PRMT7 ablation in cardiomyocytes causes cardiac hypertrophy and fibrosis through ß-catenin dysregulation.

Angiotensin II (AngII) has potent cardiac hypertrophic effects mediated through activation of hypertrophic signaling like Wnt/ß-Catenin signaling. In the current study, we examined the role of protein arginine methyltransferase 7 (PRMT7) in cardiac function. PRMT7 was greatly decreased in hypertrophic hearts chronically infused with AngII and cardiomyocytes treated with AngII. PRMT7 depletion in rat cardiomyocytes resulted in hypertrophic responses. Consistently, mice lacking PRMT7 exhibited the cardiac hypertrophy and fibrosis. PRMT7 overexpression abrogated the cellular hypertrophy elicited by AngII, while PRMT7 depletion exacerbated the hypertrophic response caused by AngII. Similar with AngII treatment, the cardiac transcriptome analysis of PRMT7-deficient hearts revealed the alteration in gene expression profile related to Wnt signaling pathway. Inhibition of PRMT7 by gene deletion or an inhibitor treatment enhanced the activity of ß-catenin. PRMT7 deficiency decreases symmetric dimethylation of ß-catenin. Mechanistic studies reveal that methylation of arginine residue 93 in ß-catenin decreases the activity of ß-catenin. Taken together, our data suggest that PRMT7 is important for normal cardiac function through suppression of ß-catenin activity.

PRMT5 Deficiency Enforces the Transcriptional and Epigenetic Programs of Klrg1+CD8+ Terminal Effector T Cells and Promotes Cancer Development.

Protein arginine methyltransferase 5 (PRMT5) participates in the symmetric dimethylation of arginine residues of proteins and contributes to a wide range of biological processes. However, how PRMT5 affects the transcriptional and epigenetic programs involved in the establishment and maintenance of T cell subset differentiation and roles in antitumor immunity is still incompletely understood. In this study, using single-cell RNA and chromatin immunoprecipitation sequencing, we found that mouse T cell-specific deletion of PRMT5 had greater effects on CD8+ than CD4+ T cell development, enforcing CD8+ T cell differentiation into Klrg1+ terminal effector cells. Mechanistically, T cell deficiency of PRMT5 activated Prdm1 by decreasing H4R3me2s and H3R8me2s deposition on its loci, which promoted the differentiation of Klrg1+CD8+ T cells. Furthermore, effector CD8+ T cells that transited to memory precursor cells were decreased in PRMT5-deficient T cells, thus causing dramatic CD8+ T cell death. In addition, in a mouse lung cancer cell line-transplanted tumor mouse model, the percentage of CD8+ T cells from T cell-specific deletion of PRMT5 mice was dramatically lost, but CD8+Foxp3+ and CD8+PDL1+ regulatory T cells were increased compared with the control group, thus accelerating tumor progression. We further verified these results in a mouse colon cancer cell line-transplanted tumor mouse model. Our study validated the importance of targeting PRMT5 in tumor treatment, because PRMT5 deficiency enforced Klrg1+ terminal CD8+ T cell development and eliminated antitumor activity.

The protein arginine methyltransferase PRMT1 promotes TBK1 activation through asymmetric arginine methylation.

TBK1 is an essential kinase for the innate immune response against viral infection. However, the key molecular mechanisms regulating the TBK1 activation remain elusive. Here, we identify PRMT1, a type I protein arginine methyltransferase, as an essential regulator of TBK1 activation. PRMT1 directly interacts with TBK1 and catalyzes asymmetric methylation of R54, R134, and R228 on TBK1. This modification enhances TBK1 oligomerization after viral infection, which subsequently promotes TBK1 phosphorylation and downstream type I interferon production. More important, myeloid-specific Prmt1 knockout mice are more susceptible to infection with DNA and RNA viruses than Prmt1fl/fl mice. Our findings reveal insights into the molecular regulation of TBK1 activation and demonstrate the essential function of protein arginine methylation in innate antiviral immunity.

