Protein Arginine Methyltransferases (PRMTs) catalyze the posttranslational methylation of arginine using S–adenosyl–methionine (SAM) as a methyl–donor. The PRMT family is widely expressed and has been implicated in biological functions such as RNA splicing, transcriptional control, signal transduction, and DNA repair. Therefore, specific inhibitors of individual PRMTs have potentially significant research and therapeutic value. In particular, PRMT1 is responsible for >85% of arginine methyltransferase activity, but currently available inhibitors of PRMT1 lack specificity, efficacy, and bioavailability. To address this limitation, we developed a high–throughput screening assay for PRMT1 that utilizes a hyper–reactive cysteine within the active–site, which is lacking in almost all other PRMTs. This assay, which monitors the kinetics of the fluorescence polarization signal increase upon PRMT1 labeling by a rhodamine–containing cysteine–reactive probe, successfully identified two novel inhibitors selective for PRMT1 over other SAM–dependent methyltransferases.
arginine methylation; PRMT1; inhibitor
Despite the emerging importance of protein arginine N-methyltransferase (PRMT) activity in regulating cellular processes, only a limited number of PRMT assays have been developed. Here, we compare several qualitative and quantitative methods that we use for measuring PRMT activity. Gel-based methods allow for the simultaneous detection of methyl transfer activity on multiple substrates, but require signals well above background in order to generate reliable data for quantitation, which can be challenging with low activity PRMTs or substrates that are poor methyl-acceptors. Techniques that measure S-adenosyl-L-homocysteine (AdoHcy) product formation suffer from a background caused by PRMT automethylation and the spontaneous formation of AdoHcy from S-adenosyl-L-methionine (AdoMet). However, when this background is controlled, this approach is useful for product inhibition studies. Methods that detect methylated arginines derived from acid hydrolysis of PRMT reaction samples can determine the absolute amounts of ω-NG-monomethylarginine (MMA), asymmetric ω-NG,NG-dimethylarginine (aDMA) or symmetric ω-NG,N′G-dimethylarginine (sDMA) to quantify PRMT activity. We describe separation methods of these methylated arginine derivatives by thin layer, reverse phase, or cation exchange chromatography, and quantification by radioactivity or mass spectrometry. The latter approach is advantageous because it does not require radiolabelled samples for detection, and activity is readily quantified with commercially available standards.
PRMT; AdoMet; AdoHcy; methylarginine; enzyme kinetics
Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.
Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical SN2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.
The protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and di-methylation of peptidyl arginine residues. PRMT1 is the founding member of the PRMT family, and this isozyme is responsible for methylating ~85% of the arginine residues in mammalian cells. Additionally, PRMT1 activity is aberrantly upregulated in heart disease and cancer. As a part of a program to develop isozyme specific PRMT inhibitors, we recently described the design and synthesis of C21, a chloroacetamidine bearing histone H4 tail analog that acts as an irreversible PRMT1 inhibitor. Given the covalent nature of the interaction, we set out to develop Activity Based Probes (ABPs) that could be used to characterize the physiological roles of PRMT1. Herein, we report the design, synthesis, and characterization of fluorescein-conjugated C21 (F-C21) and biotin-conjugated C21 (B-C21) as PRMT1-specific ABPs. Additionally, we provide the first evidence that PRMT1 activity is negatively regulated in a spatial and temporal fashion.
The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.
Arginine methylation governs important cellular processes that impact growth and proliferation, as well as differentiation and development. Through their ability to catalyze symmetric or asymmetric methylation of histones and non-histone proteins, members of the protein arginine methyltransferase (PRMT) family regulate chromatin structure and expression of a wide spectrum of target genes. Unlike other PRMTs, PRMT5 works in concert with a variety of cellular proteins including ATP-dependent chromatin remodelers and co-repressors to induce epigenetic silencing. Recent work also implicates PRMT5 in the control of growth-promoting and pro-survival pathways, demonstrating its versatility as an enzyme involved in both epigenetic regulation of anti-cancer target genes and organelle biogenesis. These studies not only provide insight into the molecular mechanisms by which PRMT5 contributes to growth control, but also justify targeting PRMT5 therapeutically.
