Cell culture and transfection
Generation of the RBPJ depleted HeLa cells was previously described . HEK293, HEK293T, Hela and Hela-S cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 61965–059) supplemented with 10% fetal bovine serum (FBS, Pan Biotech) and penicillin/ streptomycin (Gibco). Mouse leukemia pre-T cells were grown in Iscove’s Modified Dulbecco Medium (IMDM, Gibco 21980–065) and supplemented with 2% of FBS (Pan Biotech), 0.3 mg/ml Primatone, nonessential amino acids (Gibco), 5 mg/l insulin (Merck) and penicillin/streptomycin (Gibco). All cell lines were grown at 37 °C under 5% CO2.
HEK293 and HEK293T cells were transiently transfected using linear polyethylenimine (PEI, Polysciences, 23966–1) or with the ProFection mammalian transfection system (Promega). For the polyethylenimine method cells (2.5 × 106) were seeded on 10 cm plate in 10 ml of medium and incubated for 16-24 h. 14 μl of linear PEI was diluted in 309 µl of PBS, 20 µg of DNA were mixed with 325 µl of PBS, and combined together with diluted PEI. After 30 min of incubation in room temperature, DNA solution was added to the cells dropwise. The medium was changed to fresh one after 6 h of incubation in 37 °C.
For heat shock treatment cells were incubated for 1 h at 42 °C, followed by a recovery for 1 h at 37 °C.
For Luciferase assays, 20 × 104 Hela cells were seeded in 48-well plates and transfected using Lipofectamine 2000 reagent (Thermo Fisher) according to manufacturer’s instructions.
Lentiviral shRNA Knockdown in suspension cells
HEK293T cells (2.5 × 106) were transfected using linear PEI as described above. 3.33 μg of desired plasmid DNA, 2,5 μg of psPAX and 1 μg of pMD were used. 48 h after transfection, supernatant was collected, filtered and 2 µg/ml of polybrene (Merck, H9268) was added. Supernatant containing viral particles was then used to infect Beko cells (5 × 105) by centrifugation (1800 rpm, 45 min, 37 °C). Infection was repeated after 6, 24 and 30 h. Cells were selected with 1 µg/ml Puromycin (Serva, 33835) 24 h after the last infection. Knockdown experiments were performed using shRNA library (Merck).
Generation of CRISPR/Cas9 mediated depletion cells
HEK293 cells were transfected using linear PEI either with empty vector px459 V2.0 pSpCas9(BB)-2A-Puro (WT) or with the same vector containing sgRNAs targeting the coding region of RBPJ (ENSE00003633263).
HEK293 cells were treated with 10 µM SUMO inhibitor ML-792 (Medkoo, 407886) for 24 h. DMSO was used as a control.
The following antibodies were used in this study: anti-L3MBTL2 (Active Motif, 39,569), anti-E2F6 (Diagenode, C15410314), anti-RBPJ (Cell signaling, 5313), rabbit IgG (Diagenode, C15410206), mouse IgG (Santa Cruz, sc2025), anti-Flag (Merck, F3165, F4042), anti-Flag HRP (Merck, A8592), anti-GFP (Merck/Roche, 11,814,460,001), anti-VP16 (Santa Cruz, sc-7545), anti-GAPDH (Abcam, ab8245), anti-TBP (Abcam, ab818), anti-GST (kind gift from Dr. M.L. Schmitz), anti-SUMO2/3 (Abcam, ab81371), anti-VINCULIN (Abcam, ab130007), anti-mouse IgG-HRP (Cell signaling, 7076 or Amersham NXA931), anti-rabbit IgG HRP (Cell signaling, 7074), anti-rat IgG HRP (Jackson ImmunoResearch, 112-035-072), sheep anti-mouse IgG HRP (GE Healthcare, NA931V), anti-Flag (M2) conjugated agarose beads (Merck, A2220), Hemaglutinin (HA) (Covance, MMS-101P).
Protein extracts and western blotting
Whole cell extracts from HEK293 and HEK293T cells were obtained by washing cells in PBS and scraping them from the plate. The cells were washed twice in ice cold PBS and resuspended in extraction buffer (10 mM Tris/HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) freshly supplemented with 10 mM NaF, 0.5 mM sodium orthovanadate, 1 mM PMSF and 1 × protease inhibitor cocktail mix. After 30 min of incubation cells were centrifuged 20,000 rpm for 10 min in 4 °C. Protein concentrations were determined using Bradford assay (Merck). Equalled lysates were subjected for immunoprecipitation or boiled in SDS loading buffer and analysed by Western blotting.
