Cell culture and treatments
C2C12 mouse skeletal myoblasts (ATCC; CRL-1772) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/l d-glucose and Glutamax) (GIBCO) and 10% fetal bovine serum (FBS; GIBCO) with penicillin–streptomycin supplement, according to standard protocols. HEK293T (ATCC; CRL-3216) and Phoenix-Eco (ATCC; CRL-3214) were cultured in similar condition like mouse C2C12 plus 1 mM sodium pyruvate. When C2C12 reached 90–95% confluence, it was differentiated to myofibers in DMEM and 2% horse serum (GIBCO) with penicillin–streptomycin supplement.
Where indicated, cells were treated with MG-132 (Sigma, 10 μM), cycloheximide (CHX, Sigma, 100 μg ml−1). For in vivo ubiquitination assay, MG-132 was added and treated for 4 h before protein extraction. For CHX chasing assay, CHX was added and treated as indicated time under normal myotube or stressed myotube. Oxidative stress was induced following previously described protocol .
For Ezh1α-FLAG-HA, Ezh1β-FLAG-HA, EED500-FLAG-HA and EED441-FLAG-HA, full-length CDS without stop codon were amplified with corresponding primers (Additional file 5: Table S1) and ligated into pJET1.2 (Thermo Fisher Scientific) vector for Sanger sequencing. Sanger sequencing confirmed inserts were cut with XhoI/NotI and finally ligated into pOZ-C-FH vector.
For Ezh1α-2XT7, Ezh1β-2XT7, Ezh1βS560A-2XT7 and Ezh1βS560D-2XT7, full-length CDS containing stop codon were amplified using indicated primers listed in Appendix Table EV1, then, similar strategy was used to clone inserts into pOZ-C-FH vectors.
For Lenti-HA-Ubi was purchased from Addgene (Plasmid, #74218). For pOZ-HA-Ubi, HA-Ubi was amplified from Lenti-HA-Ubi and cloned into pJET1.2 (Thermo Fisher Scientific) for Sanger sequencing, then finally cloned into pOZ-C-FH vector.
Plasmid transfection, retrovirus or lentivirus packaging and infection
To package retrovirus, Phoenix-Eco (ATCC;CRL-3214) was transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to standard protocol. Transfection medium was changed to virus collection medium (DMEM plus 5% FBS) after 8 h of lipofectamine transfection. After 48 h, virus collection medium containing retrovirus was filtered with 0.45 μm filter and be ready for titer assay or transduction. Lentivirus production was performed in HEK293T (ATCC; CRL-3216) using 3rd Generation Packaging Mix kit (Abmgood) following commercially provided protocol. Validated retroviral and lentiviral vectors containing GFP protein were used as positive control during lipofectamine-mediated transfection process.
Freshly prepared and tittered retrovirus or lentivirus was used to infect C2C12 mouse skeletal myoblasts (ATCC; CRL-1772), 8 μg ml−1 polybrene was added during infection procedure. After 8 h, fresh growth medium was added to replace infection medium, after that, C2C12 was allowed to grow another 24–48 h before they reach 80% confluence. Then, positive cells were selected using Anti-CD25 beads (Invitrogen) for retrovirus infected cells, or selected using 1.6 μg ml−1 for lentivirus transduced positive cells.
pLKO shRNA lentivirus to target HUWE1, NEDD4 and FBXW8 were purchased from Sigma: HUWE1 #1 (TRCN0000092554), HUWE1 #2 (TRCN0000092555), NEDD4 #1 (TRCN0000092436), NEDD4 #2(TRCN0000092437), FBXW8 #1 (TRCN0000012731), FBXW8 #2(TRCN0000012732).
Protein extraction for co-immunoprecipitation and tandem affinity purification
Cytosolic and nuclear extracts were prepared Extracts were prepared using our previous protocol with minor modifications23. Briefly, cells were lysed in cytosolic extraction buffer (50 mM Tris–HCl, pH 8, 150 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 5% glycerol). The nuclei were collected at 1500 g and 4 °C, and the supernatant was stored as cytosolic extracts. Nuclei were washed three times in cytosolic extraction buffer and suspended in nuclear extraction buffer (50 mM Tris–HCl, pH 8, 50 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 5% glycerol), sonicated (BRANSON A250 with a 3.2-mm tapered microtip; two cycles of 30 s at 20% amplitude, 50% of duty cycle). Debris was pelleted at 16,380 g and 4 °C, and the supernatant was used for nuclear fraction extracts. Before IP, NaCl concentration would be adjusted to 150 mM.
