that phosphorylates Tyr 142 of H2A.X.
Figure 4: WSTF is critical for the maintenance of γ-H2A.X phosphorylation after DNA damage.

Similar content being viewed by others

References

  1. Khanna, K. K. & Jackson, S. P. DNA double-strand breaks: signaling, repair and the cancer connection. Nature Genet. 27, 247–254 (2001)

    Article  CAS  Google Scholar 

  2. Zhou, B. B. & Elledge, S. J. The DNA damage response: putting checkpoints in perspective. Nature 408, 433–439 (2000)

    Article  ADS  CAS  Google Scholar 

  3. Redon, C. et al. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12, 162–169 (2002)

    Article  CAS  Google Scholar 

  4. Celeste, A. et al. Genomic instability in mice lacking histone H2AX. Science 296, 922–927 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Celeste, A. et al. H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell 114, 371–383 (2003)

    Article  CAS  Google Scholar 

  6. Reina-San-Martin, B. et al. H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197, 1767–1778 (2003)

    Article  CAS  Google Scholar 

  7. Bassing, C. H. et al. Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors. Cell 114, 359–370 (2003)

    Article  CAS  Google Scholar 

  8. Unal, E. et al. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell 16, 991–1002 (2004)

    Article  Google Scholar 

  9. Harper, J. W. & Elledge, S. J. The DNA damage response: ten years after. Mol. Cell 28, 739–745 (2007)

    Article  CAS  Google Scholar 

  10. Morrison, A. J. et al. INO80 and γ-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119, 767–775 (2004)

    Article  CAS  Google Scholar 

  11. Downs, J. A. et al. Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol. Cell 16, 979–990 (2004)

    Article  CAS  Google Scholar 

  12. van Attikum, H., Fritsch, O., Hohn, B. & Gasser, S. M. Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119, 777–788 (2004)

    Article  CAS  Google Scholar 

  13. Park, J. H. et al. Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting γ-H2AX induction. EMBO J. 25, 3986–3997 (2006)

    Article  CAS  Google Scholar 

  14. Stewart, G. S., Wang, B., Bignell, C. R., Taylor, A. M. & Elledge, S. J. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966 (2003)

    Article  ADS  CAS  Google Scholar 

  15. Goldberg, M. et al. MDC1 is required for the intra-S-phase DNA damage checkpoint. Nature 421, 952–956 (2003)

    Article  ADS  CAS  Google Scholar 

  16. Lou, Z., Minter-Dykhouse, K., Wu, X. & Chen, J. MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways. Nature 421, 957–961 (2003)

    Article  ADS  CAS  Google Scholar 

  17. Stucki, M. et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226 (2005)

    Article  CAS  Google Scholar 

  18. Lou, Z. et al. MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 21, 187–200 (2006)

    Article  CAS  Google Scholar 

  19. Lee, M. S., Edwards, R. A., Thede, G. L. & Glover, J. N. Structure of the BRCT repeat domain of MDC1 and its specificity for the free COOH-terminal end of the γ-H2AX histone tail. J. Biol. Chem. 280, 32053–32056 (2005)

    Article  CAS  Google Scholar 

  20. Dimitrov, S., Dasso, M. C. & Wolffe, A. P. Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly. J. Cell Biol. 126, 591–601 (1994)

    Article  CAS  Google Scholar 

  21. Kleinschmidt, J. A. & Steinbeisser, H. DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing. EMBO J. 10, 3043–3050 (1991)

    Article  CAS  Google Scholar 

  22. Mendez, J. & Stillman, B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000)

    Article  CAS  Google Scholar 

  23. Fyodorov, D. V. & Kadonaga, J. T. The many faces of chromatin remodeling: SWItching beyond transcription. Cell 106, 523–525 (2001)

    Article  CAS  Google Scholar 

  24. Becker, P. B. & Horz, W. ATP-dependent nucleosome remodeling. Annu. Rev. Biochem. 71, 247–273 (2002)

    Article  CAS  Google Scholar 

  25. 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  Google Scholar 

  26. Poot, R. A. et al. HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J. 19, 3377–3387 (2000)

    Article  CAS  Google Scholar 

  27. Jones, M. H., Hamana, N., Nezu, J. & Shimane, M. A novel family of bromodomain genes. Genomics 63, 40–45 (2000)

    Article  CAS  Google Scholar 

  28. Poot, R. A. et al. The Williams syndrome transcription factor interacts with PCNA to target chromatin remodelling by ISWI to replication foci. Nature Cell Biol. 6, 1236–1244 (2004)

    Article  CAS  Google Scholar 

  29. Bozhenok, L., Wade, P. A. & Varga-Weisz, P. WSTF-ISWI chromatin remodeling complex targets heterochromatic replication foci. EMBO J. 21, 2231–2241 (2002)

    Article  CAS  Google Scholar 

  30. Kukimoto, I., Elderkin, S., Grimaldi, M., Oelgeschlager, T. & Varga-Weisz, P. D. The histone-fold protein complex CHRAC-15/17 enhances nucleosome sliding and assembly mediated by ACF. Mol. Cell 13, 265–277 (2004)

    Article  CAS  Google Scholar 

  31. Stopka, T. & Skoultchi, A. I. The ISWI ATPase Snf2h is required for early mouse development. Proc. Natl Acad. Sci. USA 100, 14097–14102 (2003)

    Article  ADS  CAS  Google Scholar 

  32. Dellaire, G. & Bazett-Jones, D. P. Beyond repair foci: subnuclear domains and the cellular response to DNA damage. Cell cycle 6, 1864–1872 (2007)

