Enzymatic and structural insights for substrate specificity of a family of jumonji histone lysine demethylases

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
38–43
Year published:
DOI:
doi:10.1038/nsmb.1753
Received
Accepted
Published online

Abstract

Combinatorial readout of multiple covalent histone modifications is poorly understood. We provide insights into how an activating histone mark, in combination with linked repressive marks, is differentially 'read' by two related human demethylases, PHF8 and KIAA1718 (also known as JHDM1D). Both enzymes harbor a plant homeodomain (PHD) that binds Lys4-trimethylated histone 3 (H3K4me3) and a jumonji domain that demethylates either H3K9me2 or H3K27me2. The presence of H3K4me3 on the same peptide as H3K9me2 makes the doubly methylated peptide a markedly better substrate of PHF8, whereas the presence of H3K4me3 has the opposite effect, diminishing the H3K9me2 demethylase activity of KIAA1718 without adversely affecting its H3K27me2 activity. The difference in substrate specificity between the two is explained by PHF8 adopting a bent conformation, allowing each of its domains to engage its respective target, whereas KIAA1718 adopts an extended conformation, which prevents its access to H3K9me2 by its jumonji domain when its PHD engages H3K4me3.

At a glance

Figures

  1. PHF8 PHD domain binding of H3K4me3 enhances its jumonji domain-mediated demethylation of H3K9me2.
    Figure 1: PHF8 PHD domain binding of H3K4me3 enhances its jumonji domain–mediated demethylation of H3K9me2.

    (a) Schematic representation of PHF8. (b) Effect of H3K4me3 on the demethylation of H3K9me2 by PHF8. Top panels show progression of demethylation as a function of reaction time. Supplementary Figure 11a shows representative mass spectra at various time points. Bottom panels show kinetics of PHF8 on two peptide substrates, with calculated kinetic parameters. (c) ITC measurement of binding of PHF8 to doubly methylated H31–24K4me3-K9me2 peptides, carried out under the conditions of 11 μM protein concentration and 0.2 mM peptide concentration in 100 mM NaCl and 50 mM HEPES, pH 7.0. (d) The inhibitory effect of adding an equimolar ratio of H31–12K4me3 (top) or H31–21K4me3 peptides (bottom) on the demethylation of H31–24K9me2 by PHF8. (e) The PHD (blue) and jumonji (green) collaborate in binding the H3 peptide (magenta) containing H3K4me3 and H3K9me2. Omit electron densities, FoFc (black mesh), contoured at 4σ above the mean, are shown for the trimethlyated H3K4me3 and dimethlyated H3K9me2, respectively. (f) The surface representation of PHF8, colored with blue (PHD), green (jumonji) and magenta (H3 peptide). (g) H3K4me3 binding in the cage, surrounded on four sides by Tyr14, Met20 and Trp29 of PHD (blue) and Ser354 of jumonji (green). The carbonyl oxygen of Ser354 is in van der Waals contact with one of the methyl groups. Tyr7 (in thin lines) covers the top of the cage. (h) H3K9me2 binds in the active site.

  2. KIAA1718 PHD binding of H3K4me3 inhibits its jumonji domain activity targeting H3K9me2.
    Figure 2: KIAA1718 PHD binding of H3K4me3 inhibits its jumonji domain activity targeting H3K9me2.

    (a) Effect of H3K4me3 on the demethylation of H3K9me2 by KIAA1718. Left panels show progression of demethylation as a function of reaction time. Middle panels show representative mass spectra at various time points. Right top panel shows kinetics of KIAA1718 on substrate H31–24K9me2. KM is estimated to be less than 1.2 μM (indicated by an arrow), which is the amount of enzyme used to generate sufficient fluorescence signal. Right bottom shows the IC50 value of H31–24K4me3-K9me2 peptide [I] on H31-24K9me2 [S] demethylase activity of KIAA1718 [E]. The relative abundance of the substrate [S] was measured after eight minutes incubation at 37 °C by adding varying amounts of [I] to the reaction mixture. No demethylation of the doubly methylated peptide occurred. (b) Peptide pulldown assays with peptides H31-21 that were unmodified or mono-, di- or trimethylated (1, 2 or 3) at H3K4 or H3K9 using a GST-tagged PHD domain of KIAA1718. (c) KIAA1718 contains four segments: a disordered alanine-rich sequence followed by a stretch of prolines, a PHD domain (blue) containing two zinc metals (gray balls), a rigid linker (cyan) and a jumonji domain (green) followed by a four-helix bundle. (d) The KIAA1718 PHD domain contains a surface hydrophobic cage, a presumptive site for binding of H3K4me3. In the crystal lattice, the cage is blocked by the N-terminal prolines of a crystallographic symmetry-related molecule (Supplementary Fig. 6c).

