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

Nature volume 457, pages 57–62 (2009)Cite this article

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.

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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.

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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.

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Authors and Affiliations

  1. Laboratory of Chromatin Biology, The Rockefeller University, New York, New York 10065, USA ,

    Andrew Xiao, David Shechter, Sung Hee Ahn & C. David Allis

  2. Structural Biology Program,,

    Haitao Li, Satoko Ishibe-Murakami & Dinshaw J. Patel

  3. Molecular Biology Program, Memorial-Sloan-Kettering Cancer Center, New York, New York 10065, USA ,

    Laura A. Fabrizio, Hediye Erdjument-Bromage & Paul Tempst

  4. Department of Genetics, Howard Hughes Medical Institute, Harvard Partners Center for Genetics and Genomics, Harvard Medical School, Boston, Massachusetts 02115, USA,

    Bin Wang & Stephen J. Elledge

  5. Miltenyi Biotec GmbH, 50829 Koeln, Germany

    Kay Hofmann

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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

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