PRMT7 targets of Foxm1 controls alveolar myofibroblast proliferation and differentiation during alveologenesis.

Although aberrant alveolar myofibroblasts (AMYFs) proliferation and differentiation are often associated with abnormal lung development and diseases, such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), epigenetic mechanisms regulating proliferation and differentiation of AMYFs remain poorly understood. Protein arginine methyltransferase 7 (PRMT7) is the only reported type III enzyme responsible for monomethylation of arginine residue on both histone and nonhistone substrates. Here we provide evidence for PRMT7's function in regulating AMYFs proliferation and differentiation during lung alveologenesis. In PRMT7-deficient mice, we found reduced AMYFs proliferation and differentiation, abnormal elastin deposition, and failure of alveolar septum formation. We further shown that oncogene forkhead box M1 (Foxm1) is a direct target of PRMT7 and that PRMT7-catalyzed monomethylation at histone H4 arginine 3 (H4R3me1) directly associate with chromatin of Foxm1 to activate its transcription, and thereby regulate of cell cycle-related genes to inhibit AMYFs proliferation and differentiation. Overexpression of Foxm1 in isolated myofibroblasts (MYFs) significantly rescued PRMT7-deficiency-induced cell proliferation and differentiation defects. Thus, our results reveal a novel epigenetic mechanism through which PRMT7-mediated histone arginine monomethylation activates Foxm1 transcriptional expression to regulate AMYFs proliferation and differentiation during lung alveologenesis and may represent a potential target for intervention in pulmonary diseases.

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.

PRMT7 is involved in regulation of germ cell proliferation during embryonic stage.

Arginine methylation is one of the most important post-translational modifications which is catalyzed by protein arginine methyltransferases (PRMTs). Previous studies have demonstrated that Prmt5 plays important role in germ cell development. Prmt7 is the only family member responsible for mono-methylation of arginine residue. However, whether Prmt7 is also involved in germ cell development remains unclear. In this study, we find that PRMT7 is abundantly expressed in the male germ cells during embryonic stage (from E10.5). Depletion of Prmt7 results in the defect of germ cell proliferation during embryonic stage and the number of primordial germ cells is significantly reduced in Prmt7-/- mice at E11.5. We also find that the size of testes is reduced in Prmt7-/- mice at P5 with reduced germ cell number and the diameter of seminiferous tubules. Further study reveals that the expression of BMPs and TGF-ß singling pathway is significantly changed in germ cells of Prmt7-/- mice at E12.5. However, no defect of testes development is observed in adult Prmt7-/flox; Mvh-Cre mice. Collectively, this study demonstrates that Prmt7 plays roles in male germ cell proliferation during embryonic stages and it is not required for germ cell development postnatally.

PRMT1 is critical to FEN1 expression and drug resistance in lung cancer cells.

The up-regulation of PRMT1 is critical to the cell growth and cancer progression of lung cancer cells. In our research, we found that PRMT1 is important to the DNA repair ability and drug resistance of lung cancer cells. To demonstrate the functions of PRMT1, we identified Flap endonuclease 1 (FEN1) as a post-translationally modified downstream target protein of PRMT1. As a major component of Base Excision Repair pathway, FEN1 plays an important role in DNA replication and DNA damage repair. However, the detailed mechanism of FEN1 up-regulation in lung cancer cells remains unclear. In our study, we identified PRMT1 as a key factor that maintains the high expression levels of FEN1, which is critical to the DNA repair ability and the chemotherapeutic drug resistance of lung cancer cells.

PRMT7 deficiency causes dysregulation of the HCN channels in the CA1 pyramidal cells and impairment of social behaviors.