Western blotting was performed by SDS-PAGE and proteins were transferred to nitrocellulose membranes using wet transfer. Membranes were blocked in 5% skimmed milk and subjected to overnight incubation with primary antibodies. After extensive washings in TBST (1 × TBS, 0.1% Tween 20), incubation with secondary antibody coupled to HRP was performed. The membranes were washed again using TBST and results were visualized using ECL solution and Vilber Fusion FX7 system.
HEK293T cells were co-transfected with desired plasmids using linear PEI and harvested 48 h after the transfection. Protein levels after extraction (described above) were analysed using Bradford assay and equalized. GFP TRAP (Chromotek) beads were equilibrated according to the manufacture’s protocol. Protein extracts were than incubated with GFP TRAP beads for 1 h with rotation at 4 °C. Beads were centrifuged (2500×g, 5 min, 4 °C) and washed three times in Dilution Buffer (10 mM Tris/HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA).After removing the supernatant, the beads were resuspended in SDS loading buffer and boiled.
GST proteins purification and GST pulldown
GST fusion proteins were expressed in Escherichia coli strain BL21. After purification, the lysates from whole bacterial cells were stored at − 80 °C. Proteins were in vitro translated in presence of [35S] methionine using rabbit reticulocyte lysate system (Promega L4610). GST and GST fusion proteins were immobilized on Glutathione Sepharose beads and incubated with buffer A (40 mM HEPES [pH 7.5], 0.2 mM EDTA, 5 mM MgCl2, 100 mM KCl, 0.5% NP-40) with rotation for 1 h at 4 °C. Beads were washed one time with buffer A, two times with buffer B (40 mM HEPES [pH 7.5], 0.2 mM EDTA, 5 mM MgCl2, 300 mM KCl, 0.5% NP-40) and one time with PBS. Beads were boiled with SDS loading buffer and proteins were separated in SDS-PAGE. Dried gels were exposed to X-ray films (GE Healthcare).
Modified GST pulldown
HEK293T cells were transfected using the desired expression plasmids. After 48 h the cells were collected and lysed in a lysis buffer. Protein extracts were analysed using Bradford assay and protein concentration was equalized among the samples. Glutathione Sepharose beads were washed three times (3000 rpm, 3 min, 4 °C) in ice cold PBS and then used to immobilize GST fusion proteins during 30 min incubation with rotation at 4 °C. After washing the beads 3 times, the protein extracts were added to the beads and incubated for 4 h with rotation at 4 °C. Beads were washed five times, 10 min with rotation at 4 °C, in ice cold PBS and boiled with SDS loading buffer.
In vitro protein transcription/translation (TNT-assay)
In vitro protein translations were performed using the TNT-assay from Promega according to the manufacturer's instructions. Expression of translated RBPJ proteins (wt, IV/AA, R218H) was monitored by western blotting.
Electro mobility shift assay (EMSA)
Reticulocyte lysates (2 μl and 4 μl) from in vitro translations (see above) were used for the EMSAs. Binding reaction was performed in a buffer consisting of 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT and 4% glycerol. For binding reaction, 10 ng (0.02 U) poly(dI-dC) (GE Healthcare) and approximately 0.5 ng of 32P-labelled oligonucleotides were added. The sequence of the double-stranded oligonucleotide FO-233F/FO-233R (Additional file 3: Table S2) corresponds to the two RBPJ-binding sites within the EBV TP-1 promoter. Where indicated an anti-Flag antibody (M5, Merck) where added to the binding reaction to analyse specificity of binding (supershift). DNA–protein complexes were separated in SDS-PAGE. Gels were dried and exposed to X-ray films (GE Healthcare).
For reporter gene assays Hela cells were transfected using Lipofectamine (as described above) with 250 ng of the reporter construct pGa981/6 (12 × CSL-RE-Luc) alone or together with 10 ng of expression plasmid. After 24 h luciferase activity was determined from at least six independent experiments from 20 μl of cleared lysate. Measurements were performed using a LB 9501 luminometer (Berthold) and the luciferase assay system from Promega.
