Jumonji Domain Containing-3 Research Articles

JMJD3 ablation in myeloid cells confers renoprotection in mice with DOCA/salt-induced hypertension.

Hypertension-induced renal injury is characterized by robust inflammation and tubulointerstitial fibrosis. Jumonji domain containing-3 (JMJD3) is closely linked with inflammatory response and fibrogenesis. Here we examined the effect of myeloid JMJD3 ablation on kidney inflammation and fibrosis in deoxycorticosterone acetate (DOCA)/salt hypertension. Our results showed that JMJD3 is notably induced in the kidneys with hypertensive injury. DOCA/salt stress causes an elevation in blood pressure that was no difference between myeloid specific JMJD3-deficient mice and wild-type control mice. Compared with wild-type control mice, myeloid JMJD3 ablation ameliorated kidney function and injury of mice in response to DOCA/salt challenge. Myeloid JMJD3 ablation attenuated collagen deposition, extracellular matrix proteins expression, and fibroblasts activation in injured kidneys following DOCA/salt treatment. Furthermore, myeloid JMJD3 ablation blunts inflammatory response in injured kidneys after DOCA/salt stress. Finally, myeloid JMJD3 ablation precluded myeloid myofibroblasts activation and protected against macrophages to myofibroblasts transition in injured kidneys. These beneficial effects were accompanied by reduced expression of interferon regulator factor 4. In summary, JMJD3 ablation in myeloid cells reduces kidney inflammation and fibrosis in DOCA salt-induced hypertension. Inhibition of myeloid JMJD3 may be a novel potential therapeutic target for hypertensive nephropathy. Myeloid JMJD3 deficiency reduces inflammatory response, myeloid fibroblasts activation, macrophages to myofibroblasts transition, and delays kidney fibrosis progression.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

HMGA1 Aggravates Oxidative Stress Injury and Inflammatory Responses in IL-1β-Induced Primary Chondrocytes through the JMJD3/ZEB1 Axis

Introduction: Osteoarthritis (OA) is associated with oxidative stress injury (OSI) and inflammatory responses in chondrocytes. This study sought to explore the mechanism of high mobility group A1 (HMGA1) in interleukin-1beta (IL-1β)-induced OSI and inflammatory responses in primary chondrocytes. Methods: Primary chondrocytes were cultured and treated with IL-1β to establish an OA-cell model. Levels of HMGA1, Jumonji domain-containing 3 (JMJD3), and ZEB1 in cells were determined by real-time quantitative polymerase chain reaction and Western blot analysis. Cell viability, contents of tumor necrosis factor-α, IL-6, and IL-10, reactive oxygen species level, and glutathione peroxidase activity were assessed by the cell counting kit-8 assay, enzyme-linked immunosorbent assay, and assay kits. Enrichment levels of HMGA1 on the JMJD3 promoter and enrichment levels of JMJD3 and trimethylated histone H3 at lysine 27 (H3K27me3) on the ZEB1 promoter region were determined by chromatin immunoprecipitation. Functional rescue experiments were performed to analyze the impact of ZEB1 and JMJD3 on IL-1β-induced chondrocytes. Results: IL-1β treatment induced HMGA1 upregulation, OSI, and inflammatory responses in chondrocytes. HMGA1 downregulation reduced IL-1β-induced OSI and inflammatory responses in chondrocytes. Mechanically, HMGA1 was bound to the JMJD3 promoter to promote JMJD3 transcription, and JMJD3 induced demethylation of H3K27me3 on the ZEB1 promoter to promote ZEB1 transcription. Overexpression of JMJD3 or ZEB1 neutralized the protective role of silencing HMGA1 in IL-1β-induced chondrocytes. Conclusion: HMGA1 aggravated IL-1β-induced OSI and inflammatory responses in chondrocytes through the promotion of JMJD3 and ZEB1.

Decreased Jumonji Domain-Containing 3 at the Promoter Downregulates Hematopoietic Progenitor Kinase 1 Expression and Cytoactivity of T Follicular Helper Cells from Systemic Lupus Erythematosus Patients.

