Jumonji Gene Research Articles

Mutation and Expression Analysis of Jumonji Genes in Myelodysplastic Syndrome & Acute Myeloid Leukaemia

IntroductionDiscovery of cytogenetic abnormalities together with specific aberrations in RNA splicing, cell signalling, translational regulation and tumour suppressor genes are increasingly been applied for the prognostic stratification well as understanding the pathobiology of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML).Epigenetic regulation of transcription has attracted particular attention, because of the efficacy of DNA methyltransferase and Histone deacetylase inhibitor therapies for the treatment of both MDS and AML. The Jumonji (JMJ) family of histone demethylases are epigenetic regulators that demethylate lysine residues of histones in a site and methylation state specific context. Given the importance of these in the transcriptional machinery of myeloid progenitors, we examined the role of seventeen Jumonji genes; JARID1A-1D, JARID2, JMJD3, UTX, JMJD4, JMJD2A-2D, JMJD1A-1C, JHJDM1A-1B in the bone marrow samples from patients with MDS/AML. Majority of the genes tested are involved in the demethylation of H3K4, H3K9, H3K27 or H3K36 histone tails while others associate with the polycomb repressive complex (PRC), abnormalities of which are well documented in both MDS/AML.MethodsSNP6 array karyotyping was carried out in 124 MDS patients [M/F ratio = 1:1.7, median age= 69 yrs, WHO subtypes: RCMD 21%, RAEB I/II 50 %, AML 1%, sAML/tMDS 16 %, CMML 6.5 %, MDS/MPD 5.5 %], focusing exclusively on the Jumonji genes loci to identify copy number variations [CNVs] (deletion/ gain) & Uniparental disomy. The patients with SNP6 aberrations in the Jumonji genes were examined by 454 DNA parallel sequencing and Real-time PCR for mutational analysis and alterations in gene expression respectively. The sequencing depth was 350-450 reads / amplicon.ResultsOf the 124 cases, 28 patients (22.5%) were identified with a deletion, gain or UPD at 15/17 Jumonji gene loci while two genes showed no SNP6 abnormalities. Of the 22.5 % patients, the highest frequencies of deletions were found in JMJD1B (chr 5q31.2) [41 %], JARID2 [10.7 %] and JMJD3, JMJD2D, JMJD1A [7.14 %] each. On the other hand, the highest frequency of gain was observed in JMJD2C [10.7 %], followed by JMJD2A &UTX [7.14 %] each. Interestingly, only three genes showed both deletion and amplification in different patients in our cohort (JARID2, JMJD4, and JMJD2D) while the rest were either deleted or amplified. Compared to CNVs (69 %), only twelve patients (44 %) carried UPD [telomeric, size: majority were >20Mb]. JMJD2D and JMJD4 had the highest frequency (14.3 %) while JMJD3 had [10.7 %] of UPDs. 454 DNA parallel sequencing of the fifteen Jumonji genes in the 28 patients with SNP6 abnormalities revealed no mutations. To elucidate changes in gene expression as a result of CNVs; two patients with CN gain at the UTX locus showed 5 fold increase (p value < 0.0001) in the expression of this gene while two patients with deletion at the JARID1A locus showed knock down in the expression levels as compared to patients with normal SNP6 profile (n =20).ConclusionIn summary, 22.5 % of high-risk MDS patients show SNP6 aberrations at the Jumonji gene loci. No mutations were associated with the SNP6 abnormalities even with a read depth of 350-450 reads/ amplicon. However, alteration in the expression of JARIDA and UTX was consistent with the CNVs detected on SNP6 which might have direct consequence on the methylation status of the genome or may assist as yet unidentified targets in the pathogenesis of MDS. [Display omitted] [Display omitted] DisclosuresShinde:Celgene: Research Funding.

The Jumonji family: past, present and future of histone demethylases in cancer.

The first Jumonji gene was cloned in 1995 by Takeuchi et al. [Takeuchi T, Yamazaki Y, Katoh-Fukui Y, Tsuchiya R, Kondo S, Motoyama J, Higashinakagawa T. Gene trap capture of a novel mouse gene, jumonji, required for neural tube formation. Genes Dev 1995; 9: 1211-22.]. Several genes sharing similar biological features have since been discovered, and are currently grouped into the JMJ family. Interestingly, their deregulation has been associated with cardiac disease, obesity, neurological disorders and cancer. One of the mechanisms underlying their function is gene expression modulation via histone post-translational modifications (PTMs). Increasing evidence of Jumonji deregulation in tumours such as colon, prostate, haematological and breast cancer is continually emerging, hence the need to acquire a better understanding. The Genesapiens.org database of patient arrays allows target expression levels to be investigated in a wide range of cancers, corroborating and extending the role of the JMJ family. Here, we provide an overview of the expression profile and regulation of JMJ family members in cancer, examining the most recent literature in the light of analyses drawn from this database.

