An oncogenic JMJD6-DGAT1 axis tunes the epigenetic regulation of lipid droplet formation in clear cell renal cell carcinoma.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

An HSF1-JMJD6-HSP feedback circuit promotes cell adaptation to proteotoxic stress.

Heat Shock Factor 1 (HSF1) is best known as the master transcriptional regulator of the heat-shock response (HSR), a conserved adaptive mechanism critical for protein homeostasis (proteostasis). Combining a genome-wide RNAi library with an HSR reporter, we identified Jumonji domain-containing protein 6 (JMJD6) as an essential mediator of HSF1 activity. In follow-up studies, we found that JMJD6 is itself a noncanonical transcriptional target of HSF1 which acts as a critical regulator of proteostasis. In a positive feedback circuit, HSF1 binds and promotes JMJD6 expression, which in turn reduces heat shock protein 70 (HSP70) R469 monomethylation to disrupt HSP70-HSF1 repressive complexes resulting in enhanced HSF1 activation. Thus, JMJD6 is intricately wired into the proteostasis network where it plays a critical role in cellular adaptation to proteotoxic stress.

Widespread hydroxylation of unstructured lysine-rich protein domains by JMJD6.

The Jumonji domain-containing protein JMJD6 is a 2-oxoglutarate-dependent dioxygenase associated with a broad range of biological functions. Cellular studies have implicated the enzyme in chromatin biology, transcription, DNA repair, mRNA splicing, and cotranscriptional processing. Although not all studies agree, JMJD6 has been reported to catalyze both hydroxylation of lysine residues and demethylation of arginine residues. However, despite extensive study and indirect evidence for JMJD6 catalysis in many cellular processes, direct assignment of JMJD6 catalytic substrates has been limited. Examination of a reported site of proline hydroxylation within a lysine-rich region of the tandem bromodomain protein BRD4 led us to conclude that hydroxylation was in fact on lysine and catalyzed by JMJD6. This prompted a wider search for JMJD6-catalyzed protein modifications deploying mass spectrometric methods designed to improve the analysis of such lysine-rich regions. Using lysine derivatization with propionic anhydride to improve the analysis of tryptic peptides and nontryptic proteolysis, we report 150 sites of JMJD6-catalyzed lysine hydroxylation on 48 protein substrates, including 19 sites of hydroxylation on BRD4. Most hydroxylations were within lysine-rich regions that are predicted to be unstructured; in some, multiple modifications were observed on adjacent lysine residues. Almost all of the JMJD6 substrates defined in these studies have been associated with membraneless organelle formation. Given the reported roles of lysine-rich regions in subcellular partitioning by liquid-liquid phase separation, our findings raise the possibility that JMJD6 may play a role in regulating such processes in response to stresses, including hypoxia.

Cyclin B2 impairs the p53 signaling in nasopharyngeal carcinoma.

BACKGROUND: Cyclin B2 (CCNB2), a member of the cyclin family, is an oncogene in multiple cancers, including nasopharyngeal carcinoma (NPC). However, the epigenetics mechanism for CCNB2 overexpression in NPC remains unclear. This study dissects the regulatory role of CCNB2 in NPC and the molecular mechanism. METHODS: Differentially methylated genes (DMG) and differentially expressed genes (DEG) were screened out in GSE52068 and GSE13597 databases, respectively, and candidate targets were identified by the Venn diagram. GO annotation and pathway enrichment analyses were performed on selected DMG and DEG, and a PPI network was constructed to pinpoint hub genes. PCR and qMSP were conducted to detect the expression and methylation of CCNB2 in cells. The siRNA targeting CCNB2 was transfected into NPC cells, and the migration, proliferation, cell cycle, epithelial-mesenchymal transition (EMT), tumorigenesis, and metastasis were examined. The upstream factor responsible for CCNB2 overexpression in NPC was explored. The p53 activity in NPC cells was assessed using western blot analysis. RESULTS: CCNB2 showed hypomethylation and overexpression in NPC. CCNB2 silencing inhibited cell migration, proliferation, cell cycle entry, and EMT. JMJD6 was overexpressed in NPC and upregulated CCNB2 through demethylation. JMJD6 reversed the effects of CCNB2 downregulation, resulting in elevated cellular activity in vitro and tumorigenic and metastatic activities in vivo. CCNB2 blocked the p53 pathway, while the p53 pathway inhibitor reversed the effect of CCNB2 silencing to increase the activity of NPC cells. CONCLUSIONS: JMJD6 enhanced CCNB2 transcription by demethylating CCNB2, thereby repressing the p53 pathway and promoting NPC progression.

