Jumonji Histone Demethylases Research Articles

Jumonji histone demethylases are therapeutic targets in small cell lung cancer

Small cell lung cancer (SCLC) is a recalcitrant cancer of neuroendocrine (NE) origin. Changes in therapeutic approaches against SCLC have been lacking over the decades. Here, we use preclinical models to identify a new therapeutic vulnerability in SCLC consisting of the targetable Jumonji lysine demethylase (KDM) family. We show that Jumonji demethylase inhibitors block malignant growth and that etoposide-resistant SCLC cell lines are particularly sensitive to Jumonji inhibition. Mechanistically, small molecule-mediated inhibition of Jumonji KDMs activates endoplasmic reticulum (ER) stress genes, upregulates ER stress signaling, and triggers apoptotic cell death. Furthermore, Jumonji inhibitors decrease protein levels of SCLC NE markers INSM1 and Secretogranin-3 and of driver transcription factors ASCL1 and NEUROD1. Genetic knockdown of KDM4A, a Jumonji demethylase highly expressed in SCLC and a known regulator of ER stress genes, induces ER stress response genes, decreases INSM1, Secretogranin-3, and NEUROD1 and inhibits proliferation of SCLC in vitro and in vivo. Lastly, we demonstrate that two different small molecule Jumonji KDM inhibitors (pan-inhibitor JIB-04 and KDM4 inhibitor SD70) block the growth of SCLC tumor xenografts in vivo. Our study highlights the translational potential of Jumonji KDM inhibitors against SCLC, a clinically feasible approach in light of recently opened clinical trials evaluating this drug class, and establishes KDM4A as a relevant target across SCLC subtypes.

Dysregulation of Stem-Progenitor and Differentiation Programmes through Modulation of Bivalent Chromatin Drives Leukemogenesis in Mutant IDH2 Acute Myeloid Leukaemia

Differentiation arrest is a hallmark in isocitrate dehydrogenase mutant (mIDH) acute myeloid leukaemia (AML). MIDH enzyme produces d-2-hydroxyglutarate, which inhibits α-ketoglutarate-dependent dioxygenases, including TET2 and Jumonji histone demethylases (J-KDM). The underlying leukemogenic mechanism is unclear and may be due to impaired DNA and/ or histone demethylation. Bivalent chromatin, with both K4me3 and K27me3 marks, maintain genes in a 'poised’ state and is important in modulating expression during cell differentiation. We hypothesised that this may be an important mechanism by which mIDH drives differentiation arrest. Using an mIDH2 inhibitor Enasidenib (ENA), we stimulated granulocytic differentiation of IDH2m AML progenitors (L-Prog), and using a multiomic approach, we tracked epigenetic events that reprogramme arrested L-Prog. We previously reported downregulation of stem-progenitor signatures and co-incident expression of cell cycle and granulocytic transcriptional networks during ENA-induced differentiation. We also observed increased chromatin accessibility in ENA-treated cells and hypothesised that regulation of H3K27me3 is critical in creating a more permissive epigenetic landscape (Silveira et al., Blood 2021; 138 (Supplement 1): 3467). To test this hypothesis, we performed enhanced reduced representation methylation sequencing and chromatin immunoprecipitation (ChIPseq) assay in parallel with RNAseq. Compared with controls, ENA stimulated global CpG demethylation (mean LogFC=2.01, range 1.79-2.36). However, promoter CpG islands were only modestly hypomethylated in genes upregulated by ENA. This suggests that mechanisms other than DNA hypermethylation are crucial for IDHm leukemogenesis. Focusing on histone regulation, we studied H3-K4me3, K27me3 and K27ac marks in ENA-treated L-prog from responsive (R) and non-responsive (NR) samples. We annotated gene promoters as monovalent (mono), bivalent (biv) and partial bivalent (part-K27me3 or part-K4me3), depending on relative enrichment of K27me3 and K4me3 marks, and show correlation between gene expression and repressive (mono-K27me3) to active (mono-K4me3) histone states in a continuum. Consistent with the role of bivalent states in modulating gene expression, the most dynamically differentially expressed genes (DEG, LogFC > 0.25) had the greatest proportion of bivalent promoters. Furthermore, ENA-treated R samples have significantly increased (OR range: 1.11 - 2.17, p < 10e-5) bivalent and part-K4me3 marked promoters respectively compared with DMSO, or with ENA-treated NR samples. 67-84% of ENA-upregulated genes where we detected altered chromatin states transitioned from bivalent to more active partial or monovalent K4me3 states. These transitions were 3x more likely to arise from loss of K27me3 than gain K4me3 (p < 10e-16), suggesting specific enhancement of KDM4/6 activity. Conversely, >95% of downregulated genes moved from relatively activated states (mono-K4me3 or part-K4me3) towards bivalency, mainly through gain of K27me3. These genes were highly enriched with polycomb repressor complex 2 (PRC2)/ EZH2 targets (OR range: 4.28-11.03, p < 0.01). The switch from part-H3K27me3 to part-H3K4me3 via bivalency in ENA-upregulated differentiation genes is associated with increased H3K27ac, which in turn is correlated with increased chromatin accessibility measured by single cell (sc)ATACseq. Motif usage analysis across the ENA-induced differentiation trajectory revealed that differentiation genes are dual regulated: repressed by stem-progenitor transcription factors (prog-TFs), e.g. GATA2, FOSL1 and MYC which are active in early differentiation, and then activated by granulocyte-monocyte TFs EGR1, CEBPE, JUNB/FOSB and SPI1 in late differentiation, as prog-TFs themselves are downregulated. Our model (Figure 1) of d-2HG-induced differentiation arrest places modulation of promoter chromatin accessibility and gene expression via KDM4/5 and PRC2-mediated changes to H3K27 methylation centre-stage. For the first time, we show how these chromatin changes drive dynamic transcriptional programmes that initiate and sustain restoration of granulocytic differentiation in primary AML progenitors, and in so doing, identified a novel mechanism of leukemogenesis which may be targetable in other AML subtypes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Demethylases JMJ30 and JMJ32 Modulate the Speed of Vernalization Through the Activation of FLOWERING LOCUS C in Arabidopsis thaliana.

