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Cited by 58interferon and immune responsesGenomics and Chromatin DynamicsNeurofibromatosis and Schwannoma Cases

PU.1, Interferon Regulatory Factor (IRF) 2, and the Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Cooperate to Activate NF1 Transcription in Differentiating Myeloid Cells

Open access
Huang, Weiqi, Horvath, Elizabeth, Eklund, Elizabeth A.·2007·Journal of Biological Chemistry
DOI 10.1074/jbc.m607760200
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Abstract

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper shows that cytokine-induced NF1 transcription in differentiating myeloid cells requires sequential assembly of a PU.1–IRF2–ICSBP/IRF8 complex on a composite ets/IRF cis element in the NF1 promoter. This assembly depends on specific phosphorylation-sensitive residues in PU.1, IRF2, and ICSBP/IRF8.

Key findings

  • The NF1 promoter cis element (−320 to −336 bp) binds a multiprotein complex containing PU.1, IRF2, and ICSBP/IRF8; IFNγ differentiation of U937 cells increases binding, and antibodies to PU.1, IRF2, and ICSBP/IRF8 disrupt the complex, whereas IRF1 antibody does not.
  • Assembly is sequential: PU.1 binds the NF1-cis element directly; PU.1 recruits IRF2 but not ICSBP/IRF8; the DNA-bound PU.1–IRF2 heterodimer then recruits ICSBP/IRF8. This requires PU.1 Ser-132/133 for IRF2 recruitment, PU.1 Ser-148 for ICSBP/IRF8 recruitment, IRF2 Tyr-109, and ICSBP/IRF8 Tyr-95.
  • In U937 reporter assays, IRF2 overexpression activated the NF1-cis element reporter (p < 0.0001, n = 5); PU.1 overexpression activated it in undifferentiated (p ≤ 0.04, n = 12) and IFNγ-differentiated (p < 0.0001, n = 12) cells; PU.1 plus either IRF2 or ICSBP/IRF8 increased activity over either IRF alone (p ≤ 0.002, n = 4), and all three together were greater than PU.1 plus either IRF alone (p ≤ 0.002, n = 7).
  • PU.1-specific shRNA blocked activation by overexpressed IRF2, ICSBP/IRF8, or both (p = 0.61, F = 0.54, n = 3) and decreased endogenous Nf1 expression. Mutant S132A/S133A PU.1 did not significantly activate the reporter versus control (p = 0.122 undifferentiated; p = 0.54 IFNγ-treated; n = 6), and S148A PU.1 plus IRF2 plus ICSBP/IRF8 produced less than half the activity of wild-type PU.1 plus both IRFs (p = 0.001, n = 4; p < 0.00002, n = 6 with IFNγ). IFNγ also increased PU.1 phosphorylation and IRF2 tyrosine phosphorylation, while tyrosine-phosphorylated PU.1 was not detected.

Methods

  • U937 human myelomonocytic cells were treated with or without 500 units/ml IFNγ for 24 or 48 h; nuclear proteins were analyzed by EMSA, antibody supershift, DNA affinity co-immunoprecipitation (DAPA), immunoprecipitation/Western blot, metabolic labeling with [32P]orthophosphate, and GST pulldown assays with in vitro translated proteins.
  • Transfections used 32 × 10^6 U937 cells per sample with an nf1TATACAT reporter containing four copies of the −320 to −336 NF1 cis element or control pTATACAT, plus expression vectors for wild-type or mutant PU.1, IRF2, ICSBP/IRF8, or PU.1-specific shRNA; CAT and β-galactosidase activities were measured 48 h after transfection with or without IFNγ.
  • Reporter gene results are reported as comparisons with p values and sample sizes (n) as given in the text; no additional statistical model is described.

Limitations

  • Stated limitations: the paper does not include an explicit limitations section.
  • Evident from the text: the mechanistic conclusions rely on the U937 cell line, in vitro translated proteins, GST pulldown assays, and an artificial reporter with four copies of the NF1 cis element, rather than primary myeloid cells, endogenous promoter chromatin assays, or in vivo models.
  • Evident from the text: the discussion notes that IRF2-deficient mice do not exhibit a myeloproliferative disorder, so the in vivo proliferative consequence of IRF2 regulation of NF1 remains uncertain.