    Article  CAS  Google Scholar 

  33. Bakkenist, C. J. & Kastan, M. B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003)

    Article  ADS  CAS  Google Scholar 

  34. Lee, J. H. & Paull, T. T. ATM activation by DNA double-strand breaks through the Mre11–Rad50–Nbs1 complex. Science 308, 551–554 (2005)

    Article  ADS  CAS  Google Scholar 

  35. Lee, J. H. & Paull, T. T. Direct activation of the ATM protein kinase by the Mre11/Rad50/Nbs1 complex. Science 304, 93–96 (2004)

    Article  ADS  CAS  Google Scholar 

  36. Francke, U. Williams–Beuren syndrome: genes and mechanisms. Hum. Mol. Genet. 8, 1947–1954 (1999)

    Article  CAS  Google Scholar 

  37. Pear, W., Scott, M. & Nolan, G. P. in Methods in Molecular Medicine: Gene Therapy Protocols, 41–57 (Humana, 1997)

    Google Scholar 

  38. Bellard, M., Dretzen, G., Giangrande, A. & Ramain, P. Nuclease digestion of transcriptionally active chromatin. Methods Enzymol. 170, 317–346 (1989)

    Article  CAS  Google Scholar 

  39. Erdjument-Bromage, H. et al. Examination of micro-tip reversed-phase liquid chromatographic extraction of peptide pools for mass spectrometric analysis. J. Chromatogr. A. 826, 167–181 (1998)

    Article  CAS  Google Scholar 

  40. Sebastiaan Winkler, G. et al. Isolation and mass spectrometry of transcription factor complexes. Methods 26, 260–269 (2002)

    Article  CAS  Google Scholar 

  41. Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999)

    Article  CAS  Google Scholar 

  42. Lennox, R. W. & Cohen, L. H. Analysis of histone subtypes and their modified forms by polyacrylamide gel electrophoresis. Methods Enzymol. 170, 532–549 (1989)

    Article  CAS  Google Scholar 

  43. Sambrook, J. & Russell, D. W. Molecular Cloning: a Laboratory Manual (Cold Spring Harbour Laboratory Press, 2001)

    Google Scholar 

  44. Wysocka, J. et al. A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature 442, 86–90 (2006)

    Article  ADS  CAS  Google Scholar 

  45. Tempst, P., Geromanos, S., Elicone, C. & Erdjument-Bromage, H. Improvements in microsequencer performance for low picomole sequence analysis. Methods 6, 248–261 (1994)

    Article  CAS  Google Scholar 

  46. Luger, K., Rechsteiner, T. J. & Richmond, T. J. Preparation of nucleosome core particle from recombinant histones. Methods Enzymol. 304, 3–19 (1999)

    Article  CAS  Google Scholar 

  47. Steger, D. J., Eberharter, A., John, S., Grant, P. A. & Workman, J. L. Purified histone acetyltransferase complexes stimulate HIV-1 transcription from preassembled nucleosomal arrays. Proc. Natl Acad. Sci. USA 95, 12924–12929 (1998)

    Article  ADS  CAS  Google Scholar 

  48. 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  Google Scholar 

Download references

Acknowledgements

We thank A. Nussenzweig for H2A.X-/- MEF cells, P. Varga-Weisz for WSTF constructs and antibodies, W. Herr for pBABE-puro vectors, J. Kim and R. Roeder for anti-BAF53 antibodies, D. Reinberg for anti-SNF2H antibodies, Z. Lou for anti-Mdc1 antibodies and Mdc1-/- MEF cells, L. Liang and Q. Li for their assistance in recombinant protein expression and purification, C. H. McDonald, R. G. Cook and The Rockefeller University Proteomic Core facility for H2A.X peptides. We would also like to thank the Millipore antibody development scientists for collaborating with us on the generation of H2A.X Tyr 142(ph) antibodies, catalogue number 07-1590. This study was supported by the following sources: Susan G. Komen Breast Cancer Foundation (A.X.), Abby Rockefeller Mauze Trust and Starr Foundation (H.L. and D.J.P.), The Dewitt Wallace and Maloris Foundations (H.L. and D.J.P.), The Irma T. Hirschl Trust (D.S.), NCI Cancer Center Support Grant P30 CA08748 (L.A.F., H.E.-B. and P.T.), research grants from National Institutes of Health to S.J.E., S.H.A. and C.D.A., and The Rockefeller University (C.D.A.). S.J.E. is an Investigator with the Howard Hughes Medical Institute. We are grateful to E. Bernstein and E. Duncan for critical reading of the manuscript.

Author Contributions A.X. designed the study, performed the experiments and wrote the paper; H.L. generated recombinant WSTF protein and performed CD analysis; D.S. helped with the experiments performed in Xenopus egg extracts and edited the manuscript; S.H.A. generated and analysed H2A Leu132Tyr mutant yeast strain; L.A.F., H.E.-B. and P.T. performed MS analysis; S.I.-M. provided technical assistance for protein production; B.W. and S.J.E. provided Mdc1 constructs; K.H. performed bioinformatical analysis on the WSTF kinase domain; D.J.P. provided general guidance for generating recombinant WSTF. S.J.E. also discussed results and commented on the manuscript. C.D.A. provided support and general guidance for this work.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to C. David Allis.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary References, and Supplementary Figures S1-S10 with Legends (PDF 1067 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xiao, A., Li, H., Shechter, D. et al. WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity. Nature 457, 57–62 (2009). https://doi.org/10.1038/nature07668

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07668

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

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