Constructs and vectors engineering
Full-length open reading frames of all cDNAs, as well as truncated and mutant cDNAs were PCR amplified from IMAGE cDNA clones purchased from Deutsches Ressourcenzentrum für Genomforschung GmbH (RZPD) and cloned into the GATEWAY entry vector pDONR201 (Invitrogen).
Moloney murine leukemia virus-based vectors were generated from pLNCX2 (Clontech) in which an IRES-pac cassette from pIRESpuro (Clontech) and conferring resistance to puromycin was introduced. Then, GATEWAY compatible target vectors for expression of TAP-tagged (pRP-NTAP-GW) or GFP-tagged (pRP-NGFP-GW) proteins were engineered by inserting GATEWAY and TAP (from pZome-1-N, Cellzome) or GFP (from pEGFP-N1, Clontech) cassettes, respectively. For bacterial expression of the GST-tagged proteins, a GATEWAY cassette was introduced into pGEX-4T1 (Amersham). Entry clones were recombined into suitable target vectors by GATEWAY LR reactions. A list of clones used, primer and vector sequences is available on request.
Cell culture and stable cell lines
HeLa (Human cervix epitheloid carcinoma – ECACC), L929 (Mouse cell line C3H/An connective tissue – ECACC) and HEK293 (ECACC) cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin (Invitrogen), and 100 μg/ml streptomycin (Invitrogen) at 37°C in humidity-saturated 5% CO2 atmosphere.
Retroviral stable cell lines were generated according to the following procedure. Phoenix amphotropic packaging cells (3 × 105 cells/well) were seeded in a 6-well plate and transfected 24 hours later with 0.8 μg of retroviral plasmid using Exgen 500 (Euromedex) following the instructions of the manufacturer. After 48 hours virus-containing supernatant was filtered through a 0.45-μm-pore-size filter. HeLa or L929 cells (105 each) were seeded in a six-well plate and transduced with 3 ml filtered virus supernatant in the presence of 8 μg/ml of Polybrene for an infectious round of 24 hours. Cells were then incubated for 24 hours in normal medium. The polyclonal population of cells was then selected with 1 μg/ml of puromycin. Growing cells were then tested for recombinant protein expression using immunocytochemistry for TAP-tagged protein expression or immunofluorescence for GFP-tagged protein expression.
For cell cycle synchronization, cells were seeded at a concentration of 2 × 104 cells/cm2 either in 6-well plates or in 10-cm dishes and grown to 50–70% confluence to obtain cultures in the logarithmic growth phase. Synchronization in G0 was achieved by serum deprivation; cultures were washed 3 times with PBS, once with DMEM and then cultured for 72 hours in DMEM without serum, whereas synchronization at G2/M transition was achieved using a nocodazole treatment ; cultures were grown in DMEM with 10% fetal bovine serum and 50 ng/ml nocodazole for 24 hours. All synchronization experiments based on serum deprivation or nocodazole treatment were performed in parallel to ensure accurate comparisons.
Antibodies and Western blotting
The following primary antibodies were used for Western blotting: mouse anti-HP1α antibody (2HP-2G9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1β antibody (1MOD-1A9, Euromedex; used at a dilution of 1:2 000), mouse anti-HP1γ antibody (2MOD-1G6, Euromedex; used at a dilution of 1:2 000), goat anti-CBP antibody (sc-9456, Santa Cruz Biotechnology; used at a dilution of 1:300). Secondary antibodies were: horseradish peroxidase-congugated goat anti-mouse antibody (115-035-003, Jackson ImmunoResearch; used at a dilution of 1:10 000), horseradish peroxidase-conjugated donkey anti-goat antibody (705-035-003, Jackson ImmunoResearch; used at a dilution of 1:5 000). The protein A moiety of the TAP tag was revealed with rabbit peroxidase anti-peroxidase antibody (P1291, Sigma; used at a dilution of 1:10 000).
For Western blotting, protein samples (50 μg) in SDS loading buffer were electrophoresed on 4–12% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked in 10% milk powder in PBS-T (1× PBS with 0.1% Tween20) for 1 hour at room temperature, incubated for same time with the primary antibody in PBS-T, and washed three times 10 min in PBS-T. The membranes were then incubated with the peroxidase-conjugated secondary antibody in PBS-T for 1 hour and afterward washed three times 10 min in PBS-T. Signal was detected using chemiluminescence reagent (ECL, Amersham) on imaging film (GE Healthcare).
Cell cycle analysis was performed by flow cytometry as described by Evans et al . Single-cell suspensions were obtained by trypsinization, washed twice with PBS and incubated in 80% ethanol at room temperature for 16–24 hours. Cells were rinsed with PBS, resuspended in 200 μg/ml Propidium Iodide (PI), incubated with 100 μg/ml RNase at 37°C for 30 min, and stored at least one hour at 4°C. Stained cells were analyzed with a Coulter Epics XL flow cytometer equipped with a 488-nm argon laser (Beckman Coulter, Fullerton, CA) and a 530 nm band-pass filter allowing detection of PI fluorescence. A minimum of 10,000 events were collected. GFP or PI fluorescence was expressed as a ratio of the mean channel value of the GFP or PI histogram to the mean channel value of the isotype control histogram.