The Ligand of Ate1 is intrinsically disordered and participates in nucleolar phase separation regulated by Jumonji Domain Containing 6.

The Ligand of Ate1 (Liat1) is a protein of unknown function that was originally discovered through its interaction with arginyl-tRNA protein transferase 1 (Ate1), a component of the Arg/N-degron pathway of protein degradation. Here, we characterized the functional domains of mouse Liat1 and found that its N-terminal half comprises an intrinsically disordered region (IDR) that facilitates its liquid-liquid phase separation (LLPS) in the nucleolus. Using bimolecular fluorescence complementation and immunocytochemistry, we found that Liat1 is targeted to the nucleolus by a low-complexity poly-K region within its IDR. We also found that the lysyl-hydroxylase activity of Jumonji Domain Containing 6 (Jmjd6) modifies Liat1, in a manner that requires the Liat1 poly-K region, and inhibits its nucleolar targeting and potential functions. In sum, this study reveals that Liat1 participates in nucleolar LLPS regulated by Jmjd6.

JMJD6 Autoantibodies as a Potential Biomarker for Inflammation-Related Diseases.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima-media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

JMJD6-BRD4 complex stimulates lncRNA HOTAIR transcription by binding to the promoter region of HOTAIR and induces radioresistance in liver cancer stem cells.

BACKGROUND: Long non-coding RNA (lncRNA) HOTAIR acts importantly in liver cancer development, but its effect on radioresistance remains poorly understood. Here, our study probed into the possible impact of HOTAIR in radioresistance in liver cancer stem cells (LCSCs) and to elucidate its molecular basis. METHODS: Following sorting of stem and non-stem liver cancer cells, LCSCs were identified and subjected to RNA-seq analysis for selecting differentially expressed genes. Expression of HOTAIR was determined in liver cancer tissues and CSCs. The stemness, proliferation, apoptosis and radioresistance of LCSCs were then detected in response to altered expression of HOTAIR-LSD1-JMJD6-BRD4. RESULTS: Ectopic HOTAIR expression was found to promote radioresistance of LCSCs by maintaining its stemness. Mechanistic investigations indicated that HOTAIR recruited LSD1 to the MAPK1 promoter region and reduced the level of H3K9me2 in the promoter region, thus elevating ERK2 (MAPK1) expression. JMJD6-BRD4 complex promoted HOTAIR transcription by forming a complex and positively regulated ERK2 (MAPK1) expression, maintaining the stemness of LCSCs, and ultimately promoting their radioresistance in vitro and in vivo. CONCLUSION: Collectively, our work highlights the promoting effect of the JMJD6-BRD4 complex on the radioresistance of LCSCs through a HOTAIR-dependent mechanism.

c-Jun-mediated JMJD6 restoration enhances resistance of liver cancer to radiotherapy through the IL-4-activated ERK pathway.

Radiotherapy is widely used in the treatment of liver cancer, but the efficacy can be limited by radioresistance. In this study, we attempt to delineate the possible molecular mechanism of c-Jun-regulated Jumonji domain-containing protein 6/interleukin 4/extracellular signal-regulated kinase (JMJD6/IL-4/ERK) axis in radioresistance of liver cancer. The expression of c-Jun was quantified in liver cancer tissues and cell lines, and the results indicated that c-Jun was upregulated in liver cancer tissues and cells. We further illustrated the role of c-Jun following gain- and loss-of-function strategies in malignant phenotypes of liver cancer cells. It was established that c-Jun elevated JMJD6 expression and augmented the malignancy and aggressiveness of liver cancer cells. The in vivo effects of c-Jun on radioresistance in liver cancer were validated in nude mice, in response to IL-4 knockdown or the ERK pathway inhibitor, PD98059. In the presence of JMJD6 upregulation, the expression of IL-4 was elevated in mice with liver cancer, which enhanced the radiation resistance. Moreover, knockdown of IL-4 inactivated the ERK pathway, thereby reversing the radiation resistance caused by overexpressed JMJD6 in tumor-bearing mice. Taken together, c-Jun augments the radiation resistance in liver cancer by activating the ERK pathway through JMJD6-upregulated IL-4 transcription.

JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg-1·d-1) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

The JMJD6/HURP axis promotes cell migration via NF-κB-dependent centrosome repositioning and Cdc42-mediated Golgi repositioning.

