Abstract
Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective and cell active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 resulted in drastically reduced levels of arginine monomethylation of HSP70 family members and other stress-associated proteins. Structural and biochemical analysis revealed that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibited methylation of both constitutive and inducible forms of HSP70, and led to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.
Introduction
Protein arginine methyl transferases (PRMTs) methylate arginine residues in histone and non-histone proteins in a mono, and symmetric or asymmetric dimethyl manner 1, 2. Arginine methylation of both histone and non-histone substrates plays major roles in transcription and chromatin regulation, cell signaling, DNA damage response, RNA and protein metabolism 3, 4. PRMT7, a member of the PRMT family, has been functionally implicated in a wide range of cellular processes including DNA damage signaling 5–7, imprinting 8, and regulation of pluripotency 9–11. Recently several elegant studies using Prmt7 knockout mouse models also revealed the role of this methyltransferase in maintenance of muscle satellite cell quiescence, muscle oxidative metabolism, and B cell biology 12–14. While these studies have greatly expanded our understanding of PRMT7 biology, it remains an understudied member of the PRMT family with poor understanding of its cellular substrates.
PRMT enzymes display methylation preference for RGG/RG motifs enriched at protein-protein interfaces, while PRMT7 has been reported to target RXR motifs in arginine and lysine rich regions 15, 16. PRMT7 is the sole evolutionary conserved class III PRMT enzyme, the subfamily which carries out only monomethylation of arginine 17, 18. Other PRMT family members such as PRMT1 or PRMT5 catalyse arginine dimethylation in an asymmetric or symmetric manner, respectively, playing distinctly different downstream biological roles 1. Remarkably, PRMT7-mediated monomethylation of histone H4R17 allosterically potentiates PRMT5 activity on H4R3 19. Thus, possible overlap between substrates for PRMT7 and other PRMT enzymes and their interplay is complex and for most part still largely unknown. The best-characterized PRMT7 substrates are histone proteins, such as H3, H4, H2B and H2A 1, 3, 6, 18. Additional non-histone PRMT7 substrates such as DVL320and G3BP2 21 have also been described. Proteomics studies have identified an abundance of cellular monomethyl arginine-containing proteins 22–25, however since other PRMT family members may be responsible for this methylation it is not clear which of these substrates are dependent on PRMT7 as systematic studies of PRMT7 cellular substrates are lacking.
To enable further discovery of PRMT7 biology and to better explore its potential as a therapeutic target, we developed a chemical probe of PRMT7 methyltransferase activity. SGC8158 is a potent, selective, and SAM-competitive inhibitor of PRMT7. To achieve cell permeability, we utilized a pro-drug strategy where upon conversion of SGC3027 by cellular reductases, the active component, SGC8158, potently and specifically inhibits PRMT7-driven methylation of cellular substrates. A systematic screen of arginine monomethylated proteins dependent on PRMT7 in cells identified several novel and previously described RG, RGG, and RXR motif proteins. HSP70 family members involved in stress response, apoptosis and proteostasis were further investigated as PRMT7 substrates in vitro and in cells. Our data shows that PRMT7 methylates HSPA8 (Hsc70) and HSPA1 (Hsp70) on R469, which resides in a highly conserved sequence in the substrate binding domain. SGC3027 inhibits the PRMT7-driven methylation impacting the thermotolerance and proteostatic stress response in cells.
Results
Identification and characterization of selective PRMT7 chemical probe compound
S-adenosyl methionine (SAM) is a co-factor required by all methyltransferases. To identify potential PRMT7 inhibitors, a library of SAM mimetic small molecules was assembled and screened by a scintillation proximity assay (SPA) against PRMT7 using 3H-SAM and a histone H2B peptide (residues 23-37) as a substrate (Supplementary Fig. 1). This screening resulted in the identification of SGC0911 with IC50 of 1 μM (Fig. 1a). Further derivatization of this hit yielded SGC8172, which displayed improved potency (IC50 <2.5 nM) but was not selective for PRMT7 (Supplementary Table 1). Additional optimization of the alkyl chain linking the amine moiety to the adenosine ring resulted in SGC8158 (Fig. 1a) which showed good selectivity over a panel of 35 methyltransferases including PRMTs (Fig. 1b; Supplementary Tables 2 and 3) while still retaining potent PRMT7 inhibition (IC50 <2.5 nM) (Fig. 1c). Importantly, we also developed a negative control compound (SGC8158N) which was dramatically less potent (15 ± 2 μM) against PRMT7 (Fig. 1a,c) and other protein methyl transferases (Supplementary Table 2). Binding of SGC8158 to PRMT7 was also confirmed by surface plasmon resonance (SPR; Supplementary Fig. 1b,c). From kinetic fitting, a KD value of 6.4 ± 1.2 nM, kon of 4.4 ± 1.1 × 106 M-1 s-1 and koff of 2.6 ± 0.5 × 10-2 s-1 were calculated from triplicate experiments. We next investigated the mechanism of action (MOA) of SGC8158 by determining the IC50 values at various concentrations of substrate (SAM and peptide). With increasing concentrations of SAM in the presence of a constant peptide concentration we observed higher IC50 values, which indicated a SAM competitive pattern of inhibition (Fig. 1d). However, no change in IC50 value was observed as the concentration of peptide was increased at fixed concentration of SAM indicating a noncompetitive pattern of inhibition with respect to peptide substrate (Fig. 1e).
