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

Journal name:
Nature Structural & Molecular Biology
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Published online


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


  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).

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Author information

  1. These authors contributed equally to this work.

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


  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


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.

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