T follicular helper (Tfh) cells are overactivated in systemic lupus erythematosus (SLE) patients and contribute to excessive immunity. Hematopoietic progenitor kinase 1 (HPK1), as an inhibitor of T cells, is underexpressed in SLE Tfh cells and consequently induces autoimmunity. However, the reason for downregulation of HPK1 in SLE Tfh cells remains elusive. By combining chromatin immunoprecipitation with quantitative polymerase chain reaction assays, it was found that histone H3 lysine 27 trimethylation (H3K27me3) at the HPK1 promoter in SLE Tfh cells elevated greatly. We also confirmed jumonji domain-containing 3 (JMJD3) binding at the HPK1 promoter in SLE Tfh cells reduced profoundly. Knocking down JMJD3 in normal Tfh cells with siRNA alleviated enrichments of JMJD3, H3K4me3, and mixed-lineage leukemia (MLL) 1 at the HPK1 promoter and increased H3K27me3 number in the region. HPK1 expression was lowered, while Tfh cell proliferation activity, IL-21 and IFNγ secretions in the supernatants of Tfh cells, and IgG1 and IgG3 concentrations in the supernatants of Tfh-B cell cocultures all upregulated markedly. In contrast, elevating JMJD3 amount in SLE Tfh cells by JMJD3-overexpressed plasmid showed opposite effects. The abundances of H3K4me3 and MLL1 at the HPK1 promoter in SLE Tfh cells were greatly attenuated. Our results suggest that deficient JMJD3 binding at the promoter dampens HPK1 expression in SLE Tfh cells, thus making Tfh cells overactive, and ultimately results in onset of SLE.

JMJD3-deficiency in CD4+ T cells attenuates atherosclerosis by modulating T-cell polarization

Background and Aims : Atherosclerosis is a chronic inflammatory disease, which drives the progression of luminal plaques in arteries. T cells help orchestrate the underlying inflammation through polarization into pro-atherogenic and atheroprotective subsets. Epigenetic signatures including H3K27me3 can skew T-cell polarization, which may ultimately affect atheroprogression. We hypothesize the H3K27 demethylase, Jumonji domain containing 3 (JMJD3) may contribute to atheroprogression through modulation of T helper (Th) cell polarization.

JMJD3 deficiency alleviates lipopolysaccharide‑induced acute lung injury by inhibiting alveolar epithelial ferroptosis in a Nrf2‑dependent manner.

Acute respiratory distress syndrome (ARDS) is a deadly illness which presents with severe hypoxemia as well as diffuse alveolar damage. Jumonji domain‑containing3 (JMJD3), which belongs to the UTX/UTY JmjC‑domain protein subfamily, is involved in infection, development, aging and immune disorders. However, the role of JMJD3 in acute lung injury (ALI) is still unclear. The present study explored the roles and potential mechanisms of JMJD3 in ALI. Alveolar epithelial cell‑specific knockout of JMJD3 mice and A549 alveolar epithelial cells were used to investigate the function of JMJD3 in ALI. Lipopolysaccharide (LPS) was used to establish an invivo and invitro ALI model. The expression of JMJD3 in murine lung tissue and alveolar epithelial cells was detected. Pathological injury of lung tissue and alveolar epithelial cells was also investigated following inhibition of JMJD3. The results showed that JMJD3 expression was significantly increased in murine lung tissues and in A549 cells following LPS stimulation. JMJD3‑deficient mice in alveolar epithelial cells exhibited alleviated lung pathological injury and ferroptosis following h stimulation. Mechanistically, it was found that JMJD3 knockout could increase the expression of nuclear factor erythroid‑2‑related factor‑2 (Nrf2) in lung tissues challenged with h. However, Nrf2 overexpression by adenovirus could further enhance the anti‑ferroptotic effect from JMJD3 silence in h‑treated A549 cells. Taken together, the present study revealed that JMJD3 deficiency may relieve LPS‑induced ALI by blocking alveolar epithelial ferroptosis in a Nrf2‑dependent manner, which may serve as a novel therapeutic target against ALI.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus–associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Epigenetic regulation of TGF-\u03b2-induced EMT by JMJD3/KDM6B histone H3K27 demethylase