Human PRMT7 shares 93% sequence identity with Mus musculus PRMT7 (mPRMT7), and residues involved in SAM and substrate binding sites are highly conserved between species. To provide further insight into the mechanism of action of SGC8158, we solved the crystal structure of full-length mPRMT7 in complex with SGC8158 refined to 2.94Å resolution, and compared it to that of S-Adenosyl-L-Homocysteine (SAH)-bound mPRMT7 (PDB ID: 4C4A). The ribosyl moiety of SGC8158 is almost identical in position to that of SAH and interacts similarly within the SAM binding pocket, explaining the SAM-competitive kinetics observed in our assays (Fig. 1f). Upon soaking mPRMT7-SAH cocrystals with SGC8158, the electron density corresponding to the methionyl moiety of SAH disappeared and new density emerged, leading towards the Arg311-Met315 loop and merging with the electron density of Trp314 side chain (Supplementary Fig. 2a). Interestingly, the Arg311-Met315 loop was distorted as a result of soaking with SGC8158, and extra electron density appeared beneath the side chain of Trp314 (Supplementary Fig. 2a,b). Accordingly, we reasoned that the biphenylmethylamine moiety of SGC8158 might displace the Trp314 side chain to dock itself into a hydrophobic pocket composed of Trp282, Phe146, Tyr48, Met296, Arg44 and Arg311 residues (Fig. 1f). Trp314 is part of the peptide binding pocket that accommodates the Arg3 side chain of histone H4 in peptide-bound Trypanosoma brucei PRMT7 (TbPRMT7_H4), (PDB ID: 4M38) (Fig. 1f). Comparison of the 311-315 loop region in mPRMT7 with all other PRMTs (Supplementary Fig. 2c) showed that this loop has a variable sequence and conformation suggesting that it may play a role in the selectivity of SGC8158 for PRMT7. Taken together, these data indicate that SGC8158 is a potent and selective inhibitor in vitro that binds in the adenosine region of the of SAM-binding pocket of PRMT7.
Identification of novel PRMT7 substrates
In order to evaluate pharmacological inhibition of PRMT7 in cells, we first sought to identify a cellular biomarker of its methylation activity. PRMT7 has a distinct substrate preference for RXR motifs surrounded by basic residues 17 and although RGG and RXR motifs are abundant in the proteome 16, very few have been validated in the cellular context. The fact that PRMT7 localization is mostly cytoplasmic (Fig. 2a,b), and most of the previously investigated substrates are histone proteins (ie. nuclear), led us to undertake a proteomics-based exploration of potential substrates of PRMT7. Wild-type (WT) and PRMT7 knockout (KO) HCT116 cells were labelled according to the isomethionine methyl-SILAC (iMethyl-SILAC) approach (Supplementary Fig. 3)23. Of the proteins identified, 280 had at least one monomethylation of arginine and 38 are differentially methylated with a WT/KO fold change equal/greater than 1.3 (Fig. 2c, Supplementary Table 4).
HSPA8 R469 methylation was identified as being highly dependent on PRMT7 and, due to abundant levels of this constitutive HSP70 family member, a commonly used pan monomethyl arginine antibody enabled the detection of this decrease using bulk cell lysates (Supplementary Fig. 4a,b). R469 is highly conserved amongst HSP70 family members (Fig. 2d) thus in addition to the constitutively expressed HSPA8, HSPA1, or HSPA6, inducible members of the HSP70 family were also identified as putative PRMT7 substrates. Gene pathway analysis of other identified substrates indicated enrichment of proteins associated with RNA metabolism, splicing, and stress granules (Fig. 2e).
PRMT7 methylates HSP70 in cells
In order to confirm that HSPA8 is indeed methylated by PRMT7 in cells, we investigated the endogenous regulation of HSP70 methylation in PRMT7 KO (HCT116, C2C12 cells), siRNA knockdown (HEK293 and MCF7 cells) and mouse embryonic fibroblasts (MEFs) derived from WT or Prmt7 KO mice. The PRMT7 genetic knockout or knockdown resulted in decreased methylation associated with monomethyl arginine signal coinciding with the HSP70 specific signal (Fig. 3a, Supplementary Fig 4). As HSP70 proteins are induced and play a role in heat shock response, we investigated if the induced forms of HSP70, such as HSPA6, and HSPA1, are methylated by PRMT7. Heatshock exposure resulted in increased levels of the inducible HSP70 forms, coinciding with increased abundance of the arginine monomethyled species indicating a matched rapid methylation by PRMT7 (Fig. 3b).
The re-expression of WT PRMT7 or the catalytic-dead mutant R44A in HCT116 PRMT7 KO cells indicated that only WT PRMT7 was able to methylate HSP70 proteins (Fig. 3c). Moreover, introduction of WT or R469A mutant HSP70 into HCT116 PRMT7 KO cells confirmed the methylation site of HSPA8/HSPA1 as identified in the iMethyl-SILAC dataset (Fig. 3d). R469 has previously been identified as a methylation site for PRMT4 and PRMT7 26 however, in MCF7 cells, we observed a major effect on HSP70 methylation driven by PRMT7 but not PRMT4 (Supplementary Fig. 5) possibly indicating cell-type specific effects. These results demonstrate robust PRMT7-dependent methylation of both steady state and inducible HSP70 proteins in cells.
PRMT7 methylation of HSP70 in vitro reveals a dependency on the open form of HSP70
HSP70 proteins have dynamic structures with nucleotide and substrate binding domains undergoing dramatic conformational changes upon nucleotide or substrate binding 27, 28. As R469 resides in the substrate binding domain, we examined the 3D protein structure to determine the accessibility of the R469 side chain. Available full-length structures of HSPA5 (65% sequence identity to HSPA8) and HSPA1A (86% identity) were analysed for the positioning of R469. The structures of HSPA5 in both the ATP-bound open state 29 as well as the ADP-bound closed lid state 29, 30 for the substrate binding and nucleotide binding domains, respectively, and HSPA1A 31 substrate binding domain in the ADP-bound state were used. Analysis of these HSP70 structures indicated that R469 resides in a loop of the substrate binding domain which is likely of limited accessibility in the ADP-bound form of HSP70 when the substrate binding domain is in a closed conformation (Fig. 4a-c).
