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Cited by 100Cytokine Signaling Pathways and InteractionsGenomics and Chromatin DynamicsNF-κB Signaling Pathways

Synergistic gene expression during the acute phase response is characterized by transcription factor assisted loading

Open access
Goldstein, Ido, Paakinaho, Ville, Baek, Songjoon, Sung, Myong-Hee, Hager, Gordon L.·2017·Nature Communications
DOI 10.1038/s41467-017-02055-5PMC PMC5707366
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Abstract

The cytokines interleukin 1β and 6 (IL-1β, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins. Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression. By mapping changes in enhancer landscape and transcription factor occupancy (using ChIP-seq), we show that synergistic gene induction is achieved by assisted loading of STAT3 on chromatin by NF-κB. With IL-6 treatment alone, STAT3 does not efficiently bind 20% of its coordinated binding sites. In the presence of IL-1β, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac. This facilitates STAT3 binding and synergistic gene expression. Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others. This model reconciles seemingly contradictory reports of NF-κB-STAT3 crosstalk.

AI snapshot

Written from the paper’s full text.

TL;DR

In primary mouse hepatocytes, IL-1β and IL-6 produce a bifurcated acute-phase transcriptional response, with both synergistic and antagonistic gene regulation. Genome-wide profiling shows NF-κB binds and primes a subset of enhancers via p300/CBP-dependent H3K27ac, enabling assisted STAT3 loading and synergistic acute-phase gene induction.

Key findings

  • RNA-seq in primary mouse hepatocytes identified 3,260 genes differentially expressed after at least one cytokine treatment (1.5-fold change, adjusted p ≤ 0.05); dual IL-1β + IL-6 treatment produced both synergistic and antagonistic expression patterns.
  • STAT3 ChIP-seq showed 351 sites (20% of total STAT3 binding events) with significantly increased STAT3 binding in dual treatment versus IL-6 alone; the majority, 80% (n = 1,428), were unaffected or reduced by IL-1β. Assisted sites reached STAT3 binding levels similar to unassisted sites, while IL-1β reduced STAT3 binding at unassisted sites.
  • Assisted STAT3 sites were far more likely to be near synergistic genes: the nearest gene for 63 assisted sites was a synergistic gene, compared with only 2 additive genes. NF-κB motifs and p65 binding were enriched at assisted sites; NF-κB inhibition abolished assisted STAT3 binding at 92% of sites (325/351) and abolished synergistic gene induction.
  • Acute phase signaling was the most enriched pathway in dual-induced genes (p = 1.11 × 10−24) and in synergistic genes (p = 2.15 × 10−10); p300/CBP inhibition negated H3K27ac and assisted STAT3 binding.

Methods

  • Primary mouse hepatocytes from male C57BL/6 mice were treated with IL-1β, IL-6, both (10 ng ml−1 each), or TNF (2 ng ml−1) for 2 h; nascent transcripts were measured by qPCR spanning exon-intron junctions and normalized to Tbp.
  • Genome-wide profiling used RNA-seq (CuffDiff; two replicates per treatment) and ChIP-seq for STAT3, p65, CEBPB, cJun, RNAP II, and H3K27ac, with peak calling by MACS2 and differential binding by DESeq/HOMER.
  • Perturbation experiments used STAT3 inhibitor BP-1-102, p300/CBP inhibitors C646, SGC-CBP30, and I-CBP112, and adenoviral dominant-negative NF-κB, dominant-negative AP-1, or shRNA against CEBPB; sequencing data are in GEO GSE96770.

Limitations

  • The paper does not include a dedicated limitations section.
  • Evident from the text: the main experiments use primary mouse hepatocytes in vitro, male C57BL/6 mice, and a short 2 h cytokine treatment, so the findings may not fully capture chronic inflammation, other liver cell types, or in vivo complexity.
  • Evident from the text: RNA-seq used two replicates per treatment, and the Saa1/2 qPCR primer could not differentiate between Saa1 and Saa2.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-017-02055-5

Cites

71 of 71 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

37 of 50 cited sources checked · 27 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Assisted loading events are coupled with increases in enhancer activity as measured by DNase hypersensitivity or H3K27 acetylation (H3K27ac).

    “Assisted loading events are coupled with increases in enhancer activity as measured by DNase hypersensitivity or H3K27 acetylation (H3K27ac), two well-established markers for active enhancers38–40.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [38] Rivera, C. M. & Ren, B. Mapping human epigenomes. Cell 155, 39–55 (2013). source
    • cites [39] Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad. Sci. USA 107, 21931–21936 (2010). source
    • cites [40] Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012). source

      The Introduction (page unavailable) describes DNaseI hypersensitivity as characteristic of active regulatory elements, including enhancers, and the section on stereotyped chromatin accessibility links DHS patterns to enhancer activity. This supports DNase hypersensitivity as an enhancer marker, but the cited paper does not establish H3K27ac as a marker or examine assisted-loading events. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    The claim that pioneer factors open inaccessible chromatin without the assistance of chromatin remodelers has been recently challenged.

