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Cited by 161Cytokine Signaling Pathways and InteractionsGenomics and Chromatin DynamicsNF-κB Signaling Pathways

DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1

Open access
Meyer, Thomas, Marg, Andreas, Lemke, Petra, Wiesner, Burkhard, Vinkemeier, Uwe·2003·Genes & Development
DOI 10.1101/gad.268003PMC PMC196254
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Abstract

Cytokine-dependent gene transcription greatly depends on the tyrosine phosphorylation (“activation”) of Stat proteins at the cell membrane. This rapidly leads to their accumulation in the nucleus by an unknown mechanism. We performed microinjections of recombinant Stat1 protein to show that nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation of cells. Microinjection of Stat1 antibody and treatment of cells with kinase or phosphatase inhibitors revealed that nuclear accumulation is a highly dynamic process sustained by Stat1 nucleocytoplasmic cycling and continuous kinase activity. By characterizing nuclear accumulation mutants, it is demonstrated that nuclear import and nuclear retention are two separate steps leading up to nuclear accumulation, with nonspecific DNA binding of activated Stat1 being sufficient for nuclear retention. Critical for nuclear buildup of Stat1 and the subsequent nuclear export is the point of time of tyrosine dephosphorylation, because our data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so. It is demonstrated that the inactivation of Stat1 is controlled by its exchange reaction with DNA, whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner. Thus, during nuclear accumulation, a surprisingly simple mechanism integrates central aspects of cytokine-dependent gene regulation, for example, receptor monitoring, promoter occupancy, and transcription factor inactivation.

AI snapshot

Written from the paper’s full text.

TL;DR

This paper shows that tyrosine-phosphorylated Stat1 accumulates in the nucleus without cytokine receptor activation, and that nuclear accumulation is a dynamic process requiring continuous nucleocytoplasmic shuttling and kinase activity. DNA binding, especially nonspecific DNA binding, mediates nuclear retention and protects Stat1 from tyrosine dephosphorylation, thereby controlling the duration of nuclear accumulation.

Key findings

  • Microinjected tyrosine-phosphorylated Stat1 accumulated in the nucleus of untreated HeLa and U3A cells, whereas unphosphorylated Stat1 distributed pancellularly after ∼30 min and did not accumulate even after 3 h. Nuclear export required dephosphorylation: after nuclear injection, tyrosine-phosphorylated Stat1 remained nuclear at 6 h, while unphosphorylated Stat1 became almost pancellular.
  • Nuclear accumulation was dynamic: staurosporine caused complete Stat1 dephosphorylation after 60 min and collapse of nuclear accumulation, while vanadate/H₂O₂ suppressed dephosphorylation and extended accumulation. Cytoplasmic antibody injection depleted nuclear Stat1 in ∼30 min, but vanadate prevented this clearance, indicating continuous shuttling.
  • DNA binding controlled nuclear retention and inactivation. Stat1dna minus failed to accumulate in the nucleus despite unaltered import, whereas Stat1dna plus accumulated and bound specific and nonspecific DNA with almost equal avidity. DNA protected Stat1 from TC45 dephosphorylation in a concentration- and sequence-dependent manner; GAS sites protected better than unrelated DNA.
  • FRAP showed altered intranuclear mobility: after IFNγ, wild-type Stat1 recovery was ∼18.6 ± 3.2 sec, while Stat1dna plus was ∼206.7 ± 30.1 sec; with vanadate, wild-type was ∼46.4 ± 11.2 sec and dna plus ∼233.7 ± 46.6 sec. Stat1dna plus showed prolonged nuclear accumulation, Stat1dna off showed abbreviated accumulation, but neither dna plus nor dna minus induced a GAS-driven reporter in response to IFNγ.