In order to establish if PRMT7 methylates HSP70 in vitro, we performed full characterization of PRMT7 enzyme kinetics with HSPA8 as substrate. In agreement with structural analysis, our data indicated that PRMT7 methylates HSPA8 in the presence of ATP (Fig 4d, e). Apparent kinetic parameters were then determined for HSPA8 methylation yielding a Km for SAM of 1.6 ± 0.1 μM (Fig 4d), and Km for HSPA8 of 7.2 ± 0.5 μM (Fig. 4e). To test the specificity of this activity, we also tested the activity of PRMT7 on HSPA8 with a single arginine to lysine mutation (R469K). Consistent with previous findings, PRMT7 was completely inactive with the HSPA8-R469K mutant as substrate in the presence or absence of ATP (Fig. 4f). We further tested the effect of SGC8158 and SGC8158N on PRMT7-dependent methylation of HSPA8. SGC8158 inhibited PRMT7 methylation of full length HSPA8 in vitro with IC50 = 294 ± 26 nM under balanced conditions. As expected, SGC8158N showed very poor inhibition with an IC50 value estimated to be higher than 100 μM (Fig. 5a).
SGC3027, a prodrug form of SGC8158, selectively inhibits PRMT7 in cells
Having established that PRMT7 methylates cellular HSP70, we returned to SGC8158 in order to determine its cellular activity. We observed no inhibition of cellular HSP70 methylation by this compound, likely due to its SAM-like structure and low cell permeability often associated with SAM analogs32, 33, 34. To increase the cellular permeability we employed the Trimethyl Lock prodrug strategy in which SGC8158 is derivatized with a quinonebutanoic acid that masks a secondary amine group and increases lipophilicity35. The resulting derivative, SGC3027, undergoes reduction in cells followed by rapid lactonization, releasing the active component SGC8158 (Fig. 5b).
The SGC3027 prodrug compound inhibited cellular HSP70 methylation with IC50 of 2.4 ± 0.1 μM (Fig. 5c,d) in C2C12 cells. The inactive compound had no effect at 5 μM, the cellular IC90 of SGC3027, and had a minimal effect at 10 μM (Fig. 5e). In addition SGC3027, but not SGC3027N, was effective at reducing HSP70 methylation in several commonly used cell lines (Supplementary Fig. 6), and SGC3027 was selective over PRMT4, and PRMT5 at 10 μM exposure of (Supplementary Fig. 7a,b). In Prmt7 KO MEFs, SGC3027 does not affect the methylation of HSP70 (Supplementary Fig. 7c), further confirming the on-target activity for PRMT7. Taken together these data demonstrate that SGC3027 is a selective and cell-active inhibitor of PRMT7.
PRMT7 driven methylation is cytoprotective against proteostasis perturbations
HSP70 family members play key roles in nascent protein folding and refolding, as well as distinct roles in anti-apoptotic responses 36. To determine if PRMT7-driven methylation contributes to protection from cellular toxic insults, we investigated cell survival in response to thermal and proteasome stress using the genetic Prmt7 KO models and a chemical biology approach employing the SGC3027 chemical probe. Prmt7 KO MEF cells were more sensitive to acute heat stress with fewer cells surviving the treatment (Fig. 6a) and more cells undergoing apoptosis (Fig. 6b). SGC3027, but not the negative control SGC3027N, showed a similar response (Fig. 6c,d), indicating that SGC3027 phenocopies the genetic ablation of Prmt7. We also investigated if PRMT7 stress protection extends to proteasome stress using proteasome inhibitor bortezomib. Compared to WT cells, Prmt7 KO MEFs cells were more sensitive to acute bortezomib induced cell death, and had poorer recovery after 4 or 20 h exposure to bortezomib (Fig. 6e,f). SGC3027, but not SGC3027N, sensitized the WT Prmt7 MEFs to bortezomib while neither compound affected Prmt7 KO cells (Fig. 6g,h). These results indicate that PRMT7-driven methylation plays a cytoprotective role in stress response, while inhibition of PRMT7 catalytic function can sensitize cells to toxic stimuli.
Discussion
PRMT7 is a monomethyl arginine methyltransferase that plays a role in muscle physiology and stem cell biology 3, 10–13. However, the PRMT7 substrates that mediate this biology are not well understood, and tools to selectively and temporally modulate PRMT7 catalytic activity are lacking. Here we report the first selective PRMT7 chemical probe, SGC3027, and a closely related but inactive compound, SGC3027N, for use as a specificity control for biological experiments. We also identified novel PRMT7 methylation substrates using proteomics and further validated HSPA8 and related HSP70 family members as PRMT7 substrates by employing in vitro assays, genetic methods, and chemical biology. Our data suggest that PRMT7 activity plays a role in cellular thermotolerance and proteasomal stress response. Therefore, SGC3027 maybe be a useful modulator of cellular proteostasis in stress response under physiological and pathological conditions.
