STAT1 WT-HA, STAT1 WT-Flag, STAT1 K703R-HA, STAT1 E705A-HA and STAT1 Y701F plasmids were previously described [11–13]. STAT1 E705Q mutation was created with site directed polymerase chain reaction (PCR) mutagenesis using primers: 5’-GGAACTGGATATATCAAGACTCAGTTGATTTCTGTGTC-3’ and 5’-GACACAGAAATCAACTGAGTCTTGATATATCCAGTTCC-3’.
pSG5-SUMO-1-His was provided by Dr. A. Dejean . The GAS-luciferase construct contains the IFN-γ regulated GAS element . Flag-tagged SENP1 and SENP1 C603S were previously described .
Human HeLa cells and monkey Cos-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin and 50 mg/ml streptomycin. Human fibrosarcoma U3A cells (kindly provided by Dr. I. Kerr) were cultured in DMEM supplemented with 10% Cosmic calf serum (CCS) (HyClone, Logan, UT) and 100 U/ml penicillin and 50 mg/ml streptomycin. Stable U3A-STAT1 WT-HA and U3A-STAT1 K703R-HA clones were previously described [11, 13].
Reporter gene assays
Approximately 0.2 × 106 HeLa cells were plated on to 24-well plates and transfected with 0.25 μg CMV-β-galactosidase reporter plasmid as an internal transfection efficiency control and 0.25 μg GAS-luciferase construct together with empty pcDNA3.1 vector as a control or with increasing amount of SENP1-Flag or SENP1 C603S-Flag. After 36 hours the cells were serum starved over night with 0.5% FBS in DMEM, following stimulation with 100 ng/ml human IFN-γ (Immugenex, Los Angeles, CA) for additional 6 hours and lysed in Promega’s reporter lysis buffer according to the manufacturer’s instructions. Luciferase activity was measured using Luminoscan Ascent (ThermoElectron Corporation, Finland) and normalized against β-galactosidase activity of the lysates.
Immunoprecipitation, co-immunoprecipitation and Immunoblotting
Total amount of 3 × 106 Cos-7 cells were transfected with 2 μg of STAT1-WT, 1 μg of SUMO-1, together with 2 μg of SENP1 or 2 μg of SENP1 C603S mutant. The cells were lysed in Triton X lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10% glycerol, 1% Triton X-100) supplemented with protease inhibitors including 10 mM NEM (Sigma Aldrich, St. Louis, MO). The lysates were incubated with anti-STAT1 antibody (monoclonal anti-STAT1 N-terminus, Transduction Laboratories, BD Biosciences, Franklin Lakes, NJ) and the immunocomplex was washed and subjected to SDS-PAGE electrophoresis. STAT1 and sumoylated STAT1 protein levels were determined by using anti-STAT1 (Transduction Laboratories) and anti-SUMO-1 (Zymed) antibodies. SENP1 protein levels from the whole cell lysates were determined by immunoblotting with anti-Flag antibody (Sigma-Aldrich, St Louis, MO). For co-immunoprecipitation experiments 1.6 × 106 Cos-7 cells were transiently transfected with 3 μg of STAT1-HA and 3 μg of STAT1-Flag with or without 4 μg of SUMO-1-His using L-PEI transfection reagent as described . After 48 hours cells were lysed in buffer containing 20 mM HEPES pH 8.0, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 50 mM NaF and 1mM EDTA supplemented with 10 mM NEM and protease inhibitors. Equal amounts of whole cell lysates were incubated for 3 hours in rotator at 4°C in the presence of 20 μl of anti-Flag M2 agarose beads (Sigma Aldrich, St. Louis, MO). The beads were washed 3 times with the lysis buffer and anti-Flag immunoprecipitated proteins were released from the beads by incubating them in the presence of Flag-peptide (F3290, Sigma Aldrich, St. Louis, MO) for 30 min at 4°C. Proteins were separated by SDS-PAGE and STAT1 was detected by immunoblotting using anti-HA antibody (clone 16B12, Covance, Princeton, NY). STAT1 and SENP1 protein levels from luciferase assay samples were analysed by immunoblotting using anti-STAT1 and anti-Flag antibodies, respectively.
