Increased angiotensin II coupled with decreased Adra1a expression enhances cardiac hypertrophy in pregnancy-associated hypertensive mice.

Cardiac hypertrophy is a crucial risk factor for hypertensive disorders during pregnancy, but its progression during pregnancy remains unclear. We previously showed cardiac hypertrophy in a pregnancy-associated hypertensive (PAH) mouse model, in which an increase in angiotensin II (Ang II) levels was induced by human renin and human angiotensinogen, depending on pregnancy conditions. Here, to elucidate the factors involved in the progression of cardiac hypertrophy, we performed a comprehensive analysis of changes in gene expression in the hearts of PAH mice and compared them with those in control mice. We found that alpha-1A adrenergic receptor (Adra1a) mRNA levels in the heart were significantly reduced under PAH conditions, whereas the renin-angiotensin system was upregulated. Furthermore, we found that Adra1a-deficient PAH mice exhibited more severe cardiac hypertrophy than PAH mice. Our study suggests that Adra1a levels are regulated by renin-angiotensin system and that changes in Adra1a expression are involved in progressive cardiac hypertrophy in PAH mice.

Histamine receptor agonist alleviates severe cardiorenal damages by eliciting anti-inflammatory programming.

Heart failure and chronic kidney disease are major causes of morbidity and mortality internationally. Although these dysfunctions are common and frequently coexist, the factors involved in their relationship in cardiorenal regulation are still largely unknown, mainly due to a lack of detailed molecular targets. Here, we found the increased plasma histamine in a preclinical mouse model of severe cardiac dysfunction, that had been cotreated with angiotensin II (Ang II), nephrectomy, and salt (ANS). The ANS mice exhibited impaired renal function accompanied with heart failure, and histamine depletion, by the genetic inactivation of histidine decarboxylase in mice, exacerbated the ANS-induced cardiac and renal abnormalities, including the reduction of left ventricular fractional shortening and renal glomerular and tubular injuries. Interestingly, while the pharmacological inhibition of the histamine receptor H3 facilitated heart failure and kidney injury in ANS mice, administration of the H3 agonist immethridine (Imm) was protective against cardiorenal damages. Transcriptome analysis of the kidney and biochemical examinations using blood samples illustrated that the increased inflammation in ANS mice was alleviated by Imm. Our results extend the pharmacological use of H3 agonists beyond the initial purposes of its drug development for neurogenerative diseases and have implications for therapeutic potential of H3 agonists that invoke the anti-inflammatory gene expression programming against cardiorenal damages.

Starvation-induced transcription factor CREBH negatively governs body growth by controlling GH signaling.

cAMP responsive element-binding protein H (CREBH) is a hepatic transcription factor to be activated during fasting. We generated CREBH knock-in flox mice, and then generated liver-specific CREBH transgenic (CREBH L-Tg) mice in an active form. CREBH L-Tg mice showed a delay in growth in the postnatal stage. Plasma growth hormone (GH) levels were significantly increased in CREBH L-Tg mice, but plasma insulin-like growth factor 1 (IGF1) levels were significantly decreased, indicating GH resistance. In addition, CREBH overexpression significantly increased hepatic mRNA and plasma levels of FGF21, which is thought to be as one of the causes of growth delay. However, the additional ablation of FGF21 in CREBH L-Tg mice could not correct GH resistance at all. CREBH L-Tg mice sustained GH receptor (GHR) reduction and the increase of IGF binding protein 1 (IGFBP1) in the liver regardless of FGF21. As GHR is a first step in GH signaling, the reduction of GHR leads to impairment of GH signaling. These data suggest that CREBH negatively regulates growth in the postnatal growth stage via various pathways as an abundant energy response by antagonizing GH signaling.

Loss of PRMT1 in the central nervous system (CNS) induces reactive astrocytes and microglia during postnatal brain development.

PRMT1, a major arginine methyltransferase, plays critical roles in transcription, DNA damage response, and cell proliferation. Although we have previously discovered the crucial roles of PRMT1 for oligodendrocyte lineage progression in the central nervous system of neural stem cell-specific PRMT1 conditional knockout (PRMT1-CKO) mice, the context of other glial cell states that may cause the hypomyelination phenotype in PRMT1-CKO mice has not been explored so far. Here, we performed RNA-seq of the neonatal cortices of PRMT1-CKO mice to reveal overall gene expression changes and show the up-regulation of inflammatory signaling which is generally mediated by astrocytes and microglia in advance of the myelination defects. In particular, qRT-PCR analyses revealed Interleukin-6 (Il-6), a major central nervous system cytokine, was dramatically increased in the PRMT1-CKO brains. The gene expression changes led to augmentation of glial fibrillary acidic protein and Vimentin protein levels in PRMT1-CKO mice, showing severe reactive astrogliosis after birth. We further show that IBA1-positive and CD68-positive activated microglia were increased in PRMT1-CKO mice, in spite of intact Prmt1 gene expression in purified microglia from the mutant mice. Our results indicate that PRMT1 loss in the neural stem cell lineage causes disruptive changes in all glial types perturbing postnatal brain development and myelination.