SGC8158 is a structural derivative of SAM, acts as a SAM-competitive inhibitor of PRMT7, and occupies the adenosine pocket in the SAM binding site of PRMT7. Other potent and selective SAM-like inhibitors of methyltransferases including EPZ004777 37 and SGC0946 for DOT1L 38 and LLY-283 for PRMT5 39 feature extensively modified adenosine and methionine moieties that likely enhance the cell permeability of otherwise cell impermeable SAM. We employed an alternative prodrug strategy in which adding the quinonebutanoic moiety increased cell permeability and allowed for cellular reductases to generate the active component, SGC8158. Notably, the cellular reductase-driven activation of SGC3027 to the active SGC8158 may vary among cell types depending on the abundance or activity of reductase enzymes. Thus, we recommend that the appropriate concentration range for use of these probes should be empirically evaluated in each experimental setting by monitoring a biomarker such as HSP70 methylation before evaluation of specific biological readouts.
SGC3027 sensitized cells to heat and proteasomal stresses indicating that PRMT7 catalytic function is required for normal physiological response to these stimuli. Prmt7 KO cells were also more sensitive to these stresses, indicating that the chemical probe phenocopies the genetic knockout effects. Heat stress and proteasome inhibition elicits orchestrated protective responses, with the central goal of maintenance of cell protein homeostasis, proteostasis. Failure to maintain proteostasis results in numerous diseases. For example, altered proteostatic balance due to rewiring of chaperome complexes has been observed in cancer cells, contributing to drug resistance 40, 41. PRMT7-dependent protection against cellular stress may have physiological importance in cancer cell survival, consistent with higher levels of PRMT7 that have been reported in breast cancer cell lines 42, 43. Interestingly, PRMT7 was initially identified in a screen for sensitization to topoisomerase inhibitors in cancer cells 5, suggesting a wider range of stressors against which PRMT7 may be protective. Our findings of PRMT7 inhibition leading to the sensitization of cells to bortezomib-induced cell death indicate potential therapeutic applications in cancers such as multiple myeloma and some lymphomas. It is possible that PRMT7 inhibition may lead to overcoming resistance to bortezomib or other therapies. Further work is needed to determine if PRMT7 inhibition can indeed synergize with proteasome inhibitor drugs in therapeutically relevant cell systems, and these efforts will be aided by the use of SGC3027 as a chemical probe for PRMT7.
The majority of PRMT7 substrates identified in this study were functionally associated with stress response, RNA metabolism, splicing, and stress granules. Notably G3BP2, a previously reported PRMT7 substrate 21, STAU1 44 and KHSRP 45 have all been linked to stress granule biology and cell stress response. Previous arginine methylome studies have also noted the abundance of mono and dimethyl arginine posttranslational modifications (PTMs) in proteins involved in splicing, transcription, and RNA metabolism 22–25, 46, 47. We identified and validated members of the HSP70 family as cellular substrates of PRMT7 leading us to further investigate the role of PRMT7 in stress response.
The cytoprotective function of HSP70 proteins has been attributed to the modulation of protein refolding and transport of client proteins, as well as direct regulation of apoptotic signaling pathways 40, 48. Several HSP70 inhibitors have been reported and their utility is being explored in counteracting bortezomib resistance in multiple myeloma as well as therapeutic applications in other cancers 49–52. PTMs of the HSP70 family members such as lysine methylation, ubiquitination, acetylation, and phosphorylation have been reported 53 and some of the PTMs stabilize the HSP70/HSP90/HSP40 and client protein complexes allowing the formation of antiparallel HSP70 dimers 54. K561 trimethylation by METTL21A and dimethylation by SETD1A have been reported to result in the modulation of HSP70 affinity for client proteins or potentiation of AURKB activity, respectively 55–57. Here, using genetic and pharmacological means, we identify R469 monomethylation as an abundant cellular modification that depends on PRMT7 catalytic function and correlates with the cytoprotective properties of PRMT7.
HSP70 R469 monomethylation by PRMT4 and PRMT7 was recently reported as contributing to transcriptional activation by retinoic acid receptor RAR 26. Interestingly PRMT4 methylation of HSP70 did not seem to require an ATP-driven conformational change 26 while we found that PRMT7 driven methylation occurs when HSP70 adopts an open, ATP-bound conformation that promotes R469 accessibility. In all of the examined structures of the substrate and ADP-bound forms of HSP70, R469 packs into the lid subdomain of the substrate binding domain to ensure flexible, yet stable, interaction with the client protein 31, but rendering this residue inaccessible to modifying enzymes such as PRMT7. Although the substrate binding domains and lid subdomains are more variable among HSP70 proteins than other regions, possibly due to the need of accommodating a large number of substrates 31, the region surrounding R469 is highly conserved among HSP70 family members and species. PTMs of the HSP70 lid and tail domains have been reported to disrupt the interaction with co-chaperone CHIP TPR 58. Since the R469-containing loop resides in close proximity to the CHIP binding region it is also possible that the methylation may influence HSP70 co-chaperone recruitment. Further experiments, required to completely understand the mechanistic role of R469 methylation in HSP70 function, will benefit from the use of this PRMT7 chemical probe.
We uncovered new aspects of PRMT7 biology associated with proteostasis, identified PRMT7 cellular substrates and validated HSP70 family members HSPA1/6/8 as PRMT7 substrates, whose methylation is likely to contribute to the cytoprotective effects of PRMT7. SGC3027, together with its negative control SGC3027N, will be useful tools for further understanding PRMT7 function in physiological and disease states.