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

Integrity signals

Registry

Crossref · 10.1074/jbc.m607760200

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

30 of 67 cited sources checked · 13 supported · 2 flagged · accuracy not yet measured

  • Premisecitation

    DNA-bound PU.1 interacts with IRF1.

    “DNA-bound PU.1 interacts with IRF1”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [11] Rehli, 2000, J. Biol. Chem. source

      Figure 7 and the Discussion identify ICSBP, not IRF-1, as the factor that complexes with DNA-bound PU.1 at the TLR4 promoter. The Discussion mentions IRF-1 as an interaction partner of ICSBP, rather than of PU.1. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [21] Eklund, 1998, J. Biol. Chem. source
    • cites [22] Eklund, 1999, J. Immunol. source
  • Premisecitation

    Tyrosine-phosphorylated ICSBP/IRF8 can activate PRDI-cis elements by interaction with DNA-bound IRF1.

    “In contrast, tyrosine-phosphorylated ICSBP/IRF8 can activate PRDI-cis elements by interaction with DNA-bound IRF1 (19).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Sharf, 1997, J. Biol. Chem. source

      The Discussion says tyrosine-phosphorylated ICSBP can associate with IRF-1 and bind target DNA through that association. But the introduction describes ICSBP as a repressor of PRDI-containing promoters, and the PRDI reporter assay reports a sevenfold decrease in activity with full-length ICSBP—not activation. The assay does not isolate the phosphorylated ICSBP–IRF-1 complex, but the paper's reported transcriptional effect is contrary to the claim. (LLM, conf=0.83)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premiseown data

    NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the IRF domain of ICSBP/IRF8.

    “We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Huang, 2006, Mol. Cell. Biol. source

      The Discussion (page unavailable) states that NF1 transcriptional activation requires cytokine-induced phosphorylation of specific tyrosine residues in the ICSBP IRF domain. Materials and Methods identifies these as conserved residues Y92 and Y95. The claim captures the mechanism but describes a single residue where the paper identifies two. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Acquired ICSBP/IRF8 deficiency is found in bone marrow cells from subjects with CML, AML, and MDS.

    “acquired ICSBP/IRF8 deficiency is found in bone marrow cells from subjects with chronic myeloid leukemia, AML, and MDS (11,12).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Rehli, 2000, J. Biol. Chem. source

      The Discussion mentions ICSBP-deficient mice developing a chronic myelogenous leukemia-like syndrome, but it does not report acquired ICSBP/IRF8 deficiency in human bone marrow cells. Searches for the patient, bone-marrow, AML, and MDS claim found no supporting passage in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [12] Schmidt, 1998, Blood source
  • Premisecitation

    Acquired Nf1 deficiency has been documented in myeloid cells from human subjects with AML and MDS.

    “Acquired Nf1 deficiency has been documented in myeloid cells from human subjects with acute myeloid leukemia (AML) 2 and myelodysplastic syndromes (MDS) (7).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [7] Lu, 2003, Cancer source

      The NF1 Protein Expression section reports deficient bone-marrow NF1 protein levels in 8 of 29 patients with MDS and 20 of 66 with AML. The study measured bone-marrow protein levels, but does not establish that the deficiency was acquired or confined to myeloid cells. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 26
  • May combine two claims. 22
  • The quote only partly states this claim. 3

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

    Premisecitation

    Nf1 is a 2818-amino acid protein with Ras-GAP activity encoded by the NF1 gene.

    “Neurofibromin 1 (Nf1) is 2818-amino acid protein with Ras-GAP activity encoded by the NF1 gene (1).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [1] Ballester, 1990, Cell source

      The Complementation section reports increased H-ras GTPase-stimulating activity in lysates expressing an NF1 segment, supporting the Ras-GAP activity attributed to the NF1 gene product. The Experimental Procedures describe a 2,484-residue partial NF1 sequence, not a 2,818-amino-acid full-length protein; the paper does not substantiate the claimed length. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Nf1 deficiency induces a myeloproliferative disorder in murine models.