Transforming growth factor-β (TGF-β) signaling pathways are well-recognized for their role in proliferation and epithelial–mesenchymal transition (EMT) of cancer cells, but much less is understood about their contribution to interactions with other signaling events. Recent studies have indicated that crosstalk between TGF-β and Ras signaling makes a contribution to TGF-β-mediated EMT. Here, we demonstrate that Jumonji domain containing-3 (JMJD3 also called KDM6B) promotes TGF-β-mediated Smad activation and EMT in Ras-activated lung cancer cells. JMJD3 in lung cancer patients was significantly increased and JMJD3 expression in lung tumor tissues was correlated with expression of K-Ras or H-Ras in particular, and its expression was regulated by Ras activity in lung cancer cells. JMJD3 promotes TGF-β-induced Smad activation and EMT in Ras-activated lung cancer cells through the induction of syntenin, a protein that regulates TGF-β receptor activation upon ligand binding. Tissue array and ChIP analysis revealed that JMJD3 epigenetically induces syntenin expression by directly regulating H3K27 methylation levels. Mechanical exploration identified a physical and functional association of JMJD3 with syntenin presiding over the TGF-β in Ras-activated lung cancer cells. Taken together, these findings provide new insight into the mechanisms by which JMJD3 promotes syntenin expression resulting in oncogenic Ras cooperation with TGF-β to promote EMT.

Histone demethylase JMJD3 protects against renal fibrosis by suppressing TGFβ and Notch signaling and preserving PTEN expression.

Rationale: The Jumonji domain containing-3 (JMJD3), a specific histone demethylase for trimethylation on histone H3 lysine 27 (H3K27me3), is associated with the pathogenesis of many diseases, but its role in renal fibrosis remains unexplored. Here we examined the role of JMJD3 and mechanisms involved in the activation of renal fibroblasts and development of renal fibrosis.Methods: Murine models of 5/6 surgical nephrectomy (SNx) and ureteral unilateral obstruction (UUO) were used to assess the effect of a specific JMJD3 inhibitor, GSKJ4, and genetic deletion of JMJD3 from FOXD1 stroma-derived renal interstitial cells on the development of renal fibrosis and activation of renal interstitial fibroblasts. Cultured rat renal interstitial fibroblasts (NRK-49F) and mouse renal tubular epithelial cells (mTECs) were also used to examine JMJD3-mediated activation of profibrotic signaling.Results: JMJD3 and H3K27me3 expression levels were upregulated in the kidney of mice subjected to SNx 5/6 and UUO. Pharmacological inhibition of JMJD3 with GSKJ4 or genetic deletion of JMJD3 led to worsening of renal dysfunction as well as increased deposition of extracellular matrix proteins and activation of renal interstitial fibroblasts in the injured kidney. This was coincident with decreased expression of Smad7 and enhanced expression of H3K27me3, transforming growth factor β1 (TGFβ1), Smad3, Notch1, Notch3 and Jagged1. Inhibition of JMJD3 by GSK J4 or its specific siRNA also resulted in the similar responses in cultured NRK-49F and mTECs exposed to serum or TGFβ1. Moreover, JMJD3 inhibition augmented phosphorylation of AKT and ERK1/2 in vivo and in vitro.Conclusion: These results indicate that JMJD3 confers anti-fibrotic effects by limiting activation of multiple profibrotic signaling pathways and suggest that JMJD3 modulation may have therapeutic effects for chronic kidney disease.

Modification of histone 3 lysine 27 (H3K27) trimethylation in EZH2 and JMJD3 deficient T cells attenuates atherosclerosis through polarization towards anti-inflammatory phenotypes