Methods

  • Recombinant human Stat1 variants were purified from baculovirus-infected Sf9 cells, in vitro tyrosine-phosphorylated, and microinjected into HeLa and U3A cells. Cells were treated with IFNγ, staurosporine, leptomycin B, and vanadate/H₂O₂; antibody microinjection was used to probe nucleocytoplasmic shuttling.
  • DNA-binding mutants were generated by site-directed mutagenesis and analyzed by EMSA, biotinylated-oligonucleotide pull-down, coimmunoprecipitation, Western blotting, immunocytochemistry, and luciferase reporter assays.
  • In vitro dephosphorylation used purified TC45 phosphatase with or without GAS or mutant DNA. Intranuclear mobility was measured by FRAP, and immunofluorescence intensities were quantified with Student’s t-test or ANOVA/Tukey tests.

Limitations

  • The paper states no formal limitations section. It notes that nuclear dephosphorylation of recombinant Stat1 was slow compared with endogenous protein, which could be caused by the high concentration of injected material or by the absence of Arg31 methylation. It also notes that no data are available concerning nonspecific DNA binding of previously reported Stat1 mutants.
  • Evident from the text: conclusions rely heavily on microinjection, overexpression, and purified in vitro systems, which may not fully reproduce endogenous chromatin and cellular contexts. FRAP sample sizes are small (n = 4–12 per condition), and mutant proteins could have effects beyond altered DNA binding.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1101/gad.268003

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

10 of 28 cited sources checked · 8 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Stat1dna off displays a markedly increased off-rate from GAS sites.

    “Additionally, we included another DNA binding mutant, named Stat1dna off here, that was previ- ously characterized by Darnell and coworkers (Yang et al. 2002) and displays a markedly increased off-rate from GAS sites.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Yang et al. 2002 source
  • Premisecitation

    Quantitative analysis of the Stat1-DNA exchange reaction had revealed identical affinities for various sites.

    “Quantitative analysis of the Stat1-DNA exchange reaction had revealed identical affinities for various sites, but despite having similar apparent K d values, the binding with DNA may differ significantly in rates of association with and dissociation from the Stat1 protein (Vinkemeier et al. 1996).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Vinkemeier et al. 1996 source
  • Premisecitation

    The cell line U3A does not express Stat1.

    “the cell line U3A (data not shown), which does not express Stat1 (Müller et al. 1993).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Müller et al. 1993 source
  • Premisecitation

    Despite having similar apparent Kd values, the binding with DNA may differ significantly in rates of association with and dissociation from the Stat1 protein.

    “Quantitative analysis of the Stat1-DNA exchange reaction had revealed identical affinities for various sites, but despite having similar apparent K d values, the binding with DNA may differ significantly in rates of association with and dissociation from the Stat1 protein (Vinkemeier et al. 1996).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Vinkemeier et al. 1996 source
  • Findingown data

    DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner.

    “whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner.”
    • Also stated in: § DNA-bound Stat1 is protected from tyrosine dephosphorylation · plain
      “In conclusion, Stat1 that was not bound to DNA underwent tyrosine dephosphorylation at maximum rate, whereas DNA binding protected this molecule from inactivation in a sequence-dependent manner.”
    • Also stated in: § Abstract · plain
      “DNA-bound Stat1 is protected from dephosphorylation and is thus transiently retained in the nucleus.”
    Quote in the paperSubstantial claim
  • May combine two claims. 43
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Premisecitation

    The mobility of Stat1 is much faster than core histones and similar to other inducible transcriptional regulators.

    “Compared with other studies, the mobility of Stat1 was much faster than the core histones and similar to other inducible transcriptional regulators (McNally et al. 2000; Kimura and Cook 2001; Lillemeier et al. 2001).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites McNally et al. 2000 source
    • cites Kimura and Cook 2001 source
    • cites Lillemeier et al. 2001 source

      The Discussion (page unavailable) describes STAT1–GFP as highly mobile and compares its dynamic interactions with those of the glucocorticoid receptor, an inducible transcriptional regulator. It does not compare STAT1 mobility with that of core histones, so that part of the claim is not supported by this paper. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Stat1 has a slow turnover time (t1/2 > 8 h) and its dephosphorylation is rapid (t1/2 < 15 min).

