Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA
© Koch et al.; licensee BioMed Central Ltd. 2012
Received: 26 April 2012
Accepted: 21 August 2012
Published: 24 August 2012
In interferon-γ-stimulated cells, the dimeric transcription factor STAT1 (s ignal t ransducer and a ctivator of t ranscription 1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (g amma-a ctivated s ites). However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood.
Using a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-γ. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites.
Thus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.
A variety of different transcription factors are involved in the execution of genetic programs, and sequence-specific DNA binding is a hallmark of transcriptional regulation. Central to their role as gene-specific transcription factors is their ability to recognize distinct elements in the promoter regions of responsive genes. Typically, transcription factors bind with high affinity and specificity to short motifs of DNA via protein surfaces that are complementary to a particular base sequence. Although there is often some flexibility in the nucleotide sequence that is recognized, certain key bases are crucial for interactions with the DNA-binding domain. The STAT (s ignal t ransducer and a ctivator of t ranscription) proteins constitute a paradigmatic family of evolutionary conserved transcription factors with a modular domain arrangement[1, 2]. In mammals, seven different members of the STAT family are expressed, all of which consist of three proteolytically separable structural subunits. An amino-terminal domain is separated by a protease-sensitive linker peptide from a large core structure containing the central DNA-binding domain and a carboxy-terminal transactivation domain[3–6]. The amino-terminal domain of about 130 residues is folded into a unique hook-shaped architecture that facilitates cooperative DNA binding through the formation of tetramers[7–10]. The large core domain encompasses several distinct functional domains beginning at the amino-terminal end with a coiled-coil domain, which is engaged in protein-protein interactions. The DNA-binding domain displays an immunoglobulin fold and is required for the binding of tyrosine-phosphorylated STAT dimers to semi-palindromic DNA sequences termed g amma-a ctivated s ites (GAS). The neighboring linker region consists of a unique all-alpha helical structure and assists in binding to GAS elements. The Src homology 2 (SH2) domain mediates recruitment to phospho-tyrosyl residues in the intracellular receptor tails and the formation of tyrosine-phosphorylated STAT dimers[14, 15]. The carboxy terminus containing the transactivation domain is most variable in size and sequence among the different STAT family members and is frequently subjected to alternative splicing.
The STATs are best known for their function as cytokine-activated transcription factors, which play a vital role in such diverse processes as growth control, proliferation, and immune activation[17, 18]. The triggering of cytokine receptors initiates a cascade of phosphorylation steps that causes auto-phosphorylation of non-covalently attached JAK kinases. The activated JAK kinases then phosphorylate signature tyrosine residues in the intracellular receptor tails, thereby, creating phospho-tyrosine docking sites for the STAT SH2 domain. Phosphorylation of a single tyrosine residue in the STAT carboxy terminus results in a structural change within the STAT dimer that shifts from an “antiparallel” to a DNA-bound “parallel” conformation[20–22]. Tyrosine-phosphorylated STAT enters the nucleus via importin-α/β-mediated transport[23–27] and binds to semi-palindromic GAS elements in the promoter region of cytokine-responsive genes that contain the consensus sequence 5′-TTC(N)3-4GAA-′3[3, 28, 29]. STAT proteins are then dephosphorylated by nuclear tyrosine phosphatases, some of which have been identified, such as the Tc45 phosphatase for inactivation of STAT1[30–32]. Additionally, unphosphorylated STAT1 molecules translocate constitutively between the cytoplasm and the nucleus in both directions through direct contacts with nucleoporins located in the nuclear pore complex[33–36].
In contrast to this high-affinity GAS binding, much less is known about the molecular processes that ensure the release of STAT1 dimers from DNA. In the following, we report on a novel and simple mechanism that allows STAT1 homodimers to disengage from DNA. Additionally, we show that a high dissociation rate from non-specific DNA and a preserved sequence-specific discrimination between GAS and non-GAS sites are both required for optimal transcriptional activation. Moreover, we directly confirm that DNA-bound STAT1 molecules are protected from dephosphorylation in vivo, pointing to the essential role of non-specific DNA binding in the search for cytokine-regulated promoter elements.
Mutation of two glutamyl residues in the DNA-binding domain results in increased tyrosine phosphorylation of STAT1
We then performed kinetic studies on tyrosine dephos-phorylation in IFNγ−prestimulated U3A cells which were subsequently exposed to 500 nM of the potent ATP-competitive kinase blocker staurosporine. Treatment with the kinase inhibitor resulted in a rapid and complete dephosphorylation of wild-type STAT1 within 15 min, while the E411A mutant exhibited a much lower dephosphorylation rate (Figure1C). Moreover, the ratio of tyrosine-phosphorylated STAT1 to the total intracellular STAT1 pool, which also contained unphosphorylated protein, was elevated in the mutant as compared to its wild-type counterpart (Figure1B,C). Similarly, mutation of another glutamic acid residue in position 421, which also points with its side chain in the direction of the DNA double helix (Figure1A), resulted in defective dephosphorylation and increased DNA-binding activity (Figure1E).
When we tested for cooperative DNA binding resulting from the ability to form stable tetramers on tandem GAS sites by means of EMSA analysis, we found no significant difference between the wild-type and mutant STAT1 (Figure1F). Both variants bound independently to either GAS site, resulting in both fast migrating STAT1/DNA complexes containing a single STAT1 dimer and slow migrating complexes with two dimers. When such complexes were challenged with a 750-fold molar excess of unlabeled M67 duplex oligonucleotides, the tetrameric complexes resisted displacement due to stable tetramerization. In contrast, the dimeric complexes of both wild-type and mutant STAT1 were either totally or partially displaced, indicative of cooperative DNA binding. Thus, substitution of either of the two conserved glutamyl residues in position 411 or 421 of the full-length STAT1 molecule critically impaired the continuous dephosphorylation/rephosphorylation cycle and resulted in elevated and prolonged tyrosine phosphorylation levels. However, binding to an optimal GAS site as well as cooperative DNA binding due to tetramer stabilization was unaltered.
Tyrosine-phosphorylated STAT1-E411A protects co-expressed endogenous STAT1 from inactivation
Interferon-prestimulated HeLa cells expressing endogenous STAT1, in addition to either the GFP fusion of wild-type STAT1 or its GFP-tagged mutant, were subjected to the inhibitory effect of staurosporine. In cells expressing STAT1-E411A-GFP, not only did the mutant phospho-protein resist staurosporine treatment much better, endogenous STAT1 was also partially insensitive, as revealed by its prolonged tyrosine phosphorylation (Figure2A) and enhanced DNA-binding activity (Figure2B). Thus, the presence of the E411A substitution protects also co-expressed native STAT1 protein from its rapid inactivation. This finding suggested that the mutant STAT1 protein interacts with endogenous STAT1 in a way that impairs access to the inactivating nuclear phosphatase.
Diminished nuclear export of tyrosine-phosphorylated STAT1-E411A
To confirm the altered nucleocytoplasmic shuttling properties of the mutants by a different approach, we performed a permeabilized cell transport assay (Figure3B). HeLa cells expressing GFP-tagged wild-type STAT1 or the respective glutamyl mutants were stimulated for 45 min with IFNγ to induce nuclear accumulation of the recombinant fusion proteins. Subsequently, the cells were either immediately fixed or incubated for 6 min with 50 μg/ml digitonin on ice before fixation. Treatment with digitonin at this concentration selectively permeabilized the plasma membrane, thereby, releasing cytoplasmic proteins, while the integrity of the nuclear envelope remained intact. As expected, stimulation with IFNγ resulted in the nuclear accumulation of all GFP-tagged STAT1 variants (Figure3B, top panel). However, permeabilization by digitonin completely abrogated the pre-existing nuclear presence of STAT1-WT-GFP, while the two mutants remained accumulated in the nucleus (Figure3B, bottom panel). Thus, the nuclear export rate of the mutants was critically reduced as compared to the wild-type protein.
By adding a transferable nuclear export signal (NES) to GFP-tagged STAT1, we have gathered further evidence for an altered DNA binding of the mutants. In resting cells, STAT1-NES-GFP showed a cytoplasmic redistribution as compared to the nearly pancellular localization of STAT1-GFP, which resulted from enhanced nuclear export (Figure3C). Also in contrast to STAT1-WT, the NES adduct failed to accumulate in the nuclei of interferon-stimulated cells, because the enhanced nuclear export rate competed with nuclear retention on DNA. Interestingly, however, nuclear accumulation was fully restored in the additional presence of the E411A mutation. This observation clearly confirms that high-affinity DNA-binding is the underlying phenotype of the E411A mutant.
The mutant E411A exhibits high-affinity GAS binding and has a broad repertoire of non-optimal binding sites
In order to compare the sequence requirement for DNA binding between the E411A mutant and wild-type STAT1, we used non-optimal GAS elements as molecular probes in mobility shift assays (Figure4C). Both the wild-type and the mutant bound with high affinity to oligonucleotides containing a single GAS site. However, STAT1-E411A also reacted with a mutated probe which, due to the exchange of two base pairs, contained no consensus GAS element. Although binding to this 2x nonGAS probe was weaker than to either GAS-nonGAS or tandem GAS oligos, there was a detectable formation of DNA-bound STAT1 dimers not requiring an intact GAS site for DNA binding. Thus, in the presence of excess unlabeled GAS oligos, the E411A mutant bound to DNA not only with a higher affinity than the wild-type molecule, but also showed a relaxed sequence requirement for interaction with DNA.
In vitro dephosphorylation assays, using whole cell extracts from reconstituted U3A cells in the presence of the STAT1-inactivating Tc45 phosphatase, confirmed that the two glutamyl mutants are indeed DNA-binding mutants (Figure4D). It has been shown that DNA-bound STAT1 is protected from dephosphorylation and barred from nuclear exit, and we report here that the glutamyl mutants but not the wild-type protein resisted Tc45-catalyzed inactivation. These experiments collectively demonstrate that there must be a considerable amount of mutant phospho-STAT1 interacting with genomic DNA that does not participate in nucleocytoplasmic shuttling and resists inactivation by nuclear phosphatases.
A low dissociation rate from DNA results in prolonged cytokine-induced nuclear accumulation
To exclude the possibility that the differential nuclear accumulation kinetics seen for the glutamyl mutants is an artefact resulting from the presence of the GFP domain, we confirmed this finding by means of immunocytochemical staining in U3A cells expressing recombinant, untagged STAT1 (Figure5B). Similarly to the GFP adducts expressed in HeLa cells, the respective glutamyl mutants showed an unaltered resting distribution and accumulated normally in the nuclei of IFNγ−stimulated U3A cells. However, after 60 min of staurosporine addition to the cells, the mutant STAT1 molecules were still predominantly localized in the nucleus, whereas the resting distribution of the wild-type protein had already been restored at that time point. Following 90 min of staurosporine exposure, the nuclear accumulation of both mutants had also collapsed, demonstrating that the DNA-binding mutants were less sensitive to kinase inhibition. This finding in U3A cells confirmed that the reduced dephosphorylation rate and prolonged nuclear accumulation are inherent properties of the glutamyl mutants, which result directly from their slow off-rate from DNA.
High-affinity DNA binding crucially impairs transcriptional responses
We then used real-time RT-PCR assays to probe the induction of three endogenous IFNγ−responsive genes in transfected U3A cells. Again, the mutants failed to reach the transcriptional activity of the wild-type protein (Figure6B). While constitutively expressed recombinant stat1 mRNA was detected in all samples as expected (p > 0.05), there was a significant reduction in irf1 mRNA synthesis in U3A cells expressing the E411K and E421K mutant as compared to wild-type STAT1 (p = 0.029 and p = 0.003, respectively). Induction of the gbp1 and mig1 gene was also critically impaired or even completely abolished by replacing either of the glutamyl residues (for all comparisons p < 0.03).
Previously, it was shown by in vitro studies using purified STAT1 that tyrosine-phosphorylated STAT1 dimers bound to DNA are protected from the inhibitory activity of nuclear phosphatases and barred from nuclear exit. Although the physiological significance of this finding remains unclear, it has been suggested that a slow off-rate from genomic DNA critically compromises the STAT′s functions as potent transcription factors for a limited number of target genes. The corresponding DNA-binding mutants of STAT1 created so far display either a decreased affinity for DNA or a complete failure to discriminate between GAS and non-GAS elements. Thus, not surprisingly, both resulted in defective transcriptional activity[13, 41]. However, the behavior of a hypothetical DNA-binding mutant with preserved GAS recognition and enhanced DNA-binding affinity surpassing that of the wild-type protein has not been studied so far. It is surmised that such a DNA-binding mutant would be a useful tool to dissect STAT1 signal transduction in terms of nucleocytoplasmic shuttling, cytokine-induced nuclear accumulation, and activation of target genes. As presented here, we have created such a point mutant which has allowed us to systematically investigate the transcriptional consequences of enhanced DNA binding. Using this mutant, we have revealed a simple molecular mechanism that enables STAT dimers to dissociate from non-target DNA in order to continue their search for GAS sites. We have demonstrated that a high off-rate from genomic DNA is required as a key feature for target gene finding, which allows for the efficient transmission of extracellular signals into transcriptional responses.
In an attempt to characterize the biological effects of increased protein-DNA interactions at a transcriptional level, we performed a mutational study on STAT1 and assessed the resulting mutants for their capability to bind sequence-specifically to DNA and activate interferon-responsive target genes. Most of the STAT1 point mutants generated with substitutions in the DNA-binding domain showed a reduced affinity for DNA and were, therefore, inappropriate to test the functional consequences of high-affinity DNA binding for gene expression. However, we identified two single point mutants that fulfilled our expectations for an enhanced binding to GAS sites. Replacement of two glutamic acid residues in the DNA-binding domain, although not interfering with the recognition of GAS elements, independently stabilizes preformed STAT1-DNA complexes. The presence of negatively charged residues at position 411 and 421 is required for the release of STAT1 dimers from DNA, as their substitution with either alanine or a positively charged lysyl residue remarkably reduced the dissociation rate from both GAS and GAS-like elements. The striking finding that enhanced GAS binding is associated with a dramatically reduced gene expression in cytokine-stimulated cells clearly underlines the significance of intact nucleocytoplasmic shuttling for full transcriptional activation. Moreover, it suggests that a limited residence time in the nucleus is an inherent property of STAT1 signal transduction and, conversely, a reduced dissociation rate from GAS elements results in suppressed gene induction.
Available crystallographic data have revealed that the glutamyl residue 411 does not directly contact specific nucleotide bases or the sugar-phosphodiester backbone of DNA, but in the DNA-bound form it has nevertheless free access to the DNA molecule, suggesting that there may be some minor structural flexibility within the STAT1 DNA-binding domain (see Figure1A). It has been reported that residue 421 can accept hydrogen bonds from guanine in the minor groove, although the precise interface between the surface of the STAT1 DNA-binding domain and the DNA double helix in the proximity to E421 is not known due to the superimposition of non-equivalent base pairs at these positions. The functional relevance of the two glutamyl residues can best be regarded as an off-switch to release STAT1 dimers from DNA, so that they become a readily accessible substrate for the inactivating nuclear phosphatase. The presence of a glutamic acid residue with a terminal carboxyl group adjacent to phosphate groups in the DNA backbone facilitates the fast disassembly of STAT1-DNA complexes possibly via electrostatic repulsion. Interestingly, these residues are directly engaged in the discrimination between canonical and non-canonical binding sites, since its replacement by alanine results in a mutant with preserved GAS recognition and a broadened spectrum of potential binding sites (see Figure4C). This finding suggests that the repulsive effect on DNA binding exerted by these residues is independent of the underlying DNA sequences and occurs at classical GAS, GAS-like or even non-GAS sites. The native glutamyl residues seem to facilitate the release of STAT1 dimers from DNA via electrostatic interactions, thereby increasing the number of STAT1 molecules participating in productive nucleocytoplasmic shuttling.
In the wild-type molecule, the fast dissociation from DNA contributes to the coupling of DNA release and subsequent tyrosine dephosphorylation to transcriptional activation. Under conditions of cytokine stimulation the fast release from DNA ensures that the intracellular concentration of tyrosine-phosphorylated STAT1 is always limited due to the high tyrosine phosphatase activity in the nucleoplasma. In the DNA-binding mutants E411A/K and E421K, this coupling between the recruitment to genomic DNA and their fast dephosphorylation is critically disturbed, since these mutants are more than the wild-type protein stacked on genomic DNA in complexes, which may also contain co-expressed native STAT1 (see Figures1B and2A,B). Due to the reduced number of cycling STAT1 dimers (Figure3), their cytokine-induced transcriptional response is substantially limited (Figure6). The prolonged nuclear residence time of the glutamyl mutants following cytokine stimulation of cells (Figure5) appears to directly reflect their decreased tyrosine dephosphorylation (Figure4D), suggesting that they are retained in a DNA-bound state at transcriptionally inert genomic loci. Tyrosine-phosphorylated native STAT1 molecules form heterodimers with the co-expressed recombinant STAT1 mutants as detected by gelshift experiments (Figure2B), which are integrated into DNA-bound STAT complexes and protected from fast inactivation (Figure2A). Thus, paradoxically, despite their increased GAS binding and elevated concentration in the nuclear compartment, where transcription exclusively takes place, the mutants are nevertheless weaker transcriptional activators.
Interestingly, by introducing a neutral or a positively charged functional group at position 411, we generated a graduated series of STAT1 variants (glutamate > alanine > lysine) with stepwise diminished transcriptional activity at an artificial reporter gene construct. Thus, changing the electric charge of this residue permits interference with gene induction simply by shifting the amount of STAT1 dimers to a DNA-bound state in which they are prevented from freely shuttling between cytoplasm and nucleus. From our experiments, we cannot conclude whether the impaired transcriptional activity at native target genes detected for the mutants results from a diminished exchange rate at a single promoter or merely reflects decreased promoter occupancy due to predominant deposition at low-affinity DNA-binding sites. However, we observed that cytokine stimulation leads to high nuclear concentrations of mutant STAT1, which clearly exceed that of the wild-type protein (see Figure3A). This finding suggests that mutant STAT1 preferentially deposits outside transcriptionally active sites. In this scenario, a limited number of high-affinity GAS sites compete with the virtually unlimited amount of non-GAS sequences within the entire genome for binding to STAT1. In interferon-stimulated cells, phospho-STAT dimers retained in the nucleus may not be exclusively bound to GAS sites, but are additionally recruited to an overwhelming reservoir of unspecific, low-affinity DNA-binding sites, from which they are released with very high exchange rates. Interestingly, Lerner and colleagues had previously shown that STAT3 and glucocorticoid receptor assembled at the α2-macroglobulin promoter into an enhanceosome for which continued renewal of both transcription factors was required for full transcriptional activity.
In summary, we present evidence showing that the presence of two single glutamic acid residues in the DNA-binding domain adjacent to the DNA backbone sequence-independently weakens the binding to DNA and is required for full transcriptional activation of cytokine-driven target genes. The high dissociation rate from non-GAS sites ensures that tyrosine-phosphorylated STAT1 dimers can successfully scan genomic DNA for the presence of specific GAS sites, at which they assemble into transcriptional active complexes until they are finally dephosphorylated for nuclear exit. Furthermore, we demonstrate that not a high affinity for GAS sites, but rather the inherent difference in the off-rates between specific and non-specific binding sites crucially determines the function of STAT proteins as transcriptional regulators.
HeLa cells were cultured at 37οC in a humidified 5% CO2 atmosphere in Quantum 101 medium (PAA Laboratories) supplemented with 5% fetal calf serum (FCS; Biochrom), 1% penicillin, and 1% streptomycin. STAT1-negative U3A cells, originally derived by Müller and colleagues, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 1% penicillin, 1% streptomycin, and 0.04 μg/ml puromycin. Transfection was achieved with Lipofectamine plus (Invitrogen) according to the manufacturer's recommendation. Twenty-four hours after transfection, cells were either left unstimulated or stimulated with 5 ng/ml human IFNγ (Biomol). Subsequently, cells were incubated with 500 nM staurosporine (Sigma) for the time periods indicated.
The plasmid pEGFP-N1-STAT1, which coded for full-length human STAT1 (amino acids 1–746) fused carboxy-terminally to green fluorescent protein (GFP), has been described. For the detection of untagged protein, STAT1 cDNA was cloned in the expression vector pcDNA3.1 (Invitrogen). The plasmid pSTAT1-NES-GFP contained a transferable nuclear export signal (NES) activity (amino acids 367–427 of STAT1) situated between the cDNAs for full-length STAT1 and GFP, as described. Mutations in each of these expression vectors were introduced by site-directed point mutagenesis using the QuikChange kit from Stratagene, as recommended by the manufacturer. The native glutamic acid codons at position 411 and 421 were substituted for either alanine or lysine. All mutations were verified by standard didesoxy termination DNA sequencing (Seqlab).
For direct fluorescence microscopic localization of GFP-tagged STAT1, transiently transfected cells were treated as described and subsequently fixed in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature (RT). Subsequently, nuclei were stained for 10 min with 5 μg/ml Hoechst 33258 (Sigma) and the samples were mounted in fluorescence mounting medium (Southern Biotech). Fluorescence microscopy was performed using a Leica DM5000B microscope equipped with appropriate fluorescence filters. Images were obtained with a CCD camera and further processed with the Leica QWin software.
Immunocytochemical detection of untagged STAT1 was carried out in U3A cells expressing either wild-type or mutant STAT1. Adherent cells grown on chamber slides were either left untreated or treated with IFNγ for 45 min. Interferon-stimulated cells were additionally incubated in the presence of 500 nM staurosporine for an additional 0, 60 or 90 min and then fixed with methanol for 20 min at −20°C. After two washes in PBS, the cells were permeabilized with 1.0% Triton X-100 in PBS and non-specific binding was blocked by incubation with 25% FCS/PBS for 45 min at RT. The samples were then incubated for 45 min with anti-STAT1 antibody C-24 (Santa Cruz) diluted 1:1000 in 25% FCS/PBS. After three washes in PBS they were incubated with Cy3-conjugated secondary antibody (Dianova), diluted 1:500 in PBS, for 45 min at RT followed by nuclear staining with Hoechst dye. Finally, the samples were mounted and images were captured by fluorescence microscopy.
Adherent HeLa cells expressing GFP-fusion proteins of STAT1 were stimulated for 45 min with IFNγ to induce nuclear accumulation of STAT1. Then cells were either left untreated or permeabilized in the presence of 50 μg/ml digitonin (Roche) in transport buffer (0.2% Triton X-100, 10 mM KCl, 1.5 mM MgCl2, 10 mM Hepes, pH 7.4, 1 mM DTT, Complete protease inhibitors from Roche) for 6 min on ice. After two washes in transport buffer, cells were fixed for 15 min at RT with 3.7% paraformaldehyde in PBS followed by staining with Hoechst dye. The presence of STAT1-GFP in the nuclei was probed by means of direct fluorescence microscopy.
In vitro dephosphorylation assay
For in vitro dephoshorylation assays, 10 μl of cytosolic extracts from U3A cells expressing mutant STAT1 proteins were mixed with a similar volume of dephosphorylation buffer containing 25 mM Tris–HCl, pH 7.5, 50 mM KCl, 5 mM EDTA, and 0.3 mg/ml bovine serum albumin. Then DTT was added to a final concentration of 2 mM before the samples were incubated at 30°C with 2 U of the T-cell protein tyrosine phosphatase Tc45 (Biomol International, Plymouth, USA) for 0 min, 15 min and 30 min, respectively. Dephosphorylation reactions were stopped by adding SDS sample buffer and boiling the samples for 3 min. The amount of tyrosine-phosphorylated STAT1 in each sample was tested by means of Western blotting.
Cells grown on 6-well dishes were lysed in 30 μl cytoplasmic extraction buffer (0.2% Nonidet P40, 10 mM KCl, 1 mM EDTA, 10% glycerol, 20 mM Hepes, pH 7.4, 50 mM NaF, 1 mM vanadate, 1 mM DTT, 0.1 mM PMSF, Complete protease inhibitors) for 5 min on ice. The lysates were spun at 16000 g for 10 sec at 4°C. The supernatants were recentrifuged at 16000 g for 5 min and the pellets resuspended in 30 μl nuclear extraction buffer (420 mM KCl, 1 mM EDTA, 20% glycerol, 20 mM Hepes, pH 7.4, 50 mM NaF, 1 mM vanadate, 1 mM DTT, 0.1 mM PMSF, Complete protease inhibitors) for 30 min on ice and spun for 15 min at 16000 g. The isolated or combined cytoplasmic and nuclear extraction lysates were boiled in SDS sample buffer. Proteins were then resolved by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes. The membranes were incubated with a polyclonal antibody specific for phospho-STAT1-Tyr701 (Cell Signaling) and then with a horseradish peroxidase-conjugated secondary antibody (Dako). Bound immunoreactivity was detected using the enhanced chemiluminescence reaction (Pierce). Subsequently, the blots were stripped for 60 min at 60°C in 2% SDS, 0.7% β-mercaptoethanol, and 62.5 mM Tris–HCl, pH 6.8. Finally, the blots were reprobed with the polyclonal pan-STAT1 antibody C-24 followed by incubation with secondary antibodies. The efficiency of nuclear/cytoplasmic fractionation was assessed by simultaneously incubating blot membranes with rabbit lamin A (H-102, Santa Cruz) and mouse β-tubulin antibodies (clone TUB2.1, Sigma) followed by detection with secondary IRDye 680LT and 800CW antibodies visualized on a LI-COR Odyssey imaging machine.
Electrophoretic mobility shift assay (EMSA)
HeLa or U3A cells were transiently transfected with pSTAT1-GFP or pcDNA3.1-STAT1 coding for either wild-type or mutant STAT1. The cells were allowed to recover for 24 hours and then either left unstimulated or stimulated for 45 min with 5 ng/ml of IFNγ followed by staurosporine treatment. Cell extracts were prepared as described above. To prevent dephosphorylation and proteolysis, all cell extracts contained a protease inhibitor cocktail (Roche), 1 mM vanadate, and 10 mM NaF. Four microliters of each extract were incubated with 1 ng 32P]-labeled duplex oligonucleotide probes, generated by an end-filling reaction using Klenow fragment (New England Biolabs). The following duplex oligonucleotides were used (4 bp overhangs at the 5′ ends are not included; the respective antisense oligos are not listed; GAS sites are underlined): M67, 5′-CGACATTTCCCGTAA ATCTG-′3; 2x GAS, 5′-CGTTTCCCCGAA ATTGACGGATTTCCCCGAA AC-′3; GAS-nonGAS, 5′-CGTTTCCCCGAA ATTGACGGATTTACCCCAAC-′3; and 2x nonGAS, 5′-CGTTTACCCCAAATTGACGGATTTACCCCAAC-′3. For competition experiments, the EMSA reactions were equilibrated for 15 min at RT before incubation with a 750-fold molar excess of unlabeled M67 DNA for the indicated times. In supershift assays, 20 ng of the STAT1-specific antibody C-24 were preincubated with the shift reaction for 15 min at RT. The reactions were loaded on a 4% 29:1 acrylamide:bisacrylamide gel at 4°C, as described. STAT1 DNA-binding activity was visualized with a phosphoimaging system (BAS-1000, Fujifilm) using the computer programs BAS reader and TINA version 2.08.