Posttranslational modifications (PTMs) are important strategies used by eukaryotic organisms to modulate their phenotypes. One of the well studied PTMs, arginine methylation, is catalyzed by protein arginine methyltransferases (PRMTs) with SAM as the methyl donor. The functions of PRMTs have been broadly studied in different biological processes and diseased states, but the molecular basis for arginine methylation is not well defined. In this study, we report the transient-state kinetic analysis of PRMT1 catalysis. The fast association and dissociation rates suggest that PRMT1 catalysis of histone H4 methylation follows a rapid equilibrium sequential kinetic mechanism. The data give direct evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, from the stopped-flow fluorescence measurements, we have identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. These results provide new insights into the mechanism of arginine methylation and the rational design of PRMT inhibitors.
PRMT1; arginine methylation; transient-state kinetics; conformational transition; fluorescent probe; stopped flow
PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression. Knockdown of PRMT6 expression in the human osteosarcoma cell line U2OS results in an accumulation of cells at the G2 checkpoint. Loss of PRMT6 coincides with upregulation of p21 and p27, two members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors. Gene expression and promoter analysis show that p21 and p27 are direct targets of PRMT6, which involves methylation of arginine-2 of histone H3. Our findings imply arginine methylation of histones by PRMT6 in cell cycle regulation.
Arginine methylation by protein N-arginine methyltransferases (PRMTs) is an important post-translational modification in the regulation of protein signaling. PRMT2 contains a highly conserved catalytic Ado-Met binding domain, but the enzymatic function of PRMT2 with respect to methylation is unknown. The JAK-STAT pathway is proposed to be regulated through direct arginine methylation of STAT transcription factors, and STAT3 signaling is known to be required for leptin regulation of energy balance.
To identify the potential role of STAT3 arginine methylation by PRMT2 in the regulation of leptin signaling and energy homeostasis.
Methods and Results
We identified that PRMT2-/- mice are hypophagic, lean, and have significantly reduced serum leptin levels. This lean phenotype is accompanied by resistance to food-dependent obesity and an increased sensitivity to exogenous leptin administration. PRMT2 co-localizes with STAT3 in hypothalamic nuclei, where it binds and methylates STAT3 through its Ado-Met binding domain. In vitro studies further clarified that the Ado-Met binding domain of PRMT2 induces STAT3 methylation at the Arg31 residue. Absence of PRMT2 results in decreased methylation and prolonged tyrosine phosphorylation of hypothalamic STAT3, which was associated with increased expression of hypothalamic pro-opiomelanocortin following leptin stimulation.
These data elucidate a molecular pathway that directly links arginine methylation of STAT3 by PRMT2 to the regulation of leptin signaling, suggesting a potential role for PRMT2 antagonism in the treatment of obesity and obesity-related syndromes.
PRMT2; leptin; methylation; STAT3
Arginine methylation is a common posttranslational modification that has been strongly implicated in transcriptional regulation. The arginine methyltransferases (PRMTs) were first reported as transcriptional coactivators for the estrogen and androgen receptors. Compounds that inhibit these enzymes will provide us with valuable tools for dissecting the roles of these enzymes in cells, and will possibly also have therapeutic applications. In order to identify such inhibitors of the PRMTs, we performed a high throughput screen using a small molecule library a number of years ago. We termed these compounds AMIs (arginine methyltransferase inhibitors). The majority of these inhibitors were polyphenols, and one in particular (AMI-18) shared additional features with a group of known xenoestrogens. We thus tested a panel of xenoestrogens and found that a number of them possess the ability to inhibit PRMT activity in vitro. These inhibitors primarily target CARM1, and include licochalcone A, kepone, benzyl 4-hydroxybenzoate, and tamoxifen. We developed a cell-based reporter system for CARM1 activity, and showed that tamoxifen (IC50=30 µM) inhibits this PRMT. The ability of these compounds to regulate the activity of transcriptional coactivators may be an unappreciated mechanism of action for xenoestrogens and may also explain the efficacy of high-dose tamoxifen treatment on estrogen receptor negative cancers.