HCN channels regulate excitability and rhythmicity in the hippocampal CA1 pyramidal cells. Perturbation in the HCN channel current (Ih) is associated with neuropsychiatric disorders, such as autism spectrum disorders. Recently, protein arginine methyltransferase 7 (PRMT7) was shown to be highly expressed in the hippocampus, including the CA1 region. However, the physiological function of PRMT7 in the CA1 neurons and the relationship to psychiatric disorders are unclear. Here we showed that PRMT7 knockout (KO) mice exhibit hyperactivity and deficits in social interaction. The firing frequency of the CA1 neurons in the PRMT7 KO mice was significantly higher than that in the wild-type (WT) mice. Compared with the WT CA1 neurons, the PRMT7 KO CA1 neurons showed a more hyperpolarized resting potential and a higher input resistance, which were occluded by the Ih-current inhibitor ZD7288; these findings were consistent with the decreased Ih and suggested the contribution of Ih-channel dysfunction to the PRMT7 KO phenotypes. The HCN1 protein level was decreased in the CA1 region of the PRMT7 KO mice in conjunction with a decrease in the expression of Shank3, which encodes a core scaffolding protein for HCN channel proteins. A brief application of the PRMT7 inhibitor DS437 did not reproduce the phenotype of the PRMT7 KO neurons, further indicating that PRMT7 regulates Ih by controlling the channel number rather than the open probability. Moreover, shRNA-mediated PRMT7 suppression reduced both the mRNA and protein levels of SHANK3, implying that PRMT7 deficiency might be responsible for the decrease in the HCN protein levels by altering Shank3 expression. These findings reveal a key role for PRMT7 in the regulation of HCN channel density in the CA1 pyramidal cells that may be amenable to pharmacological intervention for neuropsychiatric disorders.

Islet-specific Prmt5 excision leads to reduced insulin expression and glucose intolerance in mice.

Protein arginine methyltransferase 5 (PRMT5), a symmetric arginine methyltransferase, regulates cell functions by influencing gene transcription through posttranslational modification of histones and non-histone proteins. PRMT5 interacts with multiple partners including menin, which controls beta cell homeostasis. However, the role of Prmt5 in pancreatic islets, particularly in beta cells, remains unclear. A mouse model with an islet-specific knockout (KO) of the Prmt5 gene was generated, and the influence of the Prmt5 excision on beta cells was investigated via morphologic and functional studies. Beta cell function was evaluated by glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test. Beta cell proliferation was evaluated by immunostaining. Gene expression change was determined by real-time qPCR. Molecular mechanisms were investigated in beta cells in vitro and in vivo in Prmt5 KO mice. The results show that islet-specific KO of Prmt5 reduced expression of the insulin gene and impaired glucose tolerance and GSIS in vivo. The mechanistic study indicated that PRMT5 is involved in the regulation of insulin gene transcription, likely via histone methylation-related chromatin remodeling. The reduced expression of insulin in beta cells in the Prmt5 KO mice may contribute to impaired glucose tolerance (IGT) and deficient GSIS in the mouse model. These results will provide new insights into exploring novel strategies to treat diabetes caused by insulin insufficiency.

Prmt7 promotes myoblast differentiation via methylation of p38MAPK on arginine residue 70.

MyoD functions as a master regulator to induce muscle-specific gene expression and myogenic differentiation. Here, we demonstrate a positive role of Protein arginine methyltransferase 7 (Prmt7) in MyoD-mediated myoblast differentiation through p38MAPK activation. Prmt7 depletion in primary or C2C12 myoblasts impairs cell cycle withdrawal and myogenic differentiation. Furthermore, Prmt7 depletion decreases the MyoD-reporter activities and the MyoD-mediated myogenic conversion of fibroblasts. Together with MyoD, Prmt7 is recruited to the Myogenin promoter region and Prmt7 depletion attenuates the recruitment of MyoD and its coactivators. The mechanistic study reveals that Prmt7 methylates p38MAPKα at the arginine residue 70, thereby promoting its activation which in turn enhances MyoD activities. The arginine residue 70 to alanine mutation in p38MAPKα impedes MyoD/E47 heterodimerization and the recruitment of Prmt7, MyoD and Baf60c to the Myogenin promoter resulting in blunted Myogenin expression. In conclusion, Prmt7 promotes MyoD-mediated myoblast differentiation through methylation of p38MAPKα at arginine residue 70.

