Cell culture methods
50B11 cells were grown and propagated in culture as described previously . Briefly, growth media consisted of NeuroPlex Serum-free Neuronal Medium (Gemini Bioscience, #600-300) and supplemented with Fetal Bovine Serum (10% final, Gemini Biosciences, Cat. 100-106), 100 mM l-glutamate (275 μM final, Gibco, Cat. 25030-081), 20% glucose (0.2% v/v final), and 10X Gem21 NeuroPlex supplement (1X final, Gemini Biosciences, Cat. 400-160). 50B11 cells were grown in an incubator at 37 ℃, 5% CO2 and were passaged no more than 5 times. HEK293 and MC38 cells were grown in 1X DMEM media (Gibco, Cat. 11995-065) containing 10% v/v FBS (Gemini Biosciences, Cat. 100-106) and 1X Penn/Strep (Gibco, Cat. 10378-016) and were passaged no more than 5 times. Mouse ESCs were grown on TC-treated plates coated with 0.1% w/v gelatin (Millipore-Sigma, Cat. ES-006-B) with 1X DMEM media (Gibco, Cat. 11995-065) containing 15% v/v knock-out serum replacement (ThermoFisher, Cat. 10828010), 2-mercaptoethanol (100 μM, Gibco, Cat. 31350-010), Non-essential amino acids (1X, Sigma, Cat. M7145-100 mL), glutamine (2 mM, Gibco, Cat. 25030–081), 1X Penn/Strep as previously described. Mouse ESCs were passaged every 1–2 days to remove differentiated cells and media were changed daily. All cell types were harvested by washing cells 3X in 1X DPBS (Gibco, Cat. 14190-136), trypsinization, and spinning down at 500 rpm at 4 ℃ to pellet cells. All mammalian cell lines were STR profiled and mycoplasma tested for interspecies contamination and mycoplasma bacteria, respectively.
Generation of transgenic 50B11 cells expressing mutant histones
Plasmids encoding mammalian histone H3.1 with various lysine-to-methionine mutants were constructed using iterative rounds of mutagenic PCR. Once sanger-sequence validated, the resulting H3.1 mutant DNA sequence was subcloned into a lentiviral vector (pLVX-IRES-mCherry). To prepare lentivirus containing the construct encoding the mutant histone, HEK293 cells were plated and grown in 1X DMEM (containing 10% FBS and 1X Penn/Strep) to approximately 70% confluency. To transfect HEK293 cells, Fugene transfection reagent was mixed with 400 μL complete DMEM media and incubated at room temperature for 5 min at a ratio of 3:1 of Fugene to total DNA. To the Fugene/DMEM mixture, 5 μg of the vector encoding mutant H3.1, 3.75 μg of Ä8.9 packaging plasmid and 2.5 μg of VSV-G envelope plasmid was added and allowed to incubate at room temperature for 15 min. The resulting mixture was added to HEK293 cells and incubated at 37 ℃ for 3 h. 90 μL of 1 M sodium butyrate was added (final concentration 110 mg/mL) to open up the chromatin and allow for more efficient integration into the host genome. The following day, the media were discarded and replaced with Opti-MEM, without 1X Penn/Strep, and allowed to incubate for 12–18 h. The virus-enriched supernatant was collected and stored at 4 ℃. This process was repeated 3 times, each time pooling the viral supernatant. Pooled viral supernatant was filtered through a 0.45-μM filter to remove any HEK293 cells carried over. 0.5 mL of viral supernatant was used to transduce 50B11 cells for 24 h. Following transduction, the media were removed and cells allowed to incubate for 2 days. At this point, a small portion of the heterogeneously transduced cells were collected for analysis (Additional file 1: Fig. S4B). The remaining cells were FACS sorted for high vs low mCherry expression into wells in a 384-well plate (1 cell per well). Colonies were expanded, harvested, lysed via resuspension in 2% SDS, and western blotting was performed to evaluate FLAG expression as well as for various methyllysine histone modifications (Fig. 4B).
Generation of mouse ESCs
Wild-type and knock out mouse ESCs (G9a –/–, GLP –/–, G9a/GLP –/–) were graciously provided by the lab of Dr. Yoichi Shinkai from the Cellular Memory Laboratory at the RIKEN Advanced Science Institute in Japan and generated as previously described . KO cells were validated via western blotting. To generate whole cell lysates for western blotting, cell pellets were resuspended in 2% (w/v) SDS, aggressively vortexed to rupture the cells, and spun down at 16,000 g to clarify lysate from insoluble cellular debris. The resulting protein-rich supernatant was decanted and quantitated using a nanodrop (A280 nm).
In vitro histone methyltransferase assay
In vitro histone methyltransferase reactions were prepared in buffer containing 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 20 μM S-adenosyl methionine (Promega, Cat. V7601), 1 μg human recombinant H3.1 (New England Biolabs, Cat. M2503S) and 10 ng of either EHMT1/GLP (Active Motif, Cat. 31920) or G9a/EHMT2 (Active Motif, Cat. 31410) and allowed to incubate for 18–24 h at 25 °C. Reactions were quenched with TFA (0.012% v/v final concentration). Following quenching, samples were submitted for EthD mass spectrometry or used for western blotting analysis. To prepare samples for western blotting, 1X SDS sample buffer and 1 μL of BME were added to the reactions and the samples were incubated at 98 oC for 5 min, cooled, run on a 16% acrylamide gel, transferred to a PVDF membrane, and blotted for various histone H3 methyl-lysine marks (blotting conditions below).