Methods
Protein expression and purification
Full-length PRMT7 was expressed in Sf9 cells grown in HyQ® SFX Insect serum-free medium (ThermoScientific). Cells were harvested and lysed, and the cleared lysate was incubated with 5 mL anti-FLAG M2-Agarose (Sigma) in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol and washed with the same buffer with 500 mM NaCl. The pure recombinant protein was eluted from the column using the same buffer with 0.1 mg.mL-1 FLAG peptide (Sigma). Pure PRMT7 was flash frozen and stored at −80 °C. Full length HSPA8 was overexpressed in Escherichia coli strain BL21(DE3) V2R-pRARE2 during an overnight induction with 0.5 mM isopropyl 1-thio-d-galactopyranoside at 18 °C. Cells were suspended in 20 mM Tris-HCl (pH 8.0, 1 mM DTT, 300 mM NaCl). The clarified lysate was loaded onto a Hispur™ nickel-nitrilotriacetic acid column (Thermo Scientific) and washed with buffer. Then, protein was eluted and concentrated. Protein purity was determined by SDS-PAGE and LC-MS.
Radioactive activity assay in vitro
Assays using biotinylated H2B (23-37) as a substrate were performed in buffer (20 mM Tris-HCl, pH 8.5, 0.01% Tween-20, and 5 mM DTT) containing 5 nM PRMT7, 1.1 μM 3H-SAM (Cat.# NET155V250UC; Perkin Elmer; www.perkinelmer.com) and 0.3 μM H2B (23-37). The reaction mixtures were incubated for 60 min at 23 °C. To stop the enzymatic reactions, 10 μL of 7.5 M guanidine hydrochloride was added, followed by 180 μL of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a 96-well FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plates were incubated for 1 hour and the CPM were measured using Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background (0%). The IC50 values were determined using GraphPad Prism 7 software. For the kinetic analysis of HSPA8 methylation by PRMT7, the assay mixture contained 20 mM Tris-HCl, pH 8.5, 0.01% Tween-20, and 5 mM dithiothreitol (DTT), 1 mM MgCl2, 1 mM ATP where indicated, 250 nM PRMT7, fixed concentration (20 μM) of SAM, various concentrations (up to 40 μM) of HSPA8; or fixed concentration (10 μM) of HSPA8 with different concentrations of SAM (up to 20 μM). IC50 determinations of SGC8158 and SGC8158N were performed at 150 nM PRMT7, close to Km values of both SAM (2 μM) and substrate HSPA8 (7 μM). To determine the mode of action, the experiments were performed in the presence of fixed biotinylated H2B peptide (residues 23-37) or HSPA8 substrate concentration and increasing SAM concentration or at fixed concentration of SAM and varying concentration of the substrate. Twenty μL of reaction mixtures were incubated at 23 °C for 60 min. To stop reactions, 100 μL of 10% trichloroacetic acid (TCA) was added, mixed and transferred to filter-plates (Millipore; cat.# MSFBN6B10; www.millipore.com). Plates were centrifuged at 2000 rpm (Allegra X-15R - Beckman Coulter, Inc.) for 2 min followed by 2 additional 10% TCA washes and one ethanol wash followed by centrifugation. Plates were dried and 30 μL MicroScint-O (Perkin Elmer) was added to each well, centrifuged and removed. 50 μL of MicroScint-O was added again and CPM was measured using Topcount plate reader. The IC50 values were determined using GraphPad Prism 7 software.
SPR analysis
SPR analysis was performed using a Biacore™ T200 (GE Health Sciences Inc.) at 20 °C. Approximately 5500 response units of Bio-PRMT7 (amino acids 1-692) was fixed on a flow cell of a SA chip according to manufacturer’s protocol, while another flow cell was left empty for reference subtraction. SPR analysis was performed in HBS-EP (20 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) with 3% DMSO. Five concentrations of SGC8158 (150, 50, 16.6, 5.5 and 1.85 nM) were prepared by serial dilution. Kinetic analysis was performed using single cycle kinetics with contact time of 60 sec, off time of 300 sec, and a flow rate of 100 μL.min-1. To favor complete dissociation of compound for the next cycle, a regeneration step (300 second, 40 μL.min-1 of buffer), a stabilization period (120 sec) and two blank cycles were run between each cycle. Kinetic curves were fitted using a 1:1 binding model and the Biacore T200 Evaluation software (GE Health Sciences Inc.).
Selectivity assays
The methyltransferase selectivity was assessed at two compound concentrations of 1 and 10 μM as described previously 59–61.
Constructs, Cells and Antibodies
MCF7 (ATCC® HTB-22™), C2C12, MEF WT and MEF Prmt7KO (kind gift from Dr. Stephane Richard, McGill University), U-2 Os (ATCC®HTB-96™), HT-1080 (ATCC®CCL-121™) and HEK293T (kind gift from Sam Benchimol, York University) were grown in DMEM (Wisent), HCT116 WT (ATCC®CCL-247™), in McCoy’s (Gibco) and MDA-MB-231 (ATCC® HTB-26™) in RPMI1640 (Wisent) supplemented with 10% FBS (Wisent), penicillin (100 U.mL-1) and streptomycin (100 μg.mL-1). Anti-Rme1 (#8015), anti-Rme2s (#13222) and anti-mouse Alexa Fluor 488 (#4408) were purchased from Cell Signaling Technologies. Anti-Hsp/Hsc70 was from Enzo (#ADI-SPA-820). Antibodies for PRTMT7 (#ab179822), PRMT5 (#ab109451), and β-actin (#ab3280) were purchased form Abcam. Anti-PRMT4 (#A300-421A) was from Bethyl. Anti-GFP (#632381) used for western blot was purchased from Clontech. Anti-GFP used for IP was purchased from Invitrogen (#G10362). Anti-Flag (#F4799) was from Sigma. Anti-SmBB’ (#sc-130670) and anti-BAF155 (#sc-32763) was from Santa Cruz Biotechnologies. Anti-BAF155-R1064me2a (#ABE1339) was from Millipore. Goat-anti rabbit-IR800 (#926-32211) and donkey anti-mouse-IR680 (#926-68072) were purchased from LiCor. Full length of HSPA8, HSPA1 were cloned into pAcGFPN3 vector (Clontech) and PRMT7 were cloned into pAcGFPN3 (Clontech) or pcDNA3 (N-terminus FLAG). Site directed mutagenesis to generate PRMT7 R44A mutant, HSPA1 R469A and HSP8 R469A mutants was performed using Q5® Site-Directed Mutagenesis Kit (NEB), following manufacturer’s instructions. MEF WT and MEF Prmt7 KO were immortalized at passage 3 by transfection of SV40LT.
