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PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53

Nature Structural & Molecular Biology volume 19, pages 916–924 (2012)Cite this article

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Abstract

PHF20 is a multidomain protein and subunit of a lysine acetyltransferase complex that acetylates histone H4 and p53 but whose function is unclear. Using biochemical, biophysical and cellular approaches, we determined that PHF20 is a direct regulator of p53. A Tudor domain in PHF20 recognized p53 dimethylated at Lys370 or Lys382 and a homodimeric form of this Tudor domain could associate with the two dimethylated sites on p53 with enhanced affinity, indicating a multivalent interaction. Association with PHF20 promotes stabilization and activation of p53 by diminishing Mdm2-mediated p53 ubiquitylation and degradation. PHF20 contributes to upregulation of p53 in response to DNA damage, and ectopic expression of PHF20 in different cell lines leads to phenotypic changes that are hallmarks of p53 activation. Overall our work establishes that PHF20 functions as an effector of p53 methylation that stabilizes and activates p53.

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Figure 1: Interaction of PHF20 with p53 in vitro and in cells.
Figure 2: Structures of PHF20 Tudor1 and Tudor2.
Figure 3: Chemical dimethylation and enzymatic monomethylation of p53 at Lys370 or Lys382, and interaction of methylated p53 with PHF20 Tudor2.
Figure 4: Evidence for dual recognition of p53K370me2K382me2 by PHF20 Tudor2 dimer.
Figure 5: PHF20 interacts with the methylated region of p53 and stabilizes p53 in cells.
Figure 6: Knockdown of PHF20 reduces p53 upregulation induced by DNA damage.
Figure 7: Ectopic expression of PHF20 leads to phenotypic changes consistent with p53 activation.