    “Consistent with this, Nf1 deficiency induces a myeloproliferative disorder in murine models (8,9).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [8] Brannan, 1994, Genes Dev. source

      The Abstract and ‘NF1 as a regulator of growth’ section report cardiac malformations and hyperplasia of neural crest-derived sympathetic ganglia in Nf1-deficient embryos, not a myeloproliferative disorder. Searches for myeloproliferative, myeloid, hematopoietic, leukemia, blood cells, and bone marrow found no passage in our copy establishing the claimed disorder. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [9] Zhang, 1998, J. Exp. Med. source

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

    Premisecitation

    Activated SHP2 mutants induce cytokine hypersensitivity in myeloid progenitors.

    “Such activated SHP2 mutants also induce cytokine hypersensitivity in myeloid progenitors (15,18).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [15] Huang, 2006, Mol. Cell. Biol. source
    • cites [18] Chan, 2005, Blood source

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

    Premisecitation

    Interferon-stimulated response elements can be repressed by an interaction between ICSBP/IRF8, the ets protein Tel, and histone deacetylase 3.

    “Interferon-stimulated response elements can be repressed by an interaction between ICSBP/IRF8, the ets protein Tel, and histone deacetylase 3 (20).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [20] Kuwata, 2002, Mol. Cell. Biol. source

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

    Premisecitation

    ICSBP/IRF8 is a substrate for SHP1 and SHP2 protein-tyrosine phosphatases in undifferentiated myeloid cells.

    “ICSBP/IRF8 is a substrate for SHP1 and SHP2 protein-tyrosine phosphatases in undifferentiated myeloid cells (15,16).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [15] Huang, 2006, Mol. Cell. Biol. source
    • cites [16] Kautz, 2001, J. Biol. Chem. source

      The Discussion (page not provided) says ICSBP may be an SHP1 substrate in undifferentiated myeloid cells, but cautions that another phosphatase could be responsible. This paper does not establish ICSBP as an SHP2 substrate, and the citing sentence states both substrate relationships as fact. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Previous studies found IRF2 activates transcription of some genes involved in the innate immune response.

    “Previous studies found IRF2 activates transcription of some genes involved in the innate immune response (23, 44 -46).”
    Quote in the paperNot a substantial claim1 of 4 sources checked
    • cites [23] Xi, 2003, Mol. Immunol. source
    • cites [44] Luo, 1996, J. Biol. Chem. source
    • cites [45] Heinz, 2003, J. Biol. Chem. source
    • cites [46] Guyot, 2003, J. Leukocyte Biol. source

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

    Premisecitation

    A number of previously identified PU.1 target genes confer mature phagocyte functional competence.

    “A number of previously identified PU.1 target genes confer mature phagocyte functional competence (11,21,22,(32)(33)(34)(35)(36).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [11] Rehli, 2000, J. Biol. Chem. source
    • cites [21] Eklund, 1998, J. Biol. Chem. source
    • cites [22] Eklund, 1999, J. Immunol. source

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

    Premisecitation

    Studies of leukemia in γ-irradiated mice documented frequent deletion in the PU.1 gene.

    “Additionally, studies of leukemia in ␥-irradiated mice documented frequent deletion in the PU.1 gene (41).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] Cook, 2004, Blood source

  • May combine two claims.

    Premisecitation

    Conditional PU.1-knock-out mice develop a myeloproliferative disorder that evolves to AML over several months.

    “These animals develop a myeloproliferative disorder that evolves to AML over several months.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [42] Metcalf, 2006, Proc. Natl. Acad. Sci. U. S. A. source

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

    Premisecitation

    PU.1 is expressed at low levels in hematopoietic stem cells and is relatively more abundant during monocyte versus neutrophil differentiation.