PRMT7 cellular assay
C2C12 cells were plated and next day treated with compounds. After 48 h, cells were lysed in lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% TritonX-100, 12.5 U.mL-1 benzonase (Sigma), complete EDTA-free protease inhibitor cocktail (Roche)). After 2 min incubation at RT, SDS was added to final 1% concentration. Cell lysates were analysed in western blot for unmethylated and monomethylated Hsp70/Hsc70 levels. The IC50 values were determined using GraphPad Prism 7 software.
Western blot
Total cell lysates or cellular fractions (as indicated) were resolved in 4-12% Bis-Tris Protein Gels (Invitrogen) and transferred in for 1.5 h (80 V) onto PVDF membrane (Millipore) in Tris-Glycine transfer buffer containing 20% MeOH and 0.05% SDS. Blots were blocked for 1 h in blocking buffer (5% milk in PBS) and incubated with primary antibodies in blocking buffer (5% BSA in PBST: 0.1% Tween 20 PBS) overnight at 4 °C. After five washes with PBST the blots were incubated with goat-anti rabbit (IR800) and donkey anti-mouse (IR 680) antibodies in Odyssey Blocking Buffer (LiCor) for 1 h at RT and washed five times with PBST. The signal was read on an Odyssey scanner (LiCor) at 800 nm and 700 nm.
Knock-down
Cells were transfected with 15 nM of either non-targeting siRNA or siRNA against PRMT7, PRMT4 or PRMT5 (Dharmacon) using jetPRIME® transfection reagent (Polyplus), following manufacturer instructions. After 3 days the protein levels were measured by Western blot as described above.
Cell growth and apoptosis assay after heat shock
MEF WT and Prmt7 KO cells were heat shocked in a water bath for 20 min at 44 °C. Cell number was determined with Vybrant® DyeCycle™ Green 24 h after heat shock and apoptosis levels were determined with IncuCyte® Caspase-3/7 Reagent within 12 h after heat shock using IncuCyte™ ZOOM live cell imaging device (Essen Bioscence) and analysed with IncuCyte™ ZOOM (2015A) software.
Cell growth after bortezomib treatment
MEF WT and Prmt7 KO cells were treated with bortezomib for 4 h or 24 h and the confluency monitoring was started at 4 h after bortezomib (30 nM) treatment. For experiments with SGC3027 or SGC3027N cells were pretreated with 4 μM compounds for 2 days before bortezomib treatment (30 nM). After 20 h bortezomib, SGC3027 or SGC3027N were removed and SGC3027 or SGC3027N were replaced. Cell confluency was monitored right after bortezomib addition using IncuCyte™ ZOOM live cell imaging device (Essen Bioscence) and analysed with IncuCyte™ ZOOM (2015A) software.
Cellular fractionation
Cells were trypsinized and 1 x 106 of cells were centrifuged at 400 x g for 5 min at 4 °C. Cell pellets were resuspended in 200 μL of hypotonic buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.3 M Sucrose, 1 mM TCEP, 0.1% Triton X-100 and protease inhibitors). The cell suspensions were incubated on ice for 15 minutes followed by centrifugation at 1300 x g for 5 min at 4 °C. The supernatants were collected and cleared by centrifugation at 18,000 x g to produce the cytoplasmic fraction. The pellets were then washed in hypotonic buffer, centrifuged again and resuspended in an equal volume of lysis buffer (as described above in PRMT7 cellular assay) to produce nuclear fraction. The fractions were analysed by Western blot as described above.
Immunofluorescence
C2C12 cells were electroporated (1650 V, 10 ms, 3 pulses) with 0.5 μg of PRMT7-FLAG plasmid using Neon transfection system (Life Technologies), following manufacturer instructions. Other cells were transfected with PRMT7-FLAG using X-tremeGeneXP (Sigma), following manufacturer instructions.The next day cells were washed with PBS, fixed with 4% PFA for 10 min, permeabilized with 0.1% Tritton X-100/PBS for 5 min, blocked with 5% BSA in PBS-T (PBS, 0.1% Tween20) for 1 h and incubated with anti-FLAG antibodies (1:1000) overnight at 4 °C. Next day cells were washed with PBS-T, incubated with anti-mouse Alexa Fluor 488 in blocking buffer (1:1000) and washed with PBS-T. Nuclei were labeled with Hoechst 33342 dye (Thermo Fisher Scientific), following manufacturer instructions. The images were taken with EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific).