Vernalization is the promotion of flowering after prolonged exposure to cold. In Arabidopsis thaliana, vernalization induces epigenetic silencing of the floral repressor gene FLOWERING LOCUS C (FLC). Among the repressive epigenetic marks, the trimethylation of lysine 27 on histone H3 proteins (H3K27me3) is a critical contributor to the epigenetic silencing of FLC. The deposition of H3K27me3 is mediated by Polycomb Repressive Complex 2 (PRC2). Conversely, the elimination of H3K27me3 is mediated by histone demethylases, Jumonji-C domain-containing protein JMJ30 and its homolog JMJ32. However, the role of JMJ30 and JMJ32 in vernalization is largely unknown. In this study, we found that cold treatment dramatically reduced the expression levels of JMJ30 and did not reduce those of JMJ32. Next, by using the genetic approach, we found that the flowering of jmj30 jmj32 was accelerated under moderate vernalized conditions. Under moderate vernalized conditions, the silencing of FLC occurred more quickly in jmj30 jmj32 than in the wild type. These results suggested that the histone demethylases JMJ30 and JMJ32 brake vernalization through the activation of FLC. Our study suggested that PRC2 and Jumonji histone demethylases act in an opposing manner to regulate flowering time via epigenetic modifications.

Inhibition of Jumonji demethylases reprograms severe dilated cardiomyopathy and prolongs survival

