Protein Arginine Methyltransferase 4 Research Articles

PRMT4 interacts with NCOA4 to inhibit ferritinophagy in cisplatin-induced acute kidney injury.

Cisplatin-induced acute kidney injury (AKI) is commonly seen in the clinical practice, and ferroptosis, a type of non-apoptotic cell death, plays a pivotal role in it. Previous studies suggested that protein arginine methyltransferase 4 (PRMT4) was incorporated in various bioprocesses, but its role in renal injuries has not been investigated. Our present study showed that PRMT4 was highly expressed in renal proximal tubular cells, and it was downregulated in cisplatin-induced AKI. Besides, genetic disruption of PRMT4 exacerbated, while its overexpression attenuated, cisplatin-induced redox injuries in renal proximal epithelia. Mechanistically, our work showed that PRMT4 interacted with NCOA4 to inhibit ferritinophagy, a type of selective autophagy favoring lipid peroxidation to accelerate ferroptosis. Taken together, our study demonstrated that PRMT4 interacted with NCOA4 to attenuate ferroptosis in cisplatin-induced AKI, suggesting that PRMT4 might present as a new therapeutic target for cisplatin-related nephropathy.

PRMT4 facilitates white adipose tissue browning and thermogenesis by methylating PPARγ.

Obesity is a global health threat, and the induction of white adipose tissue (WAT) browning presents a promising therapeutic method for it. Recent publications revealed the essential role of protein arginine methyltransferase 4 (PRMT4) in lipid metabolism and adipogenesis, but its involvement in WAT browning has not been investigated. Our initial studies found that the expression of PRMT4 in adipocytes was upregulated in cold-induced WAT browning but downregulated in obesity. Besides, PRMT4 overexpression in inguinal adipose tissue accelerated WAT browning and thermogenesis to protect against high-fat diet (HFD)-induced obesity and metabolic disruptions. Mechanistically, our work demonstrated that PRMT4 methylated peroxisome proliferator-activated receptor-γ (PPARγ) on Arg240 to enhance its interaction with the co-activator PR domain-containing protein 16 (PRDM16), leading to the increased expression of thermogenic genes. Taken together, our results uncover the essential role of PRMT4/PPARγ/PRDM16 axis in the pathogenesis of WAT browning.

Protein arginine methyltransferase 4 modulates nitric oxide synthase uncoupling and cerebral blood flow in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of mortality, disability, and long-term care burden in the United States, with women comprising the majority of AD diagnoses. While AD-related dementia is associated with tau and amyloid beta accumulation, concurrent derangements in cerebral blood flow have been observed alongside these proteinopathies in humans and rodent models. The homeostatic production of nitric oxide synthases (NOS) becomes uncoupled in AD which leads to decreased NO-mediated vasodilation and oxidative stress via the production of peroxynitrite (ONOO-∙) superoxide species. Here, we investigate the role of the novel protein arginine methyltransferase 4 (PRMT4) enzyme function and its downstream product asymmetric dimethyl arginine (ADMA) as it relates to NOS dysregulation and cerebral blood flow in AD. ADMA (type-1 PRMT product) has been shown to bind NOS as a noncanonic ligand causing enzymatic dysfunction. Our results from RT-qPCR and protein analyses suggest that aged (9-12 months) female mice bearing tau- and amyloid beta-producing transgenic mutations (3xTg-AD) express higher levels of PRMT4 in the hippocampus when compared to age- and sex-matched C57BL6/J mice. In addition, we performed studies to quantify the expression and activity of different NOS isoforms. Furthermore, laser speckle contrast imaging analysis was indicative that 3xTg-AD mice have dysfunctional NOS activity, resulting in reduced production of NO metabolites, enhanced production of free-radical ONOO-, and decreased cerebral blood flow. Notably, the aforementioned phenomena can be reversed via pharmacologic PRMT4 inhibition. Together, these findings implicate the potential importance of PRMT4 signaling in the pathogenesis of Alzheimer's-related cerebrovascular derangement.

