Cell culture, synchronization and cell cycle analysis
HeLa cells and HEK 293T cells were grown in Dulbecco’s modified Eagle medium with l-glutamine and 4.5 g/L glucose (DMEM; Lonza) supplemented with 10 m % fetal calf serum (Gibco) and antibiotics [100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B (Sigma)] at 37 °C in a moist atmosphere containing 5% CO2.
For G1 synchronization cells were blocked first by 2 mM thymidine (Sigma-Aldrich) for 24 h, then released for 3 h, blocked again by 0.1 μM nocodazole (Sigma-Aldrich) for 12 h and collected 5–7 h after release. For S phase synchronization cells were blocked first by 2 mM thymidine for 17 h, then released for 12 h, blocked again by 400 μM mimosine (Sigma-Aldrich) for 14 h and collected 4.5–5 h after release. For detailed analysis of cell cycle progression, cells were synchronized in G2/M by addition of 200 ng/mL nocodazole (Sigma-Aldrich) to the medium for 18 h and then cells were collected and monitored every 2 h after release from the block.
For cytofluorometric analysis, cells were trypsinized, washed with phosphate-buffered saline (PBS) and fixed by dropwise addition to ice-cold 70% ethanol. On the day of staining, cells were washed twice with PBS, resuspended in propidium iodide staining solution [0.1% Triton X-100 in PBS, 0.2 mg/mL RNase A (Termo Scientific), 0.02 mg/mL propidium iodide (Sigma)] and incubated for at least 30 min at room temperature in the dark. Cell cycle profiles were analyzed by flow cytometry with a Guava easyCyte™ System (Merck Millipore) flow cytometer and the data was processed with InCyte Software (utilities from guavaSoft 3.1.1).
For cell proliferation tests, the cells were plated in triplicate in 12-well plates at the density of 50,000 cells/well. Then cell counts and viability were measured every 24 h for 6 days by using a Countess™ Automated Cell Counter (Life Technologies). For the MTT assay, cells were plated in triplicate in 24-well plates at 50,000 cells/well. To measure cell viability, Thiazolyl Blue Tetrazolium Bromide salt (Sigma M2128) in PBS was added to each well at 500 µg/mL final concentration. After 3 h of incubation, the formazan crystals were centrifuged at 300g for 10 min and dissolved by adding ethanol:DMSO (ratio 1:1). The absorption of the formazan solution was measured using an Infinite F200 PRO Tecan spectrophotometer at a wavelength of 570 nm. Cell viability was measured every 24 h for 6 days.
Plasmid construction, lentiviral vector production and cells transduction
A lentiviral vector for the doxycycline-inducible PC4 knockdown was constructed by inserting annealed and kinased oligonucleotides (Additional file 5: Table S2) into the AgeI/EcoRI sites of the pLKO-Tet-On plasmid (a gift from Dmitri Wiederschain; Addgene plasmid # 21915 ) to create pLKO-Tet-On-shPC4. The shRNA is expressed from this vector under the H1 promoter and is further converted into siRNA that targets nucleotides 170–188 of PC4 mRNA (numbering according to U12979.1, GCAGCAGAGATGATAACAT). A control lentiviral vector with an inducible shRNA scramble expression cassette was ordered from Addgene (a gift from David Sabatini; Addgene plasmid # 1864 ). The lentiviral expression vector encoding FLAG-tagged PC4 was constructed by amplification of the coding sequence of PC4 with a FLAG sequence added downstream of the AUG codon by using specific primers in a PCR (primer sequences available on request). FLAG-PC4 cDNA was then cloned under the EF-1 alpha promoter in into the MluI/SmaI sites of the pLV-tTR-KRAB-dsRed vector to create pLV-ttR-FLAG-PC4-dsRed. Lentiviral expression vector encoding enhanced blue fluorescence protein (EBFP) was prepared as described previously .