Pathophysiological Roles of Stress-Activated Protein Kinases in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is one of the most symptomatic progressive fibrotic lung diseases, in which patients have an extremely poor prognosis. Therefore, understanding the precise molecular mechanisms underlying pulmonary fibrosis is necessary for the development of new therapeutic options. Stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) are ubiquitously expressed in various types of cells and activated in response to cellular environmental stresses, including inflammatory and apoptotic stimuli. Type II alveolar epithelial cells, fibroblasts, and macrophages are known to participate in the progression of pulmonary fibrosis. SAPKs can control fibrogenesis by regulating the cellular processes and molecular functions in various types of lung cells (including cells of the epithelium, interstitial connective tissue, blood vessels, and hematopoietic and lymphoid tissue), all aspects of which remain to be elucidated. We recently reported that the stepwise elevation of intrinsic p38 signaling in the lungs is correlated with a worsening severity of bleomycin-induced fibrosis, indicating an importance of this pathway in the progression of pulmonary fibrosis. In addition, a transcriptome analysis of RNA-sequencing data from this unique model demonstrated that several lines of mechanisms are involved in the pathogenesis of pulmonary fibrosis, which provides a basis for further studies. Here, we review the accumulating evidence for the spatial and temporal roles of SAPKs in pulmonary fibrosis.

Transcriptomic Evaluation of Pulmonary Fibrosis-Related Genes: Utilization of Transgenic Mice with Modifying p38 Signal in the Lungs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing lung disease that is caused by the dysregulation of alveolar epithelial type II cells (AEC II). The mechanisms involved in the progression of IPF remain incompletely understood, although the immune response accompanied by p38 mitogen-activated protein kinase (MAPK) activation may contribute to some of them. This study aimed to examine the association of p38 activity in the lungs with bleomycin (BLM)-induced pulmonary fibrosis and its transcriptomic profiling. Accordingly, we evaluated BLM-induced pulmonary fibrosis during an active fibrosis phase in three genotypes of mice carrying stepwise variations in intrinsic p38 activity in the AEC II and performed RNA sequencing of their lungs. Stepwise elevation of p38 signaling in the lungs of the three genotypes was correlated with increased severity of BLM-induced pulmonary fibrosis exhibiting reduced static compliance and higher collagen content. Transcriptome analysis of these lung samples also showed that the enhanced p38 signaling in the lungs was associated with increased transcription of the genes driving the p38 MAPK pathway and differentially expressed genes elicited by BLM, including those related to fibrosis as well as the immune system. Our findings underscore the significance of p38 MAPK in the progression of pulmonary fibrosis.

The N-terminal sequence of murine PRMT5 variant 2 is required for Hsp70 interaction and CHIP ligase-mediated degradation.

Protein arginine methyltransferase PRMT5 synthesizes the symmetric dimethylarginine in nuclear and cytoplasmic proteins such as histone H2A, H4 and several non-histone proteins that are required for a variety of biological processes. Currently, two splice variants (v1 and v2) of murine PRMT5 have been deposited in the NCBI sequence database, in which PRMT5-v1 and -v2 contain different 33 and 16 amino acids at the N-terminal sequences, respectively. Here we showed that murine PRMT5-v1 is stable, but PRMT5-v2 is constantly degraded through both the ubiquitin proteasome system (UPS) and the autophagic-lysosomal pathway (ALP) in an N-terminal sequence-dependent manner. Furthermore, inhibition of UPS and ALP elevated the stability of PRMT5-v2 that made it localized in the nucleus and the cytoplasm. In addition, PRMT5-v2 exhibited the enzyme activity to catalyze histone H2A and H4 methylation. Notably, we found that the heat shock protein (Hsp) 70 specially recognizes the N-terminal sequence of PRMT5-v2 and the carboxyl terminus of Hsp70-interacting protein (CHIP) is required for poly-ubiquitination and the degradation of PRMT5-v2. These results suggest that Hsp70/CHIP chaperone-mediated protein degradation system is crucial in the regulation of PRMT5-v2 turnover, which has the potential to balance the symmetrical arginine dimethylation in cells.