Golgi apparatus (GA) and centrosome reposition toward cell leading end during directional cell migration in a coupling way, thereby determining cell polarity by transporting essential factors to the proximal plasma membrane. The study provides mechanistic insights into how GA repositioning (GR) is regulated, and how GR and centrosome repositioning (CR) are coupled. Our previous published works reveals that PRMT5 methylates HURP at R122 and the HURP m122 inhibits GR and cell migration by stabilizing GA-associated acetyl-tubulin and then rigidifying GA. The current study further shows that the demethylase JMJD6-guided demethylation of HURP at R122 promotes GR and cell migration. The HURP methylation mimicking mutant 122 F blocks JMJD6-induced GR and cell migration, suggesting JMJD6 relays GR stimulating signal to HURP. Mechanistic studies reveal that the HURP methylation deficiency mutant 122 K promotes GR through NF-κB-induced CR and subsequently CR-dependent Cdc42 upregulation, where Cdc42 couples CR to GR. Taken together, HURP methylation statuses provide a unique opportunity to understand how GR is regulated, and the GA intrinsic mechanism controlling Golgi rigidity and the GA extrinsic mechanism involving NF-κB-CR-Cdc42 cascade collectively dictate GR.

Regulatory role of JMJD6 in placental development.

Correct placental development and function are critical to both the mother's and the foetus' health during pregnancy. Placental function depends on the correct development of the vascular network, which requires proper angiogenesis. Impaired angiogenesis in the placenta can induce foetal growth restriction, preeclampsia, and even foetal death. Placental angiogenesis is finely controlled by ubiquitous and pregnancy-specific angiogenic factors. Jumonji domain-containing protein 6 (JMJD6) is a Fe (II)- and 2-oxoglutarate (2OG)-dependent oxygenase that catalyses arginine demethylation and lysine hydroxylation of histone and non-histone peptides. JMJD6 has been implicated in embryonic development, cellular proliferation and migration, self-tolerance induction in the thymus, and adipocyte differentiation. In this review we present JMJD6's structure and activity, as well as its role in angiogenesis, oxygen sensing, and adverse pregnancy outcomes related to placental development. Understanding the interaction between JMJD6 and other placental factors may identify potential therapeutic targets for correcting abnormal placental angiogenesis and function.

Design and synthesis of N-(1-(6-(substituted phenyl)-pyridazin-3-yl)-piperidine-3-yl)-amine derivatives as JMJD6 inhibitors.

JMJD6 is a member of the JmjC domain-containing family and has been identified as a promising therapeutic target for treating estrogen-induced and triple-negative breast cancer. To develop novel anti-breast cancer agents, we synthesized a class of N-(1-(6-(substituted phenyl)-pyridazine-3-yl)-piperidine-3-yl)-amine derivatives as potential JMJD6 inhibitors. Among them, the anti-cancer compound A29 was an excellent JMJD6 binder (KD = 0.75 ± 0.08 µM). It could upregulate the mRNA and protein levels of p53 and its downstream effectors p21 and PUMA by inhibiting JMJD6. Besides, A29 displayed potent anti-proliferative activities against tested breast cancer cells by the induction of cell apoptosis and cell cycle arrest. Significantly, A29 also promoted a remarkable reduction in tumor growth, with a TGI value of 66.6% (50 mg/kg, i.p.). Taken together, our findings suggest that A29 is a potent JMJD6 inhibitor bearing a new scaffold acting as a promising drug candidate for the treatment of breast cancer.

Discovery of a new class of JMJD6 inhibitors and structure-activity relationship study.

JmjC domain-containing protein 6 (JMJD6) has been thought as a potential target for various diseases particularly cancer. However, few selective JMJD6 inhibitors have been reported. In this investigation, molecular docking and biological activity evaluation were performed to retrieve new JMJD6 inhibitors, which led to the identification of a hit compound, J2. Further structural optimization and structure-activity relationship (SAR) analysis towards J2 were carried out, which gave a new potent JMJD6 inhibitor, 7p. This compound showed an IC50 value of 0.681 µM against JMJD6, but displayed no activity against other tested JmjC domain-containing protein family members, indicating good selectivity (>100 fold). Collectively, this investigation offers a selective JMJD6 inhibitor, which could be taken as a lead compound for subsequent drug discovery targeting JMJD6.

Computational and Mass Spectrometry-Based Approach Identify Deleterious Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) in JMJD6.