Immunoprecipitation
HCT116 PRMT7 KO (clone 94A) cells were cotransfected with GFP-tagged HSPA8/1 (WT or R469A mutant) and FLAG-tagged PRMT7 (WT or R44A mutant) at 1:10 ratio using JetPRIME transfection reagent (Polyplus), following manufacturer’s instructions. Cells were lysed in lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.1% TritonX-100 and complete EDTA-free protease inhibitor cocktail (Roche)) for 20 min and centrifuged 18,000 x g for 3 min. The supernatants were incubated with rabbit anti-GFP antibody (Invitrogen) overnight at 4 °C. Next day the antibody complexes were incubated with prewashed Dynabeads™ Protein G (Thermofisher) for 2 hours. Beads were washed in lysis buffer and proteins were eluted with 2 x SDS loading buffer and analyzed by Western blot, as described above.
CRISPR/Cas9 gRNA vector design for HCT116 cells
For HCT116 cells 3 guide RNA were designed on PRMT7 locus. To generate gRNA expression vectors, the annealed oligonucleotide for each targeting site and annealed scaffold oligonucleotides were ligated into pENTER/U6 vector (Life Technologies). Cas9 expression vector was prepared previously 62. Briefly, the Cas9 cDNA was synthesized by Eurofins genomics and inserted into the pCAGGS expression vector provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan) 63. Following guide RNA was used for generation of knockout cells: guideRNA/synthetic Oligonucleotide_sense/ synthetic Oligonucleotide_antisense (21: GGGACTCTTGTCAATGATGGCGG/caccGGGACTCTTGTCAATGATGGgtttta/ctctaaaacccatcattgacaagagtccc; 74: GGCATGGGTACTCCCACAGCGGG/caccGGCATGGGTACTCCCACAGCgtttta/ ctctaaaacgctgtgggagtacccatgcc; 94: GGGCAGCTCTCCACGTCAACGGG/ caccGGGCAGCTCTCCACGTCAACgtttta/ ctctaaaacgttgacgtggagagctgccc.
CRISPR/Cas9-mediated genome editing
HCT116 cells were seeded onto 6-well plates at a density of 40,000 cells.well-1, 24 hr prior to transfection. Cells were transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instruction. A total of 3 μg Cas9 expression vector, 1 μg of gRNA expression vector, and 0.4 μg of pEBMultipuro (Wako Chemicals) as a transfection marker were transfected. After 48 h of transfection, 1 μg.mL-1 puromycin was added for selection. The colonies were isolated by limiting dilution. PRMT7 destruction for the isolated clones was confirmed by Sanger sequencing and Western blotting.
CRISPR for mouse C2C12 cells
C2C12 PRMT7 KO clones were generated by cloning guideRNA (caccgGTCATGTAGCATGTCGGCAT/aaacATGCCGACATGCTACATGAC) into LentiGuide-Puro vector (from Zhang lab), following Zhang laboratory protocols. Lentivirus was prodiced using standard protocols. 48 h post transfection the supernatant was collected, filtered through 0.5 μm filter and used to infect C2C12 cells in presence of polybrene to final conc of 8 μg.ml-1. After 24 h media was changed and after 2 days puromycin was added to final concentration of 2 μg.mL-1. 5 x 104 puromycin selected cells were electroporated with 0.5 μg Cas9-GFP plasmid (1650 V, 10 ms, 3 pulses). Next day GFP positive cells were sorted and plated in 96-well plates (1 cell.well-1). The clones were analysed for PRMT7 expression and HSP70 monomethylation in western blot. The PRMT7 KO and PRMT7 catalytic mutant clones were genotyped by Sanger sequencing PCR-amplified TA-cloned genomic DNA. The premature stop codon in exon 3 or 4 resulted in PRMT7 KO. The mutation in one of the alleles delY35,A35S in addition to premature codons in exon 3 or 4 of other alleles resulted in expression of catalytically inactive PRMT7 protein (Supplementary Table 5)
Cell culture for methylome analysis
PRMT7 KO HCT116 cells (clone 94A) or parental control cells were cultivated in McCoy’s 5A medium (Gibco) containing 10% fetal bovine serum (FBS) (Hyclone), and maintained in an incubator set to 37 °C, 5% CO2. For iMethyl-SILAC-labeling of cells, DMEM without L-methionine, L-cystine and L-glutamine (Sigma) was supplemented with 0.2 mM of L-cystine and 2 mM of L-glutamine to prepare DMEM lacking only L-methionine. The L-methionine free DMEM was supplemented with 10% dialyzed fetal bovine serum (Thermofisher Scientific) and either L-methionine-13C4 (Cambridge Isotope Laboratories) or L-methionine-methyl-13CD3 (Sigma) at the final concentration of 0.1 mM. Clone 94A or control cells were labelled with medium containing L-methionine-13C4 and L-methionine-methyl-13CD3, respectively for five days.
Enrichment of mono-methylarginine peptides
Each stable isotope labeled HCT116 cell in 10 cm dishes was washed with phosphate-buffered saline (PBS), and lysed with 0.6 mL.dish-1 RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% NP-40 containing protein phosphatase inhibitor cocktail 1 and 2 (Sigma) and protease inhibitors (Sigma)]. The cell lysate was centrifuged for 15 min at 20000 x g to remove cellular debris. The recovered proteins were quantified with a BCA Protein Assay Kit (Thermofisher Scientific). Five mg of each cell lysate was combined and precipitated with 4 volumes of cold acetone and centrifuged for 15 min at 20000 x g. The precipitates were dissolved in 8 M urea/50 mM triethylammonium bicarbonate (TEAB, Wako), reduced with 5 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl, Thermofisher Scientific) and then digested with 100 μg of Lys-C (Wako) for 2 h at room temperature, followed by 5-fold dilution with Milli-Q water and digested with 100 μg of trypsin (Promega) overnight at room temperature. The protein digests were alkylated with iodoacetamide (IAA, Sigma) for 30 min at room temperature, acidified with 10% trifluoroacetic acid (TFA, Wako) and desalted on ODS column (C18MG 4.6 x 150 mm, Shiseido). The digested peptide mixtures were lyophilized to give white powders. Mono-methyarginine peptides were enriched by a PTMscan Mono-Methyl Arginine Motif [mme-RG] Kit (Cell Signaling) according to the manufacturer’s protocol followed by strong cation exchange (SCX) chromatographic fractionation.