Truncated Cables1 causes agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

Detection of choline and phosphatidic acid (PA) catalyzed by phospholipase D (PLD) using MALDI-QIT-TOF/MS with 9-aminoacridine matrix.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), the most abundant phospholipids of plasma membrane, resulting in the production of choline and phosphatidic acid (PA). Choline is a precursor of the neurotransmitter acetylcholine, whereas PA functions as an intracellular lipid mediator of diverse biological functions. For assessing PLD activity in vitro, PLD-derived choline has been often analyzed with radioactive or non-radioactive methods. In this study, we have developed a new method for detecting choline and PA with MALDI-QIT-TOF/MS by using 9-aminoacridine as a matrix. The standard calibration curves showed that choline and PA could be detected with linearity over the range from 0.05 and 1 pmol, respectively. Importantly, this method enables the concomitant detection of choline and PA as a reaction product of PC hydrolysis by PLD2 proteins. Thus, our simple and direct method would be useful to characterize the enzymatic properties of PLD, thereby providing insight into mechanisms of PLD activation.

Metabolic changes in the plasma of mild Alzheimer's disease patients treated with Hachimijiogan.

Background: Alzheimer's disease (AD), the most prevalent form of dementia, is a debilitating, progressive neurodegeneration. Amino acids play a wide variety of physiological and pathophysiological roles in the nervous system, and their levels and disorders related to their synthesis have been related to cognitive impairment, the core feature of AD. Our previous multicenter trial showed that hachimijiogan (HJG), a traditional Japanese herbal medicine (Kampo), has an adjuvant effect for Acetylcholine estelase inhibitors (AChEIs) and that it delays the deterioration of the cognitive dysfunction of female patients with mild AD. However, there are aspects of the molecular mechanism(s) by which HJG improves cognitive dysfunction that remain unclear. Objectives: To elucidate through metabolomic analysis the mechanism(s) of HJG for mild AD based on changes in plasma metabolites. Methods: Sixty-seven patients with mild AD were randomly assigned to either an HJG group taking HJG extract 7.5 g/day in addition to AChEI or to a control group treated only with AChEI (HJG:33, Control:34). Blood samples were collected before, 3 months, and 6 months after the first drug administration. Comprehensive metabolomic analyses of plasma samples were done by optimized LC-MS/MS and GC-MS/MS methods. The web-based software MetaboAnalyst 5.0 was used for partial least square-discriminant analysis (PLS-DA) to visualize and compare the dynamics of changes in the concentrations of the identified metabolites. Results: The VIP (Variable Importance in Projection) score of the PLS-DA analysis of female participants revealed a significantly higher increase in plasma metabolite levels after HJG administration for 6 months than was seen in the control group. In univariate analysis, the aspartic acid level of female participants showed a significantly higher increase from baseline after HJG administration for 6 months when compared with the control group. Conclusion: Aspartic acid was a major contributor to the difference between the female HJG and control group participants of this study. Several metabolites were shown to be related to the mechanism of HJG effectiveness for mild AD.

Multi­omics analysis of right ventricles in rat models of pulmonary arterial hypertension: Consideration of mitochondrial biogenesis by chrysin.

In pulmonary arterial hypertension (PAH), right ventricular failure is accompanied by metabolic alterations in cardiomyocytes, which may be due to mitochondrial dysfunction and decreased energy production. Chrysin (CH) is a phytochemical with pharmacological activity that is involved in the regulation of mitochondrial biogenesis. The present study investigated the role of CH in the right ventricle (RV) by analyzing the cardiac transcriptome and metabolome of a SU5416(a vascular endothelial growth factor receptor blocker, /hypoxia (Su/Hx) rat model of PAH. RNA­sequencing of the RV transcriptome between Su/Hx, Su/Hx with CH (Su/Hx + CH) and control groups, extracellular matrix (ECM) organization and ECM­receptor interaction­associated genes were upregulated in the RV of Su/Hx but not Su/Hx + CH rats. Furthermore, expression of mitochondrial function­, energy production­, oxidative phosphorylation­ and tricarboxylic acid (TCA) cycle­associated genes was decreased in the RV of Su/Hx rats; this was reverse by CH. Metabolomic profiling analysis of Su/Hx and Su/Hx + CH rats showed no significant changes in glycolysis, TCA cycle, glutathione, NADH or NADPH. By contrast, in the RV of Su/Hx rats, decreased adenylate energy charge was partially reversed by CH administration, suggesting that CH was involved in the improvement of mitochondrial biogenesis. Reverse transcription­quantitative PCR analysis revealed that expression of peroxisome proliferator­activated receptor γ, a master regulator of fatty acid metabolism and mitochondrial biogenesis, was increased in the RV of Su/Hx + CH rats. CH ameliorated cardiac abnormality, including cardiac fibrosis, RV hypertrophy and PH. The present study suggested that CH altered patterns of gene expression and levels of mitochondrial metabolites in cardiomyocytes, thus improving RV dysfunction in a Su/Hx PAH rat model.