PRMT1; CARM1; Arginine methylation; Xenoestrogens
The proper epigenetic modification of chromatin by protein arginine methyltransferases (PRMTs) is crucial for normal cell growth and health. The human SWI/SNF-associated PRMT5 is involved in the transcriptional repression of target genes by directly methylating H3R8 and H4R3. To further understand the impact of PRMT5-mediated histone methylation on cancer, we analyzed its expression in normal and transformed human B lymphocytes. Our findings reveal that PRMT5 protein levels are enhanced in various human lymphoid cancer cells, including transformed chronic lymphocytic leukemia (B-CLL) cell lines. PRMT5 overexpression is caused by the altered expression of the PRMT5-specific microRNAs 19a, 25, 32, 92, 92b, and 96 and results in the increased global symmetric methylation of H3R8 and H4R3. An evaluation of both epigenetic marks at PRMT5 target genes such as RB1 (p105), RBL1 (p107), and RBL2 (p130) showed that promoters H3R8 and H4R3 are hypermethylated, which in turn triggers pocket protein transcriptional repression. Furthermore, reducing PRMT5 expression in WaC3CD5 B-CLL cells abolishes H3R8 and H4R3 hypermethylation, restores RBL2 expression, and inhibits cancer cell proliferation. These results indicate that PRMT5 overexpression epigenetically alters the transcription of key tumor suppressor genes and suggest a causal role of the elevated symmetric methylation of H3R8 and H4R3 at the RBL2 promoter in transformed B-lymphocyte pathology.
Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.
Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.
These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.
PRMT6; HIV-1; Automethylation; Protein stability; Antiretroviral activity
Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is implicated in modulation of cellular processes including gene transcription. The role of PRMTs in the regulation of intracellular signaling pathways has remained obscure, however. We now show that PRMT1 methylates apoptosis signal-regulating kinase 1 (ASK1) at arginine residues 78 and 80 and thereby negatively regulates ASK1 signaling. PRMT1-mediated ASK1 methylation attenuated the H2O2-induced stimulation of ASK1, with this inhibitory effect of PRMT1 being abolished by replacement of arginines 78 and 80 of ASK1 with lysine. Furthermore, depletion of PRMT1 expression by RNA interference potentiated H2O2-induced stimulation of ASK1. PRMT1-mediated ASK1 methylation promoted the interaction between ASK1 and its negative regulator thioredoxin, whereas it abrogated the association of ASK1 with its positive regulator TRAF2. Moreover, PRMT1 depletion potentiated paclitaxel-induced ASK1 activation and apoptosis in human breast cancer cells. Together, our results indicate that arginine methylation of ASK1 by PRMT1 contributes to the regulation of stress-induced signaling that controls a variety of cellular events including apoptosis.
arginine methylation; ASK1; paclitaxel; PRMT1
Protein arginine methyltransferase 5 (PRMT5) is an enzyme that catalyzes transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or nonhistone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumors, its direct role in cancer growth has not been fully investigated. In this study, in human lung tissue samples, we found that PRMT5 was highly expressed in lung cancer cells whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to downregulation of fibroblast growth factor receptor signaling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.