    “Given that Stat1 has a slow turnover time (t 1/2 > 8 h; Lee et al. 1997) but dephosphorylation is rapid (t 1/2 < 15 min; Haspel et al. 1996), we wanted to determine how long Stat1 molecules remained nuclear.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Lee et al. 1997 source
    • cites Haspel et al. 1996 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Dimerization is sufficient for nuclear import of activated Stat1.

    “This observation extends the previous finding that dimerization is sufficient for nuclear import of activated Stat1 (Milocco et al. 1999).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Milocco et al. 1999 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The analysis of nucleocytoplasmic transport of Stat1 has revealed its high, energy-independent mobility in the cytoplasm, indicative of random diffusion.

    “The analysis of nucleocytoplasmic transport of Stat1 has revealed its high, energy-independent mobility in the cytoplasm, which is indicative of random diffusion (Lillemeier et al. 2001).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Lillemeier et al. 2001 source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    TC45 is a ubiquitously expressed Stat1-specific nuclear tyrosine phosphatase.

    “purified TC-PTPa (also called TC45; Ibarra-Sanchez et al. 2000), a ubiquitously expressed Stat1-specific nuclear tyrosine phosphatase (ten Hoeve et al. 2002).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Ibarra-Sanchez et al. 2000 source
    • cites ten Hoeve et al. 2002 source

      The Results identify TC45 as a mainly nuclear phosphatase that dephosphorylates Stat1. But the Discussion says the TC-PTP gene is ubiquitously expressed—not specifically that TC45 is—and the Results and Discussion also implicate TC45 in Stat3 dephosphorylation, so “Stat1-specific” overstates its selectivity. Page numbers were not available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Fluorescence recovery of all Stat1 variant proteins is complete even in the absence of phosphatase activity, and mobility continues to differ between DNA binding mutants.

    “Notably, fluorescence recovery of all Stat1 variant proteins was complete even in the absence of phosphatase activity, and the mobility continued to differ between the DNA binding mutants (Fig. 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pervanadate suppresses Stat1 dephosphorylation and extends the duration of nuclear accumulation.

    “On the contrary, inactivating tyrosine phosphatases by treatment of cells with pervanadate (Gordon 1991) not only suppressed Stat1 dephosphorylation but also extended the duration of nuclear accumulation (Fig. 2A,B, bottom panels).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Gordon 1991 source

  • May combine two claims.

    Findingown data

    Preventing tyrosine dephosphorylation has little impact on intranuclear mobility of all Stat1 proteins investigated, with recovery times increasing only by a factor of 2.5 or less.

    “Strikingly, preventing tyrosine dephosphorylation had little impact on the intranuclear mobility of all Stat1 proteins investigated (Fig. 5B), because recovery times increased only by a factor of 2.5 or less (Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stat1dna plus is much less sensitive to cessation of kinase activity, with the level of activated protein remaining high, indicating strongly reduced tyrosine dephosphorylation.

    “In contrast, Stat1dna plus was much less sensitive to the cessation of kinase activity, as the level of activated protein remained high, indicating strongly reduced tyrosine dephosphorylation (Fig. 5A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Staurosporine after IFNγ-induced nuclear accumulation results in complete dephosphorylation of Stat1 after 60 min and a collapse of nuclear accumulation that mirrors dephosphorylation kinetics.

    “This resulted in the complete dephosphorylation of Stat1 after 60 min and a collapse of nuclear accumulation that closely mirrored the kinetics of tyrosine dephosphorylation (Fig. 2A,B, top panels).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Unphosphorylated Stat1 microinjected into the cytoplasm enters the nucleus rapidly and shows a pancellular distribution after about 30 min.

    “The Stat1 protein entered the nucleus rapidly and showed a pancellular distribution after ∼30 min (Fig. 1B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Recombinant Stat1 protein purified from baculovirus-infected Sf9 cells and microinjected into cells is correctly folded and functionally equivalent to endogenous Stat1.

  • May combine two claims.

    Findingown data

    Stat1dna plus shows significantly reduced intranuclear mobility, with recovery time extended to about 200 sec.