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References

  1. Vousden, K.H. & Prives, C. Blinded by the light: the growing complexity of p53. Cell 137, 413–431 (2009).

    Article  CAS  PubMed  Google Scholar 

  2. Beckerman, R. & Prives, C. Transcriptional regulation by p53. Cold Spring Harb. Perspect. Biol. 2, a000935 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Huang, J. et al. Repression of p53 activity by Smyd2-mediated methylation. Nature 444, 629–632 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Huang, J. et al. p53 is regulated by the lysine demethylase LSD1. Nature 449, 105–108 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Shi, X. et al. Modulation of p53 function by SET8-mediated methylation at lysine 382. Mol. Cell 27, 636–646 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kachirskaia, I. et al. Role for 53BP1 Tudor domain recognition of p53 dimethylated at lysine 382 in DNA damage signaling. J. Biol. Chem. 283, 34660–34666 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Roy, S. et al. Structural insight into p53 recognition by the 53BP1 tandem Tudor domain. J. Mol. Biol. 398, 489–496 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. West, L.E. & Gozani, O. Regulation of p53 function by lysine methylation. Epigenomics 3, 361–369 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Joo, W.S. et al. Structure of the 53BP1 BRCT region bound to p53 and its comparison to the Brca1 BRCT structure. Genes Dev. 16, 583–593 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Derbyshire, D.J. et al. Crystal structure of human 53BP1 BRCT domains bound to p53 tumour suppressor. EMBO J. 21, 3863–3872 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cui, G., Botuyan, M.V. & Mer, G. Preparation of recombinant peptides with site- and degree-specific lysine 13C-methylation. Biochemistry 48, 3798–3800 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Botuyan, M.V. et al. Structural basis for the methylation state-specific recognition of histone H4–K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fischer, U. et al. Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls. Clin. Exp. Immunol. 126, 206–213 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pallasch, C.P. et al. Autoantibodies against GLEA2 and PHF3 in glioblastoma: Tumor-associated autoantibodies correlated with prolonged survival. Int. J. Cancer 117, 456–459 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Heisel, S.M. et al. Increased seroreactivity to glioma-expressed antigen 2 in brain tumor patients under radiation. PLoS ONE 3, e2164 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Park, S. et al. Identification of an Akt interaction protein, PHF20/TZP, that transcriptionally regulates p53. J. Biol. Chem. 287, 11151–11163 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dou, Y. et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 121, 873–885 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Taipale, M. et al. hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells. Mol. Cell. Biol. 25, 6798–6810 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mendjan, S. et al. Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila. Mol. Cell 21, 811–823 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Li, X., Wu, L., Corsa, C.A., Kunkel, S. & Dou, Y. Two mammalian MOF complexes regulate transcription activation by distinct mechanisms. Mol. Cell 36, 290–301 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cai, Y. et al. Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex. J. Biol. Chem. 285, 4268–4272 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Sykes, S.M. et al. Acetylation of the p53 DNA-binding domain regulates apoptosis induction. Mol. Cell 24, 841–851 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yang, W.H. et al. Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability. Nat. Cell Biol. 8, 1074–1083 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Badeaux, A.I. et al. Loss of the methyl lysine effector protein PHF20 impacts the expression of genes regulated by the lysine acetyltransferase MOF. J. Biol. Chem. 287, 429–437 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Huang, Y., Fang, J., Bedford, M.T., Zhang, Y. & Xu, R.M. Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A. Science 312, 748–751 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Lee, J., Thompson, J.R., Botuyan, M.V. & Mer, G. Distinct binding modes specify the recognition of methylated histones H3K4 and H4K20 by JMJD2A-tudor. Nat. Struct. Mol. Biol. 15, 109–111 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Brown, M.A., Sims, R.J. III, Gottlieb, P.D. & Tucker, P.W. Identification and characterization of Smyd2: a split SET/MYND domain-containing histone H3 lysine 36-specific methyltransferase that interacts with the Sin3 histone deacetylase complex. Mol. Cancer 5, 26 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Xiao, B. et al. Specificity and mechanism of the histone methyltransferase Pr-Set7. Genes Dev. 19, 1444–1454 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Couture, J.F., Collazo, E., Brunzelle, J.S. & Trievel, R.C. Structural and functional analysis of SET8, a histone H4 Lys-20 methyltransferase. Genes Dev. 19, 1455–1465 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Selenko, P. et al. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Nat. Struct. Mol. Biol. 15, 321–329 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Ito, Y. & Selenko, P. Cellular structural biology. Curr. Opin. Struct. Biol. 20, 640–648 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Liokatis, S., Dose, A., Schwarzer, D. & Selenko, P. Simultaneous detection of protein phosphorylation and acetylation by high-resolution NMR spectroscopy. J. Am. Chem. Soc. 132, 14704–14705 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Xu, C., Cui, G., Botuyan, M.V. & Mer, G. Structural basis for the recognition of methylated histone H3K36 by the Eaf3 subunit of histone deacetylase complex Rpd3S. Structure 16, 1740–1750 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Brooks, C.L. & Gu, W. p53 ubiquitination: Mdm2 and beyond. Mol. Cell 21, 307–315 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Meek, D.W. & Anderson, C.W. Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harb. Perspect. Biol. 1, a000950 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Poyurovsky, M.V. et al. The C terminus of p53 binds the N-terminal domain of MDM2. Nat. Struct. Mol. Biol. 17, 982–989 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Adams-Cioaba, M.A. et al. Structural studies of the tandem Tudor domains of fragile X mental retardation related proteins FXR1 and FXR2. PLoS ONE 5, e13559 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Tripsianes, K. et al. Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. Nat. Struct. Mol. Biol. 18, 1414–1420 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Li, B. et al. Combined action of PHD and chromo domains directs the Rpd3S HDAC to transcribed chromatin. Science 316, 1050–1054 (2007).

    Article  CAS  PubMed  Google Scholar 

  41. Ruthenburg, A.J., Li, H., Patel, D.J. & Allis, C.D. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wang, Z. & Patel, D.J. Combinatorial readout of dual histone modifications by paired chromatin-associated modules. J. Biol. Chem. 286, 18363–18368 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Horton, J.R. et al. Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases. Nat. Struct. Mol. Biol. 17, 38–43 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Benezra, R. An intermolecular disulfide bond stabilizes E2A homodimers and is required for DNA binding at physiological temperatures. Cell 79, 1057–1067 (1994).