PRMT5 methylome profiling uncovers a direct link to splicing regulation in acute myeloid leukemia.

Protein arginine methyltransferase 5 (PRMT5) has emerged as a promising cancer drug target, and three PRMT5 inhibitors are currently in clinical trials for multiple malignancies. In this study, we investigated the role of PRMT5 in human acute myeloid leukemia (AML). Using an enzymatic dead version of PRMT5 and a PRMT5-specific inhibitor, we demonstrated the requirement of the catalytic activity of PRMT5 for the survival of AML cells. We then identified PRMT5 substrates using multiplexed quantitative proteomics and investigated their role in the survival of AML cells. We found that the function of the splicing regulator SRSF1 relies on its methylation by PRMT5 and that loss of PRMT5 leads to changes in alternative splicing of multiple essential genes. Our study proposes a mechanism for the requirement of PRMT5 for leukemia cell survival and provides potential biomarkers for the treatment response to PRMT5 inhibitors.

PRMT7 deficiency enhances adipogenesis through modulation of C/EBP-ß.

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-ß 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-ß upon adipogenic induction and modulated the accumulation of C/EBP-ß at its target sites in the PPAR-γ2 promoter. Taken together, our data suggest that PRMT7 suppresses adipogenesis through modulation of C/EBP-ß activity.

Hepatocyte PRMT1 protects from alcohol induced liver injury by modulating oxidative stress responses.

Protein Arginine methyltransferase 1 (PRMT1) is the main enzyme of cellular arginine methylation. Previously we found that PRMT1 activity in the liver is altered after alcohol exposure resulting in epigenetic changes. To determine the impact of these PRMT1 changes on the liver's response to alcohol, we induced a hepatocyte specific PRMT1 knockout using AAV mediated Cre delivery in mice fed either alcohol or control Lieber-DeCarli liquid diet. We found that in alcohol fed mice, PRMT1 prevents oxidative stress and promotes hepatocyte survival. PRMT1 knockout in alcohol fed mice resulted in a dramatic increase in hepatocyte death, inflammation and fibrosis. Additionally, we found that alcohol promotes PRMT1 dephosphorylation at S297. Phosphorylation at this site is necessary for PRMT1-dependent protein arginine methylation. PRMT1 S297A, a dephosphorylation mimic of PRMT1 had reduced ability to promote gene expression of pro-inflammatory cytokines, pro-apoptotic genes BIM and TRAIL and expression of a suppressor of hepatocyte proliferation, Hnf4α. On the other hand, several functions of PRMT1 were phosphorylation-independent, including expression of oxidative stress response genes, Sod1, Sod2 and others. In vitro, both wild type and S297A PRMT1 protected hepatocytes from oxidative stress induced apoptosis, however S297D phosphorylation mimic PRMT1 promoted cell death. Taken together these data suggest that PRMT1 is an essential factor of liver adaptation to alcohol; alcohol-induced dephosphorylation shifts PRMT1 toward a less pro-inflammatory, more pro-proliferative and pro-survival form.

Arginine Methyltransferase PRMT8 Provides Cellular Stress Tolerance in Aging Motoneurons.

Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration.SIGNIFICANCE STATEMENT Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.

Prmt1 regulates craniofacial bone formation upstream of Msx1.