Abstract Background The trimethylation status of Histone 3 Lysine 27 (H3K27), which is regulated by the methylating enzyme Enchancer of zeste homolog 2 (EZH2) and the demethylating enzyme Jumonji domain containing 3 (JMJD3), is a critical epigenetic signature for the differentiation and polarization of T cells. During atherogenesis, T cells initiate and propagate lesion formation through imbalanced polarization of T helper (Th) cells leading to the accumulation of pro-atherogenic T cell subsets, including Th1, that exacerbate inflammation. Purpose We hypothesize that T-cell EZH2 and JMJD3 may contribute to inflammation and atherosclerotic plaque formation through opposing polarization of T helper cells. Methods We generated transgenic mice with either Ezh2 or Jmjd3 flanked by loxP-sites that were sensitive to Cre-mediated inactivation. Mice were backcrossed to apolipoprotein E (ApoE−/−) mice expressing Cre in T cells (Cd4Cretg) to generate hypercholesterolemic Ezh2fl/fl-Cd4Cre-ApoE−/− (Ezh2fl/fl) and Jmjd3fl/fl-Cd4Cre-ApoE−/− (Jmjd3fl/fl) mice. Following a 6 week high fat diet, the immune status and atherosclerotic progression of the mice were assessed by histology, flow cytometry, and RNA sequencing. Results Mass spectrometry of histones isolated from CD4+ T cells confirmed a decrease in H3K27 trimethylation in Ezh2fl/fl mice and an increase in Jmjd3fl/fl mice (p=0.0002 and p=0.01, respectively). In the aortic root, both Ezh2fl/fl and Jmjd3fl/fl mice developed significantly less atherosclerosis (p=0.001 and p=0.0006, respectively). A shift from naive T cells to effectors was observed in the lymph nodes and spleens in both models (p<0.0001 for all). Flow cytometric analysis revealed a systemic accumulation of Th2 in Ezh2fl/flmice (p<0.0001), which was corroborated by a 2.75 log2 fold change of IL-4 expression (padj<0.0001) (RNA-sequencing) in CD4+ T cells as well as elevated IL-4 plasma concentrations (p=0.04). Ingenuity pathway analysis revealed the canonical Th1 pathway was inhibited in Ezh2fl/fl mice. In Jmjd3fl/fl mice, flow cytometric analysis revealed a 2 fold increase of Foxp3-expressing T regulatory (Treg) cells in the blood (p=0.005), lymph nodes (p<0.0001), and spleen (p<0.0001). Conclusions Our data demonstrate deficiency of either EZH2 or JMJD3 strongly reduce lesion progression in atherosclerosis through polarization of T cells towards anti-atherogenic Th2 or Treg populations, respectively. Targeting T-cell H3K27 trimethylation may be a promising candidate for further investigation to treat atherosclerosis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG)

Histone Demethylase JMJD3 Mediated Doxorubicin-Induced Cardiomyopathy by Suppressing SESN2 Expression.

Jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, specifically catalyzes the demethylation of H3K27 (H3K27me3) and regulates gene expression. Sestrin2 (SESN2), a stress-inducible protein, protected against doxorubicin (DOX)-induced cardiomyopathy by regulating mitophagy and mitochondrial function. Here, the expression of JMJD3 was increased and that of SESN2 was decreased in both the heart samples from patients with dilated cardiomyopathy and chronic DOX-stimulation induced cardiomyopathy. Inhibition or knockdown of JMJD3 attenuated DOX-induced cardiomyocytes apoptosis, mitochondrial injury and cardiac dysfunction. However, JMJD3 overexpression aggravated DOX-induced cardiomyopathy, which were relieved by SESN2 overexpression. JMJD3 inhibited the transcription of SESN2 by reducing tri-methylation of H3K27 in the promoter region of SESN2. In conclusion, JMJD3 negatively regulated SESN2 via decreasing H3K27me3 enrichment in the promoter region of SESN2, subsequently inducing mitochondrial dysfunction and cardiomyocytes apoptosis. Targeting the JMJD3-SESN2 signaling axis may be a potential therapeutic strategy to protect against DOX-mediated cardiomyopathy.

Histone methylation and demethylation are implicated in the transient and sustained activation of the interleukin-1β gene in murine macrophages.

Macrophages play a crucial role in the development of atherosclerosis. To explore the mechanism by which macrophages attain a proinflammatory phenotype for a sustained period, we stimulated macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) and measured the interleukin-1β (IL-1β) expression. The IL-1β expression increased transiently, and its expression lasted for, at least, 1week after the cessation of LPS and IFN-γ stimulation. At the promoter region of the IL-1β gene, the demethylation of histone H3 lysine 27 (H3K27) was significantly induced for 1week after transient stimulation with LPS and IFN-γ. The expression of H3K27 demethylases ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) and jumonji domain-containing 3 (JMJD3) increased significantly for 1week after transient stimulation with LPS and IFN-γ. When the UTX expression was inhibited by using small interfering RNA (siRNA) for UTX, the IL-1β expression was significantly suppressed in both transient and sustained phases, whereas siRNA for JMJD3 significantly inhibited only the sustained phase of the IL-1β expression. These results suggested that H3K27 demethylation was implicated in the transient and sustained increase in the IL-1β expression after LPS and IFN-γ stimulation.

Jumonji domain containing-3 (JMJD3)inhibition attenuatesIL-1β-inducedchondrocytes damage in vitro and protects osteoarthritis cartilage in vivo.