Role of JMJD3 and histone modifications in intermittent hypoxia‐induced neuroinflammation (1120.6)

Microglial phenotypic plasticity enables them to sense and respond to a variety of stimuli. While much of this plasticity involves transcriptional gene regulation that is highly susceptible to epigenetic modulation, little is known about how post‐translational histone modifications regulate microglial activities. We have shown that intermittent hypoxia (IH), a hallmark of sleep apnea, induces microglial inflammation in vivo, but mechanisms underlying IH‐induced microglial activation are unknown. The Jumonji gene family of histone demethylases are hypoxia‐sensitive, and JMJD3 is known to regulate macrophage inflammatory processes, but nothing is known about its regulation of microglial activities, or its role in IH‐induced neuroinflammation. Thus, using in vivo and in vitro models we tested the hypothesis that JMJD3 is necessary for IH‐induced microglial inflammation. In vivo, JMJD3 mRNA is upregulated in microglia immunomagnetically isolated from the cortex and hippocampus of IH‐treated animals, with a time course that correlates with inflammatory gene induction. To test the requirement of JMJD3 in IH‐induced microglial gene expression, we exposed microglial cell cultures to IH in the presence and absence of the small molecule JMJD3 inhibitor GSK‐J4 (7.5μM). IH increased JMJD3 mRNA levels which correlated with a decrease in histone H3 lysine 27 (H3K27) trimethylation, the enzymatic target of JMJD3 activity. IH‐induced IL‐6, IL‐1β, iNOS, and COX‐2 pro‐inflammatory gene expression was blocked in the presence of GSK‐J4, suggesting a critical role of JMJD3 and H3K27 demethylation in IH‐induced inflammation. This is the first study to investigate a Jumonji demethylase in the context of IH, and the first to link IH‐induced microglial inflammation to JMJD3 activities.Grant Funding Source: Supported by NIH R01 HL111598 and the Graduate Women in Science Ruth Dickie Fellowship

The Jumonji gene family in Crassostrea gigas suggests evolutionary conservation of Jmj-C histone demethylases orthologues in the oyster gametogenesis and development

Jumonji (Jmj) proteins are histone demethylases, which control the identity of stem cells. Jmj genes were characterized from plants to mammals where they have been implicated in the epigenetic regulation of development. Despite the Pacific oyster Crassostrea gigas representing one of the most important aquaculture resources worldwide, the molecular mechanisms governing the embryogenesis and reproduction of this lophotrochozoan species remain poorly understood. However, annotations in the C. gigas EST library suggested the presence of putative Jumonji genes, raising the question of the conservation of this family of histone demethylases in the oyster.Using Primer walking, 5′-RACE PCR and in silico analyses, we characterized nine Jumonji orthologues in the oyster, called Cg-Jmj, bearing conserved domains critical for putative histone demethylase activity. Phylogenic analyses revealed that oyster Jumonji cluster into two distinct groups: ‘single-domain Jmj’ and ‘multi-domain Jmj’, and define 8 subgroups corresponding to each cognate orthologues in metazoans. RT-qPCR investigations showed specific regulations of Cg-Jmj mRNAs during the early development and along the reproduction cycle. Furthermore, in situ and in toto hybridizations indicate that oyster Jumonji genes are transcribed mostly within the gonad in adult oysters whereas they display a ubiquitous expression during embryonic and larval development.Our study demonstrates the presence of nine Jumonji orthologues in the oyster C. gigas. Their domain conservation and their expression profile suggest an implication during reproduction and development, questioning about the epigenetic regulation by histone methylation in lophotrochozoans.

Role of Hypoxia‐Inducible Factors in Epigenetic Regulation via Histone Demethylases

Eukaryotic chromatin is subject to multiple posttranslational histone modifications such as acetylation, methylation, phosphorylation, and ubiquitination. These various covalent modifications have been proposed to constitute a "histone code," playing important roles in the establishment of global chromatin environments, transcription, DNA repair, and DNA replication. Among these modifications, histone methylation specifies regulatory marks that delineate transcriptionally active and inactive chromatin. These histone methyl marks were considered irreversible; however, recent identification of site-specific histone demethylases demonstrates that histone methylation is dynamically regulated, which may allow cells to rapidly change chromatin conformation to adapt to environmental stresses or intrinsic stimuli. Of major interest is the observation that these histone demethylase enzymes, which are in the Jumonji gene family, require oxygen to function and, in some cases, are induced by hypoxia in an HIFalpha-dependent manner. This provides a new mechanism for regulation of the response to hypoxia.

