SETMAR methylates splicing factor snRNP70 1 A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by SETMAR

The lysine methyltransferase (KMT) SETMAR is implicated in response to and repair of DNA damage but its molecular function is not clear. More broadly, enzyme/substrate relationships are largely unknown for the dozens of predicted KMTs and hundreds or thousands of proteins modified by lysine methylation. SETMAR has been associated with di-methylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates we utilized a strategy based on quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 (K130) of mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Further, we show that SETMAR methylates snRNP70 K130 in cells. As snRNP70 is a key early regulator of 5’ splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for largescale mapping of KMT substrates. SETMAR (also called Metnase) arose in the anthropoid lineage by fusion of a lysine methyltransferase (KMT) SET domain to a copy of the Mariner family transposase Hsmar1 (1). http://www.jbc.org/cgi/doi/10.1074/jbc.M115.641530 The latest version is at JBC Papers in Press. Published on March 20, 2015 as Manuscript M115.641530 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from SETMAR methylates splicing factor snRNP70 2 SETMAR is implicated in response to or repair of DNA double strand breaks and in nonhomologous end joining through the combined activities of the SET and transposase domains (2-5), and through interactions with factors including Topoisomerase IIα (Topo IIα) and PRPF19 (5-7). SETMAR knock-out is reported to delay recovery from DNA damage in a variety of cell lines and increases sensitivity to inhibitors of Topo IIα (8,9). However, the molecular mechanism by which SETMAR impacts DNA damage responses is not wellunderstood, and in particular the enzymatic function of the SET domain remains enigmatic. Specifically, the SET domain of SETMAR has been suggested to di-methylate histone H3 lysine 36 (H3K36) but direct evidence for this activity has not been demonstrated (4). Here we perform a detailed analysis of SETMAR activity on histones and show that while SETMAR can weakly methylate histones H2B and H3 in vitro, this activity does not occur on nucleosomes, the predominant physiologic context of histones. Moreover, SETMAR-catalyzed methylation of histones does not occur on any known physiological sites of histone methylation. This suggests that there may be other substrates, likely non-histone proteins, which contribute to SETMAR’s biological function. Recent analyses of the methyl-lysine proteome have shown that hundreds or thousands of proteins are modified by lysine methylation (10-13). Only a few of these sites have been associated with specific KMTs so a large number of enzyme/substrate relationships remain to be discovered (14). Techniques for linking KMTs to non-histone substrates are limited and this remains a major challenge in understanding how lysine methylation participates in wider signaling processes (15). We recently reported a proteomic strategy to identify and measure lysine methylation by using the 3xMBT domain of L3MBTL1 as a universal affinity reagent to capture modified proteins (10,16). Here we extend that strategy into a generalized approach for identifying KMT substrates, and we use it to identify substrates of SETMAR. The approach uses in vitro methylation by an enzyme of interest on cell lysates prepared with stable isotope labeling by amino acids in cell culture (SILAC) (17,18). SILAC labeling allows lysine methylation to be compared between lysates treated with active or inactive enzyme. Proteins showing increased methylation can be tested as potential substrates through direct molecular approaches. Our proteomic approach identified two candidate SETMAR substrates, and we found that SETMAR methylates the splicing factor snRNP70 (also called U1-70K) at lysine 130 both in vitro and in its cellular context. EXPERIMENTAL PROCEDURES Protein sequences, plasmids and antibodies – Recombinant protein sequences were SETMAR (accession NP_006506.3), snRNP70 (NP_003080.2) and NSD2 SET domain (19). pGEX-6p-1 (GE Healthcare) was used to express proteins as N-terminal fusions with glutathione-S-transferase (GST) (20). For mammalian cell expression SETMAR was cloned into pCAG-FLAG and the 3xFLAG snRNP70 expression vector was a gift from Robin Reed (Harvard Medical School). lentiCRISPR v2 (AddGene plasmid #52961) (21) was used for SETMAR knock-out. Antibodies used: SETMAR (Abcam ab129455), FLAG M2 (Sigma F1804) and Anti-FLAG M2 magnetic beads (Sigma M8823), and Rabbit monoclonal H3K36 methyl-specific antibodies were from Cell Signaling Technology (antibodies 14111S and 2901S). Antibodies against methylated H3K36 were validated by probing a custom peptide array as described (22). Cell culture, SETMAR knock out and SETMAR reconstitution – HEK 293T, HT-1080 and DLD1 cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum (Gibco), Penicillin-Streptomycin (Life Technologies) and L-glutamine (Life Technologies). CRISPR/Cas9 knock-out used stably expressed lentiCRISPR v2 expressing indicated guide RNAs. Virus particles were produced by co-transfection of HEK 293T cells with pCMV-VSVG (AddGene plasmid #8454) and pCMV-dR8.2 (AddGene plasmid #8455). DLD-1 cells were transduced with virus and 4 μg/mL polybrene (Millipore) followed by selection starting at 48 hours with 4 μg/uL puromycin (Sigma). Guide sequences were: 5’ by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from