PRMT5; protein arginine methyltransferase; lung cancer; proliferation; FGFR
Post-translational methylation of arginine residues profoundly affects the structure and functions of protein and, hence, implicated in a myriad of essential cellular processes such as signal transduction, mRNA splicing and transcriptional regulation. Protein arginine methyltransferases (PRMTs), the enzymes catalyzing arginine methylation have been extensively studied in animals, yeast and, to some extent, in model plant Arabidopsis thaliana. Eight genes coding for the PRMTs were identified in Oryza sativa, previously. Here, we report that these genes show distinct expression patterns in various parts of the plant. In vivo targeting experiment demonstrated that GFP-tagged OsPRMT1, OsPRMT5 and OsPRMT10 were localized to both the cytoplasm and nucleus, whereas OsPRMT6a and OsPRMT6b were predominantly localized to the nucleus. OsPRMT1, OsPRMT4, OsPRMT5, OsPRMT6a, OsPRMT6b and OsPRMT10 exhibited in vitro arginine methyltransferase activity against myelin basic protein, glycine-arginine-rich domain of fibrillarin and calf thymus core histones. Furthermore, they depicted specificities for the arginine residues in histones H3 and H4 and were classified into type I and Type II PRMTs, based on the formation of type of dimethylarginine in the substrate proteins. The two homologs of OsPRMT6 showed direct interaction in vitro and further titrating different amounts of these proteins in the methyltransferase assay revealed that OsPRMT6a inhibits the methyltransferase activity of OsPRMT6b, probably, by the formation of heterodimer. The identification and characterization of PRMTs in rice suggests the conservation of arginine methylation in monocots and hold promise for gaining further insight into regulation of plant development.
Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.
Protein arginine methyltransferases (PRMTs) are SAM-dependent enzymes that catalyze the mono- and di-methylation of peptidyl arginine residues. Although all PRMTs produce mono-methyl arginine (MMA), type 1 PRMTs go on to form asymmetrically dimethylated arginine (ADMA), while type 2 enzymes form symmetrically dimethylated arginine (SDMA). PRMT1 is the major type 1 PRMT in vivo, thus it is the primary producer of the competitive NOS inhibitor, ADMA. Hence, potent inhibitors, which are highly selective for this particular isozyme, could serve as excellent therapeutics for heart disease. However, the design of such inhibitors is impeded by a lack of information regarding this enzyme’s kinetic and catalytic mechanisms. Herein we report an analysis of the kinetic mechanism of human PRMT1 using both an unmethylated and a mono-methylated substrate peptide based on the N-terminus of histone H4. The results of initial velocity and product and dead-end inhibition experiments indicate that PRMT1 utilize a rapid equilibrium random mechanism with the formation of dead-end EAP and EBQ complexes. This mechanism is gratifyingly consistent with previous results demonstrating that PRMT1 catalyzes substrate dimethylation in a partially processive manner.
Arginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of proteins whose functions are modulated by arginine methylation await discovery in trypanosomes. Here, we employed a yeast two-hybrid screen using as bait the major T. brucei type I PRMT, TbPRMT1, to identify potential substrates of this enzyme.
We identified a protein containing N-LIP and C-LIP domains that we term TbLpn. These domains are usually present in a family of proteins known as lipins, and involved in phospholipid biosynthesis and gene regulation. Far western and co-immunoprecipitation assays confirmed the TbPRMT1-TbLpn interaction. We also demonstrated that TbLpn is localized mainly to the cytosol, and is methylated in vivo. In addition, we showed that, similar to mammalian and yeast proteins with N-LIP and C-LIP domains, recombinant TbLpn exhibits phosphatidic acid phosphatase activity, and that two conserved aspartic acid residues present in the C-LIP domain are critical for its enzymatic activity.
This study reports the characterization of a novel trypanosome protein and provides insight into its enzymatic activity and function in phospholipid biosynthesis. It also indicates that TbLpn functions may be modulated by arginine methylation.