Hypertrophic/dilated cardiomyopathy, often a prequel to heart failure, is accompanied by maladaptive transcriptional changes that contribute to arrythmias and contractile misfunction. Transgenic mice constitutively expressing high levels of calcineurin are known to develop extreme heart hypertrophy, which progresses to dilated cardiomyopathy, and to die several weeks after birth. Here, we characterized aberrant transcriptional and epigenetic pathways in this mouse model and established a pharmacological approach to treat established cardiomyopathy. We found that H3K4me3 (trimethyl histone 3 lysine 4) and H3K9me3 (trimethyl histone 3 lysine 9) Jumonji histone demethylases are markedly increased at the protein level and show enhanced enzymatic activity in diseased hearts. These epigenetic regulators continued to increase with time, further affecting cardiac gene expression. Our findings parallel the lower H3K4me3 and H3K9me3 levels seen in human patients. Inhibition of Jumonji demethylase activities in vivo results in lower histone demethylase enzymatic function in the heart and higher histone methylation levels and leads to partial reduction of heart size, reversal of maladaptive transcriptional programs, improved heart function, and prolonged survival. At the molecular level, target genes of transcription factor myocyte enhancer factor 2 are specifically regulated in response to pharmacological or genetic inhibition of Jumonji demethylases. Similar transcriptional reversal of disease-associated genes is seen in a second disease model based on cardiac mechanical overload. Our findings validate pharmacological inhibitors of Jumonji demethylases as potential therapeutics for the treatment of cardiomyopathies across disease models and provide evidence of the reversal of maladaptive transcriptional reprogramming leading to partial restoration of cardiac function. In addition, this study defines pathways of therapeutic resistance upregulated with disease progression.

Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression

Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets.We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, ‘R‘). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R.At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation.Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog.Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs.Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. DisclosuresSilveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

The toxic side of one-carbon metabolism and epigenetics

One-carbon metabolism is a central metabolic hub that provides one-carbon units for essential biosynthetic reactions and for writing epigenetics marks. The leading role in this hub is performed by the one-carbon carrier tetrahydrofolate (THF), which accepts formaldehyde usually from serine generating one-carbon THF intermediates in a set of reactions known as the folate or one-carbon cycle. THF derivatives can feed one-carbon units into purine and thymidine synthesis, and into the methionine cycle that produces the universal methyl-donor S-adenosylmethionine (AdoMet). AdoMet delivers methyl groups for epigenetic methylations and it is metabolized to homocysteine (Hcy), which can enter the transsulfuration pathway for the production of cysteine and lastly glutathione (GSH), the main cellular antioxidant. This vital role of THF comes to an expense. THF and other folate derivatives are susceptible to oxidative breakdown releasing formaldehyde, which can damage DNA -a consequence prevented by the Fanconi Anaemia DNA repair pathway. Epigenetic demethylations catalysed by lysine-specific demethylases (LSD) and Jumonji histone demethylases can also release formaldehyde, constituting a potential threat for genome integrity. In mammals, the toxicity of formaldehyde is limited by a metabolic route centred on the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which oxidizes formaldehyde conjugated to GSH, lastly generating formate. Remarkably, this formate can be a significant source of one-carbon units, thus defining a formaldehyde cycle that likely restricts the toxicity of one-carbon metabolism and epigenetic demethylations. This work describes recent advances in one-carbon metabolism and epigenetics, focusing on the steps that involve formaldehyde flux and that might lead to cytotoxicity affecting human health.

Abstract B170: Therapeutic implications of altered epigenetics and DNA damage responses in IDH2-mutated hematologic diseases