A Jumonji (Jarid2) Protein Complex Represses cyclin D1 Expression by Methylation of Histone H3-K9

Covalent modifications of histone tails have critical roles in regulating gene expression. Previously, we identified the jumonji (jmj, Jarid2) gene, the jmjC domain, and a Jmj family. Recently, many Jmj family proteins have been shown to be histone demethylases, and jmjC is the catalytic domain. However, Jmj does not have histone demethylase activity because the jmjC domain lacks conserved residues for binding to cofactors. Independently of these studies, we previously showed that Jmj binds to the cyclin D1 promoter and represses the transcription of cyclin D1. Here, we show the mechanisms by which Jmj represses the transcription of cyclin D1. We found that a protein complex of Jmj had histone methyltransferase activity toward histone H3 lysine 9 (H3-K9). We also found that Jmj bound to the H3-K9 methyltransferases G9a and GLP. Expression of Jmj recruited G9a and GLP to the cyclin D1 promoter and increased H3-K9 methylation. Inactivation of both G9a and GLP, but not of only G9a, inhibited the methylation of H3-K9 in the cyclin D1 promoter and repression of cyclin D1 expression by Jmj. These results suggest that Jmj methylates H3-K9 and represses cyclin D1 expression through G9a and GLP, and that Jmj family proteins can regulate gene expression by not only histone demethylation but also other histone modification.

Induction by left ventricular overload and left ventricular failure of the human Jumonji gene (JARID2) encoding a protein that regulates transcription and reexpression of a protective fetal program

We identified changes in Jumonji (JARID2) expression in failing human hearts and determined its effects on expressions of atrial natriuretic factor (ANF), myosin light chain 2a (MLC2A), and alpha myosin heavy chain (MHCA), genes associated with both human heart failure and the fetal gene program. Left ventricular outflow tract cardiac biopsy samples were taken from 31 patients with aortic valvular stenosis. Hearts were grouped according to left ventricular size and function: nonfailing hearts (undilated with good function) and failing hearts (dilated with poor function). Protein levels were determined by Western blotting, and messenger RNA transcript levels by ratiometric reverse transcriptase-polymerase chain reaction. Luciferase assays in HL-2 cells were used to assess effects of Jarid2 on Anf, Mlc2a, and Mhca transcriptions. Chromatin immunoprecipitation was used to detect interaction of JARID2 with specific target-gene promoters. JARID2 and MHCA expressions were reduced in failing hearts, whereas MLC2A and ANF were increased. In HL-2 cell culture, Jarid2 suppressed Anf and Mlc2a but enhanced Mhca. Jarid2 expression was reduced by cyclic mechanical stress, with concomitant increased Anf and Mlc2a and decreased Mhca expressions, reproducing the expression profile found in decompensated human pressure overload. Jumonji expression is reduced by mechanical stress in human heart failure from aortic stenosis. JARID2 regulates ANF, MLC2A, and MHCA transcription and contributes to reexpression of the fetal gene program in decompensated aortic stenosis. JARID2 appears important in transcriptional regulation of fetal genes and may emerge as a diagnostic marker for left ventricular decompensation in aortic stenosis.

The Drosophila jumonji gene encodes a JmjC-containing nuclear protein that is required for metamorphosis

Jumonji (Jmj) is a transcriptional repressor that plays important roles in the suppression of cell proliferation and development of various tissues in the mouse. To further clarify the roles of Jmj during development and gain insight into mechanisms of Jmj-mediated transcriptional regulation, we have taken advantage of Drosophila as a model organism. Drosophila Jmj (dJmj) shares high homology with mammalian Jmj in the JmjN, JmjC and AT-rich interaction domains, as well as in the N-terminal repression domain. dJmj localizes to hundreds of euchromatic sites but not to chromocenter heterochromatin on salivary gland polytene chromosomes. In addition, dJmj is excluded from regions stained with an antibody against Ser5-phosphorylated RNA polymerase II, suggesting a function of dJmj in transcriptionally inactive chromatin. Loss of djmj results in larval and pupal lethality with phenotypes similar to those observed in mutants of ecdysone-regulated genes, implying the involvement of dJmj in the repression of gene expression in the ecdysone pathway. Transgenic mouse Jmj mostly colocalizes with dJmj and partially rescues the phenotypes of djmj mutants, indicating that dJmj is a functional homolog of mammalian Jmj. Furthermore, mutation in djmj suppresses position effect variegation of the T(2;3)Sb(V) rearrangement. These findings suggest that dJmj controls expression of developmentally important genes through modification of chromatin into a transcriptionally silenced state.