  3. Effect of the linker on the KIAA1718 jumonji activity targeting H3K9me2.
    Figure 3: Effect of the linker on the KIAA1718 jumonji activity targeting H3K9me2.

    (a) ITC measurements of binding of the KIAA1718 to doubly methylated H31–24K4me3-K9me2 peptides (left) and H31–24K9me2 peptides (right). The measurements were carried out under the conditions of 18 μM protein concentration and 0.4 mM peptide concentration in 50 mM NaCl and 50 mM HEPES, pH 7.0. (b) Superimposition of PHF8 (colored) and KIAA1718 (gray) in their respective jumonji domains. (c) The engineered hybrid enzyme of KIAA1718 carrying the PHF8 linker gains substantial activity on H31–24K3me3-K9me2 (right), faster by a factor of more than 100 than that of the wild-type enzyme (see Fig. 2a).

  4. KIAA1718 selectively demethylates H3K27me2 in the presence of H3K4me3 in cis.
    Figure 4: KIAA1718 selectively demethylates H3K27me2 in the presence of H3K4me3 in cis.

    (a) A model of KIAA1718 PHD on methylated H3K4 and its linked jumonji active site on a target lysine (left). Surface representation displayed as blue for positive, red for negative and white for neutral (right). The dashed line connects H3K4me3 bound in the aromatic cage and the target lysine in the jumonji domain. (b) The presence of H3K4 methylation in cis enhances KIAA1718 demethylase activities on H3K27me2. (c) When two peptide substrates were mixed in equimolar ratio, H31–35K27me2 (left) and H31–35K4me3-K27me2 (right), KIAA1718 selectively demethylated H31–35 peptides containing both H3K4me3 and H3K27me2 (right).