Virus production and HeLa transduction for cells with stable overexpression of FLAG-PC4 as well as PC4 shRNA and scramble shRNA was performed as follows: HEK 293T cells were transfected with pLKO-Tet-On-shPC4, scramble shRNA or pLV-ttR-FLAG-PC4-dsRed plasmids supplemented with packaging and envelope vector, psPAX2 and pMD2.G, respectively, by the calcium phosphate method . Fresh medium was added to the cells 24 h after transfection, and lentiviral supernatants were collected 72 h after transfection. For transduction, HeLa cells were incubated with lentiviral supernatants supplemented with 4 μg/ml polybrene (hexadimethrine bromide, Sigma Aldrich) for 14 h, and then fresh medium was added. Highly RFP positive cells with FLAG-PC4 overexpression were selected by fluorescence-activated cell sorting using a BD FACS Aria™III (Becton–Dickinson) flow cytometer (cell sorter). The configuration of the flow cytometer was as follows: 100 μm nozzle and 20 psi (0.138 MPa) sheath fluid pressure. The cells were characterized by two non-fluorescent parameters: forward scatter (FSC) and side scatter (SSC), and one fluorescent parameter: yellow fluorescence (PE detector) from RFP collected using 585/42 band pass filter. For excitation, a 488 nm blue laser was employed. The flow cytometric analyses were performed using logarithmic gains and specific detectors settings (10,000 events were recorded per analysis). Data were acquired in a 4-decade logarithmic scale as area signals (FSC-A, SSC-A and PE-A) and analyzed with FACS DIVA software (Becton–Dickinson).
A sub-population P5 demonstrating high levels of yellow fluorescence (as measured by PE detector) was selected for sorting. The sort region (P5) was defined on bivariate dot plot (SSC-A vs. PE-A). Cell sorting preceded a doublets discrimination procedure which used measurements of height versus width scatter (FSC and SSC) signals, in order to discriminate single cells from conglomerates. Cells from sub-population P5 were sorted into 5 mL cytometric tubes.
HeLa cells with PC4 shRNA or scramble shRNA were selected by adding puromycin to the final concentration of 0.3 µg/mL for 7 days.
RNA isolation, cDNA preparation, PCR and qPCR
RNA was isolated from cells by using TRIZOL reagent followed by DNAse treatment as described in . First strand cDNAs were synthesized in 50 μL reactions with 4.5 μg of RNA by using 400 ng random hexamers as primers and 200 U Superscript III Reverse Transcriptase (SSIII RT, Thermo Scientific), according to the manufacturer’s protocol. PCR amplifications were carried out in 25 µL reactions containing 2.5 µL of Pfu buffer, 2 mM MgCl2, nucleotide mix [0.2 mM each dNTP (Roche)], 0.5 µM primers and 2 U of Pfu DNA Polymerase (Thermo Scientific). The samples were incubated for 30 cycles under the following conditions: 95 °C for 2 min, each cycle: 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min. The reactions were completed by incubation for 10 min at 72 °C. For qPCR amplifications, 10 μL reaction mix contained 5 μL of Power SYBR Green PCR Master Mix (Applied Biosystems), 4 μL of 0.5 mM primers mix and 1 μL of 10× diluted cDNA template. The qPCR was performed under the following conditions: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min (QuantStudio™ 7 Flex Real-Time PCR Instrument). Primers used for qPCR are listed in Additional file 6: Table S3. The statistical significance of qPCR results was determined by Student’s T test.
Antibodies, protein extract preparation, immunoprecipitation
The following primary antibodies were used in this work: anti-RPB2 (Abcam, ab10338), anti-β-actin (MP Biomedicals, 691001), anti-FLAG (Sigma Aldrich, A8592), anti-PC4 (Abcam, ab72132), anti-CstF64 (Santa Cruz Biotechnology, sc-28201). The following secondary antibodies were used: goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2004, sc-2005, respectively).
For total protein extract preparation, cells were harvested by trypsinization, washed with PBS, resuspended in lysis buffer (50 mM Tris–HCl pH 7.9, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) and incubated for 10 min on ice. Supernatants containing total protein extracts were collected after 30 min centrifugation at 4 °C at 16,000g.