    “Consistent with this, PU.1 is expressed at low levels in hematopoietic stem cells and is relatively more abundant during monocyte versus neutrophil differentiation (43).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] DeKoter, 1998, EMBO J. source

      The Introduction (page unavailable) says PU.1 is expressed in various hematopoietic lineages, but does not give its level in hematopoietic stem cells. The discussion of monocytic and granulocytic development addresses PU.1’s functional roles, not whether its expression is higher during monocyte than neutrophil differentiation. Searches for stem-cell expression levels and a monocyte–neutrophil expression comparison found no passage establishing either claim. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the NF1-cis element.

    “DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element.”
    • Also stated in: § Identification of Protein/Protein/DNA Interactions Required for Assembly of the NF1-cis Element-binding Complex-Previ- · plain
      “We found that PU.1 recruits IRF2 to the NF1-cis element but not ICSBP/ IRF8.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The shifted complex is cross-immunoreactive with PU.1, ICSBP/IRF8, and IRF2 but not IRF1.

    “we found that the shifted complex is cross-immunoreactive with PU.1, ICSBP/IRF8, and IRF2 but not IRF1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Co-overexpression of PU.1, IRF2, and ICSBP/IRF8 increases expression of endogenous Nf1 in U937 cells, consistent with the reporter assays.

    “We also find that this cooverexpression increases expression of endogenous Nf1, consistent with the reporter assays.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 interaction with ICSBP/IRF8 requires PU.1 Ser-148 and PU.1 interaction with IRF2 requires PU.1 Ser-132/133.

    “In these studies, PU.1 interaction with ICSBP/IRF8 requires PU.1 Ser-148 and PU.1 interaction with IRF2 requires PU.1 Ser-132/133.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 overexpression induces a smaller but statistically significant increase in reporter expression from the NF1-cis element in undifferentiated U937 transfectants (p≤0.04, n=12) and a larger increase in differentiated transfectants (p<0.0001, n=12).

    “PU.1 overexpression induces a smaller but statistically significant increase in reporter expression from the NF1-cis element in undifferentiated transfectants (p Յ 0.04, n ϭ 12) and a larger increase in differentiated transfectants (p Ͻ 0.0001, n ϭ 12).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Overexpression of IRF2 and ICSBP/IRF8 has a more than additive impact on the NF1-cis element, indicating nonredundant roles for IRF2 and ICSBP/IRF8.

    “We found that overexpression of these two IRF proteins has a more than additive impact on the NF1-cis element, indicating nonredundant roles for IRF2 and ICSBP/IRF8.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    S148A PU.1 interacts only with IRF2 but not ICSBP/IRF8.

    “However, S148A PU.1 interacts only with IRF2 but not ICSBP/IRF8.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Overexpressed PU.1, IRF2, and ICSBP/IRF8 increase expression of endogenous Nf1.

    “Increased PU.1, IRF2, and ICSBP/IRF8 expression correlates with increase in endogenous Nf1 expression.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PU.1 knockdown decreases activity of the NF1-cis element and prevents overexpressed IRF2 (with or without ICSBP/IRF8) from activating this cis element.

    “Expression of PU.1-specific shRNA (but not scrambled control shRNA) decreases activity of the NF1-cis element and prevents overexpressed IRF2, with and without ICSBP/IRF8, for activating this cis element.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 knockdown and IRF2 or ICSBP/IRF8 overexpression do not influence reporter expression from control pTATACAT vector.

    “PU.1 knockdown and IRF2 or ICSBP/IRF8 overexpression do not influence reporter expression from control pTATACAT vector.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PU.1 is serine/threonine-phosphorylated and IRF2 is tyrosine-phosphorylated during IFNγ differentiation of U937 cells.

    “PU.1 is serine/threonine-phosphorylated and IRF2 is tyrosinephosphorylated during IFN␥ differentiation of U937 cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Nf1 is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells.

    “Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Congenital Nf1 deficiency is associated with neurofibromatosis.

    “Congenital Nf1 deficiency is associated with neurofibromatosis (5)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] DeClue, 1992, Cell source

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

    Premisecitation

    Congenital Nf1 deficiency is associated with juvenile myelomonocytic leukemia.

    “and juvenile myelomonocytic leukemia (6).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [6] Largaespada, 1996, Nat. Genet. source

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

    Premisecitation

    ICSBP/IRF8 is expressed exclusively in myeloid and B-cells.