For Co-IP, each IP was set up with 2 mg of protein in a final volume of 700 μl at a final concentration of 150 mM NaCl; then 7 μg of the appropriate primary antibodies were added and incubated with protein extracts overnight at 4 °C on the wheel. The immunocomplexes were then recovered with 70 μl (1/10 of IP volume) of magnetic Dynabeads (Protein A for primary antibody produced in Rabbit/Protein G for primary antibody produced in mice; Invitrogen) and washed with wash buffer (50 mM Tris–HCl, pH 8, 200 mM NaCl, 0.5 mM EDTA, 0.5% Triton X-100, 5% glycerol) four times, each time wash was carried out for 5 min with rotation at 4 °C. Immuno-precipitates were eluted with 2XLDS loading buffer at 95 °C for 5 min. The eluted immuno-precipitates were loaded on Bolt Bis–Tris precast gel (Invitrogen) and subjected to western blotting analysis. A list of antibodies used is provided in Appendix Table EV2.
For TAP, tagged proteins were immunoprecipitated with anti-Flag M2-agarose (Sigma), and eluted with Flag peptide (0.2 mg/ml). Further affinity purification was performed with anti-HA antibody-conjugated agarose (Pierce), and eluted with HA peptide (0.2 mg/ml). The HA and Flag peptides were prepared as 5 mg/ml stock in 50 mM Tris–Cl (pH 8.5) and 150 mM buffer, then diluted to corresponding concentration in TGEN 150 buffer (20 mM Tris at pH 7.65, 150 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.01% NP40). Between each step, beads were washed in TGEN 150 buffer three times. Complexes were resolved by SDS-PAGE and stained using the SilverQuest Silver staining kit (Invitrogen).
RNA preparation and qPCR
Total RNA was extracted with TRI Reagent (Sigma) according to manufacturer’s instructions. cDNA was prepared starting at 1 μg of RNA from each sample with a QuantiTect reverse-transcription kit (Qiagen). Real-time PCR analyses were carried out using SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) and analyzed in CFX96 Touch™ Real-Time PCR Detection System (BioRad). The primer sequences are provided (Additional file 5: Table S1).
Chromatin immunoprecipitation (ChIP) and qPCR
Cells were cross-linked in 1% formaldehyde (Thermo Fisher Scientific, 28906) for 10 min at room temperature. Cross-linked cells were lysed in lysis buffer 1 (50 mM HEPES KOH, pH 7.5, 10 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) overnight. Nuclei were collected, washed in lysis buffer 2 (10 mM Tris–HCl, pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and lysed in lysis buffer 3 (10 mM Tris–HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine). Freshly prepared 1 × protease inhibitor cocktail was added into all lysis buffers. Chromatin was sheared (BRANSON A250 with a 3.2-mm tapered microtip; four to five cycles of 2 min at 20% amplitude, 50% of duty cycle). In each IP reaction, 100 μg of chromatin DNA equivalents (DNA concentration detected at Nanodrop) were incubated overnight with 5–8 μg of antibodies. The immunocomplexes were recovered with magnetic Dynabeads (Protein A; Invitrogen) for 2 h and washed on the wheel at 4 °C for 5 min with Low-Salt (LS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 150 mM NaCl) and High-Salt (HS) wash buffer (0.1% SDS, 2 mM EDTA, 1% Triton X-100, 20 mM Tris–HCl, pH 8, 500 mM NaCl). Then, LS and HS buffers wash were repeated one more time. Final wash was carried out with TE buffer plus 150 mM NaCl twice. Precipitated DNA was eluted using elution buffer (50 mM Tris–HCl, pH 8, 10 mM EDTA, 1% SDS) at 65 °C for 15 min. For de-cross-linking, all eluted samples were incubated at 65 °C overnight. Chromatin was digested with RNase A (0.2 mg/ml) and proteinase K (0.2 mg/ml), and DNA was purified for qPCR analysis. H3K27me3 ChIP results are expressed as percentage of input. A list of oligos and antibodies used are provided in Additional file 5: Table S1, Additional file 6: Table S2.