    Article  CAS  PubMed  Google Scholar 

  45. Mayo, L.D. & Donner, D.B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA 98, 11598–11603 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Botuyan, M.V. et al. Structural basis of BACH1 phosphopeptide recognition by BRCA1 tandem BRCT domains. Structure 12, 1137–1146 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Simon, M.D. et al. The site-specific installation of methyl-lysine analogs into recombinant histones. Cell 128, 1003–1012 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Kelley, L.A. & Sternberg, M.J. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371 (2009).

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  PubMed  Google Scholar 

  53. Johnson, B.A. & Blevins, R.A. NMRView: a computer program for visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    Article  CAS  PubMed  Google Scholar 

  54. Duggan, B.M., Legge, G.B., Dyson, H.J. & Wright, P.E. SANE (Structure Assisted NOE Evaluation): an automated model-based approach for NOE assignment. J. Biomol. NMR 19, 321–329 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    Article  CAS  PubMed  Google Scholar 

  56. Wishart, D.S. & Sykes, B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994).

    Article  CAS  PubMed  Google Scholar 

  57. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  58. Case, D.A. et al. The Amber biomolecular simulation programs. J. Comput. Chem. 26, 1668–1688 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    Article  CAS  PubMed  Google Scholar 

  60. Koradi, R., Billeter, M. & Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 51–55 (1996).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We acknowledge the use of beamline 19-BM at the Advanced Photon Source (APS), Argonne National Laboratory. APS is operated by UChicago Argonne, LLC, for the US Department of Energy under contract DE-AC02-06CH11357. We are grateful to Y. Kim at APS for assistance with X-ray data collection. We acknowledge the help of A. Espejo and C. Sagum with protein microarray work and the support from the Center for Environmental and Molecular Carcinogenesis at M.D. Anderson Cancer Center. This research is supported by US National Institutes of Health (NIH) grant CA132878 (G.M.), NIH grant CA137041 and James and Esther King grant 1KG02 (J.Q.C.), institutional NIH grant ES007784 and Cancer Prevention Research Institute of Texas grant RP110471 (M.T.B.) and NIH training grant 5T32ES007247 (A.I.B.).

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Author notes

  1. Satoshi Kaneko & Zengqiang Yuan

    Present address: Present addresses: Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA (S.K.) and Institute of Biophysics, Chinese Academy of Sciences, Beijing, China (Z.Y.).,

  2. Gaofeng Cui, Sungman Park, Aimee I Badeaux and Donghwa Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota, USA

    Gaofeng Cui, Joseph Lee, Maria Victoria Botuyan & Georges Mer

  2. Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA

    Sungman Park, Donghwa Kim, Fei Yan, Satoshi Kaneko, Zengqiang Yuan & Jin Q Cheng

  3. Department of Molecular Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, Texas, USA

    Aimee I Badeaux & Mark T Bedford

  4. Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota, USA

    James R Thompson

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Contributions

G.C. designed and prepared the fusion proteins and all peptides with incorporation of methyllysine analogs, determined the crystal structure of PHF20 Tudor1 and did the NMR spectroscopy-based experiments including structure determination and binding assays. S.P. and D.K. designed and performed the in vivo mutagenesis studies, the ubiquitylation assays, the DNA damage assays and the cell cycle regulation assays. A.I.B. designed and performed the protein array assays and cell biology experiments. J.L. determined the crystal structure of PHF20 Tudor2. J.R.T. contributed to the refinement of crystal structures. F.Y., S.K. and Z.Y. contributed to the cell biology experiments. M.V.B. designed and performed the p53 methylation assay with SET8 and SMYD2 and contributed extensively to the preparation of proteins used for structural studies. M.T.B., J.Q.C. and G.M. supervised the research in their respective laboratories. G.M. wrote the manuscript with input from the other authors.

Corresponding authors

Correspondence to Mark T Bedford, Jin Q Cheng or Georges Mer.

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Cui, G., Park, S., Badeaux, A. et al. PHF20 is an effector protein of p53 double lysine methylation that stabilizes and activates p53. Nat Struct Mol Biol 19, 916–924 (2012). https://doi.org/10.1038/nsmb.2353

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