Total amount of 5 × 105 U3A cells were transfected with 6 μg of STAT1 WT-HA or STAT1 E705Q-HA or STAT1 Y701F-HA mutants together with 4 μg of SUMO-1-His using L-PEI transfection reagent. After 48 hour incubation at 37°C cells were either left unstimulated or stimulated with 100 ng/ml of human IFN-γ (Immugenex, Los Angeles, CA) for total of 1 hour and by osmotic shock (0.3 M sorbitol) for 15 minutes. The cells were lysed in lysis buffer (0.5% Triton X-100, 100 mM Tris–HCl pH 7.5, 0.2 mM EDTA, 300 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM NaF) supplemented with protease inhibitors. The lysates were diluted fourfold with dilution buffer lacking NaCl (0.1% Triton X-100, 100 mM Tris–HCl pH 7.5, 0.2 mM EDTA, 1 mM DTT, 1.0% glycerol, 1 mM NaF supplemented with protease inhibitors).
For the binding assay, a biotinylated oligonucleotide containing the GAS from the human Gbp-1 gene (5’-GGATCCTACTTTCAGTTTCATATTACTCTAAAT-3’) or STAT1 binding site from human Irf- 1 gene (5’-GGATCCCAACAGCTTGATTTCCCCGAAATGACGGCA-3’) promoter was annealed and 3 nmols of biotinylated oligonucleotide duplex were rotated for 2 hours at 4°C with Neutravidin agarose to form GAS-agarose affinity beads. Diluted cell extracts were precleared with Neutravidin beads and then incubated with GAS-agarose affinity beads for 2 hours in rotator at 4°C. The beads were then washed four times with buffer containing 0,2% Triton X-100, 10 mM HEPES pH 7.9, 2 mM EDTA, 1 mM EGTA, 150 mM KCl, 10% glycerol and 1 mM NaF. GAS-agarose affinity bead-bound proteins were subjected to SDS-PAGE and detected by immunoblotting with phospho-tyrosine (Tyr701)-specific STAT1 antibody (Cell Signaling). The Western blot membranes were stripped and reprobed with anti-HA antibody (Covance, Princeton, NY) to detect total amount of DNA-bound STAT1. Detected bands were quantified by using ImageJ image analysis software and analyzed after background subtraction.
Stable U3A-STAT1 WT-HA and U3A-STAT1 K703R-HA clones were starved and left unstimulated or stimulated with IFN-γ for 1h. Chromatin immunoprecipitation (ChIP) was performed as previously described  using anti-acetyl-histone H4 antibody (Upstate Biotech) or as a control, anti-IgG antibody (Santa Cruz Biotechnology). DNA was analyzed for human Gbp-1 promoter by quantitative-RT-PCR with the following primers: 5’-AGCTTCTGGTTGAGAAATCTTT-3’ and 5’-CCCTGGACTAATATTTCACTG-3’. Quantitative-PCR was done using SYBR green I kit (Qiagen) according to manufacturer’s instructions. The values from ChIP assays were normalized to the total input DNA.
A 3D-structure of STAT1 dimer with DNA has been built using crystal structure of tyrosine phosphorylated STAT1-DNA complex (PDB code:1BF5) . The molecular geometry of the loop 684–699 in the SH2 domain was calculated using the program Sybyl with Amber 7 FF99 force field parameters. The initial model for the loop region was constructed using the crossover loop structure from the SUMO-1-TDG (PDB code:1wyw)  as a template. First, during the energy and geometry minimization for the loop all hydrogen atoms and non-constraints were included in the protocol. Second, during the molecular dynamic refinement the constraints were on for outer part of the loop in the SH2 domain. After the loop modeling we used the deposited coordinates of SUMO-1 (PDB code:1wyw)  in our model. The SUMO-1 was set nearby the constructed loop 684–699 so that its C-terminal residue (Gly97) is in the vicinity of the Lys703 of the STAT1 and the loop can form a new β-strand to an existing antiparallel β- sheet structure in the SUMO-1. The loop 684–699 was also modeled with InsightII (2005, Accelrys Inc. InsightII - molecular modeling program. http://www.accelrys.comomain; 1wyw.pdb). The entire structure was then subjected to energy minimization using the molecular mechanics force field CVFF (consistent valence force field) and the steepest descent algorithm implemented under Insight II Discover program. During the minimization, the DNA and the atoms of the STAT1 residues 136–686 and 700–710 were fixed.