PRMT4-mediated arginine methylation promotes tyrosine phosphorylation of VEGFR-2 and regulates filopodia protrusions

SummaryThrough tightly controlled multilayer mechanisms, vascular endothelial growth factor receptor-2 (VEGFR-2) activation and its downstream signal transduction govern vasculogenesis and pathological angiogenesis, such as tumor angiogenesis. Therefore, it is critical to understand the molecular mechanisms governing VEGFR-2 signal transduction. We report that protein arginine methyltransferase 4 (PRMT4) via its highly conserved EVH1 and PH domain-like N-terminal domain binds to VEGFR-2 and mediates methylation of the juxtamembrane arginine 817 (R817) on VEGFR-2. Methylation of R817 selectively increases phosphorylation of tyrosine 820 (Y820). Phosphorylation of Y820 facilitates the c-Src binding with VEGFR-2 via Src homology domain 2 (SH2). Interfering with the methylation of R817 or phosphorylation of Y820 inhibits VEGFR-2-induced filopodia protrusions, a process that is critical for the core angiogenic responses of VEGFR-2. Methylation of R817 is an important previously unrecognized mechanism of the angiogenic signaling of VEGFR-2, with implications for the development of novel-targeted VEGFR-2 inhibitors.

PRMT4 promotes ferroptosis to aggravate doxorubicin-induced cardiomyopathy via inhibition of the Nrf2/GPX4 pathway.

Doxorubicin (DOX), a commonly used antitumor agent, is often accompanied by its dosage-dependent cardiotoxicity, which incorporates ferroptosis in its pathogenesis. Protein arginine methyltransferase 4 (PRMT4) is a transcription regulator involved in the modulation of oxidative stress and autophagy, but its role in DOX-induced cardiomyopathy (DIC) and ferroptosis remains elusive. Herein, we aimed to investigate the involvement and the underlying mechanisms of PRMT4 in the pathogenesis of DIC. Our present study revealed that the expression level of PRMT4 was markedly decreased in DOX-treated cardiomyocytes. Interestingly, it is noted that PRMT4 overexpression accelerated ferroptosis to aggravate DIC, while its gene disruption or pharmaceutical inhibition exhibited the opposite effect. Mechanistically, our observation demonstrated that PRMT4 interacted with the nuclear factor erythroid 2-related factor 2 (Nrf2) to promote its enzymatic methylation, which restricted the nuclear translocation of Nrf2 and subsequently suppressed the transcription of glutathione peroxidase 4 (GPX4). Importantly, the detrimental role of PRMT4 in DOX-induced cardiomyocyte ferroptosis was abolished by Nrf2 activation or Fer-1 administration. Collectively, our data reveal that PRMT4 inhibits Nrf2/GPX4 signaling to accelerate ferroptosis in DIC, suggesting that targeting PRMT4 may present as a potential preventive strategy against the development of DIC.

PRMT4 inhibitor TP-064 impacts both inflammatory and metabolic processes without changing the susceptibility for early atherosclerotic lesions in male apolipoprotein E knockout mice

Background and aimsAtherosclerotic cardiovascular disease is a metabolic and inflammatory disorder. In vitro studies have suggested that protein arginine methyltransferase 4 (PRMT4) may act as a transcriptional coactivator to modulate inflammatory and metabolic processes. Here we investigated the potential anti-atherogenic effect of PRMT4 inhibitor TP-064 in vivo. MethodsMale apolipoprotein E knockout mice fed a high cholesterol/high fat Western-type diet were intraperitoneally injected three times a week with 2.5 mg/kg (low dose) or 10 mg/kg (high dose) TP-064 or with DMSO control. ResultsTP-064 induced a dose-dependent decrease in lipopolysaccharide-induced ex vivo blood monocyte Tnfα secretion (p < 0.05 for trend) in the context of unchanged blood monocyte concentrations and neutrophilia induction (p < 0.01 for trend). A dose-dependent decrease in gonadal white adipose tissue expression levels of PPARγ target genes was detected, which translated into a reduced body weight gain after high dose TP-064 treatment (p < 0.05). TP-064 treatment also dose-dependently downregulated gene expression of the glycogen metabolism related protein G6pc in the liver (p < 0.001 for trend). In addition, a trend towards lower plasma insulin and higher blood glucose levels was observed, which was paralleled by a reduction in hepatic mRNA expression levels of the insulin-responsive genes Fasn (−55%; p < 0.001) and Gck (−47%; p < 0.001) in high dose-treated mice. Plasma triglyceride levels were reduced by high dose TP-064 treatment (−30%; p < 0.05). However, no change was observed in the size or composition of aortic root atherosclerotic lesions. ConclusionsThe PRMT4 inhibitor TP-064 impacts both inflammatory and metabolic processes without changing atherosclerosis susceptibility of male apolipoprotein E knockout mice.