    “This has been recently challenged by us and others59.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [59] Goldstein, I. & Hager, G.L. Dynamic enhancer function in the chromatin context. Wiley Interdiscip. Rev. Syst. Biol. Med. doi:10.1002/wsbm.1390 (2017). source
  • Premisecitation

    Chromatin loading of FoxA1 (a well-established pioneer factor) was shown to need assistance by non-pioneer TFs in a signal-dependent manner.

    “For example, the chromatin loading of FoxA1 (a well-established pioneer factor), was shown to need assistance by non-pioneer TFs in a signal-dependent manner35.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [35] Swinstead, E. E. et al. Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions. Cell 165, 593–605 (2016). source
  • Interpretationcitation

    These reports, together with ours, challenge the concept that NF-κB and other signal-activated TFs are restricted to enhancer regions pre-activated by lineage-determining factors.

    “These reports, together with ours, challenge the concept that NF-κB and other signal-activated TFs are restricted to enhancer regions pre-activated by lineage-determining factors58.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [58] Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011). source

      In “Initiating events in chromatin: pioneer factors bind first” (page unavailable), the review describes PU.1 priming enhancers and facilitating other factors’ binding, including at an enhancer regulated by NF-κB. It does not establish that NF-κB and other signal-activated factors are restricted to pre-activated enhancers; “Interaction between FoxA1 and diverse nuclear receptors” (page unavailable) even describes sites where the signal-responsive glucocorticoid receptor pioneers binding. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    Pioneer factors have been shown to be dependent on chromatin remodelers during differentiation.

    “Moreover, pioneer factors have been shown to be dependent on chromatin remodelers during differentiation60.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [60] King, H. W. & Klose, R. J. The pioneer factor OCT4 requires the chromatin remodeller BRG1 to support gene regulatory element function in mouse embryonic stem cells. Elife 6, pii: e22631 (2017). source

      The Discussion (page not available) states that OCT4 requires the chromatin remodeller BRG1 for chromatin accessibility and notes a similar dependency for GATA3. However, the direct experiments concern mouse embryonic stem cells; the Results section on reprogramming and development says only that developmental transitions may require OCT4 and BRG1. The source therefore does not establish a general dependency of pioneer factors on remodelers during differentiation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 17
  • May combine two claims. 46
  • The quote only partly states this claim. 1

  • Classified as probably not a substantial claim.

    Premisecitation

    TFs do not bind DNA for periods of time longer than a few seconds.

    “In contrast to the long-held view, an accumulating body of work suggests that TFs do not bind DNA for periods of time longer than a few seconds26, 27.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [26] Voss, T. C. & Hager, G. L. Dynamic regulation of transcriptional states by chromatin and transcription factors. Nat. Rev. Genet. 15, 69–81 (2014). source

      The ‘In vivo dynamics of transcription factors’ section reports rapid exchange for many transcription factors, supporting the contrast with long-term binding. But it also says some site-specific DNA-binding proteins have longer residence times and that transcription factors likely have a broad range of residence times. It does not support a universal limit of a few seconds. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [27] Stavreva, D. A. & Hager, G. L. Chromatin structure and gene regulation: a dynamic view of enhancer function. Nucleus 6, 442–448 (2015). source

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

    Premisereasoning

    The suggested mechanisms for the STAT3-NF-κB crosstalk are often contradictory and a consensus has not been reached.

    “Thus, the suggested mechanisms for the STAT3-NF-κB crosstalk are often contradictory and a consensus has not been reached.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [18] Yang, J. et al. Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev. 21, 1396–408 (2007). source

      The paper’s Abstract and Discussion describe several STAT3–NF-κB interactions with different effects, including enhanced transcription of some genes and inhibition of another. That supports the idea that proposed mechanisms vary, but the paper does not say they are often contradictory or that no consensus has been reached. Page numbers are unavailable in our copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [19] Yu, Z., Zhang, W. & Kone, B. C. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB. Biochem. J. 367, 97–105 (2002). source

      The introduction says relatively little is known about how transcription-factor interactions regulate iNOS, and the discussion says the domains mediating the STAT3–NF-κB interaction remain unidentified (page numbers unavailable). These passages support uncertainty about the mechanism, but the paper does not say proposed mechanisms are often contradictory or that the field lacks a consensus. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [21] Martincuks, A. et al. Nuclear translocation of STAT3 and NF-kappaB are independent of each other but NF-kappaB supports expression and activation of STAT3. Cell Signal. 32, 36–47 (2017). source

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

    Premisecitation

    Dynamic assisted loading does not require a physical interaction between TFs, but is thought to rely on one TF activating the enhancer by recruiting chromatin remodeling and histone modifying enzymes to the enhancer, thereby making it more accessible to other TFs.