    “Remarkably, the mutant Stat1dna plus showed a significantly reduced intranuclear mobility, and the recovery time was extended to ∼200 sec.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Unphosphorylated Stat1 is capable of nucleocytoplasmic shuttling, but accumulation only occurs with the tyrosine-phosphorylated species.

    “These data confirm that unphosphorylated Stat1 is capable of nucleocytoplasmic shuttling, yet accumulation only occurred with the tyrosine-phosphorylated species.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Recombinant Stat1 protein purified from baculovirus-infected Sf9 cells and microinjected into cells is correctly folded and functionally equivalent to endogenous Stat1.

  • May combine two claims.

    Findingown data

    Initial concentrations of activated Stats are similar for all variants after 30 min IFNγ treatment, indicating comparable facility to become tyrosine-phosphorylated.

    “As is shown in Figure 5A, the initial concentrations of activated Stats were similar for all variants after 30 min of IFN␥ treatment, indicating their comparable facility to become tyrosine-phosphorylated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fluorescence recovery is complete for all Stat1 variants, indicating no immobile population within the nucleus during accumulation.

    “Importantly, fluorescence recovery was complete for all Stat1 variants, indicating that no immobile population was present within the nucleus during accumulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Antibody-induced nuclear accumulation of Stat1wt and Stat1dna minus cannot be discriminated, indicating unperturbed import of the mutant.

    “Importantly, the antibody-induced nuclear accumulation of Stat1wt and Stat1dna minus could not be discriminated, thus indicating unperturbed import of the mutant.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Stat1 variants are expressed well and phosphorylated on tyrosine 701 in response to IFNγ.

    “The Stat1 variants were expressed well and phosphorylated on tyrosine 701 in response to IFN␥ (Fig. 3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Addition of a negative charge at position 327 (Thr to Glu) of Stat1dna minus results in defective nuclear accumulation, albeit less severely, with partial accumulation still inducible by IFNγ.

    “Addition of a negative charge in position 327 (Thr to Glu) of Stat1dna minus also resulted in defective nuclear accumulation, albeit less severely, because a partial accumulation could still be induced by IFN␥ (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Vanadate treatment precludes antibody-induced nuclear clearance of Stat1, with no translocation to the cytoplasm even after 90 min.

    “However, treatment of cells with the tyrosine phosphatase inhibitor vanadate precluded the antibody-induced nuclear clearance of Stat1, because even after 90 min of incubation there was no translocation of Stat1 to the cytoplasm (Fig. 2C, sec-ond row).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Wild-type Stat1, dna off, and dna minus are highly mobile in the nucleus, with recovery times of about 20 sec.

    “Wild-type Stat1 was highly mobile in the nucleus, as were the dna off and dna minus mutants.These proteins had recovery times of ∼20 sec (Fig. 5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stat1dna plus and dna off accumulate in the nucleus after IFNγ stimulation, whereas Stat1dna minus fails to accumulate.

    “First, we observed the nucleocytoplasmic distribution of GFP-fusion proteins before and after IFN␥ stimulation and found accumulation of Stat1dna plus and dna off , whereas the Stat1dna minus mutant failed to accumulate in the nucleus in response to IFN␥ (Fig. 3C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After 6 h, considerable amounts of Stat1 are found outside the nucleus, with an almost pancellular distribution.

    “However, after 6 h, considerable amounts of Stat1 were found outside of the nucleus, because an almost pancellular distribution was observed with a Stat1-specific antibody (Fig. 1D,F).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Recombinant Stat1 protein purified from baculovirus-infected Sf9 cells and microinjected into cells is correctly folded and functionally equivalent to endogenous Stat1.

  • May combine two claims.

    Findingown data

    Nuclear accumulation of Stat1dna off is shortened, despite the mutant being activated to a similar extent as wildtype Stat1 and despite its normal ability to recognize GAS sites.

    “Figure 5 demonstrates that nuclear accumu- lation of Stat1dna off is shortened, despite the fact that this mutant was activated to a similar extent as wildtype Stat1 and despite its normal ability to recognize GAS sites (Fig. 3D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tyrosine-phosphorylated Stat1 was not exported readily but was retained in the nucleus.