In vitro histone methyltransferase inhibition assay
In vitro histone methyltransferase reactions were prepared in buffer containing 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, and 10 μM UNC0642 (or the corresponding amount of DMSO as a control), 20 uM S-adenosyl methionine, 1 μg human recombinant H3.1 and 10 ng of either EHMT1/GLP or EHMT2/G9a. These reactions were allowed to incubate for 18–24 h at 25 oC. Reactions were quenched with TFA (0.012% v/v final concentration). 1X SDS sample buffer and 1 uL of BME was added to the reactions and the samples were subjected to SDS-PAGE separation (run on a 16% gel) followed by western blotting against the histone modifications of interest (see blotting conditions). To assess enzymatic turnover of SAM to SAH by the HMTs, the MTase-Glo kit (Promega, V7601) was used.
In vivo GLP/G9a inhibition with UNC0642
The corresponding mammalian cell line was cultured in the appropriate media containing 10 μM UNC0642 inhibitor (Sigma Aldrich HY-13980) or the corresponding volume of DMSO as a control for 5 days (media changed daily). All mammalian cell lines were grown at 37 ℃, 5% CO2. Upon completion of treatment, whole cell lysates were generated by resuspending cell pellets in 2% SDS, aggressively vortexed to rupture cells, spun down at 16,000 g to clarify from insoluble cellular debris, and decant off the protein-rich supernatant, which was quantitated via a nanodrop (A280 nm).
Mass spectrometry sample preparation
Each 5 μg of recombinant histone was first resuspended in 50 μl of 20 mM ammonium bicarbonate pH 8.5. Samples were then reduced by adding 5 μL of 7.5 mg/ml (DTT) and put on a heat block at 60 oC for 1 h. After cooling to room temperature samples were then alkylated with 5 μl of 18.5 mg/ml iodoacetamide for 15 min at room temperature in the dark before adding 0.5 μg of Lysyl Endopeptidase MS Grade (Wako/Fuji Osaka, Japan) to make a protein/protease ratio of 10:1. Samples were digested overnight at 37 ℃ before adding 10 ul of 10% v/v TFA and evaporated to dryness in a speed vac and stored at – 80 ℃. Prior to analysis, samples were resuspended in 100 μl of 10 mM TEAB and subjected to SPE cleanup using stage tips constructed with styrenedivinylbenzene disks (Empore SDB-XC, 3 M Corp.) under basic conditions. This was found to be necessary for retention due to the extremely hydrophilic nature of the modified peptides. Stage tips were wetted with 20 μl 100% acetonitrile followed by preparation with two 20-μl aliquots of 10 mM TEAB followed by loading of 20 μl of the resuspended solution or 1ug of LysC digested protein. This was then eluted with a solution of 10 mM TEAB in 75% v/v acetonitrile and evaporated to dryness.
Mass spectrometry analysis
Due to significant loss of these early eluting peptides, a direct on column approach was taken for the stage tipped samples. The entire 1 μg aliquot was resuspended in 5 μl of 2% v/v acetonitrile, 0.1% formic acid and loaded onto the nanoLC column (75 μm x 20 cm in house packed with ReproSil-Pur C18-AQ, 3 μm Dr. Maisch, Ammerbuch, Germany) at 500 nl/min using an EasyLC chromatography system (Thermo Scientific). Once the entire volume was loaded onto the column a shallow gradient was started at 300 nl/min into an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with electron transfer dissociation (ETD) capability. The samples were run in HCD mode at first, but none of the methylated peptides were positive hits when searched. Upon switching to EThcD, several of the isoforms of methylated peptides were found to exist in the +4, +5, and +6 charge states eluting early in the run before the bulk of the unmodified peptides. Since the methylation of the lysines creates a missed cleavage by the LysC enzyme, the peptides were found to be methylated at K18, K23, and K27 of the same peptide in a variety of permutations. Tri-methylation on K27 and K18 were easily detected, but the H3K23 tri-methyl lysine was not found in the first few runs. At that point, a permutation mass to charge list was created in Skyline (University of Washington) consisting of all peptides that contain a H3K23 tri-methylation in the +4, +5, or +6 and the list was imported as an inclusion list into the instrument acquisition parameters giving the expected peptides priority over any other m/z. The final method of acquisition ran with a 60-min gradient using a resolution of 120,000 for precursors and 60,000 for fragment ions. The instrument was run using ETD mode with a supplemental collision energy of 20 (EThcD). Since duty cycle was not an issue with the targeted run, the AGC target and maximum injection time were each set to 1000 giving maximum sensitivity for the inclusion masses.