    “This effect, termed ‘dynamic assisted loading’, does not require a physical interaction between TFs, but is thought to rely on one TF activating the enhancer by recruiting chromatin remodeling and histone modifying enzymes to the enhancer, thereby making it more accessible to other TFs29.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [29] Swinstead, E. E., Paakinaho, V., Presman, D. M. & Hager, G. L. Pioneer factors and ATP-dependent chromatin remodeling factors interact dynamically: A new perspective: Multiple transcription factors can effect chromatin pioneer functions through dynamic interactions with ATP-dependent chromatin remodeling factors. Bioessays 38, 1150–1157 (2016). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The crosstalk between STAT3 and NF-κB is an active field of research with studies showing contradictory outcomes of concomitant activation of the two TFs.

    “The crosstalk between STAT3 and NF-κB is an active field of research with studies showing contradictory outcomes of concomitant activation of the two TFs.”
    Quote in the paperSubstantial claim4 of 7 sources checked
    • cites [2] Bode, J. G., Albrecht, U., Haussinger, D., Heinrich, P. C. & Schaper, F. Hepatic acute phase proteins--regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-kappaB-dependent signaling. Eur. J. Cell Biol. 91, 496–505 (2012). source
    • cites [16] Yu, H., Pardoll, D. & Jove, R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9, 798–809 (2009). source
    • cites [17] Quinton, L. J. & Mizgerd, J. P. NF-kappaB and STAT3 signaling hubs for lung innate immunity. Cell Tissue Res. 343, 153–65 (2011). source
    • cites [18] Yang, J. et al. Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev. 21, 1396–408 (2007). source
    • cites [19] Yu, Z., Zhang, W. & Kone, B. C. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB. Biochem. J. 367, 97–105 (2002). source

      The introduction (page unavailable) describes cooperative and antagonistic interactions involving STAT proteins and NF-κB, and the paper reports that STAT3 antagonizes NF-κB signaling in activated mesangial cells. It does not establish that studies found contradictory outcomes specifically for concomitant STAT3 and NF-κB activation; its broader examples include other STAT proteins. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [20] Lee, H. et al. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell 15, 283–93 (2009). source
    • cites [21] Martincuks, A. et al. Nuclear translocation of STAT3 and NF-kappaB are independent of each other but NF-kappaB supports expression and activation of STAT3. Cell Signal. 32, 36–47 (2017). source

  • Classified as probably not a substantial claim.

    Premisecitation

    The events bringing about the hepatic immune response during inflammation remain elusive.

    “Several models have been suggested2, 16 but the events bringing about the hepatic immune response during inflammation remain elusive.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [2] Bode, J. G., Albrecht, U., Haussinger, D., Heinrich, P. C. & Schaper, F. Hepatic acute phase proteins--regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-kappaB-dependent signaling. Eur. J. Cell Biol. 91, 496–505 (2012). source
    • cites [16] Yu, H., Pardoll, D. & Jove, R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9, 798–809 (2009). source

      The section “STAT3–NF-κB in cancer inflammation” says that a mechanism of immune-gene regulation in tumours remains unresolved, but it does not address what brings about the hepatic immune response during inflammation. A search for hepatic immune-response mechanisms found no passage in our copy that makes the attributed point. (LLM, conf=0.74)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    NF-κB is able to recruit p300/CBP to chromatin and to increase H3K27ac.

    “Moreover, NF-κB is able both to recruit p300/CBP to chromatin53 and to increase H3K27ac54, 55.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [53] Zhong, H., Voll, R. E. & Ghosh, S. Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol. Cell 1, 661–71 (1998). source
    • cites [54] Jurida, L. et al. The activation of IL-1-induced enhancers depends on TAK1 kinase activity and NF-kappaB p65. Cell Rep. pii: S2211-1247(15)00002-9. doi:10.1016/j.celrep.2015.01.001 (2015). source
    • cites [55] Brown, J. D. et al. NF-kappaB directs dynamic super enhancer formation in inflammation and atherogenesis. Mol. Cell 56, 219–31 (2014). source

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

    Interpretationcitation

    These observations are consistent with a reported role for NF-κB in modifying the enhancer landscape following stimuli.

    “These observations are consistent with a reported role for NF-κB (a classic signal-activated TF) in modifying the enhancer landscape following stimuli54, 55.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [54] Jurida, L. et al. The activation of IL-1-induced enhancers depends on TAK1 kinase activity and NF-kappaB p65. Cell Rep. pii: S2211-1247(15)00002-9. doi:10.1016/j.celrep.2015.01.001 (2015). source
    • cites [55] Brown, J. D. et al. NF-kappaB directs dynamic super enhancer formation in inflammation and atherogenesis. Mol. Cell 56, 219–31 (2014). source

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

    Premisecitation

    Some studies suggest cooperation between STAT3 and NF-κB, while others show antagonism.