Endotoxin stabilizes protein arginine methyltransferase 4 (PRMT4) protein triggering death of lung epithelia

Lung epithelial cell death is a prominent feature of acute lung injury and acute respiratory distress syndrome (ALI/ARDS), which results from severe pulmonary infection leading to respiratory failure. Multiple mechanisms are believed to contribute to the death of epithelia; however, limited data propose a role for epigenetic modifiers. In this study, we report that a chromatin modulator protein arginine N-methyltransferase 4/coactivator-associated arginine methyltransferase 1 (PRMT4/CARM1) is elevated in human lung tissues with pneumonia and in experimental lung injury models. Here PRMT4 is normally targeted for its degradation by an E3 ubiquitin ligase, SCFFBXO9, that interacts with PRMT4 via a phosphodegron to ubiquitinate the chromatin modulator at K228 leading to its proteasomal degradation. Bacterial-derived endotoxin reduced levels of SCFFBXO9 thus increasing PRMT4 cellular concentrations linked to epithelial cell death. Elevated PRMT4 protein caused substantial epithelial cell death via caspase 3-mediated cell death signaling, and depletion of PRMT4 abolished LPS-mediated epithelial cell death both in cellular and murine injury models. These findings implicate a unique molecular interaction between SCFFBXO9 and PRMT4 and its regulation by endotoxin that impacts the life span of lung epithelia, which may play a key role in the pathobiology of tissue injury observed during critical respiratory illness.

Functional and Molecular Characterization of Skeletal Muscle‐Specific CARM1 Knockout Mice

Coactivator-associated arginine methyltransferase 1 [CARM1, also known as protein arginine methyltransferase 4 (PRMT4)] catalyzes the methylation of arginine residues on target proteins. Skeletal muscle CARM1 appears to have important functions during myogenesis, as well as in response to exercise and during disuse-induced phenotypic plasticity. However, a mechanistic understanding of the role(s) of CARM1 in skeletal muscle biology is lacking. In this study, we test the hypotheses that: 1) Skeletal muscle-specific CARM1 knockout (mKO) animals will have reduced strength, endurance, and motor function compared to their wild-type (WT) littermates, and 2) The acute intracellular and gene expression responses in skeletal muscle to a single bout of exercise will be altered in CARM1 mKO versus WT animals. To address our first hypothesis male 12-week-old mKO mice and WT littermates underwent a variety of functional tests. We found that mKO mice had significantly weaker maximum grip strength, increased fatigue, and slower ambulatory activity relative to their WT counterparts. Compared to WT mice, mKO animals had altered muscle mass depending on the tissue analysed. Immunofluorescence fiber type analysis revealed significant differences between male mKO and WT mice in the soleus (SOL) muscle. Male animals were then randomly assigned to one of three experimental groups: sedentary (SED), acute exercise (0AE), or acute exercise followed by 3 hours of recovery (3AE). CARM1 mKO animals ran for significantly less time and distance (~30%) than their WT counterparts. CARM1 mRNA levels remained constant across timepoints in WT animals in both the extensor digitorum longus (EDL) and SOL. EDL muscle from WT animals displayed significantly increased (737%) exercise-induced PGC-1α mRNA between SED and 3AE groups, while no increase was observed in mKO animals. PGC-1α levels were similar between all experimental groups in both WT and mKO animals in the SOL muscle. Additionally, PRMT1, PRMT5, and PRMT7 transcript expression was also consistent across timepoints, genotypes, and muscles. We observed that CARM1 protein levels were similar between experimental groups in WT animals. WT and mKO animals displayed increased phosphorylated AMPK levels at 0AE when compared to SED. Total AMPK levels were consistent across all 3 experimental groups in both WT and mKO animals, while removal of CARM1 increased AMPK content. AMPK activation status was significantly elevated from SED to 0AE, with a return to basal levels at 3AE in both the WT and mKO animals. PGC-1α protein content was not altered by exercise or by genotype and PRMT1, PRMT5, and PRMT7 levels were also similar between groups. In conclusion, this ongoing study demonstrates that the removal of CARM1 in skeletal muscle negatively impacts measures of muscle function, as well as alters the molecular response of skeletal muscle to acute physical activity. Altogether this study serves to increase our understanding of CARM1 and its role in regulating skeletal muscle plasticity.