    “Nonetheless, because tyrosine-phosphorylated Stat1 was not exported readily but was retained in the nucleus, DNA binding per se cannot account for nuclear retention.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IFNγ-induced nuclear import of Stat1dna minus proceeds unaltered, but the mutant is incapable of nuclear accumulation.

    “Judged by several criteria, such as the abundance of phosphorylated Stat1 in nuclear extracts, the antibody-induced nuclear precipitation after IFN␥ stimulation, and the rapid nuclear accumulation induced by an export block through preventing tyrosine dephosphorylation (Fig. 3), the IFN␥-induced nuclear import of Stat1dna minus proceeded unaltered, but nevertheless the mutant was incapable of nuc”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nuclear export of wild-type Stat1 is blocked after IFNγ stimulation in the presence of vanadate, whereas nuclear export of Stat1Y701F and an export reporter construct are not affected.

    “The nuclear export of wild-type Stat1 was blocked after stimulation with IFN␥ in the presence of vanadate, whereas nuclear export of both a Stat1 variant with a mutated tyrosine phosphorylation site and an export reporter construct were not affected by this treatment (Fig. 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although the protein-DNA complex had a half-life of no more than 3 min for any of the GAS sites investigated, the off-times varied at least sixfold.

    “The Stat1 protein achieves equilibrium in DNA binding very rapidly (Ӷ30 sec); however, although the protein-DNA complex had a half-life of no more than 3 min for any of the GAS sites investigated, the off-times varied at least sixfold.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The cycling rate of Stat1 is high, as cytoplasmic trapping depletes nuclear Stat1 in about 30 min and blocking kinase activity collapses nuclear accumulation in less than 60 min.

    “Nevertheless, the cycling rate was high, as cytoplasmic trapping depleted nuclear Stat1 in ∼30 min (Fig. 2C), and blocking of kinase activity led to a collapse of nuclear accumulation in <60 min (Fig. 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Stat1dna minus and dna plus have opposed nuclear accumulation abilities, neither can discriminate GAS elements from nonspecific sites, although they differ in overall DNA affinity.

    “This was exemplified by the opposed nuclear accumulation abilities of Stat1dna minus and dna plus , neither of which could discriminate GAS elements from nonspecific sites, although they differed from one another in their overall DNA affinity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so.

    “because our data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so.”
    • Also stated in: § The role of receptor activation and tyrosine phosphorylation in nucleocytoplasmic shuttling and nuclear accumulation of Stat1 · plain
      “Conversely, nuclear export required dephosphorylation.”
    • Also stated in: § Discussion · plain
      “We conclude that tyrosinephosphorylated dimers were incapable of nucleocytoplasmic shuttling and required inactivation before release into the cytoplasm.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Stat1dna plus binds DNA nonspecifically, whereas Stat1dna minus does not.

    “Thus, Stat1dna plus bound to DNA, albeit nonspecifically, whereas dna minus did not.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Stat1 not bound to DNA undergoes tyrosine dephosphorylation at maximum rate, whereas DNA binding protects it from inactivation in a sequence-dependent manner.

    “In conclusion, Stat1 that was not bound to DNA underwent tyrosine dephosphorylation at maximum rate, whereas DNA binding protected this molecule from inactivation in a sequence-dependent manner.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: In vitro dephosphorylation of recombinant Stat1 by TC45 reflects the in vivo dephosphorylation and inactivation of Stat1.

  • May combine two claims.

    Premisecitation

    Nuclear accumulation depends on the integrity of the DNA-binding domain, attributable to the need for a functional dsNLS located there.

    “Nuclear accumulation was shown to depend on the integrity of the DNA-binding domain, which can be attributed to the need for a functional dsNLS located here (Fagerlund et al. 2002;Meyer et al. 2002a).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Fagerlund et al. 2002 source
    • cites Meyer et al. 2002a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    DNA binding differences are reflected in altered nuclear mobility, with enhanced binding correlating with reduced mobility.

    “Thus, DNA binding differences were reflected in an altered mobility in the nuclear compartment, with enhanced binding correlating with reduced mobility.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Although the enzymatic reaction was required for nuclear export, it could not be the causative step for the release of Stat1 from its nuclear DNA tether.

    “Thus, although the enzymatic reaction was required for nuclear export, it could not be the causative step for the release of Stat1 from its nuclear DNA tether.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The data describe a mechanism that allows nuclear Stats to monitor receptor activity and selectively promotes occupation of target regulatory elements.

    “These data describe a mechanism that allows nuclear Stats to monitor receptor activity and that at the same time selectively promotes occupation of target regulatory elements.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    GAS sites are generally poorly conserved, making it difficult to predict actual chromatin binding sites.

    “We also point out the fact that GAS sites are generally poorly conserved, making it difficult to predict actual chromatin binding sites (Decker et al. 1997), indicating that the distinction between”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Decker et al. 1997 source

  • May combine two claims.

    Premisecitation

    Nuclear import does not necessarily result in nuclear accumulation, which is only observed with tyrosine-phosphorylated Stat1 dimers containing a functional SH2 domain.

    “Thus, nuclear import does not necessarily result in nuclear accumulation, which is only observed with tyrosine-phosphorylated Stat1 dimers containing a functional SH2 domain (Mowen and David 1998).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Mowen and David 1998 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The two TC45 preparations differ in Stat1 dephosphorylation activity, but the specificity of the reaction is unchanged by truncation.

    “The two preparations differed in their Stat1 dephosphorylation activity, but the specificity of the reaction was unchanged by the truncation (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Binding to DNA outside of consensus binding elements is common for transcriptional regulators.

    “We show that nonspecific interactions with DNA are observable for wild-type Stat1 (Fig. 3E), and binding to DNA outside of consensus binding elements is common for transcriptional regulators (Lin and Riggs 1975).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lin and Riggs 1975 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The truncated TC45 variant exhibits increased phosphatase activity.

    “This variant enzyme was demonstrated to exhibit increased phosphatase activity (Hao et al. 1997).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hao et al. 1997 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Binding to the glucocorticoid receptor enhanced DNA binding of Stat5, which correlated with its prolonged tyrosine phosphorylation in vivo.

    “Binding to the glucocorticoid receptor enhanced DNA binding of Stat5, which correlated with its prolonged tyrosine phosphorylation in vivo (Wyszomierski et al. 1999).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wyszomierski et al. 1999 source

  • May combine two claims.

    Interpretationreasoning

    DNA binding is sufficient but not necessary for nuclear accumulation.

    “For these reasons, DNA binding is sufficient but not necessary for nuclear accumulation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Fast dephosphorylation with concomitant nuclear export and reactivation in the cytoplasm are both essential for dynamic signaling.

    “Fast dephosphorylation with concomitant nuclear export and reactivation in the cytoplasm are both essential for dynamic signaling.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A negative influence of DNA binding on Stat dephosphorylation was noted earlier also for Stat5.

    “A negative influence of DNA binding on Stat dephosphorylation was noted earlier also for Stat5.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wyszomierski et al. 1999 source

  • May combine two claims.

    Interpretationown data

    Tyrosine-phosphorylated Stat1 dimers are incapable of nucleocytoplasmic shuttling and require inactivation before release into the cytoplasm.

    “We conclude that tyrosinephosphorylated dimers were incapable of nucleocytoplasmic shuttling and required inactivation before release into the cytoplasm.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    A model explains the molecular basis of signal-dependent nuclear accumulation of Stat1 and outlines constitutive cycling of Stat1 in resting cells and during IFNγ stimulation.

    “A model is shown in Figure 7 that explains the molecular basis of the signaldependent nuclear accumulation of the transcription factor Stat1 and outlines the constitutive cycling of Stat1 both in resting cells and during stimulation with IFN␥.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The process of nuclear accumulation might confer a selection advantage on Stat1 dimers bound to GAS consensus promoter sites.