    “Some suggest cooperation between them, while others show antagonism2, 16–21.”
    Quote in the paperSubstantial claim4 of 7 sources checked
    • cites [2] Bode, J. G., Albrecht, U., Haussinger, D., Heinrich, P. C. & Schaper, F. Hepatic acute phase proteins--regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-kappaB-dependent signaling. Eur. J. Cell Biol. 91, 496–505 (2012). source
    • cites [16] Yu, H., Pardoll, D. & Jove, R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9, 798–809 (2009). source
    • cites [17] Quinton, L. J. & Mizgerd, J. P. NF-kappaB and STAT3 signaling hubs for lung innate immunity. Cell Tissue Res. 343, 153–65 (2011). source
    • cites [18] Yang, J. et al. Unphosphorylated STAT3 accumulates in response to IL-6 and activates transcription by binding to NFkappaB. Genes Dev. 21, 1396–408 (2007). source
    • cites [19] Yu, Z., Zhang, W. & Kone, B. C. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB. Biochem. J. 367, 97–105 (2002). source

      The introductory passage (page unavailable) says STAT proteins and NF-κB can act cooperatively, but does not establish cooperation specifically between STAT3 and NF-κB. The paper does show that STAT3 antagonizes NF-κB-dependent transcription in activated mesangial cells, supporting the antagonism part of the claim. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [20] Lee, H. et al. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell 15, 283–93 (2009). source
    • cites [21] Martincuks, A. et al. Nuclear translocation of STAT3 and NF-kappaB are independent of each other but NF-kappaB supports expression and activation of STAT3. Cell Signal. 32, 36–47 (2017). source

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

    Premisecitation

    Pioneer factors were attributed with special properties that separated them functionally from the rest of TFs.

    “However, these TFs (termed pioneer factors) were attributed with special properties that separated them functionally from the rest of TFs58.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [58] Zaret, K. S. & Carroll, J. S. Pioneer transcription factors: establishing competence for gene expression. Genes Dev. 25, 2227–2241 (2011). source

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationown data

    The study sheds light on previously described immune-related transcriptional programs where assisted loading was not suggested but seems plausible in light of our findings.

    “Our study also sheds light on previously described immune-related transcriptional programs where assisted loading was not suggested but seems plausible in light of our findings24, 56, 57.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [24] Bevington, S. L. et al. Inducible chromatin priming is associated with the establishment of immunological memory in T cells. EMBO. J. 35, 515–535 (2016). source
    • cites [56] Ostuni, R. et al. Latent enhancers activated by stimulation in differentiated cells. Cell 152, 157–71 (2013). source
    • cites [57] Qiao, Y. et al. Synergistic activation of inflammatory cytokine genes by interferon-gamma-induced chromatin remodeling and toll-like receptor signaling. Immunity 39, 454–69 (2013). source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Differentially regulated H3K27ac or STAT3 sites were identified from three (H3K27ac) or two (STAT3) biological replicates using DESeq.

    “Differentially regulated H3K27ac or STAT3 sites were identified from three (H3K27ac) or two (STAT3) biological replicates using DESeq65 in the default parameters through HOMER66 (http://homer.salk.edu/homer/).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [65] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010). source
    • cites [66] Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010). source

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

    Premisecitation

    Assisted loading has been described in various systems.

    “Assisted loading has been described in various systems30–37.”
    Quote in the paperNot a substantial claim7 of 8 sources checked
    • cites [30] Biddie, S. C. et al. Transcription factor AP1 potentiates chromatin accessibility and glucocorticoid receptor binding. Mol. Cell 43, 145–155 (2011). source
    • cites [31] Grontved, L. et al. C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements. EMBO J 32, 1568–1583 (2013). source
    • cites [32] Miranda, T. B. et al. Reprogramming of the chromatin landscape: interplay of the estrogen and glucocorticoid receptors at the genomic level. Cancer Res. 73, 5130–5139 (2013). source
    • cites [33] Madsen, M. S., Siersbaek, R., Boergesen, M., Nielsen, R. & Mandrup, S. Peroxisome proliferator-activated receptor gamma and C/EBPalpha synergistically activate key metabolic adipocyte genes by assisted loading. Mol. Cell. Biol. 34, 939–954 (2014). source
    • cites [34] Soccio, R. E. et al. Genetic variation determines PPARgamma function and anti-diabetic drug response in vivo. Cell 162, 33–44 (2015). source
    • cites [35] Swinstead, E. E. et al. Steroid receptors reprogram FoxA1 occupancy through dynamic chromatin transitions. Cell 165, 593–605 (2016). source
    • cites [36] Zhu, B. et al. Coactivator-dependent oscillation of chromatin accessibility dictates circadian gene amplitude via REV-ERB loading. Mol. Cell 60, 769–83 (2015). source
    • cites [37] Goldstein, I. et al. Transcription factor assisted loading and enhancer dynamics dictate the hepatic fasting response. Genome Res. 27, 427–439 (2017). source

  • May combine two claims.

