A Conserved Motif in the Linker Domain of STAT1 Transcription Factor Is Required for Both Recognition and Release from High-Affinity DNA-Binding Sites

<div><p>Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (<i>s</i>ignal <i>t</i>ransducer and <i>a</i>ctivator of <i>t</i>ranscription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.</p></div

(A, B) The E559A and E563A mutants display high-affinity DNA binding and restore defective nuclear accumulation of a STAT1 variant with artificially enhanced nuclear export.

<p>HeLa cells transfected with pSTAT1-NES-GFP, which coded for a transferable nuclear export signal (NES) situated between the cDNAs for full-length STAT1 and GFP, were either left untreated or treated for 45 min with IFNγ (5 ng/ml), the CRM1 export inhibitor leptomycin B (LMB, 10 ng/ml) or a combination of the two. (A) The micrographs demonstrate the intracellular distribution of STAT1-NES-GFP and the localization of corresponding Hoechst-stained nuclei (scale bar 10 µm). (B) Histograms demonstrate the percentage of nuclear-to-total STAT1 fluorescence intensities for the various STAT1-NES proteins and stimulation modes. (C) <i>In vitro</i> dephosphorylation assays showing that DNA-bound E559A, E563A and K567A are protected from Tc45-catalyzed inactivation. Whole cell extracts from reconstituted U3A cells expressing wild-type or mutant STAT1 were incubated for 30 min with or without the recombinant Tc45 phosphatase in the absence or presence of 2xGAS, GAS-nonGAS or 2xnonGAS, as indicated. Reactions were probed for phospho-STAT1 levels by means of Western blotting (αSTAT1-P). The membrane was then stripped and re-incubated with the pan-STAT1 antibody C-24 (αSTAT1). (D) Precipitation of STAT1-WT, -E559A and -K567A with agarose-bound duplex oligonucleotides comprising tandem M67 binding sites (2xGAS), a GAS and non-GAS site (GAS-nonGAS) or a mutated sequence thereof (2xnonGAS). Beads were incubated with cell extracts from transfected U3A cells with and without IFNγ treatment. Five percent (vol/vol) of the cell extract input was blotted and probed with anti-STAT1 antibody. The precipitate (pull down) was analyzed by Western blotting with anti-Stat1 antibody. (E) Quantification of three independent pull-down experiments for each of the STAT1 variants tested, as shown in D. Significant differences between the three variants are indicated with an asterisk.</p

(A) Hyperphosphorylation of the STAT1 mutants E559A and E563A upon stimulation of cells with IFNγ.

<p>Equal cell numbers of HeLa cells expressing either wild-type or one of the indicated STAT1 mutants, all fused to GFP, were prestimulated for 45 min with 5 ng/ml IFNγand subsequently exposed to the kinase inhibitor staurosporine for increasing time periods, before tyrosine phosphorylation was tested for in cell lysates. A representative Western blot experiment using a STAT1-specific phospho-tyrosine antibody (top panel) and the corresponding re-blot after the stripping off of bound immunoreactivity and re-incubation with pan-STAT1 antibody C-24 (bottom panel) are shown. The upper band on each blot marks recombinant STAT1-GFP, whereas the lower band corresponds to endogenous STAT1. (B) Elevated and prolonged tyrosine phosphorylation levels of the E559A and E563A mutants in STAT1-reconstituted U3A cells. Similar experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g003" target="_blank">Figure 3A</a>, except that U3A cells were transfected with the respective pSTAT1-GFP constructs. Staurosporine treatment resulted in the rapid loss of tyrosine-phosphorylated STAT1-WT and -K567A, whereas the two glutamic acid mutants partially resisted the inactivating effect of the kinase blocker. (C) Quantification of immunoblots for expression of tyrosine-phosphorylated and total STAT1 in IFNγ-pretreated (45 min) U3A cells exposed to staurosporine for different time periods, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g003" target="_blank">Figure 3B</a>. Significant differences in the ratio of phosphorylated to total STAT1 between different incubation times or STAT1 variants are marked with bars and asterisks. (D) Prolonged GAS-binding activity of tyrosine-phosphorylated E559A and E563A and defective binding of the phospho-K567A mutant. Gel shift experiment with cellular extracts from STAT1-reconstituted U3A cells treated as described above. Cell lysates were equilibrated with a radioactively labelled high-affinity STAT-binding probe termed M67, before being loaded onto a non-denaturing gel. (E) GAS-binding activity from IFNγ-prestimulated U3A cells exposed to staurosporine was densitometrically analyzed. (F) The same extracts used for the EMSA experiment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g003" target="_blank">Figure 3D</a> were probed for the presence of phosphorylated and total STAT1. (G) Similar experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g003" target="_blank">Figure 3D</a>, except that STAT1-GFP-expressing HeLa cells were used and supershifts using an unspecific STAT3-antibody (αS3, lane 1) and a specific STAT1-antibody (αS1, lane 2) were included. At the right-hand margin of the gel the positions of the GFP-tagged and untagged STAT1 homodimers as well as the corresponding STAT1-GFP/STAT1 heterodimer are indicated. Asterisks mark unspecific bands.</p

The point mutants E559A, E563A and K567A display altered DNA-binding kinetics.