Tandem affinity purification
For Tandem affinity purification (TAP), cells expressing a TAP-tagged protein were expanded into forty 15-cm dishes. At confluence (about 100 mg total protein), cells were harvested, washed with PBS, resuspended in cell lysis buffer (10 mM Tris-HCl pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 25 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol [DTT] and complete protease inhibitors [Roche]) and homogenized by 20 strokes with a type B pestle. Nuclei were recovered by centrifugation 10 min at 2000 g, resupended in nuclear lysis buffer (50 mM Tris-HCl pH 7.4, 1.5 mM MgCl2, 420 mM NaCl, 20% glycerol, 25 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol [DTT] and complete protease inhibitors [Roche]), and then incubated for 1 hour in a rotation wheel at 4°C to extract nuclear proteins. Lysates were subsequently clarified by ultracentrifugation at 100,000 g, 1 hour.
Nuclear lysate was incubated with IgG agarose beads (Sigma) for 2 hours at 4°C in a rotation wheel. Bound proteins were washed with an excess of lysis buffer and then with a TEV-protease cleavage buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl and 0.2% NP-40) and eluted by addition of 30 mg TEV protease (Invitrogen) for 2 hours at 16°C. The TEV-protease cleavage product was incubated with calmodulin sepharose (Amersham) in the presence of 2 mM CaCl2 for 30 min at 4°C in a rotation wheel. After extensive washes in 100 mM Trs-HCl pH8, 100 mM NaCl, 0.5 mM EDTA, 2 mM CaCl2, calmodulin-bound proteins were eluted by boiling in SDS loading buffer.
Mass spectrometric analysis
Protein eluate was separated on a 4–12% NuPAGE Novex gel (Invitrogen) and stained with Imperial Protein Stain (Pierce). Gel was sliced into 37 bands across the entire separation range of the lane. Cut bands were reduced, alkylated with iodoacetamide, and in-gel digested with trypsin (Promega) as described previously . In brief, gel bands were destained overnight at 4°C in a solution containing 50 mM NH4HCO3 and 50% acetonitrile, dehydrated in acetonitrile, and dried in a vacuum centrifuge. Gel pieces were then rehydrated at 4°C for 45 min in a digestion buffer (25 mM NH4HCO3 and 12.5 ng/μl trypsin). The supernatant was replaced by 50 μl of 25 mM NH4HCO3, and the samples were incubated overnight at 37°C. The tryptic peptides were recovered by 10-min incubations, twice in 45% acetonitrile, 10% HCOOH and once in 95% acetonitrile, 5% HCOOH. All supernatants were pooled and dried in a vacuum centrifuge.
Each tryptic digest sample was subjected to nano-LC-nano-ESI-MS/MS analysis on an ion trap mass spectrometer (LCQ Deca XP+, Thermo Electron Corp.), equipped with a nanoelectrospray ion source, coupled with a nano-high pressure liquid chromatography system (LC Packings Dionex). Samples were resuspended in 3 μl of 0.1% HCOOH, and 1.4 μl were injected into the mass spectrometer using a Famos autosampler (LC Packings Dionex). The samples were first desalted and then concentrated on a reverse phase precolumn of 5 mm × 0.3 mm inner diameter (Dionex) by solvent A (95% H2O, 5% acetonitrile, 0.1% HCOOH) delivered by the Switchos pumping device (LC Packings Dionex) at a flow rate of 10 μl/min for 3 min. Peptides were separated on a 15 cm × 75 μm-inner diameter C18 PepMap column (Dionex). The flow rate was set at 200 nl/min. Peptides were eluted using a 5–70% linear gradient of solvent B (20% H2O, 80% acetonitrile, 0.08% HCOOH) in 45 min. Coated nanoelectrospray needles (360 μm outer diameter, 20 μm inner diameter, 10 μm tip inner diameter, standard coating) were obtained from New Objective (Woburn, MA). Spray voltage was set at 1.5 kV, and capillary temperature was set at 170°C. The mass spectrometer was operated in positive ionization mode.
Data acquisition was performed in a data-dependent mode consisting of, alternatively in a single run, a full-scan MS over the range m/z 500–2,000 and a full MS/MS of the ion selected in an exclusion dynamic mode (the most intense ion is selected and excluded for further selection for a duration of 3 min). MS/MS data were acquired using a 2 m/z unit ion isolation window and 35% relative collision energy. MS/MS .raw data files were transformed to .dta files with the Bioworks 3.1 software (Thermo Electron Corp.). The .dta files generated were next concatenated with merge.bat (a DOS batch file for Windows) to be uploaded in Mascot public interface version 2.2.03 http://www.matrixscience.com for database searches in Swiss-Prot 55.1 (359,942 sequences; 129,199,355 residues).