This study aimed to explore the effects and relative mechanism of JMJD3 on knee osteoarthritis (OA). In this study, we first analyzed the expression of JMJD3 in OA cartilage using western blot and immunohistochemistry. In an in vitro study, the effects of GSK-J4, JMJD3 inhibitor, on ATDC-5 chondrocytes were evaluated by CCK-8 assay. Real-time PCR and western blot were used to examine the inhibitory effect of GSK-J4 on the inflammation and ECM degradation of chondrocytes. NF-κB p65 phosphorylation and nuclear translocation were measured by western blot and immunofluorescence. In the animal study, twenty mice were randomized into four experimental groups: sham group, DMM-induced OA + DMSO group, OA + low-dose GSK-J4 group, and OA + high-dose GSK-J4 group. After the treatment, hematoxylin-eosin and safranin O/fast green staining were used to evaluate cartilage degradation of knee joint, with OARSI scores for quantitative assessment of cartilage damage. Our results revealed that JMJD3 was overexpressed in OA cartilage and GSK-J4 could suppress the IL-1β-induced production of pro-inflammatory cytokines and catabolic enzymes, including IL-6, IL-8, MMP-9 and ADAMTS-5. Consistent with these findings, GSK-J4 could inhibit IL-1β-induced degradation of collagen II and aggrecan. Mechanistically, GSK-J4 dramatically suppressed IL-1β-stimulated NF-κB signal pathway activation. In vivo, GSK-J4 prevented cartilage damage in mouse DMM-induced OA model. This study elucidates the important role of JMJD3 in cartilage degeneration in OA, and our results indicate that JDJM3 may become a novel therapeutic target in OA therapy.

Epigenetic Regulation of KL (Klotho) via H3K27me3 (Histone 3 Lysine [K] 27 Trimethylation) in Renal Tubule Cells.

KL (klotho) levels decline with age, which is an important mechanistic driver of aging. KL gene deficiency is associated with hypertension. The purpose of this study is to investigate the potential role of H3K27me3 (histone 3 lysine [K] 27 trimethylation) in the regulation of KL gene expression and examine the related molecular pathways that may drive kidney cell aging. Kidneys were collected from 6-month-old WT (wild type; young WT), 30-month-old WT (aged WT), and 6- (young) and 20-month-old (aged) KL mutant mice, respectively. We demonstrated that the H3K27me3 level was increased in kidneys of aged WT and KL mutant mice versus young WT mice. Elevation of H3K27me3 levels was likely due to downregulation of the H3K27 (histone H3 Lys 27)-specific demethylase JMJD3 (the Jumonji domain containing-3) in the aged kidneys. Inhibition of PRC2 (polycomb repressive complex C2; histone trimethyltransferase) decreased the H3K27me3 levels leading to an increase in the expression of KL in cultured primary renal tubule cells assessed by Western blot and KL promoter activity assays. The chromatin immunoprecipitation qPCR assay revealed that H3K27me3 was physically associated with the KL promoter region. Furthermore, aging impaired the SGK1 (serum- and glucocorticoid-induced protein kinase 1)/FOXO3a (the forkhead box class O 3a) signaling leading to upregulation of p53 and p16 (aging markers) in the kidney of aged WT mice. KL may regulate the SGK1/FOXO3 signaling, which was decreased due to KL deficiency. Thus, aging-associated downregulation of KL gene expression may be partly attributed to upregulation of H3K27me3 levels. Downregulation of KL may impair the SGK1/FOXO3 signaling contributing to kidney cell aging.

JMJD3-regulated expression of IL-6 is involved in the proliferation and chemosensitivity of acute myeloid leukemia cells.

Emerging evidence shows that histone modification and its related regulators are involved in the progression and chemoresistance of multiple tumors including acute myeloid leukemia cells (AML). Our present study found that the expression of histone lysine demethylase Jumonji domain containing-3 (JMJD3) was increased in AML cells as compared with that in human primary bone marrow (HPBM) cells. Knockdown of JMJD3 can decrease the proliferation of AML cells and increase the chemosensitivity of daunorubicin (DNR) and cytarabine (Ara-C). By screening the expression of cytokines involved in AML progression, we found that knockdown of JMJD3 can inhibit the expression of interleukin-6 (IL-6). Recombinant IL-6 (rIL-6) can attenuate si-JMJD3-suppressed proliferation of AML cells. Mechanistically, JMJD3 can positively regulate the promoter activity and transcription of IL-6 mRNA, while had no effect on its mRNA stability. Further, JMJD3 can regulate the expression of p65, which can directly bind with promoter of IL-6 to increase its transcription. Over expression of p65 significantly attenuated si-JMJD3-suppressed expression of IL-6. Collectively, we revealed that JMJD3 can regulate the proliferation and chemosensitivity of AML cells via upregulation of IL-6. It suggested that JMJD3 might be a potential therapy target for AML treatment.