SETMAR methylates splicing factor snRNP70 3 CGCCGGCGCCCTTCCAGGTA 3’ (SETMAR knock-out) and 5’ CGGCATACTCACTGCGAGTG 3’ (nontargeting control). Knock-down was verified by qPCR and Western blotting. For quantitative proteomics HT-1080 cells were prepared using SILAC with light amino acids or heavy amino acids ( 13 C6 15 N2-L-lysine/ 13 C6 15 N4-L-arginine) as described (23). Recombinant protein preparation – GST fusion proteins were expressed in BL21 E.coli by overnight culture at 20° C in LB media supplemented with 50 μM ZnCl2 and 0.1 mM IPTG (Sigma), and purified using Glutathione Sepharose 4B (GE Healthcare) as described (16). Where indicated, GST was cleaved by incubation with PreScission protease (GE Healthcare) according to the manufacturer instructions. Protein concentrations were measured using the Coomassie Plus assay (Pierce). In vitro methylation and radiolabeling reactions – Enzymes and substrates were combined in 25 μL methyltransferase buffer (50 mM Tris pH 8.0, 20 mM KCl, 5 mM MgCl2, 10% glycerol) supplemented with 1 mM S-adenosyl methionine (SAM, New England Biolabs) or deuterated SAM (CDN Isotopes), or 2 μCi tritiated SAM (American Radiolabeled Chemicals Inc.). Reactions were incubated overnight at 37° C and stopped by addition of Laemmli buffer. Radiolabeling reactions were visualized by SDS-PAGE followed by transfer to PVDF membrane, treatment with EN 3 HANCE spray (PerkinElmer) and exposure to film for at least 24 hours at -80° C. Histone peptide arrays – Histone peptides bearing specific methyl PTMs were printed in triplicate on a peptide microarray. Primary antibodies were applied to arrays at 1:500 dilution in PBS + 1% Tween-20 + 10% newborn calf serum and incubated overnight at 4° C. Chicken anti-rabbit Alexa-Fluor 647 secondary antibody (Life Technologies) was applied at 1:500 dilution, also in PBS + 1% Tween-20 + 10% newborn calf serum, for 1 hour at room temperature. Arrays were washed five times in PBS + 1% Tween-20, and rinsed in PBS and water to before imaging. Arrays were imaged using the GenePix 4000 Axon Scanner (Molecular Devices). FLAG-tagged protein transfection and immunoprecipitation – 5 μg pCAG-FLAG SETMAR or Y262 mutant along with 5 μg 3xFLAG snRNP70 vector were transfected into HEK 293T cells using TransIT-293 transfection reagent (Mirus) according to manufacturer instructions. Two days following transfection cells were lysed in RIPA buffer (50 mM Tris pH 7.5, 1% NP40, 0.1% SDS, 150 mM NaCl) with protease inhibitor cocktail (Roche). FLAGtagged proteins were isolated using Anti-FLAG M2 magnetic beads according to the manufacturer instructions. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) of recombinant and immunoprecipitated protein methylation – Recombinant histone H3 was treated to propionylate lysine prior to tryptic digest (24), while recombinant SETMAR and snRNP70 were processed by in-solution digest with trypsin (Promega) or Glu-C (New England Biolabs) according to manufacturer instructions. Immunoprecipitated proteins were separated by SDS-PAGE, stained using the SilverQuest Silver Staining Kit (Invitrogen) according to manufacturer instructions, and processed by ingel digestion with trypsin or Glu-C (16). Peptides were desalted using C18 StageTips (Thermo Scientific). Peptides were separated by high pressure liquid chromatography (HPLC) using an Ekspert nanoLC 420 (AB Sciex) and analyzed with an Orbitrap Elite mass spectrometer (Thermo Scientific). Acquisition used data-dependent selection of top 20 ions with dynamic exclusion followed by collisioninduced dissociation (CID) or higher-energy collisional dissociation (HCD) and analysis of fragment ions in the Orbitrap. Data were analyzed using MaxQuant version 1.3.0.5 (25) with 1% false-discovery rate for proteins and peptides and allowing as variable modifications methionine oxidation, acetylation of protein Ntermini and mono-, diand tri-methylation of lysine. Candidate methylation sites were verified by manual inspection. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from SETMAR methylates splicing factor snRNP70 4 Proteome-wide labeling and analysis of SETMAR substrates – SILAC labeled HT-1080 nuclear extracts were prepared by hypotonic lysis and high salt extraction of nuclei (10). Lysates were dialyzed into KMT reaction buffer (50 mM Tris pH 8.0, 20 mM KCl, 5 mM MgCl2, 10% glycerol) using Slide-A-Lyzer MINI dialysis devices with 3.5K MWCO (Pierce) and cleared by centrifugation at 15,000 g for 5 minutes at 4° C (Note: we now use 250 mM KCl in the reaction buffer to reduce protein precipitation). 20 μg recombinant SETMAR or Y262A mutant with N-terminal GST fusion was added to 375 μg light and heavy lysate supplemented with 100 μM SAM. Identical reactions were prepared in parallel with light and heavy labels reversed. The reactions were incubated overnight at 37° C and stopped by placing them on ice and adding 10 mM EDTA and 100 μM S-adenosyl homocysteine. Each pair of light and heavy lysates were combined, supplemented with 0.1% Triton-X, and incubated overnight on 30 μL GlutathioneSepharose saturated with 3xMBT (16). Bound proteins were recovered and analyzed by LCMS/MS as described above except that only the top 10 ions were selected for fragmentation and the ion trap was used for MS/MS to decrease cycle time. Data was processed using MaxQuant as described above. Proteins enriched in SETMAR relative to Y262 control from both labeling directions were selected for validation as novel methylation substrates.