Reporter gene assay
U3A cells grown on 48-well plates were transiently transfected with the following amounts of cDNAs added into a single well: 250 ng of the respective STAT1 expression plasmid, 70 ng of a β-galactosidase reporter plasmid (Stratagene), and 200 ng of an IFNγ−sensitive reporter construct. Luciferase reporters contained either a triple Ly6E STAT-binding site (termed 3xLy6E) or the 5′-region of the human intercellular adhesion molecule 1 (ICAM-1) gene 339 bp and 1352 bp relative to the transcription start site (termed pIC339 and pIC1352, respectively)[46, 47]. Twenty-four hours post-transfection, cells were either left unstimulated or treated for 6 hours with IFNγ. Whole cell extracts were prepared and measured for luciferase (Promega) and β-galactosidase activities. The data were normalized for the expression of β-galactosidase. For each STAT1 variant and stimulation mode, six independent samples were tested and the experiment was repeated at least three times. Differences in gene activation between IFNγ−stimulated cells expressing the indicated STAT1 variants were assessed using Student’s t tests and Mann-Whitney-Wilcoxon tests, where appropriate. Statistical significance was defined as p < 0.05.
The transcriptional activities of wild-type and mutant STAT1 were assessed by means of real-time PCR. Gene-specific primers for three endogenous transcripts (irf1, mig1, and gbp1) as well as for stat1 and gapdh were designed using Primer 3 software (Applied Biosystems) in order to amplify fragments of about 200 bp in length. The following primer pairs were used: IRF1F, 5′-AGCTCAGCTGTGCGAGTGTA-′3; IRF1R, 5′-TAGCTGCTGTGGTCATCAGG-′3; MIG1F, 5′-CCACCGAGATCCTTATCGAA-′3; MIG1R, 5′-CTAACCGACTTGGCTGCTTC-′3; GBP1F, 5′-GGTCCAGTTGCTGAAAGAGC-′3; GBP1R, 5′-TGACAGGAAGGCTCTGGTCT-′3; GAPDHF, 5′-GAAGGTGAAGGTCGGAGTC-′3; GAPDHR, 5′-GAAGATGGTGATGGGATTTC-′3; STAT1F, 5′-CCGTTTTCATGACCTCCTGT-′3; and STAT1R, 5′-TGAATATTCCCCGACTGAGC-′3. Twenty-four hours after transfection, U3A cells were incubated for an additional 15 hours with 1% FCS diluted in Dulbecco’s modified Eagle’s medium. The cells were then either left untreated or stimulated for 6 hours with IFNγ. RNA was isolated with the peqGold Total RNA kit (Peqlab) and first-strand cDNA synthesis was performed using the Superscript III kit from Invitrogen. The real-time PCR reactions were carried out in a total volume of 25 μl, containing 25 ng cDNA, 70 nM of each specific primer pair, and 12.5 μl SYBR Green (Thermo Fisher Scientific). The following protocol was applied: a denaturation step at 95°C for 15 min, and 45 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Following the final amplification, a melting curve analysis was run via a temperature gradient from 60°C to 95°C in 0.5°C increment steps, fluorescence being measured at each temperature for a period of 10 s. All reactions were carried out in at least triplicate for each sample. The relative expression of a transcript was normalized to the expression of gapdh as determined for each sample. Using the Realplex 1.5 software from Eppendorf, the threshold (Ct) at which the cycle numbers were measured was adjusted to areas of exponential amplification of the traces. The ΔΔCt-method was used to determine comparative relative expression levels, the formula 2-(ΔCt target - ΔCt reference sample) being applied as described previously. Statistical analysis was as described above.
The authors gratefully acknowledge the excellent technical assistance of Miriam Gehring and Sandra Sieber from the University of Marburg. We kindly thank Dr. Uwe Vinkemeier, University of Nottingham, for valuable reagents and discussions. The research on this subject was funded by a grant from the Deutsche Forschungsgemeinschaft to TM.
electrophoretic mobility shift assay
green fluorescent protein
nuclear export signal
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
polymerase chain reaction
- SH2 domain:
Src homology 2 domain
signal transducer and activator of transcription 1.
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