PRMT4 drives post-ischemic angiogenesis via YB1/VEGF signaling.

Angiogenesis is an integral process in many ischemic disorders, and vascular endothelial growth factor (VEGF) plays an important role in it. Protein arginine methyltransferase 4 (PRMT4), a member of the type I PRMT family, is involved in various biological activities, but its role in endothelial cell (EC) remains elusive. Here, we aimed to investigate the role of PRMT4 in ischemic angiogenesis and explore the possible underlying mechanism. We found that PRMT4 was upregulated in ischemic muscles, and VEGF treatment potentiated its expression in ECs. In vitro, adenovirus-mediated PRMT4 overexpression promoted, while its gene disruption inhibited, EC proliferation, migration, and tube formation. In an in vivo hindlimb ischemia model, forced expression of PRMT4 in ECs showed accelerated blood flow recovery and increased capillary density, whereas its knockdown exhibited the opposite effect. Mechanistically, PRMT4 activated the transcription of VEGF via the interaction with Y-box binding protein-1 (YB1), leading to accelerated angiogenesis. Interestingly, the loss of YB1 partially abolished PRMT4-mediated angiogenesis in vitro. Collectively, our data revealed that PRMT4 promoted angiogenesis through interacting with YB1 and the consequential VEGF upregulation, suggesting that PRMT4 may present as a potential therapeutic target in ischemic angiogenesis. KEY MESSAGES: •PRMT4 is induced by VEGF and upregulated in a hindlimb ischemia model. •PRMT4 promotes angiogenesis both in vitro and in vivo. •PRMT4 regulates VEGF expression through interacting with YB1. •YB1 knockdown retards PRMT4-mediated angiogenic effects in vitro.

Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme’s active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.

PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis.

Background: Protein arginine methyltransferase 4 (PRMT4) has been reported to play a role in several common cancers; however, the function and mechanism of PRMT4 in hepatocellular carcinoma (HCC) are not fully understood. This study aimed to investigate the role and mechanism of PRMT4 in the progression of HCC.Methods: PRMT4 expression and clinicopathological characteristics were investigated using an HCC tissue microarray (TMA) consisting of 140 patient samples analyzed by immunohistochemistry. CCK-8, crystal violet and Transwell assays were used to determine cell proliferation, colony formation, migration, and invasion of HCC cell lines in which PRMT4 was overexpressed or downregulated. The underlying mechanism of PRMT4 function was explored by Western blot assays.Results: PRMT4 was highly expressed in HCC tumor tissues compared to adjacent nontumor tissues. PRMT4 expression was significantly associated with alpha-fetoprotein levels, tumor size, satellite nodules, and microvascular invasion. Patients with higher PRMT4 expression had a shorter survival time and higher recurrence rate. Functional studies demonstrated that PRMT4 overexpression promoted HCC cell proliferation, migration, and invasion in vitro, while knocking down PRMT4 inhibited these malignant behaviors. Additional results revealed that PRMT4 promoted the progression of HCC cells via activation of the AKT/mTOR signaling pathway. Furthermore, inhibition of the AKT/mTOR signaling by MK2206 or rapamycin significantly attenuated PRMT4-mediated malignant phenotypes.Conclusions: This study suggests that PRMT4 may promote the progression of HCC cells by activating the AKT/mTOR signaling pathway, which may be a valuable biomarker and potential target for HCC.

Protein Arginine Methyltransferase 4 Regulates Adipose Tissue Lipolysis in Type 1 Diabetic Mice.

