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Histone H3 Thr 45 phosphorylation is a replication-associated post-translational modification in S. cerevisiae

Abstract

Post-translational histone modifications are crucial for the regulation of numerous DNA-templated processes, and are thought to mediate both alteration of chromatin dynamics and recruitment of effector proteins to specific regions of the genome1. In particular, histone Ser/Thr phosphorylation regulates multiple nuclear functions in the budding yeast Saccharomyces cerevisiae, including transcription, DNA damage repair, mitosis, apoptosis and sporulation2. Although modifications to chromatin during replication remain poorly understood, a number of recent studies have described acetylation of the histone H3 N-terminal α-helix (αN helix) at Lys 56 as a modification that is important for maintenance of genomic integrity during DNA replication and repair3,4. Here, we report phosphorylation of H3 Thr 45 (H3-T45), a histone modification also located within the H3 αN helix in S. cerevisiae. Thr 45 phosphorylation peaks during DNA replication, and is mediated by the S phase kinase Cdc7–Dbf4 as part of a multiprotein complex identified in this study. Furthermore, loss of phosphorylated H3-T45 causes phenotypes consistent with replicative defects, and prolonged replication stress results in H3-T45 phosphorylation accumulation over time. Notably, the phenotypes described here are independent of Lys 56 acetylation status, and combinatorial mutations to both Thr 45 and Lys 56 of H3 cause synthetic growth defects. Together, these data identify and characterize H3-T45 phosphorylation as a replication-associated histone modification in budding yeast.

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Figure 1: Identification of the Cdc7–Dbf4 histone kinase complex.
Figure 2: Mapping of Cdc7-dependent histone phosphorylation.
Figure 3: Histone H3 T45 phosphorylation is linked to replication in yeast.
Figure 4: Mutation of T45 causes sensitivity to replication stress.
Figure 5: H3-T45 phosphorylation is distinct from H3-K56 acetylation.

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References

  1. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    Article  CAS  Google Scholar 

  2. Ito, T. Role of histone modification in chromatin dynamics. J. Biochem. 141, 609–614 (2007).

    Article  CAS  Google Scholar 

  3. Masumoto, H., Hawke, D., Kobayashi, R. & Verreault, A. A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response. Nature 436, 294–298 (2005).

    Article  CAS  Google Scholar 

  4. Han, J. et al. Rtt109 acetylates histone H3 lysine 56 and functions in DNA replication. Science 315, 653–655 (2007).

    Article  CAS  Google Scholar 

  5. Grant, P. A., Berger, S. L. & Workman, J. L. Identification and analysis of native nucleosomal histone acetyltransferase complexes. Methods Mol. Biol. 119, 311–317 (1999).

    CAS  PubMed  Google Scholar 

  6. Lo, W. S. et al. Snf1—a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293, 1142–1146 (2001).

    Article  CAS  Google Scholar 

  7. Hartwell, L. H. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 115, 966–974 (1973).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Jackson, A. L., Pahl, P. M., Harrison, K., Rosamond, J. & Sclafani, R. A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein. Mol. Cell. Biol. 13, 2899–2908 (1993).

    Article  CAS  Google Scholar 

  9. Chapman, J. W. & Johnston, L. H. The yeast gene, DBF4, essential for entry into S phase is cell cycle regulated. Exp. Cell Res. 180, 419–428 (1989).

    Article  CAS  Google Scholar 

  10. Sclafani, R. A. Cdc7p-Dbf4p becomes famous in the cell cycle. J. Cell. Sci. 113 (Pt 12), 2111–2117 (2000).

    Google Scholar 

  11. Weinreich, M. & Stillman, B. Cdc7p–Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO J. 18, 5334–5346 (1999).

    Article  CAS  Google Scholar 

  12. Lei, M. et al. Mcm2 is a target of regulation by Cdc7–Dbf4 during the initiation of DNA synthesis. Genes Dev. 11, 3365–3374 (1997).

    Article  CAS  Google Scholar 

  13. Masai, H. et al. Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J. Biol. Chem. 281, 39249–39261 (2006).

    Article  CAS  Google Scholar 

  14. White, C. L., Suto, R. K. & Luger, K. Structure of the yeast nucleosome core particle reveals fundamental changes in internucleosome interactions. EMBO J. 20, 5207–5218 (2001).

    Article  CAS  Google Scholar 

  15. Ozdemir, A., Masumoto, H., Fitzjohn, P., Verreault, A. & Logie, C. Histone H3 lysine 56 acetylation: a new twist in the chromosome cycle. Cell Cycle 5, 2602–2608 (2006).

    Article  CAS  Google Scholar 

  16. Ferreira, H., Somers, J., Webster, R., Flaus, A. & Owen-Hughes, T. Histone tails and the H3 αN helix regulate nucleosome mobility and stability. Mol. Cell. Biol. 27, 4037–4048 (2007).