    Findingown data

    In the presence of IL-1β, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac.

    “In the presence of IL-1β, NF-κB is activated, binds a subset of enhancers and primes their activity, as evidenced by increasing H3K27ac.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using RNA-seq in primary hepatocytes, IL-1β and IL-6 regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression.

    “Using RNA-seq in primary hepatocytes, we show that these cytokines regulate transcription in a bifurcated manner, leading to both synergistic and antagonistic gene expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The effect of TNF on gene expression was similar but much weaker than that of IL-1β.

    “The effect of TNF on gene expression was similar but much weaker than that of IL-1β (Supplementary Fig. 2a, c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    While IL-6 is sufficient to efficiently activate STAT3 at most sites, IL-1β is required for maximal STAT3 binding in 20% of sites, while reducing it at others.

    “Taken together, these findings reveal that while IL-6 is sufficient to efficiently activate STAT3 at most sites, IL-1β is required for maximal STAT3 binding in 20% of sites, while reducing it at others.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The gene nearest to 63 assisted sites was a synergistic gene compared to only 2 additive genes.

    “Reciprocally, the gene nearest to 63 assisted sites was a synergistic gene compared to only 2 additive genes (Supplementary Data 5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Synergistic gene expression was either completely abolished or severely diminished following two of the p300/CBP inhibitors used (SGC-CBP30 and C646); the third inhibitor (I-CBP112) diminished synergy in four out of six tested genes.

    “Synergistic gene expression was either completely abolished or severely diminished following two of the p300/CBP inhibitors used (SGC-CBP30 and C646). The third inhibitor (I-CBP112) diminished synergy in four out of six tested genes (Fig. 4f, Supplementary Fig. 6).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The p300/CBP inhibitors used (SGC-CBP30, C646, I-CBP112) specifically inhibit p300/CBP-mediated H3K27 acetylation without off-target effects that could affect synergistic gene expression.

  • May combine two claims.

    Findingown data

    When p300/CBP are inhibited, both H3K27 acetylation and STAT3 binding at the Saa1/2 enhancer are negated.

    “However, when p300/CBP were inhibited, both H3K27 acetylation and STAT3 binding were negated (Fig. 7b).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The data meet assumptions of population distribution.
    • Rests on an assumption: Variance between the groups that are being statistically compared is similar.

  • May combine two claims.

    Findingown data

    STAT3 binding is very low following IL-6 treatment and only reaches maximal binding in the dual treatment at assisted sites.

    “(a) STAT3 binding is very low following IL-6 treatment and only reaches maximal binding in the dual treatment (in contrast to unassisted sites that follow the prevalent dogma, i.e. that IL-6 treatment is sufficient for maximal STAT3 binding).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Binding of cJun and CEBPB was similar throughout STAT3 binding sites (cJun binding was modestly increased in unassisted sites), while binding of the NF-κB subunit p65 was significantly enriched around assisted STAT3 sites compared to unassisted sites.

    “In line with motif occurrence, binding of cJun and CEBPB was similar throughout STAT3 binding sites (cJun binding was modestly increased in unassisted sites). Conversely, the binding of the NF-κB subunit p65, was significantly enriched around assisted STAT3 sites compared to unassisted sites (Fig. 5b, c).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    H3K27ac is low at the untreated condition and increases markedly following IL-1β treatment at assisted sites.

    “(b) H3K27ac is low at the untreated condition and increases markedly following IL-1β treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The combinatorial response of inflammatory cytokines is bifurcated, with some genes synergistically induced and others antagonized compared to single cytokine treatments.

    “The combinatorial response of inflammatory cytokines is bifurcated, with some genes synergistically induced while others are antagonized compared to the single cytokine treatments.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the presence of dominant negative NF-κB, the IL-1β-dependent increase in H3K27ac at assisted enhancers is abolished, with H3K27ac remaining similar to untreated cells.

    “Indeed, in the presence of dominant negative NF-κB, the priming effect IL-1β has on assisted enhancers was abolished, keeping H3K27ac at a level similar to that of non-treated cells (Fig. 7a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The data meet assumptions of population distribution.
    • Rests on an assumption: Variance between the groups that are being statistically compared is similar.

  • May combine two claims.

    Findingown data

    In control cells, STAT3 binding is assisted by IL-1β and is associated with a marked increase in H3K27ac.

    “Indeed, in control cells, STAT3 binding was assisted by IL-1β and associated with a marked increase in H3K27ac.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-1β-induced enhancers were enriched for motifs bound by the CEBP, AP-1 and NF-κB TFs, while IL-6-induced enhancers were enriched for STAT3 and ATF3 motifs; the top four motifs enriched in dual-induced enhancers are bound by CEBP, AP-1, NF-κB and STAT3.