Abstract Acute myeloid leukemia (AML) and angioimmunoblastic T-cell lymphoma (AITL), are hematologic diseases requiring novel approaches to patient selection and therapy. In AITL, neoplastic cells express many markers of T follicular helper (TFH) cells, while in AML, myeloid stem and progenitor cells are affected. Tumor cells of AML and AITL patients frequently bear mutations affecting genes involved in epigenetic regulation, including isocitrate dehydrogenase (IDH) and ten-eleven translocation-2 (TET2). IDH mutations drive production of the rare metabolite D-2-hydroxyglutarate (2HG), which competitively inhibits α-ketoglutarate (α-KG)-dependent dioxygenases such as the TET proteins (affecting DNA methylation) and Jumonji histone demethylases (altering histone methylation). IDH mutations occur in 30% of AML and AITL cases but the spectrum of these mutations differs. In AML, IDH1R132 (41%), IDH2R140 (44%) and IDH2R172 (15%) dominate, and IDH and TET2 mutations are mutually exclusive. In AITL, IDH2R140 (2%) and IDH2R172 (98%) dominate (no IDH1 mutations), with co-mutation of TET2 in 82.1% of cases. In AML, loss of these dioxygenase activities causes epigenetic alterations to DNA and histones, leads to abnormal gene transcription that affects hematopoietic cell differentiation, and drives myeloid disease. However, in AITL disease, the impact of the IDH2 mutation is completely unknown. My goal is to dissect the effects of IDH2 and TET2 mutations in hematologic diseases to understand how IDH2 and TET2 mutations collaborate to drive malignancy in AITL, and why they cooperate differently in myeloid and lymphoid diseases. I focus on DDR signaling and epigenetics analysis to found novel therapeutic vulnerabilities arise from the altered epigenetic regulation and DDR linked to IDH2 and TET2 mutations. I used a CD4-Cre mouse model to introduce the IDH2R172K and TET2 mutations in the T-cell compartment. This would allow me to study the collaboration between IDH2 and TET2 mutations in the context of T-cells. Mice bearing both IDH2 and TET2 mutations show decreased survival, with a median survival of approximately 8 months accompanied by splenomegaly and lymphadenopathy. This is significantly different than CD4+ control mice or single mutant mice. Double mutant mice (DM) present a disruption of spleen and lymph node architecture. To understand this phenotype, I performed comprehensive flow cytometry staining and, surprisingly, I found that the increased spleen size is not due to T-cell infiltration, but is due to increased erythropoiesis and expansion of immature erythropoietic cells (CD71+, cKit+). We can explain this result by the fact that CD4-Cre is also expressed in some progenitor cells leading to stress induced erythropoiesis. However, if we focus on the T-cell population, we see that DM mice present a T-cell phenotype including a decrease of CD4+ naïve cells and an increase of CD4+ effector memory cells as early as 5 months. So, there is an imbalance in T-cell homeostasis, but only when both the IDH2 and TET2 mutations are present, suggesting a cooperative role for both mutations in T-cell development. Since both IDH2 and TET2 affect epigenetic regulation, I want to conduct experiments to understand how these changes modulate T-cells homeostasis. Moreover, emerging data support the hypothesis that connections exist between epigenetic regulators and DDR signaling in hematologic diseases. My final aim will be to attempt to evaluate the efficacy of treatment with IDH2 inhibitors, hypomethylating agents, or DDR-targeting drugs, alone or in combination in AITL disease and identify factors involved in responses to these therapies or in the development of resistance. Finally, I will compare results obtained in AML versus AITL mouse models and clinical samples to identify mechanisms that are shared, and those that are unique to each disease. Citation Format: Julie Leca. Therapeutic implications of altered epigenetics and DNA damage responses in IDH2-mutated hematologic diseases [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B170.

L-ascorbic acid: A true substrate for HIF prolyl hydroxylase?

L-Ascorbate (L-Asc), but not D-isoascorbate (D-Asc) and N-acetylcysteine (NAC) suppress HIF1 ODD-luc reporter activation induced by various inhibitors of HIF prolyl hydroxylase (PHD). The efficiency of suppression by L-Asc was sensitive to the nature of HIF PHD inhibitor chosen for reporter activation. In particular, the inhibitors developed to compete with alpha-ketoglutarate (αKG), were less sensitive to suppression by the physiological range of L-Asc (40–100 μM) than those having a strong iron chelation motif. Challenging those HIF activators in the reporter system with D-Asc demonstrated that the D-isomer, despite exhibiting the same reducing potency with respect to ferric iron, had almost no effect compared to L-Asc. Similarly, no effect on reporter activation was observed with cell-permeable reducing agent NAC up to 1 mM. Docking of L-Asc and D-Asc acid into the HIF PHD2 crystal structure showed interference of Tyr310 with respect to D-Asc. This suggests that L-Asc is not merely a reducing agent preventing enzyme inactivation. Rather, the overall results identify L-Asc as a co-substrate of HIF PHD that may compete for the binding site of αKG in the enzyme active center. This conclusion is in agreement with the results obtained recently in cell-based systems for TET enzymes and jumonji histone demethylases, where L-Asc has been proposed to act as a co-substrate and not as a reducing agent preventing enzyme inactivation.