Abstract TP99: Hypoxia Drives Anti-Inflammatory Polarization of Microglia Through the JMJD3-IRF4 Axis

Background: Microglia, as the innate immune cells of the central nervous system, are poised to be the first responders of the immune system to ischemic stroke. In response to an injury or stimulus, microglia polarize from a resting to a pro- or anti-inflammatory phenotype. We have previously shown that hypoxia, such as that in the setting of ischemic stroke, acts as a stimulus itself to polarize microglia to an anti-inflammatory phenotype. Additionally, we have shown that Jumonji Domain Containing 3 (JMJD3) is a histone demethylase that is important for anti-inflammatory polarization of microglia. JMJD3 functions to demethylate trimethylated histone H3 lysine 27 (H3K27me3) at the Irf4 promoter to monomethylation marks (H3K27me1), resulting in transcriptional activation of Irf4 and downstream expression of anti-inflammatory genes. Hypothesis: Thus, we hypothesized that hypoxia drives microglial anti-inflammatory polarization through the JMJD3-IRF4 axis. Methods: We cultured male and female primary microglia from neonatal P0.5-P3 C57Bl/6 mice, inhibited JMJD3 with GSK-J4 (Cayman Chemical) or DMSO control, and stimulated cultures with oxygen glucose deprivation (OGD). After 8hr OGD, we isolated RNA (RNEasy Micro Plus kit, Qiagen) and performed down-stream real-time PCR array analysis (PrimePCR Arrays, Bio-Rad) or protein (RIPA Buffer, Thermo Fisher) for Western blot analysis (Bio-Rad) for JMJD3, histone H3 (Abcam), H3K27me1, and H3K27me3 (Millipore). Results: We found that OGD induced up-regulation anti-inflammatory genes, notably Csf1 , Cntf , Gdf15 , Il1rn , Irf4 , Mif , and Vegfa while down-regulating pro-inflammatory genes Tnfa , Il1b , and Il6 (n=4, p<0.05). Additionally, we found that Kdm6b (the gene encoding JMJD3) was up-regulated following 8hr OGD, suggesting JMJD3 is involved in hypoxia-induced anti-inflammatory microglial phenotype polarization. Pharmacological inhibition of JMJD3 with GSK-J4 prevented demethylation of H3K27me3 at the Irf4 promoter and blocked hypoxia-induced up-regulation of Irf4 and abrogated hypoxia-induced anti-inflammatory gene up-regulation. Conclusion: In conclusion, our data suggests that hypoxia polarizes microglia to an anti-inflammatory phenotype through the JMJD3-IRF4 axis.

Epigenetic and transcriptional modulation of WDR5, a chromatin remodeling protein, in Huntington's disease human induced pluripotent stem cell (hiPSC) model.

DNA methylation (DNAm) changes are of increasing relevance to neurodegenerative disorders, including Huntington's disease (HD). We performed genome-wide screening of possible DNAm changes occurring during striatal differentiation in human induced pluripotent stem cells derived from a HD patient (HD-hiPSCs) as cellular model. We identified 240 differentially methylated regions (DMRs) at promoters in fully differentiated HD-hiPSCs. Subsequently, we focused on the methylation differences in a subcluster of genes related to Jumonji Domain Containing 3 (JMJD3), a demethylase that epigenetically regulates neuronal differentiation and activates neuronal progenitor associated genes, which are indispensable for neuronal fate acquisition. Noticeably among these genes, WD repeat-containing protein 5 (WDR5) promoter was found hypermethylated in HD-hiPSCs, resulting in a significant down-modulation in its expression and of the encoded protein. A similar WDR5 expression decrease was seen in a small series of HD-hiPSC lines characterized by different CAG length. The decrease in WDR5 expression was particularly evident in HD-hiPSCs compared to hESCs and control-hiPSCs from healthy subjects. WDR5 is a core component of the MLL/SET1 chromatin remodeling complexes essential for H3K4me3, previously reported to play an important role in stem cells self-renewal and differentiation. These results suggest the existence of epigenetic mechanisms in HD and the identification of genes, which are able to modulate HD phenotype, is important both for biomarker discovery and therapeutic interventions.