Generation of a conditional null allele of jumonji

The jumonji (jmj) gene plays important roles in multiple organ development in mouse, including cardiovascular development. Since JMJ is expressed widely during mouse development, it is essential that conditional knockout approaches be employed to ablate JMJ in a tissue-specific manner to identify the cell lineage specific roles of JMJ. In this report, we describe the establishment of a jmj conditional null allele in mice by generating a loxP-flanked (floxed) jmj allele, which allows the in vivo ablation of jmj via Cre recombinase-mediated deletion. Gene targeting was used to introduce loxP sites flanking exon 3 of the jmj allele to mouse embryonic stem cells. Our results indicate that the jmj floxed allele converts to a null allele in a heart-specific manner when embryos homozygous for the floxed jmj allele and carrying the alpha-myosin heavy chain promoter-Cre transgene were analyzed by Southern and Northern blot analyses. Therefore, this mouse line harboring the conditional jmj null allele will provide a valuable tool for deciphering the tissue and cell lineage specific roles of JMJ.

Roles of jumonji and jumonji family genes in chromatin regulation and development

The jumonji (jmj) gene was identified by a mouse gene trap approach and has essential roles in the development of multiple tissues. The Jmj protein has a DNA binding domain, ARID, and two conserved jmj domains (jmjN and jmjC). In many diverse species including bacteria, fungi, plants, and animals, there are many jumonji family proteins that have only the jmjC domain or both jmj domains. Recently, Jmj protein was found to be a transcriptional repressor. Several proteins in the jumonji family are involved in transcriptional repression and/or chromatin regulation. Most recently, one of the human members has been shown to be a histone demethylase, and the jmjC domain is essential for the demethylase activity. Meanwhile, more and more evidence indicating that the jumonji family proteins play important roles during development is accumulating. Many proteins in the jumonji family may regulate chromatin and gene expression, and control development through various signaling pathways. Here, we highlight the roles of jmj and jumonji family proteins in chromatin regulation and development.

Roles of JUMONJI in mouse embryonic development

Cardiac development is a complex biological process requiring the integration of cell specification, differentiation, migration, proliferation, and morphogenesis. Although significant progress has been made recently in understanding the molecular basis of cardiac development, mechanisms of transcriptional control of cardiac development remain largely unknown. In search for the developmentally important genes, the jumonji gene (jmj) was identified by gene trap technology and characterized as a critical nuclear factor for mouse embryonic development. Jmj has been shown to play important roles in cardiovascular development, neural tube fusion process, hematopoiesis, and liver development in mouse embryos. The amino acid sequence of the JUMONJI protein (JMJ) reveals that JMJ belongs to the AT-rich interaction domain transcription factor family and more recently has been described as a member of the JMJ transcription factor family. Here, we review the roles of jmj in multiple organ development with a focus on cardiovascular development in mice.

Jumonji Represses Atrial Natriuretic Factor Gene Expression by Inhibiting Transcriptional Activities of Cardiac Transcription Factors

Mice with a homozygous knockout of the jumonji (jmj) gene showed abnormal heart development and defective regulation of cardiac-specific genes, including the atrial natriuretic factor (ANF). ANF is one of the earliest markers of cardiac differentiation and a hallmark for cardiac hypertrophy. Here, we show that JMJ represses ANF gene expression by inhibiting transcriptional activities of Nkx2.5 and GATA4. JMJ represses the Nkx2.5- or GATA4-dependent activation of the reporter genes containing the ANF promoter-enhancer or containing the Nkx2.5 or GATA4-binding consensus sequence. JMJ physically associates with Nkx2.5 and GATA4 in vitro and in vivo as determined by glutathione S-transferase pull-down and immunoprecipitation assays. Using mutational analyses, we mapped the protein-protein interaction domains in JMJ, Nkx2.5, and GATA4. We identified two DNA-binding sites of JMJ in the ANF enhancer by gel mobility shift assays. However, these JMJ-binding sites do not seem to mediate ANF repression by JMJ. Mutational analysis of JMJ indicates that the protein-protein interaction domain of JMJ mediates the repression of ANF gene expression. Therefore, JMJ may play important roles in the down-regulation of ANF gene expression and in heart development.

A role of jumonji gene in proliferation but not differentiation of megakaryocyte lineage cells.

In this study, megakaryocytopoiesis was investigated in the recessive mutant mouse, jumonji, obtained by a gene-trap strategy. We investigated the number of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygous embryos by in vitro colony forming assay and monitored colony formation from single megakaryocyte progenitors. We also investigated the differentiation of jumonji-deficient megakaryocytes in terms of the expression of megakaryocyte differentiation markers PF4, CD62P, and GATA-1, proplatelet formation, cytoplasmic maturation, and endomitosis. We found that the population of megakaryocyte progenitors in the fetal liver, yolk sac, and peripheral blood of jumonji homozygotes increased. A fraction of megakaryocyte progenitors derived from the fetal liver of jumonji homozygotes formed larger colonies in vitro when compared with controls. This abnormality is caused by delayed growth arrest in the progeny. Immature megakaryocyte progenitors showed this abnormality. The megakaryocytes of jumonji homozygotes expressed PF4, CD62P, and GATA-1, obtained cytoplasmic maturation, extended proplatelet-like processes, and underwent endomitosis. The loss of the jumonji gene causes an increase in the number of megakaryocyte lineage cells. Our data suggest that the jumonji gene regulates proliferation but not differentiation of megakaryocyte lineage cells.

Jumonji Is a Nuclear Protein That Participates in the Negative Regulation of Cell Growth

The jumonji (jmj) gene, obtained by a gene trap strategy, is essential for mouse embryogenesis and is suggested to play important roles in cell growth during development. The amino acid sequence of the Jmj protein includes a nuclear localization signal and a DNA binding motif called the AT-rich interaction domain (ARID). To investigate the biological functions of the Jmj protein, we prepared specific antibodies. Using these antibodies, we showed that the Jmj protein is a 160-kDa protein and localizes in the nuclei of COS-7 cells transfected with jmj cDNA and megakaryocytes from fetal liver which show strong endogenous expression of the jmj gene. Moreover, overexpression of the Jmj protein in COS-7 and NIH3T3 cells remarkably reduced cell proliferation compared with control cells transfected with vector alone. These results show that the Jmj protein acts in cell nuclei and participates in the negative regulation of cell proliferation signaling.

Jumonji gene is essential for the neurulation and cardiac development of mouse embryos with a C3H/He background

The recessive mutant mouse jumonji ( jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygous embryos with a BALB/cA and 129/Ola mixed background, but no neural tube defects with BALB/cA, C57BL/6J, and DBA/2J backgrounds. Here, we show that neural tube and cardiac defects are observed in all embryos with a C3H/HeJ background. In addition, abnormal groove formation and prominent flexure are observed on the neural plate with full penetrance, suggesting that abnormal groove formation leads to neural tube defects. We found morphogenetic abnormalities in the bulbus cordis (future outflow tract and the right ventricle) of homozygous embryo hearts. Moreover, myocytes in the ventricular trabeculae show hyperplasia with cells filling the ventricles. Together with the observation that the jmj gene is expressed in the neural epithelium of the head neural plate and in myocytes in the bulbus cordis and trabeculae, the results show that the jmj gene plays essential roles in the normal development of the neural plate, morphogenesis of bulbus cordis, and proliferation of trabecular myocytes on a C3H/He background.

Definitive But Not Primitive Hematopoiesis Is Impaired injumonji Mutant Mice

A novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonjigene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver–derived definitive erythrocytes, but not yolk sac–derived primitive erythrocytes, showed a marked reduction, suggesting thatjumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonjimutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin−/c-Kit+/Sca-1+ cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver ofjumonji mutant embryos.

Definitive But Not Primitive Hematopoiesis Is Impaired injumonji Mutant Mice

AbstractA novel gene, jumonji was identified by a mouse gene trap strategy. The jumonji gene encodes a protein containing a putative DNA binding domain. The mice homozygous for jumonjigene with a BALB/cA genetic background show hypoplasia of the fetal liver and embryonic lethality, suggesting impaired hematopoiesis. In the peripheral blood of jumonji mutant embryos, the number of fetal liver–derived definitive erythrocytes, but not yolk sac–derived primitive erythrocytes, showed a marked reduction, suggesting thatjumonji mutants die of anemia. The defects of definitive erythrocytes in jumonji mutants seemed to be caused by a decrease in the numbers of multiple hematopoietic progenitors including colony-forming unit-spleen (CFU-S) in the fetal liver. However, hematopoietic stem cells (HSCs) in the fetal liver of jumonjimutants could reconstitute the hematopoietic system of lethally irradiated recipients. In the fetal liver, the jumonji gene is expressed in fibroblastic cells and endothelial cells, but not in Lin−/c-Kit+/Sca-1+ cells known to include HSCs. These results suggest that an environmental defect induce the impaired hematopoiesis in the fetal liver ofjumonji mutant embryos.

Characterization of the human jumonji gene.

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.