For affinity purification strategy protein extracts from HeLa cells expressing MS2-tagged U7 snRNA were purified on the MS2-MBP-bound resin and eluted from the resin by mild condition using 10 mM maltose. Samples were either directly submitted to mass spectrometric analysis or first separated on a SDS polyacrylamide gel and then selected bands were cut from the gel and submitted to mass spectrometric analysis, as described in . In some cases, probes were first fractionated on 10–50% continuous glycerol gradients prior to affinity purification, as described in . As an another approach HeLa nuclear extracts were incubated with a biotinylated 2′-O-methyl RNA oligonucleotide complementary to U7 snRNA followed by purification on streptavidin-coated Dynabeads, as described in .
For co-immunoprecipitation, an amount of 250 µg of protein extracts was immunoprecipitated for 2 h at 4 °C with 3 µg of anti-PC4 antibody previously conjugated for 1 h at 4 °C with gentle rotation with 20 µL of Dynabeads® Protein A (Life Technologies) or with 20 µL of non-conjugated beads. 5% of protein extracts used for immunoprecipitation was kept in a separate tube as input. After immunoprecipitation, beads were washed three times with PBS-T and twice with lysis buffer, each time for 10 min and eluted by boiling in sample buffer (50 mM Tris–HCl pH 6.8, 10% glycerol, 2% SDS, 10 mM DTT, 0.1% bromophenol blue). After elution, the immune complexes were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was incubated for 2 h with primary antibodies in the presence of 2% milk and then detected by the enhanced chemiluminescence method (ECL, GE Healthcare) after incubation for 1.5 h with corresponding species-specific horseradish peroxidase (HRP)-coupled secondary antibody. For Phos-tag™-based mobility shift detection of phosphorylated PC4 protein electrophoresis was run according to manufacturer’s manual (Wako Pure Chemical Industries).
A total of 12 × 106 HeLa PC4 OE cells (or EBFP OE as negative control) or PC4-depleted cells (with HeLa scramble as negative control) were synchronized to G1 and S phase, as described above. Cells were trypsinized, washed with PBS and cross-linked with 1% formaldehyde for 10 min followed by quenching with 125 mM glycine for 5 min. Next, cells were washed twice with PBS and lysed in cell lysis buffer [10 mM Tris–HCl pH 8.1, 10 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, 1× EDTA-free protease inhibitor (Roche)] for 15 min on a rotating wheel at 4 °C and then centrifuged at 1200g for 5 min at 4 °C. The pellet was resuspended in nuclear lysis buffer [50 mM Tris–HCl pH 8.1, 5 mM EDTA, 0.5% sarkosyl, 1× EDTA-free protease inhibitor (Roche)] and moved to 1.5 mL DNA LoBind tubes (Eppendorf). After 10 min incubation on a rotating wheel, the nuclear lysate was sonicated with a Bioruptor® Plus Sonicator (Diagenode) to generate DNA fragments between 200 and 700 bp (usually 28 cycles, at high intensity: 30 s ON/30 s OFF at 4 °C). Each time, the sizes of DNA fragments were verified by agarose gel electrophoresis. After sonication, the cell debris were removed by centrifugation at 18,000g for 15 min at 4 °C. From this point, samples were further processed as described . Per sample, 4 µg of anti-RPB2 antibody (Abcam, ab10338) and 50 µL of a 50% slurry of Protein A-Sepharose® 4B conjugate was used. One percent of the chromatin used for immunoprecipitation was kept in a separate tube as input. As modification of the protocol, a portion of the conjugate designated for chromatin pre-clearing was blocked for 1 h. Eluted samples were used for qPCR analysis; primer pairs encompassing the “TSS region”, “histone body” and “3′ end” regions of histone genes are listed in Additional file 6: Table S3. The quantitative analysis of precipitated material was shown as a fold change normalized to input and relative to HeLa EBFP OE or scramble cells. The statistical significance of qPCR results was determined by Student’s T test.
RNAP2 ChIP and input samples were used for library generation and sequencing by Illumina HiSeq 2000 system, performed by Fasteris SA (Switzerland). The quality of generated data was verified by the FastQC software ; libraries were mapped to the human genome (GRCh38/hg38; released 2013/12/17) by bowtie ; RNAP2 enriched/depleted regions were identified using MACS software; the gene annotation was done in HOMER software ; profiles of RNAP2 occupancy were created using bedtools package .