Crystal Structure and Functional Analysis of JMJD5 Indicate an Alternate Specificity and Function

JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a β-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.     

Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role of JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21(CIP1) proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.     

Inhibitor scaffold for the histone lysine demethylase KDM4C (JMJD2C)

The human histone demethylases of the KDM4 (JMJD2) family have been associated to diseases such as prostate and breast cancer, as well as X-linked mental retardation. Therefore, these enzymes are considered oncogenes and their selective inhibition might be a possible therapeutic approach to treat cancer. Here we describe a heterocyclic ring system library screened against the histone demethylase KDM4C (JMJD2C) in the search for novel inhibitory scaffolds. A 4-hydroxypyrazole scaffold was identified as an inhibitor of KDM4C; this scaffold could be employed in the further development of novel therapeutics, as well as for the elucidation of the biological roles of KDM4C on epigenetic regulation.     

JMJD3 in the regulation of human diseases

In recent years, many studies have shown that histone methylation plays an important role in maintaining the active and silent state of gene expression in human diseases. The Jumonji domain-containing protein D3 (JMJD3), specifically demethylate di- and trimethyllysine 27 on histone H3 (H3K27me2/3), has been widely studied in immune diseases, infectious diseases, cancer, developmental diseases, and aging related diseases. We will focus on the recent advances of JMJD3 function in human diseases, and looks ahead to the future of JMJD3 gene research in this review.     

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.     

Histone demethylase JMJD2B-mediated cell proliferation regulated by hypoxia and radiation in gastric cancer cell

Histone modifying factors are functional components of chromatin and play a role in gene regulation. The expression level of JMJD2B, a histone demethylase, is notably up-regulated in cancer tissues. Upregulation of JMJD2B promotes cancer cell proliferation under hypoxic conditions through target gene expression. Here, we describe the patterns of histone methylation and JMJD2B expression under various stressed conditions, such as hypoxia and radiation, in a gastric cancer cell line. JMJD2B expression in AGS cells was actively regulated by hypoxia and radiation. Chromatin immunoprecipitation experiments demonstrated that binding of JMJD2B on the cyclin A1 (CCNA1) promoter resulted in CCNA1 upregulation under hypoxic conditions. Furthermore, we confirmed that AGS cell proliferation was directly affected by JMJD2B and CCNA1 expression by performing experiments with JMJD2B depleted cells. Interestingly, the effects of JMJD2B on cell growth under hypoxia were remarkably repressed after gamma-ray irradiation. These results suggest that JMJD2B may play a central role in gastric cancer cell growth and might constitute a novel therapeutic target to overcome hypoxia-induced radio-resistance, thereby improving the efficiency of radiation therapy.     

Ascorbate antagonizes nickel ion to regulate JMJD1A expression in kidney cancer cells

Abnormal expression of histone demethylase Jumonji domain-containing protein 1A (JMJD1A) is associated with many kinds of cancers. JMJD1A is also a hypoxic response gene and its expression is regulated by hypoxia-inducible factor-1α (HIF-1α). In this study, we determined the role of JMJD1A in development and hypoxia pathway. We also measured the expression of JMJD1A and two hypoxia factors glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) in 786-0 and HEK293 cells treated with different concentrations of NiCl(2) (2.5-100 μM) for 24 h, and found that JMJD1A mRNA and protein were up-regulated with increased concentrations of NiCl(2). We then observed that ascorbate could retard the up-regulated effect of NiCl(2)-induced JMJD1A expression in a dose-dependent manner through decreasing the stability of HIF-1α protein. Immunohistochemical analysis further demonstrated ascorbate antagonized Ni(2+)-induced up-regulation of JMJD1A expression in 786-0, HEK293, and OS-RC-2 cells. These findings suggest that both Ni(2+) and ascorbate can regulate the expression of histone demethylase JMJD1A, which is important for cancer development or inhibition.     

JMJD3 as an epigenetic regulator in development and disease

Gene expression is epigenetically regulated through DNA methylation and covalent chromatin modifications, such as acetylation, phosphorylation, ubiquitination, sumoylation, and methylation of histones. Histone methylation state is dynamically regulated by different groups of histone methyltransferases and demethylases. The trimethylation of histone 3 (H3K4) at lysine 4 is usually associated with the activation of gene expression, whereas trimethylation of histone 3 at lysine 27 (H3K27) is associated with the repression of gene expression. The polycomb repressive complex contains the H3K27 methyltransferase Ezh2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3). The Jumonji domain containing-3 (Jmjd3, KDM6B) and ubiquitously transcribed X-chromosome tetratricopeptide repeat protein (UTX, KDM6A) have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3. The role and mechanisms of both JMJD3 and UTX have been extensively studied for their involvement in development, cell plasticity, immune system, neurodegenerative disease, and cancer. In this review, we will focus on recent progresses made on understanding JMJD3 in the regulation of gene expression in development and diseases. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

Regulation of tumor suppressor p53 and HCT116 cell physiology by histone demethylase JMJD2D/KDM4D

JMJD2D, also known as KDM4D, is a histone demethylase that removes methyl moieties from lysine 9 on histone 3 and from lysine 26 on histone 1.4. Here, we demonstrate that JMJD2D forms a complex with the p53 tumor suppressor in vivo and interacts with the DNA binding domain of p53 in vitro. A luciferase reporter plasmid driven by the promoter of p21, a cell cycle inhibitor and prominent target gene of p53, was synergistically activated by p53 and JMJD2D, which was dependent on JMJD2D catalytic activity. Likewise, overexpression of JMJD2D induced p21 expression in U2OS osteosarcoma cells in the absence and presence of adriamycin, an agent that induces DNA damage. Furthermore, downregulation of JMJD2D inhibited cell proliferation in wild-type and even more so in p53(-/-) HCT116 colon cancer cells, suggesting that JMJD2D is a pro-proliferative molecule. JMJD2D depletion also induced more strongly apoptosis in p53(-/-) compared to wild-type HCT116 cells. Collectively, our results demonstrate that JMJD2D can stimulate cell proliferation and survival, suggesting that its inhibition may be helpful in the fight against cancer. Furthermore, our data imply that activation of p53 may represent a mechanism by which the pro-oncogenic functions of JMJD2D become dampened.     

Histone Demethylase KDM6B Promotes Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.     

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.     

High-throughput TR-FRET assays for identifying inhibitors of LSD1 and JMJD2C histone lysine demethylases

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 μL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.