HeLa cells were plated (1 × 105 cells/cm2) on chamber coverslips (Nunc). After 18 h, cells were transfected with 150 ng of expression plasmids for the specific GFP-fusion proteins. After 24 h, cells were rinsed with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Specimens were embedded in ProLong© Gold antifade reagent (Invitrogen) supplemented with [2-(4-carbamimidoylphenyl)-1H-indol-6-carboximidamide] (DAPI) and stored at 4 °C overnight. Cells were imaged using a fluorescence microscope (IX71, Olympus) equipped with a digital camera (C4742, Hamamatsu) and a 100-W mercury lamp (HBO 103 W/2, Osram) using the following filter sets: GFP detection, ex: HQ470/40, em: HQ525/50, DAPI detection, ex: HQ360/40, em: HQ457/50.
Oligonucleotides and constructs
The complete list of oligonucleotides used in this study is shown in Additional file 3: Table S2.
For complex purification of RBPJ, human RBPJ was cloned into pENTR/D-Topo via PCR and then transferred into a lentiviral destination vector containing an N-terminal Flag-HA-tag using Clonase (Gateway system).
The GST-L3MBTL2 expression plasmid was generated from pCMV tag4A L3MBTL2 plasmid (kindly provided by Dr. G. Suske). Fragment digested using BamHI and XhoI restriction sites was inserted into pGEX6P1 plasmid (GE Healthcare) pre-digested with the same restriction sites.
pGEX-4T-1 GST, GST SUMO1, GST SUMO2, GST SUMO3 fusion proteins were kindly provided by Dr. M.L. Schmitz.
GST-RBPJ fusion protein was previously described .
pcDNA 3.1 Flag hL3MBTL2 ΔN (aa179-705), C (aa604-705), N (aa1-178), ΔC (aa1-603) fragments used for GST pull down experiments were generated by PCR-amplification, primers used for PCR are listed in Additional file 3: Table S2. PCR products were first cloned into pSC-A-amp/kan (Agilent Technologies, 2402055), digested with desired restriction enzymes and inserted into pre-digested pcDNA 3.1 Flag2 (Invitrogen).
pN3-3xFLAG-C-Terminus L3MBTL2 was kindly provided by Dr. G. Suske. pcDNA hRBPJ N/B (aa1-315), B (aa166-334), B/C (aa166-487) fragments and pcDNA hRBPJ NTD (aa1-165) were generated by PCR as previously described [30, 43].
mRBPJ ∆NTD (aa206-526 which corresponds to human fragment aa166-487) was generated by PCR amplification, primers used for PCR are listed in Additional file 3: Table S2. PCR products were first cloned into pSC-A-amp/kan (Agilent Technologies, 2402055), digested with desired restriction enzymes and inserted into pre-digested pcDNA EGFP mRBPJ WT.
To generate expression vectors for the RBPJ IV/AA mutant, specific mouse RBPJ fragments of 721 bp EcoRV/KpnI were synthetized at BioCat GmbH and inserted using corresponding restriction sites into predigested pcDNA3 mRBPJ EGFP (wt) and pcDNA3-Flag-mRBPJ(wt). All plasmids were analysed by sequencing.
RNA extraction, reverse transcription and qPCR
Total RNA was isolated using Trizol reagent (Ambion, 15596018), following the manufacturer’s instructions. 1 µg of RNA was used for generating cDNA using random hexamers and reverse transcriptase M-MuLV (NEB). qPCR analysis were performed using cDNA, Absolute QPCR ROX Mix (Thermo Scientific, AB-1139), gene-specific oligonucleotides, double dye probes (see Additional file 3: Table S2). StepOnePlus (Applied Biosystems) was used as a sequence detector system. Data were normalized to a housekeeping gene.
The cells were collected in ice cold PBS and crosslinked at room temperature for 1 h in 10 mM DMA. After one washing in PBS the cells were additionally crosslinked for 30 min in 1% formaldehyde. Crosslinking was stopped by adding 1 M Glycine (pH 7.5) for 5 min. After two washings in PBS, cells were pelleted at 3000 rpm, 3 min in 4 °C and lysed in 1 ml of SDS Lysis Buffer for 10 min on ice. The lysates were sonicated in Covaris System (28 cycles) and the chromatin was sheared to fragments ranging from 200 to 1000 bp. After measuring concentration, sheared chromatin was aliquoted and stored for further experiments at − 80 °C.