JMJD3 Is Crucial for the Female AVPV RIP-Cre Neuron-Controlled Kisspeptin–Estrogen Feedback Loop and Reproductive Function

The hypothalamic-pituitary-gonadal axis controls development, reproduction, and metabolism. Although most studies have focused on the hierarchy from the brain to the gonad, many questions remain unresolved concerning the feedback from the gonad to the central nervous system, especially regarding the potential epigenetic modifications in hypothalamic neurons. In the present report, we generated genetically modified mice lacking histone H3 lysine 27 (H3K27) demethylase Jumonji domain-containing 3 (JMJD3) in hypothalamic rat-insulin-promoter-expressing neurons (RIP-Cre neurons). The female mutant mice displayed late-onset obesity owing to reduced locomotor activity and decreased energy expenditure. JMJD3 deficiency in RIP-Cre neurons also results in delayed pubertal onset, an irregular estrous cycle, impaired fertility, and accelerated ovarian failure in female mice owing to the dysregulation of the hypothalamic-ovarian axis. We found that JMJD3 directly regulates Kiss1 gene expression by binding to the Kiss1 promoter and triggering H3K27me3 demethylation in the anteroventral periventricular (AVPV) nucleus. Further study confirmed that the aberrations arose from impaired kisspeptin signaling in the hypothalamic AVPV nucleus and subsequent estrogen deficiency. Estrogen replacement therapy can reverse obesity in mutant mice. Moreover, we demonstrated that Jmjd3 is an estrogen target gene in the hypothalamus. These results provide direct genetic and molecular evidence that JMJD3 is a key mediator for the kisspeptin-estrogen feedback loop.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive.To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs.We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. DisclosuresNo relevant conflicts of interest to declare.

H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis.

BackgroundEpigenetic changes (i.e., chromatin modifications) occur during chondrogenesis and in osteoarthritis (OA). We investigated the effect of H3K27me3 demethylase inhibition on chondrogenesis and assessed its utility in cartilage tissue engineering and in understanding cartilage destruction in OA.MethodsWe used a high-content screen to assess the effect of epigenetic modifying compounds on collagen output during chondrogenesis of monolayer human mesenchymal stem cells (MSCs). The impact of GSK-J4 on gene expression, glycosaminoglycan output and collagen formation during differentiation of MSCs into cartilage discs was investigated. Expression of lysine (K)-specific demethylase 6A (UTX) and Jumonji domain-containing 3 (JMJD3), the HEK27Me3 demethylases targeted by GSK-J4, was measured in damaged and undamaged cartilage from patients with OA. The impact of GSK-J4 on ex vivo cartilage destruction and expression of OA-related genes in human articular chondrocytes (HACs) was assessed. H3K27Me3 demethylase regulation of transforming growth factor (TGF)-β-induced gene expression was measured in MSCs and HACs.ResultsTreatment of chondrogenic MSCs with the H3K27me3 demethylase inhibitor GSK-J4, which targets JMJD3 and UTX, inhibited collagen output; expression of chondrogenic genes, including SOX9 and COL2A1; and disrupted glycosaminoglycan and collagen synthesis. JMJD3 but not UTX expression was increased during chondrogenesis and in damaged OA cartilage, suggesting a predominant role of JMJD3 in chondrogenesis and OA. GSK-J4 prevented ex vivo cartilage destruction and expression of the OA-related genes MMP13 and PTGS2. TGF-β is a key regulator of chondrogenesis and articular cartilage homeostasis, and TGF-β-induced gene expression was inhibited by GSK-J4 treatment of both chondrogenic MSCs and HACs.ConclusionsOverall, we show that H3K27me3 demethylases modulate chondrogenesis and that enhancing this activity may improve production of tissue-engineered cartilage. In contrast, targeted inhibition of H3K27me3 demethylases could provide a novel approach in OA therapeutics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1053-7) contains supplementary material, which is available to authorized users.