Wogonin, a Compound in Scutellaria baicalensis, Activates ATF4-FGF21 Signaling in Mouse Hepatocyte AML12 Cells.

Fibroblast growth factor 21 (FGF21), which is mainly synthesized and secreted by the liver, plays a crucial role in systemic glucose and lipid metabolism, ameliorating metabolic diseases. In this study, we screened the WAKANYAKU library derived from medicinal herbs to identify compounds that can activate Fgf21 expression in mouse hepatocyte AML12 cells. We identified Scutellaria baicalensis root extract and one of its components, wogonin, as an activator of Fgf21 expression. Wogonin also enhanced the expression of activating transcription factor 4 (ATF4) by a mechanism other than ER stress. Knockdown of ATF4 by siRNA suppressed wogonin-induced Fgf21 expression, highlighting its essential role in wogonin's mode of action. Thus, our results indicate that wogonin would be a strong candidate for a therapeutic to improve metabolic diseases by enhancing hepatic FGF21 production.

The C. elegans PRMT-3 possesses a type III protein arginine methyltransferase activity.

Protein arginine methylation is a common post-translational modification in eukaryotes that is catalyzed by a family of the protein arginine methyltransferases (PRMTs). PRMTs are classified into three types: type I and type II add asymmetrically and symmetrically dimethyl groups to arginine, respectively, while type III adds solely monomethyl group to arginine. However, although the enzymatic activity of type I and type II PRMTs have been reported, the substrate specificity and the methylation activity of type III PRMTs still remains unknown. Here, we report the characterization of Caenorhabditis elegans PRMT-2 and PRMT-3, both of which are highly homologous to human PRMT7. We find that these two PRMTs can bind to S-adenosyl methionine (SAM), but only PRMT-3 has methyltransferase activity for histone H2A depending on its SAM-binding domain. Importantly, thin-layer chromatographic analysis demonstrates that PRMT-3 catalyzes the formation of monomethylated, but not dimethylated arginine. Our study thus identifies the first type III PRMT in C. elegans and provides a means to elucidate the physiological significance of arginine monomethylation in multicellular organisms.

Temporal transcriptomic profiling reveals dynamic changes in gene expression of Xenopus animal cap upon activin treatment.

Activin, a member of the transforming growth factor-ß (TGF-ß) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis.

Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.

In vivo function of CDK5 and Abl enzyme substrate 2 (Cables2), belonging to the Cables protein family, is unknown. Here, we found that targeted disruption of the entire Cables2 locus (Cables2d) caused growth retardation and enhanced apoptosis at the gastrulation stage and then induced embryonic lethality in mice. Comparative transcriptome analysis revealed disruption of Cables2, 50% down-regulation of Rps21 abutting on the Cables2 locus, and up-regulation of p53-target genes in Cables2d gastrulas. We further revealed the lethality phenotype in Rps21-deleted mice and unexpectedly, the exon 1-deleted Cables2 mice survived. Interestingly, chimeric mice derived from Cables2d ESCs carrying exogenous Cables2 and tetraploid wild-type embryo overcame gastrulation. These results suggest that the diminished expression of Rps21 and the completed lack of Cables2 expression are intricately involved in the embryonic lethality via the p53 pathway. This study sheds light on the importance of Cables2 locus in mouse embryonic development.

Transcriptomic changes involved in the dedifferentiation of myofibroblasts derived from the lung of a patient with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiology. Under pathological conditions in lungs with IPF, myofibroblasts serve a key role in fibrogenesis via the accumulation of an excessive amount of extracellular matrix. To develop effective therapeutic interventions against IPF, studies have recently focused on how to dedifferentiate established myofibroblasts. The present study revealed that JQ1, an inhibitor of bromodomain and extra­terminal proteins, markedly suppressed the expression levels of α­smooth muscle actin and ED­A­fibronectin in myofibroblasts prepared from the lung of a patient with end­stage IPF. Furthermore, these findings were supported by transcriptome analysis using RNA sequencing, in which differentially expressed genes (DEGs) downregulated by JQ1 treatment were significantly enriched in the fibrosis­related signaling pathway. On the other hand, the upregulated DEGs in response to JQ1 treatment were significantly enriched in glutathione metabolism, which may affect the cell status of fibroblast/myofibroblast. To the best of our knowledge, this was the first study to comprehensively analyze transcriptome profiles associated with dedifferentiation of IPF myofibroblasts.

