PML used in the study was classified as PML IVa (Jensen et al., 2001
). It was cloned into BamH
I and EcoR
I sites of pCMVtag2B vector (Stratagene) so that a FLAG tag was placed at the PML N-terminus. PML3m, PMLcs, PMLas, PML3mas, and PML3mds were constructed from the cloned FLAG-PML IVa plasmid using a Quikchange Site-Directed Mutagenesis Kit (Stratagene). Plasmids expressing His-tagged PML I mutants were generated by using a Quikchange mutagenesis based on pET33b-PML I from S. Bergmann. The plasmid expressing recombinant GST-SUMO1 was constructed by insertion of a PCR product of activated SUMO1 (C-terminal ended as two glycines) into BamH
I and Xho
I sites of pGEX-5X-1 (Amersham Biosciences). Retroviral vectors that express FLAG-PMLwt and FLAG-PML3m were constructed by insertion of respective PCR products into BamH
I and EcoR
I sites of a pBABE vector, and other mutants were subsequently made by Quikchange mutagenesis. All plasmids were verified by DNA sequencing. Primer sequences are available upon request. The pGFP-SUMO1 plasmid was a kind gift of P. Salomoni. Xpress-PML was previously described (Lin et al., 2004
Cell lines, Transfections, Immunoprecipitations and Western Blotting
Human 293T cells, Pml+/+ immortalized mouse embryonic fibroblast (MEFs), and Pml−/− immortalized MEFs were maintained in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamate and antibiotics (1% v/v penicillin-streptomycin). Cells were split one day before transfection in 6 well plates (for 293T cells) or in 10 cm plates (for MEFs) so that cells reach to ~70% confluency upon transfection using effectene transfection reagent (Qiagen) according to supplier’s protocol. At 36–48h after transfection, cells were rinsed with cold phosphate buffered saline (PBS), and scraped in 1 ml of cold PBS with 10 mM N-ethylmaleimide (NEM, Sigma). Cell pellets were lysed at 4 °C in 200 μl of RIPA buffer (1% NP40, 0.5% DOC, 0.1% SDS in PBS) with freshly added 10 mM NEM, protease inhibitor (Roche, 50 ml/tablet) and 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma). The cell lysates were then sonicated on ice for 3 sec and centrifuged at maximum speed for 10 min at 4 °C in an Eppendorf centrifuge. The supernatants were mixed with 10 μl of washed and packed protein G sepharose beads (Amersham Biosciences) at 4 °C for 30 min. The cleared supernatants were then mixed with 10 μl of washed and packed protein G sepharose beads and 1 μl of anti-GFP (rabbit polyclonal antibody, Clontech), or anti-FLAG antibodies (Sigma) at 4 °C overnight. The beads were washed 4 times with RIPA buffer and the immunoprecipitates together with inputs were analyzed by 8% SDS-PAGE. The stacking gels were always saved for the detection of high molecular weight SUMOylated proteins. Western blotting was performed using anti-GFP (mouse monoclonal antibody, 1:1000, Clontech), or anti-FLAG antibodies (1:1000), visualized by enhanced chemiluminescence (Amersham Biosciences).
Recombinant Protein Purification, Peptide Synthesis and In Vitro Binding Assay
His-tagged Recombinant PML and mutants were produced in Rossetta 2(DE3)pLysS cells (Novagen) upon induction with 0.5 mM IPTG (Sigma) at 37 °C for 4 h. The proteins were purified using Talon magnetic beads (Dynal Biotech) according to the supplier’s procedure. Recombinant GST-SUMO1, and GST were produced in BL21 cells (Novagen) upon induction with 0.5 mM IPTG (Sigma) at 37 °C for 4 h. The proteinswere purified using glutathione-sepharose beads (Amersham Biosciences) according to the supplier’s protocol. The eluted proteins were dialyzed, concentrated by Centriprep 10 (Millipore), aliquotted and stored at −80 °C until use. The purity and concentration of purified proteins were analyzed by SDS-PAGE and Coomassie blue staining with known amount of bovine serum albumin (BSA) as a control. PML peptides were designed to contain an N-terminal biotin and tetrapeptide linker SGSG followed by 17 mers of PML centered at the SUMO binding motif VVVI, or its mutants in which VVVI was replaced with AAAS or VVKI. The peptides were synthesized and HPLC purified by the Protein Core facility at MSKCC.