Kinetoplastid; Lipin; Arginine methylation; Phosphatidic acid phosphatase
Protein arginine methylation is a posttranslational modification that impacts cellular functions, such as RNA processing, transcription, DNA repair, and signal transduction. The majority of our knowledge regarding arginine methylation derives from studies of yeast and mammals. Here, we describe a protein arginine N-methyltransferase (PRMT), TbPRMT5, from the early-branching eukaryote Trypanosoma brucei. TbPRMT5 shares the greatest sequence similarity with PRMT5 and Skb1 type II enzymes from humans and Schizosaccharomyces pombe, respectively, although it is significantly divergent at the amino acid level from its mammalian and yeast counterparts. Recombinant TbPRMT5 displays broad substrate specificity in vitro, including methylation of a mitochondrial-gene-regulatory protein, RBP16. TbPRMT5 catalyzes the formation of ω-NG-monomethylarginine and symmetric ω-NG,NG′-dimethylarginine and does not require trypanosome cofactors for this activity. These data establish that type II PRMTs evolved early in the eukaryotic lineage. In vivo, TbPRMT5 is constitutively expressed in the bloodstream form and procyclic-form (insect host) life stages of the parasite and localizes to the cytoplasm. Genetic disruption via RNA interference in procyclic-form trypanosomes indicates that TbPRMT5 is not essential for growth in this life cycle stage. TbPRMT5-TAP ectopically expressed in procyclic-form trypanosomes is present in high-molecular-weight complexes and associates with an RG domain-containing DEAD box protein related to yeast Ded1 and two kinetoplastid-specific proteins. Thus, TbPRMT5 is likely to be involved in novel methylation-regulated functions in trypanosomes, some of which may include RNA processing and/or translation.
Arginine methylation of histones is a well-known regulator of gene expression. Protein arginine methyltransferase 6 (PRMT6) has been shown to function as a transcriptional repressor by methylating the histone H3 arginine 2 [H3R2(me2a)] repressive mark; however, few targets are known. To define the physiological role of PRMT6 and to identify its targets, we generated PRMT6−/− mouse embryo fibroblasts (MEFs). We observed that early passage PRMT6−/− MEFs had growth defects and exhibited the hallmarks of cellular senescence. PRMT6−/− MEFs displayed high transcriptional levels of p53 and its targets, p21 and PML. Generation of PRMT6−/−; p53−/− MEFs prevented the premature senescence, suggesting that the induction of senescence is p53-dependent. Using chromatin immunoprecipitation assays, we observed an enrichment of PRMT6 and H3R2(me2a) within the upstream region of Trp53. The PRMT6 association and the H3R2(me2a) mark were lost in PRMT6−/− MEFs and an increase in the H3K4(me3) activator mark was observed. Our findings define a new regulator of p53 transcriptional regulation and define a role for PRMT6 and arginine methylation in cellular senescence.
In an effort to understand the epigenetic regulation of ribosomal RNA gene (rDNA) expression we have previously demonstrated the role of DNA methyltransferases and methyl CpG binding proteins in rRNA synthesis. Here, we studied the role of protein arginine methyltransferase PRMT5 and the two methylated histones H3R8Me2 and H4R3Me2, in rDNA expression in Epstein Barr virus- transformed primary B-cells (LCLs) and in HeLa cells responding to serum-regulated growth. Chromatin immunoprecipitation assay showed that histones H3 and H4 associated with rRNA promoters were differentially methylated at arginine residues 8 and 3, respectively, depending on its transcriptional activity. Association of PRMT5 and methylated H3 with the unmethylated promoters in resting B-cells was significantly reduced in rapidly growing LCLs. Unlike PRMT5 and H3R8Me2, histone H4 associated with both methylated and unmethylated rRNA promoters in resting B-cells was methylated at the R3 residue. However, a dramatic decrease in R3 methylation of H4 recruited to the unmethylated rRNA promoters was observed in LCLs while it remained unaltered in the fraction bound to the methylated promoters. Differential interaction of PRMT5 and methylation of H3 and H4 associated with the rRNA promoters was also observed when serum starved HeLa cells were allowed to grow in serum replenished media. Ectopic expression of PRMT5 suppressed activity of both unmethylated and methylated rRNA promoter in transient transfection assay whereas siRNA mediated knockdown of PRMT5 increased rRNA synthesis in HeLa cells. These data suggest a key role of PRMT5 and the two methylated histones in regulating rRNA promoter activity.
RNA POLYMERASE 1; PRMT5; H3R8ME2; H4R3ME2; EBV TRANSFORMATION
Background: Neural stem cells generate all the cell types of the central nervous system.