<p>(A–C) Binding affinity of the indicated STAT1-GFP fusion proteins to three different duplex oligonucleotides containing two strong GAS elements in tandem arrangement (2xGAS) or mutations thereof, with a single GAS element (GAS-nonGAS) or no GAS element (2xnonGAS). The images depict representative EMSA results using whole cell extracts from reconstituted U3A cells (A) and HeLa cells (C) with the positions of tetrameric and dimeric GFP-tagged STAT1 indicated at the right-hand margin of the gel. Where indicated, anti-STAT3- (αS3) and anti-STAT1-antibodies (αS1) were included in the reactions. The additional bands in HeLa extracts correspond to homo- and heterodimers of recombinant GFP-tagged STAT1 as well as homo- and heterotetramers of native STAT1 lacking the GFP fusion. (B) The percentage of tetrameric-to-total STAT1 complexed to GAS-nonGAS was significantly elevated in the case of glutamic acid point mutants as compared to the wild-type protein. (D) Mutation of either glutamic acid 559 or 563 to alanines results in decreased dissociation rates from DNA and high-affinity GAS binding. Whole cell extracts from STAT1-GFP-reconstituted U3A cells were incubated for 15 min with [³³P]-labelled DNA containing a single STAT binding site (M67) and, subsequently, a 750-fold molar excess of unlabelled DNA was added for 0, 5 and 10 min, respectively, before the samples were loaded onto a native polyacrylamide gel. In the second lane, anti-STAT1 antibody C-24 was present in the EMSA reaction for the identification of STAT1-M67 complexes which are marked with an arrowhead. (E) Dissociation of STAT1-GFP from M67 was analyzed quantitatively confirming the high stability of DNA-bound mutant STAT1 in comparison to wild-type STAT1. (F) Similar EMSA experiment as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g004" target="_blank">Figure 4D</a>, except that STAT1-GFP-expressing HeLa cells were used. Note that not only STAT1-GFP homodimers but also heterodimers of recombinant and native STAT1 are partially protected from competition. (G, H) Reduced nuclear export kinetics of STAT1-E559A and -E563A. HeLa cells expressing GFP-tagged wild-type or mutant STAT1 (scale bar 10 µm) were prestimulated for 45 min with IFNγ to induce nuclear accumulation (top panel) and then treated for 6 min in the presence of 50 µg/ml digitonin in ice-cold transport buffer (bottom panel). Fluorescence micrographs of formaldehyde-fixed cells demonstrating the amount of nuclear STAT1-GFP and the localization of the corresponding Hoechst-stained nuclei before and after digitonin treatment are shown in (G). (H) Similar experiment as in (G), except that immunofluorescence staining of transfected U3A cells was performed. (I) STAT1-K567A is a DNA-binding mutant. DNA binding activity to a [³³P]-labelled native <i>ccl2</i> promoter element (CCL2 native), but not to a mutated version thereof (CCL2 mutant), was detected in extracts from reconstituted U3A cells by means of EMSA. (J) Weak binding of tetrameric STAT1-K567A to [³³P]-labelled DNA containing a tandem GAS site. The reactions were either left unchallenged (−) or challenged for 20 min with a 750-fold molar excess of a single, unlabelled GAS site (+ competition). At the margin of the EMSA gel, the positions of tetrameric (top arrowhead) and dimeric (bottom arrowhead) STAT1 are marked. Asterisks label a non-specific band.</p

Gene-specific expression pattern of STAT1-E559A and -E563A and lack of transcriptional activation by the K567A mutant.

<p>(A) Luciferase reporter gene assay in U3A cells expressing the indicated STAT1 variants normalized to the expression of constitutively co-expressed β-galactosidase. The reporter constructs used in this experiment contained a triple GAS site from the Ly6E promoter (3xLy6E) in the luciferase gene. Cells were treated for 0 h (white columns), 2 h (bright grey), 4 h (dark grey) and 6 h (black) with 5 ng/ml IFNγ before, in whole cell extracts, luciferase luminescence and β-galactosidase activity were measured. The experiment was repeated at least three times in six independent transfections. (B) Endogenous gene induction by the STAT1 mutants was determined by real-time PCR assays. Histograms depict expression levels of the <i>gbp1</i>, <i>irf1</i>, <i>mig1</i>, <i>ccl2</i> and for control <i>stat1</i> gene before (grey columns) and after 6 h stimulation with 5 ng/ml IFNγ (black columns). Specific gene induction was normalized to the expression of the house-keeping gene <i>gapdh</i>. The data are presented as means and standard deviations from at least three independent experiments. Statistical significance between the groups of IFNγ-stimulated cells expressing the indicated STAT1 variants is marked by asterisks.</p

Replacement of glutamine residues in position 559 and 563 results in STAT1 mutants with a prolonged IFNγ-induced nuclear accumulation.

<p>(A, B) HeLa cells expressing GFP fusion proteins with either STAT1-WT, -E559A, -E563A, or -K567A were untreated or stimulated for 45 min with 5 ng/ml human IFNγ followed by exposure to staurosporine (500 nM) for 0 min, 60 min and 120 min, respectively. The fluorescence micrographs show the intracellular distribution of the GFP-tagged fusion proteins and the localization of the corresponding Hoechst-stained nuclei (scale bar 10 µm). (B) Quantification of the nucleocytoplasmic STAT1-GFP distribution in untreated and IFNγ-pretreated cells exposed to staurosporine as determined from experiment (A). The histograms demonstrate the ratio of nuclear-to-total cellular fluorescence intensities as measured for the indicated STAT1 variants and stimulation modes. (C) Indirect fluorescence microscopy confirming the decreased nuclear export rates of STAT1-E559A and -E563A. STAT1-negative U3A cells, reconstituted with untagged wild-type or mutant STAT1, were treated as above, except that exposure times to staurosporine were shortened (0 min, 30 min, and 60 min). Intracellular distribution of recombinant STAT1 in the fixed, Hoechst-stained cells was monitored immunocytochemically using anti-STAT1 C-24 and Cy3-labelled secondary antibodies (scale bar 10 µm). (D) Nucleocytoplasmic distribution of the STAT1 mutants, as quantified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097633#pone-0097633-g002" target="_blank">Figure 2C</a>, with bars and asterisks indicating significant differences between wild-type and the respective mutant.</p