    “IL-1β-induced enhancers were enriched for motifs bound by the CEBP, AP-1 and NF-κB TFs, while IL-6-induced enhancers were enriched for STAT3 and ATF3 motifs. The top four motifs enriched in dual-induced enhancers are bound by CEBP, AP-1, NF-κB and STAT3 (Supplementary Fig. 4d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-1β does not lead to 'super' STAT3 binding at assisted sites; rather, IL-1β allows STAT3 binding at assisted sites to reach the level of unassisted sites.

    “When examining the extent of STAT3 binding, we found that IL-1β does not lead to ‘super’ STAT3 binding at assisted sites. Rather, IL-1β allows STAT3 binding at assisted sites to reach the level of unassisted sites (Figs. 4b, c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At unassisted sites, NF-κB does not enhance (and in some cases even perturbs) STAT3 binding.

    “In contrast, at unassisted sites, NF-κB does not enhance (and in some cases even perturbs) STAT3 binding (Fig. 8).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    351 sites (20% of total STAT3 binding events) showed significantly increased STAT3 binding in the dual treatment compared to IL-6, while the majority of sites (80%, n = 1428) were unaffected, or even reduced by addition of IL-1β.

    “When quantifying this phenomenon, we found 351 sites (20% of total STAT3 binding events) where STAT3 binding was significantly increased in the dual treatment compared to IL-6. In contrast, the majority of sites (80%, n = 1428) were unaffected, or even reduced by addition of IL-1β (Fig. 4a, Supplementary Data 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The induction of many genes induced in the individual treatments was negated in the dual treatment, and reciprocally, a significant portion of dual-induced genes was not induced in individual treatments.

    “Notably, the induction of many genes induced in the individual treatments was negated in the dual treatment. Reciprocally, a significant portion of dual-induced genes was not induced in individual treatments (Fig. 1b, Supplementary Data 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    These TFs do not cooperate, and even antagonize each other's activity, in other enhancers and target genes.

    “Conversely, these TFs do not cooperate, and even antagonize each other’s activity, in other enhancers and target genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-6 induces the gene levels of STAT3 (3.3-fold), cJun (1.8-fold) and CEBPB (1.9-fold), while IL-1β induces the gene level of p65 (1.7-fold).

    “Interestingly, IL-6 induces the gene levels of STAT3 (3.3-fold), cJun (the major subunit of AP-1, 1.8-fold) and CEBPB (1.9-fold) while IL-1β induces the gene level of p65 (the principal hepatic NF-κB subunit7, 1.7-fold, Supplementary Data 2).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Acute phase signaling was the most enriched pathway in dual-induced genes (p-value = 1.11 × 10−24, Fisher's exact test) as well as in the sub-group of synergistic genes (p-value = 2.15 × 10−10, Fisher's exact test).

    “Acute phase signaling was the most enriched pathway in dual-induced genes (p-value = 1.11 × 10−24 , Fisher’s exact test) as well as in the sub-group of synergistic genes (p-value = 2.15 × 10−10 Fisher’s exact test).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    K-means clustering of all differentially expressed genes revealed three patterns when comparing dual to single treatments: two antagonistic patterns wherein IL-1β and TNF reduced the expression of IL-6-induced genes, and reciprocally, one profile wherein IL-6 reduces the expression of IL-1β- and TNF-induced genes; in addition, a synergistic pattern in which the cytokines enhance each other's gene-

    “With regards to gene induction, three patterns were observed when comparing the dual to single treatments: two ‘antagonistic’ patterns wherein IL-1β and TNF reduced the expression of IL-6-induced genes, and reciprocally, one profile wherein IL-6 reduces the expression of IL-1β- and TNF-induced genes. In addition, a synergistic pattern in which the cytokines enhance each other’s gene induction”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The level of STAT3 binding at unassisted sites was unaffected (and even sometimes increased) by DN-NFκB, suggesting a highly enhancer-specific augmenting effect of STAT3 binding by NF-κB.

    “In stark contrast, the level of STAT3 binding at unassisted sites was unaffected (and even sometimes increased) by DN-NFκB, suggesting a highly enhancer-specific augmenting effect of STAT3 binding by NF-κB (Fig. 6c, d, Supplementary Fig. 8b, c).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The dominant negative NF-κB peptide specifically inhibits NF-κB activity without affecting other pathways or STAT3 directly.

  • May combine two claims.

    Findingown data

    Synergistic gene induction was unaffected by perturbed CEBPB or AP-1, but was abolished following NF-κB inhibition.

    “Synergistic gene induction was unaffected by perturbed CEBPB or AP-1, but was abolished following NF-κB inhibition (Fig. 6a, Supplementary Fig. 7a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The dominant negative NF-κB peptide specifically inhibits NF-κB activity without affecting other pathways or STAT3 directly.

  • May combine two claims.

    Findingown data

    Synergistic genes showed more RNAP II occupancy compared to the average occupancy of all dual-induced genes combined and compared to single treatments.

    “Interestingly, synergistic genes showed more RNAP II occupancy compared to the average occupancy of all dual-induced genes combined and compared to single treatments (Fig. 2a, Supplementary Fig. 2e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The CEBP and AP-1 motifs occurred at a similar frequency in assisted and unassisted sites, while the NF-κB motif was much more prevalent in assisted sites.

    “While the CEBP and AP-1 motifs occurred at a similar frequency in assisted and unassisted sites, the NF-κB motif was much more prevalent in assisted sites (Fig. 5a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The H3K27ac signal at unassisted sites was similar between the non-treated and IL-1β-treated conditions, while the H3K27ac signal at assisted sites only reached a level similar to unassisted sites following IL-1β treatment.

    “The H3K27ac signal at unassisted sites was similar between the non-treated and IL-1β-treated conditions. However, the H3K27ac signal at assisted sites only reached a level similar to unassisted sites following IL-1β treatment (Figs. 3c, 4e, Supplementary Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-1β treatment did not lead to increases in the total gene or protein levels of STAT3 and did not increase the levels of STAT3 phosphorylation on Tyr705.

    “This enhancer-specific, rather than systemic effect is supported by the finding that IL-1β treatment did not lead to increases in the total gene or protein levels of STAT3 and did not increase the levels of STAT3 phosphorylation on Tyr705, a modification correlated with activity (Supplementary Fig. 8d, Supplementary Data 2).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The rest of STAT3 binding sites are either unaffected or even suppressed by NF-κB.

    “Conversely, the rest of STAT3 binding sites are either unaffected or even suppressed by NF-κB.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither STAT3 gene and protein expression, nor total chromatin-binding levels are affected by IL-1β in our system.

    “Indeed, neither STAT3 gene and protein expression, nor total chromatin-binding levels are affected by IL-1β in our system.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    IL-1β and IL-6 mediate the acute phase response (APR) and regulate the secretion of acute phase proteins in liver.

    “The cytokines interleukin 1β and 6 (IL-1β, IL-6) mediate the acute phase response (APR). In liver, they regulate the secretion of acute phase proteins.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Gabay, C. & Kushner, I. Acute-phase proteins and other systemic responses to inflammation. N. Engl. J. Med. 340, 448–54 (1999). source

  • May combine two claims.

    Interpretationown data

    This facilitates STAT3 binding and synergistic gene expression.

    “This facilitates STAT3 binding and synergistic gene expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    NF-κB enables STAT3 binding at some enhancers while perturbing it at others.

    “Our findings reveal an enhancer-specific crosstalk whereby NF-κB enables STAT3 binding at some enhancers while perturbing it at others.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    These findings correlate NF-κB, but not CEBPB or AP-1, to assisted loading of STAT3.

    “These findings correlate NF-κB, but not CEBPB or AP-1, to assisted loading of STAT3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The enhancers where assisted sites reside are less active prior to IL-1β treatment, a condition unfavorable to STAT3 binding.

    “These data imply that the enhancers where assisted sites reside are less active prior to IL-1β treatment, a condition unfavorable to STAT3 binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Upon IL-1β stimulation, NF-κB is activated and binds enhancers near synergistic genes.

    “Collectively, these observations support a model whereby upon IL-1β stimulation, NF-κB is activated and binds enhancers near synergistic genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    STAT3 binding at assisted sites is perturbed in the lack of IL-1β due to diminished enhancer activity but is increased following enhancer activation by an IL-1β-activated TF, resulting in synergistic gene expression.

    “Collectively, these findings support a scenario whereby STAT3 binding at assisted sites is perturbed in the lack of IL-1β due to diminished enhancer activity but is increased following enhancer activation by an IL-1β-activated TF, resulting in synergistic gene expression.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: IL-1β does not directly activate STAT3.

  • May combine two claims.

    Interpretationown data

    Genes induced following dual IL-1β + IL-6 treatment in primary mouse hepatocytes faithfully represent gene expression patterns seen in acute and chronic inflammation.

    “We conclude that genes induced following dual IL-1β + IL-6 treatment in primary mouse hepatocytes faithfully represent gene expression patterns seen in acute and chronic inflammation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    This enhancer-specific crosstalk between TFs reconciles the seemingly contradictory observations of pro-inflammatory TFs in liver and reveals the mode of action behind inflammatory gene regulation.

    “This enhancer-specific crosstalk between TFs reconciles the seemingly contradictory observations of pro-inflammatory TFs in liver2 and reveals the mode of action behind inflammatory gene regulation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Bode, J. G., Albrecht, U., Haussinger, D., Heinrich, P. C. & Schaper, F. Hepatic acute phase proteins--regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-kappaB-dependent signaling. Eur. J. Cell Biol. 91, 496–505 (2012). source

  • May combine two claims.

    Interpretationown data

    Enhancers harboring assisted sites are somewhat quiescent in the untreated condition and are 'primed' for STAT3 binding by IL-1β.