Protein arginine methylation has been recently identified as an important form of post-translational modification (PTM). It is carried out by the protein arginine methyltransferase (PRMT) family of enzymes, which in mammals consists of nine members. Among them, PRMT1 is the major arginine methyltransferase and participates in transcription, signal transduction, development and cancer. The function of PRMT1 in craniofacial development remains unclear. We generated Wnt1-Cre;Prmt1fl/fl mice with cranial neural crest (CNC)-specific deletion of Prmt1 and compared CNC-derived craniofacial bones from newborn control and Wnt1-Cre;Prmt1fl/fl mice. The size, surface area and volume of the premaxilla, maxilla, palatine bone, frontal bone, and mandible were analyzed using three-dimensional (3D) micro-computed tomography (microCT). We found that Prmt1 deficiency led to alterations in craniofacial bones including the premaxilla, maxilla, palatine bone, frontal bone, and mandible, as well as defects in the incisor and alveolar bone, recapitulating changes seen in Msx1-deficient mice. We further determined that Prmt1 depletion resulted in significant downregulation of Msx1 in calvaria-derived preosteoblast and primordium of frontal bone and mandible. Our study reveals critical roles of PRMT1 in the formation of CNC-derived craniofacial bones and suggests that Prmt1 is an upstream regulator of Msx1 in craniofacial bone development.

Network-based characterization of the synaptic proteome reveals that removal of epigenetic regulator Prmt8 restricts proteins associated with synaptic maturation.

The brain adapts to dynamic environmental conditions by altering its epigenetic state, thereby influencing neuronal transcriptional programs. An example of an epigenetic modification is protein methylation, catalyzed by protein arginine methyltransferases (PRMT). One member, Prmt8, is selectively expressed in the central nervous system during a crucial phase of early development, but little else is known regarding its function. We hypothesize Prmt8 plays a role in synaptic maturation during development. To evaluate this, we used a proteome-wide approach to characterize the synaptic proteome of Prmt8 knockout versus wild-type mice. Through comparative network-based analyses, proteins and functional clusters related to neurite development were identified to be differentially regulated between the two genotypes. One interesting protein that was differentially regulated was tenascin-R (TNR). Chromatin immunoprecipitation demonstrated binding of PRMT8 to the tenascin-r (Tnr) promoter. TNR, a component of perineuronal nets, preserves structural integrity of synaptic connections within neuronal networks during the development of visual-somatosensory cortices. On closer inspection, Prmt8 removal increased net formation and decreased inhibitory parvalbumin-positive (PV+) puncta on pyramidal neurons, thereby hindering the maturation of circuits. Consequently, visual acuity of the knockout mice was reduced. Our results demonstrated Prmt8's involvement in synaptic maturation and its prospect as an epigenetic modulator of developmental neuroplasticity by regulating structural elements such as the perineuronal nets.

Angiodysplasia in embryo lacking protein arginine methyltransferase 1 in vascular endothelial cells.

Protein arginine methyltransferase 1 (PRMT1) is involved in multiple cellular functions including proliferation and differentiation. Although PRMT1 is expressed in vascular endothelial cells (ECs), which are responsible for angiogenesis during embryonic development, its role has remained elusive. In this study, we generated endothelial-specific prmt1-knockout (Prmt1-ECKO) mice, and found that they died before embryonic day 15. The superficial temporal arteries in these embryos were poorly perfused with blood, and whole-mount 3D imaging revealed dilated and segmentalized luminal structures in Prmt1-ECKO fetuses in comparison with those of controls. Our findings provide evidence that PRMT1 is important for embryonic vascular formation.

Differential regulation of T-cell dependent and T-cell independent antibody responses through arginine methyltransferase PRMT1 in vivo.

Protein arginine methyltransferase 1 (PRMT1), a major PRMT in mammalian cells, has been shown to play a crucial role in multiple biological functions in vitro. To explore the role of PRMT1 in B cells in vivo, we generated B cell-specific PRMT1-deficient (Prmt1(-/-) ) mice using a Cre-loxP system. Prmt1(-/-) mice showed a defect in B-cell development with diminished levels of serum antibodies. Antibody responses in Prmt1(-/-) mice were absent after stimulation with the type 2 T cell-independent antigen NP-Ficoll but intact after stimulation with the T cell-dependent antigen NP-OVA. Our findings comprise the first evidence showing that PRMT1 is necessary for lymphocyte functions in vivo.

PRMT7 Preserves Satellite Cell Regenerative Capacity.

Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity.