Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1
- Juha Grönholm†1,
- Sari Vanhatupa†1,
- Daniela Ungureanu†1,
- Jouni Väliaho1,
- Tuomo Laitinen2,
- Jarkko Valjakka1 and
- Olli Silvennoinen1, 3Email author
© Grönholm et al.; licensee BioMed Central Ltd. 2012
Received: 10 September 2012
Accepted: 1 October 2012
Published: 8 October 2012
STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood.
Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1.
Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.
KeywordsSignal transduction Transcription factors Sumoylation Signal transducers and activators of transcription (STATs) Interferon
STAT1 (Signal transducer and activator of transcription 1) is the founder member of the STAT family of transcription factors and plays a critical role in interferon (IFN) regulated gene responses. IFN-γ activates STAT1 through Janus kinase (JAK)-mediated phosphorylation of Tyr701. Activated STAT1 homodimerizes and translocates to the nucleus where it binds to DNA and initiates transcription of IFN-γ-regulated genes [1, 2]. The X-ray structure (1bf5.pdb) of the DNA-bound STAT1 dimer shows a contiguous C-shaped clamp around DNA, that is mediated by specific interactions between the SH2 domain and the tyrosine (Tyr701) phosphorylated C-terminal tail segment (residues 700–708) of the monomers .
Small ubiquitin-like modifier (SUMO) proteins belong to the family of ubiquitin-like protein modifiers, collectively termed Ubls, that are covalently attached to substrate proteins by a cascade of enzymatic reactions . The conjugation is regulated by distinct SUMO specific enzymes such as E1 activating enzyme Aos1/Uba2 and the E2 conjugase Ubc9. The protein inhibitor of activated STAT (PIAS) family of proteins, PIAS1, PIAS3, PIASx and PIASy have been shown to function as E3-type ligases to promote SUMO conjugation to the target proteins [5–7]. PIAS1 functions as a negative regulator of STAT1-mediated transcription through interaction with the dimerized STAT1 and by inhibiting STAT1 DNA-binding [8, 9]. Interestingly, PIAS proteins have also been shown to promote sumoylation of STAT1 at single Lys703 amino acid residue within the SUMO consensus sequence (ψKxE, where ψ indicates large hydrophobic amino acid and x refers to any amino acid) 702IKTE705 in the C-terminal region of STAT1 [10, 11]. Furthermore, it has been shown that mitogen activated protein kinase (MAPK)-induced phosphorylation of Ser727 in STAT1 promotes interaction of STAT1 with PIAS1 and leads to enhanced STAT1 sumoylation .
Several studies suggest that sumoylation has a negative effect on STAT1-mediated gene responses [11, 13, 14]. Sumoylation site Lys703 is in a close proximity to Tyr701 that is required for STAT1 activation, and sumoylation has been shown to directly inhibit STAT1 Tyr701 phosphorylation [15–17]. Sumoylation has also been shown to prevent condensation of STAT1 oligomers in the nuclear paracrystals, and thereby increase the solubility of STAT1 and promote its dephosphorylation . Recently, it was discovered that in addition to human STAT1, also murine STAT5 and Drosophila Stat92E are regulated through SUMO conjugation, confirming that sumoylation has an evolutionary conserved role in regulation of the cytokine signaling [18, 19].
This study was aimed to investigate the mechanism by which sumoylation regulates STAT1 activity. Inspection of molecular model indicates that SUMO consensus site is well exposed in STAT1 dimer, and it is accessible for propitious interactions with regulatory proteins. The constructed molecular model of SUMO-1 conjugated STAT1 dimer further suggested that SUMO-1 moiety is oriented towards DNA, thus able to affect the DNA-binding properties of STAT1 with its presence. The molecular model was supported by experimental evidence, and oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant display higher DNA-binding activity on STAT1 target gene promoters when compared to STAT1 wild-type (WT). Furthermore, sumoylation deficient STAT1 mutant showed enhanced histone H4 acetylation at the Gbp-1 promoter.
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’.
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.
SENP1 deconjugates SUMO-1 from STAT1 and enhances STAT1-mediated gene expression
The identification of STAT1 as a substrate for SENP1 prompted us to investigate whether SENP1-mediated desumoylation affects the transcriptional activity of STAT1. For this purpose, we analysed the activity of STAT1-responsive GAS-luciferace reporter in HeLa cells transfected with different concentrations of SENP1 WT or SENP1 C603S mutant. As shown in Figure 1B, overexpression of SENP1 significantly increased the transcriptional activity of endogenous STAT1. In contrast, overexpression of catalytically inactive SENP1 C603S resulted in a dose-dependent decrease in GAS-luciferase activity, most probably by blocking the interaction of endogenous SENP1 with STAT1, leading to increased level of SUMO-modified STAT1 in the cells. This effect is also seen in Figure 1A, where STAT1 sumoylation is significantly increased when SENP1 C603S is co-transfected into the Cos-7 cells. Collectively, these results indicate that desumoylation of STAT1 enhances its transcriptional activity. These results are in line with previously reported results that sumoylation deficient STAT1 mutants display higher transcriptional activity at STAT1 target gene promoters [13, 14].
Molecular model of the SUMO conjugated STAT1 dimer
The crystal structure of thymine DNA glycosylase (TDG) conjugated to SUMO-1 has revealed that TDG forms two dissimilar molecular interfaces with SUMO-1. The covalent contact to SUMO-1 occurs at the Lys330 residue, but another interface is a β-sheet structure formed by β-strands of TDG and SUMO-1 . The structure of STAT1 dimer (PDB code: 1bf5) has a linker region (aa 684–699; loop structure) that is invisible in the electron density maps . The immediate vicinity of sumoylation site to residues in both ‘ends’ of the loop structure pointed us to investigate and remodel this loop. To get insight on this, we constructed a model of sumoylated STAT1 dimer using previously published coordinates of conjugated SUMO-1 . The loop amino acids 684–699 was reconstructed using two programs Sybyl with Amber7 FF99 force field (Figure 2C) and InsightII, and the analysis resulted in two highly similar loop models. SUMO-1 was positioned on conjugation distance, and the constructed loop structure was presented adjacent to β-sheet structure of SUMO-1. This model proposes that interface between SUMO-1 and the loop structure of STAT1 can direct the SUMO-1 moiety towards DNA, creating a steric hindrance that can affect DNA-binding of sumoylated STAT1 dimer.
Sumoylation deficient STAT1 shows increased DNA-binding activity
The molecular model suggested that sumoylation may alter the DNA-binding properties of STAT1. Mutation of Lys703 (K703R) or Glu705 (E705A) within the sumoylation consensus sequence in STAT1 abolish sumoylation of STAT1 and leads to enhanced STAT1 transcriptional activity [11, 13]. Thus, we wanted to investigate if the DNA-binding activity of sumoylation deficient STAT1 mutants differ from the DNA-binding properties of the WT STAT1. Amino acids essential to SUMO conjugation reside in the close proximity of the STAT1 activating Tyr701 phosphorylation site and therefore the mutations in the sumoylation site may affect to the tyrosine phosphorylation or dephosphorylation properties of STAT1. E705Q mutation in STAT1 is predicted to have minimal structural consequences to STAT1 but it abolishes STAT1 sumoylation (Additional file 2: Figure S2) . To analyse the phosphorylation of different sumoylation deficient STAT1 mutants, U3A cells lacking endogenous STAT1 were transfected either with STAT1 WT or with sumoylation deficient K703R, E705A or E705Q mutants. Phosphorylation deficient STAT1 Y701F mutant was used as a negative control. After IFN-γ stimulation cells were lysed and equal amounts of protein were separated in SDS-PAGE and Tyr701 phosphorylation was analyzed by using phospho-Tyr701-STAT1 specific antibody (anti-pSTAT1, Rabbit polyclonal antibody, Cell Signaling). The pSTAT1 antibody detected the Tyr701 phosphorylation of STAT1 E705Q, while the STAT1 E705A showed only a weak signal and the antibody failed to detect IFN-γ stimulated STAT1 K703R (Additional file 3: Figure S3A). The difference is likely to be caused by the altered amino acid sequence within or in the proximity of the epitope for the pSTAT1 antibody since another pSTAT1 antibody (New England Biolabs) readily detected also K703R and E705A mutants after pervanadate stimulation (data not shown). The SUMO deficient STAT1 mutant E705Q was chosen for further DNA-binding studies.
Promoter bound STAT1 dimer is known to interact with histone acetyl transferase CREB-binding protein (CBP) and acetylation of histones is essential for STAT1-mediated transcriptional activation . Next, we wanted to investigate whether the enhanced DNA-binding of sumoylation deficient STAT1 has functional consequences at the promoter level. To this end, we performed chromatin immunoprecipitation (ChIP) assays on the STAT1 target gene Gbp-1. U3A cells stably overexpressing STAT1 WT or sumoylation deficient STAT1 mutant were either left unstimulated or stimulated with human IFN-γ. Immunoprecipitation of cross-linked and scattered chromatin was performed with anti-acetylated histone H4 antibody or anti-rabbit IgG antibody as a control. STAT1 K703R expressing cells showed increased acetylation of histone H4 when compared to STAT1 WT (Figure 3C). This result suggests that the enhanced promoter binding of sumoylation defective STAT1 results in enhanced association of histone acetyl transferases to the promoter leading to increased histone H4 acetylation.
Sumoylation does not prevent STAT1 dimerization
Sumoylation is a common post-translational modification of transcription factors, but in several proteins the physiological functions and molecular mechanisms of this modification have remained enigmatic. Several lines of evidence support the concept that SUMO serves as a negative regulator of STAT1 [10, 11, 13, 14]. Furthermore, the results demonstrating that sumoylation also negatively regulates STAT5-mediated signaling and the only STAT transcription factor in Drosophila melanogaster, Stat92E, indicates that sumoylation is an evolutionary conserved post-translational modification for some STAT transcription factors [18, 19].
Sumoylation is a highly reversible covalent modification that is regulated through conjugating and deconjugating enzymes. Several studies support the importance of PIAS1-mediated sumoylation of the proteins. Recently, it was shown that PIAS1 regulates oncogenic signaling by sumoylating promyelocytic tumor suppressor (PML) that leads to its ubiquitination and proteosomal degradation . PIAS1 has also been shown to associate with protein-tyrosine phosphatase 1B (PTP1B) and to catalyze its sumoylation, which resulted in down-regulation of the phosphatase activity and inhibited dephosphorylation of the insulin receptor. PIAS1-mediated negative regulation of PTP1B was reversed by SENP1, an isopeptidase that was also shown to regulate sumoylation of STAT5 [18, 28]. Senp1 knock out mice were found to have severe defects in early T and B cell development. The defect in lymphoid development was likely caused by enhanced level of STAT5 sumoylation that subsequently led to decreased STAT5 transcriptional activity . In our experiments removal of conjugated SUMO-1 by SENP1 increased STAT1-mediated reporter gene expression, thus confirming the negative regulatory role of sumoylation for STAT1 (Figure 1B).
STAT1 homodimerization is required for the optimal IFN-γ-mediated gene activation and STAT1 homodimers form a nutcracker-like structure that binds to DNA. The monomers are held together by interface between Tyr701 phosphorylated C-terminal tail segment of one monomer and the SH2 domain of the other . The Lys703 is located adjacent to the dimerization interface and this prompted us to investigate if sumoylation of the Lys703 could affect dimerization or DNA-binding of STAT1. Analysis of the SUMO conjugation consensus site in STAT1 dimer revealed that side chain of Lys703 formed a projection towards DNA. Both Lys703 and Glu705 residues have hydrophilic side chains, which are converted away from the hydrophobic core of SH2 interface. Additionally, the β-sheet structure between two C-tail segments of STAT1 dimer is not likely to be affected by Lys703 mutation to Arg, while this mutation will interrupt the formation of covalent bond with SUMO. The structural analysis revealed that side chain of Lys703 has an interaction phase with Glu632 residue in the SH2 domain of the adjacent monomer. Most probably this interface prevents rotation of this flexible side chain and keeps orientation favorable for the SUMO conjugation. The finding of controlled position of Lys703 also supports the importance of Lys703 as an SUMO acceptor site (Additional file 2: Figure S2). Sumoylation has been shown to impede Tyr701 phosphorylation of STAT1 and subsequent SH2 domain-phospho-Tyr701-mediated homodimerization, leading to formation of semi-phosphorylated dimers that interact through their N-terminal domains [15–17]. Our experimental data indicated that sumoylated STAT1 can form dimers, but it remains to be determined if the interaction is mediated through their N-terminal domains or through the SH2 domains (Figure 4).
To predict how SUMO-1 would structurally orientate in SH2 domain-phospho-Tyr701 interaction-mediated STAT1 dimers, we reconstructed the structure of the disordered loop 684–699 of STAT1, and made a molecular model of sumoylated STAT1 dimer using x-ray structure of TDG-SUMO-1 as a template . This model suggests that the position of SUMO under the loop structure is directed towards DNA and can inhibit interaction with nucleic acids (Figure 2C). Furthermore, results from the oligonucleotide pull down experiments indicated that sumoylation interferes STAT1 binding to STAT1-responsive promoters, as sumoylation deficient STAT1 E705Q showed increased DNA-binding to both Gbp-1- and Irf-1-oligos (Figure 3A and B), and as SUMO-1 overexpression hindered STAT1 binding to Irf-1-oligo (Additional file 3: Figure 3F). The difference in the DNA-binding properties between STAT1 WT and E705Q mutant was not caused by altered Tyr701 phosphorylation (Figure 3A and B) [15–17]. In addition, another sumoylation deficient STAT1 mutant K703R also showed increased binding to Gbp-1 oligo (data not shown). Taken together, the oligoprecipation experiments are supporting the molecular model where SUMO moiety interferes with DNA-binding of STAT1 (Figure 3A and B, Additional file 3: Figure S3F).
Sumoylated STAT1 was not detected in our oligoprecipitation experiments (Additional file 3: Figure S3G) and this result is consistent with results by Song et al. (2006), showing that sumoylated STAT1 does not bind to DNA, or that the bound fraction is very small . In their EMSA studies Song et al. also found that sumoylation deficient STAT1 K703R mutant shows prolonged binding to GAS-probe, but unexpectedly sumoylation deficient E705A mutant had similar DNA-binding profile than STAT1 WT . We chose to use STAT1 E705Q mutant in the DNA-binding experiments because the mutant has been reported to have minimal SUMO-independent effects on STAT1 when compared to K703R and E705A mutations . Our results with STAT1 E705Q suggest that sumoylation inhibits DNA-binding properties of STAT1. Supporting this and previously published results of Song et al. (2006), we observed that STAT1 K703R has enhanced binding to Gbp-1-oligo when compared to STAT1 WT as well (data not shown). Furthermore, sumoylation deficient STAT1 showed enhanced histone H4 acetylation on Gbp-1 promoter (Figure 3C), thus functionally confirming the enhanced STAT1 promoter binding. Whether sumoylation also alters the interaction with histone acetyl transferases, such as CBP, remains to be determined.
It has become evident that sumoylation participates in regulation of STATs and the precise molecular mechanisms and physiological functions are gradually being revealed. Several studies have analysed sumoylation in STAT1, and sumoylation has been shown to inhibit STAT1 activity by different mechanisms. SUMO conjugation to Lys703 inhibits phosphorylation of Tyr701 [15, 16] and prevents paracrystal formation, thereby increasing solubility of STAT1 which subjects STAT1 for dephosphorylation [14, 17]. Our results suggest an additional regulatory mechanism for sumoylation and indicate that SUMO moiety is directed towards DNA and can inhibit DNA-binding of STAT1.
SUMO conjugation to STAT1 has been shown to negatively regulate STAT1-mediated gene responses [11, 13, 14]. This study was aimed to investigate further the mechanism by which sumoylation regulates STAT1. The inhibitory role of SUMO-1 on STAT1 was confirmed by showing that overexpression of desumoylating enzyme SENP1 increases STAT1-mediated transcriptional activity. A molecular model of sumoylated STAT1 dimer suggested that SUMO-1 is directed towards DNA creating steric hindrance that is able to affect DNA-binding properties of STAT1. Oligoprecipitation experiments were consistent with this model and showed that sumoylation deficient STAT1 mutant has enhanced binding to two independent STAT1 target gene promoters. The difference in DNA-binding was not attributed to the level of Tyr701 phosphorylation of STAT1. Consequently, sumoylation defective STAT1 mutant displayed increased histone H4 acetylation of Gbp-1 promoter. Taken together, these findings suggest that sumoylation functions as a negative regulator of STAT1 responses by modulating the DNA-binding properties of STAT1.
Dulbecco’s modified eagle’s medium
Ethylene diamine tetraacetic acid
Ethylene glycol tetraacetic acid
Fetal bovine serum
Guanylate binding protein
Interferon regulatory factor
Protein inhibitor of activated STAT
Sodium dodecylsulfate polyacrylamide gel electrophoresis
Signal transducer and activator of transcription 1
Small ubiquitin-like modifier
We thank M. Lehtinen for her huge contribution to this work and P. Kosonen for excellent technical assistance. Dr. Juha Saarikettu is acknowledged for the critical comments on the manuscript. This work was supported by grants from the Medical Research Council of the Academy of Finland, the Emil Aaltonen Foundation, Centre for Laboratory Medicine and Medical Research Foundation of Tampere University Hospital, The Finnish Cultural Foundation, the Finnish Foundation for Cancer Research, the Tampere Tuberculosis Foundation and the Sigrid Jusélius Foundation.
- Shuai K, Schindler C, Prezioso VR, Darnell JE: Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science. 1992, 258: 1808-1812. 10.1126/science.1281555.PubMedView ArticleGoogle Scholar
- Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD: How cells respond to interferons. Annu Rev Biochem. 1998, 67: 227-264. 10.1146/annurev.biochem.67.1.227.PubMedView ArticleGoogle Scholar
- Chen X, Vinkemeier U, Zhao Y, Jeruzalmi D, Darnell JE, Kuriyan J: Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA. Cell. 1998, 93: 827-839. 10.1016/S0092-8674(00)81443-9.PubMedView ArticleGoogle Scholar
- Melchior F: SUMO–nonclassical ubiquitin. Annu Rev Cell Dev Biol. 2000, 16: 591-626. 10.1146/annurev.cellbio.16.1.591.PubMedView ArticleGoogle Scholar
- Kotaja N, Karvonen U, Jänne OA, Palvimo JJ: PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol Cell Biol. 2002, 22: 5222-5234. 10.1128/MCB.22.14.5222-5234.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Jackson PK: A new RING for SUMO: wrestling transcriptional responses into nuclear bodies with PIAS family E3 SUMO ligases. Genes Dev. 2001, 15: 3053-3058. 10.1101/gad.955501.PubMedView ArticleGoogle Scholar
- Hochstrasser M: SP-RING for SUMO: new functions bloom for a ubiquitin-like protein. Cell. 2001, 107: 5-8. 10.1016/S0092-8674(01)00519-0.PubMedView ArticleGoogle Scholar
- Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, Shuai K: Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci USA. 1998, 95: 10626-10631. 10.1073/pnas.95.18.10626.PubMedPubMed CentralView ArticleGoogle Scholar
- Liao J, Fu Y, Shuai K: Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1-Stat1 interaction. Proc Natl Acad Sci USA. 2000, 97: 5267-5272. 10.1073/pnas.97.10.5267.PubMedPubMed CentralView ArticleGoogle Scholar
- Rogers RS, Horvath CM, Matunis MJ: SUMO modification of STAT1 and its role in PIAS-mediated inhibition of gene activation. J Biol Chem. 2003, 278: 30091-30097. 10.1074/jbc.M301344200.PubMedView ArticleGoogle Scholar
- Ungureanu D, Vanhatupa S, Kotaja N, Yang J, Aittomaki S, Jänne OA, Palvimo JJ, Silvennoinen O: PIAS proteins promote SUMO-1 conjugation to STAT1. Blood. 2003, 102: 3311-3313. 10.1182/blood-2002-12-3816.PubMedView ArticleGoogle Scholar
- Vanhatupa S, Ungureanu D, Paakkunainen M, Silvennoinen O: MAPK-induced Ser727 phosphorylation promotes SUMOylation of STAT1. Biochem J. 2008, 409: 179-185. 10.1042/BJ20070620.PubMedView ArticleGoogle Scholar
- Ungureanu D, Vanhatupa S, Grönholm J, Palvimo JJ, Silvennoinen O: SUMO-1 conjugation selectively modulates STAT1-mediated gene responses. Blood. 2005, 106: 224-226. 10.1182/blood-2004-11-4514.PubMedView ArticleGoogle Scholar
- Begitt A, Droescher M, Knobeloch KP, Vinkemeier U: SUMO conjugation of STAT1 protects cells from hyperresponsiveness to IFNγ. Blood. 2011, 118: 1002-1007. 10.1182/blood-2011-04-347930.PubMedView ArticleGoogle Scholar