The jumonji domain-containing protein 6 (JMJD6) gene catalyzes the arginine demethylation and lysine hydroxylation of histone and a growing list of its known substrate molecules, including p53 and U2AF65, suggesting a possible role in mRNA splicing and transcription in cancer progression. Mass spectrometry-based technology offers the opportunity to detect SNP variants accurately and effectively. In our study, we conducted a combined computational and filtration workflow to predict the nonsynonymous single nucleotide polymorphisms (nsSNPs) present in JMJD6, followed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and validation. The computational approaches SIFT, PolyPhen-2, SNAP, I-Mutant 2.0, PhD-SNP, PANTHER, and SNPS&GO were integrated to screen out the predicted damaging/deleterious nsSNPs. Through the three-dimensional structure of JMJD6, H187R (rs1159480887) was selected as a candidate for validation. The validation experiments showed that the mutation of this nsSNP in JMJD6 obviously affected mRNA splicing or the transcription of downstream genes through the reduced lysyl-hydroxylase activity of its substrates, U2AF65 and p53, further indicating the accuracy of this prediction method. This research provides an effective computational workflow for researchers with an opportunity to select prominent deleterious nsSNPs and, thus, remains promising for examining the dysfunction of proteins.

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6.

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.

The potential underlying mechanism of the leukemia caused by MLL-fusion and potential treatments.

A majority of infant and pediatric leukemias are caused by the mixed-lineage leukemia gene (MLL) fused with a variety of candidates. Several underlying mechanisms have been proposed. One currently popular view is that truncated MLL1 fusion and its associated complex constitutively hijacks super elongation complex, including positive transcription elongation factor b, CDK9, and cyclin T1 complex and DOT1L, to enhance the expression of transcription factors that maintain or restore stemness of leukocytes, as well as prevent the differentiation of hematopoietic progenitor cells. An alternative emerging view proposes that MLL1-fusion promotes the recruitment of TATA binding protein and RNA polymerase II (Pol II) initiation complex, so as to increase the expression levels of target genes. The fundamental mechanism of both theories are gain of function for truncated MLL1 fusions, either through Pol II elongation or initiation. Our recent progress in transcription regulation of paused Pol II through JMJD5, JMJD6, and JMJD7, combined with the repressive role of H3K4me3 revealed by others, prompted us to introduce a contrarian hypothesis: the failure to shut down transcribing units by MLL-fusions triggers the transformation: loss of function of truncated MLL1 fusions coupled with the loss of conversion of H3K4me1 to H3K4me3, leading to the constitutive expression of transcription factors that are in charge of maintenance of hematopoietic progenitor cells, may trigger the transformation of normal cells into cancer cells. Following this track, a potential treatment to eliminate these fusion proteins, which may ultimately cure the disease, is proposed.

JMJD6 promotes hepatocellular carcinoma carcinogenesis by targeting CDK4.

Jumonji domain-containing protein 6 (JMJD6), a histone arginine demethylase, plays a multifaceted and significant role in embryonic development and cancer progression. However, the function of JMJD6 and its precise mechanism in regulating hepatocellular carcinoma (HCC) remain unknown. Here, we show that aberrant JMJD6 overexpression is associated with poor prognosis and aggressive characteristics of HCC. In hepatoma cell lines, we demonstrated that knockdown of JMJD6 inhibited hepatoma cell migration and proliferation. JMJD6 overexpression displays the opposite effects. Interestingly, JMJD6 regulates hepatoma cell cycle and apoptosis progression. Moreover, there was a positive correlation between cell cycle regulatory protein CDK4 and JMJD6 level. Mechanism analysis suggested JMJD6 promotes CDK4 expression by directly targeting to its promoter, and interacts with PCAF to regulate the histone modifications on the promoter of CDK4. Furthermore, we found that inhibiting CDK4 abolished the ability of JMJD6 in enhancing cell proliferation. Taken together, for the first, we demonstrated that JMJD6 is critically involved in HCC carcinogenesis, and indicated that JMJD6 may be a novel potential biomarker for HCC.

LncRNA-MALAT1 promotes CPC proliferation and migration in hypoxia by up-regulation of JMJD6 via sponging miR-125.