25 μg to 50 μg of chromatin were diluted in ratio 1:6 in ChIP dilution buffer (0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris HCl [pH 8.1], 167 mM NaCl, 1,1% Trition X-100) and precleared with pre-saturated protein-A-Sepharose beads (GE Healthcare 17-5280-02) for 30 min at 4 °C with rotation. Protein-A-Sepharose beads were pelleted at 3000 rpm, 3 min in 4 °C. Diluted chromatin was transferred to a new tube. The input was transferred to a separate tube and stored in 4 °C for the next day. Desired antibodies used for immunoprecipitation were added to diluted chromatin and incubated overnight in 4 °C with rotation. Antibody–protein–DNA complexes were bound to saturated protein A beads and washed each time for 5 min at 4 °C with rotation. The washings steps were as follows: 1 × low salt washing buffer (20 mM Tris/HCl [pH 8.1], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), 1 × high salt washing buffer (20 mM Tris/HCl [pH 8.1], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), 1 × LiCl washing buffer (10 mM Tris/HCl [pH 8.1], 250 mM LiCl, 1 mM EDTA, 1% NP-40), 3 × TE buffer (10 mM Tris/HCl [pH 7.9], 1 mM EDTA) or 1 × low salt washing buffer, 2 × high salt washing buffer, 2 × LiCl washing buffer, 3 × TE buffer in case of RBPJ ChIP. DNA bound to the proteins was eluted in two steps using freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at room temperature. After combining two elutions, 20 μl of 5 M NaCl was added and samples were reverse crosslinked overnight at 65 °C. Bound proteins were digested using Proteinase K for 1 h at 45 °C. DNA was purified using phenol–chloroform extraction and precipitated overnight at − 20 °C in isopropanol in presence of tRNA and glycogen. Samples were centrifuged 13,000 rpm, 30 min at 4 °C and washed in 70% ethanol. After centrifugation samples were dried in the concentrator and resuspended in 20 μl of TE buffer.
RBPJ complex purification and mass spectrometry
Flag-HA-tagged human RBPJ was expressed after lentiviral infection of HeLa-S. The cells were expanded to 8L and harvested. All following steps were performed at 4 °C. The cells were lysed via incubating with 5 cell pellet volume hypotonic buffer (10 mM Tris/HCl, pH 7.3, 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF and 10 mM β-Mercaptethanol, Protease inhibitor) for 30 min and douncing. The mixture was centrifuged (4000 rpm) and the nuclei pellet was washed twice with hypotonic buffer. Nuclear extract was made by low salt/high salt extraction. The nuclei were resuspended in 1 nuclei pellet volume low salt buffer (20 mM Tris/HCl, pH7.3, 20 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 10 mM β-mercaptoethanol, protease inhibitor). Subsequently, 0.6 nuclei pellet volume high salt buffer (20 mM Tris/HCl, pH 7.3, 1.2 M KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Gyclerol, 10 mM β-Mercaptethanol, Protease inhibitor) was added dropwise under stirring. After adding the high salt buffer, the mixture was incubated for further 30 min, stirring. The mixture was centrifuged at 13,000 rpm, and the supernatant was taken. The gained nuclear extract was split into two fractions. To one fraction the wild type (2 µg) and mutated oligonucleotide (16 µg) was added. The other fraction remained untouched (see Fig. 1a, c). Both fractions were dialyzed overnight in (20 mM Tris/HCl, pH7.3, 100 mM KCl, 0.2 mM EDTA, 20% Gyclerol, 10 mM β-Mercaptethanol, 1 mM DTT, Protease inhibitor). The RBPJ complexes were then purified using anti-Flag (M2) conjugated agarose beads by incubation in TAP buffer (50 mM Tris–HCl pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 1 mM DTT, and protease inhibitors) for 4 h and 3 times washing with TAP buffer. Flag-HA-RBPJ was eluted from the beads with Flag peptides. The obtained samples were TCA precipitated and peptides were identified via LC–MS/MS at the Taplin Core facility (Harvard Medical School).