- Jakobs A, Koehnke J, Himstedt F, Funk M, Korn B, Gaestel M, Niedenthal R: Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation. Nat Methods. 2007, 4: 245-250. 10.1038/nmeth1006.PubMedView ArticleGoogle Scholar
- Zimnik S, Gaestel M, Niedenthal R: Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation. Nucleic Acids Res. 2009, 37: e30-PubMedPubMed CentralView ArticleGoogle Scholar
- Droescher M, Begitt A, Marg A, Zacharias M, Vinkemeier U: Cytokine-induced paracrystals prolong the activity of signal transducers and activators of transcription (STAT) and provide a model for the regulation of protein solubility by small ubiquitin-like modifier (SUMO). J Biol Chem. 2011, 286: 18731-18746. 10.1074/jbc.M111.235978.PubMedPubMed CentralView ArticleGoogle Scholar
- Van Nguyen T, Angkasekwinai P, Dou H, Lin FM, Lu LS, Cheng J, Chin YE, Dong C, Yeh ET: SUMO-specific protease 1 is critical for early lymphoid development through regulation of STAT5 activation. Mol Cell. 2012, 45: 210-221. 10.1016/j.molcel.2011.12.026.PubMedPubMed CentralView ArticleGoogle Scholar
- Grönholm J, Ungureanu D, Vanhatupa S, Rämet M, Silvennoinen O: Sumoylation of drosophila transcription factor STAT92E. J Innate Immun. 2010, 2: 618-624. 10.1159/000318676.PubMedView ArticleGoogle Scholar
- Muller S, Berger M, Lehembre F, Seeler JS, Haupt Y, Dejean A: c-Jun and p53 activity is modulated by SUMO-1 modification. J Biol Chem. 2000, 275: 13321-13329. 10.1074/jbc.275.18.13321.PubMedView ArticleGoogle Scholar
- Pine R, Canova A, Schindler C: Tyrosine phosphorylated p91 binds to a single element in the ISGF2/IRF-1 promoter to mediate induction by IFN alpha and IFN gamma, and is likely to autoregulate the p91 gene. EMBO J. 1994, 13: 158-167.PubMedPubMed CentralGoogle Scholar
- Bailey D, O’Hare P: Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1. J Biol Chem. 2004, 279: 692-703.PubMedView ArticleGoogle Scholar
- Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP: A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA. 1995, 92: 7297-7301. 10.1073/pnas.92.16.7297.PubMedPubMed CentralView ArticleGoogle Scholar
- Välineva T, Yang J, Palovuori R, Silvennoinen O: The transcriptional co-activator protein p100 recruits histone acetyltransferase activity to STAT6 and mediates interaction between the CREB-binding protein and STAT6. J Biol Chem. 2005, 280: 14989-14996. 10.1074/jbc.M410465200.PubMedView ArticleGoogle Scholar
- Baba D, Maita N, Jee JG, Uchimura Y, Saitoh H, Sugasawa K, Hanaoka F, Tochio H, Hiroaki H, Shirakawa M: Crystal structure of thymine DNA glycosylase conjugated to SUMO-1. Nature. 2005, 435: 979-982. 10.1038/nature03634.PubMedView ArticleGoogle Scholar
- Zhang JJ, Vinkemeier U, Gu W, Chakravarti D, Horvath CM, Darnell JE: Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling. Proc Natl Acad Sci USA. 1996, 93: 15092-15096. 10.1073/pnas.93.26.15092.PubMedPubMed CentralView ArticleGoogle Scholar
- Rabellino A, Carter B, Konstantinidou G, Wu SY, Rimessi A, Byers LA, Heymach JV, Girard L, Chiang CM, Teruya-Feldstein J, Scaglioni PP: The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA. Cancer Res. 2012, 72: 2275-2284. 10.1158/0008-5472.CAN-11-3159.PubMedPubMed CentralView ArticleGoogle Scholar
- Dadke S, Cotteret S, Yip SC, Jaffer ZM, Haj F, Ivanov A, Rauscher F, Shuai K, Ng T, Neel BG, Chernoff J: Regulation of protein tyrosine phosphatase 1B by sumoylation. Nat Cell Biol. 2007, 9: 80-85. 10.1038/ncb1522.PubMedView ArticleGoogle Scholar
- Song L, Bhattacharya S, Yunus AA, Lima CD, Schindler C: Stat1 and SUMO modification. Blood. 2006, 108: 3237-3244. 10.1182/blood-2006-04-020271.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.