    Article  CAS  Google Scholar 

  17. Ozdemir, A. et al. Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae. J. Biol. Chem. 280, 25949–25952 (2005).

    Article  CAS  Google Scholar 

  18. Driscoll, R., Hudson, A. & Jackson, S. P. Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315, 649–652 (2007).

    Article  CAS  Google Scholar 

  19. Hardy, C. F., Dryga, O., Seematter, S., Pahl, P. M. & Sclafani, R. A. mcm5/cdc46–bob1 bypasses the requirement for the S phase activator Cdc7p. Proc. Natl Acad. Sci. USA 94, 3151–3155 (1997).

    Article  CAS  Google Scholar 

  20. Duncker, B. P. & Brown, G. W. Cdc7 kinases (DDKs) and checkpoint responses: lessons from two yeasts. Mutat. Res. 532, 21–27 (2003).

    Article  CAS  Google Scholar 

  21. Tsuji, T., Lau, E., Chiang, G. G. & Jiang, W. The role of Dbf4/Drf1-dependent kinase Cdc7 in DNA-damage checkpoint control. Mol. Cell 32, 862–869 (2008).

    Article  CAS  Google Scholar 

  22. Hurd, P. J. et al. Phosphorylation of histone H3 Thr-45 is linked to apoptosis. J. Biol. Chem. 284, 16575–16583 (2009).

    Article  CAS  Google Scholar 

  23. Nowak, S. J. & Corces V. G. Phosphorylation of histone H3: a balancing act between chromosome condensation and transcriptional activation. Trends Genet. 20, 214–220 (2004).

    Article  CAS  Google Scholar 

  24. Ahn, S. H. et al. Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae. Cell 120, 25–36 (2005).

    Article  CAS  Google Scholar 

  25. Owen-Hughes, T. et al. Analysis of nucleosome disruption by ATP-driven chromatin remodeling complexes. Methods Mol. Biol. 119, 319–331 (1999).

    CAS  PubMed  Google Scholar 

  26. Grant, P. A., Berger, S. L. & Workman, J. L. Identification and analysis of native nucleosomal histone acetyltransferase complexes. Methods Mol. Biol. 119, 311–317 (1999).

    CAS  PubMed  Google Scholar 

  27. Keogh, M. C. et al. A phosphatase complex that dephosphorylates γH2AX regulates DNA damage checkpoint recovery. Nature 439, 497–501 (2006).

    Article  CAS  Google Scholar 

  28. Haase, S. B. & Reed, S. I. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1, 132–136 (2002).

    Article  CAS  Google Scholar 

  29. Megee, P. C., Morgan, B. A., Mittman, B. A. & Smith, M. M. Genetic analysis of histone H4: essential role of lysines subject to reversible acetylation. Science 247, 841–845 (1990).

    Article  CAS  Google Scholar 

  30. Slater, M. L., Sharrow, S. O. & Gart, J. J. Cell cycle of Saccharomyces cerevisiae in populations growing at different rates. Proc Natl Acad Sci USA 74, 3850–3854 (1977).

    Article  CAS  Google Scholar 

  31. Grant, P. A. et al. Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 11, 1640–1650 (1997).

    Article  CAS  Google Scholar 

  32. Pray-Grant, M. G., Daniel, J. A., Schieltz, D., Yates, J. R., 3rd & Grant, P. A. Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation. Nature 433, 434–438 (2005).

    Article  CAS  Google Scholar 

  33. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank members of the Grant and Hunt labs for helpful discussion and technical assistance; R. Sclafani for yeast strains and reagents; G. Kupfer for reagents; J. Smith and J. Reese for yeast strains; and D. Auble for yeast strains, helpful discussion and reading of this manuscript. We also thank J. Bone (Active Motif) for assistance with antibody generation. S.P.B. was supported in part by NIH pre-doctoral cancer training grant no. 5 T32 CA009109-30. This work was supported by grants from the NIH to P.A.G. (5 P30 CA044579-18 and R56 DK082673-01), D.F.H. (GM37537), J.R.Y. (P41 RR011823), and M.M.S. (GM60444).

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S.P.B. performed purifications of TAP-tagged Cdc7 complex, recombinant Cdc7 and Dbf4 expression and purification, in vitro histone and peptide kinase assays, mass spectrometric identification of Thr 45 phosphorylation, antibody characterization, yeast strain generation, mutagenesis, growth assays, synchronization experiments, inductions, and associated western blotting. J.P. identified and isolated the native Cdc7 histone kinase complex, generated the Cdc7–TAP strain and performed the Cdc7 immunoprecipitation and associated kinase assays. S.A. and J.R.Y. performed the mass spectrometric identification of the kinase complex subunits and subsequent data analysis. Q.Q. and M.M.S. performed and analysed the FACS data. J.S. and D.F.H. assisted with the mass spectrometric mapping of the histone phosphorylation mark and data analysis. P.A.G. conducted experimental design and analysis. S.P.B. and P.A.G. wrote the manuscript.

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Correspondence to Patrick A. Grant.

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Baker, S., Phillips, J., Anderson, S. et al. Histone H3 Thr 45 phosphorylation is a replication-associated post-translational modification in S. cerevisiae. Nat Cell Biol 12, 294–298 (2010). https://doi.org/10.1038/ncb2030

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