PurposeHypertriglyceridemia is considered to be driven by increased lipolysis in type 1 diabetes mellitus (T1DM). However, information regarding the transcriptional circuitry that governs lipolysis remains incomplete in T1DM. Protein arginine methyltransferase 4 (PRMT4), a transcriptional coactivation factor, promotes autophagy and may play an important role in lipolysis. We wonder whether activated lipolysis in T1DM is regulated by PRMT4.Materials and MethodsRecombinant adeno-associated virus was adopted to overexpress PRMT4 in adipose tissue of mice. Streptozotocin (150 mg/kg) was injected intraperitoneally into mice to induce T1DM. Plasma insulin, triglycerides, free fatty acids (FFAs) levels were determined using commercial assay kits. Differentiated adipocytes were applied to verify the regulation of PRMT4 on lipolysis.ResultsElevated serum triglycerides and FFAs were observed in PRMT4-overexpressed T1DM mice. We also observed that PRMT4 over-expression induced the decrease of fat pads weights and adipocyte sizes. Moreover, expression levels of lipolysis-related molecules, including ATGL, HSL, and MAGL, and HSL phosphorylation levels were increased in PRMT4-overexpressed mice when compared to those of control mice. In vitro, PRMT4 promoted FFAs release and activated HSL phosphorylation, whereas PRMT4 knockdown inhibited these processes.ConclusionPRMT4 promotes lipolysis and increases serum triglyceride in T1DM.

PRMT4 overexpression aggravates cardiac remodeling following myocardial infarction by promoting cardiomyocyte apoptosis

Myocardial infarction due to coronary artery occlusion leads to adverse cardiac remodeling and heart failure. Apoptotic loss of cardiomyocytes near the ischemia area enlarges infarct area and promotes cardiac remodeling. Protein arginine methyltransferase 4 (PRMT4), a type I protein arginine methyltransferase, is involved in many cellular processes. Here we aimed to investigate the role of PRMT4 in cardiomyocyte apoptosis and myocardial infarction. We found that PRMT4 expression was markedly increased in ischemic heart and hypoxic cardiomyocytes. In vivo, cardiac-specific overexpression of PRMT4 in mice resulted in decreased survival rate, reduced left ventricular function, and aggravated cardiac remodeling following myocardial infarction. Mechanistically, PRMT4 overexpression promoted hypoxia-induced cardiomyocytes apoptosis, while its inhibition abolished these effects. Taken together, our work suggested an essential role of PRMT4 in myocardial infarction and cardiomyocyte apoptosis.

PRMT7 deficiency enhances adipogenesis through modulation of C/EBP-β

Obesity that is critically correlated with the initiation and development of metabolic syndrome and cardiovascular diseases has increased worldwide. Adipogenesis is coordinated through multi-steps involving adipogenic commitment, mitotic clonal expansion (MCE) and differentiation. Recently, protein arginine methyltransferase 4 (PRMT4) and PRMT5 have been implicated in modulation of adipogenesis via regulation of C/EBP-β activity or PPAR-γ2 expression. In the current study, we demonstrate a suppressive role of PRMT7 in adipogenesis. PRMT7-depleted preadipocytes or PRMT7−/− mouse embryonic fibroblasts (MEFs) displayed increased adipogenesis while PRMT7 overexpression attenuated it. PRMT7 depletion in preadipocytes promoted MCE, an initial step of adipogenesis. Furthermore, we found that PRMT7 interacted with and methylated a key adipogenic factor C/EBP-β upon adipogenic induction and modulated the accumulation of C/EBP-β at its target sites in the PPAR-γ2 promoter. Taken together, our data suggest that PRMT7 suppresses adipogenesis through modulation of C/EBP-β activity.

Identification and insilico structural analysis of Gallus gallus protein arginine methyltransferase 4 (PRMT4).

Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4‐specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT4 (ggPRMT4) transcript encompassing the complete open reading frame. Recombinant ggPRMT4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR/Cas9‐mediated deletion of ggPRMT4 demonstrated that the transcript identified here encodes avian PRMT4. Combining protein–protein docking and homology modeling based on published crystal structures of murine PRMT4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT4. Strikingly, in silico structural comparison of the N‐terminal Pleckstrin homology (PH) domain of avian and murine PRMT4 identified strictly conserved amino acids that are involved in an interaction interface toward the catalytic core domain, facilitating for the first time a prediction of the relative spatial arrangement of these two domains. Our novel findings are particularly exciting in light of the essential function of the PH domain in substrate recognition and methylation by PRMT4.