SCX fractionation
SCX chromatographic fractionation was performed on Shimazu HPLC system using a polysulfoethyl A SCX column (2.1 x 35 mm, 5 μm, 300 A, PolyLC). Mono-methyarginine peptide eluates were diluted with solvent A by 10 fold, and loaded to an equilibrated column with 80% acetonitrile (ACN) containing 0.1% HCO2H (Solvent A). After 10 min in 100% A, peptides were eluted with 30% ACN containing 350 mM NH4HCO3, pH 3 (Solvent B) using a gradient from 0% to 10% B in 20 min, 10% to 20% B in 10 min, 20% to 40% B in 5 min, 40% to 80% B in 5 min, followed by 80% to 100% B in 1 min and then maintained at 100% B for 30 min at a flow rate of 0.1 mL.min-1. Fractions were collected at 2 min interval (0.2 mL each) from 20 min to 60 min after sample injection and polled at various intervals to bring total fraction (fr) number to 8; SCX08 : fr. 1-8, SCX10 : fr. 9-10, SCX12 : fr. 11-12, SCX14 : fr. 13-14, SCX16 : fr. 15-16, SCX18 : fr. 17-18, SCX19 : fr. 19, SCX20 : fr. 20.
LC-MS/MS analysis
The SCX column fractionated peptides were dissolved in 0.1% TFA with 2% ACN and analyzed by on-line nano LC using an Easy nLC1000 System (Thermo Fisher Scientific) coupled to Fusion Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were loaded onto a trap column (C18 Pepmap100, 3 μm, 0.075 x 20 mm) and resolved on an analytical column (Reprosil-Pur C18AQ 3 μm 0.075 x 150 mm, Nikkyo Technos) at 300 nL.min-1 over 45 min. The mass spectrometer was operated in a top-20 mode with dynamic exclusion of 10 s collecting MS spectra in the Orbitrap mass analyzer at a resolution of 120000 and data-dependent collision-induced dissociation MS/MS spectra in the ion trap with normalized collision energy of 30.
Database Search
Peak lists extraction from Xcalibur raw files were automatically performed using Proteome Discoverer 1.4 software (Thermofisher Scientific). Peak lists were searched against Uniprot using Mascot software (version 2.5, Matrix Science). The methylation level of arginine was calculated by the ratio of L/H (light/heavy) peptide peaks using Proteome Discoverer. The mass tolerance of precursor and fragment were set to 10 ppm and 0.45 Da. Up to two missed trypsin cleavages were allowed. For Mascot search, the following parameters were set: carbamidomethylation cysteine was selected as a fixed modification; oxidation of methionine, monomethylation of arginine, [2H(3)13C(1)] labeled monomethylation of arginine, [13C(4)] labeled methionine, and [2H(3)13C(1)] labeled methionine were selected as variable modifications. Peptide identification data are routinely filtered to 1% false-discovery rate using the target-decoy strategy. The LC/MS data were also searched using Peaks 8.5 software (Bioinformatics Solutions Inc.) against the human Uniprot database (downloaded on July 2018) and using the same database search parameters as above.
Purification, crystallization and structural determination
mPRMT7 was expressed and purified according to the previously published protocol 64. mPRMT7 was first mixed with 5-fold molar excess of SAH and incubated for 30 min on ice. Good diffracting crystals were obtained in vapour-diffusion sitting drops, in a precipitant solution containing 0.04 M citric acid, 0.06 M Bis-Tris propane [pH 6.4], 20% (v/v) PEG 3350. The mPRMT7 crystals were soaked into a 1 μL drop containing the same precipitant solution supplemented with 1 mM SGC8158 and 20% (v/v) glycerol for 5 days at 20 °C. Crystals were then cryo-cooled in liquid nitrogen. The mPRMT7_ SGC8158 dataset was collected at the 24ID-E beamline at the Advanced Photon Source (APS). Dataset was processed with XDS 65 and merged with Aimless 66. Initial phases were obtained by using mPRMT7 (PDB ID:4C4A) as initial model in Fourier transform with refmac5 67. Model building was performed in COOT 68 and the structure was validated with Molprobity 69. SGC8158 restraints were generated using Grade Web Server (http://grade.globalphasing.org). Images were prepared with PyMol Software (www.pymol.org).
Statistical Testing
Statistical significance was assessed using GraphPad Prism 7 software via Student’s t test (unpaired, 95% confidence interval). p values < 0.05 were considered statistically significant.
Data Availability
The mass spectrometry data have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository. The dataset accession number is PXD012119. mPRMT7_SGC8158 structure has been deposited in PDB (PDB ID: 6NPG).
Acknowledgements
The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z].
This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on 24-ID-E beam line is funded by a NIH-ORIP HEI grant (S10OD021527). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
We thank Kumar Singh Saikatendu, Charles E. Grimshaw, Nobuo Cho, Toshiyuki Nomura, and Atsushi Kiba for suggestions and support throughout the project and Michiko Tawada and Sachiko Itono for suggestions and chemistry support. We thank Alma Seitova, Ashley Hutchinson, Hong Zheng for recombinant protein production and Aiping Dong for crystallography support.