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. 2011 Dec 2;286(48):41616-25.
doi: 10.1074/jbc.M111.283689. Epub 2011 Sep 13.

Structural and Evolutionary Basis for the Dual Substrate Selectivity of Human KDM4 Histone Demethylase Family

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Free PMC article

Structural and Evolutionary Basis for the Dual Substrate Selectivity of Human KDM4 Histone Demethylase Family

Lars Hillringhaus et al. J Biol Chem. .
Free PMC article

Abstract

N(ε)-Methylations of histone lysine residues play critical roles in cell biology by "marking" chromatin for transcriptional activation or repression. Lysine demethylases reverse N(ε)-methylation in a sequence- and methylation-selective manner. The determinants of sequence selectivity for histone demethylases have been unclear. The human JMJD2 (KDM4) H3K9 and H3K36 demethylases can be divided into members that act on both H3K9 and H3K36 and H3K9 alone. Kinetic, crystallographic, and mutagenetic studies in vitro and in cells on KDM4A-E reveal that selectivity is determined by multiple interactions within the catalytic domain but outside the active site. Structurally informed phylogenetic analyses reveal that KDM4A-C orthologues exist in all genome-sequenced vertebrates with earlier animals containing only a single KDM4 enzyme. KDM4D orthologues only exist in eutherians (placental mammals) where they are conserved, including proposed substrate sequence-determining residues. The results will be useful for the identification of inhibitors for specific histone demethylases.

Figures

FIGURE 1.
FIGURE 1.
Substrate selectivity of the human KDM4 histone demethylase subfamily. A, results using H31–9K4me3, H31–15K9me3, H31–15K9me2, H31–15K9me1, H324–33K27me3, H330–41K36me3, and H330–41K36me2 as substrates. The extent of demethylation was measured after a 30-min incubation (37 °C). B, results of competition assay between H36–17K9me3 and H330–41K36me3. C, results of competition assays between H31–15K9me3[13C]G12-[13C]G13 and H31–15K9me2 peptides. Pairs of competing peptides are color-coded. In each case, incubations (37 °C) were analyzed by MS after quenching with MeOH (1:1). Errors represent S.D. of three replicates.
FIGURE 2.
FIGURE 2.
Comparison of the Zn(II)-binding sites of human and yeast KDM4 members as observed in crystal structures for KDM4A,C,D,E and Rph1. Note that in case of the KDM4D structure (blue, PDB code 3DXU) there are substantial differences in secondary structure affecting parts of the Zn(II) (dark sphere) binding loop. The zinc-binding site in KDM4E is disordered possibly due to loss of zinc in the crystallization process. NOG, N-oxalylglycine.
FIGURE 3.
FIGURE 3.
Structural comparisons of the KDM4 subfamily reveal potential determinants of sequence selectivity. A, residue differences between KDM4D/E (dark gray) and KDM4A/B/C (light gray) subgroups in the H3K36 peptide binding region as observed in KDM4A-substrate crystal structures. Proposed interactions in brackets were not observed in these crystal structures likely due to missing electron density for the Arg-40H3 side chain. B, comparison of proposed substrate binding interactions as observed in KDM4A (PDB code 2OS2), KDM4C (PDB code 2XML), and KDM4E (PDB code 2W2I) structures. KDM4A/C/E are in gray, H3K9me3 in turquoise, and H3K36me3 in yellow. The predicted substrate binding surfaces are in red. For KDM4C/E the substrate conformations observed in KDM4A were docked into the active sites.
FIGURE 4.
FIGURE 4.
Demethylation of histones by wild type KDM4A and variants. A, demethylation of H31–15K9me3, H31–15K9me2, H330–41K36me3, and H330–41K36me2 by wild type KDM4A and variants. Reactions were at 37 °C and quenched by MeOH (1:1) after 60 min. The amount of demethylation was measured by MS. Errors represent S.D. of three replicates. B and C, kinetic analysis of the KDM4A quintuple variant with H37–14K9me3 and H37–14K9me2 employing a HCHO release assay. Errors represent S.D. of three replicates. D, demethylation of bulk histones by KDM4A variants. Reactions were at 37 °C and quenched by addition of Laemmli buffer after 5 h. KDM4A quintuple variant (quint. var.) = I71L, N86H, I87K, Q88K, and R309G.
FIGURE 5.
FIGURE 5.
Demethylation of H3K9me3 and H3K36me3 in human cells. Wild type KDM4E, KDM4A, and variants were expressed in HeLa cells as FLAG fusion proteins. Immunofluorescence assays with antibodies against methylated histone (left panels) or FLAG (middle panels) were used to analyze the activity of the proteins. DAPI (4,6-diamidino-2-phenylindole) staining (right panels) indicates location of nuclei. A and C, overexpression of KDM4A, KDM4E, and the I71L, R309G, and quintuple KDM4A variant enzymes led to a substantial loss of H3K9me3. B and C, KDM4A wild type and the KDM4A R309G variant showed a substantial loss of H3K36me3; the KDM4A I71L variant showed slightly reduced H3K36me3 levels; KDM4E and the KDM4A quintuple variants (quint. var.) do not reduce H3K36me3 levels. Errors represent S.D. of three replicates.
FIGURE 6.
FIGURE 6.
Evolutionary analysis of the KDM4 demethylase subfamily. A, phylogenetic domain analysis of eukaryotic KDM4 orthologues. Some orthologues in invertebrates lack the PHD and Tudor domains found in vertebrates. The yeast orthologues Rph1 and Gis1 carry two C-terminal zinc fingers and lack the PHD and Tudor domains. Ovals represent genome duplications. Branch lengths and domain sizes are not to scale. Likely pseudogenes (e.g. KDM4E/F in humans) are not shown. Note that the JmjC domain of the KDM4 enzymes itself contains a Zn(II)-binding motif. B, sequence alignment of eutherian KDM4D orthologues. Fe(II)-binding residues are in red and 2OG-binding residues in blue. Residues proposed to determine sequence specificity for H3K9 are in green and conserved in all analyzed eutherians. JmjN, Jumonji N; JmjC, Jumonji C; PHD, plant homeobox domain; Tudor, Tudor domain; bosta, Bos taurus; eleph, Loxodonta africana; mondo, Monodelphis domestica; chick, Gallus gallus; xentr, Xenopus tropicalis; danre, Danio rerio; brafl, Branchiostoma floridae; cioin, Ciona intestinalis; strpu, Strongylocentrotus purpuratus; drome, Drosophila melanogaster; caeel, Caenorhabditis elegans; triad, Trichoplax adherens; monbr, Monosiga brevicollis.

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