    “ICSBP/IRF8 is expressed exclusively in myeloid and B-cells (10)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Nelson, 1993, Mol. Cell. Biol. source

  • May combine two claims.

    Premisecitation

    NF1 transcription and Nf1 expression increase during cytokine-induced differentiation of myeloid leukemia cell lines or murine myeloid progenitor cells.

    “Previously, we found that NF1 transcription and Nf1 expression increase during cytokine-induced differentiation of myeloid leukemia cell lines or murine myeloid progenitor cells (4).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] Zhu, 2004, J. Biol. Chem. source

  • May combine two claims.

    Premisecitation

    NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation.

    “NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [4] Zhu, 2004, J. Biol. Chem. source
    • cites [13] Holtchke, 1996, Cell source
    • cites [14] Scheller, 1999, Blood source

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

    Study design and conductown data

    GAPDH was used to control for loading in Western blots.

    “Western blots were serially probed with antibodies to Nf1, ICSBP/IRF8, IRF2, PU.1, and GAPDH (to control for loading).”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    p-CMVβgal was used to control for transfection efficiency.

    “and p-CMV␤gal (to control for transfection efficiency).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Mutant clones were sequenced on both strands to verify that only intended mutations had been introduced.

    “Mutant clones were sequenced on both strands to verify that only intended mutations had been introduced.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    PU.1 binds to the NF1-cis element and recruits IRF2.

    “In these studies, we determine that PU.1 binds to the NF1-cis element and recruits IRF2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Because PU.1 and IRF2 undergo post-translational modification during myelopoiesis, the studies identify an additional mechanism that regulates NF1 transcription in differentiating myeloid cells.

    “Because PU.1 and IRF2 undergo post-translational modification during myelopoiesis, our studies identify an additional mechanism that regulates NF1 transcription in differentiating myeloid cells.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Expression of wild type PU.1 and various serine mutants is equivalent in such transfection experiments.

    “Previous studies and our own preliminary data indicate that expression of wild type PU.1 and various serine mutants is equivalent in such transfection experiments (not shown in this study) (26).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Pongubala, 1991, Mol. Cell. Biol. source

  • May combine two claims.

    Interpretationown data

    S132A/S133A PU.1 prevents endogenous PU.1 from binding to the NF1-cis element, thereby preventing interaction with overexpressed IRF2.

    “These results support the hypothesis that S132A/ S133A PU.1 prevents endogenous PU.1 from binding to the NF1-cis element, therefore preventing interaction with overexpressed IRF2.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Premiseown data

    PU.1 bound to the CYBB or NCF2 composite ets/IRF-cis elements interacts with IRF1 and this heterodimer recruits ICSBP/IRF8 in differentiated myeloid cells.

    “Our previous studies suggest that PU.1 bound to the CYBB or NCF2 composite ets/IRF-cis elements interacts with IRF1 and that this heterodimer recruits ICSBP/IRF8 in differentiated myeloid cells.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    Identification of NF1 as an IRF2 target gene suggests that IRF2 deficiency might be expected to result in cytokine hypersensitivity.

    “Identification of NF1 as IRF2 target genes suggests that IRF2 deficiency might be expected to result in cytokine hypersensitivity.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: No IRF2 target genes that regulate cellular proliferation or cell cycle progression have been identified previously.

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

    Interpretationreasoning

    PU.1 function is partly regulated by protein abundance.

    “These results suggest that PU.1 function is partly regulated by protein abundance.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    IRF2 activates the gene encoding histone H4 during G1/S transition.

    “IRF2 also activates the gene encoding histone H4 during G 1 /S transition (5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] DeClue, 1992, Cell source

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

    Premisecitation

    Immunodeficiency is found in PU.1-deficient murine models.

    “This is consistent with immunodeficiency found in PU.1-deficient murine models (37,38).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] McKercher, 1996, EMBO J. source
    • cites [38] Scott, 1994, Science source

  • May combine two claims.

    Interpretationown data

    PU.1 binding to the NF1 composite ets/IRF-cis element recruits IRF2 and this heterodimer recruits ICSBP/IRF8.