For the in vitro binding assay using synthetic peptides, 3 μg of recombinant GST-SUMO1 or GST were incubated with 15 μg of biotinylated PML peptide or its mutant peptides at 4 °C overnight in 300 μl of interaction buffer containing 20 mM Hepes, pH 7.9, 150 mM KCl, 5 mM EDTA, 0.5 mM DTT, 0.1% (V/V) Nonidet P-40, 0.1% (W/V) BSA, 1 mM PMSF, 10% glycerol. Then 20 μl of streptavidin beads (Pierce) was added and incubated for 1 h. After three washes with PBS, the bound proteins were determined by Western blot analysis using antibodies against GST (1: 1000, Sigma) or SUMO1 (1: 500, Santa Cruz Biotechnology).
For the in vitro binding assay using full length recombinant proteins, 1 μg of purified His-tagged PML or its mutants was incubated with equivalent amounts of purified GST-SUMO1 and GST in 20 mM Hepes, pH7.5, 150 mM NaCl, 0.1% Tween-20 at 4 °C for 4 h. Then the mixtures were mixed with 10 μl of washed Talon magnetic beads with rotation at 4 °C for 1 h. The input and unbound fractions, together with the bound fractions after three washes, were analyzed by Western blot using antibodies against His (1:1000, Sigma) or GST (1:1000, Sigma). The intensities of His-tagged PML and GST-SUMO1 bands in the bound fractions were quantified using MacBAS V2.5-1 software and the ratios of GST-SUMO1 to PML calculated.
Pml−/− MEFs were grown in slide chambers and transfected with PML or its mutants and GFP-SUMO1 using effectene transfection reagent (Qiagen) according to the supplier’s protocol. After 36–48h, the cells were washed with PBS and fixed in 4% paraformaldehyde at RT for 20 min. The cells were then permeabilized in PBS containing 10% FCS and 0.1% Triton X-100. Rabbit anti-PML antibodies (PGM3, Santa Cruz Biotechnolgy) were added (1:500) and incubated in the same buffer at RT for 1 h, followed by incubation with goat anti-rabbit IgG conjugated with Alexa fluor 568 (Molecular Probes, 1:500), together with DAPI (0.1 μg/ml) at RT for 30 min. After washes, the slides were mounted. Images were captured by laser-scanning microscope (Leica) and processed in Adobe Photoshop 7.0. For the detection of endogenous protein markers, GFP-SUMO1 was omitted in transfections. These proteins include: SUMO1 (1:50, Santa Cruz Biotechnolgy), Daxx (1:25, Santa Cruz Biotechnolgy), Fibrillarin (1:100, Abcam), and SC35 (1:100, Abcam). They are all polyclonal antibodies and were used with anti-rabbit antibodies conjugated with Alexa fluor 488 (Molecular Probes, 1:500).
Retroviral production, infection and apoptosis analysis
Phoenix cells grown on 10 cm lysine coated plates (Biocoat Inc.) at 70% confluency were transfected with retroviral vectors expressing FLAG-PMLwt or its mutants using Lipofectamine 2000 (Invitrogen) according to the supplier’s protocol. After 48 h, the viruses containing media were collected, passed through a 0.45 μM filter, and directly used in the infection of Pml−/− MEFs for 48 h in the presence of 4 μg/ml of polybrene (Sigma). Infected cells were then selected for one week in the presence of 2.5 μg/ml of puromycin with 2–3 medium changes. The resulting cells were seeded on 6 well plates at 70% confluency. After 48 h, the cells were collected in PBS by trypsinization, together with dead cells in the medium. The cells were washed with PBS, fixed in 70% ethanol at 4 °C overnight. After washing with PBS, the cells were stained in PBS containing 2 μg/ml of propidium iodide (PI, BD Biosciences) and 0.5 μg/ml of DNase-free RNase (Roche) at RT for 1 h. The PI stained cells were subjected to Fluorescence Activated Cell Sorting (FACS) analysis. The results are presented in log scale to magnify the sub-G1 population of cells.