    “This way, the process of nuclear accumulation might confer a selection advantage on Stat1 dimers bound to GAS consensus promoter sites.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Homogeneous tyrosine phosphorylation of the final preparation was verified by MALDI analysis, as was the absence of Arg 31 methylation.

    “Homogeneous tyrosine phosphorylation of the final preparation was verified by MALDI analysis, as was the absence of Arg 31 methylation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Full-length and truncated TC45 dephosphorylated pNPP with identical rates.

    “As expected, full-length and truncated TC45 dephosphorylated pNPP with identical rates (Hao et al. 1997).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hao et al. 1997 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Dephosphorylation of the control substrate p-nitrophenyl phosphate (pNPP) was not influenced by DNA (25 nM GAS).

    “Dephosphorylation of the control substrate p-nitrophenyl phosphate (pNPP) was not influenced by DNA (25 nM GAS; data not shown)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Mutations were verified by DNA sequencing.

    “Mutations were verified by DNA sequencing.”
    Quote in the paperNot a substantial claim
52 of 52

  • The paper relies on this without support.

    Recombinant Stat1 protein purified from baculovirus-infected Sf9 cells and microinjected into cells is correctly folded and functionally equivalent to endogenous Stat1.

    “To investigate nuclear translocation, we purified the human Stat1 protein from baculovirus-infected Sf9 cells (Fig. 1A).”
    “These proteins were microinjected into HeLa cells and the cell line U3A (data not shown), which does not express Stat1 (Müller et al. 1993).”

  • The paper relies on this without support.

    In vitro dephosphorylation of recombinant Stat1 by TC45 reflects the in vivo dephosphorylation and inactivation of Stat1.

    “At first we therefore examined the influence of DNA binding on the dephosphorylation of Stat1 variants in vitro.”
    “In conjunction with the in vitro dephosphorylation data, we conclude that the release of Stat1 from DNA did not require enzymatic activity.”
2 of 2

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Authors
Meyer, T., Marg, A., Lemke, P., Wiesner, B., & Vinkemeier, U.
Affiliations
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (DE)
Corresponding author
Vinkemeier, Uwe
ORCID
Venue
Genes & Development
Publisher
Cold Spring Harbor Laboratory
Published
2003-08-15
Volume
17
Issue
16
Pages
1992-2005
ISSN
  • 0890-9369 (ISSN-L)
  • 1549-5477
DOI
10.1101/gad.268003
PMC
PMC196254
OpenAlex
W2099132542
Semantic Scholar
229b280fb881046a1375087eb563a61164547ec3
Cited by
161
References
48
Citations by year
2026: 2 · 2025: 1 · 2024: 1 · 2023: 4 · 2022: 4 · 2021: 5 · 2020: 4 · 2019: 6 · 2018: 5 · 2017: 7 · 2016: 8 · 2015: 4 · 2014: 9 · 2013: 9 · 2012: 7
Type
posted content
Language
en
Open access
Locations
  • Genes & Development (publishedVersion)
  • PubMed (publishedVersion)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
  • Israel Cancer Research Fund
  • Bundesministerium für Bildung und Forschung
  • Université de Strasbourg
Topics
  • Health Sciences › Medicine › Oncology › Cytokine Signaling Pathways and Interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
Keywords
STAT1, DNA binding, tyrosine phosphorylation, tyrosine dephosphorylation, nucleocytoplasmic shuttling, nuclear import, nuclear retention, nuclear export, transcription factor inactivation, sequence-specific DNA binding, phosphatase activity
MeSH terms
Antibodies, Monoclonal, Cell Nucleus, Cytoplasm, DNA, DNA-Binding Proteins, Enzyme Inhibitors, HeLa Cells, Humans, Interferon-gamma, Models, Biological, Mutation, Phosphorylation, Recombinant Proteins, Transcription, Genetic, Tumor Cells, Cultured, Tyrosine, Vanadates, Transcriptional Activation, Trans-Activators, Active Transport, Cell Nucleus, STAT1 Transcription Factor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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