Specificity and mechanism of JMJD2A, a trimethyllysine-specific histone demethylase

JMJD2A is a JmjC histone demethylase (HDM) that catalyzes the demethylation of di- and trimethylated Lys9 and Lys36 in histone H3 (H3K9me2/3 and H3K36me2/3). Here we present the crystal structures of the JMJD2A catalytic domain in complex with H3K9me3, H3K36me2 and H3K36me3 peptides. The structures reveal that histone substrates are recognized through a network of backbone hydrogen bonds and hydrophobic interactions that deposit the trimethyllysine into the active site. The trimethylated epsilon-ammonium cation is coordinated within a methylammonium-binding pocket through carbon-oxygen (CH...O) hydrogen bonds that position one of the zeta-methyl groups adjacent to the Fe(II) center for hydroxylation and demethylation. Mutations of the residues comprising this pocket abrogate demethylation by JMJD2A, with the exception of an S288A substitution, which augments activity, particularly toward H3K9me2. We propose that this residue modulates the methylation-state specificities of JMJD2 enzymes and other trimethyllysine-specific JmjC HDMs.     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

Engineering human JMJD2A tudor domains for an improved understanding of histone peptide recognition

JMJD2A is a histone lysine demethylase which recognizes and demethylates H3K9me3 and H3K36me3 residues and is overexpressed in various cancers. It utilizes a tandem tudor domain to facilitate its own recruitment to histone sites, recognizing various di- and tri-methyl lysine residues with moderate affinity. In this study, we successfully engineered the tudor domain of JMJD2A to specifically bind to H4K20me3 with a 20-fold increase of affinity and improved selectivity. To reveal the molecular basis, we performed molecular dynamics and free energy decomposition analysis on the human JMJD2A tandem tudor domains bound to H4K20me2, H4K20me3, and H3K23me3 peptides to uncover the residues and conformational changes important for the enhanced binding affinity and selectivity toward H4K20me2/3. These analyses revealed new insights into understanding chromatin reader domains recognizing histone modifications and improving binding affinity and selectivity of these domains. Furthermore, we showed that the tight binding of JMJD2A to H4K20me2/3 is not sufficient to improve the efficiency of CRISPR-CAS9 mediated homology directed repair (HDR), suggesting a complicated relationship between JMJD2A and the DNA damage response beyond binding affinity toward the H4K20me2/3 mark.     

JMJD3 is a histone H3K27 demethylase

Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction oftrimethyl H3K27, but had no effect on di-and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.     

Identification of histone demethylases in Saccharomyces cerevisiae

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.     

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A–C and JMJD2A–D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.     

A comparative molecular dynamics study of methylation state specificity of JMJD2A

Histone modifications have great importance in epigenetic regulation. JMJD2A is a histone demethylase which is selective for di- and trimethyl forms of residues Lys9 and Lys36 of Histone 3 tail (H3K9 and H3K36). We present a molecular dynamics simulations of mono-, di- and trimethylated histone tails in complex with JMJD2A catalytic domain to gain insight into how JMJD2A discriminates between the methylation states of H3K9. The methyl groups are located at specific distances and orientations with respect to Fe(II) in methylammonium binding pocket. For the trimethyllysine the mechanism which provides the effectual orientation of methyl groups is the symmetry, whereas for the dimethyllysine case the determining factors are the interactions between methyllysine head and its environment and subsequently the restriction on angular motion. The occurrence frequency of methyl groups in a certain proximity of Fe(II) comes out as the explanation of the enzyme activity difference on di- and tri-methylated peptides. Energy analysis suggests that recognition is mostly driven by van der Waals and followed by Coulombic interactions in the enzyme-substrate interface. The number (mono, di or tri) and orientations of methyl groups and water molecules significantly affect the extent of van der Waals interaction strengths. Hydrogen bonding analysis suggests that the interaction between JMJD2A and its substrates mainly comes from main chain-side chain interactions. Binding free energy analysis points out Arg8 as an important residue forming an intra-substrate hydrogen bond with tri and dimethylated Lys9 of the H3 chain. Our study provides new insights into how JMJD2A discriminates between its substrates from both a structural and dynamical point of view.