The death of cardiomyocytes after myocardial infarction (MI) often leads to ventricular remodeling as well as heart failure (HF). The cardiac progenitor cells (CPCs) have the ability to regenerate functional heart muscle in patients after MI, which provides a promising method for MI-induced HF therapy. However, to date, CPCs can easily lose their proliferation ability in the infarcted myocardium. Therefore, exploring the mechanism for CPC proliferation is essential for CPC-based therapy in MI-induced HF. A previous study indicated that a hypoxic environment is essential for CPC proliferation, but the mechanism is not yet clear. In this work, we discovered that CoCl2-induced hypoxia can promote CPC proliferation and migration. Additionally, long non-coding RNA MALAT1 expression was significantly up-regulated in the CoCl2-induced hypoxia CPC model. MALAT1 suppression inhibited CPC proliferation and migration under hypoxic conditions. In addition, MALAT1 acted as a sponge for miR-125. The miR-125 inhibitor restored the proliferation and migration potentials of CPCs after a MALAT1 knockdown in hypoxia. A further study demonstrated that JMJD6 was a target of miR-125 whose expression was negatively regulated by miR-125. JMJD6 knockdown blocked miR-125 inhibitor's protective effect on CPC function in hypoxia. Ultimately, our finding demonstrated that MALAT1 can modulate CPC proliferation and migration potential through the miR-125/JMJD6 axis in hypoxia. Our finding provided a new regulatory mechanism for CPC proliferation in hypoxia, which provided a new target for MI-induced HF therapy.

JMJD6 promotes melanoma carcinogenesis through regulation of the alternative splicing of PAK1, a key MAPK signaling component.

BACKGROUND: Melanoma, originated from melanocytes located on the basal membrane of the epithelial tissue, is the most aggressive form of skin cancer that accounts for 75% of skin cancer-related death. Although it is believed that BRAF mutation and the mitogen-activated protein kinase (MAPK) pathway play critical roles in the pathogenesis of melanoma, how the MAPK signaling is regulated in melanoma carcinogenesis is still not fully understood. METHODS: We characterized JMJD6 expression in melanoma tissue array by immunohistochemistry analysis. We used human melanoma A375, 451Lu and SK-MEL-1 cell lines for in vitro proliferation and invasion experiments, and xenograft transplanted mice using murine melanoma B16F10 cells by bioluminescence imaging for in vivo tumor growth and pulmonary metastasis assessments. Endothelial tube formation assay, chicken yolk sac membrane assay and matrigel plug assay were performed to test the effect of JMJD6 on the angiogenic potential in vitro and in vivo. RESULTS: Here we report that the jumonji C domain-containing demethylase/hydroxylase JMJD6 is markedly up-regulated in melanoma. We found that high expression of JMJD6 is closely correlated with advanced clinicopathologic stage, aggressiveness, and poor prognosis of melanoma. RNA-seq showed that knockdown of JMJD6 affects the alternative splicing of a panel of transcripts including that encoding for PAK1, a key component in MAPK signaling pathway. We demonstrated that JMJD6 enhances the MAPK signaling and promotes multiple cellular processes including melanogenesis, proliferation, invasion, and angiogenesis in melanoma cells. Interestingly, JMJD6 is transcriptionally activated by c-Jun, generating a feedforward loop to drive the development and progression of melanoma. CONCLUSIONS: Our results indicate that JMJD6 is critically involved in melanoma carcinogenesis, supporting the pursuit of JMJD6 as a potential biomarker for melanoma aggressiveness and a target for melanoma intervention.

Thymic epithelial cell-specific deletion of Jmjd6 reduces Aire protein expression and exacerbates disease development in a mouse model of autoimmune diabetes.

Thymic epithelial cells (TECs) establish spatially distinct microenvironments in which developing T cells are selected to mature or die. A unique property of medullary TECs is their expression of thousands of tissue-restricted self-antigens that is largely under the control of the transcriptional regulator Aire. We previously showed that Jmjd6, a lysyl hydroxylase for splicing regulatory proteins, is important for Aire protein expression and that transplantation of Jmjd6-deficient thymic stroma into athymic nude mice resulted in multiorgan autoimmunity. Here we report that TEC-specific deletion of Jmjd6 exacerbates development of autoimmune diabetes in a mouse model, which express both ovalbumin (OVA) under the control of the rat insulin gene promoter and OT-I T cell receptor specific for OVA peptide bound to major histocompatibility complex class I Kb molecules. We found that Aire protein expression in mTECs was reduced in the absence of Jmjd6, with retention of intron 2 in Aire transcripts. Our results thus demonstrate the importance of Jmjd6 in establishment of immunological tolerance in a more physiological setting.