Accession codes

Primary accessions

Referenced accessions

Protein Data Bank

References

  1. Rea, S. et al. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593599 (2000).
  2. van Leeuwen, F., Gafken, P.R. & Gottschling, D.E. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 109, 745756 (2002).
  3. Shi, Y. et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941953 (2004).
  4. Tsukada, Y. et al. Histone demethylation by a family of JmjC domain-containing proteins. Nature 439, 811816 (2006).
  5. Yamane, K. et al. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 125, 483495 (2006).
  6. Whetstine, J.R. et al. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125, 467481 (2006).
  7. Wysocka, J. et al. PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling. Nature 442, 8690 (2006).
  8. Li, H. et al. Molecular basis for site-specific readout of histone H3K4me3 by the BPTF PHD finger of NURF. Nature 442, 9195 (2006).
  9. Shi, X. et al. ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression. Nature 442, 9699 (2006).
  10. Pena, P.V. et al. Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2. Nature 442, 100103 (2006).
  11. Lan, F. et al. Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression. Nature 448, 718722 (2007).
  12. Ooi, S.K.T. et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448, 714717 (2007).
  13. Klose, R.J., Kallin, E.M. & Zhang, Y. JmjC-domain-containing proteins and histone demethylation. Nat. Rev. Genet. 7, 715727 (2006).
  14. Couture, J.F., Collazo, E., Ortiz-Tello, P.A., Brunzelle, J.S. & Trievel, R.C. Specificity and mechanism of JMJD2A, a trimethyllysine-specific histone demethylase. Nat. Struct. Mol. Biol. 14, 689695 (2007).
  15. Ng, S.S. et al. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature 448, 8791 (2007).
  16. Chen, Z. et al. Structural basis of the recognition of a methylated histone tail by JMJD2A. Proc. Natl. Acad. Sci. USA 104, 1081810823 (2007).
  17. 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, 748751 (2006).
  18. 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, 109111 (2008).
  19. Fatemi, M., Hermann, A., Pradhan, S. & Jeltsch, A. The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. J. Mol. Biol. 309, 11891199 (2001).
  20. Pradhan, M. et al. CXXC domain of human DNMT1 is essential for enzymatic activity. Biochemistry 47, 1000010009 (2008).
  21. Loenarz, C. et al. PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nϵ-dimethyl lysine demethylase. Hum. Mol. Genet. Published online, doi:10.1093/hmg/ddp480 (19 October 2009).
  22. Abidi, F.E., Miano, M.G., Murray, J.C. & Schwartz, C.E. A novel mutation in the PHF8 gene is associated with X-linked mental retardation with cleft lip/cleft palate. Clin. Genet. 72, 1922 (2007).
  23. Koivisto, A.M. et al. Screening of mutations in the PHF8 gene and identification of a novel mutation in a Finnish family with XLMR and cleft lip/cleft palate. Clin. Genet. 72, 145149 (2007).
  24. Laumonnier, F. et al. Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. J. Med. Genet. 42, 780786 (2005).
  25. Couture, J.F., Hauk, G., Thompson, M.J., Blackburn, G.M. & Trievel, R.C. Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases. J. Biol. Chem. 281, 1928019287 (2006).
  26. Bernstein, B.E. et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169181 (2005).
  27. Azuara, V. et al. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532538 (2006).
  28. Zhao, X.D. et al. Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell Stem Cell 1, 286298 (2007).
  29. Pan, G. et al. Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 1, 299312 (2007).
  30. Strahl, B.D. & Allis, C.D. The language of covalent histone modifications. Nature 403, 4145 (2000).
  31. Jenuwein, T. & Allis, C.D. Translating the histone code. Science 293, 10741080 (2001).
  32. Turner, B.M. Defining an epigenetic code. Nat. Cell Biol. 9, 26 (2007).
  33. Suganuma, T. & Workman, J.L. Crosstalk among histone modifications. Cell 135, 604607 (2008).
  34. Iwase, S. et al. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 128, 10771088 (2007).
  35. Li, F. et al. Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin. Cell 135, 272283 (2008).
  36. Collins, R.E. et al. The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules. Nat. Struct. Mol. Biol. 15, 245250 (2008).
  37. Zhang, K., Mosch, K., Fischle, W. & Grewal, S.I. Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin. Nat. Struct. Mol. Biol. 15, 381388 (2008).
  38. Berger, S.L. The complex language of chromatin regulation during transcription. Nature 447, 407412 (2007).
  39. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207234 (2005).
  40. Storoni, L.C., McCoy, A.J. & Read, R.J. Likelihood-enhanced fast rotation functions. Acta Crystallogr. D Biol. Crystallogr. 60, 432438 (2004).
  41. Kelley, L.A., MacCallum, R.M. & Sternberg, M.J. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299, 499520 (2000).
  42. Han, Z., Liu, P., Gu, L., Zhang, Y., Chen, S. & Chai, J. Structure basis for histone demethylation by JHDM1. Frontier Science 1, 5267 (2007).
  43. Jones, T.., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110119 (1991).
  44. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 21262132 (2004).
  45. Brunger, A.T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905921 (1998).
  46. Roy, T.W. & Bhagwat, A.S. Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation. Nucleic Acids Res. 35, e147 (2007).

Download references

Author information

  1. These authors contributed equally to this work.

    • John R Horton,
    • Anup K Upadhyay &
    • Hank H Qi

Affiliations

  1. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

    • John R Horton,
    • Anup K Upadhyay,
    • Xing Zhang &
    • Xiaodong Cheng
  2. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Hank H Qi &
    • Yang Shi
  3. Division of Newborn Medicine, Department of Medicine, Children's Hospital, Boston, Massachusetts, USA.

    • Hank H Qi &
    • Yang Shi

Contributions

J.R.H. performed crystallographic experiments; A.K.U. performed kinetic experiments; H.H.Q. and Y.S. provided initial expression constructs and the knowledge of specificities of individual PHD and jumonji domains; X.Z. generated hybrid enzymes; X.C. organized and designed the scope of the study and wrote the manuscript, and all others helped in analyzing data and revising the manuscript.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (16M)

    Supplementary Figures 1–12

Additional data