Abstract 3016: Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a rapidly progressing disease associated with less than 10% 5-year survival rate. Various treatment regimens failed to improve survival of PDAC patients, thus a critical need exists to identify druggable targets essential for tumor maintenance. We developed a powerful in vivo platform that enables the identification of new molecular drivers in the PDAC context where activating mutation of KRAS gene and loss of p53 dominate the genetic landscape. Through an in vivo loss of function screen performed in KRAS/p53 mutant PDAC primary patient models, we identify protein arginine methyltransferase 1 (PRMT1) as top scoring hit. This novel dependency in PDAC was subsequently validated in multiple PDAC models using both shRNA mediated as well as CRISPR base genetic inhibition and we demonstrated that PRMT1 knockdown induces a significant growth inhibition in vitro. Methylation of arginine 3 on histone H4 (H4R3me2a) as well as global arginine methylation was also evaluated and showed a dramatic reduction upon PRMT1 knockdown, correlating observed phenotype with target engagement. To further confirm a role for PRMT1 in tumor maintenance, we developed inducible PRMT1 knockdown in a primary patient model and showed a dramatic tumor growth inhibition (TGI) in vivo upon PRMT1 knockdown. PRMT1 is the primary enzyme responsible for arginine asymmetric demethylation, however other members of the Type I family are also involved in this process and we evaluated the role of protein arginine methyltransferase 4 (PRMT4) and 6 (PRMT6) in our workhorse model. Surprisingly, no significant phenotypic response was observed upon genetic inhibition of PRMT4 or PRMT6 suggesting no redundancy between different PRMT type I and a unique dependency on PRMT1. To strengthen and complement the genetic validation, we leveraged a PRMT Type I inhibitor and confirmed in vitro results as well as in vivo efficacy at tolerated doses (xenograft vs allograft). Key models have been prioritized in order to inform on PRMT1 dependency and to refine responder population. Our research has identified and validated for the first time an arginine methyltransferase as a novel genetic vulnerability in PDAC and strongly suggest PRMT1 as a new therapeutic opportunity in PDAC cancers. Citation Format: Virginia Giuliani, Bhavatarini Vangamudi, Erika Suzuki, Meredith Miller, Chiu-Yi Liu, Alessandro Carugo, Christopher Bristow, Guang Gao, Jing Han, Yuting Sun, Ningping Feng, Edward Chang, Joseph Marszalek, Jeffrey Kovacs, Maria Emilia Di Francesco, Carlo Toniatti, Timothy Heffernan, Philip Jones, Giulio Draetta. Identification of protein arginine methyltransferase 1 as novel epigenetic vulnerability in KRAS/p53 mutant PDAC primary patient models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3016. doi:10.1158/1538-7445.AM2017-3016

Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration.

Oxidative stress impacts normal cellular function leading to the pathogenesis of various diseases including pulmonary illnesses. Protein arginine methyltransferase 4 (PRMT4) is critical for normal lung alveolar epithelial cell development; however, the regulation of PRMT4 within such pulmonary diseases has yet to be elucidated. Using biochemical approaches, we uncovered that peroxide (H2O2) treatment decreases PRMT4 protein stability in murine lung epithelial (MLE12) cells to impede cell migration. Protein kinase glycogen synthase kinase 3β (GSK-3β) interacts with PRMT4 and catalyzes PRMT4 T132 phosphorylation that protects PRMT4 from ubiquitin proteasomal degradation. H2O2 downregulates GSK-3β to reduce PRMT4 at protein level. PRMT4 promotes cell migration and H2O2 degrades PRMT4 to inhibit lung epithelial cell migration. These observations demonstrate that oxidative stress destabilizes PRMT4 via GSK-3β signaling to impede lung epithelial cell migration that may hinder the lung repair and regeneration process.