Mass spectrometry data analysis
Mass spec data were searched against the UniProt version 5640 Homo sapiens database using Mascot search engine version 2.8.0 through Proteome Discoverer version 2.5. The search parameters were set to include up to 5 missed cleavages with LysC as the enzyme and EThcD as the instrument type. Mass tolerances were set to 5 ppm for precursors and 20 ppm for fragments, although this was later filtered to 2 ppm for all conclusive spectra. Dynamic modifications were set as methyl K, di-methyl K, and tri-methyl K, in addition to oxidation on methionine and deamidation on asparagine and glutamine. Carbamidomethylation was set as a static modification. Database hits were filtered at the 1% FDR level using target-decoy validation.
In vitro peptide pulldowns
To prepare beads for the peptide pulldown, 25 μL of M280 Streptavidin-conjugated Dynabeads (Invitrogen, Cat. # 11205D) were washed 3 times with 1X PBS-T (0.1% Triton-X 100) and resuspended in 500 μL of 1X PBS-T. 1 μg of biotinylated peptide, listed in Table 2, was added and allowed to incubate for 1 h at room temperature. Following peptide conjugation, the peptide-conjugated beads were washed with 1X PBS-T three times to displace any unconjugated peptide. Following the washes, 2 μg of the recombinantly expressed EHMT2/G9a (Active Motif 31410) or 5 μg of recombinantly expressed EHMT1/GLP (Active Motif 31920) was incubated with the peptide-conjugated dynabeads overnight at 4 ℃ with mild agitation. Following incubation, the beads were washed five times with buffer containing 300 mM KCl, 0.2% v/v Triton-X 100, 1 mM PMSF. Tubes containing the beads were changed on the fifth wash to eliminate any contaminating protein remaining on the sides of the tube. Following the tube-change, a sixth wash was performed. Following the last wash, the beads were resuspended in 1X SDS loading buffer, boiled and subjected to SDS-PAGE for analysis. Flag-tagged EHMT2/G9a was blotted using 1:10,000 anti-Flag antibody (Sigma F1804) followed by a 1:10,000 anti-mouse secondary incubation (GE Healthcare, NA9310). GST-tagged EHMT1/GLP was blotted using anti-GST antibody 1:10,000 (GE Healthcare, 27457701) followed by a rabbit anti-goat HRP secondary incubation (Invitrogen, A27014). The biotinylated peptides were blotted using streptavidin-HRP conjugate (Sigma, #RABHRP3, 1:10,000, 1 h room temperature). All western blots were imaged using an Acura Biosystems Imager.
ELISA antibody validation
Histone methyl-lysine peptides, listed in Table 2, were serially diluted in high-bind, hydrophobic 96-well plates (Sigma, #M9410) and allowed to incubate 37 ℃, 100 rpm, overnight. The following day, plates were washed with 1X PBS twice, and blocked with 1X PBS containing 4% BSA for 2 h, 37 ℃. Following blocking, plates were washed twice with 1X PBS containing 0.15% Tween-20. All antibodies were diluted 1:10,000 in 1X PBS (containing 4% BSA and 0.15% v/v Tween-20), added to plates and allowed to incubate at 37 ℃ for 2 h. Peptide competition reactions were pre-incubated 2 h at room temperature with the respective antibody prior to addition to the ELISA plates. Following incubation with primary antibody incubation, the plates were washed 3X with 1X PBS containing 0.15% v/v Tween-20 and incubated with secondary antibody (1:10,000) for 2 h at 37 °C. Following incubation in secondary antibody, the plates were washed 3X with 1X PBS containing 0.15% v/v Tween-20. The plates were then incubated with buffer containing dibasic sodium phosphate (20 mM), citric acid (10 mM) and fresh hydrogen peroxide (final concentration: 0.0012%), and incubated for 30 min at room temperature in the dark. The reaction was quenched with 1 M H2SO4 and imaged on a spectrometer at 492 nm. All data are represented in Additional file 1: Fig. S6.
Western blotting conditions for in vitro HMT and in vivo inhibition assays
Anti-H3K9me1; Abcam 8896; 1:5,000
Anti-H3K9me2; Active motif 39753; 1:5000
Anti-H3K9me3; Abcam 8898; 1:5000
Anti-H3K18me1; Diagenode C15410290; 1:500
Anti-H3K18me2; Diagenode C15410291; 1:500
Anti-H3K18me3; Diagenode C15410292; 1:500
Anti-H3K23me1; Active motif 39387; 1:10,000 (1:500 for in vivo)
Anti-H3K23me2; Abcam 214654; 1:2000
Anti-H3K23me3; Active motif 61499; 1:500
Anti-H3K27me1; Active motif 61015; 1:500
Anti-H3K27me2; Active motif 61435; 1:1000
Anti-H3K27me3; Millipore-Sigma 07-449; 1:5000
Anti-EHMT1/GLP; Abcam ab41969; 1:500
Anti-EHMT2/GLP; Abcam ab185050; 1:500
Anti-Actin; Thermo Fisher MA1-91399; 1:2000
Anti-H3; Abcam ab1791; 1:5000
Anti-GST; GE Healthcare, 27457701; 1:10,000
Anti-Flag; Sigma Aldrich F1804; 1:10,000.