    “Thus, enhancers harboring assisted sites are somewhat quiescent in the untreated condition and are ‘primed’ for STAT3 binding by IL-1β.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    The primer used in this study could not differentiate between Saa1 and Saa2 due to their highly similar sequence.

    “Due to the highly similar sequence of the Saa1 and Saa2 genes, the primer used in this study could not differentiate between them (and neither did any other primer we tested).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Assisted loading of STAT3 by NF-κB is at play during inflammation, allowing a bifurcated gene regulation program and a fine-tuned crosstalk between the two TFs.

    “We suggest that assisted loading of STAT3 by NF-κB is at play during inflammation, allowing a bifurcated gene regulation program and a fine-tuned crosstalk between the two TFs.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Two replicates were used for each treatment in RNA-seq.

    “Two replicates were used for each treatment.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All qPCR experiments were replicated at least three independent times.

    “All qPCR experiments were replicated at least three independent times.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    This phenomenon resolves some of the confusion regarding the complex crosstalk between the two TFs and unfolds critical regulatory events in acute and chronic inflammation.

    “This phenomenon resolves some of the confusion regarding the complex crosstalk between the two TFs and unfolds critical regulatory events in acute and chronic inflammation.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All RNA-seq and ChIP-seq p-values were adjusted for multiple comparisons.

    “All RNA-seq and ChIP-seq p-values were adjusted for multiple comparisons.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The primers used in this study (except for Tbp) were designed to amplify nascent transcripts spanning exon-intron junctions as a proxy for transcription to avoid confounding post-transcriptional events.

    “The primers used in this study (except for Tbp) were designed to amplify nascent transcripts (i.e., the amplified region span exon-intron junctions) as a proxy for transcription to avoid confounding post-transcriptional events.”
    Quote in the paperSubstantial claim
59 of 59

  • The paper relies on this without support.

    IL-1β does not directly activate STAT3.

    “Because IL-1β does not directly activate STAT3 but is still essential for maximal STAT3 binding at a subset of sites, we termed these ‘assisted sites,’”

  • The paper relies on this without support.

    The p300/CBP inhibitors used (SGC-CBP30, C646, I-CBP112) specifically inhibit p300/CBP-mediated H3K27 acetylation without off-target effects that could affect synergistic gene expression.

    “To that end, we inhibited the two major enzymes catalyzing H3K27 acetylation, p300 and CREB binding protein (CBP).”

  • The paper relies on this without support.

    The dominant negative NF-κB peptide specifically inhibits NF-κB activity without affecting other pathways or STAT3 directly.

    “Next, we profiled STAT3 binding in the presence of a NF-κB dominant negative peptide (DN-NFκB).”

  • The paper relies on this without support.

    The data meet assumptions of population distribution.

    “The data meet assumptions of population distribution.”

  • The paper relies on this without support.

    Variance between the groups that are being statistically compared is similar.

    “Variance between the groups that are being statistically compared is similar.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

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.

  • Different cutoffs are given for differential STAT3 binding sites

    Major

    The Fig. 4 caption defines assisted STAT3 sites using a fold-change cutoff of 1.7 and p-value cutoff of 0.01. The Methods instead specify a fold-change cutoff of 2 and an adjusted p-value cutoff of 0.1 for differential STAT3 sites, leaving the criteria for this central group inconsistent.

  • Non-treated STAT3 peak percentage does not match the stated total

    Minor

    Of 2,975 peaks, 39 are 1.31%, which rounds to 1.3%, not the reported 1.2%.

    Reported 1.2 · recomputed 1.310924

2 of 2

Cites

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Related

Authors
Goldstein, I., Paakinaho, V., Baek, S., Sung, M.-H., & Hager, G. L.
Affiliations
National Institutes of Health (US)
Corresponding author
  • Goldstein, Ido
  • Hager, Gordon L.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-11-29
Volume
8
Issue
1
Pages
1849-1849
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-02055-5
PMC
PMC5707366
OpenAlex
W2770530503
Cited by
97
References
71
Citations by year
2026: 12 · 2025: 8 · 2024: 17 · 2023: 8 · 2022: 14 · 2021: 11 · 2020: 13 · 2019: 10 · 2018: 4
FWCI
2.91
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Sigrid Juséliuksen Säätiö
  • National Institutes of Health
  • National Institute on Aging
  • National Cancer Institute
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
acute phase response, IL-1β, interleukin-6, STAT3, NF-κB, synergistic gene expression, hepatocytes, acute phase proteins, H3K27ac, transcription factor binding, chromatin occupancy
MeSH terms
Acute-Phase Reaction, Animals, Binding Sites, Cells, Cultured, Enhancer Elements, Genetic, Gene Expression Regulation, Male, Mice, Inbred C57BL, RNA Polymerase II, Transcription Factors, Interleukin-6, NF-kappa B, Hepatocytes, Chromatin Immunoprecipitation, STAT3 Transcription Factor, Interleukin-1beta

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.

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