PRMT4-mediated arginine methylation negatively regulates retinoblastoma tumor suppressor protein and promotes E2F-1 dissociation.

The retinoblastoma protein (pRb/p105) tumor suppressor plays a pivotal role in cell cycle regulation by blockage of the G1-to-S-phase transition. pRb tumor suppressor activity is governed by a variety of posttranslational modifications, most notably phosphorylation by cyclin-dependent kinase (Cdk) complexes. Here we report a novel regulation of pRb through protein arginine methyltransferase 4 (PRMT4)-mediated arginine methylation, which parallels phosphorylation. PRMT4 specifically methylates pRb at the pRb C-terminal domain (pRb C(term)) on arginine (R) residues R775, R787, and R798 in vitro and R787 in vivo. Arginine methylation is important for efficient pRb C(term) phosphorylation, as manifested by the reduced phosphorylation of a methylation-impaired mutant, pRb (R3K). A methylmimetic form of pRb, pRb (R3F), disrupts the formation of the E2F-1/DP1-pRb complex in cells as well as in an isolated system. Finally, studies using a Gal4-E2F-1 reporter system show that pRb (R3F) expression reduces the ability of pRb to repress E2F-1 transcriptional activation, while pRb (R3K) expression further represses E2F-1 transcriptional activation relative to that for cells expressing wild-type pRb. Together, our results suggest that arginine methylation negatively regulates the tumor suppressor function of pRb during cell cycle control, in part by creating a better substrate for Cdk complex phosphorylation and disrupting the interaction of pRb with E2F-1.

PRMT4 Is a Novel Coactivator of c-Myb-Dependent Transcription in Haematopoietic Cell Lines

Protein arginine methyltransferase 4 (PRMT4)–dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2β, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/β interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/β in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling.

CARM1/PRMT4 is necessary for the glycogen gene expression programme in skeletal muscle cells

CARM1 (co-activator-associated arginine methyltransferase 1)/PRMT4 (protein arginine methyltransferase 4), functions as a co-activator for transcription factors that are regulators of muscle fibre type and oxidative metabolism, including PGC (peroxisome-proliferator-activated receptor γ co-activator)-1α and MEF2 (myocyte enhancer factor 2). We observed significantly higher Prmt4 mRNA expression in comparison with Prmt1-Prmt6 mRNA expression in mouse muscle (in vitro and in vivo). Transfection of Prmt4 siRNA (small interfering RNA) into mouse skeletal muscle C2C12 cells attenuated PRMT4 mRNA and protein expression. We subsequently performed additional qPCR (quantitative PCR) analysis (in the context of metabolism) to examine the effect of Prmt4 siRNA expression on >200 critical genes that control (and are involved in) lipid, glucose and energy homoeostasis, and circadian rhythm. This analysis revealed a strikingly specific metabolic expression footprint, and revealed that PRMT4 is necessary for the expression of genes involved in glycogen metabolism in skeletal muscle cells. Prmt4 siRNA expression selectively suppressed the mRNAs encoding Gys1 (glycogen synthase 1), Pgam2 (muscle phosphoglycerate mutase 2) and Pygm (muscle glycogen phosphorylase). Significantly, PGAM, PYGM and GYS1 deficiency in humans causes glycogen storage diseases type X, type V/McArdle's disease and type 0 respectively. Attenuation of PRMT4 was also associated with decreased expression of the mRNAs encoding AMPK (AMP-activated protein kinase) α2/γ3 (Prkaa2 and Prkag3) and p38 MAPK (mitogen-activated protein kinase), previously implicated in Wolff-Parkinson-White syndrome and Pompe Disease (glycogen storage disease type II). Furthermore, stable transfection of two PRMT4-site-specific (methyltransferase deficient) mutants (CARM1/PRMT4 VLD and CARM1E267Q) significantly repressed the expression of Gys1, Pgam2 and AMPKγ3. Finally, in concordance, we observed increased and decreased glycogen levels in PRMT4 (native)- and VLD (methylation deficient mutant)-transfected skeletal muscle cells respectively. This demonstrated that PRMT4 expression and the associated methyltransferase activity is necessary for the gene expression programme involved in glycogen metabolism and human glycogen storage diseases.