    “In contrast, our current studies indicate that PU.1 binding to the NF1 composite ets/IRFcis element recruits IRF2 and that this heterodimer recruits ICSBP/IRF8.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationown data

    Specific PU.1 serine residues (S132/S133 and S148) are required for cooperation with IRF2 and ICSBP/IRF8 and activation of the NF1-cis element.

    “Specific PU.1 serine residues are required for cooperation with IRF2 and ICSBP and activation of the NF1-cis element.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    GAPDH was used to control for protein loading in Western blots.

    “GAPDH (to control for protein loading)”
    • Also stated in: § DISCUSSION · plain
      “GAPDH (to control for loading)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    Empty vector control was used for overexpression experiments.

    “empty vector control (pTATACAT)”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Actin was used to control for protein loading in Western blots.

    “actin (to control for loading)”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Phospho-ERK was used as a control for IFNγ treatment and total ERK as a loading control.

    “Blots were also probed with antibody to phospho-ERK as a control for IFN␥ treatment and with total ERK as a loading control.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Scrambled control shRNA was used as a control for PU.1-specific shRNA.

    “PU.1-specific or scrambled control shRNA”
    Quote in the paperSubstantial claim
46 of 46

  • The paper relies on this without support.

    The reporter construct containing four copies of the NF1-cis element reflects regulation of endogenous Nf1 transcription.

    “U937 cells were transfected with a reporter construct containing a minimal promoter with or without four copies of the NF1-cis element (nf1TATACAT and pTATACAT control, respectively).”

  • The paper relies on this without support.

    PU.1 is a key transcription factor in myelopoiesis.

    “PU.1 is a key transcription factor in myelopoiesis.”

  • The paper relies on this without support.

    Target genes responsible for dysregulated myeloproliferation in PU.1-deficient cells had not been identified prior to this study.

    “However, target genes responsible for dysregulated myeloproliferation in PU.1-deficient cells had not been identified prior to this study.”

  • The paper relies on this without support.

    Relatively few IRF2 target genes have been identified in myeloid cells.

    “Relatively few IRF2 target genes have been identified in myeloid cells.”

  • The paper relies on this without support.

    No IRF2 target genes that regulate cellular proliferation or cell cycle progression have been identified previously.

    “However, no IRF2 target genes that regulate cellular proliferation or cell cycle progression have been identified previously.”
5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 6A gives conflicting PU.1 mutant positions

    Minor

    The Figure 6A legend identifies the tested double mutant as S132A/S133A PU.1, but later calls it S132A/S33A PU.1. Serine 33 and serine 133 are different positions.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. Nelson, 1993, Mol. Cell. Biol., 13, 588
  2. Pongubala, 1991, Mol. Cell. Biol., 12, 368
  3. Iida, 2000, Int. J. Hematol., 71, 153

Cited by

Sorted by citations

Related

Authors
Huang, W., Horvath, E., & Eklund, E. A.
Affiliations
Northwestern University (US)
Corresponding author
Eklund, Elizabeth A.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2007-03
Volume
282
Issue
9
Pages
6629-6643
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m607760200
OpenAlex
W1965828533
Semantic Scholar
8bdd306f5666c355c10ce2a3e1676e4cec6dcc1e
Cited by
58
References
48
Citations by year
2026: 1 · 2024: 1 · 2023: 1 · 2022: 1 · 2021: 5 · 2020: 3 · 2019: 1 · 2018: 2 · 2017: 2 · 2016: 3 · 2015: 2 · 2014: 4 · 2013: 3 · 2012: 5
FWCI
2.31
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Neurology › Neurofibromatosis and Schwannoma Cases
Keywords
neurofibromatosis type 1, neurofibromin 1, PU.1, IRF2, transcriptional activation, myeloid differentiation, protein-protein interaction, tyrosine phosphorylation
MeSH terms
Interferon Regulatory Factor-8, Proto-Oncogene Protein Spi-1, Cell Differentiation, Cell Line, Humans, Promoter Regions, Genetic, Proto-Oncogene Proteins, Transcription, Genetic, Trans-Activators

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 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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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