Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

Abstract

The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N(ϵ)-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N(ϵ)-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N(ϵ)-methyllysine of an H3K27me3 peptide binds similarly to N(ϵ)-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N(ϵ)-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.

Keywords: 2OG oxygenases; 2OG, 2-oxoglutarate; Epigenetics; FIH, Factor Inhibiting HIF; H3, histone 3; HIF, Hypoxia Inducible Factor; JmjC histone demethylase; JmjC, Jumonji C-terminal; JmjN, Jumonji N-terminal; KDM, Lysine Demethylase; LSD, Lysine Specific Demethylase; MALDI-TOF MS, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry; MINA53, Myc-Induced Nuclear Antigen with a molecular mass of 53 kDa; NO66, Nucleolar protein 66; PHD, Plant Homeodomain; Rp, Ribosomal protein; TPR, Tetratricopeptide repeat; demethylation; histone; methyllysine.

Figure 1.

JmjC Oxygenases share sequence homology and catalytic mechanisms. (A) Phylogenetic analysis of the catalytic domains of human JmjC oxygenases. Reported catalytic functions are indicated by colored circles. MINA53, NO66 and JMJD6 have been reported to be demethylases but have subsequently been shown to have hydroxylase activities. KDM6C (UTY) was recently identified as a histone demethylase in vitro, acting on H3 peptide fragments methylated at H3K27. HR is Hairless Protein, a recently identified H3K9 demethylase. Enzymes used in this work are underlined. (B) Outline mechanism of JmjC oxygenase catalysis. Oxidative decarboxylation of 2-oxoglutarate (2OG) in the active site forms a highly reactive iron(IV)-oxo intermediate, which hydroxylates the substrate. In the case of demethylation (X = N), the hydroxylated product is unstable and fragments to produce the demethylated species and formaldehyde. The exact protonation states of water molecules complexed to the iron(II) species are unknown.

Figure 2.

JmjC Oxygenases MINA53, NO66 and JMJD5 do not catalyze demethylation of histone peptides. In addition to putative demethylation activities, MINA53 and NO66 have been characterized as hydroxylases acting on ribosomal proteins Rpl27a and Rpl8 respectively. Hydroxylation activities were observed for MINA53 and NO66, acting on Rpl27a and Rpl8 peptide fragments respectively (A and C); no demethylation was observed with methylated histone peptides (B, D and E). Prime-substrate uncoupled turnover of 2OG by JMJD5 (residues 1–416) was observed in a [¹⁴C]-labeled 2OG assay, which was dependent on the presence of iron(II) and inhibited by the broad-spectrum 2OG oxygenase inhibitor 2,4-pyridinedicarboxylic acid (2,4 PDA) (F). However, demethylation of an H3K36me2 histone peptide was not observed (G). Control reactions without added protein are in red.

Figure 3.

KDM4A catalyzes demethylation of histone fragment peptides methylated at H3K27. MALDI-TOF spectra of KDM4A (1–359) catalyzed demethylation of (A) H3K27me3 peptide (Biotin-Ahx(aminohexanoic acid)-KAPRKQLATKAARKme3SAPATGG), (B) H3K9me3 peptide (Biotin-Ahx-ARTKQTARKme3STGGKAPRKQLA). MALDI-TOF spectra of full-length KDM4A (1–1064) catalyzed demethylation of (C) H3K27me3 peptide (KAPRKQLATKAARKme3SAPATGG) and (D) H3K9me3 peptide (Biotin-Ahx-ARTKQTARKme3STGGKAPRKQLA). FLAG-tagged full-length KDM4A (1–1064) was produced in HEK293T cells and purified from cell lysates using anti-FLAG beads prior to reaction with the histone peptide. MALDI-TOF spectra of reactions with (standard conditions, black) and without added enzyme (red) for (A)–(D) are shown. (E) Kinetic parameters determined for KDM4A (1–359) catalyzed demethylation of N^ε-trimethylated H3K9, H3K27 and H3K36 fragment peptides using FDH assay (Figure S16).

Figure 4.

View from an X-ray crystal structure of the catalytic domain of KDM4A in complex with an H3K27me3 fragment peptide (PDB ID: 4V2W). The active site residues are highlighted in yellow. The visible residues of the 25mer H3_10–35K27me3 (green) peptide is shown overlaid with H3K9me3 (pink), as complexed with KDM4A (PDB ID: 2OQ6, nickel substituted for iron, and N-oxalylglycine substituted for 2OG). The position of the H3K27me3 residue of the fragment peptide correlates closely with that of H3K9me3 (and H3K36me3, Figure S18). However, in the H3_10–25K27me3 derived structure only the electron density for the tri-methyl lysine and the residues either side (H3R26 and H3S28) are clearly defined, suggesting the other residues are bound less tightly than the comparable H3K9 and H3K36 substrates. A second structure of a shorter 5 residue H3_25–29K27me3 peptide in complex with KDM4A (PDB ID: 4V2V) overlays well with that of the 25 residue peptide (Figure S19). The sequences for the H3K9, K27 and K36 peptides present in these crystal structures are included with residues for which electron density is observed in italics. The red lysine residue marks the position of the N^ε-trimethylated lysine.