Results: Transcription factor, PRDM4, recruits protein arginine methyltransferase 5 (PRMT5) to control the timing of neurogenesis.
Conclusion: PRDM4- and PRMT5-mediated histone arginine methylation controls neural stem cell proliferation and differentiation.
Significance: Histone arginine methylation is a novel epigenetic mechanism that regulates neural stem cell reprogramming.
During development of the cerebral cortex, neural stem cells (NSCs) undergo a temporal switch from proliferative (symmetric) to neuron-generating (asymmetric) divisions. We investigated the role of Schwann cell factor 1 (SC1/PRDM4), a member of the PRDM family of transcription factors, in this critical transition. We discovered that SC1 recruits the chromatin modifier PRMT5, an arginine methyltransferase that catalyzes symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) and that this modification is preferentially associated with undifferentiated cortical NSCs. Overexpressing SC1 in embryonic NSCs led to an increase in the number of Nestin-expressing precursors; mutational analysis of SC1 showed that this was dependent on recruitment of PRMT5. We found that SC1 protein levels are down-regulated at the onset of neurogenesis and that experimental knockdown of SC1 in primary NSCs triggers precocious neuronal differentiation. We propose that SC1 and PRMT5 are components of an epigenetic regulatory complex that maintains the “stem-like” cellular state of the NSC by preserving their proliferative capacity and modulating their cell cycle progression. Our findings provide evidence that histone arginine methylation regulates NSC differentiation.
Epigenetics; Histone Methylation; Neural Stem Cell; Neurodevelopment; Protein Methylation; PRDM; PRMT5; Protein Arginine Methylation
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.
Our manuscript deals with the Protein arginine methyltransferase 4 (PRMT4), which modifies arginine residues in histones and other chromatin-associated proteins and plays an important role in the regulation of gene expression. We addressed the question of how the transcriptional function of PRMT4 might contribute to cell lineage specification despite its ubiquitious expression pattern and how this could explain its involvement in tumorigenesis. As protein associations are likely to provide an answer to this question, we attempted to identify novel interaction partners of PRMT4 using a biochemical approach. By this means, we found that PRMT4 binds Mi2α and its close relative Mi2β. In the search for the biological role of this interaction, we found that PRMT4 and Mi2α/β interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. Depletion of PRMT4 or Mi2α/β in human erythroleukemia cells resulted in deregulated cell proliferation and differentiation resembling the effects caused by c-Myb depletion. Our findings unravel an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling and gene activation and identify both coregulators as attractive targets for leukaemia research and therapy in the future.
Protein arginine methyltransferases (PRMTs) have been implicated in transcriptional activation and repression, but their role in controlling cell growth and proliferation remains obscure. We have recently shown that PRMT5 can interact with flag-tagged BRG1- and hBRM-based hSWI/SNF chromatin remodelers and that both complexes can specifically methylate histones H3 and H4. Here we report that PRMT5 can be found in association with endogenous hSWI/SNF complexes, which can methylate H3 and H4 N-terminal tails, and show that H3 arginine 8 and H4 arginine 3 are preferred sites of methylation by recombinant and hSWI/SNF-associated PRMT5. To elucidate the role played by PRMT5 in gene regulation, we have established a PRMT5 antisense cell line and determined by microarray analysis that more genes are derepressed when PRMT5 levels are reduced. Among the affected genes, we show that suppressor of tumorigenicity 7 (ST7) and nonmetastatic 23 (NM23) are direct targets of PRMT5-containing BRG1 and hBRM complexes. Furthermore, we demonstrate that expression of ST7 and NM23 is reduced in a cell line that overexpresses PRMT5 and that this decrease in expression correlates with H3R8 methylation, H3K9 deacetylation, and increased transformation of NIH 3T3 cells. These findings suggest that the BRG1- and hBRM-associated PRMT5 regulates cell growth and proliferation by controlling expression of genes involved in tumor suppression.