Systematic discovery of genetic modulation by Jumonji histone demethylases in Drosophila

Jumonji (JmjC) domain proteins influence gene expression and chromatin organization by way of histone demethylation, which provides a means to regulate the activity of genes across the genome. JmjC proteins have been associated with many human diseases including various cancers, developmental and neurological disorders, however, the shared biology and possible common contribution to organismal development and tissue homeostasis of all JmjC proteins remains unclear. Here, we systematically tested the function of all 13 Drosophila JmjC genes. Generation of molecularly defined null mutants revealed that loss of 8 out of 13 JmjC genes modify position effect variegation (PEV) phenotypes, consistent with their ascribed role in regulating chromatin organization. However, most JmjC genes do not critically regulate development, as 10 members are viable and fertile with no obvious developmental defects. Rather, we find that different JmjC mutants specifically alter the phenotypic outcomes in various sensitized genetic backgrounds. Our data demonstrate that, rather than controlling essential gene expression programs, Drosophila JmjC proteins generally act to “fine-tune” different biological processes.

Macrophage Polarization In The Tumor Microenvironment

Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells. We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets. Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used. Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented. We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.

Metabolism and epigenetics in the nervous system: Creating cellular fitness and resistance to neuronal death in neurological conditions via modulation of oxygen-, iron-, and 2-oxoglutarate-dependent dioxygenases

Modern definitions of epigenetics incorporate models for transient but biologically important changes in gene expression that are unrelated to DNA code but responsive to environmental changes such as injury-induced stress. In this scheme, changes in oxygen levels (hypoxia) and/or metabolic co-factors (iron deficiency or diminished 2-oxoglutarate levels) are transduced into broad genetic programs that return the cell and the organism to a homeostatic set point. Over the past two decades, exciting studies have identified a superfamily of iron-, oxygen-, and 2-oxoglutarate-dependent dioxygenases that sit in the nucleus as modulators of transcription factor stability, co-activator function, histone demethylases, and DNA demethylases. These studies have provided a concrete molecular scheme for how changes in metabolism observed in a host of neurological conditions, including stroke, traumatic brain injury, and Alzheimer׳s disease, could be transduced into adaptive gene expression to protect the nervous system. We will discuss these enzymes in this short review, focusing primarily on the ten eleven translocation (TET) DNA demethylases, the jumonji (JmJc) histone demethylases, and the oxygen-sensing prolyl hydroxylase domain enzymes (HIF PHDs).This article is part of a Special Issue entitled SI: Neuroprotection.

Cell-type-specific Jumonji histone demethylase gene expression in the healthy rat CNS: detection by a novel flow cytometry method.

Our understanding of how histone demethylation contributes to the regulation of basal gene expression in the brain is largely unknown in any injury model, and especially in the healthy adult brain. Although Jumonji genes are often regulated transcriptionally, cell-specific gene expression of Jumonji histone demethylases in the brain remains poorly understood. Thus, in the present study we profiled the mRNA levels of 26 Jumonji genes in microglia (CD11b+), neurons (NeuN+) and astrocytes (GFAP+) from the healthy adult rat brain. We optimized a method combining a mZBF (modified zinc-based fixative) and FCM (flow cytometry) to simultaneously sort cells from non-transgenic animals. We evaluated cell-surface, intracellular and nuclear proteins, including histones, as well as messenger- and micro-RNAs in different cell types simultaneously from a single-sorted sample. We found that 12 Jumonji genes were differentially expressed between adult microglia, neurons and astrocytes. While JMJD2D was neuron-restricted, PHF8 and JMJD1C were expressed in all three cell types although the expression was highest in neurons. JMJD3 and JMJD5 were expressed in all cell types, but were highly enriched in microglia; astrocytes had the lowest expression of UTX and JHDM1D. Levels of global H3K27 (H3 lysine 27) methylation varied among cell types and appeared to be lowest in microglia, indicating that differences in basal gene expression of specific Jumonji histone demethylases may contribute to cell-specific gene expression in the CNS (central nervous system). This multiparametric technique will be valuable for simultaneously assaying chromatin modifications and gene regulation in the adult CNS.

Le (R)-2-hydroxyglutarate ou (R)-2HG est bien un oncométabolite

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo and, in this way, may further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases.

Reconstitution of Nucleosome Demethylation and Catalytic Properties of a Jumonji Histone Demethylase

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo and, in this way, may further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases.

Investigating Roles of Reader Domains in Regulating Activity of Jumonji Histone Demethylases

Methylation of histones, protein subunits of nucleosome particles within chromatin, has a profound impact on regulation of transcription. This post‐translational modification is controlled by opposing action of histone methyltransferases and histone demethylases, enzymes that install and remove histone methyl marks, respectively. Jumonji histone demethylases are Fe(II)‐ and alpha‐ketoglutarate‐ dependent oxygenases that remove methyl marks from side chain amino groups of lysines. It is postulated that, in analogy to other members of the superfamily, demethylation proceeds by a radical mechanism where hydrogen atom abstraction from the methyl group, followed by oxygen rebound, results in the formation of a hemiaminal intermediate which fragments to release formaldehyde and demethylated product. The precise control of the activity of demethylases is crucial, and misregulation of demethylation is associated with various human diseases. In addition to a catalytic domain that harbors the iron binding site, several families of demethylases also contain reader domains which recognize chromatin modifications. In this talk, our finding on regulation of the catalytic activity of jumonji histone demethylases by their reader domains will be presented.

Pre-Clinical Studies of Epigenetic Therapies Targeting Histone Modifiers in Lung Cancer

Treatment options for lung cancer patients have been generally limited to standard therapies or targeted interventions which involve a small number of known mutations. Although the targeted therapies are initially successful, they most often result in drug resistance, relapse, and mortality. We now know that the complexity of lung cancer comes not only from genomic changes, but also from aberrant epigenetic regulatory events. Epigenetic therapies have shown promise as single agents in the treatment of hematological malignancies but have yet to meet this expectation in solid tumors thus fostering researchers to pursue new approaches in the development and use of epigenetic interventions. Here, we review some recent pre-clinical findings involving the use of drugs targeting histone modifying enzymes both as single agents and as co-therapies against lung cancer. A greater understanding of the impact of these epigenetic compounds in lung cancer signaling is needed and further evaluation in vivo is warranted in several cases based on the pre-clinical activity of a subset of compounds discussed in this review, including drugs co-targeting HDACs and EGF receptor, targeting Brd4 and targeting Jumonji histone demethylases.

An inhibitor of histone demethylases specifically blocks cancer growth in vitro and in vivo

Histone modifying enzymes are highly deregulated in cancers, leading to aberrant transcriptional programs and thus constitute key therapeutic targets. Using an unbiased cell‐based screen, we have identified a novel compound that modulates the activity of Jumonji histone demethylases. Here, we report the anti‐cancer properties and the mode of action of this small molecule called Elizib. We show, through viability measurements across a large panel of human cancer and normal epithelial cells, that this drug has remarkable specificity for cancer with IC50 values as low as 2 nM. In vivo, it effectively reduces tumor burden in mouse xenograft models. Elizib causes cancer‐cell specific changes in cell cycle progression, upregulates pro‐apoptotic and differentiation genes and downregulates proliferative genes, partially re‐establishing a normal transcriptional program in cancer cells, leading to their demise. Enzyme activity assays and chromatin immunoprecipitation studies demonstrate that our small molecule exerts these activities in cell culture and in vivo at least in part through inhibition of demethylases of the Jumonji family. Thus, we have identified a novel epigenetic inhibitor with specific anti‐cancer activity in cells and in vivo. Elizib opens up a new therapeutic venue and is a novel chemical tool to unravel aspects of epigenetic transcriptional regulation in cancer.

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 pO 2 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 miniaturized screen for inhibitors of Jumonji histone demethylases

2-Oxoglutarate- and Fe(ii)-dependent oxygenases are a major class of N(epsilon)-methyl lysine demethylases that are involved in epigenetic regulation. Assays suitable for implementation in a high-throughput manner have been lacking for these enzymes. Here, we describe the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of JMJD2E using a formaldehyde dehydrogenase-coupled reaction with real-time fluorescence detection. Reactant compatibility studies resulted in simplification of the assay scheme to the mixing of two reagent solutions, both of which were stable overnight. The assay was miniaturized to a 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Inhibitors identified by the screen were further characterized in secondary assays including FDH counterscreen and demethylation assays that monitored demethylation by MALDI-TOF MS. The assay developed here will enable the screening of large compound libraries against the Jumonji demethylases in a robust and automated fashion.