Stable C2C12 cell lines constitutively expressing Ezh1α-FH or Ezh1β-FH were cultured for myoblast or differentiated to myotubes were fixed with 4% PFA for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 1% BSA solution. Primary antibody staining was performed for 1 h at room temperature in a 1% BSA solution at dilutions of 1:200 for HA (Roche; 3F10) and 1:500 for MHC/MF-20 (DSHB; 051320). After three times washes with 0.1% PBS, secondary antibody staining was carried out at room temperature in a 1% BSA solution (1:500). Secondary antibodies conjugated Alexa Fluor 488 (Invitrogen, A-11006) or Alexa Fluor 568 (Invitrogen, A-11031). Mounting medium containing DAPI (Sigma, F6057) was used to counterstain nuclei localization. Images were obtained with a Leica TCS SP5 confocal microscope with an HCX PL APO 63.0×/1.40-NA oil-immersion objective.
Protein digestion and peptide fractionation
HA peptide eluted samples from TAP assay were diluted in 8 M urea in 0.1 M Tris–HCl followed by protein digestion with trypsin according to the FASP protocol . After an overnight digestion peptides were eluted from the filters with 25 mM ammonium bicarbonate buffer. Eluted peptide was processed desalting step by using Sep-Pag C18 Column (Waters) based on manufacture’s instruction.
Liquid chromatography–mass spectrometry (LC–MS) analysis and MS data analysis
The peptide mixture was measured on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) coupled with an UltiMate™ 3000 UHPLC (Thermo Fisher Scientific). Peptides were separated using an Acclaim PepMap100 C18 column (75 um I.D. X 25 cm, 3 μm particle sizes, 100 Å pore sizes) with a flow rate of 300 nl/min. A 75-minute gradient was established using mobile phase A (0.1% FA) and mobile phase B (0.1% FA in 80% ACN): 5–40% B for 55 min, 5-min ramping to 90% B, 90% B for 5 min, and 2% B for 10-minute column conditioning. The sample was introduced into mass spectrometer through a Nanospray Flex (Thermo Fisher Scientific) with an electrospray potential of 1.5 kV. The ion transfer tube temperature was set at 160 °C. The Q Exactive was set to perform data acquisition in DDA mode. A full MS scan (350–1400 m/z range) was acquired in the Orbitrap at a resolution of 60,000 (at 200 m/z) in a profile mode, a maximum ion accumulation time of 100 ms and a target value of 3 × e6. Charge state screening for precursor ion was activated. The ten most intense ions above a 2e4 threshold and carrying multiple charges were selected for fragmentation using higher energy collision dissociation (HCD). The resolution was set as 15,000. Dynamic exclusion for HCD fragmentation was 20 s. Other setting for fragment ions included a maximum ion accumulation time of 100 ms, a target value of 1 × e5, a normalized collision energy at 28%, and isolation width of 1.8.
The MS RAW files from Q-Exactive HF were converted to.mgf files using Proteome discoverer (V1.4) and analyzed using Mascot (Version 2.4) against mouse database (Uniprot Mus musculus database). The Mascot search results were further processed using Scaffold (Version 4.1, Proteomesoftware Inc., Portland, OR, USA) for validation of protein identification and quantitative assessment. For protein identification, it requires a minimal 99% possibility for protein and with at least one peptide having a possibility greater than 95% according to the PeptideProphet  and ProteinProphet . The label-free quantification of proteins and phosphorylation peptides were performed using Maxquant LFQ . Detailed ratio calculation of phosphorylated Ezh1b Serine 560 formula has been described previously .
Quantification of western blots
Band intensity of immunoblots was quantified using ImageJ software. Quantification was calculated by normalization to appropriate indicated internal references. For CHX half-life experiments, the maximum was scaled to 1 by dividing all normalized time points by the normalized control.
Samples were compared using two-tailed, unpaired Student’s t test, unless otherwise stated. Error bars were represented by SD± as indicated.