Emerging impacts of biological methylation on genetic information.

The central dogma of molecular biology explains the fundamental flow of genetic information for life. Although genome sequence (DNA) itself is a static chemical signature, it includes multiple layers of information composed of mRNA, tRNA, rRNA and small RNAs, all of which are involved in protein synthesis and is passing from parents to offspring via DNA. Methylation is a biologically important modification, because DNA, RNAs and proteins, components of the central dogma, are methylated by a set of methyltransferases. Recent works focused on understanding a variety of biological methylation have shed light on new regulation of cellular functions. In this review, we briefly discuss some of those recent findings of methylation, including DNA, RNAs and proteins.

KDM5D-mediated H3K4 demethylation is required for sexually dimorphic gene expression in mouse embryonic fibroblasts.

Males and females share the same genetic code, but gene expression profile often displays differences between two sexes. Mouse embryonic fibroblasts (MEFs) have been used to experiment as a useful tool to test gene function. They have also been characterized by gender-based differences in expressed genes such as Y-linked Sry or X-linked Hprt. However, there is no report on sex differences in global gene expression. Here, using the next-generation RNA sequencing, we compared the comprehensive transcriptome of MEFs derived from two sexes. In comparison with the female group, the male group up-regulated 27 differentially expressed genes (DEGs), in which a male-specific histone demethylase KDM5D gene is included, and 7 DEGs were down-regulated. Based on the results by searching the ENCODE analysis, it was shown that the expression of 15 genes identified is potentially regulated by the methylation of H3K4me1 or H3K4me3. Interestingly, we demonstrated that both of H3K4 methylation are induced by knocking down KDM5D, which causes changes in patterns of eight DEGs found in male MEFs. Collectively, these data not only suggest an importance of KDM5D-mediated demethylation of H3K4 involved in the sexually dimorphic gene expression in male MEFs, but also may provide information regarding sex-dependent changes in gene expression when MEFs are used for experiments.

Cooperative action of APJ and α1A-adrenergic receptor in vascular smooth muscle cells induces vasoconstriction.

The apelin receptor (APJ), a receptor for apelin and elabela/apela, induces vasodilation and vasoconstriction in blood vessels. However, the prolonged effects of increased APJ-mediated signalling, involving vasoconstriction, in smooth muscle cells have not been fully characterized. Here, we investigated the vasoactive effects of APJ gain of function under the control of the smooth muscle actin (SMA) gene promoter in mice. Transgenic overexpression of APJ (SMA-APJ) conferred sensitivity to blood pressure and vascular contraction induced by apelin administration in vivo. Interestingly, ex vivo experiments showed that apelin markedly increased the vasoconstriction of isolated aorta induced by noradrenaline (NA), an agonist for α- and ß-adrenergic receptors, or phenylephrine, a specific agonist for α1-adrenergic receptor (α1-AR). In addition, intracellular calcium influx was augmented by apelin with NA in HEK293T cells expressing APJ and α1A-AR. To examine the cooperative action of APJ and α1A-AR in the regulation of vasoconstriction, we developed α1A-AR deficient mice using a genome-editing technique, and then established SMA-APJ/α1A-AR-KO mice. In the latter mouse line, aortic vasoconstriction induced by a specific agonist for α1A-AR, A-61603, were significantly less than in SMA-APJ mice. These results suggest that the APJ-enhanced response requires α1A-AR to contract vessels coordinately.

EWS is a substrate of type I protein arginine methyltransferase, PRMT8.

EWS, a pro-oncoprotein which is encoded by the Ewing sarcoma (EWS) gene, contains arginine-glycine-glycine repeats (RGG box) in its COOH-terminus. We previously found that the RGG box of EWS is a target for dimethylation catalyzed by protein arginine methyltransferases (PRMTs). Although it has been observed that arginine residues in EWS are dimethylated in vivo, the endogenous enzyme(s) responsible for this reaction have not been identified to date. In the present study, we determined that EWS was physically associated with PRMT8, the novel eighth member of the PRMT family, through the COOH-terminal region of EWS including RGG3 with the NH2-terminal region of PRMT8 encompassing the S-adenosyl-L-methionine binding domain, and that arginine residues in EWS were asymmetrically dimethylated by PRMT8 using amino acid analysis with thin-layer chromatography. These results suggested that EWS is a substrate for PRMT8, as efficient as for PRMT1.