Search parameters in human sequences were: three allowed missed cleavages, methionine oxidation and cysteine carbamidomethylation as variable modifications, 2 Da for peptide tolerance, and 0.8 Da for MS/MS tolerance. Results were scored using the probability-based Mowse score [the protein score is -10 × log(p), where p is the probability that the observed match is a random event]. Most proteins were unambiguously identified by the sequencing of several independent peptides. Identifications with Mascot individual ion score < 38 or with the significance threshold p > 5% (indicate identity or extensive homology) were categorically rejected. In addition, because a shared sequence may represent a problem, for single peptide identifications, all sequences obtained by MS/MS analysis were checked using the Basic Local Alignment Search Tool (BLAST) public interface (version 2.2.18) to exclude that sequence sharing with other proteins could interfere with the reliability of the identification.
In vitro GST protein binding assays
GST fusion proteins, were expressed in E. coli BL21 (DE3) and purified on glutathione-Sepharose 4B (GE Healthcare) according to the manufacturer's instructions. GST-proteins were then fixed on glutathione-Sepharose 4B and stored in STE buffer (10 mM Tris-HCl pH8, 150 mM NaCl, 1 mM EDTA and complete protease inhibitors [Roche]). After preclearing with empty beads, nuclear extracts (~500 μg proteins) were incubated with immobilized GST-fusion proteins overnight at 4°C. Beads were washed four times with E1A Buffer (50 mM Hepes, pH 7.9, 1 mM EGTA, 250 mM NaCl, 1 mM DTT, 1 mM EDTA and complete protease inhibitors [Roche]) and bound proteins were recovered with the Elution Buffer (10 mM glutathione in 50 mM Tris-HCl pH 8.0), resolved by 4–12% gradient SDS-PAGE (Invitrogen) and visualized by Western blotting using the peroxidase-anti-peroxidase (PAP) antibody (Sigma) which recognizes the protein A moiety of the TAP tag. Input material corresponds to 2% of the material used in the binding assays.
Nuclear extracts used for GST-pull downs were prepared from HEK293 cells transiently expressing TAP-tagged proteins. Forty eight hours after transfection, cells were lysed in low salt buffer (10 mM Tris-HCl pH 7.4, 25 mM NaCl, 2 mM MgOAc, 1 mM DTT, 1 mM EDTA, 0.05% NP-40 and complete protease inhibitors [Roche]) for 15 min at 4°C. Nuclei were pelleted by centrifugation at 4°C for 10 min at 800 g, and lysed in E1A buffer during 2 hours by gently shaking at 4°C. Nuclear proteins were recovered by centrifugation at 2500 g, 10 min at 4°C.
In vitro translated proteins used for GST-pull downs were produced with the TnT7 Quick Coupled Transcription/Translation System (Promega). Immobilized GST-fusion proteins were incubated with in vitro translated proteins overnight at 4°C in IP buffer (50 mM Tris-HCl pH7.5, 500 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol and complete protease inhibitors [Roche]). The beads were washed four times with IP buffer and resuspended in loading buffer. Bound proteins were resolved by SDS-PAGE and visualized by Western blotting.
Fluorescence recovery after photobleaching
FRAP experiments were performed in living cells and dynamic parameters determined according to McNally . Briefly, GFP-expressing L929 cells were plated on coverslip surfaces, grown in L15 medium (Invitrogen), and maintained at 37°C in a Leica live-cell chamber. A Leica SP2 confocal microscope equipped with an oil 100× NA 1.4 plan Apo lens objective, a 488 nm mono-ray laser line, and a linear regime detector in intensity measurements were used for FRAP experiments . Five pre-bleach acquisitions were performed to measure background, fluorescence fading, and pre-bleach fluorescence intensity. The laser power was calibrated using an acousto-optical tunable filter (AOTF). Pre-bleach and post-bleach imaging were performed with an AOTF setting at 5% (5 mW), whereas photobleaching was at 100% AOTF (100 mW). The bleached area was chosen circular for simplicity of the equations available to analyze fluorescence recovery for such a shape . The bleached regions had a size of 1 to 1.3 μm of diameter, and were subjected to five excitation pulses of 336 ms each at high laser power. Post-bleach images were collected at 20 time intervals of 336 ms, 15 time intervals of 1,336 s and 15 time intervals of 2,336 s.
Relative fluorescence intensities within the bleached area were plotted as a function of time, yielding the RAW FRAP curve. The recovery curves were corrected for background, fluorescence fading, and decrease in fluorescence during photobleaching. The t1/2 value was defined as the time required for reaching half-maximum recovery and was calculated from the corrected recovery curves obtained using an in-house modified MATLAB fitting tool (Mathworks) using fit curves where the intensity recovered in time follows the exponential law,
according to McNally and colleagues [36, 39].